Time of day determines Arabidopsis transcriptome and growth dynamics under mild drought
ABSTRACT: RNA sequencing on the third true Arabidopsis leaves of plants exposed to mild drought stress from 12 days after stratification onwards. Samples were harvested at 4AM, 12PM and 8PM on day 14, and at 4AM on day 15.
Project description:Detailed information: Rice (*Oryza sativa* L. cv. Nipponbare) is a drought-susceptible species which is well suited for studies of abiotic stress response because of the comprehensive bioinformatics resource available. By withholding water from the entire root system of young rice plants, or half the root system only, it was possible to infer the relative impact of signals arriving from roots growing in wet and dry soil on the shoot proteome. The global proteome of shoots had 685 proteins in common to all three drought treatments but there were major shifts in abundance of individual proteins within 16 functional categories. The dominant changes were analyzed more deeply. First, we investigated transport and cell component organization, where some proteins were up-regulated by drought but many more down-regulated. Proteins involved in protein metabolism were up-regulated in general by drought when they were responsible for protein degradation but those involved in protein synthesis were down-regulated when water was withheld. Stress-related proteins behaved very consistently by increasing in droughted plants but notably some proteins were most abundant when roots of the same plant were growing in both wet and dry soil. This suggests that drought signals are complex interactions and not simply the additive effect of water supply to the roots. Changes in carbohydrate-processing proteins were consistent with the passive accumulation of soluble sugars in shoots under drought, with hydrolysis of sucrose and starch synthesis both enhanced. Data analysis information: The result raw files were converted to mzXML format and processed through the global proteome machine (GPM) software (version 2.1.1) of the X!Tandem algorithm (freely available at http://www.thegpm.org). The 16 gel fractions were processed serially for each experiment and the output files were generated as non-redundant, merged files with protein identifications with log (e) values less than -1, for each individual gel fraction. A protein database compiled from NCBI *O*. *sativa* with 26938 protein sequences (August 2011) was used in GPM to search the tandem mass spectra; the database also included common trypsin and human peptide contaminants. False discovery rates (FDR) were evaluated by searching against a reversed sequence database. Search parameters included MS and MS/MS tolerances of +2 Da and +0.2 Da, carbamidomethylation of cysteine as fixed modifications, oxidation of methionine as variable modifications and tolerance of two missed tryptic cleavages and K/R-P cleavages.
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:DNA methylation is an important biological form of epigenetic modification, playing key roles in plant development and environmental responses. In this study, we examined single-base resolution methylomes of Populus under control and drought stress conditions using high-throughput bisulfite sequencing for the first time. Our data showed methylation levels of methylated cytosines, upstream2kp, downstream2kb, and repeatitive sequences significantly increased after drought treatment in Populus. Interestingly, methylation in 100 bp upstream of the transcriptional start site (TSS) repressed gene expression, while methylations in 100 – 2000bp upstream of TSS and within the gene body were positively associated with gene expression. Integrated with the transcriptomic data, we found that all cis-splicing genes were non-methylated, suggesting that DNA methylation may not associate with cis-splicing. However, our results showed that 80% of trans-splicing genes were methylated. Moreover, we found 1156 transcription factors (TFs) with reduced methylation and expression levels and 690 TFs with increased methylation and expression levels after drought treatment. These TFs may play important roles in Populus drought stress responses through the changes of DNA methylation. Taken together, these findings may provide valuable new insight into our understanding of the interaction between gene expression and methylation of drought responses in Populus. Methylomes of Poplar response to drought
Project description:RNA-seq data of Arabidopsis thaliana accessions exposed to mild drought or control treatments. The sampled tissue is the third leaf at the last day of proliferation (cell division phase).
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
Project description:Plants require a distinctive cohort of enzymes to coordinate division and cell expansion. Proteomic analysis now enables interrogation of immature leaf bases where these processes occur. Hence we investigated proteins in tissues sampled from leaves of a drought-tolerant rice (IAC1131) to provide insights into the effect of soil drying on gene expression when compared with the drought-sensitive Nipponbare. Shoot growth zones were dissected to count dividing cells and extract protein for subsequent Tandem Mass Tags (TMT) quantitative proteomic analysis. Gene Ontology (GO) annotations of differentially expressed proteins provided insights into responses of Nipponbare and IAC1131 to drought. Soil drying did not affect the proportion of mitotic cells in IAC1131. More than 800 proteins across most functional categories were up-regulated in drought (and down-regulated on re-watering) in IAC1131, including those involved in organization of the meristem and subsequent cell formation. On the other hand, the proportion of dividing cells in Nipponbare was severely impaired during drought and fewer than 200 proteins responded in abundance when the growing zones underwent a drying cycle. However, those proteins involved in oxidation state and response to external stimuli were more likely to be upregulated by drought, even in Nipponbare.
Project description:We also used microarray analysis to examine transcriptomic changes under drought, identifying thousands of genes that potentially mediate drought responses in the flower, including genes encoding transcription factors that likely play crucial regulatory roles. Arabidopsis were well-watered until after just bolting (after 24 days growth with the main stem about 1 cm high) when drought treatment was started by withholding water (defined as day 0 for drought treatment). The relative soil moisture content decreased sharply and, after about 80 hours (defined as day 3 for drought treatment), the relative soil moisture content was near 35% of the soil water-holding capacity. The soil water condition was maintained by daily watering until almost all the fruits were mature and ready to harvest (about 50 days). For well-watered (control) plants, 90% of the soil water-holding capacity was maintained until tissue harvest or after seed maturation (pots were weighed and watered twice per day). Unopened flower samples were collected, from both treated and control plants, at day 0, 3, 4, 5, 10.
Project description:We used microarray analysis to examine transcriptomic changes upon dreb1a under drought, identifying hundreds of genes that potentially function downstream of DREB1A and mediate drought responses in the flower, including genes encoding transcription factors that likely play crucial regulatory roles. DREB1a mutant (CS872453) were well-watered until after just bolting (after 24 days growth with the main stem about 1 cm high) when drought treatment was started by withholding water (defined as day 0 for drought treatment). The relative soil moisture content decreased sharply and, after about 80 hours (defined as day 3 for drought treatment), the relative soil moisture content was near 35% of the soil water-holding capacity. The soil water condition was maintained by daily watering until almost all the fruits were mature and ready to harvest. Unopened flower samples were collected from drought treated plants, at day 3, 4, 5.
Project description:We also used microarray analysis to examine transcriptomic changes under moderate drought, identifying thousends of genes that potentially mediate moderate drought responses in the flower, including genes encoding transcription factors that likely play crucial regulatory roles. Arabidopsis were well-watered until after just bolting (after 24 days growth with the main stem about 1 cm high) when moderate drought treatment was started by withholding water (defined as day 0 for moderate drought treatment). The relative soil moisture content decreased rapidly and, after about 48 hours the relative soil moisture content was near 50% of the soil water-holding capacity (first moderate drough treated sample M3 were collected at day 3 (72 h after withholding water)). The soil water condition was maintained by daily watering until almost all the fruits were mature and ready to harvest (about 50 days). For well-watered (control) plants, 90% of the soil water-holding capacity was maintained until tissue harvest or after seed maturation (pots were weighed and watered twice per day). Unopened floral bud samples were collected at day 0, 3, 4, 5, 10.