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RNA-Seq analysis of Col-0, VQ28OE9 and VQ28KO12

ABSTRACT: The goal of the RNA sequencing is to find out the signal pathway the VQ28 gene involved in. Uninfected 30-day-old detached Arabidopsis rosette leaves of wild type Col-0, knock-out line VQ28KO12 and overexpression line VQ28OE9 were collected to isolate total RNA. Then, we created a final cDNA library and performed the 2×150bp paired-end sequencing on an Illumina Novaseq™ 6000. After removed the low quality bases and undetermined bases, we mapped reads to the genome. The differentially expressed mRNAs were selected with fold change > 2 or fold change < 0.5 and p value < 0.05, and then analysis GO enrichment and KEGG enrichment to the differentially expressed mRNAs. In comparison with Col-0, constitutively overexpressing VQ28 caused the enrichment of most genes in MAPK signaling pathway and plant hormone signal transduction. Among the 11 genes up-regulated in VQ28OE9 but down-regulated in VQ28KO12, examining their expression by querying the Arabidopsis eFP Browser which contains an extensive collection of gene expression microarray data, six of them were involved in the response to abscisic acid (ABA). qRT-PCR analyses of expression presented that the six ABA-related genes were up-regulated in VQ28OE9 without infection.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE189933 | GEO | 2022/12/30

REPOSITORIES: GEO

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Project description:Abscisic acid (ABA) is a key hormone for plant growth, development and stress adaptation. Perception of ABA through four types of receptors has been reported. We show here that impairment of ABA perception through the PYR/PYL/RCAR branch reduces vegetative growth and seed production, and leads to a severely open stomata and dramatic ABA insensitive phenotype, even though other branches for ABA perception remain functional. Arabidopsis sextuple mutant impaired in 6 PYR/PYL receptors, namely PYR1, PYL1, PYL2, PYL4, PYL5 and PYL8, was able to germinate and grow even on 100 mM ABA. Whole-rosette stomatal conductance (Gst) measurements revealed that leaf transpiration in the sextuple pyr/pyl mutant was higher than in the ABA-deficient aba3-1 or ABA-insensitive snrk2.6 mutants. The gradually increasing Gst values of plants lacking three, four, five and six PYR/PYLs indicate quantitative regulation of stomatal aperture by this family of receptors. The sextuple mutant lacked ABA-mediated activation of SnRK2s and ABA-responsive gene expression was dramatically impaired as was reported in snrk2.2/2.3/2.6. In summary, these results show that ABA perception by PYR/PYLs plays a major role to regulate seed germination and establishment, basal ABA signaling required for vegetative and reproductive growth, stomatal aperture and transcriptional response to the hormone. Four biological replicates were generated for the 3 genotypes, pyr1pyl1pyl2pyl4pyl5pyl8 sextuple mutant, snrk2.2/2.3/2.6 triple mutant, and Col-0. All the samples were treated with ABA for 3 hours before harvesting. The four Col-0 samples were mix to create an unique reference Col-0 sample. Two comparasion were made, fisrt one, PYR/PYL sextuple mutant versus Col-0 reference sample and second one, SnRK2 triple mutant versus Col-0 reference sample. In each comparasion four biological replicates were made. Replicas number 1 and 2 were labeled with Cy5 for the mutant sample and Cy3 for the Col-0 reference sample, while the other two replicas,#3 and #4, were reversed-labeled.

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RNA-Seq analysis of Col-0, VQ28OE9 and VQ28KO12

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