RNA-seq of germinating barley embryos in control and ABA
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB74509 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR12831800_1.fastq.gz | Fastqsanger.gz | |||
| ERR12831800_2.fastq.gz | Fastqsanger.gz | |||
| ERR12831801_1.fastq.gz | Fastqsanger.gz | |||
| ERR12831801_2.fastq.gz | Fastqsanger.gz | |||
| ERR12831802_1.fastq.gz | Fastqsanger.gz |
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Project description:To address the lack of information on the function of CBC in the ABA signaling pathway during seed germination in barley, we employed TILLING to identify mutants of the genes hvcbp20.ab and hvcbp80.b and subsequently created the double mutant hvcbp20.ab/hvcbp80.b. We found that mutations in both CBC subunits resulted in a unique ABA response that differed from that of single mutants. Through transcriptomic analyses, we identified differential gene expression and alternative splicing patterns. Notably, the hvcbp20.ab/hvcbp80.b double mutant exhibited altered AS regulation and significant changes in brassinosteroid signaling following ABA exposure. Seeds were sterilized in 20% sodium hypochlorite solution for 20 min and washed with sterilized water for 5 min. They were then placed in Petri dishes with three layers of filters and treated with either sterilized water or sterilized water containing 75 μM ABA (cis-trans-abscisic acid; catalog no. 862169; Sigma-Aldrich). The seeds were stratified for 4 d at 4°C and then transferred to a growth chamber. Germination was defined as the visible emergence of the radicle through the seed coat and was assessed on 1 and 7 DAI (Days After Imbibition). The assay was performed in three biological replicates, each comprising 30 seeds of each genotype per petri dish. At 1 DAI, the embryos were isolated from the endosperm using a sharp scalpel blade, placed in microcentrifuge tubes (Eppendorf) containing RNAlater reagent, and stored at 4°C until RNA isolation. RNA was isolated using the mirVana™ Isolation Kit (Ambion, USA) following the manufacturer’s instructions. RNA was isolated in four biological replicates, each consisting of 20 embryos. In total, 32 RNA samples were extracted. RNA concentration and quality were assessed using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, USA) and Agilent Bioanalyzer (Agilent Technologies, Santa Clara, USA). We used grains and embryos of barley TILLING mutants: the hvcbp20.ab, in the CBP20 gene (CAP-BINDING PROTEIN 20; BaRT2v18chr6HG306340; 41,42), and hvcbp80.b, in the CBP80 gene (CAP-BINDING PROTEIN 80; BaRT2v18chr4HG195950), single mutants, the hvcbp20.ab/hvcbp80.b double mutant, and the 'Sebastian' wild-type (WT), all sourced from the HorTILLUS population 91. Single mutants were identified through TILLING in the M2 generation (then backcrossed with WT to clean the genetic background), and the double mutant was isolated from the F2 progeny following crossbreeding of the single mutants
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Project description:During germination, the availability of sugars, oxygen, or cellular energy fluctuates under dynamic environmental conditions, and the global RNA profile of rice genes can be affected by their availabilities. In the aerobically germinating rice embryos, most sugar-regulated genes are responsive to low energy and anaerobic conditions, indicating that sugar-regulation is closely associated with energy and anaerobic signaling. The interference pattern of sugar-regulation by either anaerobic or low energy conditions indicates that induction is likely the more prevalent regulatory mechanism than repression for the alteration in the expression of sugar-regulated genes in aerobically germinating rice embryos. Among the aerobically sugar-regulated genes, limited genes exhibit sugar regulation under anaerobic conditions, indicating that anaerobic conditions strongly influence sugar-regulated gene expression. Anaerobically responsive genes are highly overlapped with low-energy responsive genes. In particular, the expression levels of anaerobically downregulated genes are consistent with those induced by low energy-conditions, suggesting that anaerobic downregulation results from the prevention of aerobic respiration due to the absence of the final electron acceptor, i.e., molecular oxygen. It was noted that abscisic acid (ABA)-responsive genes were over-representative of the genes upregulated under low energy conditions, in contrast to the downregulated genes. This suggests that either ABA itself or upstream signaling components of the ABA signaling pathway are likely to be involved in the signaling pathways activated by low energy conditions.
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