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.

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.

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.

Effects of growth temperature and carbon dioxide enrichment on soybean seed components at different stages of development.

Soybean plants were grown to maturity in controlled environment chambers and at the onset of flowering three temperature treatments were imposed that provided optimum [28/24 °C], low [22/18 °C] or high [36/32 °C] chamber air temperatures. In addition, plants were treated continuously with either 400 or 800 µmol mol-1 CO2. Seeds were harvested at 42, 53, 69 and 95 days after planting (i.e., final maturity). This study quantified 51 metabolites in developing soybean seeds, plus total lipids and proteins were measured at maturity. About 80% of measured soluble carbohydrates, amines and organic acids decreased to low levels in mature seeds, although important exceptions were raffinose, ribose/arabinose, citrate and all eight fatty acids. This suggested that the metabolism of young seeds supported lipid and protein synthesis. A total of 35 and 9 metabolites differed among temperature and CO2 treatments, respectively, and treatment effects were predominately observed on the first and second samplings. However, shikimate, pinitol and oleate were increased by high temperature treatments in mature seeds. The above results indicated that CO2 enrichment primarily altered metabolite levels during the initial stages of seed development and this was likely due to enhanced photosynthate formation in leaves.

Pathogenesis-Related Protein 1b1 (PR1b1) Is a Major Tomato Fruit Protein Responsive to Chilling Temperature and Upregulated in High Polyamine Transgenic Genotypes.

Plants execute an array of mechanisms in response to stress which include upregulation of defense-related proteins and changes in specific metabolites. Polyamines - putrescine (Put), spermidine (Spd), and spermine (Spm) - are metabolites commonly found associated with abiotic stresses such as chilling stress. We have generated two transgenic tomato lines (556HO and 579HO) that express yeast S-adenosylmethionine decarboxylase and specifically accumulate Spd and Spm in fruits in comparison to fruits from control (556AZ) plants (Mehta et al., 2002). Tomato fruits undergo chilling injury at temperatures below 13°C. The high Spd and Spm tomato together with the control azygous line were utilized to address role(s) of polyamines in chilling-injury signaling. Exposure to chilling temperature (2°C) led to several-fold increase in the Put content in all the lines. Upon re-warming of the fruits at 20°C, the levels of Spd and Spm increased further in the fruit of the two transgenic lines, the higher levels remaining stable for 15 days after re-warming as compared to the fruit from the control line. Profiling their steady state proteins before and after re-warming highlighted a protein of ∼14 kD. Using proteomics approach, protein sequencing and immunoblotting, the ∼14-kD protein was identified as the pathogenesis related protein 1b1 (PR1b1). The PR1b1 protein accumulated transiently in the control fruit whose level was barely detectable at d 15 post-warming while in the fruit from both the 556HO and 579HO transgenic lines PR1b1 abundance increased and remained stable till d 15 post warming. PR1b1 gene transcripts were found low in the control fruit with a visible accumulation only on d 15 post warming; however, in both the transgenic lines it accumulated and increased soon after rewarming being several-fold higher on day 2 while in 556HO line this increase continued until d 6 than the control fruit. The chilling-induced increase in PR1b1 protein seems independent of ethylene and methyl jasmonate signaling but may be linked to salicylic acid. We propose that polyamine-mediated sustained accumulation of PR1b1 protein in post-warmed chilled tomato fruit is a pre-emptive cold stress response and possibly a defense response mechanism related to Cold Stress-Induced Disease Resistance (SIDR) phenomenon.

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.

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.

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.

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.

Combined effects of CO2 enrichment, diurnal light levels and water stress on foliar metabolites of potato plants grown in naturally sunlit controlled environment chambers.

Experiments were conducted in outdoor, naturally sunlit, soil-plant-atmosphere research (SPAR) chambers using plants grown in pots. Drought treatments were imposed on potato plants (Solanum tuberosum cv. Kennebec) beginning 10 days after tuber initiation. A total of 23 out of 37 foliar metabolites were affected by drought when measured 11 days after initiating water stress treatments. Compounds that accumulated in response to drought were hexoses, polyols, branched chain amino acids (BCAAs) and aromatic amino acids, such as proline. Conversely, leaf starch, alanine, aspartate and several organic acids involved in respiratory metabolism decreased with drought. Depending upon harvest date, a maximum of 12 and 17 foliar metabolites also responded to either CO2 enrichment or diurnal treatments, respectively. In addition, about 20% of the measured metabolites in potato leaflets were simultaneously affected by drought, CO2 enrichment and diurnal factors combined. This group contained BCAAs, hexoses, leaf starch and malate. Polyols and proline accumulated in response to water stress but did not vary diurnally. Water stress also amplified diurnal variations of hexoses and starch in comparison to control samples. Consequently, specific drought responsive metabolites in potato leaflets were dramatically affected by daily changes of photosynthetic carbon metabolism.

Differential gene expression by Moniliophthora roreri while overcoming cacao tolerance in the field.

Frosty pod rot (FPR) of Theobroma cacao (cacao) is caused by the hemibiotrophic fungus Moniliophthora roreri. Cacao clones tolerant to FPR are being planted throughout Central America. To determine whether M. roreri shows a differential molecular response during successful infections of tolerant clones, we collected field-infected pods at all stages of symptomatology for two highly susceptible clones (Pound-7 and CATIE-1000) and three tolerant clones (UF-273, CATIE-R7 and CATIE-R4). Metabolite analysis was carried out on clones Pound-7, CATIE-1000, CATIE-R7 and CATIE-R4. As FPR progressed, the concentrations of sugars in pods dropped, whereas the levels of trehalose and mannitol increased. Associations between symptoms and fungal loads and some organic and amino acid concentrations varied depending on the clone. RNA-Seq analysis identified 873 M. roreri genes that were differentially expressed between clones, with the primary difference being whether the clone was susceptible or tolerant. Genes encoding transcription factors, heat shock proteins, transporters, enzymes modifying membranes or cell walls and metabolic enzymes, such as malate synthase and alternative oxidase, were differentially expressed. The differential expression between clones of 43 M. roreri genes was validated by real-time quantitative reverse transcription polymerase chain reaction. The expression profiles of some genes were similar in susceptible and tolerant clones (other than CATIE-R4) and varied with the biotrophic/necrotropic shift. Moniliophthora roreri genes associated with stress metabolism and responses to heat shock and anoxia were induced early in tolerant clones, their expression profiles resembling that of the necrotrophic phase. Moniliophthora roreri stress response genes, induced during the infection of tolerant clones, may benefit the fungus in overcoming cacao defense mechanisms.

Successful pod infections by Moniliophthora roreri result in differential Theobroma cacao gene expression depending on the clone's level of tolerance.

An understanding of the tolerance mechanisms of Theobroma cacao used against Moniliophthora roreri, the causal agent of frosty pod rot, is important for the generation of stable disease-tolerant clones. A comparative view was obtained of transcript populations of infected pods from two susceptible and two tolerant clones using RNA sequence (RNA-Seq) analysis. A total of 3009 transcripts showed differential expression among clones. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of differentially expressed genes indicated shifts in 152 different metabolic pathways between the tolerant and susceptible clones. Real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) analyses of 36 genes verified the differential expression. Regression analysis validated a uniform progression in gene expression in association with infection levels and fungal loads in the susceptible clones. Expression patterns observed in the susceptible clones diverged in tolerant clones, with many genes showing higher expression at a low level of infection and fungal load. Principal coordinate analyses of real-time qRT-PCR data separated the gene expression patterns between susceptible and tolerant clones for pods showing malformation. Although some genes were constitutively differentially expressed between clones, most results suggested that defence responses were induced at low fungal load in the tolerant clones. Several elicitor-responsive genes were highly expressed in tolerant clones, suggesting rapid recognition of the pathogen and induction of defence genes. Expression patterns suggested that the jasmonic acid-ethylene- and/or salicylic acid-mediated defence pathways were activated in the tolerant clones, being enhanced by reduced brassinosteroid (BR) biosynthesis and catabolic inactivation of both BR and abscisic acids. Finally, several genes associated with hypersensitive response-like cell death were also induced in tolerant clones.

Drought responses of foliar metabolites in three maize hybrids differing in water stress tolerance.

Maize (Zea mays L.) hybrids varying in drought tolerance were treated with water stress in controlled environments. Experiments were performed during vegetative growth and water was withheld for 19 days beginning 17 days after sowing. Genotypic comparisons used measured changes of leaf water potential or results were expressed by time of treatment. Total dry matter of the drought tolerant hybrid on the final harvest was 53% less than that of the intermediate and susceptible maize hybrids when plants were water sufficient. This showed that maize hybrids selected for extreme drought tolerance possessed a dwarf phenotype that affected soil water contents and leaf water potentials. Changes of shoot and root growth, leaf water potential, net photosynthesis and stomatal conductance in response to the time of water stress treatment were diminished when comparing the drought tolerant to the intermediate or susceptible maize hybrids. Genotypic differences were observed in 26 of 40 total foliar metabolites during water stress treatments. Hierarchical clustering revealed that the tolerant maize hybrid initiated the accumulation of stress related metabolites at higher leaf water potentials than either the susceptible or intermediate hybrids. Opposite results occurred when changes of metabolites in maize leaves were expressed temporally. The above results demonstrated that genotypic differences were readily observed by comparing maize hybrids differing in drought tolerance based on either time of treatment or measured leaf water potential. Current findings provided new and potentially important insights into the mechanisms of drought tolerance in maize.

Combined effects of CO2 enrichment and elevated growth temperatures on metabolites in soybean leaflets: evidence for dynamic changes of TCA cycle intermediates.

Soybean (Glycine max [Merr.] L.) was grown in indoor chambers with ambient (38 Pa) and elevated (70 Pa) CO2 and day/night temperature treatments of 28/20, 32/24 and 36/28 °C. We hypothesized that CO2 enrichment would mitigate the deleterious effects of elevated growth temperatures on metabolites in soybean leaflets. Net CO2 assimilation rates increased incrementally with growth temperature and were enhanced up to 24 % on average by CO2 enrichment. Stomatal conductance about doubled from the lowest to highest temperature but this was partially reversed by CO2 enrichment. Metabolites were measured thrice daily and 19 and 28 of 43 total leaf metabolites were altered by the 32/24 and 36/28 °C temperature treatments, respectively, in both CO2 treatments. Polyols, raffinose and GABA increased and 23 nonstructural carbohydrates, organic acids and amino acids decreased when the temperature was increased from 28 to 36 °C under ambient CO2. Citrate, aconitate and 2-oxoglutarate decreased over 90 % in the 36/28 °C compared to the 28/20 °C temperature treatment. Temperature-dependent changes of sugars, organic acids and all but three amino acids were almost completely eliminated by CO2 enrichment. The above findings suggested that specific TCA cycle intermediates were highly depleted by heat stress under ambient CO2. Mitigating effects of CO2 enrichment on soybean leaflet metabolites were attributed to altered rates of photosynthesis, photorespiration, dark respiration, the anaplerotic pathway and to possible changes of gene expression.

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.

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.

Carbon partitioning and the impact of starch deficiency on the initial response of Arabidopsis to chilling temperatures.

Metabolites and stress related transcripts were measured in Arabidopsis thaliana in response to chilling temperatures. Rates of carbon assimilation increased 17% on average in response to cold treatment. Sucrose, glucose and fructose accumulation consumed 42% of the carbon from A but leaf starch only could synthesize ~10% of observed changes in soluble sugars. Carbohydrates were the only major class of metabolites that accumulated during the first 24 h of cold treatment. Except maltose and raffinose, carbohydrate accumulation was abolished when cold treatments were in darkness. Starch hydrolysis was correlated with maltose accumulation and increased expression of BAM3, which encodes a ß-amylase necessary for starch mobilization. Hexose accumulation was delayed 6 h and raffinose accumulation was not observed in a starchless (pgm1) mutant. Changes of expression of five stress-induced transcripts in response to cold were similar in the wild type and in the pgm1 mutant. Three of five stress related transcripts had decreased expression when cold treatments were performed in the dark compared to the light. Therefore, starch hydrolysis may augment hexose and raffinose accumulations during the first 24 h after a cold shock and a partial cold stress response was observed in Arabidopsis during cold treatments in the dark.

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.