Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of the radicle and micropylar endosperm cap of Lepidium seeds during germination in response to temperature

ABSTRACT: An experiment was performed to compare global transcription patterns in two tissues of Lepidium sativum seeds at different temperatures during imbibition for 10h. RNA extractions from the two tissues were hybridized to Affymetrix ATH1 Arabidopsis microarrays. The two tissues were the radicle and the micropylar endosperm cap that covers it. The interaction of these two tissues is thought to control the timing of germination though alterations in the extensibility of the radicle and the physical resistance of the micropylar endosperm cap to radicle penetration. Only seeds without endosperm rupture were sampled.

ORGANISM(S): Lepidium sativum

SUBMITTER: Gerhard Leubner

PROVIDER: E-MTAB-1811 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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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Transcription profiling of the radicle and micropylar endosperm cap of Lepidium seeds during germination in response to temperature

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