AtDGA1 Is Involved In ABA-suppression Of Seed Germination And Post-germination Growth

Abscisic acid(ABA)is one of the most important phytohormones.It plays a very important role in many aspects of plant growth and development,which includes promoting stomatal closure and inhibiting stomatal opening;promoting senescence and inhibiting growth;regulating flowering time and seed production;and inhibiting seed germination and post-germination growth.Seed germination that begins with imbibition and ends with radicle emergence is a crucial stage in the early development of higher plant.The structures of cells are changed during seed imbibition.The biomacromolecules such as damaged proteins and DNA and cell membranes,organelles and cytoskeletons can be repaired during seed imbibition.Meanwhile,a lot of metabolic activities in seed can be activated,including some metabolisms related to sugars,amino acids,organic acids and lipids.Structure alteration and metabolism recovery in a cell are necessary to seed germination.ABA can suppress seed germination by inhibiting structure alteration and metabolism recovery.Cotyledon greening is also included in seed germination in a broader term.It is a key developmental event in post-germination growth,which marks the formation of plant photosynthesis ability and the starting point of photoautotrophic growth.Radicle emergency and cotyledon greening are often used as statistical indicators in seed germination experiment.After cotyledon greening,plant starts to accelerate growth:the main root elongates rapidly,euphylla is gradually emerging and expanding quickly and the fresh weight increases rapidly.Both endogenous ABA content and its signal transduction have a close relationship with seed germination and post-germination growth.Many mutants involved in ABA metabolic levels or signal transduction have phenotype related to seed germination.aba2,cyp707a,and from abil to abi5 are such mutants.As transcription factors,ABI4 and ABI5 suppress seed germination through their downstream target genes which specifically influence seed germination vigour.Besides,some ABA related abiotic stresses,such as salt,osmotic and glucose stresses can also inhibit seed germination and post-germination growth,because they can significantly enhance endogenous ABA content.Gibberellin acid can antagonize inhibition effect of ABA on seed germination.Both GA and ABA are two most important phytohormones that can regulate seed germination.Other phytohormones can also influence seed germination through cross talk with them.Exogenous application of ABA in high concentration(such as 10 ?M)can completely inhibit seed germination while ABA in low concentration(such as 0.25 ?M)can only delay but not suppress seed germination,which indicates there are some unknown mechanisms that can relieve or impair inhibition effect of ABA on seed germination.Then,how does the cell remove the ABA inhibition effect during seed germination?Is there any other unknown gene involved in ABA signalling with a negative role?In order to seek such genes and to further study the mechanism of ABA-suppression of seed germination,a dgal(Delay of Germination under ABA condition 1)mutant was screened out from a group of T-DNA insertion mutants.In presence of exogenous ABA,emerged radicles rates,green cotyledons rates,relative root elongation and relative fresh weight of dgal mutant were much lower than those of wide type(WT),which suggested that DGA1 was involved in ABA-suppression of seed germination and post-germination growth.DGA1 is a novel gene without any report,so an in-depth study was carried out and the main results are as follows:1.Phenotype analysis indicated that dgal mutant was much more sensitive to ABA and its relative abiotic stresses such as salt,osmotic and glucose stresses in seed germination and post-germination growth.No other phenotype could be observed.2.DGA1 encoding production was predicted as a protein with a signal peptide,a B-lectin domain,a epidermal growth factor(EGF)domain,a transmembrane domain and a kinase domain.Subcellular localization experiments showed that the full length of DGA1 was localized in cytosol,nucleus,and cell membrane;the signal peptide and B-lectin domain(DGA1_SB)was localized in endoplasmic reticulum(ER);the DGA1?N(the fragment without signal peptide,B-lectin domain and EGF domain)and the kinase domain(DGA1_KD)had the same localization with the full length of DGA1.These results indicated that DGA1 was a protein which was localized in cytosol,nucleus,and cell membrane and had a signal peptide and B-lectin domain that could guide it to enter into ER,which suggested that the B-lectin domain may undergo a glycosylation reaction in ER.3.In vitro phosphorylation assay demonstrated that autophosphorylation could occurred in DGA1_KD,which suggested DGA1 was a kinase protein.4.qRT-PCR and GUS staining experiments indicated that DGA1 was mainly expressed in radicles of young seedlings and could be up-regulated by ABA and its relative abiotic stresses such as salt and osmotic stresses.5.Protoplast transfection and Yeast one hybrid assay indicated that ABI4 could bind to DGA1 promoter and significantly enhance its promoter activity;ABI5 could not bind to DGA1 promoter but could significantly enhance its promoter activity by cooperating with ABI4.6.qRT-PCR,protoplast transfection and RNA-seq experiments indicated that:DGA1 could not affect ABA metabolism but could inhibit ABA signal transduction;DGA1 could affect ABA-inhibition on GA2oxl and GA2ox2 expression;DGA1 could affect expression of genes involved in metabolic pathways relative to seed germination;DGA1 could affect expression of genes involved in chlorophyll biosynthesis and change chlorophyll content in seedlings.7.Yeast two hybrid and BiFC experiments indicated that DGA1 could interact with PUB9,a U-box E3 ligase.ABA could strengthen the interaction between them and change their localization from cytosol to cell membrane.In vitro phosphorylation assay couldn't detect phosphorylated PUB9 by DGA1_KD,indicating that DGA1_KD couldn't phosphorylate PUB9 in vitro or other unknown cofactors were needed for the phosphorylation of PUB9 by DGA1_KD.