Dataset Information

Labratory Germination SWATH-MS proteomics

ABSTRACT: Identification and relative quantification of proteins present during germination

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Hordeum Vulgare (barley)

TISSUE(S): Endosperm

SUBMITTER: Edward Kerr

LAB HEAD: Benjamin Luke Schulz

PROVIDER: PXD019383 | Pride | 2021-07-27

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20160725_001_0_1_IDA.wiff	Wiff
	20160725_001_0_1_IDA.wiff.scan	Wiff
	20160725_002_G-24_1_IDA.wiff	Wiff
	20160725_002_G-24_1_IDA.wiff.scan	Wiff
	20160725_003_F-72_1_IDA.wiff	Wiff

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Publications

Proteomics reveals commitment to germination in barley seeds is marked by loss of stress response proteins and mobilisation of nutrient reservoirs.

Osama Sarah K SK Kerr Edward D ED Yousif Adel M AM Phung Toan K TK Kelly Alison M AM Fox Glen P GP Schulz Benjamin L BL

Journal of proteomics 20210415

Germination is a critical process in the reproduction and propagation of flowering plants, and is also the key stage of industrial grain malting. Germination commences when seeds are steeped in water, followed by degradation of the endosperm cell walls, enzymatic digestion of starch and proteins to provide nutrients for the growing plant, and emergence of the radicle from the seed. Dormancy is a state where seeds fail to germinate upon steeping, but which prevents inappropriate premature germina ...[more]

PMID: 33866056

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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