Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Leaf age determines flood resilience through distinct submergence and recovery effects

ABSTRACT: RNA sequencing (RNA-seq) was performed to investigate potential roles of the unfolded protein response in submergence and recovery responses of Arabidopsis thaliana. Plants were grown to the 10-leaf stage and then subjected to 4 days of complete submergence. Samples were collected at four time points: prior to submergence (T0), after 1 day of submergence (1d), after 4 days of submergence (4d - end of submergence period), and 3 hours after de-submergence (recovery). For each time point, rosettes were collected and divided into two developmental zones: (i) old leaves (leaves 1 to 5) and (ii) young leaves (leaves 6 to 10 including the shoot apical meristem (SAM)). There were three biological replicates per tissue type of each genotype at each time point. Each biological replicate consisted of tissue pooled from two plants. Two genotypes were examined: Col-0 (wild type) and the bzip60/bzip28 double mutant, which is impaired in unfolded protein response signaling. RNA-seq libraries were prepared using the Illumina TruSeq Stranded mRNA protocol and sequenced on an Illumina platform to generate paired-end reads.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Hans van Veen

PROVIDER: E-MTAB-15812 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: The goal of this study is to compare the transcriptomes expressed during submergence stress of two Brachypodium distachyon ecotypes with contrasting survival under this stress. Bd21 is a submergence sensitive ecotype with EC50 of 2.5 days and Bd2-3 is a tolerant ecotype with EC50 of 4 days. Methods (Stress): Brachypodium Bd21 and Bd2-3 plants (14-day-old, 6 leaves stage) were submerged in a water column of 30 cm inside opaque-wall plastic tanks. Light still reached the plants at 40 uE m-2 s-??1. Ecotypes were submerged side-by-side in a randomized manner; only plants submerged in the same tank were compared. Controls were grown in plastic tanks without a water column. Submergence stress started at ZT14 (2h before night, long-day regime 16h light, 8h dark). Above ground tissue was collected after 48 h submergence stress in liquid nitrogen and stored at -80C in an ultra freezer until further processing. Tissue was ground to powder with mortar, pestle and liquid nitrogen avoiding thawing. Control and submerged total RNA was extracted with TRIzol reagent (Invitrogen, 15596018), purified with Direct-zol RNA mini prep columns (Zymo Research, R2050) and digested in-column with DNAse I (ThermoScientific, EN0521). RNA integrity and concentration was verified in denaturing 1.0% agarose gels, Nanodrop 2000 (ThermoScientific) and in a Bioanalyzer 2100 (Agilent) with the integrated software 2100 Expert, samples had a RNA Integrity Number (RIN) between 6.4-7.2 characteristic of aerial plant tissue (Babu and Gassman, 2011). Total RNA extracted from control and submerged tissue from three independent experiments consisting each of four individuals were used to construct cDNA indexed libraries and sequenced in a HiSeq2500 (Illumina) at 1x50 format, making a total of 12 sequenced libraries (tolerant and intolerant ecotype, control and submerged, all experimental triplicates) in a 2-lane format. RNA integrity, library construction and sequencing was performed as a service at the Unidad Universitaria de Secuenciacion Masiva, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico (IBT-UNAM). Differential Gene Expression (DGE) analysis was performed with edgeR using a generalized linear model and false discovery rate <0.05 (FDR). To group differentially expressed transcripts a logFC value of 1.5 (up-regulated) or -1.5 (down-regulated) and a FDR <0.05x10-5 were selected. GO analysis of differential transcripts was performed at phytozome.org Results: We identified commonly up-regulated genes (317) and exclusively up-regulated in Bd2-3 (466) or Bd21 (706). Regarding down-regulation, 330 transcripts were common, an exclusively 851 and 1026 for Bd2-3 and Bd21, respectively. GO analysis indicated that oxidative stress, pathogen responses and nitric oxide homeostasis were the most differential characteristics of tolerant ecotype Bd2-3. Conclusions: The use of triplicate RNAseq data of transcriptomes expressed in ecotypes with contrasting tolerance to submergence under long-day light regime, allowed us to identify common responsive routes such as SUSY, glycolysis, anaerobic routes (alanine, ethanol, lactate, GABA) and glyoxylate cycle. It also enabled us to discover integrated oxidative stress and NO homeostasis pathways that are differentially expressed in the tolerant ecotype. We expect that this information can be translated to agricultural relevant plants to increase our knowledge and biotechnological possibilities on plant submergence stress. Sequenced libraries (triplicates, HiSeq2500 Illumina, 1x50 format) of aerial tissue (control and 48h submergence stress) of Brachypodium distachyon Bd21 (sensitive) and Bd2-3 (tolerant).

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Leaf age determines flood resilience through distinct submergence and recovery effects

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