Dataset Information

Submergence Stress Alters the Expression of Clock Genes and Configures New Zeniths and Expression of Outputs in Brachypodium distachyon.

ABSTRACT: Plant networks of oscillating genes coordinate internal processes with external cues, contributing to increased fitness. We hypothesized that the response to submergence stress may dynamically change during different times of the day. In this work, we determined the transcriptome (RNA sequencing) of the model monocotyledonous plant, Brachypodium distachyon, during a day of submergence stress, low light, and normal growth. Two ecotypes of differential tolerance, Bd21 (sensitive) and Bd21-3 (tolerant), were included. We submerged 15-day-old plants under a long-day diurnal cycle (16 h light/8 h dark) and collected samples after 8 h of submergence at ZT0 (dawn), ZT8 (midday), ZT16 (dusk), ZT20 (midnight), and ZT24 (dawn). Rhythmic processes were enriched both with up- and down-regulated genes, and clustering highlighted that the morning and daytime oscillator components (PRRs) show peak expression in the night, and a decrease in the amplitude of the clock genes (GI, LHY, RVE) was observed. Outputs included photosynthesis-related genes losing their known rhythmic expression. Up-regulated genes included oscillating suppressors of growth, hormone-related genes with new late zeniths (e.g., JAZ1, ZEP), and mitochondrial and carbohydrate signaling genes with shifted zeniths. The results highlighted genes up-regulated in the tolerant ecotype such as METALLOTHIONEIN3 and ATPase INHIBITOR FACTOR. Finally, we show by luciferase assays that Arabidopsis thaliana clock genes are also altered by submergence changing their amplitude and phase. This study can guide the research of chronocultural strategies and diurnal-associated tolerance mechanisms.

SUBMITTER: Medina-Chavez L

PROVIDER: S-EPMC10218231 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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Submergence Stress Alters the Expression of Clock Genes and Configures New Zeniths and Expression of Outputs in <i>Brachypodium distachyon</i>.

Medina-Chávez Lucisabel L Camacho Christian C Martínez-Rodríguez Jorge Arturo JA Barrera-Figueroa Blanca Estela BE Nagel Dawn H DH Juntawong Piyada P Peña-Castro Julián Mario JM

International journal of molecular sciences 20230510 10

Plant networks of oscillating genes coordinate internal processes with external cues, contributing to increased fitness. We hypothesized that the response to submergence stress may dynamically change during different times of the day. In this work, we determined the transcriptome (RNA sequencing) of the model monocotyledonous plant, <i>Brachypodium distachyon</i>, during a day of submergence stress, low light, and normal growth. Two ecotypes of differential tolerance, Bd21 (sensitive) and Bd21-3 ...[more]

PMID: 37239900

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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 μE 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 -80ºC 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 Secuenciación Masiva, Instituto de Biotecnología, Universidad Nacional Autónoma de México (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.

Project description:BackgroundBrassinosteroids (BRs) are steroidal phytohormones that are involved in diverse physiological processes and affect many important traits, such as plant stature, stress tolerance, leaf angle, fertility, and grain filling. BR signaling and biosynthetic pathways have been studied in various plants, such as the model dicot Arabidopsis thaliana; however, relatively little is known about these pathways in monocots.ResultsTo characterize BR-related processes in the model grass Brachypodium distachyon, we studied the response of these plants to the specific BR biosynthesis inhibitor, propiconazole (Pcz). We found that treatments with Pcz produced a dwarf phenotype in B. distachyon seedlings, similar to that observed in Pcz-treated Arabidopsis plants and in characterized BR-deficient mutants. Through bioinformatics analysis, we identified a list of putative homologs of genes known to be involved in BR biosynthesis and signaling in Arabidopsis, such as DWF4, BR6OX2, CPD, BRI1, and BIN2. Evaluating the response of these genes to Pcz treatments revealed that candidates for BdDWF4, BR6OX2 and, CPD were under feedback regulation. In addition, Arabidopsis plants heterologously expressing BdDWF4 displayed tall statures and elongated petioles, as would be expected in plants with elevated levels of BRs. Moreover, heterologous expression of BdBIN2 in Arabidopsis resulted in dwarfism, suggesting that BdBIN2 functions as a negative regulator of BR signaling. However, the dwarf phenotypes of Arabidopsis bri1-5, a weak BRI1 mutant allele, were not complemented by overexpression of BdBRI1, indicating that BdBRI1 and BRI1 are not functionally equivalent.ConclusionWe identified components of the BR biosynthetic and signaling pathways in Brachypodium, and provided examples of both similarities and differences in the BR biology of these two plants. Our results suggest a framework for understanding BR biology in monocot crop plants such as Zea mays (maize) and Oryza sativa (rice).

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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Submergence Stress Alters the Expression of Clock Genes and Configures New Zeniths and Expression of Outputs in Brachypodium distachyon.

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Submergence Stress Alters the Expression of Clock Genes and Configures New Zeniths and Expression of Outputs in <i>Brachypodium distachyon</i>.

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