Varying Atmospheric CO2 Mediates the Cold-Induced CBF-Dependent Signaling Pathway and Freezing Tolerance in Arabidopsis.

Changes in the stomatal aperture in response to CO2 levels allow plants to manage water usage, optimize CO2 uptake and adjust to environmental stimuli. The current study reports that sub-ambient CO2 up-regulated the low temperature induction of the C-repeat Binding Factor (CBF)-dependent cold signaling pathway in Arabidopsis (Arabidopsis thaliana) and the opposite occurred in response to supra-ambient CO2. Accordingly, cold induction of various downstream cold-responsive genes was modified by CO2 treatments and expression changes were either partially or fully CBF-dependent. Changes in electrolyte leakage during freezing tests were correlated with CO2's effects on CBF expression. Cold treatments were also performed on Arabidopsis mutants with altered stomatal responses to CO2, i.e., high leaf temperature 1-2 (ht1-2, CO2 hypersensitive) and ß-carbonic anhydrase 1 and 4 (ca1ca4, CO2 insensitive). The cold-induced expression of CBF and downstream CBF target genes plus freezing tolerance of ht1-2 was consistently less than that for Col-0, suggesting that HT1 is a positive modulator of cold signaling. The ca1ca4 mutant had diminished CBF expression during cold treatment but the downstream expression of cold-responsive genes was either similar to or greater than that of Col-0. This finding suggested that ßCA1/4 modulates the expression of certain cold-responsive genes in a CBF-independent manner. Stomatal conductance measurements demonstrated that low temperatures overrode low CO2-induced stomatal opening and this process was delayed in the cold tolerant mutant, ca1ca4, compared to the cold sensitive mutant, ht1-2. The similar stomatal responses were evident from freezing tolerant line, Ox-CBF, overexpression of CBF3, compared to wild-type ecotype Ws-2. Together, these results indicate that CO2 signaling in stomata and CBF-mediated cold signaling work coordinately in Arabidopsis to manage abiotic stress.

Physiological and Metabolic Responses of Rice to Reduced Soil Moisture: Relationship of Water Stress Tolerance and Grain Production.

Access to adequate irrigation resources is critical for sustained agricultural production, and rice, a staple cereal grain for half of the world population, is one of the biggest users of irrigation. To reduce water use, several water saving irrigation systems have been developed for rice production, but a reliable system to evaluate cultivars for water stress tolerance is still lacking. Here, seven rice cultivars that have diverse yield potential under water stress were evaluated in a field study using four continuous irrigation regimes varying from saturation to wilting point. To understand the relationship between water stress and yield potential, the physiological and leaf metabolic responses were investigated at the critical transition between vegetative and reproductive growth stages. Twenty-nine metabolite markers including carbohydrates, amino acids and organic acids were found to significantly differ among the seven cultivars in response to increasing water stress levels with amino acids increasing but organic acids and carbohydrates showing mixed responses. Overall, our data suggest that, in response to increasing water stress, rice cultivars that do not show a significant yield loss accumulate carbohydrates (fructose, glucose, and myo-inositol), and this is associated with a moderate reduction in stomatal conductance (gs), particularly under milder stress conditions. In contrast, cultivars that had significant yield loss due to water stress had the greatest reduction in gs, relatively lower accumulation of carbohydrates, and relatively high increases in relative chlorophyll content (SPAD) and leaf temperature (Tm). These data demonstrate the existence of genetic variation in yield under different water stress levels which results from a suite of physiological and biochemical responses to water stress. Our study, therefore, suggests that in rice there are different physiological and metabolic strategies that result in tolerance to water stress that should be considered in developing new cultivars for deficit irrigation production systems that use less water.

Impact of carbon dioxide enrichment on the responses of maize leaf transcripts and metabolites to water stress.

Maize (Zea mays) was grown in indoor chambers with ambient (38 Pa) and elevated (70 Pa) CO(2) . Drought treatments were imposed 17 days after sowing by withholding nutrient solution. Decreases of soil water content, leaf water potential, net CO(2) assimilation and stomatal conductance as a result of drought were delayed approximately 2 days by CO(2) enrichment. Concentrations of 28 of 33 leaf metabolites were altered by drought. Soluble carbohydrates, aconitate, shikimate, serine, glycine, proline and eight other amino acids increased, and leaf starch, malate, fumarate, 2-oxoglutarate and seven amino acids decreased with drought. Drought-dependent decreases of nitrate, alanine and aspartate were impacted by limiting nitrogen. Transcript levels of 14 stress-related maize genes responded to drought but this was delayed or modified by CO(2) enrichment. Overall, CO(2) enrichment eliminated many early responses of maize metabolites and transcripts to water stress but was less effective when drought was severe. Four metabolite groupings were identified by clustering analysis. These groupings included compounds that decreased with water stress, compounds involved in osmotic adjustment and aromatic compounds that alleviate oxidative stress. Metabolite changes also supported the suggestion that water stress inhibited C(4) photosynthesis and induced photorespiration.

Protein and metabolite composition of xylem sap from field-grown soybeans (Glycine max).

The xylem, in addition to transporting water, nutrients and metabolites, is also involved in long-distance signaling in response to pathogens, symbionts and environmental stresses. Xylem sap has been shown to contain a number of proteins including metabolic enzymes, stress-related proteins, signal transduction proteins and putative transcription factors. Previous studies on xylem sap have mostly utilized plants grown in controlled environmental chambers. However, plants in the field are subjected to high light and to environmental stress that is not normally found in growth chambers. In this study, we have examined the protein and metabolite composition of xylem sap from field-grown cultivated soybean plants. One-dimensional gel electrophoresis of xylem sap from determinate, indeterminate, nodulating and non-nodulating soybean cultivars revealed similar protein profiles consisting of about 8-10 prominent polypeptides. Two-dimensional gel electrophoresis of soybean xylem sap resulted in the visualization of about 60 distinct protein spots. A total of 38 protein spots were identified using MALDI-TOF MS and LC-MS/MS. The most abundant proteins present in the xylem sap were identified as 31 and 28 kDa vegetative storage proteins. In addition, several proteins that are conserved among different plant species were also identified. Diurnal changes in the metabolite profile of xylem sap collected during a 24-h cycle revealed that asparagine and aspartate were the two predominant amino acids irrespective of the time collected. Pinitol (D-3-O-methyl-chiro-inositol) was the most abundant carbohydrate present. The possible roles of xylem sap proteins and metabolites as nutrient reserves for sink tissue and as an indicator of biotic stress are also discussed.

The beneficial endophyte Trichoderma hamatum isolate DIS 219b promotes growth and delays the onset of the drought response in Theobroma cacao.

Theobroma cacao (cacao) is cultivated in tropical climates and is exposed to drought stress. The impact of the endophytic fungus Trichoderma hamatum isolate DIS 219b on cacao's response to drought was studied. Colonization by DIS 219b delayed drought-induced changes in stomatal conductance, net photosynthesis, and green fluorescence emissions. The altered expression of 19 expressed sequence tags (ESTs) (seven in leaves and 17 in roots with some overlap) by drought was detected using quantitative real-time reverse transcription PCR. Roots tended to respond earlier to drought than leaves, with the drought-induced changes in expression of seven ESTs being observed after 7 d of withholding water. Changes in gene expression in leaves were not observed until after 10 d of withholding water. DIS 219b colonization delayed the drought-altered expression of all seven ESTs responsive to drought in leaves by > or = 3 d, but had less influence on the expression pattern of the drought-responsive ESTs in roots. DIS 219b colonization had minimal direct influence on the expression of drought-responsive ESTs in 32-d-old seedlings. By contrast, DIS 219b colonization of 9-d-old seedlings altered expression of drought-responsive ESTs, sometimes in patterns opposite of that observed in response to drought. Drought induced an increase in the concentration of many amino acids in cacao leaves, while DIS 219b colonization caused a decrease in aspartic acid and glutamic acid concentrations and an increase in alanine and gamma-aminobutyric acid concentrations. With or without exposure to drought conditions, colonization by DIS 219b promoted seedling growth, the most consistent effects being an increase in root fresh weight, root dry weight, and root water content. Colonized seedlings were slower to wilt in response to drought as measured by a decrease in the leaf angle drop. The primary direct effect of DIS 219b colonization was promotion of root growth, regardless of water status, and an increase in water content which it is proposed caused a delay in many aspects of the drought response of cacao.

Growth, photosynthesis, nitrogen partitioning and responses to CO2 enrichment in a barley mutant lacking NADH-dependent nitrate reductase activity.

Plant growth, photosynthesis and leaf constituents were examined in the wild-type (WT) and mutant nar1 of barley (Hordeum vulgare L. cv. Steptoe) that contains a defective structural gene encoding NADH-dependent nitrate reductase (NADH-NAR). In controlled environment experiments, total biomass, rates of photosynthesis, stomatal conductance, intercellular CO(2) concentrations and foliar non-structural carbohydrate levels were unchanged or differed slightly in the mutant compared with the WT. Both genotypes displayed accelerated plant growth rates when the CO(2) partial pressure was increased from 36 to 98 Pa. Total NADH-NAR activity was 90% lower in the mutant than in the WT, and this was further decreased by CO(2) enrichment in both genotypes. Inorganic nitrate was greater in the mutant than in the WT, whereas in situ nitrate assimilation by excised leaves was two-fold greater for the WT than for the mutant. Foliar ammonia was 50% lower in the mutant than in the WT under ambient CO(2). Ammonia levels in the WT were decreased by about one-half by CO(2) enrichment, whereas ammonia was unaffected by elevated CO(2) in mutant leaves. Total soluble amino acid concentrations in WT and mutant plants grown in the ambient CO(2) treatment were 30.1 and 28.4 micromol g(-1) FW, respectively, when measured at the onset of the light period. Seven of the twelve individual amino acids reported here increased during the first 12 h of light in the ambient CO(2) treatment, leading to a doubling of total soluble amino acids in the WT. The most striking effect of the mutation was to eliminate increases of glutamine, aspartate and alanine during the latter half of the photoperiod in the ambient CO(2) treatment. Growth in elevated CO(2) decreased levels of total soluble amino acids on a diurnal basis in the WT but not in mutant barley leaves. The above results indicated that a defect in NADH-NAR primarily affected nitrogenous leaf constituents in barley. Also, we did not observe synergistic effects of CO(2) enrichment and decreased foliar NADH-NAR activity on most N-containing compounds.

The drought response of Theobroma cacao (cacao) and the regulation of genes involved in polyamine biosynthesis by drought and other stresses.

Drought can negatively impact pod production despite the fact that cacao production usually occurs in tropical areas having high rainfall. Polyamines (PAs) have been associated with the response of plants to drought in addition to their roles in responses to many other stresses. The constitutive and drought inducible expression patterns of genes encoding enzymes involved in PA biosynthesis were determined: an ornithine decarboxylase (TcODC), an arginine decarboxylase (TcADC), an S-adenosylmethionine decarboxylase (TcSAMDC), a spermidine synthase (TcSPDS), and a spermine synthase (TcSPMS). Expression analysis using quantitative real-time reverse transcription-PCR (QPCR) results showed that the PA biosynthesis genes were expressed in all plant tissues examined. Constitutive expression of PA biosynthesis genes was generally highest in mature leaves and open flowers. Expression of TcODC, TcADC, and TcSAMDC was induced with the onset of drought and correlated with changes in stomatal conductance, photosynthesis, photosystem II efficiency, leaf water potential and altered emission of blue-green fluorescence from cacao leaves. Induction of TcSAMDC in leaves was most closely correlated with changes in water potential. The earliest measured responses to drought were enhanced expression of TcADC and TcSAMDC in roots along with decreases in stomatal conductance, photosynthesis, and photosystem II efficiency. Elevated levels of putrescine, spermidine, and spermine were detected in cacao leaves 13days after the onset of drought. Expression of all five PA associated transcripts was enhanced (1.5-3-fold) in response to treatment with abscisic acid. TcODC and TcADC, were also responsive to mechanical wounding, infection by Phytophthora megakarya (a causal agent of black pod disease in cacao), the necrosis- and ethylene-inducing protein (Nep1) of Fusarium oxysporum, and flower abscission. TcSAMDC expression was responsive to all stresses except flower abscission. TcODC, although constitutively expressed at much lower levels than TcADC, TcSAMDC, TcSPDS, and TcSPMS, was highly inducible by the fungal protein Nep1 (135-fold) and the cacao pathogen Phytophthora megakarya (671-fold). The full length cDNA for ODC was cloned and characterized. Among the genes studied, TcODC, TcADC, and TcSAMDC were most sensitive to induction by drought in addition to other abiotic and biotic stresses. TcODC, TcADC, and TcSAMDC may share signal transduction pathways and/or the stress induced signal induction pathways may converge at these three genes leading to similar although not identical patterns of expression. It is possible altering PA levels in cacao will result in enhanced tolerance to multiple stresses including drought and disease as has been demonstrated in other crops.

Systematic biology analysis on photosynthetic carbon metabolism of maize leaf following sudden heat shock under elevated CO2.

Plants would experience more complex environments, such as sudden heat shock (SHS) stress combined with elevated CO2 in the future, and might adapt to this stressful condition by optimizing photosynthetic carbon metabolism (PCM). It is interesting to understand whether this acclimation process would be altered in different genotypes of maize under elevated CO2, and which metabolites represent key indicators reflecting the photosynthetic rates (PN) following SHS. Although B76 had greater reduction in PN during SHS treatment, our results indicated that PN in genotype B76, displayed faster recovery after SHS treatment under elevated CO2 than in genotype B106. Furthermore, we employed a stepwise feature extraction approach by partial linear regression model. Our findings demonstrated that 9 key metabolites over the total (35 metabolites) can largely explain the variance of PN during recovery from SHS across two maize genotypes and two CO2 grown conditions. Of these key metabolites, malate, valine, isoleucine, glucose and starch are positively correlated with recovery pattern of PN. Malate metabolites responses to SHS were further discussed by incorporating with the activities and gene expression of three C4 photosynthesis-related key enzymes. We highlighted the importance of malate metabolism during photosynthesis recovery from short-term SHS, and data integration analysis to better comprehend the regulatory framework of PCM in response to abiotic stress.

An attempt to interpret a biochemical mechanism of C4 photosynthetic thermo-tolerance under sudden heat shock on detached leaf in elevated CO2 grown maize.

Detached leaves at top canopy structures always experience higher solar irradiance and leaf temperature under natural conditions. The ability of tolerance to high temperature represents thermotolerance potential of whole-plants, but was less of concern. In this study, we used a heat-tolerant (B76) and a heat-susceptible (B106) maize inbred line to assess the possible mitigation of sudden heat shock (SHS) effects on photosynthesis (PN) and C4 assimilation pathway by elevated [CO2]. Two maize lines were grown in field-based open top chambers (OTCs) at ambient and elevated (+180 ppm) [CO2]. Top-expanded leaves for 30 days after emergence were suddenly exposed to a 45°C SHS for 2 hours in midday during measurements. Analysis on thermostability of cellular membrane showed there was 20% greater electrolyte leakage in response to the SHS in B106 compared to B76, in agreement with prior studies. Elevated [CO2] protected PN from SHS in B76 but not B106. The responses of PN to SHS among the two lines and grown CO2 treatments were closely correlated with measured decreases of NADP-ME enzyme activity and also to its reduced transcript abundance. The SHS treatments induced starch depletion, the accumulation of hexoses and also disrupted the TCA cycle as well as the C4 assimilation pathway in the both lines. Elevated [CO2] reversed SHS effects on citrate and related TCA cycle metabolites in B106 but the effects of elevated [CO2] were small in B76. These findings suggested that heat stress tolerance is a complex trait, and it is difficult to identify biochemical, physiological or molecular markers that accurately and consistently predict heat stress tolerance.

Varying Response of the Concentration and Yield of Soybean Seed Mineral Elements, Carbohydrates, Organic Acids, Amino Acids, Protein, and Oil to Phosphorus Starvation and CO2 Enrichment.

A detailed investigation of the concentration (e.g., mg g-1 seed) and total yield (e.g., g plant-1) of seed mineral elements and metabolic profile under phosphorus (P) starvation at ambient (aCO2) and elevated carbon dioxide (eCO2) in soybean is limited. Soybean plants were grown in a controlled environment at either sufficient (0.50 mM P, control) or deficient (0.10 and 0.01 mM, P-stress) levels of P under aCO2 and eCO2 (400 and 800 µmol mol-1, respectively). Both the concentration and yield of 36 out of 38 seed components responded to P treatment and on average 25 and 11 components increased and decreased, respectively, in response to P starvation. Concentrations of carbohydrates (e.g., glucose, sugar alcohols), organic acids (e.g., succinate, glycerate) and amino acids increased while oil, and several minerals declined under P deficiency. However, the yield of the majority of seed components declined except several amino acids (e.g., phenylalanine, serine) under P deficiency. The concentration-based relationship between seed protein and oil was negative (r2 = 0.96), whereas yield-based relationship was positive (r2 = 0.99) across treatments. The CO2 treatment also altered the concentration of 28 out of 38 seed components, of which 23 showed decreasing (e.g., sucrose, glucose, citrate, aconitate, several minerals, and amino acids) while C, iron, Mn, glycerate, and oil showed increasing trends at eCO2. Despite a decreased concentration, yields of the majority of seed components were increased in response to eCO2, which was attributable to the increased seed production especially near sufficient P nutrition. The P × CO2 interactions for the concentration of amino acids and the yield of several components were due to the lack of their response to eCO2 under control or the severe P starvation, respectively. Thus, P deficiency primarily reduced the concentration of oil and mineral elements but enhanced a majority of other components. However, seed components yield consistently declined under P starvation except for several amino acids. The study highlighted a P nutritional-status dependent response of soybean seed components to eCO2 suggesting the requirement of an adequate P supply to obtain the beneficial effects of eCO2 on the overall yield of various seed components.

Transcriptome Analysis of Soybean Leaf Abscission Identifies Transcriptional Regulators of Organ Polarity and Cell Fate.

Abscission, organ separation, is a developmental process that is modulated by endogenous and environmental factors. To better understand the molecular events underlying the progression of abscission in soybean, an agriculturally important legume, we performed RNA sequencing (RNA-seq) of RNA isolated from the leaf abscission zones (LAZ) and petioles (Non-AZ, NAZ) after treating stem/petiole explants with ethylene for 0, 12, 24, 48, and 72 h. As expected, expression of several families of cell wall modifying enzymes and many pathogenesis-related (PR) genes specifically increased in the LAZ as abscission progressed. Here, we focus on the 5,206 soybean genes we identified as encoding transcription factors (TFs). Of the 5,206 TFs, 1,088 were differentially up- or down-regulated more than eight-fold in the LAZ over time, and, within this group, 188 of the TFs were differentially regulated more than eight-fold in the LAZ relative to the NAZ. These 188 abscission-specific TFs include several TFs containing domains for homeobox, MYB, Zinc finger, bHLH, AP2, NAC, WRKY, YABBY, and auxin-related motifs. To discover the connectivity among the TFs and highlight developmental processes that support organ separation, the 188 abscission-specific TFs were then clustered based on a >four-fold up- or down-regulation in two consecutive time points (i.e., 0 and 12 h, 12 and 24 h, 24 and 48 h, or 48 and 72 h). By requiring a sustained change in expression over two consecutive time intervals and not just one or several time intervals, we could better tie changes in TFs to a particular process or phase of abscission. The greatest number of TFs clustered into the 0 and 12 h group. Transcriptional network analysis for these abscission-specific TFs indicated that most of these TFs are known as key determinants in the maintenance of organ polarity, lateral organ growth, and cell fate. The abscission-specific expression of these TFs prior to the onset of abscission and their functional properties as defined by studies in Arabidopsis indicate that these TFs are involved in defining the separation cells and initiation of separation within the AZ by balancing organ polarity, roles of plant hormones, and cell differentiation.

Soybean grown under elevated CO2 benefits more under low temperature than high temperature stress: Varying response of photosynthetic limitations, leaf metabolites, growth, and seed yield.

To evaluate the combined effect of temperature and CO2 on photosynthetic processes, leaf metabolites and growth, soybean was grown under a controlled environment at low (22/18°C, LT), optimum (28/24°C, OT) and high (36/32°C HT) temperatures under ambient (400µmolmol-1; aCO2) or elevated (800µmolmol-1; eCO2) CO2 concentrations during the reproductive stage. In general, the rate of photosynthesis (A), stomatal (gs) and mesophyll (gm) conductance, quantum yield of photosystem II, rates of maximum carboxylation (VCmax), and electron transport (J) increased with temperature across CO2 levels. However, compared with OT, the percentage increases in these parameters at HT were lower than the observed decline at LT. The photosynthetic limitation at LT and OT was primarily caused by photo-biochemical processes (49-58%, Lb) followed by stomatal (27-32%, Ls) and mesophyll (15-19%, Lm) limitations. However, at HT, it was primarily caused by Ls (41%) followed by Lb (33%) and Lm (26%). The dominance of Lb at LT and OT was associated with the accumulation of non-structural carbohydrates (e.g., starch) and several organic acids, whereas this accumulation did not occur at HT, indicating increased metabolic activities. Compared with OT, biomass and seed yield declined more at HT than at LT. The eCO2 treatment compensated for the temperature-stress effects on biomass but only partially compensated for the effects on seed yield, especially at HT. Photosynthetic downregulation at eCO2 was possibly due to the accumulation of non-structural carbohydrates and the decrease in gs and Astd (standard A measured at 400µmolmol-1 sub-stomatal CO2 concentration), as well as the lack of CO2 effect on gm, VCmax, and J, and photosynthetic limitation. Thus, the photosynthetic limitation was temperature-dependent and was primarily influenced by the alteration in photo-biochemical processes and metabolic activities. Despite the inconsistent response of photosynthesis (or biomass accumulation) and seed yield, eCO2 tended to fully or partially compensate for the adverse effect of the respective LT and HT stresses under well-watered and sufficient nutrient conditions.

Temperature Shift Experiments Suggest That Metabolic Impairment and Enhanced Rates of Photorespiration Decrease Organic Acid Levels in Soybean Leaflets Exposed to Supra-Optimal Growth Temperatures.

Elevated growth temperatures are known to affect foliar organic acid concentrations in various plant species. In the current study, citrate, malate, malonate, fumarate and succinate decreased 40 to 80% in soybean leaflets when plants were grown continuously in controlled environment chambers at 36/28 compared to 28/20 °C. Temperature effects on the above mentioned organic acids were partially reversed three days after plants were transferred among optimal and supra-optimal growth temperatures. In addition, CO2 enrichment increased foliar malate, malonate and fumarate concentrations in the supra-optimal temperature treatment, thereby mitigating effects of high temperature on respiratory metabolism. Glycerate, which functions in the photorespiratory pathway, decreased in response to CO2 enrichment at both growth temperatures. The above findings suggested that diminished levels of organic acids in soybean leaflets upon exposure to high growth temperatures were attributable to metabolic impairment and to changes of photorespiratory flux. Leaf development rates differed among temperature and CO2 treatments, which affected foliar organic acid levels. Additionally, we report that large decreases of foliar organic acids in response to elevated growth temperatures were observed in legume species.

Effects of CO2 enrichment and drought pretreatment on metabolite responses to water stress and subsequent rehydration using potato tubers from plants grown in sunlit chambers.

Experiments were performed using naturally sunlit Soil-Plant-Atmosphere Research chambers that provided ambient or twice ambient CO2. Potato plants were grown in pots that were water sufficient (W), water insufficient for 12-18 days during both vegetative and tuber development stages (VR), or water insufficient solely during tuber development (R). In the ambient CO2 treatment, a total of 17 and 20 out of 31 tuber metabolites differed when comparing the W to the R and VR treatments, respectively. Hexoses, raffinose, mannitol, branched chain amino acids, phenylalanine and proline increased, although most organic acids remained unchanged or decreased in response to drought. Osmolytes, including glucose, branched chain amino acids and proline, remained elevated following 2 weeks of rehydration in both the ambient and elevated CO2 treatments, whereas fructose, raffinose, mannitol and some organic acids reverted to control levels. Failure of desiccated plant tissues to mobilize specific osmolytes after rehydration was unexpected and was likely because tubers function as terminal sinks. Tuber metabolite responses to single or double drought treatments were similar under the same CO2 levels but important differences were noted when CO2 level was varied. We also found that metabolite changes to water insufficiency and/or CO2 enrichment were very distinct between sink and source tissues, and total metabolite changes to stress were generally greater in leaflets than tubers.

Adjustments of net photosynthesis in Solanum tuberosum in response to reciprocal changes in ambient and elevated growth CO2 partial pressures.

Single leaf photosynthetic rates and various leaf components of potato (Solanum tuberosum L.) were studied 1-3 days after reciprocally transferring plants between the ambient and elevated growth CO2 treatments. Plants were raised from individual tuber sections in controlled environment chambers at either ambient (36 Pa) or elevated (72 Pa) CO2. One half of the plants in each growth CO2 treatment were transferred to the opposite CO2 treatment 34 days after sowing (DAS). Net photosynthesis (Pn) rates and various leaf components were then measured 34, 35 and 37 DAS at both 36 and 72 Pa CO2. Three-day means of single leaf Pn rates, leaf starch, glucose, initial and total Rubisco activity, Rubisco protein, chlorophyll (a+b), chlorophyll (a/b), alpha-amino N, and nitrate levels differed significantly in the continuous ambient and elevated CO2 treatments. Acclimation of single leaf Pn rates was partially to completely reversed 3 days after elevated CO2-grown plants were shifted to ambient CO2, whereas there was little evidence of photosynthetic acclimation 3 days after ambient CO2-grown plants were shifted to elevated CO2. In a four-way comparison of the 36, 72, 36 to 72 (shifted up) and 72 to 36 (shifted down) Pa CO2 treatments 37 DAS, leaf starch, soluble carbohydrates, Rubisco protein and nitrate were the only photosynthetic factors that differed significantly. Simple and multiple regression analyses suggested that negative changes of Pn in response to growth CO2 treatment were most closely correlated with increased leaf starch levels.

Responses of growth and primary metabolism of water-stressed barley roots to rehydration.

Barley seedlings were grown in pots in controlled environment chambers and progressive drought treatments were imposed 11 d after sowing. Soil water content decreased from 92 to 10% following 14 d without watering. Increases of biomass in shoots and roots slowed after 4 and 9 d of water stress, respectively. Thirty barley root metabolites were monitored in this study and 85% were significantly altered by drought. Sucrose, raffinose, glucose, fructose, maltose, malate, asparagine and proline increased and myo-inositol, glycerate, alanine, serine, glycine and glutamate decreased during drought. Primary metabolism was likely involved in various crucial processes during water stress including, osmotic adjustment, nitrogen sequestration and ammonia detoxification. Rates of photosynthesis and stomatal conductance recovered in 2 d and shoot growth commenced the 3rd day after rehydration. Root growth also exhibited a lag after rehydration but this was attributed to high nutrient concentrations during water stress. Malate and proline recovered within 1 d but serine was only partially reversed 6 d after rehydration. Malate, aspartate and raffinose decreased below well-watered, control levels following rehydration. Variation in the magnitude and time necessary for individual compounds to fully recover after rehydration suggested the complexity of metabolic processes initiated by re-watering.

Necrosis- and ethylene-inducing peptide from Fusarium oxysporum induces a complex cascade of transcripts associated with signal transduction and cell death in Arabidopsis.

Treatment of Arabidopsis (Arabidopsis thaliana) with a necrosis- and ethylene-inducing peptide (Nep1) from Fusarium oxysporum inhibited both root and cotyledon growth and triggered cell death, thereby generating necrotic spots. Nep1-like proteins are produced by divergent microbes, many of which are plant pathogens. Nep1 in the plant was localized to the cell wall and cytosol based on immunolocalization results. The ratio of chlorophyll a fluorescence (F685 nm/F730 nm) significantly decreased after 75-min treatment with Nep1 in comparison to the control. This suggested that a short-term compensation of photosynthesis occurred in response to localized damage to cells. The concentrations of most water-soluble metabolites analyzed were reduced in Arabidopsis seedlings after 6 h of Nep1 treatment, indicating that the integrity of cellular membranes had failed. Microarray results showed that short-term treatment with Nep1 altered expression of numerous genes encoding proteins putatively localized to organelles, especially the chloroplast and mitochondria. Short-term treatment with Nep1 induced multiple classes of genes involved in reactive oxygen species production, signal transduction, ethylene biosynthesis, membrane modification, apoptosis, and stress. Quantitative PCR was used to confirm the induction of genes localized in the chloroplast, mitochondria, and plasma membrane, and genes responsive to calcium/calmodulin complexes, ethylene, jasmonate, ethylene biosynthesis, WRKY, and cell death. The majority of Nep1-induced genes has been associated with general stress responses but has not been critically linked to resistance to plant disease. These results are consistent with Nep1 facilitating cell death as a component of diseases caused by necrotrophic plant pathogens.

Biomass and toxicity responses of poison ivy (Toxicodendron radicans) to elevated atmospheric CO2.

Contact with poison ivy (Toxicodendron radicans) is one of the most widely reported ailments at poison centers in the United States, and this plant has been introduced throughout the world, where it occurs with other allergenic members of the cashew family (Anacardiaceae). Approximately 80% of humans develop dermatitis upon exposure to the carbon-based active compound, urushiol. It is not known how poison ivy might respond to increasing concentrations of atmospheric carbon dioxide (CO(2)), but previous work done in controlled growth chambers shows that other vines exhibit large growth enhancement from elevated CO(2). Rising CO(2) is potentially responsible for the increased vine abundance that is inhibiting forest regeneration and increasing tree mortality around the world. In this 6-year study at the Duke University Free-Air CO(2) Enrichment experiment, we show that elevated atmospheric CO(2) in an intact forest ecosystem increases photosynthesis, water use efficiency, growth, and population biomass of poison ivy. The CO(2) growth stimulation exceeds that of most other woody species. Furthermore, high-CO(2) plants produce a more allergenic form of urushiol. Our results indicate that Toxicodendron taxa will become more abundant and more "toxic" in the future, potentially affecting global forest dynamics and human health.

Apple leaf sucrose-phosphate synthase is inhibited by sorbitol-6-phosphate.

Sucrose-phosphate synthase (SPS) from mature apple (Malus domestica Borhk. cv. Gala) leaves was purified 34-fold to a final specific activity of 15.3 µmol mg-1 protein h-1. The enzyme showed hyperbolic saturation kinetics for both fructose-6-phosphate (F6P) (Km= 0.36 mM) and uridine-5'-diphosphoglucose (UDPG) (Km = 6.49 mM). Glucose-6-phosphate (G6P) was found to be an activator of apple SPS, and the activation was dependent upon the F6P concentration. At a concentration of 2 mM, G6P significantly decreased the Km for F6P and increased SPS activity. However, higher concentrations of G6P did not further stimulate SPS activity. In contrast to SPS from other plant species, inorganic phosphate (Pi) had little or no inhibitory effect on apple SPS. The apple leaf enzyme was inhibited 7-10% by 10 mM Pi when F6P concentrations were in the range of 2-10 mM. We observed that sorbitol-6-phosphate, an intermediate metabolite in sorbitol biosynthesis, was a competitive inhibitor of SPS with a Ki of 1.83 mM. Sorbitol-6-phosphate also inhibited G6P activation of SPS. Our results suggest that sucrose biosynthesis may be altered by the products of sorbitol biosynthesis in apple leaves.

Regulation of apple leaf aldose-6-phosphate reductase activity by inorganic phosphate and divalent cations.

Aldose-6-phosphate reductase (A6PR), a key enzyme in sorbitol biosynthesis, has been purified to apparent homogeneity from fully developed apple (Malus domestica Borkh. cv. Gala) leaves. Inorganic phosphate inhibited A6PR by decreasing the maximum velocity of the enzyme and by increasing the Km for the substrate, glucose-6-phosphate (Glc6P). Divalent cations including Ca2+, Mg2+, Zn2+ and Cu2+ altered A6PR activity. Effects of Ca2+ and Mg2+ on A6PR activity were dependent upon both the metal ion concentration and the concentration of Glc6P. The activity of A6PR was increased by 0.5-5 mM Ca2+ or Mg2+ when Glc6P concentration was below 10 mM. However, these same metal ions decreased A6PR activity at greater Glc6P concentrations or in the presence of higher metal ion concentrations. A6PR displayed Michaelis-Menten kinetics either in the presence or absence of 2.5 mM MgCl2, but the apparent Km for Glc6P decreased from 11.3 mM for the control to 5.1 mM in the presence of 2.5 mM MgCl2 in the assay mixture. By contrast, Zn2+ and Cu2+ dramatically inactivated A6PR activity. A6PR activity was decreased approximately 50 and 70%, respectively, when the enzyme was pre-incubated with 2 mM Zn2+ or Cu2+ for 60 min at room temperature. This inactivation was partially reversed by dialysis or by chelation with 20 mM EDTA. NADPH and NADP+, which are substrates for A6PR in the oxidative and reductive directions, respectively, partially protected A6PR from inactivation by Zn2+. The above results suggest that both Mg2+ and Ca2+ were mixed-type, non-essential activators of A6PR that decreased the Km for sugar-phosphates but lowered the overall Vmax. The physiological significance of these findings is also discussed.