A Novel Protein PGABH Mediates Seed Germination And Post Germination Growth Arrest By Abscisic Acid

In flowering plant, the completion of seed germination represents a critical ecologicaland agronomic trait, determining when plants enter ecosystems. Seed germination isclassically described by three phases of water uptake. Initially, the dry seed imbibes andreinitiates metabolic processes (phase I); subsequently, it enters a lag phase of water uptake(phase II), which is followed by further water uptake, resulting in the emergence of the radicle(phase III). The process of germination is highly regulated by both internal and external cuesthat determine the optimum time for plant establishment, including plant hormones.The plant hormone abscisic acid (ABA) is a negative regulator of germination. Exposureof seeds to ABA during germination leads to rapid but reversible arrest in development.ABA-mediated postgermination arrest allows germinated seedlings to be more tolerant toearly water stress. The transcription factors ABI3and ABI5are known to be importantregulators of ABA-dependent growth arrest during germination. Here, we studied PGABHand the relationship between PGABH and ABI3and ABI5. The results are as following:(1) By the analysis of germination rates and green cotyledons of WT and pgabh mutantduring germination and post germination, we found that the germination rates of pgabhmutant were reduced significantly than WT. The higher ABA concentration, the significantinhibition of germination of pgabh mutant. The green cotyledons of pgabh mutant were alsoreduced than WT. The higher ABA concentration, the significant inhibition of greencotyledons of pgabh mutant. These results indicated that PGABH negatively regulatedABA-mediated germination and post germination growth in Arabidopsis;(2) During germination, PGABH RNAi line showed the same responses as pgabh mutantto ABA, while PGABH OE lines showed the contrast responses to ABA. These results furtherindicated that PGABH negatively regulated ABA-mediated germination and post germinationgrowth in Arabidopsis;(3) We analyzed the expression pattern of PGABH in Arabidopsis by qRT-PCR. theresults indicated that PGABH were higher during1to3days of germination, and the expression levels were reduced with the germination time elongation. The PGABHpromoter::GUS analysis also showed the same results. These indicated that PGABH mainlyexpressed in the early stages during germination;(4) By analysis the GFP fusion protein of PGABH throght confocal, we found thatPGABH localized to the chloroplast membrane;(5) The expression of ABI3and ABI5were induced higher in the pgabh mutant than thatin WT in the presence of ABA by qRT-PCR analysis. The higher protein accumulation ofABI3and ABI5in the pgabh mutant than in WT in the presence of ABA were also illustratedby Western Blot. These results indicated that PGABH negatively regulated ABI3and ABI5toregulate the ABA signaling during germination;(6) We further obtained the double mutant of pgabh abi5Unlike the pgabh mutant, thegermination rates of pgabh abi5were higher than WT. This indicated that PGABH functionedupstream of ABI5during ABA mediated germination of Arabidopsis;(7) We also carried out the germination experiments under high osmotic stress. Thegermination rates of pgabh mutant were higher than WT, indicating the involvement ofPGABH in response to osmotic stress.