Abstract: • 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 (13) C- and (15) N-labelled glycine or glutamine. I quantified (15) N and (13) C excess in leaves and roots and intact labelled amino acids in leaves, roots and stem xylem sap. A tunable diode laser quantified fluxes of (12) CO(2) and (13) CO(2) from leaves and soil. • 60-360 min after addition of amino acid, intact molecules of U-(13) C,(15) N glutamine were < 5% of (15) N excess in roots, whereas U-(13) C,(15) N glycine was 30-100% of (15) N 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, (13) C and (15) N may not reflect rates of uptake. Future experiments should focus on reconciling discrepancies between intact amino acid, (13) C and (15) N 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.
Abstract: 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 PÂ 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.
Abstract: 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 g(m) 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 (13)C 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 (13)C discrimination was also estimated. Not taking this contribution into account may lead to a 50% underestimation of g(m) but had little effect on the CO(2)- and irradiance-induced changes. In conclusion, (i) the observed responses of g(m) to CO(2) and irradiance were not artefactual; (ii) the respiratory term is important to assess absolute values of g(m) 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 g(m) in Eucalyptus seedlings.
Abstract: Exposure to an elevated CO(2) concentration ([CO(2)]) generally decreases leaf N content per unit area (N(area)) and stomatal density, and increases leaf thickness. Mature leaves can 'sense' elevated [CO(2)] and this regulates stomatal development of expanding leaves (systemic regulation). It is unclear if systemic regulation is involved in determination of leaf thickness and N(area)-traits that are significantly correlated with photosynthetic capacity. A cuvette system was used whereby [CO(2)] 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)] (720 μmol mol(-1)) while the remaining mature leaves inside the cuvette were under ambient [CO(2)] of 360 μmol mol(-1). Reverse treatments were performed. Exposure of newly developing leaves to elevated [CO(2)] increased their thickness, but when mature leaves were exposed to elevated [CO(2)] the increase in thickness of new leaves was less pronounced. The largest response to [CO(2)] was reflected in the palisade tissue thickness (as opposed to the spongy tissue) of new leaves. The N(area) of new leaves was unaffected by the local [CO(2)] where the new leaves developed, but decreased following the exposure of mature leaves to elevated [CO(2)]. The volume fraction of mesophyll cells compared with total leaf and the mesophyll cell density changed in a manner similar to the response of N(area). These results suggest that N(area) is controlled independently of the leaf thickness, and suggest that N(area) is under systemic regulation by [CO(2)] signals from mature leaves that control mesophyll cell division.
Abstract: Leaf internal, or mesophyll, conductance to CO(2) (g(m)) is a significant and variable limitation of photosynthesis that also affects leaf transpiration efficiency (TE). Genotypic variation in g(m) and the effect of g(m) on TE were assessed in six barley genotypes (four Hordeum vulgare and two H. bulbosum). Significant variation in g(m) was found between genotypes, and was correlated with photosynthetic rate. The genotype with the highest g(m) also had the highest TE and the lowest carbon isotope discrimination as recorded in leaf tissue (Delta(p)). These results suggest g(m) has unexplored potential to provide TE improvement within crop breeding programmes.
Abstract: Studies of water stress commonly examine either gas exchange or leaf metabolites, and many fail to quantify the concentration of CO₂ in the chloroplasts (C(c)). We redress these limitations by quantifying C(c) 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 (Ψ(pre-dawn) = -1.7 to -2.3 MPa). 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 C(c), 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.
Abstract: 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 delta(13)C and sugar concentrations were compared to delta(13)C 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 delta(13)C in response to altered water availability. Phloem sap delta(13)C was more sensitive to changes of water availability than the delta(13)C 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 delta(13)C 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.
Abstract: Defoliation can reduce net fixation of atmospheric CO(2) by the canopy, but increase the intensity and duration of photosynthetically active radiation on stems. Stem CO(2) 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 CO(2) fluxes (both in the dark and light). Defoliated field-grown seedlings showed similar stem CO(2) 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 CO(2) (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.
Abstract: 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 4-5 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 (13)C and (15)N enrichment in plants receiving glycine indicated uptake of 1.5 (13)C for every (15)N at the 4-h harvest (versus 2:1 (13)C:(15)N in labeled glycine), suggesting intact uptake of around 75% of glycine. Gas chromatography-mass spectrometry analysis detected intact (13)C(2),(15)N-glycine in roots, but the pool of (13)C(2),(15)N-glycine was 10-500 times smaller than (13)C and (15)N excess, and no (13)C(2),(15)N-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.
Abstract: Mesophyll diffusion conductance to CO(2) (g(m)) is an important leaf characteristic determining the drawdown of CO(2) from substomatal cavities (C(i)) to chloroplasts (C(C)). Finite g(m) results in modifications in the shape of the net assimilation (A) versus C(i) response curves, with the final outcome of reduced maximal carboxylase activity of Rubisco (V(cmax)), and a greater ratio of the capacity for photosynthetic electron transport to V(cmax) (J(max)/V(cmax)) and alterations in mitochondrial respiration rate (R(d)) when estimated from A/C(i) responses without considering g(m). The influence of different Farquhar et al. model parameterizations on daily photosynthesis under non-stressed (C(i) kept constant throughout the day) and stressed conditions (mid-day reduction in C(i)) was compared. The model was parameterized on the basis of A/C(C) curves and A/C(i) curves using both the conventional fitting procedure (V(cmax) and R(d) fitted separately to the linear part of the response curve and J(max) to the saturating part) and a procedure that fitted all parameters simultaneously. The analyses demonstrated that A/C(i) parameterizations overestimated daily assimilation by 6-8% for high g(m) values, while they underestimated if by up to 70% for low g(m) values. Qualitative differences between the A/C(i) and A/C(C) parameterizations were observed under stressed conditions, when underestimated V(cmax) and overestimated R(d) of A/C(i) 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/C(i) parameterizations. While A/C(i) parameterization predicted negative carbon balance at mid-day, actual measurements and simulations with the A/C(C) approach yielded positive carbon gain under these conditions. In addition, overall variation captured by the best A/C(i) parameterization was poor compared with the A/C(C) approach. This analysis strongly suggests that for correct parameterization of daily time-courses of photosynthesis under realistic field conditions, g(m) must be included in photosynthesis models.
Abstract: Limited mesophyll diffusion conductance to CO(2) (g(m)) can significantly constrain plant photosynthesis, but the extent of g(m)-limitation is still imperfectly known. As g(m) scales positively with foliage photosynthetic capacity (A), the CO(2) drawdown from substomatal cavities (C(i)) to chloroplasts (C(C), C(i)-C(C)=A/g(m)) rather than g(m) alone characterizes the mesophyll diffusion limitations of photosynthesis. The dependencies of g(m) on A, foliage structure (leaf dry mass per unit area, M(A)), and the resulting drawdowns across a dataset of 81 species of contrasting foliage structure and photosynthetic potentials measured under non-stressed conditions were analysed to describe the structure-driven potential photosynthetic limitations due to g(m). Further the effects of key environmental stress factors and leaf and plant developmental alterations on g(m) and CO(2) drawdown were evaluated and the implications of varying g(m) on foliage photosynthesis in the field were simulated. The meta-analysis demonstrated that g(m) of non-stressed leaves was negatively correlated with M(A), and despite the positive relationship between g(m) and A, the CO(2) drawdown was larger in leaves with more robust structure. The correlations were stronger with mass-based g(m) and A, probably reflecting the circumstance that mesophyll diffusion is a complex three-dimensional process that scales better with mesophyll volume-weighted than with leaf area-weighted traits. The analysis of key environmental stress effects on g(m) and CO(2) drawdowns demonstrated that the effect of individual stresses on CO(2) drawdowns varies depending on the stress effects on foliage structure and assimilation rates. Leaf diffusion limitations are larger in non-senescent older leaves and also in senescent leaves, again reflecting more robust leaf structure and/or non-co-ordinated alterations in leaf photosynthesis and g(m). According to simulation analyses, in plants with a larger part of the overall diffusion conductance from the ambient atmosphere to the chloroplasts in the mesophyll, photosynthesis is less sensitive to changes in stomatal conductance. Accordingly, in harsher environments that support vegetation with tougher long-living stress-tolerant leaves with lower g(m), reductions in stomatal conductance that are common during stress periods are expected to alter photosynthesis less than in species where a larger part of the total diffusion limitation is determined by stomata. While structural robustness improves plant performance under environmental stress, low g(m) and inherently large CO(2) drawdown in robust leaves limits the photosynthesis of these plants more severely under favourable conditions when stomatal conductance is high. The differences in overall responsiveness to environmental modifications of plants with varying g(m) need consideration in current large-scale ecosystem productivity models.
Abstract: 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 CO2 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 N0 trees at all canopy positions. Chlorophyll was approximately 80% higher in lower- and mid-canopy needles of N+ trees than of N0 trees, but only approximately 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.
Abstract: Internal conductance describes the movement of CO(2) 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 CO(2) 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. C(i)-C(c)) 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 CO(2) 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.
Abstract: Internal conductance to CO(2) transfer from intercellular spaces to chloroplasts (g(i)) 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 degrees 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 degrees C for 6 weeks before g(i) 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 degrees C, the maximum rate of net photosynthesis peaked at around 30 or 35 degrees 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 degrees C and increased threefold from 10 to 35 degrees C. There was some evidence that g(i) was greater in seedlings acclimated to 15 than to 30 degrees C, which resulted in seedlings acclimated to 15 degrees C having, if anything, a smaller relative limitation due to g(i) than seedlings acclimated to 30 degrees C. Stomatal limitations were also smaller in seedlings acclimated to 15 degrees C than in seedlings acclimated to 30 degrees C. Based on chloroplast CO(2) concentration, neither maximum rates of carboxylation nor RuBP-limited rate of electron transport peaked between 10 and 35 degrees 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 g(i) to be positively related to measurement temperature.
Abstract: 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 approximately 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.
Abstract: 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.
Abstract: 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.
Abstract: 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.
Abstract: Two of the ways in which plants cope with water deficits are stomatal closure and "osmotic 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 CO(2) concentrations between treatments, or in chloroplast CO(2) 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.
Abstract: 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 micromol l(-1)) of (15)N-nitrate, (15)N-ammonium and 2-(13)C(2) (15)N-glycine. Species did not differ in their preference for different forms of N (species x 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 micromol g(DM) (-1) h(-1); mean +/- SD, n = 108), uptake of glycine occurred at less than half this rate (4.4 +/- 2.6 micromol g(DM) (-1) h(-1)), whereas uptake of nitrate occurred at one-tenth of this rate (0.9 +/- 1.2 micromol g(DM) (-1) h(-1)). The strong positive relationship between (15)N and (13)C uptake indicated that at least 72% of glycine-N was taken up intact. These findings indicate the potential for considerable uptake of organic N in the field.
Abstract: 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 (O(3)) 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 O(3)) or an experimentally elevated twice-ambient O(3) regime (2 x O(3)). The elevated 2 x O (3) regime was provided by means of a free-air O(3) canopy exposure system. The hypotheses were tested that (1) gas exchange is negatively affected by O(3) and (2) the effects of O(3) are dose-dependent and thus the sizes of differences between treatments are positively related to COU. Gas exchange (light-saturated CO(2) uptake rate A(max), stomatal conductance g (s), maximum rate of carboxylation Vc (max), ribulose-1,5-bisphosphate turnover limited rate of photosynthesis J (max), CO(2) compensation point CP, apparent quantum yield of net CO(2) uptake AQ, carboxylation efficiency CE, day- and nighttime 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), J(max), and CE were lower in trees receiving 2 x O(3) compared with the ambient O(3) regime (1 x O(3)). Treatment differences in Vc (max), J (max), CE were rather small in 2004 (i.e., parameter levels were lower by 10 - 30 % in 2 x O(3) than 1 x O(3)) and not significant in 2003. In 2004 COU was positively correlated with the difference between treatments in A (max), g (s), and ETR (i.e., consistent with the dose-dependence of O(3)'s deleterious effects). However, in 2003, differences in A(max), g (s), and ETR between the two O(3) 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 O(3) did not always have significant effects on leaf gas exchange. In view of the key findings, both hypotheses were to be rejected.
Abstract: The internal conductance to CO(2) 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 degrees C and 35 degrees 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 (C(i)*) and rate of mitochondrial respiration in the light (R(d)), were estimated by the Laisk method. These were used to calculate the chloroplastic photocompensation point (Gamma*) in a simultaneous equation with g(i). 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/C(i) curve. The temperature responses of the rate of Rubisco carboxylation (V(cmax)) and the RuBP limited rate of electron transport (J(max)) were determined from chloroplastic CO(2) concentrations. The rate of net photosynthesis peaked at 24 degrees C. C(i)* was similar to reports for other species with a C(i)* of 39 micromol mol(-1) at 25 degrees C and an activation energy of 34 kJ mol(-1). Gamma* was very similar to the published temperature response for Spinacia oleracea from 20 degrees C to 35 degrees C, but was slightly greater at 10 degrees C and 15 degrees C. J(max) peaked at 30 degrees C, whereas V(cmax) did not reach a maximum between 10 degrees C and 35 degrees C. Activation energies were 49 kJ mol(-1) for V(cmax) and 100 kJ mol(-1) for J(max). Both methods showed that internal conductance doubled from 10 degrees C to 20 degrees C, and then was nearly temperature-independent from 20 degrees C to 35 degrees C. Hence, the temperature response of internal conductance could not be fitted to an Arrhenius function. The best fit to estimated g(i) was obtained with a three-parameter log normal function (R(2)=0.98), with a maximum g(i) of 0.19 mol m(-2) s(-1) at 29 degrees C.
Abstract: 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 (delta). 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 gi-photosynthesis relationship contributes to variation in photosynthetic 'use' efficiency of N (PNUE) and water (WUE). Delta 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, delta may vary by up to 3 per thousand. This is as large or larger than is commonly reported in many interspecific comparisons of delta, and adds to previous warnings about simplistic interpretations of WUE based on delta. 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.
Abstract: 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.
Abstract: 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 (r2=0.77). Interspecific differences in RGR were strongly related to LMR (positive relationship, r2=0.50) and the rate of dry matter production per mol of leaf nitrogen (positive relationship, r2=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.
Abstract: 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 micromol 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%.
Abstract: 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 (CO2) 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 gi 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.
Abstract: The internal conductance to CO(2) 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-C(i) curves. The aim of this paper was to examine the response of g(i) 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 g(i) 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 V(cmax) that were 60% of the true value, which, in turn, led to an underestimation of in vivo Rubisco specific activity (as V(cmax)/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.
Abstract: 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: Eucalyptus 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.
Abstract: 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 (A(max)) declined to low rates in control seedlings, whereas A(max) increased in N-pulse seedlings. Nevertheless, treatment and temporal trends in N and Rubisco content per unit area were poorly related to A(max), and it seems likely that photosynthesis was limited by additional factors, perhaps thylakoid proteins or an inadequate supply of other nutrients.
Abstract: 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, gs, 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 (Psil) was maintained near -0.9 MPa over a wide range of D, whereas Psil of Douglas-fir decreased from -1.2 to -1.5 MPa as D increased. The tighter (isohydric) regulation of Psil 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 Psil 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.
Abstract: 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 % 15NH4 15NO3). 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 (approximately 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.
Abstract: 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.
Abstract: The stable carbon isotope composition (delta(13)C) 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 delta(13)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 delta(13)C. The strong relationship between delta(13)C and branch length was a function of hydraulic conductance, which was negatively correlated with branch length (r(2) = 0.84). Variation in P and ET among sites was reflected in delta(13)C, which was negatively correlated with P/ET (r(2) = 0.66). However, this analysis was confounded by differences in branch length. If the effects of branch length on delta(13)C were first removed, then the 'residual' delta(13)C was more closely related to P/ET (r(2) = 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 delta(13)C in foliage can only be reliably assessed if deconvoluted from hydraulic constraints.
Abstract: 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.
Abstract: 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 delta(13)C values to the Jayme-Wise method for wood of Eucalyptus globulus Labill., Pinus radiata D. Don and Pinus pinaster Ait. The relationship between delta(13)C of wood and crude cellulose is as strong as that observed between wood and alpha-cellulose and stronger than that observed between wood and holocellulose in other species. These relationships suggest that variation in delta(13)C 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 delta(13)C of cellulose resulting from residual lignin is corrected for, then the relationship between delta(13)C of wood and crude cellulose may be used to predict delta(13)C of cellulose from a small sub-sample. The method is well suited to species with low concentrations of extractives, but further testing is needed to assess its general applicability.
