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Warren, Charles R.; Manzoni, Stefano
When dry soil is re-wet, trehalose is respired instead of supporting microbial growth Journal Article
In: Soil Biology and Biochemistry, 2023, ISSN: 0038-0717.
Liang, Grace H.; Butler, Orpheus M.; Warren, Charles R.
Lipid profiles of plants and soil microbial communities are shaped by soil parent material in Australian sclerophyll forests Journal Article
In: Plant Soil, 2023, ISSN: 1573-5036.
Plant and soil microbes can reduce their phosphorus (P)-requirements by replacing phospholipids with non-P containing lipids (e.g., galactolipids, sulfolipids, and betaine lipids). There have been few studies of this process in the field (i.e., in natural ecosystems); thus, it is unclear whether a similar replacement of phospholipids with non-P lipids occurs across natural gradients of soil P-availability.
We compared the membrane lipid profiles of plant leaves, roots, and soil microbial communities between two adjacent native Australian sclerophyll forest ecosystems—one situated on a severely P-deficient sandstone-derived soil and the other on a comparatively P-rich shale-derived soil. The herbaceous species,
The phospholipid content of plant leaves and soil microbes was two- to three-fold greater in the shale site than the sandstone site, but non-P lipid content did not differ between sites.
Our results indicate that plants and soil microbes can have a lower investment of P into phospholipids in response to P-deficiency without a concomitant increase in non-P lipid content. Modulations in phospholipid concentration occurred across all plant- and soil microbial-associated phospholipid classes.
Pasut, Chiara; Tang, Fiona H. M.; Minasny, Budiman; Warren, Charles R.; Dijkstra, Feike A.; Riley, William J.; Maggi, Federico
Seasonal biotic processes vary the carbon turnover by up to one order of magnitude in wetlands Journal Article
In: Global Biogeochemical Cycles, 2023.
Soil Organic Carbon (SOC) turnover τ in wetlands and the corresponding governing processes are still poorly represented in numerical models. τ is a proxy to the carbon storage potential in each SOC pool and C fluxes within the whole ecosystem; however, it has not been comprehensively quantified in wetlands globally. Here, we quantify the turnover time τ of various SOC pools and the governing biotic and abiotic processes in global wetlands using a comprehensively-tested process-based biogeochemical model. Globally, we found that τ ranges between 1 and 1,000 years and is controlled by anaerobic (78% of wetlands area) and aerobic (15%) respiration, and by abiotic destabilization from soil minerals (5%). τ in the remaining 2% of wetlands is controlled by denitrification, sulfur reduction, and leaching below the subsoil. τ can vary by up to one order of magnitude in temperate, continental, and polar regions due to seasonal temperature, and can shift from being aerobically-controlled to anaerobically-controlled. Our findings of seasonal variability in SOC turnover suggest that wetlands are susceptible to climate-induced shifts in seasonality, thus requiring better accounting of seasonal fluctuations at geographic scales to estimate C exchanges between land and atmosphere.
Warren, Charles R.; Butler, Orpheus M.
Turnover of non-polymeric leaf lipids in a loamy grassland soil Journal Article
In: Plant and Soil, 2023.
Leaves constitute a large input of lipids to soil, yet comparatively little is known about the fate of leaf lipids in soil. Our aim was to explore the initial stages of degradation of leaf lipids, both the loss of intact lipid and subsequent mineralisation. We focussed on intracellular lipids – triacylglycerols implicated in storage, membrane lipids such as phospholipids and galactolipids, and pigments – because they collectively constitute more than 1% of leaf mass.
A mixture of U-13C lipids was extracted from leaves of wheat grown with 13CO2. The lipid mixture included the range of plant lipids soluble in organic solvent (e.g. free fatty acids, acylglycerols, pigments) but not polymeric lipids such as cutin and suberin. Mineralisation was deduced from 13CO2 efflux, while LC–MS examined degradation of intact 13C lipids.
There was no delay before lipids were mineralised. Instead, mineralisation was significant within minutes and reached a maximum within three hours. There was rapid loss (i.e. degradation) of a broad range of intact lipids including phospholipids, galactolipids, pigments (chlorophylls), and triacylglycerols. Around two-thirds of added lipid-C was respired over the course of 15 days, with one-third of lipid-C persisting in soil.
Our study indicates that non-polymeric leaf lipids degrade quickly in soil, yet a fraction of lipid-C likely persisted in degradation products and/or microbial biomass. Persistence of lipid-C probably also reflected the presence of lipids that are more resistant to degradation (e.g. phaeophytins), and a fraction of added lipid being protected (e.g. by interaction with clays).
Warren, Charles R.
LC-MS analysis of D2O-labelled soil suggests a large fraction of membrane lipid exists within slow growing microbes Journal Article
In: Soil Biology and Biochemistry, vol. 177, pp. 108912, 2023.
Measuring the growth of microbes is essential for understanding soil function, yet it remains challenging. Knowing the rate of synthesis of polar lipids that comprise microbial membranes would serve as an indicator of microbial growth, and also improve understanding of the biogeochemical cycling of polar lipids. The aim of this study was to develop D2O-labelling of intact polar lipids (D2O-intact lipid-SIP) as a culture- and gene-independent way of probing synthesis of membrane lipids by soil microbes. To test the feasibility of coupling D2O labelling with liquid chromatography-mass spectrometry (LC-MS) quantification, and determine appropriate incubation durations, we incubated two soils with 50 atom% D2O for durations between 3 h and 46 days. We first improved the chromatographic separation to reduce the number of co-eluting lipids and enable easier quantification of D2O enrichment. Increasing column length from 150 mm to 500 mm and the gradient separation from 45 to 300 min increased 3.4-fold the number of resolved peaks, and meant that chromatographically co-eluting lipids were generally separated in the mass dimension by a minimum of ten Da. Quantification of 2H enrichment of a broad spectrum of lipids was possible in soils incubated with 50 atom% D2O for 27 h. Enrichment and rate of lipid synthesis were determined for 34–38 membrane lipids that collectively accounted for >80% of the lipid pool. There was ten-fold variation among individual lipids in rates of synthesis and turnover, which presumably reflects differences in growth rate and abundance of soil taxa. Turnover differed among classes from 1.2 to 1.5 years in phosphatidylcholine, 0.54–0.98 years in diacylglyceryl-N,N,N-trimethylhomoserine, and 0.42–0.72 years in phosphatidylethanolamine. By quantifying the isotopologue distribution, LC-MS revealed the average turnover comprised a large population of lipid that remained unlabelled (93–95% unlabelled after 27 h and 66–75% unlabelled after 46 days), and a small population of lipid that had incorporated varying numbers of 2H. These observations are consistent with much of the microbial lipid pool residing in microbes with negligible growth, and a smaller proportion in microbes with a range of growth rates. Accordingly turnover calculated solely for the active fraction of lipid was a more rapid 20–30 days. The slow average turnover of membrane lipids likely also reflects efficient microbial recycling of lipids reducing the amount of lipid synthesis required to support growth.
Grover, Samantha; Grover, Samantha; Tate, Jack; Tate, Jack; Warren, Charles; Warren, Charles; Venn, Susanna; Venn, Susanna
Nitrogen dynamics in alpine soils of south-eastern Australia Journal Article
In: Soil Research, 2023.
Context: The Australian Alps are recognised by UNESCO as a globally significant mountain range. Soils underpin all of these ecosystem services. However, sparse data exists on alpine soils.
Aims and methods: We explored nitrogen dynamics of soils from four high mountain sites, using a combination of new and established field and laboratory techniques.
Key results: Organic and inorganic N were of the same order of magnitude, with around twice as much inorganic N as organic N. Forty three small (<250 Da) organic N compounds were detected, with concentrations 30 times greater in microbial and salt-extractable pools than free in the soil solution. The net N mineralisation rate decreased four-fold over the growing season. The organic matter decomposition rate was close to the global mean (k = 0.017), while the stabilisation factor was high (0.28) in comparison with other ecosystems globally.
Conclusions: These results begin to illuminate the complexity of the belowground processes that have formed the high C soils of the Australian Alps. The combination of moderate turnover times and high stabilization of organic matter support Costin’s theory that these mountain soils formed in place as a result of biological activity, rather than reflecting their geological substrata. The pools of organic N adsorbed to mineral soil surfaces and bound up within microbes lend support to a theory of tight N cycling, with little organic or inorganic N free in the soil solution.
Implications: This new knowledge of soil N dynamics can support land managers to design successful restoration works to preserve alpine soil ecosystem services impacted by climate change, feral animal disturbance, weed invasion and the increase in summer tourism infrastructure.
Rabbi, Sheikh M. F.; Warren, Charles R.; Macdonald, Catriona; Trethowan, Richard M.; Young, Iain M.
Soil-root interaction in the rhizosheath regulates the water uptake of wheat Journal Article
In: Rhizosphere, vol. 21, pp. 100462, 2022.
The development of crops better adapted to endure weather extremes requires knowledge of the belowground traits that have potential to improve plant water uptake. Recent evidence has indicated that root induced modification of soil pore geometry of the rhizosheath is linked with drought tolerance of crops. Here we sought to understand whether the regulation of plant water uptake by rhizosheath is mediated through the rhizodeposits present at the soil-root interface. We compared eight wheat cultivars and demonstrated that cultivars with longer root hairs and greater rhizosheath mass and cover transpired more water. The wheat cultivars with less rhizosheath had the highest concentration of trehalose in the rhizosheath. Trehalose may increase the surface tension of soil water, which could further modify the transpiration of cultivars with less rhizosheath. Moreover, rhizosheath mass and cover had significant negative association with the bacterial abundance, indicating a link between metabolite concentration and bacterial abundance in the rhizosheath. We propose that the complex interactions of root, microbes and rhizodeposits in the rhizosheath have the potential to regulate water uptake by crops. The root and rhizosheath traits highlighted here can be targeted to develop crops with better ability to uptake water.
Warren, Charles R.
D2O labelling reveals synthesis of small, water-soluble metabolites in soil Journal Article
In: Soil Biology and Biochemistry, vol. 165, pp. 108543, 2022.
Small, water-soluble metabolites (e.g. amino acids, sugars) occupy central positions in biogeochemical cycling, yet for most metabolites we do not know rates of de novo synthesis. The aim of this study was to estimate synthesis of water-soluble metabolites by developing a workflow based on D2O labelling and mass spectrometry quantification of 2H-enrichment. Initial tests established that labelling up to 87 atom% soil water with D2O did not affect soil respiration, suggesting that the smaller (typically ≤60 atom% D2O) labelling of most experiments was unlikely to affect microbial activity. To examine 2H incorporation from D2O, four contrasting soils were spiked with 99.9% D2O leading to 46–61 atom% water present as D2O. Soils were incubated from ten to 240 days before extraction, then isotopologues of a broad range of water-soluble metabolites were quantified by gas chromatography-mass spectrometry and capillary electrophoresis-mass spectrometry. There were dramatic differences among metabolites in the rate of 2H-enrichment. Mannitol, Asp, Asn, Glu, Gln, approached the theoretical maximum 2H enrichment after ten days, indicating that turnover of the metabolite pool due to synthesis occurred at a time scale of around ten days; whereas muramic acid was less than 20% of maximum 2H enrichment after 240 days suggesting turnover due to de novo synthesis was likely on the time scale of several years. Previous studies of the same and similar water-soluble metabolites reported rapid metabolite fluxes into and out of the soil solution led to residence time in solution of 1 h or less. The slow turnover of metabolite pools due to synthesis indicates the fluxes of metabolites in and out of cells is dominated by community-level recycling.
Israel, D; Khan, S; Warren, C R; Zwiazek, J J; Robson, M T
The contribution of PIP2-type aquaporins to photosynthetic response to increased vapour pressure deficit. Journal Article
In: Journal of Experimental Botany, vol. 72, pp. 5066-5078, 2021.
The roles of different plasma membrane aquaporins (PIPs) in leaf-level gas exchange of Arabidopsis thaliana were examined using knockout mutants. Since multiple Arabidopsis PIPs are implicated in CO2 transport across cell membranes, we focused on identifying the effects of the knockout mutations on photosynthesis, and whether they are mediated through the control of stomatal conductance of water vapour (gs), mesophyll conductance of CO2 (gm), or both. We grew Arabidopsis plants in low and high humidity environments and found that the contribution of PIPs to gs was larger under low air humidity when the evaporative demand was high, whereas any effect of a lack of PIP function was minimal under higher humidity. The pip2;4 knockout mutant had 44% higher gs than wild-type plants under low humidity, which in turn resulted in an increased net photosynthetic rate (Anet). We also observed a 23% increase in whole-plant transpiration (E) for this knockout mutant. The lack of functional plasma membrane aquaporin AtPIP2;5 did not affect gs or E, but resulted in homeostasis of gm despite changes in humidity, indicating a possible role in regulating CO2 membrane permeability. CO2 transport measurements in yeast expressing AtPIP2;5 confirmed that this aquaporin is indeed permeable to CO2.
What are the products of enzymatic cleavage of organic N? Journal Article
In: Soil Biology and Biochemistry, vol. 154, 2021.
Nitrogen commonly limits productivity, yet most soils contain a substantial pool of N in organic forms that are too large for rapid direct uptake. These larger forms of organic N generally become available only after they have been cleaved into smaller units by extracellular enzymes. Enzymatic cleavage of organic N is often equated with complete depolymerisation of protein to amino acids, yet it is likely that small peptides are also products of depolymerisation and there is a suite of other N-containing compounds that could be produced by enzymatic cleavage. The aims of this study were to a) characterize in a range of soils the pools of organic N that represent the substrates and products of enzymatic cleavage, viz., hydrolysable N and dissolved organic nitrogen (DON); b) develop a workflow for the untargeted identification and quantification of the products of enzymatic cleavage of organic N, and c) build a picture of the quantitative significance of different compounds for N cycling by determining their rates of production by enzymatic cleavage. Among a range of soils the pool of <3 kDa DON was quantitatively dominated by protein amino acids (69% of <3 kDa DON in H2O and K2SO4 extracts) and small peptides (15% of DON in H2O extracts and 26% in K2SO4 extracts). Only a small fraction of total hydrolysable N and DON was accounted for by compounds that originate from microbial cell walls (viz. hexosamines, muramic acid, diaminopimelic acid). To determine the products of enzymatic cleavage, soils were vacuum infiltrated with chloroform gas for five days to halt microbial metabolism, then enzymatic cleavage was assessed by incubating with water + casein (potential protein depolymerisation) or water alone (enzymatic cleavage of endogenous organic matter). Reaction products were identified and quantified using mass spectrometry, with rates of production determined from the increase between 30 min and 24 h of incubation. When extra substrate was added in the form of casein, small peptides were the primary products of depolymerisation, suggesting that assays of potential protease should be interpreted in terms of partial depolymerisation to peptides. Enzymatic cleavage of endogenous organic matter resulted in production of small peptides (generally < 500 Da) and amino acids in approximately equal amounts. Hexosamines, muramic acid, and diaminopimelic acid accounted for less than 1% of the products of enzymatic cleavage, while at least 2% was accounted for by nucleobases + nucleosides and headgroups of membrane lipids. We conclude that the workflow described here coupling chloroform gas infiltration with untargeted mass spectrometry of reaction products can reveal the diversity of compounds produced during enzymatic cleavage of organic matter. Our analysis confirmed that amino acids and peptides quantitatively dominate depolymerisation, and that decomposition of nucleic acids and lipids may supply significant amounts of N and ought to be the subject of further study. © 2021 Elsevier Ltd
Rabbi, S M F; Tighe, M K; Warren, C R; Zhou, Y; Denton, M D; Barbour, M M; Young, I M
High water availability in drought tolerant crops is driven by root engineering of the soil micro-habitat Journal Article
In: Geoderma, vol. 383, 2021.
Improving our understanding of drought tolerance of crops is essential in light of future predicted changes in rainfall, decreased groundwater availability, and increasing temperatures. With a focus on above ground traits, significant improvements in drought tolerance of plants has occurred. With such gains plateauing, we have sought to quantify the belowground functional interactions between plant roots and soil in relation to drought tolerance. Using physical, chemical and biological approaches, we compared drought tolerant and sensitive model plants to demonstrate that a tolerant plant alters both the surrounding pore geometry and the relative abundance of bacteria and upregulates the development of a slow wetting rhizosheath, which increases water uptake under drought conditions. We propose that such rhizosheath traits can be targeted to modify the biophysical properties of the rhizosheath to access water in drought conditions. © 2020 Elsevier B.V.
Warren, C R
Altitudinal transects reveal large differences in intact lipid composition among soils Journal Article
In: Soil Research, 2021.
Fatty acid-based lipids comprise a small but important component of soil organic matter. Lipids are indispensable components of soil microbes due to their function as components of membranes and as stores of energy and C. Hence, lipid composition is likely under strong selection pressure and there ought to be strong associations between lipid composition of microbial communities and environmental conditions. Associations between microbial lipids and environment likely involve an integrated combination of differences in lipid headgroups (classes) and fatty acyl chains. However, past studies examining associations between soil lipid composition and environmental conditions have focussed on fatty acids hydrolysed from polar lipids and less is known about headgroups (classes) of polar lipids. The aim of this study was to examine associations between environmental conditions changing with altitude and the intact polar and non-polar lipids of soil microbial communities. We used two altitudinal transects, both spanning from forest through to above the alpine treeline, but separated from one another by ∼700 km. Liquid chromatography-mass spectrometry identified 174 intact lipids to the level of class and sum composition. Approximately half of the pool of fatty acid-based lipids was accounted for by two classes of non-polar lipids (diacylglycerol and triacylglycerols), while the other half was dominated by three classes of polar lipids (phosphatidylethanolamine, phosphatidylcholine and diacylglyceryl-N,N,N-trimethylhomoserine). There were large differences among sites in the relative amounts of lipid classes. For example, diacylglyceryl-N,N,N-trimethylhomoserine varied among sites from 5 to 41% of the polar lipid pool, phosphatidylcholine from 31 to 60% of the polar lipid pool, and diacylglycerols from 9 to 53% of the total non-polar pool. Relationships of lipid composition with altitude were weak or differed between transects, and pH was the variable most strongly associated with lipid composition. Variation among sites in the relative abundance of phosphatidylcholine were positively associated with pH, while relative and absolute abundance of diacylglycerol was negatively related to pH. We suggest that the accumulation of diacylglycerol at low pH represents slowed hydrolysis and/or microbial utilisation. A large fraction of variance among sites in lipid composition remained unexplained, which highlights the need for additional research on processes leading to production and consumption of fatty acid-based lipids. © 2021 CSIRO.
Manzoni, Stefano; Ding, Yang; Warren, Charles; Banfield, Callum C.; Dippold, Michaela A.; Mason-Jones, Kyle
Intracellular Storage Reduces Stoichiometric Imbalances in Soil Microbial Biomass – A Theoretical Exploration Journal Article
In: Frontiers in Ecology and Evolution, vol. 9, pp. 663, 2021, ISSN: 2296-701X.
Microbial intracellular storage is key to defining microbial resource use strategies and could contribute to carbon (C) and nutrient cycling. However, little attention has been devoted to the role of intracellular storage in soil processes, in particular from a theoretical perspective. Here we fill this gap by integrating intracellular storage dynamics into a microbially explicit soil C and nutrient cycling model. Two ecologically relevant modes of storage are considered: reserve storage, in which elements are routed to a storage compartment in proportion to their uptake rate, and surplus storage, in which elements in excess of microbial stoichiometric requirements are stored and limiting elements are remobilized from storage to fuel growth and microbial maintenance. Our aim is to explore with this model how these different storage modes affect the retention of C and nutrients in active microbial biomass under idealized conditions mimicking a substrate pulse experiment. As a case study, we describe C and phosphorus (P) dynamics using literature data to estimate model parameters. Both storage modes enhance the retention of elements in microbial biomass, but the surplus storage mode is more effective to selectively store or remobilize C and nutrients according to microbial needs. Enhancement of microbial growth by both storage modes is largest when the substrate C:nutrient ratio is high (causing nutrient limitation after substrate addition) and the amount of added substrate is large. Moreover, storage increases biomass nutrient retention and growth more effectively when resources are supplied in a few large pulses compared to several smaller pulses (mimicking a nearly constant supply), which suggests storage to be particularly relevant in highly dynamic soil microhabitats. Overall, our results indicate that storage dynamics are most important under conditions of strong stoichiometric imbalance and may be of high ecological relevance in soil environments experiencing large variations in C and nutrient supply.
AusTraits, a curated plant trait database for the Australian flora. Journal Article
In: Scientific data, 2021.
We introduce the AusTraits database - a compilation of values of plant traits for taxa in the Australian flora (hereafter AusTraits). AusTraits synthesises data on 448 traits across 28,640 taxa from field campaigns, published literature, taxonomic monographs, and individual taxon descriptions. Traits vary in scope from physiological measures of performance (e.g. photosynthetic gas exchange, water-use efficiency) to morphological attributes (e.g. leaf area, seed mass, plant height) which link to aspects of ecological variation. AusTraits contains curated and harmonised individual- and species-level measurements coupled to, where available, contextual information on site properties and experimental conditions. This article provides information on version 3.0.2 of AusTraits which contains data for 997,808 trait-by-taxon combinations. We envision AusTraits as an ongoing collaborative initiative for easily archiving and sharing trait data, which also provides a template for other national or regional initiatives globally to fill persistent gaps in trait knowledge.
Warren, C R
Pools and fluxes of osmolytes in moist soil and dry soil that has been re-wet Journal Article
In: Soil Biology and Biochemistry, vol. 150, 2020.
When dry soils are re-wet there is a pulse of soil respiration that could be partially fuelled by the osmolytes that had accumulated within microbial biomass during the preceding drying phase. We hypothesize that if the pulse of respiration in re-wet soil is because microbes respond to the hypo-osmotic shock of re-wetting by expelling osmolytes and then re-capture osmolytes for use as respiratory substrates: a) rewetting will lead to a massive flux of osmolytes from microbial biomass into the extracellular fraction of soil, and b) following rewetting there will be a decrease in the total pool of osmolytes (i.e. in microbial biomass + the extracellular fraction of soil) that is proportional to the extra C that is respired by rewet soil compared to permanently moist soil. We used mass spectrometry and isotope pool dilution to estimate intracellular and extracellular pools, and extracellular fluxes, of seven putative osmolytes (trehalose, betaine, carnitine, hercynine, acetyl-carnitine, choline, proline) and the protein amino acid alanine in a soil that was permanently moist and a soil that had been slowly dehydrated and was then re-wet to the same water content as the permanently moist soil. In moist soil, the influx and efflux of trehalose through the extracellular pool was around 50% faster than of alanine and proline, and all three compounds had mean residence times in the extracellular solution of less than 10 min. Quaternary ammonium compounds (betaine, carnitine, hercynine, acetyl-carnitine, choline) had gross fluxes 5 to 33 times slower than for trehalose, but despite these slower fluxes mean residence times were 45 min or less. Microbial biomass of dry soil had a three times larger pool of osmolyte-C than moist soil, while the extracellular fraction of dry soil had a six times larger pool of osmolyte-C than the moist soil. Microbial biomass of dry soil was 2.5–4 times smaller than for permanently moist soil, and thus per unit microbial biomass the amount of osmolyte-C in dry soil was 7.5 to 12 times greater than the moist soil. The contribution of intracellular osmolytes to solute potential was likely too small to maintain turgor in the dry soil, and thus it is possible the large extracellular and intracellular accumulation of osmolytes was functioning to stabilise membranes and foster desiccation tolerance. The fluxes of osmolytes into and out of solution resumed rapidly after re-wetting. There was no evidence that rewetting led to an increased flux of osmolytes from microbial to extracellular pools, as would be expected if re-wetting caused microbes to expel osmolytes or be lysed. Extracellular pools of most osmolytes changed little following re-wetting, whereas the extracellular pool of trehalose decreased significantly. Isotope pool dilution revealed that the constant extracellular concentrations of most osmolytes following rewetting was because rates of removal from solution balanced the rates of entry, while the decreasing extracellular pool of trehalose reflected that it was removed from solution almost three times faster than it entered solution. The decreasing extracellular and intracellular pools of osmolytes, especially trehalose, following rewetting could theoretically account for the faster respiration of re-wet soil than permanently moist soil for several hours. However, respiration of re-wet soil was faster than permanently moist soil for five days following re-wetting and over this longer time scale the respiration pulse must be fuelled by sources of C in addition to osmolytes. © 2020 Elsevier Ltd
Chavan, S G; Maier, C; Alagoz, Y; Filipe, J C; Warren, C R; Lin, H; Jia, B; Loik, M E; Cazzonelli, C I; Chen, Z H; Ghannoum, O; Tissue, D T
Light-limited photosynthesis under energy-saving film decreases eggplant yield Journal Article
In: Food and Energy Security, vol. 9, no. 4, 2020.
Glasshouse films with adjustable light transmittance and energy-efficient designs have the potential to reduce (up to 80%) the high energy cost for greenhouse horticulture operations. Whether these films compromise the quantity and quality of light transmission for photosynthesis and crop yield remains unclear. A “Smart Glass” film ULR-80 (SG) was applied to a high-tech greenhouse horticulture facility, and two experimental trials were conducted by growing eggplant (Solanum melongena) using commercial vertical cultivation and management practices. SG blocked 85% of ultraviolet (UV), 58% of far-red, and 26% of red light, leading to an overall reduction of 19% in photosynthetically active radiation (PAR, 380–699 nm) and a 25% reduction in total season fruit yield. There was a 53% (season mean) reduction in net short-wave radiation (radiometer range, 385–2,105 nm upward; 295–2,685 nm downward) that generated a net reduction of 8% in heat load and reduced water and nutrient consumption by 18%, leading to improved energy and resource use efficiency. Eggplant adjusted to the altered SG light environment via decreased maximum light-saturated photosynthetic rates (Amax) and lower xanthophyll de-epoxidation state. The shift in light characteristics under SG led to reduced photosynthesis, which may have reduced source (leaf) to sink (fruit) carbon distribution, increased fruit abortion and decreased fruit yield, but did not affect nutritional quality. We conclude that SG increases energy and resource use efficiency, without affecting fruit quality, but the reduction in photosynthesis and eggplant yield is high. The solution is to re-engineer the SG to increase penetration of UV and PAR, while maintaining blockage of glasshouse heat gain. © 2020 The Authors. Food and Energy Security published by John Wiley & Sons Ltd on behalf of Association of Applied Biologists
Warren, C R
Soil microbial populations substitute phospholipids with betaine lipids in response to low P availability Journal Article
In: Soil Biology and Biochemistry, vol. 140, 2020.
Soil microbes can be limited by phosphorus availability. A significant proportion of cellular P is accounted for by the phospholipids that comprise membranes. Experiments with aquatic microbial communities and isolated microbes have shown one way microbes can reduce their cellular P requirements is by substituting phospholipids with P-free polar lipids. We do not know if soil microbial communities can also spare P by using P-free lipids instead of phospholipids. The aims of this study were to examine a range of contrasting soils and a) determine the profile of polar fatty-acid-based lipids; and b) examine if P-free polar lipids are more abundant in P-poor soils. To obtain a broad range in P availability we examined soils from 16 sites that encompassed a range of geologies and vegetation types. Intact lipids were identified and quantified using liquid-chromatography-mass spectrometry. An independent estimate of polar lipid classes was obtained by hydrolysing lipids and quantifying headgroups with capillary electrophoresis-mass spectrometry. Among sites soil P status varied from supra-optimal (N:P = 2.4 g g−1) through to severely deficient (N:P = 80 g g−1). The dominant polar lipids among sites were phosphatidylethanolamine (PE), phosphatidylcholine (PC), and the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS). Among sites as P availability declined a larger proportion of polar lipids was accounted for by betaine lipids such that in the most severely P-deficient soils phospholipids were only 61% of total polar lipids. If we assume that phospholipids account for one-third of cellular P, the measured substitution of phospholipids with betaine lipids would reduce the amount of P required for growth by around 10%. The substantial P saving due to lipid substitution could contribute to the selection of taxa under P deficiency. © 2019
Warren, C R
Isotope pool dilution reveals rapid turnover of small quaternary ammonium compounds Journal Article
In: Soil Biology and Biochemistry, vol. 131, pp. 90-99, 2019.
Studies of how nitrogen (N) becomes available have tended to focus on depolymerisation of proteins and subsequent uptake of amino acids, yet amino acids and their polymers are not the only organic N compounds in soil. For example, concentrations of small quaternary ammonium compounds in the extracellular fraction of soil are often comparable to those of amino acids. We know that there is rapid turnover of amino acids in the extracellular fraction of soil, but there are no comparable data for small quaternary ammonium compounds. The most common function of small quaternary ammonium compounds is as intracellular osmolytes, which leads to the hypothesis that at constant osmolarity microbes would neither efflux nor take up osmolytes and thus there would be negligible flux of quaternary ammonium compounds through the extracellular fraction of soil. To test this hypothesis isotope pool dilution was used to compare gross fluxes of three quaternary ammonium compounds (betaine, carnitine and hercynine) with gross fluxes of two amino acids (glycine, alanine) in three different soils. Contrary to our hypothesis extracellular pools of betaine, carnitine and hercynine were highly dynamic and turned over rapidly. For example, in one soil the mean residence times of all quaternary ammonium compounds varied between 6 and 14 min such that pools would have turned over 100 to 240 times per day. Mean residence times of quaternary ammonium compounds were notably longer in soils from two other sites, yet remained shorter than 60 min such that pools would turnover many tens of time per day. Gross fluxes of betaine, carnitine and hercynine through the extracellular pool were 3–75 times slower than for the amino acid alanine, and thus fluxes of small quaternary ammonium compounds likely account for only a small fraction of the bio-available N that is produced and consumed. The combination of small concentrations and short residence times suggest there is intense microbial competition and active uptake of betaine, carnitine and hercynine from the extracellular fraction of soil. © 2019 Elsevier Ltd
Warren, C R
Does silica solid-phase extraction of soil lipids isolate a pure phospholipid fraction? Journal Article
In: Soil Biology and Biochemistry, vol. 128, pp. 175-178, 2019.
Phospholipids are isolated from crude lipid extracts by silica solid phase extraction (SPE), but for soils we don't know if phospholipids are the only fatty acid-based lipids present in the polar lipid fraction. Lipids extracted from three soils were fractionated with a silica SPE protocol commonly used for soils, with “neutrals” eluted by chloroform, “glycolipids” eluted by acetone, and “phospholipids” eluted by methanol. Fatty acid-based lipids were identified and quantified by liquid chromatography-mass spectrometry. Phospholipids were recovered in the methanol fraction, but this fraction also included betaine lipids. In two soils the methanol fraction was 3–6% betaine lipid while in one soil betaine lipids accounted for 48% of lipids in the methanol fraction. Clearly the fraction obtained by eluting lipids from silica with methanol is not purely phospholipid but can contain significant amounts of other polar lipids. © 2018 Elsevier Ltd
Warren, C R
A liquid chromatography–mass spectrometry method for analysis of intact fatty-acid-based lipids extracted from soil Journal Article
In: European Journal of Soil Science, vol. 69, no. 5, pp. 791-803, 2018.
Fatty-acid-based lipids are considered to be indicators of the structure and function of soil microbial biomass. Identifying and quantifying intact fatty-acid-based lipids in soil extracts is challenging because lipids range from polar (e.g. phospholipids and other membrane lipids) to non-polar (e.g. triacylglycerols involved in energy storage). The aim of this study was to develop a liquid chromatography–mass spectrometry (LC–MS) method to identify and quantify intact fatty-acid-based lipids in soil extracts. Reversed-phase LC separated fatty-acid-based lipids ranging from polar to non-polar in one chromatographic analysis. Within classes, individual lipid species were separated predictably from one another on the basis of carbon numbers and degree of unsaturation, such that chromatographic retention time could be used to aid identification. Lipid species were identified based on intact mass of the lipid and by fragmentation into structure-specific fragments that enabled compound classes and fatty acyl chains to be determined. Detection limits in positive and negative mode were better than 10 nmol l−1 for most lipid standards. In an extract of soil, 74 lipids from 10 classes were identified. The main non-polar lipids were 12 diacylglyerol species and 16 triacylglycerol species. Polar lipids included phosphatidylcholines, phosphatidylethanolamines, monomethyl-phosphatidylethanolamines, phosphatidylinositols and three classes of betaine lipid (diacylgycerol-N-trimethylhomoserine; diacylglycerylhydroxymethy-N,N,N-trimethyl-β-alanine; and diacylglycerylcarboxyhydroxymethylcholine). The LC–MS method developed provides a powerful analytical tool for analysis of fatty-acid-based lipids ranging from polar to non-polar and should be useful for the culture- and gene-independent assay of soil function. Highlights: A novel LC–MS method is described for identification of soil lipids. Polar and non-polar lipids were separated in one chromatographic analysis 74 lipids from 10 classes were identified Lipids identified included 25 species of betaine lipid. © 2018 British Society of Soil Science
Warren, C R
Development of online microdialysis-mass spectrometry for continuous minimally invasive measurement of soil solution dynamics Journal Article
In: Soil Biology and Biochemistry, vol. 123, pp. 266-275, 2018.
The rate that amino acids are removed from the soil solution is poorly known but vitally important. It is possible to determine the time course of soil solution concentrations by extracting soils at different time points after adding labelled compounds, but this approach either lacks sufficient temporal resolution or generates large number of samples that require subsequent offline analysis. The aim of this study was to develop online microdialysis-mass spectrometry to enable the minimally invasive measurement of the time-course of isotope labelled amino acid added to soil. The method was subsequently tested by examining the fate of isotope labelled L- and D-alanine added to sterile and non-sterile soils. One concern with application of microdialysis to soil is if calibrations are affected by inorganic ion composition of the perfusate and the external (soil) solution. Tests showed that the presence/absence of inorganic ions in perfusate and external solution did not affect dialysate concentrations, suggesting that perfusing with an artificial soil solution matching the inorganic ion composition of the external solution does not convey any benefits. Hence water was used as perfusate for development of online microdialysis-mass spectrometry. The online system took around one minute to equilibrate to step-changes in concentration and had detection limits around 0.5 μmol L−1 for alanine. Addition of isotope labelled alanine to soils led to an almost instantaneous increase and subsequent decrease in dialysate alanine concentration. With sterile soils there was a slow abiotic decrease in dialysate concentrations, presumably due to development of a depletion shell around the microdialysis probe and adsorption of alanine to the soil. For non-sterile soils there was an additional more rapid biotic decrease in dialysate concentrations that presumably reflected microbial uptake. For L-alanine added to non-sterile soil much of the compound was taken up before it reached the probe surface and concentrations decreased to below detection limits within 5–20 min. Thus microdialysis afforded a graphic illustration of the ephemeral nature of intact L-alanine in non-sterile soil, while parallel measurements showed that added D-alanine was removed from soil solution several times more slowly. © 2018 Elsevier Ltd
Warren, C R
Variation in small organic N compounds and amino acid enantiomers along an altitudinal gradient Journal Article
In: Soil Biology and Biochemistry, vol. 115, pp. 197-212, 2017.
The absolute and relative concentration of small organic N compounds varies among soils, yet we have little idea what drives this variation among soils. Previous studies have noted differences in DON/DIN and amino acid profiles among sites differing in altitude and/or productivity, and thus it seemed plausible that similar factors would have broader effects on the molecular composition of the pool of small organic N. To test this idea we used an altitudinal transect that ranged from a low altitude forest of Eucalyptus regnans with a canopy height averaging 65 m through to coniferous shrubbery that was above the alpine treeline and had a canopy less than 50 cm high. From low to high altitude mean annual temperature decreased 7 °C such that turnover was likely twice as slow at the highest site than the lowest. Capillary electrophoresis-mass spectrometry was used to identify and quantify the main small organic N compounds in free, adsorbed and microbial fractions of the soil; while chiral liquid chromatography-mass spectrometry was used to quantify amino acid enantiomers in hydrolysed soil and the free, adsorbed and microbial fractions of soil. CE-MS detected 66 small (<250 Da) organic N compounds of which 63 could be positively identified. Protein amino acids were a large fraction of the pool of small organic N, but there were also large amounts of non-protein amino acids, quaternary ammonium compounds and alkylamines. There were differences among sites in the profile of small organic N, but these differences were not monotonically related to altitude and there was no evidence pools of small organic N were larger or enriched in recalcitrant compounds at cooler high altitude sites. Among sites there was only modest variation in the molecular composition of the protein amino acid pool probably because protein amino acids are primarily derived from a common source (i.e. depolymerisation of soil proteins). In contrast, there was substantially larger variation within pools of non-protein amino acids, alkylamines and quaternary ammonium compounds; which is probably because compounds from these classes are primarily products of de novo synthesis by specific organisms, and thus molecular composition varies among sites depending on composition of the microbial community. D-enantiomers of amino acids were at low concentrations relative to L enantiomers such that in soil extracts the summed concentration of D-amino acids was 0.5–0.6% of L amino acids, while in hydrolysates D-enantiomers were 0.99% of L-enantiomers. There was no evidence that absolute or relative concentrations of D-enantiomers in free solution, microbial biomass or hydrolysates were larger at high altitude sites, despite turnover likely being slower at the cooler high altitude sites. The absence of an effect of altitude on D/L probably indicates that the turnover of soil proteins is comparatively rapid and thus soil proteins are similarly young even among sites in which mean annual temperature differs by 7 °C. © 2017
Simpson, J; Warren, C; Adams, P
Potential protease activity and organic nitrogen concentration are rapid tests and accurate indicators of N-availability in Tasmanian Eucalyptus nitens plantations Journal Article
In: Soil Biology and Biochemistry, vol. 115, pp. 152-160, 2017.
There are a variety of soil-based tests used to gauge the availability of N, with some of the more common tests being based on pools of inorganic N and/or N mineralization. Implicit assumptions of these tests are: 1) plants take up inorganic nitrogen compounds only, and 2) mineralisation is the rate-limiting step in producing available N. However, there is mounting evidence that 1) plants can take up organic nitrogen and 2) the rate-limiting step of nitrogen availability is more likely depolymerisation. The aim of this study was to test if pools of amino acids and potential protease activity could be used to predict nutrient limitation. To test this idea we set up nine experimental field sites with widely divergent nutrient availability. Sites were planted with the plantation tree species Eucalyptus nitens and we measured growth response to fertiliser to gauge the extent of nutrient limitation. Among the nine sites the size of growth responses to fertiliser were strongly correlated with potential protease activity and amino acid concentration. Data were consistent with the notion that depolymerisation (as indicated by protease activity) could be the rate-limiting step in generating available forms of N. We suggest that potential protease activity may be a useful indicator of nitrogen availability and could be used to tailor fertiliser supply to demand. © 2017 Elsevier Ltd
Warren, C R
Changes in small organic N during early stages of soil development Journal Article
In: Soil Biology and Biochemistry, vol. 110, pp. 44-55, 2017.
During the early stages of ecosystem development there are increases in plant and soil microbial biomass, nutrient availability and rates of nutrient cycling; but little is known about how pools of small organic N vary during the initial stages of soil development. The aim of this study was to examine how the pool of small organic N compounds varies during the initial stages of soil development, and if age differentially affects D- and L-enantiomers of protein amino acids. Measurements were made at a soil chronosequence on the east coast of Tasmania that comprised a series of sub-parallel beach dunes and ridges varying in age from <100 years to 5500 years. Capillary electrophoresis-mass spectrometry was used to identify and quantify the main small organic N compounds in free, adsorbed and microbial fractions of the soil; while chiral liquid chromatography-mass spectrometry was used to quantify amino acid enantiomers in hydrolysed soil and the free, adsorbed and microbial fractions of soil. CE-MS detected 66 small (<250 Da) organic N compounds of which 63 could be positively identified. Small organic N was dominated by protein amino acids, while there were also large amounts of quaternary ammonium compounds and alkylamines. There were differences among chronosequence sites in the profile of small organic N, but these differences were not monotonically related to age and there was no evidence for a build-up of recalcitrant compounds over time. Differences were instead site-specific and related to presence/absence of particular non-protein amino acids which probably related to the presence/absence of specific plants and/or microbes that produce and/or can metabolise different non-protein amino acids. In free solution and microbial biomass D enantiomers of many amino acids were below detection limits (i.e. < 0.125 nmol g−1) and D-enantiomers were at low concentrations relative to L enantiomers such that across all ages and replicates the summed concentration of D-amino acids was 0-3-0.6% of L amino acids. There was no evidence that absolute or relative concentrations of D-enantiomers in free solution, microbial biomass or hydrolysates were larger at the older chronosequence sites. The consistent lack of an effect of soil age on D/L probably indicates that the turnover of soil proteins is comparatively rapid and thus soil proteins are similarly young even among sites in which soil age is vastly different. © 2017 Elsevier Ltd
Uscola, M; Villar-Salvador, P; Oliet, J; Warren, C R
Root uptake of inorganic and organic N chemical forms in two coexisting Mediterranean forest trees Journal Article
In: Plant and Soil, vol. 415, no. 1-2, pp. 387-392, 2017.
Background and aims: Plants differ in their ability to use different nitrogen (N) chemical forms, these differences can be related to their ecology and drive community structure. The capacity to uptake intact organic N has been observed in plants of several ecosystems. However, soil organic N uptake by Mediterranean plants is unknown despite organic N being abundant in Mediterranean ecosystems. We compare the uptake of different N forms in two widespread coexisting Mediterranean forest trees with contrasting ecophysiological characteristics: Quercus ilex and Pinus halepensis. Methods: To estimate root uptake rate of each N form we used equimolar solutions (1 mM N) of 15NO3 −, 15NH4 + and 15N-13C glycine. Results: NH4 + and glycine were taken up at a similar rate, but faster than NO3 − in both species. Intact dual labeled glycine was found in both species, demonstrating that both species can absorb intact organic N. Conclusions: Despite their ecological differences, both species had similar preference for N forms suggesting no fundamental niche complementarity for N uptake. The higher preference for NH4 + and glycine over NO3 − possibly reflects adaptation to the differing proportions of N forms in Mediterranean soils. © 2017, Springer International Publishing Switzerland.
Warren, C R
Simultaneous efflux and uptake of metabolites by roots of wheat Journal Article
In: Plant and Soil, vol. 406, no. 1-2, pp. 359-374, 2016.
Background &amp; aims: Some metabolites (e.g. amino acids) present in root exudates can be taken up by roots, but we do not know if this ability extends to the broader suite of metabolites found in exudates. The aim of this study was to examine the ability of wheat (Triticum aestivum L.) to efflux and take up a broad suite of small metabolites. Methods: Four-week-old plants were placed into an uptake solution that contained a broad suite of 13C-labelled metabolites. Uptake was estimated from the decrease in concentration of the 13C isotopologue in the solution; and from the appearance of 13C isotopologues within roots. Efflux was estimated from appearance of 12C isotopologues in solution. Results: When wheat plants were placed in 13C-metabolite solutions the concentration of U-13C isotopologues of 37 metabolites decreased – as would be expected if plants were taking up the U-13C metabolites. After 4 h immersion in 13C metabolite solution, roots contained detectable amounts of U-13C isotopologues of 55 metabolites. U-13C isotopologues of organic acids were not detected within roots. Conclusions: These findings indicate that wheat can take up a broad suite of N-containing metabolites and some sugars, but there was no evidence for uptake of organic acids. © 2016, Springer International Publishing Switzerland.
Warren, C R
Do microbial osmolytes or extracellular depolymerisation products accumulate as soil dries? Journal Article
In: Soil Biology and Biochemistry, vol. 98, pp. 54-63, 2016.
When dry soil is re-wet there is a pulse of C mineralization. It is likely that the pulse of mineralization is fuelled by soluble C that accumulates as soil is drying. When soil is drying soluble C could accumulate as products of exo-enzyme mediated depolymerisation (i.e. protein amino acids and small carbohydrates) in the extracellular fraction of the soil (i.e. adsorbed and in free solution). Alternatively there could be an accumulation of osmolytes within the microbial biomass. To test whether extracellular depolymerisation products and/or microbial osmolytes accumulate as soil dries, soil from a Themeda triandra grassland was dehydrated and then depolymerisation products and osmolytes were quantified by capillary electrophoresis-mass spectrometry and gas chromatography-mass spectrometry. A secondary aim of this experiment was to determine if the response of soil to drying is the same when soil is dehydrated rapidly in the laboratory as when an intact soil containing plants is slowly dehydrated by withholding water from large (200 L) mesocosms.The responses of soil to dehydration differed between lab incubations and mesocosms, despite involving the same soil being dehydrated to the same final water content. When soil was dehydrated slowly in 200-L mesocosms the accumulation of osmolytes was more quantitatively significant than accumulation of depolymerisation products, whereas when soil was dehydrated more rapidly in lab incubations there was negligible accumulation of osmolytes but large accumulation of depolymerisation products. This study has highlighted that when soil dries the accumulation of osmolytes within microbial biomass and depolymerisation products within the extracellular fraction of soil are both quantitatively important and likely underpin the flush of soil CO2 efflux when dried soil is re-wet. © 2016 Elsevier Ltd.
Kawasaki, A; Warren, C R; Kertesz, M A
Specific influence of white clover on the rhizosphere microbial community in response to polycyclic aromatic hydrocarbon (PAH) contamination Journal Article
In: Plant and Soil, vol. 401, no. 1-2, pp. 365-379, 2016.
Background and aims: Legumes respond to PAH-contamination in a systemic manner and influence the overall rhizosphere microbial community structure, but the effect on the functional microbial community is unknown. In this study, plant-mediated PAH effects on specific bacterial taxa and the PAH-degraders in the rhizosphere were examined. Methods: White clover was cultivated using a split-root system, with one side exposed to phenanthrene or pyrene, and the other side uncontaminated. Rhizosphere microbial diversity and activity were assessed with DGGE and qPCR, and changes in the root exudation were analyzed with GC-MS and HPLC. Results: PAH contamination of one side of the rhizosphere significantly influenced the community structure of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Verrucomicrobia in the uncontaminated side of the rhizosphere. This indirect PAH-effect also influenced the diversity of bacterial PAH dioxygenase genes present, though the expression levels of these genes was not affected. No significant difference in the root exudation of general metabolites (amino acids, organic acids, sugars and sugar alcohols) and a flavonoid was observed. Conclusions: In response to PAH-stress, white clover specifically influenced the diversity of the PAH-degrading community in its rhizosphere, but the abundance and activity of these PAH-degraders was not enhanced by the indirect PAH-effect. The plant-mediated response therefore does not appear to be directed towards enhanced removal of PAH for plant protection. © 2015, Springer International Publishing Switzerland.
Warren, C R
Wheat roots efflux a diverse array of organic N compounds and are highly proficient at their recapture Journal Article
In: Plant and Soil, vol. 397, no. 1-2, pp. 147-162, 2015.
Background & aims: Small organic N compounds could contribute to N nutrition, but an alternative view is that root uptake may serve to recapture compounds that efflux out of roots. However, it is unclear if plants can recapture leaked organic N compounds because no studies have examined quantitative relationships between efflux and uptake at sub-micromolar concentrations. Methods: This study examines efflux and uptake of a broad suite of small organic N compounds by wheat (Triticum aestivum L.). 15N-labeling and capillary electrophoresis-mass spectrometry were used to estimate efflux and uptake. Results: One hundred and ten organic N compounds were detected in exudates. Amino acids were abundant but accounted for less than half of organic N. Other abundant compound classes were amines and polyamines, quaternary ammonium compounds, nucleobases and nucleosides. Uptake occurred simultaneously with efflux for all 45 compounds for which rates of efflux could be reliably determined, even though concentrations were 0.01 to 0.5 μM. Conclusions: These findings indicate that wheat is highly proficient at recapturing much of the diverse array of organic N compounds in root exudates. The ability to salvage effluxed compounds present at very low concentrations means that wheat might also be able to take up organic N compounds from the soil solution. © 2015, The Author(s).
Mawad, D; Warren, C; Barton, M; Mahns, D; Morley, J; Pham, B T T; Pham, N T H; Kueh, S; Lauto, A
Lysozyme depolymerization of photo-activated chitosan adhesive films Journal Article
In: Carbohydrate Polymers, vol. 121, pp. 56-63, 2015.
Effective tissue bioadhesion of rose bengal-chitosan films can be achieved by photoactivation using a green laser. In this study, lysozyme was incorporated in these films to enhance the rate of depolymerization and assess the laser impact on lysozyme. The lysozyme loaded films exhibited a 21% mass loss after 4 weeks implantation in rats while control films (without lysozyme) had only 7% mass loss. Capillary electrophoresis-mass spectroscopy showed that chitosan degraded into monomers and oligomers of glucosamine and N-acetyl-glucosamine. Irradiation with laser did not affect the depolymerization of adhesive by lysozyme suggesting that the inclusion of lysozyme in the bioadhesive is a viable technique for tailoring the depolymerization. © 2014 Elsevier Ltd. All rights reserved.
Warren, C R
Comparison of methods for extraction of organic N monomers from soil microbial biomass Journal Article
In: Soil Biology and Biochemistry, vol. 81, pp. 67-76, 2015.
One way of investigating the function of soil is via the pool of low molecular weight organic compounds in the soil microbial biomass. This is because low molecular weight organic compounds have key roles in metabolism of soil microbes, can function in osmotic adjustment and other stress responses, and are intermediates in the breakdown of polymers to inorganic nutrients. Methods for measuring low molecular weight microbial metabolites in soil rely upon extracting total metabolites and then subtracting the contribution from metabolites in the soil extracellular matrix (i.e. microbial=total-extracellular). Recent studies have tested methods for extracting organic N monomers from the extracellular matrix of soil, but there has not been similar testing of methods for extracting total organic N monomers. The aims of this study were to examine methods for extracting total organic N monomers by a) contrasting chloroform gas fumigation with chloroform direct extraction, and b) examining whether it is possible to extract soil with two methods that combine quenching of metabolic activity with extraction, namely cold methanol/chloroform/water and hot aqueous ethanol. To evaluate methods, organic N compounds were extracted from soil and then capillary electrophoresis-mass spectrometry identified and quantified 42 organic N monomers including amino acids, quaternary ammonium compounds, nucleobases and nucleosides, amines and polyamines. Absolute concentrations of 32 out of the 42 quantified organic N monomers were significantly different between soil extracted by chloroform gas fumigation and chloroform direct extraction. These differences were probably a function of gains and losses of compounds due to oxidation, hydrolysis and deamidation during the two-day chloroform gas fumigation. Cold methanol/chloroform/water yielded large amounts of the extremely labile compound ergothioneine, probably because the extraction method rapidly quenched metabolic activity. The primary limitation of extraction with methanol/chloroform/water is that it was ineffective at extracting strongly cationic compounds (e.g. polyamines). Extraction with hot aqueous ethanol was unsuccessful with soil presumably because soil microbes are difficult to lyse. It is recommended that future studies examining organic N monomers in soil microbial biomass use chloroform direct extraction or cold methanol/chloroform/water rather than chloroform gas fumigation. © 2014 Elsevier Ltd.
Cano, F J; López, R; Warren, C R
Implications of the mesophyll conductance to CO2 for photosynthesis and water-use efficiency during long-term water stress and recovery in two contrasting Eucalyptus species Journal Article
In: Plant Cell and Environment, vol. 37, no. 11, pp. 2470-2490, 2014.
Water stress (WS) slows growth and photosynthesis (An), but most knowledge comes from short-time studies that do not account for longer term acclimation processes that are especially relevant in tree species. Using two Eucalyptus species that contrast in drought tolerance, we induced moderate and severe water deficits by withholding water until stomatal conductance (gsw) decreased to two pre-defined values for 24d, WS was maintained at the target gsw for 29d and then plants were re-watered. Additionally, we developed new equations to simulate the effect on mesophyll conductance (gm) of accounting for the resistance to refixation of CO2. The diffusive limitations to CO2, dominated by the stomata, were the most important constraints to An. Full recovery of An was reached after re-watering, characterized by quick recovery of gm and even higher biochemical capacity, in contrast to the slower recovery of gsw. The acclimation to long-term WS led to decreased mesophyll and biochemical limitations, in contrast to studies in which stress was imposed more rapidly. Finally, we provide evidence that higher gm under WS contributes to higher intrinsic water-use efficiency (iWUE) and reduces the leaf oxidative stress, highlighting the importance of gm as a target for breeding/genetic engineering. © 2014 John Wiley & Sons Ltd.
Warren, C R
Response of osmolytes in soil to drying and rewetting Journal Article
In: Soil Biology and Biochemistry, vol. 70, pp. 22-32, 2014.
The accumulation and subsequent release of microbial osmolytes in response to drying and rewetting are thought to be key players in C and N dynamics, yet studies on soils have failed to support this hypothesis. The aim of this experiment was to determine how low-molecular weight compounds, and osmolytes in particular, are affected by drying and rewetting. Water deficits were imposed slowly by withholding water for 21 weeks from large (200L) mesocosms vegetated with a globally widespread grass Themeda triandra. A broad spectrum of small molecules in extracts was identified and quantified by capillary electrophoresis-mass spectrometry and gas chromatography-mass spectrometry. Compared with controls, drought-stressed mesocosms contained >10-fold larger amounts of known microbial osmolytes: ectoine, hydroxyectoine, betaine, proline-betaine, trigonelline, proline, trehalose, arabitol. The pool of osmolytes accounted for 3.6% of CHCl3 labile TOC in control mesocosms and 17% of CHCl3 labile TOC in drought-stressed mesocosms. There was no evidence that rewatering led to a large pulse of osmolytes in free solution. Instead osmolytes decreased to control concentrations within 1-3h of rewatering - probably indicating rapid uptake by microbes and plants. Results of this study suggest that osmolytes can account for a substantial fraction of microbial C, and are at least one of the ways that soil microbes cope with water deficits. © 2013 Elsevier Ltd.
Warren, C R
Organic N molecules in the soil solution: What is known, what is unknown and the path forwards Journal Article
In: Plant and Soil, vol. 375, no. 1-2, pp. 1-19, 2014.
Background: Many biological questions about N availability and the N cycle require knowledge of the abundance and identity of molecules comprising the pool of organic N. Moreover, basic knowledge of the molecular composition of the soil solution can give rise to new hypotheses via data-driven or inductive reasoning. Scope: This paper examines the composition of organic N molecules in the soil solution. Our perception of organic N in the soil solution is shaped by analytical approaches, and thus I briefly review approaches for sampling and analysis of the soil solution. I give examples of hypotheses generated by knowledge of the molecular composition of organic N and conclude by suggesting priorities for future research. Conclusions: Studies of the molecular composition of organic N are very much in their infancy. Amino acids, their oligomers and polymers are consistently large components of the pool of organic N. The soil solution also contains organic N compounds from at least another 12 compound classes, but almost nothing is known about their functional significance. Uncovering the role of these other compounds in the N cycle would enrich our understanding of organic N and the N cycle, and place studies of amino acids and their polymers in a broader context. © 2013 Springer Science+Business Media Dordrecht.
Buckley, T N; Warren, C R
The role of mesophyll conductance in the economics of nitrogen and water use in photosynthesis Journal Article
In: Photosynthesis Research, vol. 119, no. 1-2, pp. 77-88, 2014.
A recent resurgence of interest in formal optimisation theory has begun to improve our understanding of how variations in stomatal conductance and photosynthetic capacity control the response of whole plant photosynthesis and growth to the environment. However, mesophyll conductance exhibits similar variation and has similar impact on photosynthesis as stomatal conductance; yet, the role of mesophyll conductance in the economics of photosynthetic resource use has not been thoroughly explored. In this article, we first briefly summarise the knowledge of how mesophyll conductance varies in relation to environmental factors that also affect stomatal conductance and photosynthetic capacity, and then we use a simple analytical approach to begin to explore how these important controls on photosynthesis should mutually co-vary in a plant canopy in the optimum. Our analysis predicts that when either stomatal or mesophyll conductance is limited by fundamental biophysical constraints in some areas of a canopy, e.g. reduced stomatal conductance in upper canopy leaves due to reduced water potential, the other of the two conductances should increase in those leaves, while photosynthetic capacity should decrease. Our analysis also predicts that if mesophyll conductance depends on nitrogen investment in one or more proteins, then nitrogen investment should shift away from Rubisco and towards mesophyll conductance if hydraulic or other constraints cause chloroplastic CO2 concentration to decline. Thorough exploration of these issues awaits better knowledge of whether and how mesophyll conductance is itself limited by nitrogen investment, and about how these determinants of photosynthetic CO2 supply and demand co-vary among leaves in real plant canopies. © 2013 Springer Science+Business Media Dordrecht.
Warren, C R
Development of liquid chromatography mass spectrometry method for analysis of organic N monomers in soil Journal Article
In: Soil Biology and Biochemistry, vol. 78, pp. 233-242, 2014.
The aim of this study was to develop an analytical procedure based on liquid chromatography-mass spectrometry (LC-MS) for analysis of monomeric organic N compounds in soil extracts. To benchmark the developed LC-MS method it was compared with a capillary electrophoresis-mass spectrometry (CE-MS) method recently used for analysis of small organic N monomers in soil. The separation was optimized and analytical performance assessed with 69 purified standards, then the LC-MS method was used to analyse soil extracts. Sixty-two out of 69 standards were analysable by LC-MS with separation on a hydrophilic interaction liquid chromatography column. The seven compounds that could not be analysed were strongly cationic polyamines. Limits of detection for a 5μL injection ranged between 0.002 and 0.38μmolL-1, with the majority (49 out of 62) having limits of detection better than 0.05μmolL-1. The overall profile and concentration of small organic N monomers in soil extracts was broadly similar between LC-MS and CE-MS, with the notable exception of four ureides that were detected by LC-MS only. In soil extracts that had been concentrated ten-fold the detection and quantification of (some) organic N compounds was compromised by the presence of large amounts of inorganic salts. The developed LC-MS method offered advantages and disadvantages relative to CE-MS, and a combination of the two methods would achieve the broadest possible coverage of organic N in soil extracts. © 2014 Elsevier Ltd.
Warren, C R
Response of organic N monomers in a sub-alpine soil to a dry-wet cycle Journal Article
In: Soil Biology and Biochemistry, vol. 77, pp. 233-242, 2014.
Cycles of soil drying followed by rewetting occur in most terrestrial ecosystems, but there is conflicting evidence as to the role of osmolytes in dry-wet cycles. The broad aim of this experiment was to determine how N-containing osmolytes and other organic N monomers are affected by rewetting of a moderately dry soil. In a sub-alpine grassland, experimental plots were irrigated with 50mm of water near the conclusion of a typical late-summer drying cycle. Twelve putative osmolytes (proline, 8 quaternary ammonium compounds, trimethylamine N-oxide, ectoine, hydroxyectoine) and 60 other organic N monomers were identified and quantified by capillary electrophoresis-mass spectrometry of the free/exchangeable pool of soil water (0.5M K2SO4 extracts) and microbial biomass (via chloroform fumigation extraction). The total concentration of organic N monomers was 25-times greater in fumigated than unfumigated extracts. Differences in relative abundance of compound classes and compounds between fumigated and unfumigated extracts suggested some compounds were localized to the free/exchangeable pool; others were predominantly microbial, whereas many were shared between pools. A striking feature of the free/exchangeable pool was that on an N-basis alkylamines were the most abundant compound class and accounted for 34% of the pool of organic N monomers. There was no evidence that osmolytes were the primary means soil microbes coped with dry-wet cycles. Instead, the pool of osmolytes was an invariant 4% of the pool of CE-MS detected monomers in K2SO4 extracts and 7% of the pool of CE-MS detected monomers in the chloroform-labile (microbial) fraction. The absence of substantial amounts of osmolytes may be because water stress was too mild or brief, or because osmolyte synthesis was limited by availability of energy, N or C and some alternative strategy was used to cope with water deficits. © 2014 Elsevier Ltd.
Uscola, M; Villar-Salvador, P; Oliet, J; Warren, C R
Foliar absorption and root translocation of nitrogen from different chemical forms in seedlings of two Mediterranean trees Journal Article
In: Environmental and Experimental Botany, vol. 104, pp. 34-43, 2014.
Along with root uptake, plants can also absorb N through leaves. There are few comparative studies on the foliar absorption of N from different chemical forms of N in forest tree species. We compared the foliar N absorption capacity in seedlings of two forest trees widespread in the Mediterranean basin, Quercus ilex and Pinus halepensis. Plants were sprayed with the following individual N forms at 40mM N: 15N-nitrate (NO3-), 15N-ammonium (NH4+), 15N-urea or 13C and 15N dual-labeled glycine. Cuticular conductance was used as a surrogate of cuticle permeability to water. Q. ilex had higher N foliar absorption than P. hapelensis. Neither cuticular conductance nor shoot surface area explained N differences in absorption rate between species, which were instead likely linked to differences in stomatal density and presence of trichomes. In both species, foliar N absorption rate and N recovery differed among N forms: urea>NH4+≥glycine≥NO3-. Differences in N absorption rate among N forms were correlated with their physico-chemical properties. The strong positive relationship between 15N and 13C uptake together with detection in shoots of intact dual-labeled glycine (measured by gas chromatography-mass spectrometry), indicated that a significant fraction of glycine was absorbed intact by the seedlings. In both species, higher cuticular conductance was related to faster N absorption from all forms except NO3-. Cuticular conductance had a stronger effect on N absorption from urea and NH4+ than N absorption from glycine, and the effects were more intense in Q. ilex than in P. halepensis. This suggests that variations in cuticle permeability in both species are determined by different mechanisms and that each N form was differently affected. Absorbed N was rapidly translocated to roots, with a larger proportion of N from organic forms being translocated than N from inorganic forms. Foliar fertilization increased plant N content, especially in urea fertilized plants, but direct foliar absorption only explained up to 10% of N content increase. This study demonstrates that two important Mediterranean forest tree species can absorb through their leaves both, inorganic and organic N forms. This has important ecological and applied implications, because all chemical forms of N are present in natural N deposition. Also results show that foliar N fertilization can play an important role for seedling N nutrition, and that the effect will have different impacts depending on the species. © 2014 Elsevier B.V. All rights reserved.
Cano, F J; Sánchez-Gómez, D; Rodríguez-Calcerrada, J; Warren, C R; Gil, L; Aranda, I
Effects of drought on mesophyll conductance and photosynthetic limitations at different tree canopy layers Journal Article
In: Plant, Cell and Environment, vol. 36, no. 11, pp. 1961-1980, 2013.
In recent years, many studies have focused on the limiting role of mesophyll conductance (gm) to photosynthesis (An) under water stress, but no studies have examined the effect of drought on gm through the forest canopy. We investigated limitations to An on leaves at different heights in a mixed adult stand of sessile oak (Quercus petraea) and beech (Fagus sylvatica) trees during a moderately dry summer. Moderate drought decreased An of top and lowest beech canopy leaves much more than in leaves located in the mid canopy; whereas in oak, An of the lower canopy was decreased more than in sunlit leaves. The decrease of An was probably not due to leaf-level biochemistry given that VCmax was generally unaffected by drought. The reduction in An was instead associated with reduction in stomatal and mesophyll conductances. Drought-induced increases in stomatal limitations were largest in leaves from the top canopy, whereas drought-induced increases in mesophyll limitations were largest in leaves from the lowest canopy. Sensitivity analysis highlighted the need to decompose the canopy into different leaf layers and to incorporate the limitation imposed by gm when assessing the impact of drought on the gas exchange of tree canopies. Mesophyll conductance (gm) is now recognised as a key limitation of photosynthesis that should be incorporated into canopy-level models of C exchange. Variation in gm through the canopy of trees has been investigated, but we do not know if water stress affects equally gm of all canopy layers. We investigated limitations to photosynthesis at different heights in a mixed adult stand of Quercus petraea and Fagus sylvatica trees during a moderately dry summer. Drought-induced increases in stomatal limitations were largest in leaves from the top canopy, whereas drought-induced increases in mesophyll limitations were largest in leaves from the lowest canopy. © 2013 John Wiley & Sons Ltd.
Warren, C R
Development of a capillary electrophoresis-mass spectrometry method for small peptides in the soil solution Journal Article
In: Soil Biology and Biochemistry, vol. 63, pp. 80-84, 2013.
Small peptides are being investigated for their role in ecosystem cycling and plant uptake of organic N, but little is known of molecular forms in the soil solution. The aim of this study was to develop a capillary electrophoresis-tandem mass spectrometry procedure for profiling small peptides in the soil solution. Capillary electrophoresis-mass spectrometry was capable of separating and detecting a range of small peptide standards. Adequate recovery (>90%) of standard peptides spiked into samples of soil solution indicated that separation and detection were robust and not significantly affected by the sample matrix. The method was applied to samples of soil solution from grassland mesocosms filled with clay-loam soil from an abruptic lixisol. Soil solution (ultrafiltered <3kDa) contained at least 298 putatively identified peptides with most being smaller than 600Da. Less than 5% of small peptides contained basic amino acids, which may reflect their preferential adsorption to the soil stationary phase versus peptides comprised of acid or neutral amino acids. Capillary electrophoresis-mass spectrometry of small peptides is robust and has already yielded novel findings with its first proof of concept measurements on the soil solution. © 2013 Elsevier Ltd.
Warren, C R
Quaternary ammonium compounds can be abundant in some soils and are taken up as intact molecules by plants Journal Article
In: New Phytologist, vol. 198, no. 2, pp. 476-485, 2013.
Studies of organic nitrogen (N) cycling and uptake by plants have focused on protein amino acids, but the soil solution includes organic N compounds from many other compound classes. The two aims of this study were to characterize the 30-50 most abundant molecules of small (< 250 Da), nonpeptide organic N in the soil solution from six soils, and to determine if two ecologically disparate species (nonmycorrhizal Banksia oblongifolia and mycorrhizal Triticum aestivum) have the ability to take up intact molecules of three quaternary ammonium compounds (betaine, carnitine and acetyl-carnitine). Protein amino acids were dominant components of the pool of small nonpeptide organic N in all soils. The most abundant other compound classes were quaternary ammonium compounds (1-28% of nonpeptide small organic N) and nonprotein amino acids (3-19% of nonpeptide small organic N). B. oblongifolia and T. aestivum took up intact quaternary ammonium compounds from dilute hydroponic solution, while T. aestivum growing in field soil took up intact quaternary ammonium compounds injected into soil. Results of this study show that the pool of organic N in soil is more diverse and plants have an even broader palate than is suggested by most of the literature on organic N. © 2013 New Phytologist Trust.
Warren, C R
High diversity of small organic N observed in soil water Journal Article
In: Soil Biology and Biochemistry, vol. 57, pp. 444-450, 2013.
The pool of organic N molecules in the soil solution reflects the activity of plants, microbes and other biological processes, and thus is likely to provide information important for ecosystem N and C cycling (e.g. organic N uptake by plants). Amino acids in soil water have often been a target of study, but few previous studies have attempted to examine a broader range of organic N molecules. The aim of this study was to develop a capillary electrophoresis-mass spectrometry (CE-MS) procedure for profiling of those small (<250 Da) organic N molecules in soil water that are amenable to analysis by CE-MS (viz., cationic at low pH, ionisable by electrospray). Centrifugal extracts of soil water from a sub-alpine grassland contained approximately 100 non-redundant peaks of small organic cations, 58 of which have been identified. Consistent with earlier studies, protein amino acids and common non-protein amino acids were among the most abundant compounds. Soil water also contained large amounts of several quaternary ammonium compounds (e.g. carnitine, acetyl carnitine, betaine, choline, ergothioneine) with the pool of quaternary ammonium compounds approximately 25% of the size of the pool of common amino acids. The large amounts of quaternary ammonium compounds in soil probably reflects their dual roles in central metabolism and osmoprotection in plants and microbes. Other identified compounds included unusual amino acids (e.g. β-alanine, pipecolic acid), heterocyclic compounds derived from aromatic amino acids (e.g. 4-(hydroxymethyl)imidazole, urocanate, nicotinic acid), amines (ethanolamine, spermine), sugar amines (glucosamine), and additional putative osmolytes of microbial or plant origin (trimethylamine N oxide, ectoine). Results of this study indicate that the pool of small organic N in soil water is more diverse than generally appreciated and not necessarily dominated by protein amino acids and common non-protein amino acids. © 2012 Elsevier Ltd.
Warren, C R
Use of chemical ionization for GC-MS metabolite profiling Journal Article
In: Metabolomics, vol. 9, no. SUPPL.1, pp. 110-120, 2013.
Metabolite profiling is commonly performed by GC-MS of methoximated trimethylsilyl derivatives. The popularity of this technique owes much to the robust, library searchable spectra produced by electron ionization (EI). However, due to extensive fragmentation, EI spectra of trimethylsilyl derivatives are commonly dominated by trimethylsilyl fragments (e. g. m/z 73 and 147) and higher m/z fragment ions with structural information are at low abundance. Consequently different metabolites can have similar EI spectra, and this presents problems for identification of ünknowns" and the detection and deconvolution of overlapping peaks. The aim of this work is to explore use of positive chemical ionization (CI) as an adjunct to EI for GC-MS metabolite profiling. Two reagent gases differing in proton affinity (CH4 and NH3) were used to analyse 111 metabolite standards and extracts from plant samples. NH3-CI mass spectra were simple and generally dominated by [MH]+ and/or the adduct [M+NH4]+. For the 111 metabolite standards, m/z 73 and 147 were less than 3% of basepeak in NH3-CI and less than 30% of basepeak in CH4-CI. With CH4-CI, [MH]+ was generally present but at lower relative abundance than for NH3-CI. CH4-CI spectra were commonly dominated by losses of CH4 [M+1-16]+, 1-3 TMSOH [M+1-nx90]+, and combinations of CH4 and TMSOH losses [M+1-nx90-16]+. CH4-CI and NH3-CI mass spectra are presented for 111 common metabolites, and CI is used with real samples to help identify overlapping peaks and aid identification via determination of the pseudomolecular ion with NH3-CI and structural information with CH4-CI. © 2011 Springer Science+Business Media, LLC.
Flexas, J; Barbour, M M; Brendel, O; Cabrera, H M; Carriquí, M; Díaz-Espejo, A; Douthe, C; Dreyerc, E; Ferrio, J P; Gago, J; Gallé, A; Galmés, J; Kodama, N; Medrano, H; Niinemets, Ü; Peguero-Pina, J J; Pou, A; Ribas-Carbó, M; Tomás, M; Tosens, T; Warren, C R
Corrigendum to 'Mesophyll diffusion conductance to CO2: An unappreciated central player in photosynthesis' [Plant Sci. 193-194 (2012) 70-84] Journal Article
In: Plant Science, vol. 196, pp. 31, 2012.
Flexas, J; Barbour, M M; Brendel, O; Cabrera, H M; Carriquí, M; Díaz-Espejo, A; Douthe, C; Dreyer, E; Ferrio, J P; Gago, J; Gallé, A; Galmés, J; Kodama, N; Medrano, H; Niinemets, Ü; Peguero-Pina, J J; Pou, A; Ribas-Carbó, M; Tomás, M; Tosens, T; Warren, C R
Mesophyll diffusion conductance to CO2: An unappreciated central player in photosynthesis Journal Article
In: Plant Science, vol. 193-194, pp. 70-84, 2012.
Mesophyll diffusion conductance to CO2 is a key photosynthetic trait that has been studied intensively in the past years. The intention of the present review is to update knowledge of gm, and highlight the important unknown and controversial aspects that require future work. The photosynthetic limitation imposed by mesophyll conductance is large, and under certain conditions can be the most significant photosynthetic limitation. New evidence shows that anatomical traits, such as cell wall thickness and chloroplast distribution are amongst the stronger determinants of mesophyll conductance, although rapid variations in response to environmental changes might be regulated by other factors such as aquaporin conductance.Gaps in knowledge that should be research priorities for the near future include: how different is mesophyll conductance among phylogenetically distant groups and how has it evolved? Can mesophyll conductance be uncoupled from regulation of the water path? What are the main drivers of mesophyll conductance? The need for mechanistic and phenomenological models of mesophyll conductance and its incorporation in process-based photosynthesis models is also highlighted. © 2012 Elsevier Ireland Ltd.
Forrester, D I; Lancaster, K; Collopy, J J; Warren, C R; Tausz, M
Photosynthetic capacity of Eucalyptus globulus is higher when grown in mixture with Acacia mearnsii Journal Article
In: Trees - Structure and Function, vol. 26, no. 4, pp. 1203-1213, 2012.
Mixed species plantations of Eucalyptus and N 2-fixing species can be significantly more productive than monocultures. The aim of this study was to determine whether the improved growth resulted from increases in photosynthesis, light absorption and light-use efficiency, in addition to previously measured increases in leaf area, water-use efficiency and higher ratios of annual above-ground net primary production per unit of total annual below-ground carbon allocation in 1:1 mixtures near Cann River, Victoria, Australia. Light-saturated photosynthetic rate (A max), electron transport (J), stomatal conductance (g s) and foliar nitrogen concentrations were higher for Eucalyptus globulus trees growing in mixtures than those in monocultures. Similar increases in maximum rates of carboxylation (V cmax), Rubisco, chlorophyll, and phosphorus concentrations were not significant. In contrast, A max, V cmax and J did not vary between mixtures and monocultures for A. mearnsii, whose growth was negligible by age 15 years. Mixtures also absorbed 24 and 41% more light than E. globulus and A. mearnsii., respectively, and were 38 and 154% more light-use efficient in the mixtures compared to monocultures. The increased nutrient availability in mixtures appeared to increase productivity of E. globulus by increasing the photosynthetic capacity of the foliage, as well as the leaf area, light absorption and light-use efficiency of the canopy. © 2012 Springer-Verlag.
Douthe, C; Dreyer, E; Brendel, O; Warren, C R
Is mesophyll conductance to CO2 in leaves of three Eucalyptus species sensitive to short-term changes of irradiance under ambient as well as low O2? Journal Article
In: Functional Plant Biology, vol. 39, no. 5, pp. 435-448, 2012.
Mesophyll conductance to CO2 (g m) limits the diffusion of CO2 to the sites of carboxylation, and may respond rapidly (within minutes) to abiotic factors. Using three Eucalyptus species, we tested the rapid response of g m to irradiance under 21% and 1% O2. We used simultaneous measurements of leaf gas exchange and discrimination against 13CO2 with a tuneable diode laser absorption spectrometer. Measurements under 1% O2 were used to limit uncertainties due to 13C-12C fractionation occurring during photorespiration. Switching irradiance from 600 to 200μmolm-2s-1 led to a ≈60% decrease of gm within minutes in all species under both 21% O2 and 1% O2. The gm response to irradiance is unlikely to be a computation artefact since using different values for the parameters of the discrimination model changed the absolute values of g m but did not affect the relative response to irradiance. Simulations showed that possible rapid changes of any parameter were unable to explain the observed variations of g m with irradiance, except for 13C-12C fractionation during carboxylation (b), which, in turn, is dependent on the fraction of leaf C assimilated by phospho-enol pyruvate carboxylase (PEPc) (β). gm apparently increased by ≈30% when O2 was switched from 21% to 1% O2. Again, possible changes of with O 2 could explain this apparent g m response to O2. Nevertheless, large irradiance or O2-induced changes in would be required to fully explain the observed changes in gm, reinforcing the hypothesis that gm is responsive to irradiance and possibly also to O2. © 2012 CSIRO.
Warren, C R; Aranda, I; Cano, F J
Metabolomics demonstrates divergent responses of two Eucalyptus species to water stress Journal Article
In: Metabolomics, vol. 8, no. 2, pp. 186-200, 2012.
Past studies of water stress in Eucalyptus spp. generally highlighted the role of fewer than five "important" metabolites, whereas recent metabolomic studies on other genera have shown tens of compounds are affected. There are currently no metabolite profiling data for responses of stress-tolerant species to water stress. We used GC-MS metabolite profiling to examine the response of leaf metabolites to a long (2 month) and severe (Ψ predawn < -2 MPa) water stress in two species of the perennial tree genus Eucalyptus (the mesic Eucalyptus pauciflora and the semi-arid Eucalyptus dumosa). Polar metabolites in leaves were analysed by GC-MS and inorganic ions by capillary electrophoresis. Pressure-volume curves and metabolite measurements showed that water stress led to more negative osmotic potential and increased total osmotically active solutes in leaves of both species. Water stress affected around 30-40% of measured metabolites in E. dumosa and 10-15% in E. pauciflora. There were many metabolites that were affected in E. dumosa but not E. pauciflora, and some that had opposite responses in the two species. For example, in E. dumosa there were increases in five acyclic sugar alcohols and four lowabundance carbohydrates that were unaffected by water stress in E. pauciflora. Re-watering increased osmotic potential and decreased total osmotically active solutes in E. pauciflora, whereas in E. dumosa re-watering led to further decreases in osmotic potential and increases in total osmotically active solutes. This experiment has added several extra dimensions to previous targeted analyses of water stress responses in Eucalyptus, and highlights that even species that are closely related (e. g. congeners) may respond differently to water stress and re-watering. © 2011 Springer Science+Business Media, LLC.
Forrester, D I; Collopy, J J; Beadle, C L; Warren, C R; Baker, T G
Effect of thinning, pruning and nitrogen fertiliser application on transpiration, photosynthesis and water-use efficiency in a young Eucalyptus nitens plantation Journal Article
In: Forest Ecology and Management, vol. 266, pp. 286-300, 2012.
Interactions between thinning, pruning and fertiliser application in forestry are rarely examined, even though these treatments are often applied simultaneously in practice. Understanding these interactions can facilitate the design of regimes to best exploit such silvicultural interventions. The effects of these treatments on stand transpiration (E), photosynthesis and water-use efficiency (AGB-WUE, defined as the above-ground biomass production per unit transpiration) were measured in a Eucalyptus nitens plantation in south-eastern Australia. Two levels of each treatment were applied at age 3.2years and transpiration was measured between ages 5.3 and 6.3years. Treatments were: unthinned, or thinned from ca. 900 to 300treesha -1; unpruned, or 50% of the live crown length pruned of the 300 largest-diameter potential sawlog crop treesha -1; and nil, or 300kgha -1 N fertiliser. There were no significant treatment interactions on growth, E or AGB-WUE. Thinning and pruning reduced E by 45% and 12%, respectively, and fertiliser application increased E by 21%. Transpiration was linearly related to stand leaf area, which explained more than 90% of the variation across treatments. Thinning and pruning also increased AGB-WUE by 23% and 21%, respectively, while fertiliser application had no significant effect. There was a small increase in AGB-WUE with increasing tree size, such that in unthinned stands the largest 50% of trees were 7% more efficient than the smallest 50% of trees. Thinning increased AGB-WUE by increasing the light available to the lower canopy and pruning increased AGB-WUE by removing the least efficient lower canopy foliage and increasing the efficiency of the remaining foliage. All treatments also modified the hydraulic architecture of the trees by changing leaf area to sapwood area ratios and radial sap flux density profiles. This study shows how silvicultural treatments can be used to modify stand transpiration and AGB-WUE of E. nitens plantations, potentially reducing their drought susceptibility while making more efficient use of the sites water resources. © 2011 Elsevier B.V.
Warren, C R
Post-uptake metabolism affects quantification of amino acid uptake Journal Article
In: New Phytologist, vol. 193, no. 2, pp. 522-531, 2012.
• The quantitative significance of amino acids to plant nutrition remains controversial. This experiment determined whether post-uptake metabolism and root to shoot export differ between glycine and glutamine, and examined implications for estimation of amino acid uptake. • Field soil containing a Eucalyptus pauciflora seedling was injected with uniformly 13C- and 15N-labelled glycine or glutamine. I quantified 15N and 13C excess in leaves and roots and intact labelled amino acids in leaves, roots and stem xylem sap. A tunable diode laser quantified fluxes of 12CO 2 and 13CO 2 from leaves and soil. • 60-360min after addition of amino acid, intact molecules of U- 13C, 15N glutamine were < 5% of 15N excess in roots, whereas U- 13C, 15N glycine was 30-100% of 15N excess in roots. Intact molecules of glutamine, but not glycine, were exported from roots to shoots. • Post-uptake metabolism and transport complicate interpretation of isotope labelling such that root and shoot contents of intact amino acid, 13C and 15N may not reflect rates of uptake. Future experiments should focus on reconciling discrepancies between intact amino acid, 13C and 15N by determining the turnover of amino acids within roots. Alternatively, post-uptake metabolism and transport could be minimized by harvesting plants within minutes of isotope addition. © 2011 The Author. New Phytologist © 2011 New Phytologist Trust.
Warren, C R; García-Plazaola, J I; Niinemets, Ü
Ecophysiology of photosynthesis in temperate forests Book Chapter
In: Flexas, Jaume; Loreto, Francesco; Medrano, HipólitoEditors (Ed.): Terrestrial Photosynthesis in a Changing Environment: A Molecular, Physiological, and Ecological Approach, pp. 465–487, Cambridge University Press, 2012.
Flexas, J; Brugnoli, E; Warren, C R
Mesophyll conductance to CO2 Book Chapter
In: Flexas, Jaume; Loreto, Francesco; Medrano, HipólitoEditors (Ed.): Terrestrial Photosynthesis in a Changing Environment: A Molecular, Physiological, and Ecological Approach, pp. 169–185, Cambridge University Press, 2012.
Morales, F; Warren, C R
Photosynthetic responses to nutrient deprivation and toxicities Book Chapter
In: Flexas, Jaume; Loreto, Francesco; Medrano, HipólitoEditors (Ed.): Terrestrial Photosynthesis in a Changing Environment: A Molecular, Physiological, and Ecological Approach, pp. 312–330, Cambridge University Press, 2012.
Cano, F J; Sánchez-Gómez, D; Gascó, A; Rodríguez-Calcerrada, J; Gil, L; Warren, C R; Aranda, I
Light acclimation at the end of the growing season in two broadleaved oak species Journal Article
In: Photosynthetica, vol. 49, no. 4, pp. 581-592, 2011.
The ability of plants to increase their net CO 2 assimilation rate in response to increased irradiance is due to morphological and physiological changes, which might be related to their shade tolerance and leaf ontogeny, but few studies have considered morphology and physiology. Two sympatric oak species (the shade-tolerant Q. petraea and the comparatively shade-intolerant Q. pyrenaica) were grown in hydroponic solution in low-light (LL) and high-light (HL) conditions. 5 months after leaf expansion under these conditions, half of the LL plants were transferred to high light (TLH). Transfer of Q. pyrenaica, from low- to high light led to photoinhibition and after 21 days in higher light there was little acclimation of the maximum rate of carboxylation (V Cmax) or the maximum rate of electron transport (J max). Q. pyrenaica TLH plants showed lower stomatal conductance at all times compared to plants growing in LL. Stomatal closure was the main limitation to photosynthesis after transfer in Q. pyrenaica. The increase in evaporative demand upon TLH did not affect hydraulic conductivity of Q. pyrenaica. In contrast, the more shade-tolerant Q. petraea showed a greater degree of acclimation of gas exchange in TLH than Q. pyrenaica and two weeks after transfer gas-exchange rates were as high as in LL plants. In Q. petraea, the most important changes occurred at the level of leaf biochemistry with significant increase in V Cmax that decreased the J max/V Cmax ratio below values recorded in HL plants. However, this potential increase in photosynthesis was at least partially hamstrung by a decrease in internal conductance, which highlights the importance of internal conductance in acclimation to higher light in mature leaves. Neither oak species reached the photosynthetic rates of HL plants; however a trend towards leaf acclimation was observed in Q. petraea while the transfer was harmful to the leaves of Q. pyrenaica developed in the shade. © 2011 Springer Science+Business Media B.V.
Douthe, C; Dreyer, E; Epron, D; Warren, C R
Mesophyll conductance to CO 2, assessed from online TDL-AS records of 13CO 2 discrimination, displays small but significant short-term responses to CO 2 and irradiance in Eucalyptus seedlings Journal Article
In: Journal of Experimental Botany, vol. 62, no. 15, pp. 5335-5346, 2011.
Mesophyll conductance (g m) is now recognized as an important limiting process for photosynthesis, as it results in a significant decrease of CO 2 diffusion from substomatal cavities where water evaporation occurs, to chloroplast stroma. Over the past decade, an increasing number of studies proposed that gm can vary in the short term (e.g. minutes), but these variations are still controversial, especially those potentially induced by changing CO 2 and irradiance. In this study, g m data estimated with online 13C discrimination recorded with a tunable diode laser absorption spectrometer (TDL-AS) during leaf gas exchange measurements, and based on the single point method, are presented. The data were obtained with three Eucalyptus species. A 50% decrease in g m was observed when the CO 2 mole fraction was increased from 300 μmol mol -1 to 900 μmol mol -1, and a 60% increase when irradiance was increased from 200 μmol mol -1 to 1100 μmol mol -1 photosynthetic photon flux density (PPFD). The relative contribution of respiration and photorespiration to overall 13C discrimination was also estimated. Not taking this contribution into account may lead to a 50% underestimation of gm but had little effect on the CO 2-and irradiance-induced changes. In conclusion, (i) the observed responses of gm to CO 2 and irradiance were not artefactual; (ii) the respiratory term is important to assess absolute values of gm but has no impact on the responses to CO 2 and PPFD; and (iii) increasing irradiance and reducing the CO 2 mole fraction results in rapid increases in gm in Eucalyptus seedlings. © 2011 The Author.
Warren, C R; Aranda, I; Cano, F J
Responses to water stress of gas exchange and metabolites in Eucalyptus and Acacia spp. Journal Article
In: Plant, Cell and Environment, vol. 34, no. 10, pp. 1609-1629, 2011.
Studies of water stress commonly examine either gas exchange or leaf metabolites, and many fail to quantify the concentration of CO2 in the chloroplasts (Cc). We redress these limitations by quantifying Cc from discrimination against 13CO2 and using gas chromatography-mass spectrometry (GC-MS) for leaf metabolite profiling. Five Eucalyptus and two Acacia species from semi-arid to mesic habitats were subjected to a 2 month water stress treatment (Ψpre-dawn=-1.7 to -2.3MPa). Carbohydrates dominated the leaf metabolite profiles of species from dry areas, whereas organic acids dominated the metabolite profiles of species from wet areas. Water stress caused large decreases in photosynthesis and Cc, increases in 17-33 metabolites and decreases in 0-9 metabolites. In most species, fructose, glucose and sucrose made major contributions to osmotic adjustment. In Acacia, significant osmotic adjustment was also caused by increases in pinitol, pipecolic acid and trans-4-hydroxypipecolic acid. There were also increases in low-abundance metabolites (e.g. proline and erythritol), and metabolites that are indicative of stress-induced changes in metabolism [e.g. γ-aminobutyric acid (GABA) shunt, photorespiration, phenylpropanoid pathway]. The response of gas exchange to water stress and rewatering is rather consistent among species originating from mesic to semi-arid habitats, and the general response of metabolites to water stress is rather similar, although the specific metabolites involved may vary. Studies of water stress commonly examine either gas exchange or leaf metabolites, and many gas exchange studies fail to quantify the concentration of CO2 in the chloroplasts (Cc). This study provides a more holistic picture of how water stress affects metabolism by quantifying Cc from discrimination against 13CO2 and using GC-MS for leaf metabolite profiling. We show that in a range of Eucalyptus and Acacia species water stress decreases photosynthesis and Cc, increases amounts of 17-33 metabolites and decreases amounts of 0-9 metabolites. In addition to the well-known abundant metabolites that gave rise to osmotic adjustment, there were also changes in low-abundance metabolites that likely help plants cope with drought via non-osmotic roles (e.g. stabilisation of membranes and proteins), and metabolites that are indicative of stress-induced changes in metabolism (e.g. GABA shunt, photorespiration, pinitol synthesis, phenylpropanoid pathway). © 2011 Blackwell Publishing Ltd.
Warren, C R
How does P affect photosynthesis and metabolite profiles of Eucalyptus globulus? Journal Article
In: Tree Physiology, vol. 31, no. 7, pp. 727-739, 2011.
Phosphorus (P) has multiple effects on plant metabolism, but there are many unresolved questions especially for evergreen trees. For example, we do not know the general effects of P on metabolism, or if P affects photosynthesis via the internal conductance to CO 2 transfer from sub-stomatal cavities to chloroplast or amounts of Rubisco. This study investigates how P deficiency affects seedlings of the evergreen tree Eucalyptus globulus grown for 2.5 months with four nutrient solutions differing in P concentration. To determine why photosynthesis was affected by P supply, Rubisco was quantified by capillary electrophoresis, internal conductance was quantified from gas exchange and carbon isotope discrimination, and biochemical parameters of photosynthesis were estimated from A/C c responses. Additional insights into the effect of P on metabolism were provided by gas chromatography-mass spectrometry (GC-MS) metabolite profiling. Larger concentrations of P in the nutrient solution led to significantly faster rates of photosynthesis. There was no evidence that stomatal or internal conductances contributed to the effect of P supply on photosynthesis. The increase in photosynthesis with P supply was correlated with V cmax, and amounts of P, phosphate and fructose 6-phosphate (6-P). Phosphorous supply affected approximately one-third of the 90 aqueous metabolites quantified by GC-MS, but the effect size was generally smaller than reported for experiments on herbaceous species. Phosphorus deficiency decreased concentrations of phosphate, glucose 6-P and fructose 6-P more than it decreased photosynthesis, suggesting faster turnover of smaller pools of phosphate and phosphorylated intermediates. The effect of P supply on most amino acids was small, with the exception of arginine and glutamine, which increased dramatically under P deficiency. P deficiency had small or non-significant effects on carbohydrates and organic acids of the tricarboxylic acid (TCA) cycle. The small effect of P on carbohydrates, organic acids and (most) amino acids likely reflects a functional homeostasis among C metabolism (glycolysis, TCA and pentose P cycles), rates of photosynthesis and growth. The strong functional homeostasis in E. globulus may reflect a conservative, long-term growth and metabolic strategy of evergreen trees. © The Author 2011. Published by Oxford University Press. All rights reserved.
Miyazawa, S -I; Warren, C R; Turpin, D H; Livingston, N J
Determination of the site of CO2 sensing in poplar: Is the area-based N content and anatomy of new leaves determined by their immediate CO2 environment or by the CO2 environment of mature leaves? Journal Article
In: Journal of Experimental Botany, vol. 62, no. 8, pp. 2787-2796, 2011.
Exposure to an elevated CO2 concentration ([CO2]) generally decreases leaf N content per unit area (Narea) and stomatal density, and increases leaf thickness. Mature leaves can 'sense' elevated [CO2] and this regulates stomatal development of expanding leaves (systemic regulation). It is unclear if systemic regulation is involved in determination of leaf thickness and Narea - traits that are significantly correlated with photosynthetic capacity. A cuvette system was used whereby [CO2] around mature leaves was controlled separately from that around expanding leaves. Expanding leaves of poplar (Populus trichocarpa×P. deltoides) seedlings were exposed to elevated [CO 2] (720μmol mol-1) while the remaining mature leaves inside the cuvette were under ambient [CO2] of 360μmol mol -1. Reverse treatments were performed. Exposure of newly developing leaves to elevated [CO2] increased their thickness, but when mature leaves were exposed to elevated [CO2] the increase in thickness of new leaves was less pronounced. The largest response to [CO2] was reflected in the palisade tissue thickness (as opposed to the spongy tissue) of new leaves. The Narea of new leaves was unaffected by the local [CO2] where the new leaves developed, but decreased following the exposure of mature leaves to elevated [CO2]. The volume fraction of mesophyll cells compared with total leaf and the mesophyll cell density changed in a manner similar to the response of Narea. These results suggest that Narea is controlled independently of the leaf thickness, and suggest that Narea is under systemic regulation by [CO2] signals from mature leaves that control mesophyll cell division. © 2011 The Author(s).
Warren, C; Taranto, M
Ecosystem respiration in a seasonally snow-covered subalpine Grassland Journal Article
In: Arctic, Antarctic, and Alpine Research, vol. 43, no. 1, pp. 137-146, 2011.
Ecosystem respiration is important because it is the small imbalances between CO2 uptake via photosynthesis and CO2 release by ecosystem respiration that determine the effect of the biosphere on atmospheric CO2. For subalpine grasslands with mild winters we do not know the size of under-snow respiration relative to the total annual ecosystem respiration. This study determines the contribution of respiration through snow to total annual respiration, and models annual ecosystem respiration based on relationships with soil temperature and water content. Measurements were made monthly for two years in an unmanaged subalpine grassland in the Snowy Mountains of Australia. The vegetation is sparse (aboveground mass = 355-605 g m -2, belowground mass = 570-1010 g m-2) and dominated by native perennial C3 grasses and sedges. Ecosystem respiration was positively related to temperature, and there was some evidence that ecosystem respiration was more temperature sensitive at temperatures between 0 and 2°C than at warmer temperatures. Annual ecosystem respiration was 12.1 Mg C ha-1 yr-1 in 2007/2008 and 10.5 Mg C ha-1 yr-1 in 2008/2009. Maximum daily rates of ecosystem respiration of 7 mmol CO2 m-2 s-1 occurred during summer, while minimum rates occurred under snow cover and were 0.2 to 0.9 μmol CO2 m -2 s-1. The duration of permanent snow cover was 60-68 days (equivalent to 16-18% of the year) and ecosystem respiration under snow was 4.1 to 4.3% of annual ecosystem respiration, which is smaller than the 10-50% commonly reported from studies in areas with longer snow-covered periods.
Morales, F; Warren, C R
Photosynthetic responses to nutrient deprivation and toxicities Book
Plant growth requires the incorporation of elements (nutrients) into plant organs. In non-woody plants, 15–20% of fresh weight is made from such elements, the rest being water. There are two criteria to consider an element as essential. First, an element is essential if a plant cannot complete its lifecycle (till viable seeds) in its absence. Second, an element is essential if necessary to synthesise molecules that cannot be replaced by other element(s) (for example, N in proteins). In natural ecosystems, soil-nutrient availability is rather heterogeneous, and plants may adapt their growth to nutrients taken up by roots exploring a determined soil volume. In agricultural areas, the situation is different. Lack of or excess nutrients are frequent, owing to soil characteristics (which may immobilise nutrients) or to growers’ applications, respectively. An idealised representation of plant growth-rate response to availability of any given nutrient would show three different zones: namely (1) deficient; (2) adequate; and (3) toxic. In the range of low nutrient concentrations (deficient zone), growth and the plant nutrient concentration markedly increase as soil nutrient availability increases. As availability increases further the so-called critical concentration is reached. This corresponds to the lowest concentration of nutrient in plant tissue that gives almost maximal growth. Above this point, increases in soil nutrient availability do not affect growth (adequate zone). In the adequate zone, there is a plentiful supply of nutrients, and the excess nutrients may be taken up and stored in leaf vacuoles, special storage proteins in bark or uptake may be downregulated so as to avoid taking up excess nutrients. This zone is fairly wide for macronutrients, but narrower for micronutrients. If nutrient availability increases more and uptake cannot be controlled, toxicity appears and growth is reduced (toxic zone). © Cambridge University Press 2012.
Warren, C R; García-Plazaola, J I; Niinemets, Ü
Ecophysiology of photosynthesis in temperate forests Book
The Temperate-Forest Environment The temperate zone is characterised by pronounced seasonality with temperatures of the warmest month generally higher than 10°C and temperatures of the coldest month generally between –10 and 10°C (Köppen, 1936; Russell, 1931). Temperature is arguably the most important climatic variable in temperate forests. Temperatures during warm and cold periods are strongly variable within the temperate-forest biome, depending on continentality, latitude and topography (Fig. 30.1). Total precipitation is generally greater than 50–75 cm year–1 and is more uniformly distributed over the year than in arid (Chapter 28) and in semi-arid (including Mediterranean ecosystems) (Chapter 29) habitats. The annual input of solar radiation is between 2500–6000 MJ m–2, varying with site latitude, cloudiness and topography (Jarvis and Leverenz, 1983). Temperate forests are dominated by deciduous trees in oceanic and continental areas of the Northern hemisphere, while evergreens dominate in warmer locations and in the Southern hemisphere. In the edges of temperate biome, mixed forest may appear. Thus, on the cold border the transition to steppes is characterised by open conifer or deciduous forests, while there are mixed conifer-deciduous woodlands in the transition to the boreal biome. In the warm border, the transition is characterised by subtropical evergreen forests in humid locations and by the presence of deciduous Mediterranean oaks in more arid sites. © Cambridge University Press 2012.
Flexas, J; Brugnoli, E; Warren, C R
Mesophyll conductance to co2 Book
During photosynthesis, CO2has to move from the atmosphere surrounding the leaf, across a boundary layer in the air above the foliage surface, to the sub-stomatal internal cavities through the stomata (Fig. 12.1A) and from there to the site of carboxylation inside the chloroplast stroma through the leaf mesophyll. © Cambridge University Press 2012.
Barbour, M; Warren, C; Farquhar, G; Forrester, G; Brown, H
Variability in mesophyll conductance between barley genotypes, and effects on transpiration efficiency and carbon isotope discrimination Journal Article
In: Plant, Cell and Environment, vol. 33, no. 7, pp. 1176-1185, 2010.
Leaf internal, or mesophyll, conductance to CO2 (gm) is a significant and variable limitation of photosynthesis that also affects leaf transpiration efficiency (TE). Genotypic variation in gm and the effect of gm on TE were assessed in six barley genotypes (four Hordeum vulgare and two H. bulbosum). Significant variation in gm was found between genotypes, and was correlated with photosynthetic rate. The genotype with the highest gm also had the highest TE and the lowest carbon isotope discrimination as recorded in leaf tissue (Δp). These results suggest gm has unexplored potential to provide TE improvement within crop breeding programmes. © 2010 Blackwell Publishing Ltd.
Paulding, E M; Baker, A J M; Warren, C R
Competition for nitrogen by three sympatric species of Eucalyptus Journal Article
In: Annals of Forest Science, vol. 67, no. 4, 2010.
Nitrogen (N) exists in the soil in a variety of different forms and thus plants may avoid competition by taking up N as different chemical forms. This study examined the uptake of nitrate, ammonium and glycine by three co-occurring species of Eucalyptus (E. obliqua, E. radiata and E. rubida) from dry sclerophyll forest in south-eastern Australia. Species preference for N forms was determined by measuring uptake of glycine, nitrate and ammonium from 15N-labelled solutions containing equimolar 100 μmol L -1 concentrations of all three N forms. KCl extracts were used to assess the relative abundance of the different forms of N in the soil's exchangeable pool. KCl extracts of soil indicated that amino acids comprised 30-40% of the soluble non-protein N, while ammonium varied from 10-70% and nitrate from 5-70%. In all species, ammonium was the preferred source of nitrogen and was taken up 2.5-4.5 times faster than glycine, and 30-50 times faster than nitrate. Species did not differ in preference for N-forms (species*N-form interaction, not significant). This study indicates that nitrate, ammonium and amino acids are all present in soil, and thus there is the potential for niche differentiation based on chemical forms of N. However, there is no evidence that co-occurring Eucalyptus avoid competition for N by taking up different chemical forms. © INRA, EDP Sciences, 2010.
Merchant, A; Peuke, A D; Keitel, C; MacFarlane, C; Warren, C R; Adams, M A
Phloem sap and leaf δ13C, carbohydrates, and amino acid concentrations in Eucalyptus globulus change systematically according to flooding and water deficit treatment Journal Article
In: Journal of Experimental Botany, vol. 61, no. 6, pp. 1785-1793, 2010.
Phloem is a central conduit for the distribution of photoassimilate, nutrients, and signals among plant organs. A revised technique was used to collect phloem sap from small woody plants in order to assess changes in composition induced by water deficit and flooding. Bled phloem sap δ13C and sugar concentrations were compared to δ13C of bulk material, soluble carbon extracts, and the neutral sugar fraction from leaves. Amino acid composition and inorganic ions of the phloem sap was also analysed. Quantitative, systematic changes were detected in phloem sap composition and δ13C in response to altered water availability. Phloem sap δ13C was more sensitive to changes of water availability than the δ13C of bulk leaf, the soluble carbon fraction, and the neutral soluble fraction of leaves. Changes in water availability also resulted in significant changes in phloem sugar (sucrose and raffinose), inorganic nutrient (potassium), and amino acid (phenylalanine) concentrations with important implications for the maintenance of phloem function and biomass partitioning. The differences in carbohydrate and amino acid composition as well as the δ13C in the phloem, along with a new model system for phloem research, offer an improved understanding of the phloem-mediated signal, nutrient, and photoassimilate transduction in relation to water availability. © 2010 The Author(s).
Warren, C R; Taranto, M T
Temporal variation in pools of amino acids, inorganic and microbial N in a temperate grassland soil Journal Article
In: Soil Biology and Biochemistry, vol. 42, no. 2, pp. 353-359, 2010.
Plants can take up intact amino acids, even in competition with soil microbes, yet we lack detailed information on which amino acids dominate the soil and whether amino acid composition varies seasonally. This study tested the hypotheses that 1) the pool of amino acid N is generally larger than inorganic N; 2) temporal changes in the concentration of amino acid N is related to changes in the size of the microbial N pool; and 3) amino acid N is dominated by simple, neutral amino acids during warm months, whereas during cold months the amino acid N is dominated by more complex aromatic and basic amino acids. Approximately every month for two years we collected soil from a temperate, sub-alpine grassland in the Snowy Mountains of Australia. We quantified exchangeable pools of amino acids, nitrate and ammonium in 1 M KCl extracts. Microbial N was quantified by chloroform fumigation. Averaged across the 21 monthly samples, nitrate was 13% of the quantified pool of soluble non-protein N, ammonium was 34% and amino acid N was 53%. These data are consistent with our hypothesis that the pool of amino acid N is larger than inorganic N. There was substantial variation between months in concentrations of amino acids and inorganic N, but no clear temporal pattern. Microbial N did not vary between months, and thus changes in amino acid N were unrelated to microbial N. Principal components analysis indicated multivariate groupings of the different pools of N that were broadly indicative of function and/or biosynthetic relationships. Thus PCA identified a grouping of aromatic amino acids (Phe and Try) with amino acids derived from oxaloacetate (Asp, Ala, Val, Leu, Ile), and a second group comprising microbial N, nitrate and glycine. The pool of exchangeable amino acid N was dominated by Arg (26% of amino N) Val (20%) Gln (18%), Try (8%) and Asn (8%). Contrary to our hypothesis, the composition of the amino acid pool did not vary in a consistent way between months, and there was no evidence simple amino acids were relatively more abundant in warm months and complex amino acids in cool months. © 2009 Elsevier Ltd. All rights reserved.
Eyles, A; Pinkard, E A; O'Grady, A P; Worledge, D; Warren, C R
Role of corticular photosynthesis following defoliation in Eucalyptus globulus Journal Article
In: Plant, Cell and Environment, vol. 32, no. 8, pp. 1004-1014, 2009.
Defoliation can reduce net fixation of atmospheric CO2 by the canopy, but increase the intensity and duration of photosynthetically active radiation on stems. Stem CO2 flux and leaf gas exchange in young Eucalyptus globulus seedlings were measured to assess the impact of defoliation on these processes and to determine the potential contribution of re-fixation by photosynthetic inner bark in offsetting the effects of defoliation in a woody species. Pot and field trials examined how artificial defoliation of the canopy affected the photosynthetic characteristics of main stems of young Eucalyptus globulus seedlings. Defoliated potted seedlings were characterized by transient increases in foliar photosynthetic rates and concomitant decreases in stem CO2 fluxes (both in the dark and light). Defoliated field-grown seedlings showed similar stem CO2 flux responses, but of reduced magnitude. Despite demonstrating increased re-fixation capability, defoliated potted-seedlings had slowed stem growth. The green stem of seedlings exhibited largely shade-adapted characteristics. Defoliation reduced stem chlorophyll a/b ratio and increased carotenoid concentration. An increased capacity to re-fix internally respired CO2 (up to 96%) suggested that stem re-fixation represents a previously unexplored mechanism to minimize the impact of foliar loss by maximizing the contribution of all photosynthetic tissues, particularly for young seedlings. © 2009 Blackwell Publishing Ltd.
Niinemets, U; Díaz-Espejo, A; Flexas, J; Galmés, J; Warren, C R
Role of mesophyll diffusion conductance in constraining potential photosynthetic productivity in the field Journal Article
In: Journal of Experimental Botany, vol. 60, no. 8, pp. 2249-2270, 2009.
Limited mesophyll diffusion conductance to CO2 (gm) can significantly constrain plant photosynthesis, but the extent of gm-limitation is still imperfectly known. As gm scales positively with foliage photosynthetic capacity (A), the CO2 drawdown from substomatal cavities (Ci) to chloroplasts (CC
Niinemets, U; Díaz-Espejo, A; Flexas, J; Galmés, J; Warren, C R
Importance of mesophyll diffusion conductance in estimation of plant photosynthesis in the field Journal Article
In: Journal of Experimental Botany, vol. 60, no. 8, pp. 2271-2282, 2009.
Mesophyll diffusion conductance to CO2 (gm) is an important leaf characteristic determining the drawdown of CO2 from substomatal cavities (Ci) to chloroplasts (CC). Finite gm results in modifications in the shape of the net assimilation (A) versus Ci response curves, with the final outcome of reduced maximal carboxylase activity of Rubisco (Vcmax), and a greater ratio of the capacity for photosynthetic electron transport to Vcmax (Jmax/Vcmax) and alterations in mitochondrial respiration rate (Rd) when estimated from A/Ci responses without considering gm. The influence of different Farquhar et al. model parameterizations on daily photosynthesis under non-stressed (Ci kept constant throughout the day) and stressed conditions (mid-day reduction in Ci) was compared. The model was parameterized on the basis of A/CC curves and A/Ci curves using both the conventional fitting procedure (Vcmax and Rd fitted separately to the linear part of the response curve and Jmax to the saturating part) and a procedure that fitted all parameters simultaneously. The analyses demonstrated that A/Ci parameterizations overestimated daily assimilation by 6-8% for high gm values, while they underestimated if by up to 70% for low gm values. Qualitative differences between the A/Ci and A/CC parameterizations were observed under stressed conditions, when underestimated Vcmax and overestimated Rd of A/Ci parameterizations resulted in excessive mid-day depression of photosynthesis. Comparison with measured diurnal assimilation rates in the Mediterranean sclerophyll species Quercus ilex under drought further supported this bias of A/Ci parameterizations. While A/Ci parameterization predicted negative carbon balance at mid-day, actual measurements and simulations with the A/CC approach yielded positive carbon gain under these conditions. In addition, overall variation captured by the best A/Ci parameterization was poor compared with the A/CC approach. This analysis strongly suggests that for correct parameterization of daily time-courses of photosynthesis under realistic field conditions, gm must be included in photosynthesis models.
Warren, C R
Does nitrogen concentration affect relative uptake rates of nitrate, ammonium, and glycine? Journal Article
In: Journal of Plant Nutrition and Soil Science, vol. 172, no. 2, pp. 224-229, 2009.
Plant roots are exposed to a variety of nitrogen forms (e.g., nitrate, ammonium, amino acids) and take up these forms at different rates. Many studies have investigated whether plants prefer nitrate, ammonium, or amino acids; but studies may not be comparable because they used substrate concentrations between 100 and 2000 μmol L-1. This study tests the hypothesis that substrate concentrations from 10 to 1750 μmol L-1 affect plant preference for N forms. Nitrogen uptake by the herb Ocimum basilicum and the evergreen tree Eucalyptus regnans was examined by placing roots of intact seedlings in equimolar mixtures of nitrate, ammonium, and glycine in which one of the N forms was 15N-labelled (and 13C-labelled in the case of glycine). In both species, preference for N forms was affected by substrate concentration. At 10 μmol L-1 (O. basilicum) or 10 and 50 μmol L-1 (E. regnans), rates of N uptake did not differ among N forms. At substrate concentrations of 50 μmol L-1 and greater O. basilicum took up ammonium the fastest, glycine the slowest, and nitrate at an intermediate rate. At substrate concentrations from 100 to 1750 μmol L -1, E. regnans took up ammonium the fastest with glycine and nitrate taken up at slower rates. The absence of significant differences at lower concentrations was a true biological effect rather than a function of larger relative errors. This study demonstrates that substrate concentration has a large effect on plant preference for N forms, and sounds a warning for studies of N nutrition that do not consider the concentration-dependence of plant preference for N forms. © 2009 WILEY-VCH Verlag GmbH & Co. KGaA.
Warren, C R
Why does temperature affect relative uptake rates of nitrate, ammonium and glycine: A test with Eucalyptus pauciflora Journal Article
In: Soil Biology and Biochemistry, vol. 41, no. 4, pp. 778-784, 2009.
Few studies have examined how temperature affects uptake of nitrate, ammonium and amino acids from soil. This study tests the hypothesis that cool temperatures favour uptake of the amino acid glycine while warm temperatures favour uptake of inorganic forms of N such as nitrate. We used glasshouse-grown ectomycorrhizal seedlings of the sub-alpine tree species Eucalyptus pauciflora Sieber ex Spreng. Seedlings were grown in soil (humic umbrosol, from species' habitat) that was dominated by amino acids and ammonium with only small amounts of nitrate. To examine if root physiology affects temperature responses of N uptake, we measured uptake from 15N-labelled hydrosolutions containing equimolar 100 μmol L-1 mixtures of ammonium, nitrate and glycine at temperatures from 5 to 35 °C. We also examined if the effect of temperature on uptake of N forms was due to plant-microbe competition by following the fate of equimolar amounts of labelled ammonium, nitrate and glycine injected into the soil at temperatures of 5 °C and 25 °C. Hydrosolution experiments showed that uptake of glycine was favoured by warm temperatures and inorganic N by cool temperatures. In contrast, when 15N was injected into soil the uptake of glycine was favoured by low temperatures and nitrate by warm temperatures. At 25 °C, glycine was 17% of the N taken up from soil and nitrate was 51%; whereas at 5 °C glycine was 30% of the N taken up from soil and nitrate was 23%. Microbes were better competitors than seedlings for all forms of N, but temperature did not affect microbial preference for the different N forms. Hence, while microbes limit N available for plant uptake, they do not seem to be the cause of the greater plant uptake of glycine at cool temperatures and nitrate at warm temperatures. Intact uptake of glycine by plants was suggested by the positive relationship between uptake of 13C and 15N and detection by GC-MS of intact 13C2, 15N glycine molecules in roots. In conclusion, uptake of glycine is favoured by cool temperatures and nitrate by warm temperatures, but this is apparently not a function of root physiology or competition with soil microbes. © 2009 Elsevier Ltd. All rights reserved.
Warren, C R
Uptake of inorganic and amino acid nitrogen from soil by Eucalyptus regnans and Eucalyptus pauciflora seedlings Journal Article
In: Tree Physiology, vol. 29, no. 3, pp. 401-409, 2009.
This study examined whether two species of Eucalyptus can take up the amino acid glycine from soil and compared the uptake rate of glycine with the uptake rates of nitrate and ammonium. Ectomycorrhizal seedlings of two ecologically disparate species were studied: Eucalyptus regnans F. Muell., a fast-growing forest tree from low altitudes; and Eucalyptus pauciflora Sieber ex Spreng., a slow-growing tree that forms the alpine treeline. Seedlings were grown from seeds in field soil. When seedlings were 45 months old, soils were injected with equimolar mixtures of isotope-labeled glycine, ammonium and nitrate. Seedlings and soil were harvested 4 and 48 h later. Isotope ratio mass spectrometry analysis of 13C and 15N enrichment in plants receiving glycine indicated uptake of 1.5 13C for every 15N at the 4-h harvest (versus 2:1 13C:15N in labeled glycine), suggesting intact uptake of around 75% of glycine. Gas chromatographymass spectrometry analysis detected intact 13C2,15N-glycine in roots, but the pool of 13C2,15N-glycine was 10500 times smaller than 13C and 15N excess, and no 13C2,15N-glycine was detected in shoots. This is consistent with glycine being taken up as an intact molecule that is subsequently metabolized rapidly. Both species took up more nitrate than ammonium, and glycine was the least preferred form of nitrogen (N). Microbes took up more N than seedlings, and their preference for N forms was the mirror image of the plant preferences. These data suggest that patterns of microbial uptake may determine plant preference for forms of N. © 2009 Walter de Gruyter.
Posch, S; Warren, C R; Adams, M A; Guttenberger, H
Photoprotective carotenoids and antioxidants are more affected by canopy position than by nitrogen supply in 21-year-old Pinus radiata Journal Article
In: Functional Plant Biology, vol. 35, no. 6, pp. 470-482, 2008.
Photoprotection, light harvesting and light utilisation were investigated as a function of variation in N supply and canopy position in 21-year-old Pinus radiata D. Don. Chlorophyll fluorescence, gas exchange and photoprotective compounds were measured on lower, middle and upper canopy needles in trees receiving N fertiliser and in control trees not receiving N fertiliser. Irrespective of canopy height, additional N increased the light-harvesting capacity through greater contents of chlorophyll, neoxanthin and lutein, but did not affect light-utilisation processes, such as effective quantum yield of PSII or rates of net CO2 assimilation. Additional N fertiliser did not affect the concentrations of the measured photoprotective carotenoids (violaxanthin, antheraxanthin, zeaxanthin, α-carotene and β-carotene) or antioxidants (ascorbic acid, glutathione and α-tocopherol); however, carotenoids and antioxidants were strongly affected by canopy height and increased in concentration with increasing canopy height. The present study found that pools of photoprotective carotenoids and antioxidants were not driven by imbalances in light-harvesting and light-utilisation processes, but rather by gradients in light. © CSIRO 2008.
Warren, C R
Rapid measurement of chlorophylls with a microplate reader Journal Article
In: Journal of Plant Nutrition, vol. 31, no. 7, pp. 1321-1332, 2008.
This paper describes a robust and rapid procedure for measuring chlorophyll using a microplate reader. The procedure enables direct translation of any published equation developed on a 1-cm pathlength spectrophotometer to any format microplate with any sample volume. Chlorophylls were extracted with methanol because it is compatible with cheap polystyrene plates; however, the principles apply to any solvent system. Keys to obtaining accurate and precise results include determining pathlength in the same solvent used to extract chlorophylls, and avoiding evaporation. After taking these precautions, there was a strong linear 1:1 relationship (R2 = 0.98-0.99) between microplate and spectrophotometer estimates of chlorophyll concentrations. Copyright © Taylor & Francis Group, LLC.
Warren, C R
Stand aside stomata, another actor deserves centre stage: The forgotten role of the internal conductance to CO2 transfer Journal Article
In: Journal of Experimental Botany, vol. 59, no. 7, pp. 1475-1487, 2008.
Internal conductance describes the movement of CO2 from substomatal cavities to sites of carboxylation. Internal conductance has now been measured in approximately 50 species, and in all of these species it is a large limitation of photosynthesis. It accounts for somewhat less than half of the decrease in CO2 concentrations from the atmosphere to sites of carboxylation. There have been two major findings in the past decade. First, the limitation due to internal conductance (i.e. Ci-Cc) is not fixed but varies among species and functional groups. Second, internal conductance is affected by some environmental variables and can change rapidly, for example, in response to leaf temperature, drought stress or CO2 concentration. Biochemical factors such as carbonic anhydrase or aquaporins are probably responsible for these rapid changes. The determinants of internal conductance remain elusive, but are probably a combination of leaf anatomy, morphology, and biochemical factors. In most plants, the gas phase component of internal conductance is negligible with the majority of resistance resting in the liquid phase from cell walls to sites of carboxylation. The internal conductance story is far from complete and many exciting challenges remain. Internal conductance ought to be included in models of canopy photosynthesis, but before this is feasible additional data on the variation in internal conductance among and within species are urgently required. Future research should also focus on teasing apart the different steps in the diffusion pathway (intercellular spaces, cell wall, plasmalemma, cytosol, and chloroplast envelope) since it is likely that this will provide clues as to what determines internal conductance. © The Author . Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
Warren, C R
Rapid and sensitive quantification of amino acids in soil extracts by capillary electrophoresis with laser-induced fluorescence Journal Article
In: Soil Biology and Biochemistry, vol. 40, no. 4, pp. 916-923, 2008.
A growing body of research is arguing that amino acids are key components of the soil nitrogen cycle. For example, we now know that many plants can take up intact amino acids, even in competition with soil microbes. Our growing recognition of the importance of amino acids is not matched by knowledge of the amounts and type of amino acids in the soil, certainly not in comparison with our encyclopaedic knowledge of inorganic N. The primary reason that less is known about amino acids than inorganic N is that measuring the amounts of individual amino acids with conventional chromatographic techniques is slow and typically requires extensive sample clean-up if KCl extracts are analysed. The aim of this study was to develop capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) as a more rapid alternative for measuring individual amino acids in KCl extracts of soil. The CE-LIF method separated 17 common amino acids within 12 min, with detection limits between 7 and 250 nM. One molar KCl extracts could be analysed without any sample clean-up or de-salting, and spike and recovery tests indicated that the complex matrix of soil extracts did not affect quantification. Further evidence of the suitability and robustness of the method came from the repeated analysis (n=5) of the same soil KCl extract. The relative standard deviation of migration times for replicate analyses were <0.2% while relative standard deviations for peak areas were <5%. To demonstrate application of the CE-LIF method to real world problems it was used to analyse amino acids in 1 M KCl extracts from a sub-alpine grassland and a Eucalyptus regnans forest. The most abundant amino acids were Ala, Gly and Arg. Other amino acids present at smaller concentrations or in a minority of samples were Asn, Cit, GABA, Glu, His, Phe, Leu, and Lys. The proposed CE-LIF method offers significant advantages over chromatographic methods via its rapidity, reproducibility and, most importantly, its ability to analyse crude KCl extracts. © 2007 Elsevier Ltd. All rights reserved.
Warren, C R
Soil water deficits decrease the internal conductance to CO2 transfer but atmospheric water deficits do not Journal Article
In: Journal of Experimental Botany, vol. 59, no. 2, pp. 327-334, 2008.
The internal conductance to CO2 supply from substomatal cavities to sites of carboxylation poses a large limitation to photosynthesis. It is known that internal conductance is decreased by soil water deficits, but it is not known if it is affected by atmospheric water deficits (i.e. leaf to air vapour pressure deficit, VPD). The aim of this paper was to examine the responses of internal conductance to atmospheric and soil water deficits in seedlings of the evergreen perennial Eucalyptus regnans F. Muell and the herbaceous plants Solanum lycopersicum (formerly Lycopersicon esculentum) Mill. and Phaseolus vulgaris L. Internal conductance was estimated with the variable J method from concurrent measurements of gas exchange and fluorescence. In all three species steady-state stomatal conductance decreased by ∼30% as VPD increased from 1 kPa to 2 kPa. In no species was internal conductance affected by VPD despite large effects on stomatal conductance. In contrast, soil water deficits decreased stomatal conductance and internal conductance of all three species. Decreases in stomatal and internal conductance under water deficit were proportional, but this proportionality differed among species, and thus the relationship between stomatal and internal conductance differed among species. These findings indicate that soil water deficits affect internal conductance while atmospheric water deficits do not. The reasons for this distinction are unknown but are consistent with soil and atmospheric water deficits having differing effects on leaf physiology and/or root-shoot communication. © 2008 The Author(s).
Warren, C R
Does growth temperature affect the temperature responses of photosynthesis and internal conductance to CO2? A test with Eucalyptus regnans Journal Article
In: Tree Physiology, vol. 28, no. 1, pp. 11-19, 2008.
Internal conductance to CO2 transfer from intercellular spaces to chloroplasts (gi) poses a major limitation to photosynthesis, but only three studies have investigated the temperature dependance of g i. The aim of this study was to determine whether acclimation to 15 versus 30°C affects the temperature response of photosynthesis and g i in seedlings of the evergreen tree species Eucalyptus regnans F. Muell. Six-month-old seedlings were acclimated to 15 or 30°C for 6 weeks before gi was estimated by simultaneous measurements of gas exchange and chlorophyll fluorescence (variable J method). There was little evidence for acclimation of photosynthesis to growth temperature. In seedlings acclimated to either 15 or 30°C, the maximum rate of net photosynthesis peaked at around 30 or 35°C. Such lack of temperature acclimation may be related to the constant day and night temperature acclimation regime, which differed from most other studies in which night temperatures were lower than day temperatures. Internal conductance averaged 0.25 mol m-2 s-1 at 25°C and increased threefold from 10 to 35°C. There was some evidence that gi was greater in seedlings acclimated to 15 than to 30°C, which resulted in seedlings acclimated to 15°C having, if anything, a smaller relative limitation due to gi than seedlings acclimated to 30°C. Stomatal limitations were also smaller in seedlings acclimated to 15°C than in seedlings acclimated to 30°C. Based on chloroplast CO2 concentration, neither maximum rates of carboxylation nor RuBP-limited rate of electron transport peaked between 10 and 35°C. Both were described well by an Arrhenius function and had similar activation energies (57-70 kJ mol -1). These findings confirm previous studies showing gi to be positively related to measurement temperature. © 2008 Heron Publishing.
Posch, S; Warren, C R; Kruse, J; Guttenberger, H; Adams, M A
Nitrogen allocation and the fate of absorbed light in 21-year-old Pinus radiata Journal Article
In: Tree Physiology, vol. 28, no. 3, pp. 375-384, 2008.
We investigated effects of nitrogen (N) fertilizer and canopy position on the allocation of N to Rubisco and chlorophyll as well as the distribution of absorbed light among thermal energy dissipation, photochemistry, net CO 2 assimilation and alternative electron sinks such as the Mehler reaction and photorespiration. The relative reduction state of the primary quinone receptor of photosystem II (QA) was used as a surrogate for photosystem II (PSII) vulnerability to photoinactivation. Measurements were made on needles from the lower, mid and upper canopy of 21-year-old Pinus radiata D. Don trees grown with (N+) and without (N0) added N fertilizer. Rubisco was 45 to 60% higher in needles of N+ trees than in needles of NO trees at all canopy positions. Chlorophyll was ∼80% higher in lower- and mid-canopy needles of N+ trees than of N0 trees, but only ∼20% higher in upper-canopy needles. Physiological differences between N+ and N0 trees were found only in the lower- and mid- canopy positions. Needles of N+ trees dissipated up to 30% less light energy as heat than needles of N0 trees and had correspondingly more reduced QA. Net CO2 assimilation and the proportions of electrons used by alternative electron sinks such as the Mehler reaction and photorespiration were unaffected by N treatment regardless of canopy position. We conclude that the application of N fertilizer mainly affected the biochemistry and light-use physiology in lower- and mid-canopy needles by increasing the amount of chlorophyll and hence the amount of light harvested. This, however, did not improve photochemistry or safe dissipation, but increased PSII vulnerability to photoinactivation, an effect with likely significant consequences during sunflecks or sudden gap formation. © 2008 Heron Publishing.
Turnbull, T L; Warren, C R; Adams, M A
Novel mannose-sequestration technique reveals variation in subcellular orthophosphate pools do not explain the effects of phosphorus nutrition on photosynthesis in Eucalyptus globulus seedlings Journal Article
In: New Phytologist, vol. 176, no. 4, pp. 849-861, 2007.
• Although only a small proportion of plant phosphorus (P) is used for photosynthesis, the relationships between P and photosynthesis can be strong. It was hypothesized, in this study, that variation in the allocation of orthophosphate (Pi) between active (cytoplasmic) and nonactive (vacuolar) pools would underpin differences in rates of photosynthesis in 4-month-old Eucalyptus globulus seedlings grown with a varying P supply. • Photosynthetic biochemistry was assessed by the response of net photosynthesis to increasing intercellular [CO2]. Cytoplasmic Pi was sequestered as mannose 6-phosphate. • Total P and the proportion of P as Pi were positively related to P supply. The ratios of active : stored Pi (10-24%) varied little over the range of treatments. Active Pi was positively related to P supply, as was photosynthesis (7 μmol CO2 m-2 s-1 with 0 mm P vs. 16 μmol CO2 m-2 s-1 with 0.32 mm P). Positive relationships between P supply and photosynthesis were explained best by leaf P content, not by active pools of Pi. • The distribution of P i between the vacuole and the cytoplasm had little impact on the photosynthetic phosphorus-use efficiency (PPUE), and reductions in cytoplasmic Pi had little effect on photosynthesis. Hence, PPUE is an unsuitable guide for assessing plant responses to increasingly unavailable P in the environment. © The Authors (2007).
Warren, C R; Bleby, T; Adams, M A
Changes in gas exchange versus leaf solutes as a means to cope with summer drought in Eucalyptus marginata Journal Article
In: Oecologia, vol. 154, no. 1, pp. 1-10, 2007.
Two of the ways in which plants cope with water deficits are stomatal closure and ösmotic adjustment". We sought to assess the contributions of these processes to maintenance of leaf hydration in field-grown, 7-year-old Eucalyptus marginata. Plants were exposed to their normal summer drought (controls) or supplied with additional water (irrigated). Irrigation increased photosynthesis by 30% in E. marginata. These increases in photosynthesis were related to an 80% increase in g s. However, there was no difference in substomatal CO2 concentrations between treatments, or in chloroplast CO2 concentrations, as indicated by carbon isotope composition of leaf soluble sugars. This suggests that impaired mesophyll metabolism may partially explain slower rates of photosynthesis in plants exposed to their normal summer drought. There was no difference in concentrations of solutes or osmotic potential between non-irrigated and irrigated individuals, perhaps because relative water content was the same in non-irrigated and irrigated plants due to stomatal sensitivity to water deficits. Irrespective of the absence of osmotic adjustment, analysis of leaf solutes gave a clear indication of the major groups of compounds responsible for maintaining cell osmotic potential. Soluble sugars were three times as abundant as amino acids. Proline, a putatively osmotically active amino acid, contributed less than 1% of total solutes. These patterns of solutes in E. marginata are consistent with a growing body of literature arguing a greater role for carbohydrates and cyclitols and lesser role for amino acids in maintaining osmotic potential. Our data suggest the primary mechanism by which E. marginata coped with drought was partial stomatal closure; however, we cannot discount the possibility of osmotic adjustment under more severe water deficits. © 2007 Springer-Verlag.
Löw, M; Häberle, K -H; Warren, C R; Matyssek, R
O3 flux-related responsiveness of photosynthesis, respiration, and stomatal conductance of adult Fagus sylvatica to experimentally enhanced free-air O3 exposure Journal Article
In: Plant Biology, vol. 9, no. 2, pp. 197-206, 2007.
Knowledge of responses of photosynthesis, respiration, and stomatal conductance to cumulative ozone uptake (COU) is still scarce, and this is particularly the case for adult trees. The effect of ozone (O3) exposure on trees was examined with 60-year-old beech trees (Fagus sylvatica) at a forest site of southern Germany. Trees were exposed to the ambient O 3 regime (1 x O3) or an experimentally elevated twice-ambient O3 regime (2 x O3). The elevated 2 x O 3 regime was provided by means of a free-air O3 canopy exposure system. The hypotheses were tested that (1) gas exchange is negatively affected by O3 and (2) the effects of O3 are dose-dependent and thus the sizes of differences between treatments are positively related to COU. Gas exchange (light-saturated CO2 uptake rate Amax, stomatal conductance gs, maximum rate of carboxylation Vcmax, ribulose-1,5-bisphosphate turnover limited rate of photosynthesis Jmax, CO2 compensation point CP, apparent quantum yield of net CO2 uptake AQ, carboxylation efficiency CE, day- and night-time respiration) and chlorophyll fluorescence (electron transfer rate, ETR) were measured in situ on attached sun and shade leaves. Measurements were made periodically throughout the growing seasons of 2003 (an exceptionally dry year) and 2004 (a year with average rainfall). In 2004 Vc max, Jmax, and CE were lower in trees receiving 2 x O 3 compared with the ambient O3 regime (1 x O3). Treatment differences in Vcmax, Jmax, CE were rather small in 2004 (i.e., parameter levels were lower by 10-30% in 2 x O3 than 1 x O3) and not significant in 2003. In 2004 COU was positively correlated with the difference between treatments in Amax, g s, and ETR (i.e., consistent with the dose-dependence of O 3's deleterious effects). However, in 2003, differences in A max, gs, and ETR between the two O3 regimes were smaller at the end of the dry summer 2003 (i.e., when COU was greatest). The relationship of COU with effects on gas exchange can apparently be complex and, in fact, varied between years and within the growing season. In addition, high doses of O3 did not always have significant effects on leaf gas exchange. In view of the key findings, both hypotheses were to be rejected. © Georg Thieme Verlag KG Stuttgart.
Warren, C R; Löw, M; Matyssek, R; Tausz, M
Internal conductance to CO2 transfer of adult Fagus sylvatica: Variation between sun and shade leaves and due to free-air ozone fumigation Journal Article
In: Environmental and Experimental Botany, vol. 59, no. 2, pp. 130-138, 2007.
Two separate objectives were considered in this study. We examined (1) internal conductance to CO2 (gi) and photosynthetic limitations in sun and shade leaves of 60-year-old Fagus sylvatica, and (2) whether free-air ozone fumigation affects gi and photosynthetic limitations. gi and photosynthetic limitations were estimated in situ from simultaneous measurements of gas exchange and chlorophyll fluorescence on attached sun and shade leaves of F. sylvatica. Trees were exposed to ambient air (1× O3) and air with twice the ambient ozone concentration (2× O3) in a free-air ozone canopy fumigation system in southern Germany (Kranzberg Forest). gi varied between 0.12 and 0.24 mol m-2 s-1 and decreased CO2 concentrations from intercellular spaces (Ci) to chloroplastic (Cc) by approximately 55 μmol mol-1. The maximum rate of carboxylation (Vcmax) was 22-39% lower when calculated on a Ci basis compared with a Cc basis. gi was approximately twice as large in sun leaves compared to shade leaves. Relationships among net photosynthesis, stomatal conductance and gi were very similar in sun and shade leaves. This proportional scaling meant that neither Ci nor Cc varied between sun and shade leaves. Rates of net photosynthesis and stomatal conductance were about 25% lower in the 2× O3 treatment compared with 1× O3, while Vcmax was unaffected. There was no evidence that gi was affected by ozone. © 2006 Elsevier B.V. All rights reserved.
Erratum: Estimating the internal conductance to CO2 movement (Functional Plant Biology 33, 5 (431-442)) Journal Article
In: Functional Plant Biology, vol. 34, no. 1, pp. 82, 2007.
Warren, C R; Adams, P R
Uptake of nitrate, ammonium and glycine by plants of Tasmanian wet eucalypt forests Journal Article
In: Tree Physiology, vol. 27, no. 3, pp. 413-419, 2007.
A central assumption of ecosystem N cycling has been that organic N must be converted to inorganic N to be available for plant uptake, but this has been questioned by recent studies. We examined uptake of nitrate, ammonium and the amino acid glycine in three species from Eucalyptus obliqua L'Her. wet forest in Tasmania, south-eastern Australia, to test the hypothesis that all three species can take up glycine, and to compare rates of glycine uptake with rates of uptake of nitrate and ammonium uptake. The alternative hypothesis that species vary in their preference for nitrate, ammonium and glycine ("niche differentiation") was also examined. Measurements were made on the canopy dominant Eucalyptus obliqua, and two rain forest tree species found in the understory or as sub-dominants of the canopy, Nothofagus cunninghamii (Hook.) Oerst. and Phyllocladus aspleniifolius (Labill.) Hook.f. Nitrogen uptake was examined in situ with attached roots placed in uptake solutions containing equimolar concentrations (100 μmol 1-1) of 15N-nitrate, 15N-ammonium and 2-13C2 15N- glycine. Species did not differ in their preference for different forms of N (species × N form interaction, P > 0.05), and thus there was no evidence of niche differentiation. In all species, rates of uptake were highest for ammonium (11 ± 5 μmol gDM-1 h-1; mean ± SD
Turnbull, T L; Adams, M A; Warren, C R
Increased photosynthesis following partial defoliation of field-grown Eucalyptus globulus seedlings is not caused by increased leaf nitrogen Journal Article
In: Tree Physiology, vol. 27, no. 10, pp. 1481-1492, 2007.
Increased photosynthetic rates following partial defoliation may arise from changes in leaf biochemistry, water relations or nutrient status. Twelve-month-old field-grown Eucalyptus globulus Labill. seedlings were pruned from below to reduce the green crown depth by 50 (D50) or 70% (D70). Photosynthetic responses to light and CO2 concentration were examined before and one, three and five weeks after partial defoliation. One week after defoliation, photosynthetic rates were greater in seedlings in the D50 (21 μmol m-2 s-1 ) and D70 (23 μmol m-2 s-1) treatments than in control seedlings (15 μmol m-2 s-1); however, there was little difference in photosynthetic rates between partially defoliated seedlings and control seedlings after 5 weeks. An analysis of the sensitivity of photosynthesis to biochemical parameters revealed that the transient increase in photosynthetic rate in response to partial defoliation was largely a function of the maximum rate of carboxylation (85-87%) and the maximum rate of RuBP regeneration (55-60%) rather than stomatal conductance (12-13%). Nitrogen increased in leaves following partial defoliation (increases of 0.6 and 1.2 g m-2 for D50 and D70, respectively), but was accumulated in a non-photosynthetic form (i.e., there was no increase in nitrogen concentration of Rubisco or chlorophyll). Increased photosynthetic rates immediately following partial defoliation were primarily a result of increased activity rather than amount of photosynthetic machinery. There was no evidence that phosphorus was responsible for the increase in photosynthetic rates after partial defoliation. © 2007 Heron Publishing.
Turnbull, T L; Kelly, N; Adams, M A; Warren, C R
Within-canopy nitrogen and photosynthetic gradients are unaffected by soil fertility in field-grown Eucalyptus globulus Journal Article
In: Tree Physiology, vol. 27, no. 11, pp. 1607-1617, 2007.
A significant and well-supported hypothesis is that increased growth following nitrogen (N) fertilization is a function of the relationships among photosynthesis, tissue N content and the light environment - specifically, the within-canopy allocation of N among leaves and the within-leaf allocation of N between Rubisco and chlorophyll. We tested this hypothesis in a field trial that included annual applications of N,P,K fertilizer (from planting) to a Eucalyptus globulus Labill. plantation growing on uniform leached sands. Growth of 4-year-old E. globulus increased significantly in response to fertilization. Leaf N and phosphorus concentrations were 0.1-0.5 g m-2 and 0.4-0.5 g m-2 higher in fertilized trees compared to unfertilized trees, respectively. Stomatal conductance (gs) at the maximum photosynthetic rate (Amax) was between 0.2 and 0.4 mol m-2 s -1 higher in fertilized trees, but Amax and the concentration of Rubisco (Ruba) were unaffected by fertilization. This seeming paradox, where there was no response of Amax to fertilization despite increases in gs and leaf N concentration, was explained by reduced in vivo specific activity of Rubisco in fertilized trees. Acclimation to light, measured by redistribution of N between Rubisco and chlorophyll, was unaffected by fertilization. Distribution of leaf N followed irradiance gradients, but Amax did not. Maximum photosynthetic rate was correlated with leaf N concentration only in unfertilized trees. These findings indicate that the relationships among photosynthesis, N and the light environment in E. globulus are affected by N,P,K fertilization. © 2007 Heron Publishing.
Warren, C R; Dreyer, E; Tausz, M; Adams, M A
Ecotype adaptation and acclimation of leaf traits to rainfall in 29 species of 16-year-old Eucalyptus at two common gardens Journal Article
In: Functional Ecology, vol. 20, no. 6, pp. 929-940, 2006.
1. Relationships of leaf traits with rainfall at the place of origin of seed (RPO) are a function of acclimation and adaptation. To disentangle these effects we studied 29 species of 16-year-old Eucalyptus at a productive and an unproductive common garden (mean annual increments of above-ground stem volume = 21 ± 11 and 8 ± 5 m3 ha-1 years -1, respectively).We tested three hypotheses: (i) leaf traits vary between sites, but relationships among them do not; (ii) relationships of leaf traits with RPO do not vary between sites; and (iii) ecotypes originating from low-rainfall areas allocate a small fraction of nitrogen to thylakoid proteins and Rubisco, and have small SLA, small and narrow leaves, and large water-use efficiency (WUE). 2. Eleven leaf traits (leaf area, leaf thickness, leaf width/length, specific leaf area, fresh weight/dry weight, N, chlorophyll a/b, carotenoids/chlorophyll, thylakoid N% Rubisco N%, WUE derived from 13C content) were measured in 1-year-old sun leaves. 3. Site had a large effect on not only the absolute values of leaf traits, but also relationships between pairs of traits. There were 20 significant correlations between pairs of traits. Three of the correlations had different slopes between sites, while a further nine had different intercepts. Hence the majority of significant correlations were not independent of site. 4. Leaf area and leaf width/length were the only traits related to RPO. There was no evidence that N allocation to Rubisco or thylakoid proteins was related to RPO, or that WUE was greater in ecotypes from dry areas. 5. For Eucalyptus, and perhaps other genera, physiological leaf traits may play a minor role in adaptation to water availability. There is large phenotypic plasticity in many leaf traits affecting not only the absolute values of traits, but also relationships among them. © 2006 The Authors.
Warren, C R; Dreyer, E
Temperature response of photosynthesis and internal conductance to CO 2: Results from two independent approaches Journal Article
In: Journal of Experimental Botany, vol. 57, no. 12, pp. 3057-3067, 2006.
The internal conductance to CO2 transfer from intercellular spaces to chloroplasts poses a major limitation to photosynthesis, but few studies have investigated its temperature response. The aim of this study was to determine the temperature response of photosynthesis and internal conductance between 10°C and 35°C in seedlings of a deciduous forest tree species, Quercus canariensis. Internal conductance was estimated via simultaneous measurements of gas exchange and chlorophyll fluorescence ('variable J method'). Two of the required parameters, the intercellular photocompensation point (Ci*) and rate of mitochondrial respiration in the light (Rd), were estimated by the Laisk method. These were used to calculate the chloroplastic photocompensation point (Γ*) in a simultaneous equation with gi. An independent estimate of internal conductance was obtained by a novel curve-fitting method based on the curvature of the initial Rubisco-limited portion of an A/Ci curve. The temperature responses of the rate of Rubisco carboxylation (Vcmax) and the RuBP limited rate of electron transport (Jmax) were determined from chloroplastic CO2 concentrations. The rate of net photosynthesis peaked at 24°C. Ci* was similar to reports for other species with a Ci* of 39 μmol mol-1 at 25°C and an activation energy of 34 kJ mol-1. Γ*was very similar to the published temperature response for Spinacia oleracea from 20°C to 35°C, but was slightly greater at 10°C and 15°C. J max peaked at 30°C, whereas Vcmax did not reach a maximum between 10°C and 35°C. Activation energies were 49 kJ mol -1 for Vcmax and 100 kJ mol-1 for J max. Both methods showed that internal conductance doubled from 10°C to 20°C, and then was nearly temperature-independent from 20°C to 35°C. Hence, the temperature response of internal conductance could not be fitted to an Arrhenius function. The best fit to estimated gi was obtained with a three-parameter log normal function (R2=0.98), with a maximum gi of 0.19 mol m-2 s-1 at 29°C. © The Author . Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
Warren, C R
Potential organic and inorganic N uptake by six Eucalyptus species Journal Article
In: Functional Plant Biology, vol. 33, no. 7, pp. 653-660, 2006.
There are no published studies of organic N uptake by species of south-eastern Australia (e.g. Eucalyptus) despite several studies of ecosystem N cycling. This study examines uptake of nitrate, ammonium and glycine (an amino acid) by six species of 16-year-old Eucalyptus growing at two plantations ('common gardens'). By using two plantations, one xeric/oligotrophic and one mesic/eutrophic, I was able to disentangle genotypic from phenotypic differences in preference for N forms. Measurements were made on three separate occasions during spring. N uptake was examined in situ with attached roots placed in uptake solutions containing equimolar 100 μmol L-1 concentrations of 15N-nitrate, 15N-ammonium and 2-13C2 15N-glycine. Water and KCl extracts were used to determine the relative abundances of nitrate, ammonium and amino acids at the two plantations. Nitrate dominated at the eutrophic site, but was nearly absent at the oligotrophic site. N at the oligotrophic site was dominated by ammonium and amino acids which were present in similar concentrations. The rate of uptake of ammonium (6.3 ± 0.4 μmol g h-1; mean ± s.e.
Estimating the internal conductance to CO2 movement Journal Article
In: Functional Plant Biology, vol. 33, no. 5, pp. 431-442, 2006.
The concentration of CO2 in the chloroplast is less than atmospheric owing to a series of gas-phase and liquid-phase resistances. For a long time it was assumed that the concentration of CO2 in the chloroplasts is the same as in the intercellular spaces (e.g. as measured by gas exchange). There is mounting evidence that this assumption is invalid and that CO2 concentrations in the chloroplasts are significantly less than intercellular CO2. It is now generally accepted that internal conductance (gi) is a significant limitation to photosynthesis, often as large as that due to stomata. Internal conductance describes this decrease in CO2 concentration between the intercellular spaces and chloroplasts as a function of net photosynthesis [gi = A / (C i - Cc)]. Internal conductance is commonly estimated by simultaneous measurements of gas exchange and chlorophyll a fluorescence or instantaneous discrimination against 13CO2. These common methods are complemented by three alternative methods based on (a) the difference between intercellular and chloroplastic CO2 photocompensation points, (b) the curvature of an A / Ci curve, and (c) the initial slope of an A / Ci curve v. the estimated initial slope of an A / Cc curve. The theoretical basis and protocols for estimating internal conductance are described. The common methods have poor precision with relative standard deviations commonly >10%; much less is known of the precision of the three alternative methods. Accuracy of the methods is largely unknown because all methods share some common assumptions and no truly independent and assumption-free method exists. Some assumptions can and should be tested (e.g. the relationship of fluorescence with electron transport). Methods generally require knowledge of either the kinetic parameters of Rubisco, or isotopic fractionation by Rubisco. These parameters are difficult to measure, and thus are generally assumed a priori. For parameters such as these a sensitivity analysis is recommended. One means of improving confidence in gi estimates is by using two or more methods, but it is essential that the methods chosen share as few common assumptions as possible. All methods require accurate and precise measurements of A and Ci - these are best achieved by minimising leaks, maximising the signal-to-noise ratio by using a large leaf area and moderate flow rate, and by taking into account cuticular and boundary layer conductances. © CSIRO 2006.
Warren, C R
Why does photosynthesis decrease with needle age in Pinus pinaster? Journal Article
In: Trees - Structure and Function, vol. 20, no. 2, pp. 157-164, 2006.
Rates of photosynthesis vary with foliage age and typically decline from full-leaf expansion until senescence occurs. This age-related decline in photosynthesis is especially important in species that retain foliage for several years, yet it is not known whether the internal conductance to CO 2 movement (gi) plays any role. More generally, g i has been measured in only a few conifers and has never been measured in leaves or needles older than 1 year. The effect of ageing on g i was investigated in Pinus pinaster, a species that retains needle for 4 or more years. Measurements were made in autumn when trees were not water limited and after leaf expansion was complete. Rates of net photosynthesis decreased with needle age, from 8 μmol m-2 s-1 in fully expanded current-year needles to 4.4 μmol m-2 s-1 in 3-year-old needles. The relative limitation due to internal conductance (0.24-0.35 out of 1) was in all cases larger than that due to stomatal conductance (0.13-0.19 out of 1). Internal conductance and stomatal conductance approximately scaled with rates of photosynthesis. Hence, there was no difference among year-classes in the relative limitations posed by internal and stomatal conductance or evidence that they cause the age-related decline in photosynthesis. There was little evidence that the age-related decline in photosynthesis was due to decreases in contents of N or Rubisco. The decrease in rates of photosynthesis from current-year to older needles was instead related to a twofold decrease in rates of photosynthesis per unit nitrogen and V cmax/Rubisco (i.e., in vivo specific activity). © Springer-Verlag 2006.
Warren, C R; Adams, M A
Internal conductance does not scale with photosynthetic capacity: Implications for carbon isotope discrimination and the economics of water and nitrogen use in photosynthesis Journal Article
In: Plant, Cell and Environment, vol. 29, no. 2, pp. 192-201, 2006.
Central paradigms of ecophysiology are that there are recognizable and even explicit and predictable patterns among species, genera, and life forms in the economics of water and nitrogen use in photosynthesis and in carbon isotope discrimination (Δ). However most previous examinations have implicitly assumed an infinite internal conductance (gi) and/or that internal conductance scales with the biochemical capacity for photosynthesis. Examination of published data for 54 species and a detailed examination for three well-characterized species -Eucalyptus globulus, Pseudotsuga menziesii and Phaseolus vulgaris- show these assumptions to be incorrect. The reduction in concentration of CO2 between the substomatal cavity (Ci) and the site of carbon fixation (Cc) varies greatly among species. Photosynthesis does not scale perfectly with gi and there is a general trend for plants with low gi to have a larger draw-down from Ci to Cc, further confounding efforts to scale photosynthesis and other attributes with gi. Variation in the g i-photosynthesis relationship contributes to variation in photosynthetic 'use' efficiency of N (PNUE) and water (WUE). Δ is an information-rich signal, but for many species only about two-thirds of this information relates to A/gs with the remaining one-third related to A/gi. Using data for three well-studied species we demonstrate that at common WUE, Δ may vary by up to 3‰. This is as large or larger than is commonly reported in many interspecific comparisons of Δ, and adds to previous warnings about simplistic interpretations of WUE based on Δ. A priority for future research should be elucidation of relationships between gi and gs and how these vary in response to environmental conditions (e.g. soil water, leaf-to-air vapour pressure deficit, temperature) and among species. © 2006 Blackwell Publishing Ltd.
Warren, C R; Adams, M A
What determines interspecific variation in relative growth rate of Eucalyptus seedlings? Journal Article
In: Oecologia, vol. 144, no. 3, pp. 373-381, 2005.
The present study examines relative growth rate (RGR) and its determinants in seedlings of nine Eucalyptus species. Species were selected from mesic (1,800 mm a-1 rainfall) through to semi-arid habitats (300 mm a -1), and thus, notionally vary in "stress" tolerance. Seedlings were grown in a glasshouse during early summer and received between 33 mol and 41 mol PAR m-2 day-1 . The mean RGR varied among species-from a minimum of 66 mg g-1 day-1 in E. hypochlamydea to a maximum of 106 mg g-1 day-1 in E. delegatensis. RGR was positively related to rainfall at the sites of seed collection. Neither specific leaf area (SLA) nor net assimilation rate was related to rainfall or RGR. While the absence of relationships with SLA and net assimilation rate contrasts with other studies and species, we cannot rule out the effects of sample size (n=9 species) and modest ranges in SLA and RGR. The ratio of leaf mass to total mass (LMR) varied from 0.49±0.07 g g -1 in E. socialis to 0.74±0.04 g g-1 in E. delegatensis and was strongly positively related with rainfall (r 2=0.77). Interspecific differences in RGR were strongly related to LMR (positive relationship, r 2=0.50) and the rate of dry matter production per mol of leaf nitrogen (positive relationship, r 2=0.64). Hence, the slow RGR of low-rainfall species was functionally related to a lower growth rate per mol of leaf nitrogen than high-rainfall species. Furthermore, slow RGR of low-rainfall species was related to greater allocation to roots at the expense of leaves. Increasing allocation to roots versus leaves is likely an adaptation to soil and atmospheric water deficits, but one that comes at the expense of a slow RGR. © Springer-Verlag 2005.
Tausz, M; Warren, C R; Adams, M A
Is the bark of shining gum (Eucalyptus nitens) a sun or a shade leaf? Journal Article
In: Trees - Structure and Function, vol. 19, no. 4, pp. 415-421, 2005.
Photoinhibition and pigment composition of green stem tissues of field-grown adult Eucalyptus nitens were measured on clear spring days with low morning temperatures-conditions that cause photoinhibition in leaves of many plant species. The sun-exposed (north-facing) bark contained less chlorophyll a+b (531 vs 748 μmol m-2), neoxanthin (29 vs 41), and β-carotene (54 vs 73), more xanthophyll cycle pigments per unit surface area and per unit chlorophyll (71 vs 53 μmol m-2 and 141 vs 66 mmol mol-1 chlorophyll), and less lutein per unit chlorophyll (239 vs 190) than the shaded (southern) side. Maximum electron flow rates were 60 μmol m-2 s-1 on the sun-exposed side, and about 10 μmol m-2 s-1 on the shaded side. Fv/F m was always lower than 0.8 on the sun-exposed side and the de-epoxidation state (DEPS) of the xanthophyll cycle was dominated by zeaxanthin in midday samples. Fv/Fm increased quickly after darkening, but DEPS recovered more slowly to 40% overnight. This suggested that rapidly reversible pH-dependent quenching was responsible for the bulk of changes in PS II efficiency. Fv/Fm remained below 0.8 overnight, which may well be indicative of photo-damage to PSII. In contrast, DEPS of the shaded side was lower, and Fv/Fm was higher, than for the sun-exposed side. We conclude that E. nitens chlorenchyma on the sun-exposed stem side has a photosynthetic pigment composition similar to sun leaves and it experiences significant photoinhibition in the field. © Springer-Verlag 2005.
Warren, C R; Tausz, M; Adams, M A
Does rainfall explain variation in leaf morphology and physiology among populations of red ironbark (Eucalyptus sideroxylon subsp. tricarpa) grown in a common garden? Journal Article
In: Tree Physiology, vol. 25, no. 11, pp. 1369-1378, 2005.
We investigated adaptation of leaf morphology and physiology of red ironbark (Eucalyptus sideroxylon Cunn. Ex. Wools subsp. tricarpa L.A.S. Johnson) in a common garden experiment. Fifteen populations, representing a rainfall range of 500 to 1055 mm per annum at the sites of seed collection, were grown at the same site. Because environmental variables other than rainfall did not vary significantly among populations, we were able to test if leaf morphology and physiology were related to seed-source rainfall. There were large differences among and within populations in all measured variables. Most univariate relationships with seed-source rainfall were not significant. Notable exceptions were the weak positive correlation of specific leaf area with seed-source rainfall - consistent with expectations - and the weak negative correlation of photosynthesis and stomatal conductance with seed-source rainfall - the opposite of what we predicted. In many cases, populations collected from sites of similar rainfall differed greatly in leaf morphology and physiology. Principal component analysis (PCA) reduced the 13 input variables to five principal components (PC) explaining 73.0% of the total variance in the original data. Some of the PC axes could be interpreted in terms of adaptation to drought (i.e., to seed-source rainfall), but relationships of accumulated variables (the PC axes) with seed-source rainfall were significant for only one PC axis. Hence, among red ironbark populations grown in a common garden, there was significant genetic variation in leaf morphology and physiology, but for most traits, this variation was unrelated to rainfall at the site of seed collection. This study adds to a growing body of common garden literature showing weak within-species relationships of leaf morphology and physiology with seed-source rainfall, in contrast to the consistently stronger relationships among species growing at different points along broad environmental gradients. © 2005 Heron Publishing.
Warren, C R; McGrath, J F; Adams, M A
Differential effects of N, P and K on photosynthesis and partitioning of N in Pinus pinaster needles Journal Article
In: Annals of Forest Science, vol. 62, no. 1, pp. 1-8, 2005.
We investigated the response of one-year-old Pinus pinaster Ait. growing on a sandy, nutrient-poor soil to the factorial addition of N, P and K. We tested two hypotheses: (1) fertiliser application increases rates of light-saturated photosynthesis (Amax) and leaf-level water-use efficiency (WUE, as indicated by carbon isotope composition), and (2) greater concentrations of N and Rubisco explain fertiliser-promoted increases in Amax and WUE. Two years after fertiliser application, the height of P. pinaster was significantly greater in plots to which P was added (127 ± 7 cm; mean ± SE of three replicate plots) compared to those without added P (70 ± 10 cm), but was unaffected by addition of N or K. The fraction of N present as Rubisco varied between 4 and 18% and was unrelated to N concentrations, but positively related to P and K concentrations. A max and δ13C did not vary significantly among treatments and were unrelated to concentrations of Rubisco, N, P or K. We conclude that fertiliser application two years previously has little effect on current Amax and δ13C of P. pinaster, and thus the previously reported effects of fertiliser on gas exchange are likely ephemeral. © INRA, EDP Sciences, 2005.
Tausz, M; Warren, C R; Adams, M A
Dynamic light use and protection from excess light in upper canopy and coppice leaves of Nothofagus cunninghamii in an old growth, cool temperate rainforest in Victoria, Australia Journal Article
In: New Phytologist, vol. 165, no. 1, pp. 143-156, 2005.
Responses to simulated sunflecks were examined in upper canopy and coppice leaves of Nothofagus cunninghamii growing in an old-growth rainforest gully in Victoria, Australia. Shaded leaves were exposed to a sudden increase in irradiance from 20 to 1500 μmol m-2 s-1. Gas exchange and chlorophyll fluorescence were measured during a 10 min simulated sunfleck and, in the ensuing dark treatment, we examined the recovery of PS II efficiency and the conversion state of xanthophyll cycle pigments. Photosynthetic induction was rapid compared with tropical and northern hemisphere species. Stomatal conductance was relatively high in the shade and stomata did not directly control photosynthetic induction under these conditions. During simulated sunflecks, zeaxanthin was formed rapidly and photochemical efficiency was reduced. These processes were reversed within 30 min in coppice leaves, but this took longer in upper canopy leaves. Poor drought tolerance and achieving a positive carbon balance in a shaded canopy may be functionally related to high stomatal conductance in the shade in N. cunninghamii. The more persistent reduction in photochemical efficiency of upper canopy leaves, which means less efficient light use in subsequent shade periods, but stronger protection from high light, may be related to the generally higher irradiance and longer duration of sunflecks in the upper canopy, but potentially reduces carbon gain during shade periods by 30%. © New Phytologist (2004).
Warren, C R; Adams, M A
What determines rates of photosynthesis per unit nitrogen in Eucalyptus seedlings? Journal Article
In: Functional Plant Biology, vol. 31, no. 12, pp. 1169-1178, 2004.
Species originating from xeric sites are characterised by slower rates of photosynthesis per unit nitrogen (PNUE) than species from mesic sites, but we lack mechanistic explanations for these interspecific differences. We examined N allocation to Rubisco and chlorophyll, and photosynthetic characteristics in seedlings of nine Eucalyptus species grown in a fully sunlit glasshouse with an optimal supply of nutrients. Species were selected from mesic (1800 mm year -1 rainfall) through to semi-arid habitats (300 mm year -1). All species were characterised by allocation of a large proportion of N to Rubisco (32-48%) with high in vivo specific activity. Intercellular CO2 concentration (Ci) varied between 260 and 300 μmol mol-1, and thus, stomatal limitations were low in all species. This combination of traits resulted in a PNUE (172-335 μmol mol-1 s-1) that was higher than is commonly observed in tree species and which may be related to the rapid growth, water-spender strategy of Eucalyptus seedlings. There were significant differences in photosynthetic parameters and N allocation among species, but these were only weakly related to rainfall at the site of seed origin. There were correlations of Ci with PNUE but a sensitivity analysis suggested that interspecific variation in Ci explained at most 7% of variation in PNUE. Photosynthesis and PNUE were also rather insensitive to large interspecific differences in RuBP-limited rate of electron transport per unit N (Jmax / N), because photosynthesis was primarily limited by the maximum rate of carboxylation (Vcmax). PNUE was most sensitive to changes in N allocation to Rubisco and Vcmax/Rubisco.
Warren, C R; Adams, M A
Capillary electrophoresis of the major anions and cations in leaf extracts of woody species Journal Article
In: Phytochemical Analysis, vol. 15, no. 6, pp. 407-413, 2004.
Capillary electrophoresis methods are described for the analysis of the major inorganic anions (nitrite, nitrate, chloride, sulphate, phosphate), organic acids (oxalate, malate, citrate, succinate) and inorganic cations (ammonium, potassium, sodium, calcium, magnesium) in leaf extracts. Analytical performance was validated for extracts from leaves of four sclerophyllous species: Eucalytus globulus, E. cladocalyx, E. nitens and Pinus radiata. Inorganic anions and organic acids were analysed in a single run within 5 min using a background electrolyte of 2,6-pyridinedicarboxylic acid (20 mM) and cetyltrimethylammonium bromide (0.5 mM). Cations were analysed in a separate run also within 5 min using imidazole (10 mM) and 18-crown-6 (2 mM) as background electrolyte. Replicate injections were highly repeatable when the capillary was rinsed between runs with hydrochloric acid (0.25 M) and background electrolyte, but not when the acid rinse was omitted or replaced by a rinse with sodium hydroxide (0.25 M). Standard curves for all analytes were linear over the range of 0.05-1 mM. Standard curves constructed by serial dilution of a leaf extract were also highly linear, and this, combined with the excellent recovery of added solutes in a spike and recovery test, suggests quantification was unaffected by the complex matrix that is present in un-purified, hot water extracts of leaves. There were significant differences in concentrations of the major anions and cations between the species studied. Copyright © 2004 John Wiley & Sons, Ltd.
Warren, C R
The photosynthetic limitation posed by internal conductance to CO 2 movement is increased by nutrient supply Journal Article
In: Journal of Experimental Botany, vol. 55, no. 406, pp. 2313-2321, 2004.
The internal conductance to CO2 supply from substomatal cavities to sites of carboxylation may pose a large limitation to photosynthesis, but little is known of how it is affected by nutrient supply. Knowing how internal conductance responds to nutrient supply is critical for interpreting the biochemical responses from A-Ci curves. The aim of this paper was to examine the response of gi and photosynthetic parameters to nutrient supply in glasshouse-grown seedlings of the evergreen perennial Eucalyptus globulus Labill. Seedlings were grown with five different nutrient treatments and gi was estimated from concurrent measurements of gas exchange and fluorescence. Internal conductance varied between 0.12 and 0.19 mol m -2 s-1 and the relative limitation of photosynthesis due to internal conductance was greater than the stomatal limitation. In most species these two limitations are rather similar, but in E. globulus stomatal limitations were abnormally low due to high stomatal conductance (0.31 to 0.39 mol m-2 s-1). The large positive response of photosynthesis to nutrient supply was not matched by changes in internal conductance, and thus the relative limitation of photosynthesis due to internal conductance increased with increasing nutrient supply. Failure to account for finite internal conductance led to estimates of Vcmax that were 60% of the true value, which, in turn, led to an underestimation of in vivo Rubisco specific activity (as Vcmax/Rubisco content). The specific activity of Rubisco in E. globulus (21 mol mol-1 s-1) was close to the maximum published estimates, and thus, despite these leaves containing a large fraction of N as Rubisco (38-44%) there was no evidence that Rubisco activity was down-regulated or that the enzyme was in excess.
Warren, C R; Adams, M A
Evergreen trees do not maximize instantaneous photosynthesis Journal Article
In: Trends in Plant Science, vol. 9, no. 6, pp. 270-274, 2004.
The lifestyles of plants affect their approach to photosynthesis. The long-lived evergreen trees must cope with wide temporal variation in water and nutrient availability that herbaceous species largely avoid. Evergreen trees have thus evolved mechanisms to conserve water and nutrients, and to tolerate water and nutrient stress. However, some of these adaptations have a cost. For example, evergreen trees have lower rates of photosynthesis per unit nitrogen, which can be partly explained by the use of Rubisco for nitrogen storage and the higher internal resistance to CO2 diffusion. The high internal resistance to diffusion of CO2 in evergreen trees might be an unavoidable consequence of anatomical and biochemical traits associated with leaf longevity, nutrient conservation and/or drought tolerance.
Warren, C R; Livingston, N J; Turpin, D H
Photosynthetic responses and N allocation in Douglas-fir needles following a brief pulse of nutrients Journal Article
In: Tree Physiology, vol. 24, no. 6, pp. 601-608, 2004.
The temporal distribution of soil nutrients is heterogeneous, and thus the uptake, storage and later remobilization of brief nutrient pulses may be critical for growth in nutrient-limited habitats. We investigated the response of photosynthesis and the major nitrogen (N) fractions in needles of 2-year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings to a 15-day nutrient pulse (containing 250 ppm N). The nutrient pulse (N pulse) was imposed in late July, toward the end of the seedlings' third growing season, and subsequent changes in photosynthesis and needle N fractions were examined over the following 3 months. Needles are sites of photosynthesis and putative storage organs. Thus we tested two hypotheses: (1) N from the N pulse is quickly synthesized from soluble non-protein N into soluble proteins, especially Rubisco, and (2) the N pulse increases photosynthetic rates and thus growth. We also examined an alternative hypothesis that Rubisco functions also as a storage protein, in which case we would predict increases in amount of Rubisco in response to the N pulse without concomitant increases in photosynthesis. Soluble non-protein N was the most dynamic N pool and may have constituted a temporary storage reservoir; however, the quantitative significance of soluble non-protein N is questionable because this pool was at most only 7% of total N. Concentrations of Rubisco were unaffected by the N-pulse treatment and there was little evidence that Rubisco served as a storage protein. Nutrient-pulse seedlings added twice as much dry mass as controls during the 3 months post-treatment (Warren et al. 2003a). Over the same period, the maximum rate of light-saturated photosynthesis (Amax) declined to low rates in control seedlings, whereas Amax increased in N-pulse seedlings. Nevertheless, treatment and temporal trends in N and Rubisco content per unit area were poorly related to Amax, and it seems likely that photosynthesis was limited by additional factors, perhaps thylakoid proteins or an inadequate supply of other nutrients.
Warren, C R; Livingston, N J; Turpin, D H
Water stress decreases the transfer conductance of Douglas-fir (Pseudotsuga menziesii) seedlings Journal Article
In: Tree Physiology, vol. 24, no. 9, pp. 971-979, 2004.
We tested the hypothesis that transfer conductance (gi) of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings is reduced by water stress. Seedlings were irrigated with a solution of 25% polyethylene glycol so as to impose water stress rapidly, thereby limiting acclimatory responses. Transfer conductance was measured pre-treatment and post-treatment by two methods. Water stress reduced net photosynthesis by 20-50%. The initial slope of the rate of photosynthesis (A) over the intercellular carbon dioxide (CO 2) concentration (Ci) response was reduced by water stress, indicating that reduced photosynthesis was not wholly accounted for by reduced stomatal conductance. The carbon isotope and chlorophyll fluorescence methods both indicated that water stress decreased gi. From isotopic measurements with 1% O2, gi was 0.076 ± 0.009 (mean ± SE) mol m-2 s-1 in well-watered seedlings and 0.044 ± 0.004 mol m-2 s-1 in water-stressed seedlings. Fluorescence estimates of gi were 0.08 ± 0.01 mol m-2 s-1 in well-watered seedlings and 0.044 ± 0.004 mol m-2 s-1 in water-stressed seedlings. The drought-induced reduction in & was responsible for the reduction in slope of the A/Ci response, and thus there was no difference in the slope of the A over the chloroplastic CO2 concentration (Cc) response between treatments and no indication of impaired mesophyll metabolism. These data illustrate that impairments of mesophyll metabolism can be revealed only from analysis of the A/Cc response.
Warren, C R; Ethier, G J; Livingston, N J; Grant, N J; Turpin, D H; Harrison, D L; Black, T A
Transfer conductance in second growth Douglas-fir (Pseudotsuga menziesii (Mirb.)Franco) canopies Journal Article
In: Plant, Cell and Environment, vol. 26, no. 8, pp. 1215-1227, 2003.
The internal conductance from intercellular spaces to the sites of carboxylation (g1) has only been measured in a few tree species and not in conifers, despite the fact it may impose a large limitation on photosynthesis. The present study provides the first estimates of g1 for a coniferous species, and examines variation in g1 with height and its relationships to anatomical, biochemical and physiological traits. Measurements were made on upper and lower canopy current-year needles of 50-year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco). Needle thickness and specific leaf area decreased by 30% from the top to bottom of the canopy. These anatomical/morphological changes were accompanied by modest variation in allocation of N to chlorophyll and the chlorophyll alb ratio. Allocation of N to Rubisco did not vary with height, but the ratio of Rubisco to chlorophyll did owing to the aforementioned changes in allocation to chlorophyll. The value of g1 was estimated in one tree from concurrent measurements of carbon isotope discrimination and net photosynthesis. To examine the variation in g1 among trees a second independent method based on day respiration and the difference between the chloroplastic and intercellular photocompensation points (photocompensation point method) was used. Estimates of gi obtained by the two methods agreed well with values varying between 0.14 and 0.20 mol m-2 s-1. It is estimated that g1 limits photosynthesis by approximately 20% as compared to an approximately 30% stomatal limitation (under well-watered conditions). The value of g1 scaled approximately with maximum rates of photosynthesis, which were significantly greater in upper canopy needles. Nevertheless, gi did not vary significantly with canopy height, owing to greater variability in g1 than photosynthesis.
Warren, C R; Livingston, N J; Turpin, D H
Response of Douglas-fir seedlings to a brief pulse of 15N- labeled nutrients Journal Article
In: Tree Physiology, vol. 23, no. 17, pp. 1193-1200, 2003.
The temporal distribution of soil nutrients is heterogeneous, and thus the uptake, storage and later remobilization of brief nutrient pulses may be critical for growth in nutrient-limited habitats. We investigated the response of 2-year-old Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings receiving a low nutrient supply to a 15-day nutrient pulse (containing 250 ppm nitrogen (N) as 10 atom % 15NH415NO 3). The nutrient pulse was imposed in late July, toward the end of the seedlings' third growing season, and subsequent changes in dry mass and N content over the following 3 months were determined from destructive harvests. We tested three hypotheses: (1) N from the nutrient pulse is rapidly assimilated and accumulated primarily in needles and roots; (2) this accumulated N is later remobilized to support new growth; and (3) the nutrient pulse leads to a larger second flush of shoot growth. Seedlings increased their N content by 175 mg (67%) in response to the nutrient pulse. Nitrogen was taken up preferentially into younger tissues, especially the secondary flush and current-year roots. Immediately after the nutrient pulse, tissue N concentrations were high and supported subsequent increases in dry mass. Over 3 months, seedlings receiving the nutrient pulse added twice as much dry mass as control seedlings, and even after 3 months of growth, N concentrations remained greater than in controls. Current-year and older needles were the only components whose dry mass did not increase over this period. The nutrient pulse increased the size of the second flush, but it was still a minor component of increments in dry mass (∼10% of the total dry mass increment) and N content (23%). The relatively modest increases in N content during autumn could be accounted for by soil uptake and there was no evidence that N was remobilized to support growth of new tissues. Short-term (15 days) elevated N uptake led to sustained growth in the long term (>3 months), and thus growth rate was to a large extent decoupled from current nutrient supply.
Warren, C R; Livingston, N J; Turpin, D H
Responses of gas exchange to reversible changes in whole-plant transpiration rate in two conifer species Journal Article
In: Tree Physiology, vol. 23, no. 12, pp. 793-803, 2003.
This study examined the autonomy of branches with respect to the control of transpiration (E) in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and western red cedar (Thuja plicata Donn) seedlings. Experiments were conducted on whole seedlings in a gas exchange system with a dual-cuvette that permitted independent manipulation and measurement of E in the upper and lower cuvettes. The value of E in one cuvette was manipulated by varying vapor pressure deficit (D) between 2.2 and 0.2 kPa, whereas D in the other cuvette was held at 2.2 kPa. Reducing D, while increasing stomatal conductance (gs), resulted in an overall decrease in E. In western red cedar, this decrease was almost threefold, and in Douglas-fir, approximately fourfold. In well-watered western red cedar, a reduction of whole-plant E by 46% (brought about by reducing D in the upper cuvette) resulted in a 12% increase in gs, a 12% increase in E and a 7% increase in net assimilation (A) of untreated foliage in the lower cuvette. Responses of gs, E and A of untreated foliage were similar irrespective of whether foliage was at the top or bottom of the seedling. When D in the treatment cuvette was restored to 2.2 kPa, g s, E and A of foliage in the untreated cuvette returned to pretreatment values. In contrast, in well-watered Douglas-fir, there was almost no change in gs, E or A of untreated foliage in one cuvette when D in the other cuvette was reduced, causing a 52% reduction in whole-plant E. However, similar manipulations on drought-stressed Douglas-fir led to 7-19% increases in gs, E and A of untreated foliage. In well-watered western red cedar, daytime leaf water potential (Ψ1) was maintained near -0.9 MPa over a wide range of D, whereas Ψ1 of Douglas-fir decreased from -1.2 to -1.5 MPa as D increased. The tighter (isohydric) regulation of Ψ1 in western red cedar may partly explain its greater stomatal response to D and variation in whole-plant E compared with Douglas-fir. In response to a reduction in E, measured increases in Ψ1 and gs of unmanipulated foliage were less than predicted by a model assuming complete hydraulic connectivity of foliage. Our results suggest the foliage of both species is partially autonomous with respect to water.
Warren, C R; Dreyer, E; Adams, M A
Photosynthesis-Rubisco relationships in foliage of Pinus sylvestris in response to nitrogen supply and the proposed role of Rubisco and amino acids as nitrogen stores Journal Article
In: Trees - Structure and Function, vol. 17, no. 4, pp. 359-366, 2003.
Relationships between photosynthetic capacity, and needle contents of N, Rubisco and amino acids were investigated in potted Pinus sylvestris L. trees. Three-year-old seedlings of P. sylvestris were grown for 4 years with three nutrient regimes. Concentrations of N, amino acids, amides and Rubisco were measured and expressed on a needle area basis, and the in vivo performances of Rubisco (maximum rate of carboxylation, Vcmax) and of electron transport (maximum light driven electron flux, Jmax) were estimated via a biochemically based model of photosynthesis. Needle content of Rubisco-N was at least six times that of amino acid + amide-N and was positively related to Narea. The estimated in vivo specific activity of Rubisco (Vcmax/Rubisco content per unit area) was low and negatively related to N content per unit area (Narea). Jmax/Rubisco content was negatively related to Narea, whereas Vcmax/Jmax was unrelated to Narea. Hence, Rubisco content was in excess of the amount required for photosynthesis and this excess was positively related to Narea. These data support the hypothesis that with increasing Narea, Rubisco functions increasingly as a storage protein in addition to its catalytic functions.
Warren, C R; Adams, M A
Possible causes of slow growth of nitrate-supplied Pinus pinaster Journal Article
In: Canadian Journal of Forest Research, vol. 32, no. 4, pp. 569-580, 2002.
We investigated possible causes of slower growth of Pinus pinaster Ait. supplied with nitrate, as compared with ammonium or with ammonium nitrate mixtures. Six-month-old mycorrhizal seedlings of P. pinaster were grown in sand culture for 4 months at four concentrations of N (0.125, 0.5, 2.0, or 8.0 mM) as nitrate or ammonium or with an ammonium-nitrate mixture at 0.125 or 2.0 mM. After 4 months of nutrient addition, we measured light-saturated rates of photosynthesis (Amax), rates of ammonium and nitrate uptake, growth, macro- and micro-elements, and patterns of N allocation in needles. Dry mass was unaffected by N form at 0.125 or 0.5 mM N. In contrast, dry mass of seedlings supplied with ammonium or ammonium nitrate at 2.0 and 8.0 mM N, was approximately threefold greater than seedlings supplied with nitrate alone. Concentrations of N in foliage and Amax were unaffected by the form or concentration of N supplied. Furthermore, concentrations of amino acid N were greater in seedlings supplied with nitrate, suggesting rates of uptake were not limiting growth. Foliage concentrations of zinc were low with nitrate supplied at a concentration of 0.5 mM or greater, and seedlings displayed symptoms typical of zinc deficiency when nitrate was supplied at 2.0 or 8.0 mM. Slower growth with nitrate could not be explained solely by either slower root uptake of nitrate N or lesser Amax. Instead, aspects of N metabolism postuptake coupled with other factors such as nutrient deficiencies may limit growth with nitrate as the sole N source.
Warren, C R; Adams, M A
Phosphorus affects growth and partitioning of nitrogen to Rubisco in Pinus pinaster Journal Article
In: Tree Physiology, vol. 22, no. 1, pp. 11-19, 2002.
We tested the hypothesis that photosynthetic and growth responses to phosphorus (P) are functions of differences in the partitioning of nitrogen (N) among different compounds, particularly ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). We tested this hypothesis in: (a) a greenhouse experiment with mycorrhizal seedlings of Pinus pinaster Ait. grown in sand culture for 4 months with six factorial combinations of N (0.125 and 2.0 mM) and P (0.02, 0.08 and 0.34 mM); and (b) a field study in which P was applied at five rates (up to 175 kg ha-1) to 2-year-old P. pinaster growing on P-deficient siliceous sand. After 4 months of nutrient addition or 2 years after fertilizer application, we measured light-saturated rates of photosynthesis, growth, and N and P allocation in needles. Growth of P. pinaster increased significantly with increasing concentrations of P, as did the concentration of P in needles. Concentrations of P and Rubisco were positively related, whereas those of N and Rubisco were unrelated. At low-P supply, the Rubisco/Chl ratio varied between 8.5 and 12 mmol mol-1. With P supply in excess of requirement (needle N:P ratio = 2-12) the Rubisco/Chl ratio increased to between 24 and 26 mmol mol-1. Rates of light-saturated photosynthesis were unaffected by P supply because adequate concentrations of P were maintained in plants in all treatments. Orthophosphate accumulated in needles of plants receiving a high P supply, which may allow growth to continue for periods under P deficiency, provided that other nutrients also accumulate. In the case of N, Rubisco may fill this role.
Warren, C R; Adams, M A
Distribution of N, Rubisco and photosynthesis in Pinus pinaster and acclimation to light Journal Article
In: Plant, Cell and Environment, vol. 24, no. 6, pp. 597-609, 2001.
The light-nitrogen hypothesis suggests canopy photosynthesis is maximized when there is a positive relationship between irradiance received by foliage, its nitrogen content (per unit area Narea), and maximum rate of photosynthesis (Amax). Relationships among relative irradiance and Narea, allocation of nitrogen within the photosynthetic apparatus to Rubisco and chlorophyll, and Amax were examined in Pinus pinaster Ait. needles up to 6 years of age. Measurements were made before bud break in August 1998, and in May 1999 after the first 'winter' rains. In August, Narea in P. pinaster needles decreased from 5.1 to 5.7 g m-2 in sunlit 1-year-old needles to 2.3 g m-2 in shaded 6-year-old needles. In May, Narea was 5-40% less but spatial trends were the same. At both sampling dates, Amax was less in old shaded needles compared with young sunlit needles, and was thus consistent with the light-nitrogen hypothesis. Relationships between Narea and Amax were positive at both dates yet varied in strength and form. Allocation of nitrogen within the photosynthetic apparatus was qualitatively consistent with acclimation to light (i.e. Rubisco/Chl decreased with shading), but quantitatively suboptimal with respect to photosynthesis owing to consistent over-investment in Rubisco. This over-investment increased with height in the canopy and was greater in May than in August.
Warren, C R; McGrath, J F; Adams, M A
Water availability and carbon isotope discrimination in conifers Journal Article
In: Oecologia, vol. 127, no. 4, pp. 476-486, 2001.
The stable C isotope composition (δ13C) of leaf and wood tissue has been used as an index of water availability at both the species and landscape level. However, the generality of this relationship across species has received little attention. We compiled literature data for a range of conifers and examined relationships among landscape and environmental variables (altitude, precipitation, evaporation) and δ13C. A significant component of the variation in δ13C was related to altitude (discrimination decreased with altitude in stemwood, 2.53‰ km-1 altitude
Warren, C R; Chen, Z -L; Adams, M A
Effect of N source on concentration of Rubisco in Eucalyptus diversicolor, as measured by capillary electrophoresis Journal Article
In: Physiologia Plantarum, vol. 110, no. 1, pp. 52-58, 2000.
Proteins in plant tissues have been extensively characterised by conventional methods such as liquid chromatography and polyacrylamide gel electrophoresis - methods that are tedious and time-consuming. Capillary electrophoresis is potentially a more simple and cost-effective method (with respect to time and consumables) but needs substantial development, especially for native plants which are frequently poor in protein and rich in interfering substances (oils, tannins, phenols). We report here the development of capillary electrophoresis (CE) for the separation of SDS-protein complexes (by molecular mass) and their quantification in plant tissues. In leaf extracts, two peaks dominated the electropherograms, these peaks had migration times corresponding to the small and large subunits of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 220.127.116.11) and co-migrated with added purified Rubisco. Linearity of peak area, reproducibility of migration time and peak areas for the small and large subunit were excellent, suggesting Rubisco could be quantified with a high degree of accuracy. We determined how the concentration (0.5 or 4 mM) and form of N applied (nitrate versus ammonium) affects partitioning of N to Rubisco in seedlings of Eucalyptus diversicolor. Analysis of extracts from leaves of E. diversicolor was only possible after precipitation of proteins with trichloroacetic acid (TCA). Precipitation with TCA was highly reproducible and recovery of added Rubisco through procedures of extraction, precipitation and analysis were close to 100% for both subunits. An 8-fold difference in the concentration of N applied did not affect total N, the concentration of Rubisco or the fraction of N present as Rubisco. The similarity of total N may well reflect faster rates of growth in those plants receiving 4 mM N, and a subsequent 'dilution' of tissue N. The N source did not affect total N, the concentration of Rubisco or the fraction of N present as Rubisco. Despite similar Rubisco concentrations, the total concentration of soluble proteins was greater in ammonium-grown plants.
Warren, C R; Adams, M A
Water availability and branch length determine δ13C in foliage of Pinus pinaster Journal Article
In: Tree Physiology, vol. 20, no. 10, pp. 637-643, 2000.
The stable carbon isotope composition (δ13C) of foliage integrates signals resulting from environmental and hydraulic constraints on water movement and photosynthesis. We used branch length as a simple predictor of hydraulic constraints to water fluxes and determined the response of δ13 C to varying water availability. Foliage up to 6 years old was taken from Pinus pinaster Ait. trees growing at four sites differing in precipitation (P; 414-984 mm year-1) and potential evaporation (ET; 1091-1750 mm year-1). Branch length was the principal determinant of temporal trends in δ13C. The strong relationship between δ13C and branch length was a function of hydraulic conductance, which was negatively correlated with branch length (r2 = 0.84). Variation in P and ET among sites was reflected in δ13C, which was negatively correlated with P/ET (r2 = 0.66). However, this analysis was confounded by differences in branch length. If the effects of branch length on δ13C were first removed, then the 'residual' δ13C was more closely related to P/ET (r2 = 0.99), highlighting the importance of accounting for variation in hydraulic constraints to water flux between sites and years. For plant species that exhibit considerable phenotypic plasticity in response to changes in environment (e.g., variation in leaf area, branch length and number, or stem form), the environmental effects on δ13C in foliage can only be reliably assessed if deconvoluted from hydraulic constraints.
Warren, C R; Adams, M A
Capillary electrophoresis for the determination of major amino acids and sugars in foliage: Application to the nitrogen nutrition of sclerophyllous species Journal Article
In: Journal of Experimental Botany, vol. 51, no. 347, pp. 1147-1157, 2000.
Amino acids and sugars are probably the most commonly measured solutes in plant fluids and tissue extracts. Chromatographic techniques used for the measurement of such solutes require complex derivatization procedures, analysis times are long and separate analyses are required for sugars and amino acids. Two methods were developed for the analysis of underivatized sugars and amino acids by capillary electrophoresis (CE). Separation of a range of sugars and amino acids was achieved in under 30 min, with good reproducibility and linearity. In general, there was close agreement between amino acid analyses by CE and HPLC with post-column derivatization. An alternative, more rapid method was optimized for the common neutral sugars. Separation of a mixture of fructose, glucose, sucrose, and fucose (internal standard) was achieved in less than 5 min. How the source of N applied (nitrate or ammonium) and its concentration (8.0 or 0.5 mM) affects the amino acid and sugar composition of leaves from Banksia grandis Willd. and Hakea prostrata R. Br. was investigated. The amino acid pool of Banksia and Hakea were dominated by seven amino acids (aspartic acid, glutamic acid, asparagine, glutamine, serine, proline, and arginine). Of these, asparagaine and glutamine dominated at low N-supply, whereas at high N-supply the concentration of arginine increased and dominated amino-N. Plants grown with nitrate had a greater concentration of proline relative to plants with ammonium. In Banksia the concentration of amides was greatest and arginine least with a nitrate N-source, whereas in Hakea amides were least and arginine greatest with nitrate N-source. The concentration of sugars was greater in Banksia than Hakea and in both species at greater N-supply.
Warren, C R; Adams, M A
Trade-offs between the persistence of foliage and productivity in two Pinus species Journal Article
In: Oecologia, vol. 124, no. 4, pp. 487-494, 2000.
We investigated interspecific variation in leaf lifespan (persistence) and consequent differences in leaf biochemistry, anatomy, morphology, patterns of whole-tree carbon allocation and stand productivity. We tested the hypothesis that a species with short-lived foliage, Pinus radiata D. Don (mean leaf lifespan 2.5 years), grows faster than P. pinaster Ait., a species with more persistent foliage (leaf lifespan 5.6 years), and that the faster growth rate of P. radiata is associated with a greater allocation of nitrogen and carbon to photosynthetic tissues across a range of scales. In fully sunlit foliage, the proportion of leaf N in the major photosynthetic enzyme Rubisco (ribulose-1, 5-bisphosphate carboxylase) was greater in P. radiata than in P. pinaster, and, in mid-canopy foliage, the proportion of leaf N in thylakoid proteins was greater in P. radiata. A lesser proportion of needle cross-sectional area was occupied by structural tissue in P. radiata compared to P. pinaster. Foliage mass in stands of P. radiata was 9.7 t ha-1 compared with 18.2 t ha-1 in P. pinaster while leaf area index of both species was similar at 4.6 m2 m-2, owing to the compensating effect of differences in specific leaf area. Hence trade-offs between persistence and productivity were apparent as interspecific differences in patterns of whole-tree carbon allocation, needle morphology, anatomy and biochemistry. However, these interspecific differences did not translate into differences at the stand scale since rates of biomass accumulation were similar in both species (P. radiata 6,9±0.9 kg year-1 tree-1; P. pinaster 7.4±0.9 kg year-1 tree-1). The similarities in performance at larger scales suggest that leaf area index (and radiation interception) determines growth and productivity.
Warren, C R; Adams, M A; Chen, Z
Is photosynthesis related to concentrations of nitrogen and Rubisco in leaves of Australian native plants? Journal Article
In: Australian Journal of Plant Physiology, vol. 27, no. 5, pp. 407-416, 2000.
The relationships among light-saturated photosynthesis and concentrations of nitrogen and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 18.104.22.168) in Australian native plants are poorly known, primarily due to the difficulty of extracting and analysing Rubisco from such species. Rubisco may be rapidly quantified in crude extracts of plant tissue by capillary electrophoresis (CE); however, the presence of phenolic compounds in many Australian native plants limits the use of these methods. The addition of insoluble polyvinylpolypyrrolidone (PVPP) during leaf extractions effectively removed phenols permitting quantitation of Rubisco. Relationships among maximum rates of photosynthesis and concentrations of nitrogen and Rubisco were then investigated in ten species native to Australia. Total nitrogen and the major pools of N in foliage varied greatly between species. Equally, within species N-partitioning was highly plastic, as affected by different concentrations and forms of N applied in sand culture (0.5 or 8 mM, nitrate or ammonium). In Hakea prostrata, for example, the proportion of total N present as soluble proteins varied between 43 and 71%, while the proportion of total N present as Rubisco N ranged between 9.4 and 30.0%, and the contribution of Rubisco to soluble proteins varied between 21 and 42%. The measured concentration of Rubisco varied between 40% and 600% of that estimated from enzyme kinetics and measured rates of photosynthesis. Generally there was a large éxcess' of Rubisco, and in only two cases was the measured concentration of Rubisco significantly less than predicted. Total N, soluble protein and Rubisco concentrations were poorly related to maximum rates of photosynthesis, while the relationship between photosynthesis and Rubisco was worse than that with N, primarily due to the plants' variable over-investment in Rubisco.
Chen, Z L; Warren, C R; Adams, M A
Separation of amino acids in plant tissue extracts by capillary zone electrophoresis with indirect UV detection using aromatic carboxylates as background electrolytes Journal Article
In: Chromatographia, vol. 51, no. 3-4, pp. 180-186, 2000.
Amino acids in extracts of plant tissue were separated and detected by capillary zone electrophoresis (CZE) with indirect UV detection. Various aromatic carboxylates such as salicylate, benzoate, phthalate and trimellitate were investigated as background electrolytes (BGEs). A BGE of benzoate gave the best resolution and detector response. Amino acids were separated at a highly alkaline pH to charge amino acids negatively. Separation was achieved by the co-electroosmotic flow (Co-EOF) by the addition of the cationic surfactant myristyltrimethylammonium bromide (MTAB) to the electrolyte. The conditions affecting the separation of amino acids, including electrolyte pH, concentrations of both benzoate and MTAB, were investigated and optimised. Separation of a mixture of 17 amino acids at pH 11.20 with indirect UV detection at 225 nm was achieved with a BGE of 10 mM benzoate containing 1.0 mM MTAB at pH of 11.20. Detection limits ranged between 10 and 50 μM. The proposed method was demonstrated by the determination of amino acids in extracts of Eucalypt leaves with direct injection of samples.
Chen, Z; Warren, C R; Adams, M A
Separation of rubisco in extracts of plant leaves by capillary electrophoresis with sieving polymers Journal Article
In: Analytical Letters, vol. 33, no. 4, pp. 579-587, 2000.
Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 22.214.171.124) was separated in extracts of plant leaves by capillary electrophoresis with replaceable polymers. Rubisco extracted from plant leaves was fully denatured into small (15.8 kDa) and large (52.9 kDa) subunits in the presence of excess sodium dodecylsulfate (SDS). SDS-Rubisco complexes were separated by using an uncoated fused silica capillary filled with a replaceable polymer solution, with detection at 220 nm. A calibration plot of Rubisco concentration in plant extract versus peak area was linear over the concentration range of 0.09-1.2 mg mL-1 with a correlation coefficient (r2) of 0.995 and a detection limit of 0.05 mg mL-1. The reproducibility of migration times was less than 0.27% RSD (n=5) and reproducibility of peak area was better than 6.9%. The recovery of Rubisco in leaf tissues through the extraction and analysis procedure was 78.2% (±4.8) and through the analysis procedure alone was 96.9% (±5.1). The proposed method was used for the quantification of Rubisco in protein extracts from leaves of a variety of plant species.
Macfarlane, C; Warren, C R; White, D A; Adams, M A
A rapid and simple method for processing wood to crude cellulose for analysis of stable carbon isotopes in tree rings Journal Article
In: Tree Physiology, vol. 19, no. 12, pp. 831-835, 1999.
For analysis of carbon isotope discrimination in wood, cellulose or holocellulose is often preferred to whole tissue because of the variability in isotopic composition of different wood components and the relative immobility of cellulose. Most currently used methods for the preparation of wood components for stable isotope analysis (e.g., the Jayme-Wise method) produce a residue of holocellulose. The Jayme-Wise method was initially developed to extract holocellulose from small (~1 g) samples of wood, and despite subsequent modifications, the method requires specialized glassware, considerable time, and entails the risk of sample loss. For carbon isotope analysis, we adapted an acid-catalyzed solvolytic method for preparing crude cellulose by treating wood meal with acidified di-glycol methyl ether (diglyme). The one-step process requires no special glassware, is complete within 24 hours, and enables over 100 samples to be processed in a day. This method gives similar δ13C values to the Jayme-Wise method for wood of Eucalyptus globulus Labill., Pinus radiata D. Don, and Pinus pinaster Ait. The relationship between δ13C of wood and crude cellulose is as strong as that observed between wood and α-cellulose and stronger than that observed between wood and holocellulose in other species. These relationships suggest that variation in δ13C of wood may result from hemicellulose and that analysis of stable carbon isotopes in crude cellulose is preferable. If the consistent -0.3‰ bias in the value of δ13C of cellulose resulting from residual lignin is corrected for, then the relationship between δ13C of wood and crude cellulose may be used to predict δ13C of cellulose from a small subsample. The method is well suited to species with low concentrations of extractives, but further testing is needed to assess its general applicability.
Warren, C R; Hovenden, M J; Davidson, N J; Beadle, C L
Cold hardening reduces photoinhibition of Eucalypts nitens and E. pauciflora at frost temperatures Journal Article
In: Oecologia, vol. 113, no. 3, pp. 350-359, 1998.
Photoinhibition of photosynthesis at low temperatures was investigated in two species of subalpine eucalypt, Eucalypts nitens (Deane and Maiden) Maiden and E. pauciflora Sieb. ex Spreng. Imposition of an artificial cold-hardening treatment increased the frost tolerance of leaf tissue and increased tolerance to excess light. Cold-hardened seedlings of both species had a higher photosynthetic capacity than non-hardened seedlings at 6 and 16°C and lower levels of non-photochemical quenching (NPQ) at 20 and 5°C. Furthermore, hardened seedlings had faster rates of NPQ development at 5 and -3.5°C. An increase in minimal fluorescence, which indicates slowly reversible photoinhibition, was evident in all seedlings at -1.5 and -3.5°C but was less pronounced in hardened seedlings, with a threefold faster rate of development of NPQ, at -3.5°C than nonhardened seedlings. Hardened seedlings also recovered faster from photoinhibition at -3.5°C. Thus cold hardening increased tolerance to high light in these species. Differences between E. nitens and E. pauciflora in their response to excess light were small and significant only at -3.5°C. Faster recovery from photoinhibition of E. pauciflora was consistent with its occurrence in colder habitats than E. nitens.
Hovenden, M J; Warren, C R
Photochemistry, energy dissipation and cold-hardening in Eucalyptus nitens and E. pauciflora Journal Article
In: Australian Journal of Plant Physiology, vol. 25, no. 5, pp. 581-589, 1998.
The allocation of absorbed photon energy to thermal energy dissipation and photosynthetic electron transport was investigated as a function of photosynthetic photon flux density (PPFD) and temperature in two species of subalpine eucalypt, Eucalyptus nitens (Deane et Maiden) Maiden and E. pauciflora Sieb. ex Spreng. The proportion of absorbed light utilised in photosynthetic electron transport decreased with increasing PPFD, and the decrease was more pronounced the lower the temperature. The proportion diverted into dissipation processes increased with increasing PPFD to a maximum where it reached a plateau. This maximum increased with decreasing temperature. Exposure to a succession of cold (4°C) nights increased the photochemical quantum yield of photosystem II and decreased the allocation of excitation energy to thermal dissipation processes in conditions of excess light, particularly at low temperatures. Consequently, the photosynthetic electron transport rate (ETR) was higher and heat dissipation rate (HDR) was lower in hardened plants than in non-hardened plants at low temperatures. At 20°C, ETR was generally higher than HDR in all plants, but as the temperature decreased, HDR became the dominant process. The PPFD at which HDR exceeded ETR decreased with decreasing temperature, and at low temperatures was always lower in non-hardened plants than hardened plants, although quite similar between species.