Plant Cell and Environment

Published by Wiley
Online ISSN: 1365-3040
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Article
Seventy one cultivars of sweet sorghum (Sorghum bicolor L.) were screened for aluminium (Al) tolerance by measuring relative root growth (RRG). Two contrasting cultivars, ROMA (Al-tolerant) and POTCHETSTRM (Al-sensitive), were selected to study shorter-term responses to Al stress. POTCHETSTRM had higher callose synthase activity, lower β-1,3-glucanase activity and more callose deposition in the root apices during Al treatment compared with ROMA. We monitored the expression of twelve genes involved in callose synthesis and degradation and found that one of these, SbGlu1 (Sb03g045630.1), which encodes a β-1,3-glucanase enzyme, best explained the contrasting deposition of callose in ROMA and POTCHETSTRM during Al treatment. Full length cDNAs of SbGlu1 was prepared from ROMA and POTCHETSTRM and expressed in Arabidopsis thaliana using the constitutive CaMV 35S promoter. Independent transgenic lines displayed significantly greater Al tolerance than wild-type plants and vector-only controls. This phenotype was associated with greater total β-1,3-glucanase activity, less Al accumulation and reduced callose deposition in the roots. These results suggest that callose production is not just an early indicator of Al stress in plants but likely to be part of the toxicity pathway that leads to the inhibition of root growth.
 
Article
Plants sense light and gravity to orient their direction of growth. One common component in the early events of both phototropic and gravitropic signal transduction is activation of phospholipase C (PLC), which leads to an increase in inositol 1,4,5-triphosphate (InsP(3)) levels. The InsP(3) signal is terminated by hydrolysis of InsP(3) through inositolpolyphosphate-5-phosphatases (InsP 5-ptases). Arabidopsis plants expressing a heterologous InsP 5-ptase have low basal InsP(3) levels and exhibit reduced gravitropic and phototropic bending. Downstream effects of InsP(3)-mediated signalling are not understood. We used comparative transcript profiling to characterize gene expression changes in gravity- or light-stimulated Arabidopsis root apices that were manipulated in their InsP(3) metabolism either through inhibition of PLC activity or expression of InsP 5-ptase. We identified InsP(3)-dependent and InsP(3)-independent co-regulated gene sets in response to gravity or light stimulation. Inhibition of PLC activity in wild-type plants caused similar changes in transcript abundance in response to gravitropic and phototropic stimulation as in the transgenic lines. Therefore, we conclude that changes in gene expression in response to gravitropic and phototropic stimulation are mediated by two signal transduction pathways that vary in their dependence on changes in InsP(3).
 
Article
This study was conducted to unravel a mechanism for the gravitropic curvature response in oat (Avena sativa) shoot pulvini. For this purpose, we examined the downward movement of starch-filled chloroplast gravisensors, differential changes in inositol 1,4,5-trisphosphate (IP(3)) levels, transport of indole-3-acetic acid (IAA) and gravitropic curvature. Upon gravistimulation, the ratio for IAA levels in lower halves versus those in upper halves (L/U) increased from 1.0 at 0 h and reached a maximum value of 1.45 at 8 h. When shoots were grown in the dark for 10 d, to deplete starch in the chloroplast, the gravity-induced L/U of IAA was reduced to 1.0. N-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), both auxin transport inhibitors, significantly reduced the amount of gravitropic curvature and gravity-induced lateral IAA transport, but did not reduce the gravity-induced late change in the L/U ratio of IP(3) levels. U73122, a specific phospholipase C (PLC) inhibitor, decreased gravity-induced curvature. Because U73122 reduced the ratio of L/U of IAA imposed by gravistimulation, it is clear that IAA transport is correlated with changes in IP(3) levels upon gravistimulation. These results indicate that gravistimulation-induced differential lateral IAA transport may result from the onset of graviperception in the chloroplast gravisensors coupled with gravity-induced asymmetric changes in IP(3) levels in oat shoot pulvini.
 
Article
The activity of the photosynthetic carbon-fixing enzyme, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), is partially inhibited by arsenite in the millimolar concentration range. However, micromolar arsenite can fully inhibit Rubisco in the presence of a potentiating monothiol such as cysteine, cysteamine, 2-mercaptoethanol or N-acetylcysteine, but not glutathione. Arsenite reacts specifically with the vicinal Cys172-Cys192 from the large subunit of Rubisco and with the monothiol to establish a ternary complex which is suggested to be a trithioarsenical. The stability of the complex is strongly dependent on the nature of the monothiol. Enzyme activity is fully recovered through the disassembly of the complex after eliminating arsenite and/or the thiol from the medium. The synergic combination of arsenite and a monothiol acts also in vivo stopping carbon dioxide fixation in illuminated cultures of Chlamydomonas reinhardtii. Again, this effect may be reverted by washing the cells. However, in vivo inhibition does not result from the blocking of Rubisco since mutant strains carrying Rubiscos with Cys172 and/or Cys192 substitutions (which are insensitive to arsenite in vitro) are also arrested. This suggests the existence of a specific sensor controlling carbon fixation that is even more sensitive than Rubisco to the arsenite-thiol synergism.
 
Article
Cyclic electron flow around photosystem I (CEF1) is thought to augment chloroplast ATP production to meet metabolic needs. Very little is known about the induction and regulation of CEF1. We investigated the effects on CEF1 of antisense suppression of the Calvin-Benson enzymes glyceraldehyde-3-phosphate dehydrogenase (gapR), and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (SSU), in tobacco (Nicotiana tabacum cv. Wisconsin 38). The gapR, but not ssuR, mutants showed substantial increases in CEF1, demonstrating that specific intermediates, rather than slowing of assimilation, induce CEF1. Both types of mutant showed increases in steady-state transthylakoid proton motive force (pmf) and subsequent activation of the photoprotective q(E) response. With gapR, the increased pmf was caused both by up-regulation of CEF1 and down-regulation of the ATP synthase. In ssuR, the increased pmf was attributed entirely to a decrease in ATP synthase activity, as previously seen in wild-type plants when CO₂ levels were decreased. Comparison of major stromal metabolites in gapR, ssuR and hcef1, a mutant with decreased fructose 1,6-bisphosphatase activity, showed that neither the ATP/ADP ratio, nor major Calvin-Benson cycle intermediates can directly account for the activation of CEF1, suggesting that chloroplast redox status or reactive oxygen species regulate CEF1.
 
Article
Although ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was discovered nearly 60 years ago, the associated chemical mechanism of the reaction is still incompletely understood. The catalytic cycle consists of four major steps: ribulose-1,5-bisphosphate binding, enolization, CO(2) or O(2) addition and hydration and cleavage of the intermediate. The use of individual rate constants for these elemental steps yields mathematical expressions for usual kinetic constants (k(cat) , K(m) ), CO(2) vs. O(2) specificity (S(c/o) ) as well as other chemical parameters such as the (12) C/(13) C isotope effect. That said, most of them are not simple and thus the interpretation of experimental, observed values of k(cat) , K(m) and S(c/o) may be more complicated than expected. That is, Rubisco effective catalysis depends on several kinetic parameters which are influenced by both the biological origin and the cellular medium (that can in turn vary with environmental conditions). In this brief review, we present the basic model of Rubisco kinetics and describe how subtle biochemical changes (which may have occurred along Evolution) can easily modify Rubisco catalysis.
 
Article
Chloroplast protrusions (CPs) are often observed under environmental stresses, but their role has not been elucidated. The formation of CPs was observed in the leaf of rice plants treated with 75 mm NaCl for 14 d. Some CPs were almost separated from the main chloroplast body. In some CPs, inner membrane structures and crystalline inclusions were included. Similar structures surrounded by double membranes were observed in the cytoplasm and vacuole. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) was detected in CPs and the similar structures in the cytoplasm and vacuole. These results suggest that CP is one of the pathways of Rubisco exclusion from chloroplasts into the cytoplasm under salinity, and the exclusions could be transported to vacuole for their degradation.
 
Article
D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the first step in photosynthetic carbon assimilation and represents the largest sink for nitrogen in plants. Improvement of its kinetic properties or the efficiency with which it is used in planta would benefit photosynthesis, nitrogen and water use efficiency, and yield. This paper presents a new non-radioactive microplate-based assay, which determines the product [3-phosphoglycerate (3-PGA)] in an enzymic cycle between glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate oxidase. High sensitivity permits use of highly diluted extracts, and a short reaction time to avoid problems due to fall-off. Throughput was several hundreds of samples per person per day. Sensitivity and convenience compared favourably with radioisotopic assays, which were previously used to assay Rubisco. Its use is illustrated in three applications. (1) Maximal and initial activities and the K(m) for ribulose-1,5-bisphosphate were determined in raw extracts of leaves from several species. Similar values were obtained from those in the literature. (2) Diurnal changes were compared in rosettes of wild-type (WT) Arabidopsis and the starchless pgm mutant. Despite these dramatic differences in carbon metabolism, Rubisco activity and activation were similar in both genotypes. (3) A preliminary association mapping study was performed with 118 Arabidopsis accessions, using 183 markers that probably cover approximately 3-8% of the total genome. At a P-value < 0.005, two, two and no quantitative trait loci (QTL) were found for Rubisco maximal activity, initial activity and activation state, respectively. Inspection of the genomic regions that span these markers revealed these QTL involved genes not previously implicated in the regulation of Rubisco expression or activity.
 
Article
During photosynthesis, triose-phosphates (trioseP) exported from the chloroplast to the cytosol are converted to sucrose via cytosolic fructose-1,6-bisphosphatase (cFBPase). Expression analysis in rice suggests that OscFBP1 plays a major role in the cytosolic conversion of trioseP to sucrose in leaves during the day. The isolated OscFBP1 mutants exhibited markedly decreased photosynthetic rates and severe growth retardation with reduced chlorophyll content, which results in plant death. Analysis of primary carbon metabolites revealed both significantly reduced levels of sucrose, glucose, fructose and starch in leaves of these mutants, and a high accumulation of sucrose to starch in leaves of rice plants. In the oscfbp1 mutants, products of glycolysis and the TCA cycle were significantly increased. A partitioning experiment of (14)C-labelled photoassimilates revealed altered carbon distributions including a slight increase in the insoluble fraction representing transitory starch, a significant decrease in the neutral fraction corresponding to soluble sugars and a high accumulation of phosphorylated intermediates and carboxylic acid fractions in the oscfbp1 mutants. These results indicate that the impaired synthesis of sucrose in rice cannot be sufficiently compensated for by the transitory starch-mediated pathways that have been found to facilitate plant growth in the equivalent Arabidopsis mutants.
 
Article
A full-length FBPase cDNA has been isolated from Fragaria x ananassa (strawberry) corresponding to a novel putative chloroplastic FBPase but lacking the regulatory redox domain, a characteristic of the plastidial isoenzyme (cpFBPaseI). Another outstanding feature of this novel isoform, called cpFBPaseII, is the absence of the canonical active site. Enzymatic assays with cpFBPaseII evidenced clear Mg(2+)-dependent FBPase activity and a K(m) for fructose-1,6-bisphosphate (FBP) of 1.3 mM. Immunolocalization experiments and chloroplast isolation confirmed that the new isoenzyme is located in the stroma. Nevertheless, unlike cpFBPaseI, which is redox activated, cpFBPaseII did not increase its activity in the presence of either DTT or thioredoxin f (TRX f) and is resistant to H(2)O(2) inactivation. Additionally, the novel isoform was able to complement the growth deficiency of the yeast FBP1 deletion fed with a non-fermentable carbon source. Furthermore, orthologues are restricted to land plants, suggesting that cpFBPaseII is a novel and an intriguing chloroplastic FBPase that emerged late in the evolution of photosynthetic organisms, possibly because of a pressing need of land plants.
 
Article
The role of cysteines 449 (Cys449) and 459 (Cys459) from the large subunit (LS) of ribulose 1-5-bisphosphate carboxylase/oxygenase (Rubisco) in the reduction-oxidation (redox) regulation of the enzyme was assessed by site-directed mutagenesis of these residues and chloroplast transformation of Chlamydomonas reinhardtii. In vitro studies indicated that mutations C449S, C459S or C449S/ C459S do not affect the activity and proteolytic susceptibility of the enzyme in the reduced state. However, when oxidized, the mutant enzymes differed from the wild type (WT), showing an increased resistance to inactivation and, in the case of the double mutant (DM), an altered structural conformation as reflected by the kinetics of proteolysis with subtilisin. The response of the DM strain to saline stress revealed that the absence of Cys449 and Cys459 intensifies Rubisco degradation and the covalent disulfide and non-disulfide polymerization of the enzyme in vivo. Saline stress also induced Rubisco translocation to a membrane (M) fraction that contained only covalently polymerized enzyme. Rubisco mobilization to this M fraction was enhanced also in the DM strain. Altogether, these results indicate that Cys449 and Cys459 participate in the modulation of the conformational changes promoted by oxidative modifications retarding processes related to the catabolism of the enzyme in vivo.
 
Article
Photosynthesis depends on the diffusion of gaseous CO(2) inside the leaf spaces from the stomatal entry point to the mesophyll cell walls. Although most research considers only the vertical diffusion from stomata on upper and/or leaf lower surfaces, some of the gas will diffuse in the lateral (paradermal) direction. The importance of lateral CO(2) diffusion is reviewed, and the anatomical characteristics of leaves, including the variation of air space volume between species and conditions are discussed. The contribution of the air space conductance to the limitation of photosynthesis by the overall CO(2) diffusion pathway is usually ignored. However, the need to consider three-dimensional diffusion at the small scale of a few stomata is emphasized because stomata are discrete, and separated by 20-300 microm. At the large scale of 100s of micrometres, there may be barriers to CO(2) caused by the vascular tissue, particularly if there are bundle sheath extensions. The possible extent and controls on CO(2) lateral and vertical diffusion in different species and conditions are illustrated using chlorophyll a fluorescence imaging techniques. It is clear that there is a range of effective lateral permeabilities depending on the particular vascular patterns and cell arrangements, and that species cannot be simply divided into homobaric and heterobaric anatomies. Lateral diffusion in more permeable leaves can be sufficient to affect measurements of leaf gas exchange, particularly when fluxes are low, although its contribution to leaf photosynthesis in natural conditions needs clarification.
 
Article
Leaf area expansion is affected by environmental conditions because of differences in cell number and/or cell size. Increases in the DNA content (ploidy) of a cell by endoreduplication are related to its size. The aim of this work was to determine how cell ploidy interacts with the regulation of cell size and with leaf area expansion. The approach used was to grow Arabidopsis thaliana plants performing increased or decreased rounds of endoreduplication under shading and water deficit. The shading and water deficit treatments reduced final leaf area and cell number; however, cell area was increased and decreased, respectively. These differences in cell size were unrelated to alterations of the endocycle, which was reduced by these treatments. The genetic modification of the extent of endoreduplication altered leaf growth responses to shading and water deficit. An increase in the extent of endoreduplication in a leaf rendered it more sensitive to the shade treatment but less sensitive to water deficit conditions. The link between the control of whole organ and individual cell expansion under different environmental conditions was demonstrated by the correlation between the plasticity of cell size and the changes in the duration of leaf expansion.
 
Article
While there is currently intense effort to examine the (13)C signal of CO(2) evolved in the dark, less is known on the isotope composition of day-respired CO(2). This lack of knowledge stems from technical difficulties to measure the pure respiratory isotopic signal: day respiration is mixed up with photorespiration, and there is no obvious way to separate photosynthetic fractionation (pure c(i)/c(a) effect) from respiratory effect (production of CO(2) with a different delta(13)C value from that of net-fixed CO(2)) at the ecosystem level. Here, we took advantage of new simple equations, and applied them to sunflower canopies grown under low and high [CO(2)]. We show that whole mesocosm-respired CO(2) is slightly (13)C depleted in the light at the mesocosm level (by 0.2-0.8 per thousand), while it is slightly (13)C enriched in darkness (by 1.5-3.2 per thousand). The turnover of the respiratory carbon pool after labelling appears similar in the light and in the dark, and accordingly, a hierarchical clustering analysis shows a close correlation between the (13)C abundance in day- and night-evolved CO(2). We conclude that the carbon source for respiration is similar in the dark and in the light, but the metabolic pathways associated with CO(2) production may change, thereby explaining the different (12)C/(13)C respiratory fractionations in the light and in the dark.
 
Article
In leaves, although it is accepted that CO(2) evolved by dark respiration after illumination is naturally (13) C-enriched compared to organic matter or substrate sucrose, much uncertainty remains on whether day respiration produces (13) C-depleted or (13) C-enriched CO(2). Here, we applied equations described previously for mesocosm CO(2) exchange to investigate the carbon isotope composition of CO(2) respired by autotrophic and heterotrophic tissues of Pelargonium × hortorum leaves, taking advantage of leaf variegation. Day-respired CO(2) was slightly (13) C-depleted compared to organic matter both under 21% O(2) and 2% O(2). Furthermore, most, if not all CO(2) molecules evolved in the light came from carbon atoms that had been fixed previously before the experiments, in both variegated and green leaves. We conclude that the usual definition of day respiratory fractionation, that assumes carbon fixed by current net photosynthesis is the respiratory substrate, is not valid in Pelargonium leaves under our conditions. In variegated leaves, total organic matter was slightly (13) C-depleted in white areas and so were most primary metabolites. This small isotopic difference between white and green areas probably came from the small contribution of photosynthetic CO(2) refixation and the specific nitrogen metabolism in white leaf areas.
 
Article
Combined δ13C and δ18O analyses of leaf material were used to infer changes in photosynthetic capacity (Amax) and stomatal conductance (gl) in Fagus sylvatica and Picea abies trees growing under natural and controlled conditions. Correlation between gl and δ18O in leaf cellulose (δ18Ocel) allowed us to apply a semi-quantitative model to infer gl from δ18Ocel and also interpret variation in δ13C as reflecting variation in Amax. Extraction of leaf cellulose was necessary, because δ18O from leaf organic matter (δ18OLOM) and δ18Ocel was not reliably correlated. In juvenile trees, the model predicted elevated carbon dioxide (CO2) to reduce Amax in both species, whereas ozone (O3) only affected beech by reducing CO2 uptake via lowered gl. In adult trees, Amax declined with decreasing light level as gl was unchanged. O3 did not significantly affect isotopic signatures in leaves of adult trees, reflecting the higher O3 susceptibility of juvenile trees under controlled conditions. The isotopic analysis compared favourably to the performance of leaf gas exchange, underlining that the semi-quantitative model approach provides a robust way to gather time-integrated information on photosynthetic performance of trees under multi-faced ecological scenarios, in particular when information needed for quantitative modelling is only scarcely available.
 
Article
We characterized differences in carbon isotopic content (delta(13)C) and sugar concentrations in phloem exudates from Eucalyptus globulus (Labill) plantations across a rainfall gradient in south-western Australia. Phloem sap delta(13)C and sugar concentrations varied with season and annual rainfall. Annual bole growth was negatively related to phloem sap delta(13)C during summer, suggesting a water limitation, yet was positively related in winter. We conclude that when water is abundant, variations in carboxylation rates become significant to overall growth. Concentrations of sucrose in phloem sap varied across sites by up to 600 mm, and raffinose by 300 mm. These compounds play significant roles in maintaining osmotic balance and facilitating carbon movement into the phloem, and their relative abundances contribute strongly to overall delta(13)C of phloem sap. Taken together, the delta(13)C and concentrations of specific sugars in phloem sap provide significant insights to functions supporting growth at the tree, site and landscape scale.
 
Article
(13) C discrimination between atmosphere and bulk leaf matter (Δ(13) C(lb) ) is frequently used as a proxy for transpiration efficiency (TE). Nevertheless, its relevance is challenged due to: (1) potential deviations from the theoretical discrimination model, and (2) complex time integration and upscaling from leaf to whole plant. Six hybrid genotypes of Populus deltoides×nigra genotypes were grown in climate chambers and tested for whole-plant TE (i.e. accumulated biomass/water transpired). Net CO(2) assimilation rates (A) and stomatal conductance (g(s) ) were recorded in parallel to: (1) (13) C in leaf bulk material (δ(13) C(lb) ) and in soluble sugars (δ(13) C(ss) ) and (2) (18) O in leaf water and bulk leaf material. Genotypic means of δ(13) C(lb) and δ(13) C(ss) were tightly correlated. Discrimination between atmosphere and soluble sugars was correlated with daily intrinsic TE at leaf level (daily mean A/g(s) ), and with whole-plant TE. Finally, g(s) was positively correlated to (18) O enrichment of bulk matter or water of leaves at individual level, but not at genotype level. We conclude that Δ(13) C(lb) captures efficiently the genetic variability of whole-plant TE in poplar. Nevertheless, scaling from leaf level to whole-plant TE requires to take into account water losses and respiration independent of photosynthesis, which remain poorly documented.
 
Article
Little is known about the dynamics of concentrations and carbon isotope ratios of individual carbohydrates in leaves in response to climatic and physiological factors. Improved knowledge of the isotopic ratio in sugars will enhance our understanding of the tree ring isotope ratio and will help to decipher environmental conditions in retrospect more reliably. Carbohydrate samples from larch (Larix gmelinii) needles of two sites in the continuous permafrost zone of Siberia with differing growth conditions were analyzed with the Compound-Specific Isotope Analysis (CSIA). We compared concentrations and carbon isotope values (δ(13) C) of sucrose, fructose, glucose and pinitol combined with phenological data. The results for the variability of the needle carbohydrates show high dynamics with distinct seasonal characteristics between and within the studied years with a clear link to the climatic conditions, particularly vapor pressure deficit. Compound-specific differences in δ(13) C values as a response to climate were detected. The δ(13) C of pinitol, which contributes up to 50% of total soluble carbohydrates, was almost invariant during the whole growing season. Our study provides the first in depth characterization of compound-specific needle carbohydrate isotope variability, identifies involved mechanisms and shows the potential of such results for linking tree physiological responses to different climatic conditions. This article is protected by copyright. All rights reserved.
 
Article
Isotopic labelling experiments were conducted to assess relationships among (13)C of recently assimilated carbon (deltaC(A)), foliage respiration (deltaC(F)), soluble carbohydrate (deltaC(SC)), leaf waxes (deltaC(LW)) and bulk organic matter (deltaC(OM)). Slash pine, sweetgum and maize were grown under (13)C depleted CO(2) to label biomass and then placed under ambient conditions to monitor the loss of label. In pine and sweetgum, deltaC(F) of labelled plants (approximately -44 and -35 per thousand, respectively) rapidly approached control values but remained depleted by approximately 4-6 per thousand after 3-4 months. For these tree species, no or minimal label was lost from deltaC(SC), deltaC(LW) and deltaC(OM) during the observation periods. deltaC(F) and deltaC(SC) of labelled maize plants rapidly changed and were indistinguishable from controls after 1 month, while deltaC(LW) and deltaC(OM) more slowly approached control values and remained depleted by 2-6 per thousand. Changes in deltaC(F) in slash pine and sweetgum fit a two-pool exponential model, with the fast turnover metabolic pool (approximately 3-4 d half-life) constituting only 1-2% of the total. In maize, change in deltaC(F) fits a single pool model with a half-life of 6.4 d. The (13)C of foliage respiration and biochemical pools reflect temporally integrated values of deltaC(A), with change in isotopic composition dampened by the size of metabolic carbon reserves and turnover rates.
 
Article
Water-use efficiency and stable isotope composition were studied in three tropical tree species. Seedlings of Tectona grandis, Swietenia macrophylla and Platymiscium pinnatum were grown at either high or low water supply, and with or without added fertilizer. These three species previously exhibited low, intermediate and high whole-plant water-use efficiency (TE) when grown at high water supply in unfertilized soil. Responses of TE to water and nutrient availability varied among species. The TE was calculated as experiment-long dry matter production divided by cumulative water use. Species-specific offsets were observed in relationships between TE and whole-plant (13)C discrimination (Delta(13)C(p)). These offsets could be attributed to a breakdown in the relationship between Delta(13)C(p) and the ratio of intercellular to ambient CO(2) partial pressures (c(i)/c(a)) in P. pinnatum, and to variation among species in the leaf-to-air vapour pressure difference (v). Thus, a plot of v.TE against c(i)/c(a) showed a general relationship among species. Relationships between delta(18)O of stem dry matter and stomatal conductance ranged from strongly negative for S. macrophylla to no relationship for T. grandis. Results suggest inter-specific variation among tropical tree species in relationships between stable isotope ratios (delta(13)C and delta(18)O) and the gas exchange processes thought to affect them.
 
Article
In many tree species, physiological adaptations to drought include the accumulation of osmotically active substances and/or the presence of particular compatible solutes, among them cyclitols. Recently, the cyclitol quercitol was identified in species of Eucalyptus, a diverse genus whose speciation is probably driven by adaptation to water availability. We subjected seedlings of 13 Eucalyptus species from different ecosystems ('mesic' and 'xeric') and different sub-generic taxonomic groups to 10 weeks of water deficit (WD) treatment. Pre-dawn water potentials (psi(pdwn)) and relative water content (RWC) were determined in shoots, and total osmolality, soluble low-molecular-weight carbohydrates and cyclitols were measured in leaves and roots. Responses to water deficit followed two distinct patterns: Eucalyptus species from 'mesic' environments adjusted concentrations of sucrose (through increased levels of sucrose and decreases in RWC) in response to water deficit, whereas 'xeric' species increased concentrations of quercitol (through reductions in RWC). In root tissues, only species from xeric environments contained high levels of quercitol and mannitol, increasing under WD conditions. We suggest that the former (mesic) strategy may be beneficial to respond to short-lasting drought conditions, because sucrose is easily metabolized, whereas the latter (xeric) strategy may relate to an effective acclimation to longer-lasting drought. These physiological response groups are also related to taxonomic groups within the genus.
 
Article
A deeper understanding of the drought response and genetic improvement of the cultivated crops for better tolerance requires attention because of the complexity of the drought response syndrome and the loss of genetic diversity during domestication. We initially screened about 200 wild emmer wheat genotypes and then focused on 26 of these lines, which led to the selection of two genotypes with contrasting responses to water deficiency. Six subtractive cDNA libraries were constructed, and over 13 000 expressed sequence tags (ESTs) were sequenced using leaf and root tissues of wild emmer wheat genotypes TR39477 (tolerant) and TTD-22 (sensitive), and modern wheat variety Kiziltan drought stressed for 7 d. Clustering and assembly of ESTs resulted in 2376 unique sequences (1159 without hypothetical proteins and no hits), 75% of which were represented only once. At this level of EST sampling, each tissue shared a very low percentage of transcripts (13-26%). The data obtained indicated that the genotypes shared common elements of drought stress as well as distinctly differential expression patterns that might be illustrative of their contrasting ability to tolerate water deficiencies. The new EST data generated here provide a highly diverse and rich source for gene discovery in wheat and other grasses.
 
Article
We report diurnal variations in (13)C discrimination ((13)Delta) of Picea sitchensis (Bong.) Carr. branches measured in the field using a branch chamber technique. The observations were compared to predicted (13)Delta based on concurrent measurements of branch gas exchange. Observed (13)Delta values were described well by the classical model of (13)Delta including isotope effects during photorespiration, day respiration and CO(2) transfer through a series of resistances to the sites of carboxylation. A simplified linear of model (13)Delta did not capture the observed diurnal variability. At dawn and dusk, we measured very high (13)Delta values that were not predicted by either of the said models. Exploring the sensitivity of (13)Delta to possible respiratory isotope effects, we conclude that isotopic disequilibria between the gross fluxes of photosynthesis and day respiration can explain the high observed (13)Delta values during net photosynthetic gas exchange. Based on the classical model, a revised formulation incorporating an isotopically distinct substrate for day respiration was able to account well for the high observed dawn and dusk (13)Delta values.
 
Article
Drought-induced forest decline, like the Scots pine mortality in inner-Alpine valleys, will gain in importance as the frequency and severity of drought events are expected to increase. To understand how chronic drought affects tree growth and tree-ring delta(13)C values, we studied mature Scots pine in an irrigation experiment in an inner-Alpine valley. Tree growth and isotope analyses were carried out at the annual and seasonal scale. At the seasonal scale, maximum delta(13)C values were measured after the hottest and driest period of the year, and were associated with decreasing growth rates. Inter-annual delta(13)C values in early- and latewood showed a strong correlation with annual climatic conditions and an immediate decrease as a response to irrigation. This indicates a tight coupling between wood formation and the freshly produced assimilates for trees exposed to chronic drought. This rapid appearance of the isotopic signal is a strong indication for an immediate and direct transfer of newly synthesized assimilates for biomass production. The fast appearance and the distinct isotopic signal suggest a low availability of old stored carbohydrates. If this was a sign for C-storage depletion, an increasing mortality could be expected when stressors increase the need for carbohydrate for defence, repair or regeneration.
 
Article
Movement of photoassimilates from leaves to phloem is an important step for the flux of carbon through plants. Fractionation of carbon isotopes during this process may influence their abundance in heterotrophic tissues. We subjected Eucalyptus globulus to 20, 25 and 28 °C ambient growth temperatures and measured compound-specific δ(13)C of carbohydrates obtained from leaves and bled phloem sap. We compared δ(13)C of sucrose and raffinose obtained from leaf or phloem and of total leaf soluble carbon, with modelled values predicted by leaf gas exchange. Changes in δ(13)C of sucrose and raffinose obtained from either leaves or phloem sap were more tightly coupled to changes in c(i)/c(a) than was δ(13)C of leaf soluble carbon. At 25 and 28 °C, sucrose and raffinose were enriched in (13)C compared to leaf soluble carbon and predicted values - irrespective of tissue type. Phloem sucrose was depleted and raffinose enriched in (13)C compared to leaf extracts. Intermolecular and tissue-specific δ(13)C reveal that multiple systematic factors influence (13)C composition during export to phloem. Predicting sensitivity of these factors to changes in plant physiological status will improve our ability to infer plant function at a range of temporal and spatial scales.
 
Article
Efforts to understand the cause of ¹²C versus ¹³C isotope fractionation in plants during photosynthesis and post-photosynthetic metabolism are frustrated by the lack of data on the intramolecular ¹³C-distribution in metabolites and its variation with environmental conditions. We have exploited isotopic carbon-13 nuclear magnetic resonance (¹³C NMR) spectrometry to measure the positional isotope composition (δ¹³C(i) , ‰) in ethanol samples from different origins: European wines, liquors and sugars from C₃, C₄ and crassulacean acid metabolism (CAM) plants. In C₃-ethanol samples, the methylene group was always ¹³C-enriched (∼2‰) relative to the methyl group. In wines, this pattern was correlated with both air temperature and δ(18)O of wine water, indicating that water vapour deficit may be a critical defining factor. Furthermore, in C₄-ethanol, the reverse relationship was observed (methylene-C relatively ¹³C-depleted), supporting the concept that photorespiration is the key metabolic process leading to the ¹³C distribution in C₃-ethanol. By contrast, in CAM-ethanol, the isotopic pattern was similar to but stronger than C₃-ethanol, with a relative ¹³C-enrichment in the methylene-C of up to 13‰. Plausible causes of this ¹³C-pattern are briefly discussed. As the intramolecular δ¹³C(i) -values in ethanol reflect that in source glucose, our data point out the crucial impact on the ratio of metabolic pathways sustaining glucose synthesis.
 
Article
Boreal forests emit a large amount of monoterpenes into the atmosphere. Traditionally these emissions are assumed to originate as evaporation from large storage pools. Thus, their diurnal cycle would depend mostly on temperature. However, there is indication that a significant part of the monoterpene emission would originate directly from de novo synthesis. By applying 13CO2 fumigation and analyzing the isotope fractions with proton transfer reaction mass spectrometry (PTR-MS) and classical GC-MS, we determined the fractions of monoterpene emissions originating from de novo biosynthesis in Pinus sylvestris (58%), Picea abies (33.5%), Larix decidua (9.8%) and Betula pendula (100%). Application of the observed split between de novo and pool emissions from P. sylvestris in a hybrid emission algorithm resulted in a better description of ecosystem scale monoterpene emissions from a boreal Scots pine forest stand.
 
Article
Post-photosynthetic fractionation processes during translocation, storage and remobilization of photoassimilate are closely related to intra-annual sigma13C of tree rings, and understanding how these processes affect tree-ring sigma13C is therefore indispensable for improving the quality of climate reconstruction. Our first objective was to study the relationship between translocation path and phloem grain. We pulse-labelled a branch of Larix gmelinii (Rupr.) Rupr. and later analysed the sigma13C distribution in the stem. A 13C spiral translocation path closely related to the spiral grain was observed. Our second objective was to study the use of remobilized storage material for earlywood formation in spring, which is a suspected cause of the autocorrelation (correlation of ring parameters to the climate in the previous year) observed in (isotope) dendroclimatology. We pulse-labelled whole trees to study how spring, summer and autumn photoassimilate is later used for both earlywood and latewood formation. Analysis of intra-annual sigma13C of the tree rings formed after the labelling revealed that earlywood contained photoassimilate from the previous summer and autumn as well as from the current spring. Latewood was mainly composed of photoassimilate from the current year's summer/autumn, although it also relied on stored material in some cases. These results emphasize the need for separating earlywood and latewood for climate reconstruction work with narrow boreal tree rings.
 
Article
Nitrogen-13 (t(1/2) 9.97 m), a radioactive isotope of nitrogen, offers unique opportunities to explore plant nitrogen utilization over short time periods. Here we describe a method for administering (13)N as gaseous (13)NH(3) to intact leaves of Nicotiana tabacum L. (cv Samsun), and measuring the labelled amino acids using radio high-performance liquid chromatography (HPLC) on tissue extract. We used this method to study the effects of defence induction on plant nitrogen utilization by applying treatments of methyl jasmonate (MeJA), a potent defence elicitor. MeJA caused a significant increase relative to controls in key [(13)N]amino acids, including serine, glycine and alanine by 4 h post-treatment, yet had no effect on (13)NH(3) incorporation, a process that is primarily under the control of the glutamine synthatase/glutamate synthase pathway (GS/GOGAT) in cellular photorespiration. We suggest that the reconfiguration of nitrogen metabolism may reflect induction of non-photorespiratory sources of nitrogen to better serve the plant's defences.
 
Article
The function in plants of the non-protein amino acid, gamma-aminobutyric acid (GABA) is poorly understood. In this study, we show that GABA down-regulates the expression of a large subset of 14-3-3 gene family members in Arabidopsis thaliana seedlings in a calcium, ethylene and abscisic acid (ABA)-dependent manner. Gene expression is not affected when seedlings are supplied with glutamate (GLU), a precursor of GABA. The repression of 14-3-3 gene expression by GABA is dependent on functional ethylene and ABA signalling pathways, because the response is lost in the etr1-1, abi1-1 and abi2-1 mutants. Calcium measurements show that in contrast to GLU, GABA does not elicit a cytoplasmic calcium elevation, suggesting that the GABA response is unlikely to be mediated by GLU receptors (GLRs), as has been suggested previously. We suggest that in addition to its role as a stress-related metabolite, GABA may regulate gene expression in A. thaliana, including members of the 14-3-3 gene family.
 
Article
The regulation of the system responsible for the production of reactive oxygen species (ROS) during plant-micro-organism interaction is still largely unknown. The protein NtrbohD has been recently demonstrated as the plasma membrane oxidase responsible for ROS production in elicited tobacco cells. Here, its C-terminus part was used as a bait in a two-hybrid screen in order to identify putative regulators of this system. This led to the isolation of a cDNA coding for a member of the 14-3-3 protein family. The corresponding transcript was induced after infiltration of tobacco leaves with the fungal elicitor cryptogein. Tobacco cells transformed with an antisense construct of this 14-3-3 no longer accumulated ROS, which constitutes a functional validation of the two-hybrid screen. This work provides new insights to the understanding of the regulation of ROS production in a signalling context and gives a new light to the possible role of 14-3-3 proteins in plant-micro-organisms interactions.
 
Article
14-3-3 proteins are a large family of proteins but exact roles of their members in plant response to abiotic stresses are not clear, especially under nutrient deficiency. We investigated the expressions of all the tomato 14-3-3 gene family members (TFT1-TFT12) under low phosphorus stress (LP) and found that TFT6 belongs to the later responsive gene while TFT7 belongs to the early responsive gene. When the two genes were separately introduced into Arabidopsis and overexpressed, their plant growth under LP was much enhanced compared with wild-type plant. TFT6 overexpressing plants showed reduced starch synthase activity, reduced starch content but enhanced sucrose loading into phloem in the shoot under LP. TFT7 overexpressing plants had much enhanced H⁺ flux along their root tip and activity of plasma membrane H⁺-ATPase in the roots under LP. Our results suggest that TFT6 and TFT7 play different roles in plant adaption to LP. TFT6 acts mainly in leaves and is involved in the systemic response to LP by regulating leaf carbon allocation and increasing phloem sucrose transport to promote root growth, while TFT7 directly functions in root by activating root plasma membrane H⁺-ATPase to release more protons under LP.
 
Article
The balance of gibberellins [gibberellic acid (GA)] and abscisic acid (ABA) is a determining factor during transition of embryogenesis and seed germination. Recently, we showed that 14-3-3 proteins are important in ABA signalling in barley aleurone cells. Using 14-3-3 RNAi constructs in the barley aleurone transient expression system, we demonstrate here that silencing of each 14-3-3 isoform suppresses GA induction of the alpha-amylase gene. 14-3-3 Proteins interact with ABA-responsive element (ABRE) binding factors HvABF1, 2 and 3, and here we show that these transcription factors also interact with the ABA-responsive kinase PKABA1, a kinase that mediates cross-talk between the GA and ABA pathway. ABF1 and ABF2 have a function in both signalling pathways as: (1) ectopic expression of wild-type ABF1 and mutant ABF2, lacking the 14-3-3 interaction domain, transactivates the ABA inducible HVA1 gene; and (2) GA induction of the alpha-amylase gene is repressed by ectopic expression of wild-type ABF1 and 2. Mutant ABF1 and 2 were still effective repressors of GA signalling. In summary, our data provide evidence that 14-3-3 proteins and members of the ABF transcription factor family have a regulatory function in the GA pathway and suggest that PKABA1 and ABF transcription factors are cross-talk intermediates in ABA and GA signalling.
 
13 N retention in roots and accumulation in shoots of Arabidopsis thaliana WT and
13 NO 3 -influx into roots and relative expression of AtNRT2.5 in WT, Atnrt2.5-1 mutant
Tissue nitrate concentration of Arabidopsis thaliana WT and Atnrt2.5-1 plants, grown
Article
In plants that have been deprived of nitrate for a significant length of time, a constitutive high-affinity nitrate transport system (cHATS) is responsible for initial nitrate uptake. This absorbed nitrate leads to the induction of the major nitrate transporters and enzymes involved in nitrate assimilation. By use of (13) NO3 (-) influx measurements and Blue-Native PAGE we examined the role of AtNRT2.5 in cHATS in WT and various T-DNA mutants of Arabidopsis thaliana. We demonstrate that AtNRT2.5 is predominantly expressed in roots of nitrate-deprived WT plants as a 150 kDa molecular complex with AtNAR2.1. This complex represents the major contributor to cHATS influx, which is reduced by 63 % compared to WT in roots of Atnrt2.5 mutants. The remaining cHATS nitrate influx in these mutants is due to a residual contribution by the inducible high-affinity transporter, encoded by AtNRT2.1/AtNAR2.1. Estimates of the kinetic properties of the NRT2.5 transporter reveal that its low Km for nitrate makes this transporter ideally suited to detect and respond to trace quantities of nitrate in the root environment. This article is protected by copyright. All rights reserved.
 
Article
The Arabidopsis thaliana AtNRT2.1 gene, which encodes a NO(3)(-) transporter involved in high-affinity uptake by the roots, is a molecular target of several mechanisms responsible for the regulation of root NO(3)(-) acquisition by the N status of the plant. All levels of AtNRT2.1 expression (promoter activity, transcript level, protein accumulation, transport activity) are coordinately up-regulated in the presence of NO(3)(-), and repressed by downstream N metabolites. Transgenic plants expressing the GUS reporter gene under the control of upstream sequences of AtNRT2.1 have been studied to identify elements targeted by these two regulatory mechanisms. A 150 bp sequence located upstream of the TATA box that is required for both stimulation by NO(3)(-) and repression by N metabolites of the promoter has been identified. This sequence is able to confer these two regulations to a minimal promoter. Split-root experiments indicate that the stimulation of the chimaeric promoter by NO(3)(-) occurs only at the local level, whereas its repression by N metabolites is mediated by a systemic signal spread to the whole plant. The activity of the cis-acting 150 bp element is also regulated by sucrose supply to the roots, suggesting a possible interaction between N and C signalling within this short region. Accordingly, multiple motifs potentially involved in regulations by N and/or C status are identified within this sequence by bioinformatic approaches. This is the first report of such a cis-acting element in higher plants.
 
Article
Limited information is available on the genetic variation and control for plant growth response to elevated CO(2) (e[CO(2)]). Such information is necessary to understand plant adaptation and evolution in future rising CO(2). Here, quantitative trait loci (QTL) for leaf growth, development, quality and leaf senescence were determined in a tree pedigree - an F(2) hybrid of Populus trichocarpa T. & G and Populus deltoides Marsh, following season-long exposure to either current day ambient carbon dioxide (a[CO(2)]) or e[CO(2)] at 600 microL L(-1). Leaf growth and development differed between the grandparents such that P. trichocarpa showed greater response to e[CO(2)]. In the F(2) generation, leaf development and quality traits including leaf area, leaf shape, epidermal cell area, and stomatal number, specific leaf area (SLA), and the phenology trait, canopy senescence index, were sensitive to e[CO(2)]. Sixty-nine QTL were mapped for the 19 traits of plants in a[CO(2)] while 60 QTL were mapped for plants in e[CO(2)]. The results suggest that although many QTL mapped to common positions in a[CO(2)] and e[CO(2)], confirming their importance in determining growth, there was also differential genetic control for a number of traits including leaf senescence. Candidate genes were shown to collocate to regions where response QTL mapped. This study is the first to identify candidate genes that may be important in determining plant adaptation to future high-CO(2) world.
 
Article
Previous mangrove tree ring studies attempted, unsuccessfully, to relate the δ(18) O of trunk cellulose (δ(18) O(CELL) ) to the δ(18) O of source water (δ(18) O(SW) ). Here, we tested whether biochemical fractionation associated with one of the oxygen in the cellulose glucose moiety or variation in leaf water oxygen isotope fractionation (Δ(LW) ) can interfere with the δ(18) O(SW) signal as it is recorded in the δ(18) O(CELL) of mangrove (saltwater) and hammock (freshwater) plants. We selected two transects experiencing a salinity gradient, located in the Florida Keys, USA. The δ(18) O(CELL) throughout both transects did not show the pattern expected based on that of the δ(18) O(SW) . We found that in one of the transects, biochemical fractionation interfered with the δ(18) O(SW) signal, while in the other transect Δ(LW) differed between mangrove and hammock plants. Observed differences in Δ(LW) between mangroves and hammocks were caused by a longer effective leaf mixing length (L) of the water pathway in mangrove leaves compared to those of hammock leaves. Changes in L could have caused the δ(18) O(CELL) to record not only variations in the δ(18) O(SW) but also in Δ(LW) making it impossible to isolate the δ(18) O(SW) signal.
 
Article
Using both oxygen isotope ratios of leaf water (δ(18) OL ) and cellulose (δ(18) OC ) of T. usneoides in situ, this paper examined how short- and long-term responses to environmental variation and model parameterization affected the reconstruction of the atmospheric water vapor (δ(18) Oa ). During sample-intensive field campaigns, predictions of δ(18) OL matched observations well using a non-steady-state model, but the model required data-rich parameterization. Predictions from the more-easily-parameterized maximum-enrichment model (δ(18) OL-M ) model matched observed δ(18) OL and observed δ(18) Oa when leaf water turnover was less than 3.5 days. Using the δ(18) OL-M model and weekly samples of δ(18) OL across two growing seasons in Florida, USA, reconstructed δ(18) Oa was -12.6 ± 0.3 ‰. This is compared to a δ(18) Oa of -12.4 ± 0.2 ‰ resolved from the growing-season-weighted δ(18) OC . Both of these values were similar to δ(18) Oa in equilibrium with precipitation, -12.9 ‰. δ(18) Oa was also reconstructed through a large-scale transect with δ(18) OL and the growing-season-integrated δ(18) OC across the southeastern USA. There was considerable large-scale variation, but there was regional, weather-induced coherence in δ(18) Oa when using δ(18) OL . The reconstruction of δ(18) Oa with δ(18) OC generally supported the assumption of δ(18) Oa being in equilibrium with precipitation δ(18) O (δ(18) Oppt ), but the pool of δ(18) Oppt with which δ(18) Oa was in equilibrium- growing season versus annual δ(18) Oppt - changed with latitude.
 
Article
Stable oxygen isotope ratios (delta18O) have become a valuable tool in the plant and ecosystem sciences. The interpretation of delta18O values in plant material is, however, still complicated owing to the complex interactions among factors that influence leaf water enrichment. This study investigated the interplay among environmental parameters, leaf physiological properties and leaf water relations as drivers of the isotopic enrichment of leaf water across 17 Eucalyptus species growing in a common garden. We observed large differences in maximum daily leaf water delta18O across the 17 species. By fitting different leaf water models to these empirical data, we determined that differences in leaf water delta18O across species are largely explained by variation in the Péclet effect across species. Our analyses also revealed that species-specific differences in transpiration do not explain the observed differences in delta18O while the unconstrained fitting parameter 'effective path length' (L) was highly correlated with delta18O. None of the leaf morphological or leaf water related parameters we quantified in this study correlated with the L values we determined even though L was typically interpreted as a leaf morphological/anatomical property. A sensitivity analysis supported the importance of L for explaining the variability in leaf water delta18O across different species. Our investigation highlighted the importance of future studies to quantify the leaf properties that influence L. Obtaining such information will significantly improve our understanding of what ultimately determines the delta18O values of leaf water across different plant species.
 
Article
Leaf water becomes enriched in the heavy isotope (18) O through the process of transpiration. The leaf water (18) O signal is recorded in plant organic material (Barbour 2007), in atmospheric CO(2) via the activity of carbonic anhydrase (Farquhar et al. 1993), and in atmospheric O(2) through photosynthetic O(2) evolution (Guy, Fogel & Berry 1993). These signals can be harnessed for a range of applications in global change research and plant science, including reconstruction of climate, estimation of primary productivity, and analysis of environmental and genetic effects on stomatal conductance. Mechanistic models of leaf water (18) O enrichment are required for these applications.
 
Article
Leaf gas exchange and leaf water (18)O enrichment (Delta(18)O(L)) were measured in three Clusia species under field conditions during dry and wet seasons and in Miconia argentea during the dry season in the Republic of Panama. During the dry season, all three Clusia species used crassulacean acid metabolism (CAM); during the wet season Clusia pratensis operated in the C(3) mode, while Clusia uvitana and Clusia rosea used CAM. Large departures from isotopic steady state were observed in daytime Delta(18)O(L) of the Clusia species, especially during the dry season. In contrast, daytime Delta(18)O(L) was near isotopic steady state in the C(3) tree M. argentea. Across the full data set, non-steady-state predictions explained 49% of variation in observed Delta(18)O(L), whereas steady-state predictions explained only 14%. During the wet season, when Delta(18)O(L) could be compared with Clusia individuals operating in both C(3) and CAM modes, steady-state and non-steady-state models gave contrasting predictions with respect to interspecific variation in daytime Delta(18)O(L). The observed Delta(18)O(L) pattern matched that predicted for the non-steady state. The results provided a clear example of how non-steady-state control of leaf water (18)O dynamics can shift the slope of the relationship between transpiration rate and daytime Delta(18)O(L) from negative to positive.
 
Diel variation of photosynthetically active radiation (PAR; panel a), vapour pressure deficit (VPD; panel b), stomatal conductance (gs, panel c) and δ18O in different ecosystem compartments in June 2005 (panel d), including atmospheric water vapour (circles), xylem sap (diamonds), needle water (triangles) and water-soluble organic matter (OM, squares) in current year needles (N, closed symbols) and previous year needles (N-1, open symbols), and trunk phloem sap sampled at 10 m height (crosses). Bars represent ±  SE, n = 3.
Relationship between the transpiration rate of current year (N, circles) and previous year (N-1, triangles) needles and the discrepancy between the observed enrichment of mean lamina leaf water (Δ18OL) and the steady state prediction of water enrichment at evaporative sites (Δ18Oes), during daytime (open symbols, 1200 and 1800 h) and nighttime (closed symbols, 2400 and 0600 h) of the precipitation-free period. The predicted values are based on scaled effective path length of 0.15 and 0.05 m for N and N-1 needles, respectively.
Article
Understanding ecosystem water fluxes has gained increasing attention, as climate scenarios predict a drier environment for many parts of the world. Evaporative enrichment of (18)O (Delta(18)O) of leaf water and subsequent enrichment of plant organic matter can be used to characterize environmental and physiological factors that control evaporation, based on a recently established mechanistic model. In a Pinus sylvestris forest, we measured the dynamics of oxygen isotopic composition (delta(18)O) every 6 h for 4 d in atmospheric water vapour, xylem sap, leaf water and water-soluble organic matter in current (N) and previous year (N-1) needles, phloem sap, together with leaf gas exchange for pooled N and N-1 needles, and relevant micrometeorological variables. Leaf water delta(18)O showed strong diel periodicity, while delta(18)O in atmospheric water vapour and in xylem sap showed little variation. The Delta(18)O was consistently lower for N than for N-1 needles, possibly related to phenological stage. Modelled leaf water Delta(18)O showed good agreement with measured values when applying a non-steady state evaporative enrichment model including a Péclet effect. We determined the time lags between delta(18)O signals from leaf water to water-soluble foliar organic matter and to phloem sap at different locations down the trunk, which clearly demonstrated the relevance of considering these time-lag effects for carbon transport, source-sink and carbon flux partitioning studies.
 
Article
We report diurnal variations in 18O discrimination (18 delta) during photosynthesis (18 delta A) and respiration (18 delta R) of Picea sitchensis branches measured in branch chambers in the field. These observations were compared with predicted 18 delta (18 delta pred) based on concurrent measurements of branch gas exchange to evaluate steady state and non-steady state (NSS) models of foliage water 18O enrichment for predicting the impact of this ecosystem on the Delta 18O of atmospheric CO2. The non-steady state approach substantially improved the agreement between 18 delta pred and observed 18 delta (18 delta obs) compared with the assumption of isotopic steady state (ISS) for the Delta 18O signature of foliage water. In addition, we found direct observational evidence for NSS effects: extremely high apparent 18 delta values at dusk, dawn and during nocturnal respiration. Our experiments also show the importance of bidirectional foliage gas exchange at night (isotopic equilibration in addition to the net flux). Taken together, neglecting these effects leads to an underestimation of daily net canopy isofluxes from this forest by up to 30%. We expect NSS effects to be most pronounced in species with high specific leaf water content such as conifers and when stomata are open at night or when there is high relative humidity, and we suggest modifications to ecosystem and global models of delta 18O of CO2.
 
Article
Metabolic profiling using phosphorus nuclear magnetic resonance ((31)P-NMR) revealed that the leaves of different herbs and trees accumulate 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcDP), an intermediate of the methylerythritol 4-phosphate (MEP) pathway, during bright and hot days. In spinach (Spinacia oleracea L.) leaves, its accumulation closely depended on irradiance and temperature. MEcDP was the only (31)P-NMR-detected MEP pathway intermediate. It remained in chloroplasts and was a sink for phosphate. The accumulation of MEcDP suggested that its conversion rate into 4-hydroxy-3-methylbut-2-enyl diphosphate, catalysed by (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE), was limiting under oxidative stress. Indeed, O(2) and ROS produced by photosynthesis damage this O(2)-hypersensitive [4Fe-4S]-protein. Nevertheless, as isoprenoid synthesis was not inhibited, damages were supposed to be continuously repaired. On the contrary, in the presence of cadmium that reinforced MEcDP accumulation, the MEP pathway was blocked. In vitro studies showed that Cd(2+) does not react directly with fully assembled GcpE, but interferes with its reconstitution from recombinant GcpE apoprotein and prosthetic group. Our results suggest that MEcDP accumulation in leaves may originate from both GcpE sensitivity to oxidative environment and limitations of its repair. We propose a model wherein GcpE turnover represents a bottleneck of the MEP pathway in plant leaves simultaneously exposed to high irradiance and hot temperature.
 
Article
In this work the differential response of adult and young leaves from pea (Pisum sativum L.) plants to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) (23 mm) applied by foliar spraying was investigated. The concentration of 2,4-D (23 mm) and the time of treatment (72 h) were previously optimized in order to visualize its toxic effects on pea plants. Under these conditions, the herbicide induced severe disturbances in mesophyll cells structure and proliferation of vascular tissue in young leaves and increased acyl-CoA oxidase (ACX), xanthine oxidase (XOD) and lipoxygenase (LOX) activities in young leaves, and only ACX and LOX in adult leaves. This situation produced reactive oxygen species (ROS) over-accumulation favoured by the absence of significant changes in the enzymatic antioxidants, giving rise to oxidative damages to proteins and membrane lipids. An increase of ethylene took place in both young and adult leaves and the induction of genes encoding the stress proteins, PRP4A and HSP 71,2, was observed mainly in young leaves. These results suggest that ROS overproduction is a key factor in the effect of high concentrations of 2,4-D, and ROS can trigger a differential response in young and adult leaves, either epinasty development in young leaves or senescence processes in adult tissues.
 
Article
The methylerythritol 4-phosphate (MEP) and the mevalonate pathways are the unique synthesis routes for the precursors of all isoprenoids. An original mean to measure the carbon flux through the MEP pathway in plants is proposed by using cadmium as a total short-term inhibitor of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) reductase (GcpE) and measuring the accumulation rate of its substrate MEcDP by (31) P-NMR spectroscopy. The MEP pathway metabolic flux was determined in spinach (Spinacia oleracea), pea (Pisum sativum), Oregon grape (Mahonia aquifolium) and boxwood (Buxus sempervirens) leaves. In spinach, flux values were compared with the synthesis rate of major isoprenoids. The flux increases with light intensity (fourfold in the 200-1200 µmol m(-2) s(-1) PPFR range) and temperature (sevenfold in the 25-37 °C range). The relationship with the light and the temperature dependency of isoprenoid production downstream of the MEP pathway is discussed.
 
Plant response to salinity at different time scales. The effects on a salt-tolerant plant are basically identical to those due to soil water
Spatial distributions of Na + , Cl-and K + concentrations in the growing leaf 4 on the mainstem of wheat grown in soil with 0 and 120 m M NaCl. Plants were harvested at 3 h into a 16 h photoperiod. Arrows indicate the end of the growth zone and leaf sheath. Error bars ( n = 2) present standard errors and fit within the plot symbol if not otherwise shown. Adapted from Hu & Schmidhalter (1998).
Plasmolysis in root cells. Diagram shows an epidermal 
Onion scale leaf epidermal cells plasmolysed in 0·75 M mannitol. Stretched strands of plasma membrane connect the protoplast to the cell wall; small sections of the plasma membrane and peripheral cytoplasm remain tethered to the wall. Confocal projection, ∼× 750. Photograph courtesy of Brian Gunning.
Growth of wheat roots at 24 h after sudden addition of mannitol (rate averaged over 18 and 30 h after transfer). The osmotic pressure of 200 mM mannitol is 0·5 MPa, so plasmolysis would have occurred at the higher concentrations (see legend of Figure 4). Data from Pritchard et al. (1991).
Article
Plant responses to salt and water stress have much in common. Salinity reduces the ability of plants to take up water, and this quickly causes reductions in growth rate, along with a suite of metabolic changes identical to those caused by water stress. The initial reduction in shoot growth is probably due to hormonal signals generated by the roots. There may be salt-specific effects that later have an impact on growth; if excessive amounts of salt enter the plant, salt will eventually rise to toxic levels in the older transpiring leaves, causing premature senescence, and reduce the photosynthetic leaf area of the plant to a level that cannot sustain growth. These effects take time to develop. Salt-tolerant plants differ from salt-sensitive ones in having a low rate of Na+ and Cl-- transport to leaves, and the ability to compartmentalize these ions in vacuoles to prevent their build-up in cytoplasm or cell walls and thus avoid salt toxicity. In order to understand the processes that give rise to tolerance of salt, as distinct from tolerance of osmotic stress, and to identify genes that control the transport of salt across membranes, it is important to avoid treatments that induce cell plasmolysis, and to design experiments that distinguish between tolerance of salt and tolerance of water stress.
 
Article
It has long been believed that maintenance of low Na+ : K+ ratios in the cytosol of plant cells is critical to the plant's ability to tolerate salinity stress. Direct measurements of such ratios, however, have been few. Here we apply the non-invasive technique of compartmental analysis, using the short-lived radiotracers 42K+ and 22Na+, in intact seedlings of barley (Hordeum vulgare L.), to evaluate unidirectional plasma membrane fluxes and cytosolic concentrations of K+ and Na+ in root tissues, under eight nutritional conditions varying in levels of salinity and K+ supply. We show that Na+ : K+ ratios in the cytosol of root cells adjust significantly across the conditions tested, and that these ratios are poor predictors of the plant's growth response to salinity. Our study further demonstrates that Na+ is subject to rapid and futile cycling at the plasma membrane at all levels of Na+ supply, independently of external K+, while K+ influx is reduced by Na+, from a similar baseline, and to a similar extent, at both low and high K+ supply. We compare our results to those of other groups, and conclude that the maintenance of the cytosolic Na+ : K+ ratio is not central to plant survival under NaCl stress. We offer alternative explanations for sodium sensitivity in relation to the primary acquisition mechanisms of Na+ and K+.
 
Top-cited authors
Rana Munns
  • University of Western Australia
John Sperry
  • University of Utah
Christine H Foyer
  • University of Birmingham
Graham Noctor
  • Université Paris-Sud 11
Gad Miller
  • Bar Ilan University