The Molecular Mechanism Of RALFL32 Involved In Seed Germination In Arabidopsis

Seed germination is a crucial stage of the life cycle in higher plants.Seed germination is regulated by numerous environmental factors,including light,temperature and nutrients.The regulation of seed germination under adverse abiotic conditions,like salt stress,is important for the survival of the plant and the accomplishment of their life cycle.The phytohormone ABA can arrest seed germination to protect tender embryos by activating ABA signaling under stresses,which is a critical mechanism for protecting plants.So far most studies of ABA-mediated regulation of seed germination focus on functional analysis of different components in ABA signaling pathway and the interaction between ABA and other hormones.Whether there are other factors participated in these physiological processes needs to be further dissected.RALFs are small signaling peptides that mainly function on inhibition of plant cell growth by causing cell wall alkalinization.Previous studies on some RALFs from different plant species suggest that not all of them are negative regulators of plant cell growth,indicating the complexity and diversity of their functions.The microarray data show that the transcription levels of some RALFs are regulated by ABA.Thus,whether RALFs is involved in seed germination and their underlying regulatory mechanisms remain to be dissected.In this study,RALFL32(also known as RALF32),one member from RALF family in Arabidopsis,is functionally identified,which is involved in regulating seed germination that is regulated by ABA signaling.During seed germination,RALFL32 expression is up-regulated by exogenous ABA.The RALFL32-overexpressing lines are more sensitive to salt and ABA treatments than wild-type plants during seed germination,whereas the ralfl32 mutants exhibit obvious NaCl and ABA-resistant phenotypes of seed germination.These results demonstrated that RALFL32 is involved in seed germination under salt stress and ABA treatment.The investigation of RALFL32 in several transcriptional-factor mutants in ABA signaling(including abi3,abi4 and abi5)shows that the up-regulation of RALFL32 expression by ABA is related to ABI5.Furthermore,transient expression assay in tobacco leaves shows that ABI5 induces the expression of RALFL32.Bioinformatics analysis reveals that there are four ABRE motifs in the sequence of the RALFL32 promoter,which can be specifically recognized and bound by ABI5.EMSA and Y1H assays show that ABI5 can directly bind to the promoter of RALFL32.ChIP and real-time PCR analysis show that ABI5 binds to the promoter of RALFL32 in vivo.These results show that RALFL32 is targeted by ABI5 and involved in regulating seed germination under stresses.Because RALFL32 transcription is up-regulated by ABA and targeted by ABI5,we predicted that overexpressing RALFL32 was capable of partially rescuing abi5 insensitive to ABA on seed germination.The analysis of seed germination shows that the overexpression of RALFL32 can partially rescue the ABA-insensitive phenotype of abi5 during seed germination,further demonstrating that RALFL32 is a target gene by ABI5 and is involved in regulation of seed germination mediated by ABI5.The preliminary analysis of the mechanism regarding RALFL32 participating in seed germination under adverse conditions shows that The RAZFL32-overexpressing lines and ralfl32 mutant do not exhibit obvious phenotype on seed germination when grown on the different pH medium compared with wildtype,suggesting that alteration of pH may not participate in RALFL32-mediated seed germination.In addition,in-vitro pull-down analysis shows that RALFL32 does not bind to FERONIA(FER)which is known as a receptor kinase of RALF1.Therefore,identification of the receptor will provide important information for understanding the molecular mechanism of RALFL32 on seed germination in the future.In summary,this study demonstrated that a small signaling peptide RALFL32 is involved in seed germination mediated by ABA signaling under abiotic stresses.And it also reveals a transcription regulatory pathway in seed germination,in which RALFL32 is targeted by ABI5 and functions as a positive regulator in ABA-inhibited seed germination.