Project description:To gain insights into molecular mechanisms of tolerance to heat stress, we conducted a transcript profiling experiment to identify heat-responsive genes in contrasting peanut mini core accessions, either un-acclimated or acclimated to heat stress. Plants at reproductive stage were exposed to 28 °C (control), 45 °C for 15 d (un-acclimated) or 45 °C for 1 d followed by 7 d recovery and 15 d stress (acclimated). Two contrasting genotypes showing diverse response to stress were selected based on a bioassay involving chlorophyll fluorescence yield under elevated respiratory demand and membrane thermostability. Transcript profiling was performed using 8 x 15k custom oligo microarrays containing 15k peanut EST sequences. Gene enrichment analysis was performed using Blast2GO program and genes with homology to known proteins were categorized into detailed molecular functional groups. Majority of stress-responsive genes assigned to KEGG pathways belonged to starch, sucrose and galactose metabolism followed by aminoacid metabolism, and secondary metabolite biosynthesis. Differentially expressed transcripts from samples were validated in the samples from second year by quantitative real-time PCR. Transcripts of eight genes involved in terpenoid and flavanoid biosynthesis were induced after second and seventh day, respectively in leaves under heat stress. Metabolite analysis confirmed increases in metabolites of selected pathways under heat stress. The heat up-regulated genes in tolerant COC041 mini-core accession are potential candidate genes for engineering stress-tolerant peanuts and unraveling molecular mechanisms of peanut adaptation to heat stress. We used Agilent peanut microarrays to identify putative heat stress-responsive genes. Acclimated leaf tissues of the peanut genotypes COC041 (tolerant) and COC166 (susceptible) were used in the study. Three replications of microarray experiments were carried out by hybridizing the cRNA from different time points and stress conditions in a loop design on 8 x 15k microarray.

Project description:To gain insights into molecular mechanisms of tolerance to heat stress, we conducted a transcript profiling experiment to identify heat-responsive genes in contrasting peanut mini core accessions, either un-acclimated or acclimated to heat stress. Plants at reproductive stage were exposed to 28 °C (control), 45 °C for 15 d (un-acclimated) or 45 °C for 1 d followed by 7 d recovery and 15 d stress (acclimated). Two contrasting genotypes showing diverse response to stress were selected based on a bioassay involving chlorophyll fluorescence yield under elevated respiratory demand and membrane thermostability. Transcript profiling was performed using 4 x 44k custom oligo microarrays containing 22k peanut EST sequences. The microarray analysis identified 710 stress-induced and 770 stress-repressed putative heat-responsive transcripts in the tolerant genotype. Gene enrichment analysis was performed using Blast2GO program and genes with homology to known proteins were categorized into detailed molecular functional groups. Majority of stress-responsive genes assigned to KEGG pathways belonged to starch, sucrose and galactose metabolism followed by amino acid metabolism, and secondary metabolite biosynthesis. Differentially expressed transcripts from samples obtained from first year’s experiment were validated in the samples from second year by quantitative real-time PCR. Transcripts of eight genes involved in terpenoid and flavanoid biosynthesis were induced after second and seventh day, respectively, in leaves under heat stress. Metabolite analysis confirmed increases in metabolites of selected pathways under heat stress. The heat up-regulated genes in tolerant COC041 mini-core accession are potential candidate genes for engineering stress-tolerant peanuts and unraveling molecular mechanisms of peanut adaptation to heat stress. We used Agilent peanut microarrays to identify putative heat stress-responsive genes. Directly heat-stressed leaf tissues of the peanut genotypes COC041 (tolerant) and COC166 (susceptible) were used in the study. Three replications of microarray experiments were carried out by hybridizing the cRNA from different time points and stress conditions in a loop design on 4 x 44k microarray.

Project description:Engineered abiotic stress resistance is an important target for increasing agricultural productivity.There are concerns, however, regarding possible ecological impacts of transgenic crops. In contrast to the first wave of transgenic crops, many abiotic stress resistance genes can initiate complex downstream changes. Transcriptome profiling has been suggested as a comprehensive non-targeted approach to examine secondary effects. We compared phenotypic and transcriptomic effects of constitutive expression of genes intended to confer salt stress tolerance by three different mechanisms: a transcription factor, CBF3/DREB1a; a metabolic gene, M6PR, for mannitol biosynthesis; and the Na+/H+ antiporter, SOS1. Transgenic CBF3, M6PR, and SOS1 Arabidopsis thaliana were grown together in the growth chamber, greenhouse and field. In the absence of salt, M6PR and SOS1 lines performed comparably to wild type; CBF3 lines exhibited dwarfing as reported previously. All three transgenes conferred fitness advantage when subjected to 100 mM NaCl in the growth chamber. CBF3 and M6PR affected transcription of numerous abiotic stress- related genes as measured by Affymetrix microarray analysis. M6PR additionally modified expression of biotic- and oxidative- stress genes. Transcriptional effects of SOS1 in the absence of salt were smaller and primarily limited to redox-related genes. The extent of transcriptome change, however, did not correlate with effects on growth and reproduction. Thus, magnitude of global transcriptome differences may not predict phenotypic differences upon which environment and selection act to influence fitness. These observations have implications for interpretation of transcriptome analyses in the context of risk assessment and emphasize importance of evaluation within a phenotypic context. Both transgenic plants and relative WT plants were grown in the growth chamber in the absence and presence of salt stress. Plants from 20 days after sowing (6 days after salt treatment) were used for RNA extraction and hybridization on Affymetrix microarrays. There were two biological replicates for each genotype and salt treatment combination.

Project description:Mannitol is a sugar alcohol that serves as a compatible solute contributing to exceptional salt tolerance in several plant species. Arabidopsis plants transformed with the mannose-6-phosphate reductase (M6PR) gene from celery were dramatically more salt tolerant. Following treatment with 100mM NaCl, transgenic plants, relative to wild type (WT), were more successful in bolting, flowering, and production of viable seed, were less chlorotic/necrotic, and with less inhibition of growth (leaf number, rosette diameter, plant height, stalk number, and dry weight). When irrigated with 200 mM NaCl, both transformants and WT plants died without producing seeds, but M6PR transformants bolted and showed significantly less chlorosis and necrosis and had higher survival rates and dry weights than those of WT. Under normal growth conditions there were no negative effects of the M6PR transgene on overall growth (leaf number, rosette diameter, plant height, stalk number, and dry weight), or time to bolting/flowering or seed production compared to wild type (WT). Despite the lack of effects on phenotype in the absence of salt stress, genome wide expression analyses indicated that expression levels of more than 2000 genes were altered by presence of the M6PR transgene: however, there were many fewer differences observed between the M6PR and non-transgenic plants in the presence of salt stress (459), suggesting that M6PR pre-conditioned the plants for exposure to salinity. Gene categories most affected in the M6PR transgenic plants were involved in DNA binding, signal transduction, metabolism/energy, cell structure, membrane transport, defense response, and transcription. Notably, a large number of known stress genes were induced, including those related to cell walls, biotic stress, and ABA- and ethylene-responses. Our work and that in other labs has suggested that mannitol acts as an osmoprotectant, but mannitol levels are invariably quite low, and perhaps inadequate to explain its effects as an osmoprotectant. The gene expression data here indicate that stress tolerance of mannitol-producing Arabidopsis is also due, at least in part, to enhanced expression of a number of stress inducible genes related to both biotic and abiotic stress tolerance. Both M6PR transgenic and Col WT plants were grown in the growth chamber in the absence and presence of salt stress. Plants from 20 days after sowing (6 days after salt treatment) were used for RNA extraction and hybridization on Affymetrix microarrays. There were two biological replicates for each genotype and salt treatment combination.

Project description:Transcriptional variation, also called expression level polymorphism (ELP), contributes to intra-specific phenotypic variation in many organisms. Differentially expressed transcripts are typically enriched for stress-related genes, suggesting that differences in response to the environment are a particularly common point of divergence among gentoypes. Analysis of ELPs also has been suggested as a way to assess unintended consequences of transgene introduction; however, it is important that interpretation of transcriptional changes be performed within the context of potential fitness effects. In these studies we sought to examine differential gene expression in response to salinity for two widely used Arabidopsis thaliana ecotypes, Wassilewskija (Ws) and Columbia (Col), and a single gene mutation (glabrous, gl1-1) in the Col background (Col(gl)), in relation to genetic, phenotypic, and fitness differences. Growth analyses were performed with seedlings germinated on culture media and growth chamber-grown plants carried through the full life cycle. Transcriptome analyses were performed with salt treated and control growth-chamber grown plants six days post initiation of salt stress. Ws plants had the least salt injury and highest dry matter accumulation and seed production in salt stressed conditions. ELPs among genoytypes and in response to 100 mM NaCl were enriched for genes associated with response to stress, including stress-associated transcription factors, heat shock and redox metabolism genes, and R genes. Application of salt resulted in many more transcripts up- or down-regulated in Col and Ws than in Col(gl). Many of the transcripts influenced by salt in Col were already altered in gl1-1 plants in the absence of salt, although Col(gl) plants did not show any detectable signs of stress, or effects on fecundity in the absence of salt treatment. The majority of salt-induced transcriptional changes that occurred in Ws also occurred in Col, suggesting common salt stress responses in these two ecotypes. Many more genes were affected by salt in Col than Ws, however, possibly reflecting the greater salt injury observed for Col. There was minimal overlap between the transcripts that differed for Ws and Col prior to salt treatment and those that were subsequently affected by salt stress. Thus, many genes conferring comparative salt stress tolerance in Ws likely differ from those whose expression levels are modified in response to salt stress. These studies demonstrate transcriptional variation among Arabidopsis genotypes in response to salt stress. Greater transcriptome differences did not necessarily correspond with greater genetic difference or phenotypic differences in morphology, fecundity, and resistance to salt stress. These results suggest that depending on circumstance, transcriptional changes can reflect response to injury, facilitate adaptive expression of fitness-associated traits, or allow for phenotypic buffering to minimize the impact of genetic changes. Three Arabidopsis genotypes were grown in the growth chamber in the absence and presence of salt stress. Plants from 20 days after sowing (6 days after salt treatment) were used for RNA extraction and hybridization on Affymetrix microarrays. There were two biological replicates for each genotype and salt treatment combination.

Project description:Constituting the final growth phase during the lifecycle of maize (Zea Mays L.), leaf senescence plays an important biological role in grain yield in crops. We undertook proteomic and physiological analyses in inbred line Yu816 in order to unravel the underlying mechanisms of leaf senescence induced by preventing pollination. A total of 6,941 proteins were identified by Isobaric tags for Relative and Absolute Quantitation (iTRAQ) analysis. Proteomic analyses between pollinated (POL) and non-pollinated (NONPOL) plants indicated that 973 different proteins accumulated in NONPOL plants. The accumulated proteins were classified into various groups, including response to stimuli, cellular processes, cell death and metabolic processes using functional analysis. Furthermore, in accordance with the changes in these different accumulated proteins, analysis of changes in leaf total soluble sugars and starch content showed that the prevention of pollination can disturb endogenousplant hormone and sugar metabolism and lead to ROS bursts, protein degradation and photosystem breakdown, eventually resulting in leaf senescence. This represents the first attempt at global proteome profiling in response to induced leaf senescence by preventing pollination in maize, and provides a better understanding of the molecular mechanisms involved in induced leaf senescence.Constituting the final growth phase during the lifecycle of maize (Zea Mays L.), leaf senescence plays an important biological role in grain yield in crops. We undertook proteomic and physiological analyses in inbred line Yu816 in order to unravel the underlying mechanisms of leaf senescence induced by preventing pollination. A total of 6,941 proteins were identified by Isobaric tags for Relative and Absolute Quantitation (iTRAQ) analysis. Proteomic analyses between pollinated (POL) and non-pollinated (NONPOL) plants indicated that 973 different proteins accumulated in NONPOL plants. The accumulated proteins were classified into various groups, including response to stimuli, cellular processes, cell death and metabolic processes using functional analysis. Furthermore, in accordance with the changes in these different accumulated proteins, analysis of changes in leaf total soluble sugars and starch content showed that the prevention of pollination can disturb endogenousplant hormone and sugar metabolism and lead to ROS bursts, protein degradation and photosystem breakdown, eventually resulting in leaf senescence. This represents the first attempt at global proteome profiling in response to induced leaf senescence by preventing pollination in maize, and provides a better understanding of the molecular mechanisms involved in induced leaf senescence.

Project description:Genome-wide transcriptional profiling of Arabidopsis thaliana to a combination of heatwave and drought under ambient and elevated CO2. Goal of this study was elucidate the transcriptional responses to a combination of heat wave and drought, and to see how these responses are modifed under future climate (high) CO2. Climate changes increasingly threaten plant growth and productivity. Such changes are complex and involve multiple environmental factors, including rising CO2 levels and climate extreme events. As the molecular and physiological mechanisms underlying plant responses to realistic future climate extreme conditions are still poorly understood, a multiple organizational level-analysis (i.e. eco-physiological, biochemical and transcriptional) was performed, using Arabidopsis exposed to incremental heat wave and water deficit under elevated CO2.The climate extreme resulted in biomass reduction, photosynthesis inhibition, and considerable increases in stress parameters. Photosynthesis was a major target as demonstrated at the physiological and transcriptional levels. In contrast, the climate extreme treatment induced a protective effect on oxidative membrane damage, most likely as a result of strongly increased lipophilic antioxidants and membrane-protecting enzymes. Elevated CO2 significantly mitigated the negative impact of a combined heat and drought, as apparent in biomass reduction, photosynthesis inhibition, chlorophyll fluorescence decline, H2O2 production and protein oxidation. Analysis of enzymatic and molecular antioxidants revealed that the stress-mitigating CO2 effect operates through up-regulation of antioxidant defense metabolism, as well as by reduced photorespiration resulting in lowered oxidative pressure. Therefore, exposure to future climate extreme episodes will negatively impact plant growth and production, but elevated CO2 is likely to mitigate this effect. Transcriptome analysis was performed by Agilent Arabidopsis (V4) 4x44K platform which represented all known genes in the Arabidopsisgenome. Experiments were performed using a modified loop design (Knapen et al., 2009). This design consisted of total 8 arrays; sample from each treatment was labelled once and has 4 biological replicates, two of which were labelled in red and two in green

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Expression profiling of the heat stress response in acclimated and unacclimated peanut

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