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Effect of nitrogen compounds on imbibed mutant seeds impaired in glucosinolate biosynthesis

ABSTRACT: These RNAseq data are from germinating cyp79B2, cyp79B3, myb28, myb29 (qko), cyp79B2, cyp79B3 (cyp) and myb28 myb29 (myb) mutant seeds. Col-0 genotype was used as the Arabidopsis' WT reference ecotype and all mutant seeds are from the Col-0 genetic background. Samples were sown on agarose 0,8%, supplemented or not by nitrate KNO3 10Mm or potassium thiocyanate KSCN 8µM. RNA isolation was performed using NucleoSpin® RNA Plant and Fungi kit. RNA quantification was obtained with a NanoDrop ND-100 (NanoDrop Technologies, DE, USA) and the RNA Integrity was measured with a 4100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). cDNA library construction and sequencing were performed with DNBseq™ technology at Beijing Genomics Institute BGI. FastQC was used to check read quality, mapping analysis and transcript quantiifcation were performed using a quasi-mapping alignment from Salmon.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE221567 | GEO | 2022/12/26

REPOSITORIES: GEO

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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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Effect of nitrogen compounds on imbibed mutant seeds impaired in glucosinolate biosynthesis

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