Legumes are different: Leaf nitrogen, photosynthesis, and water use efficiency.

Using robust, pairwise comparisons and a global dataset, we show that nitrogen concentration per unit leaf mass for nitrogen-fixing plants (N2FP; mainly legumes plus some actinorhizal species) in nonagricultural ecosystems is universally greater (43-100%) than that for other plants (OP). This difference is maintained across Koppen climate zones and growth forms and strongest in the wet tropics and within deciduous angiosperms. N2FP mostly show a similar advantage over OP in nitrogen per leaf area (Narea), even in arid climates, despite diazotrophy being sensitive to drought. We also show that, for most N2FP, carbon fixation by photosynthesis (Asat) and stomatal conductance (gs) are not related to Narea-in distinct challenge to current theories that place the leaf nitrogen-Asat relationship at the center of explanations of plant fitness and competitive ability. Among N2FP, only forbs displayed an Narea-gs relationship similar to that for OP, whereas intrinsic water use efficiency (WUEi; Asat/gs) was positively related to Narea for woody N2FP. Enhanced foliar nitrogen (relative to OP) contributes strongly to other evolutionarily advantageous attributes of legumes, such as seed nitrogen and herbivore defense. These alternate explanations of clear differences in leaf N between N2FP and OP have significant implications (e.g., for global models of carbon fluxes based on relationships between leaf N and Asat). Combined, greater WUE and leaf nitrogen-in a variety of forms-enhance fitness and survival of genomes of N2FP, particularly in arid and semiarid climates.

Contrasting responses of crop legumes and cereals to nitrogen availability.

In nonagricultural systems, the relationship between intrinsic water-use efficiency (WUEi ) and leaf nitrogen (Narea ) is known to be stronger for legumes than for nonlegumes. We tested whether these relationships are retained for major agricultural legumes and nonlegumes. We compared the response to N nutrition of WUEi (and its component parts, photosynthesis (Asat ) and stomatal conductance (gs )) for legumes Cicer arietinum, Glycine max, Lupinus alba and Vicia faba, nonlegume dicots Brassica napus and Helianthus annus, and nonlegume cereals Hordeum vulgare and Triticum aestivum. Surprisingly, and in contrast to studied cereals and nonlegume dicots, Narea was positively related to photosynthesis in the legumes, explaining nearly half of the variance in Asat . WUEi was tightly coupled to Narea for agricultural legumes and nonlegume dicots, but not for cereal crops. Our analysis suggests that breeding efforts to reduce gs in legumes could increase WUEi by 120-218% while maintaining Asat at nonlegume values. Physiologically informed breeding of legumes can enhance sustainable agriculture by reducing requirements for water and N.

Does triacylglycerol (TAG) serve a photoprotective function in plant leaves? An examination of leaf lipids under shading and drought.

Plant survival in many ecosystems requires tolerance of large radiation loads, unreliable water supply and suboptimal soil fertility. We hypothesized that increased production of neutral lipids (triacylglycerols, TAGs) in plant leaves is a mechanism for dissipating excess radiation energy. In a greenhouse experiment, we combined drought and shade treatments and examined responses among four species differing in life form, habitat, and drought- and shade-tolerance. We also present a lipid extraction protocol suitable for sclerophyllous leaves of native Australian trees (e.g. Acacia, Eucalyptus). Fluorescence measurements indicated that plants exposed to full sunlight experienced mild photoinhibition during our experiment. Accumulation of TAGs did not follow photosynthetic capacity, but instead, TAG concentration increased with non-photochemical quenching. This suggests that plants under oxidative stress may increase biosynthesis of TAGs. Moderate drought stress resulted in a 60% reduction in TAG concentration in wheat (Triticum aestivum). Shading had no effect on TAGs, but increased concentrations of polar lipids in leaves; for example, acclimation to shade in Austrodanthonia spp., a native Australian grass, resulted in a 60% increase in associated polar lipids and higher foliar chlorophyll concentrations. Shading also reduced the digalactosyldiacylglycerol:monogalactosyldiacylglycerol (DGDG:MGDG) ratio in leaves, with a corresponding increase in the degree of unsaturation and thus fluidity of thylakoid membranes of chloroplasts. Our results suggest that prevention of photodamage may be coordinated with accumulation of TAGs, although further research is required to determine if TAGs serve a photoprotective function in plant leaves.

Stomatal structure and physiology do not explain differences in water use among montane eucalypts.

Understanding the regulation of water use at the whole-tree scale is critical to advancing the utility of physiological ecology, for example in its role in predictive hydrology of forested catchments. For three eucalypt species that dominate high-elevation catchments in south-eastern Australia, we examined if whole-tree water use could be related to three widely discussed regulators of water use: stomatal anatomy, sensitivity of stomata [i.e. stomatal conductance (g(s))] to environmental influences, and sapwood area. While daily tree water use varied sixfold among species, sap velocity and sapwood area varied in parallel. Combined, stomatal structure and physiology could not explain differences in species-specific water use. Species which exhibited the fastest (Eucalyptus delegatensis) and slowest (Eucalyptus pauciflora) rates of water use both exhibited greater capacity for physiological control of g(s) [indicated by sensitivity to vapour pressure deficit (VPD)] and a reduced capacity to limit g(s) anatomically [indicated by greater potential g(s) (g(max))]. Conversely, g(s) was insensitive to VPD and g(max) was lowest for Eucalyptus radiata, the species showing intermediate rates of water use. Improved knowledge of stomatal anatomy will help us to understand the capacity of species to regulate leaf-level water loss, but seems likely to remain of limited use for explaining rates of whole-tree water use in montane eucalypts at the catchment scale.

Anatomical and physiological regulation of post-fire carbon and water exchange in canopies of two resprouting Eucalyptus species.

The great majority of Eucalyptus spp. are facultative resprouters, and they dominate the eucalypt forests of Australia. Despite this numeric and geographic dominance, there is a general lack of knowledge of their capacity for carbon capture and water loss during canopy reinstation. After a crown-removing fire, we measured leaf-level determinants of carbon and water flux in resprouting canopies of Eucalyptus dives and E. radiata over the 3 years that followed. Leaf anatomy and physiology changed markedly during canopy reinstation, and leaves produced in the second year (2010) were distinct from those produced later. Leaves produced in 2010 were thicker (all measures of leaf anatomy), yet more porous (increased intercellular airspace), causing specific leaf area also to be greater. Indicators of heterotrophic activity, leaf respiration rate and light compensation point, were twofold greater in 2010, whereas all measures of photosynthetic capacity were greatest in leaves produced in 2011 and 2012. Whilst stomatal density, vein density and leaf hydraulic conductance all progressively decreased with time, neither leaf water status nor carbon isotope discrimination were affected. We conclude that canopy reinstation is primarily limited by pre-fire carbon stores, rather than by post-fire edaphic conditions (e.g., water availability), and thus argue that capacity for recovery is directly linked to pre-fire forest health.

Photosynthetic benefits of ultraviolet-A to Pimelea ligustrina, a woody shrub of sub-alpine Australia.

The definition of photosynthetically active radiation (Q) as the visible waveband (λ 400-700 nm) is a core assumption of much of modern plant biology and global models of carbon and water fluxes. On the other hand, much research has focused on potential mutation and damage to leaves caused by ultraviolet (UV) radiation (280-400 nm), and anatomical and physiological adaptations that help avoid such damage. Even so, plant responses to UV-A are poorly described and, until now, photosynthetic utilization of UV-A has not been elucidated under full light conditions in the field. We found that the UV-A content of sunlight increased photosynthetic rates in situ by 12% in Pimelea ligustrina Labill., a common and indigenous woody shrub of alpine ecosystems of the Southern Hemisphere. Compared to companion shrubs, UV-A-induced photosynthesis in P. ligustrina resulted from reduced physical and chemical capacities to screen UV-A at the leaf surface (illustrated by a lack of cuticle and reduced phenol index) and the resulting ability of UV-A to excite chlorophyll (Chl) a directly, and via energy provided by the carotenoid lutein. A screening of 55 additional sub-alpine species showed that 47% of the plant taxa also display Chl a fluorescence under UV-A. If Chl a fluorescence indicates potential for photosynthetic gain, continued exclusion of UV-A from definitions of Q in this ecosystem could result in underestimates of measured and modeled rates of photosynthesis and miscalculation of potential for carbon sequestration. We suggest that carbon gain for alpine environs across the globe could be similarly underestimated given that UV-A radiation increases with altitude and that the frequently dominant herb and grass life-forms often transmit UV-A through the epidermis.

Disentangling respiratory acclimation and adaptation to growth temperature by Eucalyptus.

• Respiratory acclimation to growth temperature differs between species, but underlying mechanisms are poorly understood. In the present study, we tested the hypothesis that respiratory acclimation of CO(2) release is a consequence of growth regulation such that growth rates of young foliage of Eucalyptus spp. are similar at contrasting growth temperatures. Further, we tested whether such a response is affected by adaptation of Eucalyptus to different thermal environments via growth at different altitudes in the Australian Alps. • We employed calorimetric methods to relate rates of CO(2) release (mainly from substrate oxidation) and rates of O(2) reduction to conservation of energy. Temperature responses of these processes provided insight into mechanisms that control energy conservation and expenditure, and helped define 'instantaneous enthalpic growth capacity' (CapG). • CapG increased with altitude, but was counteracted by other factors in species adapted to highland habitats. The acclimation response was partly driven by changes in respiratory capacity (CapR(CO2)), and partly by more pronounced dynamic responses of CO(2) release (δ(R(CO2))) to measurement temperature. We observed enhanced temperature sensitivity of O(2) reduction (E(o)(R(O2))) at higher altitudes. • Adaptation to growth temperature included differences in respiration and growth capacities, but there was little evidence that Eucalyptus species vary in metabolic flexibility.

Simple models for stomatal conductance derived from a process model: cross-validation against sap flux data.

Representation of stomatal physiology in models of plant-atmosphere gas exchange is minimal, and direct application of process-based models is limited by difficulty of parameter estimation. We derived simple models of stomatal conductance from a recent process-based model, and cross-validated them against measurements of sap flux (176-365 d in length) in 36 individual trees of two age classes for two Eucalyptus species across seven sites in the mountains of southeastern Australia. The derived models - which are driven by irradiance and evaporative demand and have two to four parameters that represent sums and products of biophysical parameters in the process model - reproduced a median 83-89% of observed variance in half-hourly and diurnally averaged sap flux, and performed similarly whether fitted using a random sample of all data or using 1 month of data from spring or autumn. Our simple models are an advance in predicting plant water use because their parameters are transparently related to reduced processes and properties, enabling easy accommodation of improved knowledge about how those parameters respond to environmental change and differ among species.

Site-specific responses to short-term environmental variation are reflected in leaf and phloem-sap carbon isotopic abundance of field grown Eucalyptus globulus.

The carbon isotopic composition (δ(13) C) of plant material has been used extensively as an indirect measure of carbon fixation per volume of water used. More recently, the δ(13) C of phloem sap (δ(13) C(phl) ) has been used as a surrogate measure of short-term, canopy scale δ(13) C. Using a combination of δ(13) C physiological, structural and chemical indices from leaves and phloem sap of Eucalyptus globulus at sites of contrasting water availability, we sought to identify short-term, canopy scale resource limitations. Results illustrate that δ(13) C(phl) offers valid reflections of short-term, canopy scale values of leaf δ(13) C and tree water status. Under conditions limited by water, leaf and phloem sap photoassimilates differ in (13) C abundance of a magnitude large enough to significantly influence predictions of water use efficiency. This pattern was not detected among trees with adequate water supply indicating fractionation into heterotrophic tissues that may be sensitive to plant water status. Trees employed a range of physiological, biochemical and structural adaptations to acclimate to resource limitation that differed among sites providing a useful context upon which to interpret patterns in δ(13) C. Our results highlight that such easily characterized properties are ideal for use as minimally invasive tools to monitor growth and resilience of plants to variations in resource availability.

CO2, nitrogen deposition and a discontinuous climate response drive water use efficiency in global forests.

Reduced stomatal conductance is a common plant response to rising atmospheric CO2 and increases water use efficiency (W). At the leaf-scale, W depends on water and nitrogen availability in addition to atmospheric CO2. In hydroclimate models W is a key driver of rainfall, droughts, and streamflow extremes. We used global climate data to derive Aridity Indices (AI) for forests over the period 1965-2015 and synthesised those with data for nitrogen deposition and W derived from stable isotopes in tree rings. AI and atmospheric CO2 account for most of the variance in W of trees across the globe, while cumulative nitrogen deposition has a significant effect only in regions without strong legacies of atmospheric pollution. The relation of aridity and W displays a clear discontinuity. W and AI are strongly related below a threshold value of AI ≈ 1 but are not related where AI > 1. Tree ring data emphasise that effective demarcation of water-limited from non-water-limited behaviour of stomata is critical to improving hydrological models that operate at regional to global scales.

Rainfall drives variation in rates of change in intrinsic water use efficiency of tropical forests.

Rates of change in intrinsic water use efficiency (W) of trees relative to those in atmospheric [CO2] (ca) have been mostly assessed via short-term studies (e.g., leaf analysis, flux analysis) and/or step increases in ca (e.g., FACE studies). Here we use compiled data for abundances of carbon isotopes in tree stems to show that on decadal scales, rates of change (dW/dca) vary with location and rainfall within the global tropics. For the period 1915-1995, and including corrections for mesophyll conductance and photorespiration, dW/dca for drier tropical forests (receiving ~ 1000 mm rainfall) were at least twice that of the wettest (receiving ~ 4000 mm). The data also empirically confirm theorized roles of tropical forests in changes in atmospheric 13C/12C ratios (the 13C Suess Effect). Further formal analysis of geographic variation in decade-to-century scale dW/dca will be needed to refine current models that predict increases in carbon uptake by forests without hydrological cost.

A standardization method to disentangle environmental information from axial trends of xylem anatomical traits.

Anatomical traits such as xylem conduit diameter and vessel connectivity are fundamental characteristics of the hydraulic architecture of vascular plants. Stem xylem conduits are narrow at the stem apex, and this confers resistance to embolisms that might otherwise be induced by large, negative water potentials at the top of tall trees. Below the apex, conduits progressively widen and this characteristic minimizes effects of path length on total hydraulic resistance. While interconnections among xylem vessels have been noted for decades, their role(s) are not fully clarified. For example, we do not know if they allow water to bypass embolized vessels, or increase the risk of spread of embolisms, or how their arrangement varies within a tree. Here we demonstrate the benefit of removing the independent effect of stem length on assessment of effects of external (e.g., climatic) factors on such xylem traits. We measured the hydraulic diameter (Dh) and vessel conductivity index (VCI) along the stem of 21 shrubs/trees of similar height (1.19 < H < 5.45 m) belonging to seven Acacia species, across a wide aridity gradient in Australia. All trees showed similar scaling exponents of Dh (b = 0.33) and VCI (b = 0.53) vs axial distance from the apex (L), thus conforming with general patterns in woody plants. After de-trending for L, neither Dh (P = 0.21) nor VCI (P = 0.109) differed across the aridity gradient. We found that across a wide gradient of aridity, climate had no effect on xylem anatomy of Acacia spp, which was instead dictated by axial distances from stem apices. We argue that the use of standardization procedures to filter out intrinsic patterns of vascular traits is an essential step in assessing climate-driven modifications of xylem architecture.

Crops, Nitrogen, Water: Are Legumes Friend, Foe, or Misunderstood Ally?

Biological nitrogen fixation (BNF) by crop legumes reduces demand for industrial nitrogen fixation (INF). Nonetheless, rates of BNF in agriculture remain low, with strong negative feedback to BNF from reactive soil nitrogen (N) and drought. We show that breeding for yield has resulted in strong relationships between photosynthesis and leaf N in non-leguminous crops, whereas grain legumes show strong relations between leaf N and water use efficiency (WUE). We contrast these understandings with other studies that draw attention to the water costs of grain legume crops, and their potential for polluting the biosphere with N. We propose that breeding grain legumes for reduced stomatal conductance can increase WUE without compromising production or BNF. Legume crops remain a better bet than relying on INF.

Increased photosynthesis following partial defoliation of field-grown Eucalyptus globulus seedlings is not caused by increased leaf nitrogen.

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 micromol m(-2) s(-1)) and D70 (23 micromol m(-2) s(-1)) treatments than in control seedlings (15 micromol 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.

Within-canopy nitrogen and photosynthetic gradients are unaffected by soil fertility in field-grown Eucalyptus globulus.

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 (g(s)) at the maximum photosynthetic rate (A(max)) was between 0.2 and 0.4 mol m(-2) s(-1) higher in fertilized trees, but A(max) and the concentration of Rubisco (Rub(a)) were unaffected by fertilization. This seeming paradox, where there was no response of A(max) to fertilization despite increases in g(s) 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 A(max) 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.

Nocturnal water loss in mature subalpine Eucalyptus delegatensis tall open forests and adjacent E. pauciflora woodlands.

We measured sap flux (S) and environmental variables in four monospecific stands of alpine ash (Eucalyptus delegatensis R. Baker, AA) and snowgum (E. pauciflora Sieb. ex Spreng., SG) in Australia's Victorian Alps. Nocturnal S was 11.8 ± 0.8% of diel totals. We separated transpiration (E) and refilling components of S using a novel modeling approach based on refilling time constants. The nocturnal fraction of diel water loss (f(n)) averaged 8.6 ± 0.6% for AA and 9.8 ± 1.7% for SG; f(n) differed among sites but not species. Evaporative demand (D) was the strongest driver of nocturnal E (E(n)). The ratio E(n)/D (G(n)) was positively correlated to soil moisture in most cases, whereas correlations between wind speed and G(n) varied widely in sign and strength. Our results suggest (1) the large, mature trees at our subalpine sites have greater f(n) than the few Australian native tree species that have been studied at lower elevations, (2) AA and SG exhibit similar f(n) despite very different size and life history, and (3) f(n) may differ substantially among sites, so future work should be replicated across differing sites. Our novel approach to quantifying f(n) can be applied to S measurements obtained by any method.

Diurnal patterns of water use in Eucalyptus victrix indicate pronounced desiccation-rehydration cycles despite unlimited water supply.

Knowledge about nocturnal transpiration (E(night)) of trees is increasing and its impact on regional water and carbon balance has been recognized. Most of this knowledge has been generated in temperate or equatorial regions. Yet, little is known about E(night) and tree water use (Q) in semi-arid regions. We investigated the influence of atmospheric conditions on daytime (Q(day)) and nighttime water transport (Q(night)) of Eucalyptus victrix L.A.S. Johnson & K.D. Hill growing over shallow groundwater (not >1.5 m in depth) in semi-arid tropical Australia. We recorded Q(day) and Q(night) at different tree heights in conjunction with measurements of stomatal conductance (g(s)) and partitioned E(night) from refilling processes. Q of average-sized trees (200-400 mm diameter) was 1000-3000 l month(-1), but increased exponentially with diameter such that large trees (>500 mm diameter) used up to 8000 l month(-1). Q was remarkably stable across seasons. Water flux densities (J(s)) varied significantly at different tree heights during day and night. We show that g(s) remained significantly different from zero and E(night) was always greater than zero due to vapor pressure deficits (D) that remained >1.5 kPa at night throughout the year. Q(night) reached a maximum of 50% of Q(day) and was >0.03 mm h(-1) averaged across seasons. Refilling began during afternoon hours and continued well into the night. Q(night) eventually stabilized and closely tracked D(night). Coupling of Q(night) and D(night) was particularly strong during the wet season (R2 = 0.95). We suggest that these trees have developed the capacity to withstand a pronounced desiccation-rehydration cycle in a semi-arid environment. Such a cycle has important implications for local and regional hydrological budgets of semi-arid landscapes, as large nighttime water fluxes must be included in any accounting.

Novel mannose-sequestration technique reveals variation in subcellular orthophosphate pools do not explain the effects of phosphorus nutrition on photosynthesis in Eucalyptus globulus seedlings.

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 micromol CO2 m(-2) s(-1) with 0 mM P vs. 16 micromol 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 Pi 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.