| Higher plants usually undergo a series of distinct phases of embryonic,vegetative and reproductive development to complete their life cycles.The vegetative phase consists of a juvenile vegetative phase and an adult vegetative phase.The juvenile-toadult transition is referred to as vegetative phase change,while the vegetative-toreproductive transition is commonly known as flowering.Vegetative phase change regulates a plethora of physiologically,biochemically,and economically important processes.The vegetative phase is the basis of reproductive development,and it also regulates biomass production,plant architecture,biotic and abiotic responses,the production of adventitious root,the biosynthesis of plant secondary metabolites,and so on.Therefore,it is of great theoretical and practical significance to study the molecular mechanism of JAT in plants.It has been shown that JAT is regulated by the miR156SPL(SQUAMOSA PROMOTER BINDING PROTEIN-lIKE)pathway.Although the transcriptional regulation of miR156 and its SPL target genes has been reported,there are only few reports on how external environmental factors or factors upstream and downstream integrate into the miR156-SPL pathway to modulate JAT.Plant hormones regulate multiple important plant growth and developmental processes.Previous studies have shown that GA(Gibberellin),JA(Jasmonic acid)and CK(Cytokinin)play a role in vegetative phase change by integration into the miR156-SPL pathway.However,whether other phytohormones play a role in this process remains unknown.To explore and identify more regulatory factors involved in JAT,we performed a forward genetic screen to identify mutants with abnormal transition from the juvenile to the adult phase in an EMS-mutagenized Arabidopsis Col-0 seeds,and we recovered a mutant exhibiting a delayed vegetative phase change phenotype,which we named del2(delayed juvenile-to-adult phase transition mutant 2).In the present study,the molecular mechanism of how DEL2 regulates plant JAT is investigated by using genetic,molecular biology,biochemical and physiological approaches.The results are as follows:1.Del2 exhibited a characteristic delayed JAT phenotype,including delayed abaxial trichome production on leaves,a smaller leaf L/W ratio,a slower leaf initiation rate,dwarf,and late flowering compared with WT.The mutation was narrowed down to a region between primer ciwl and F19K16(A)on chromosome I through map-based cloning.We cloned and sequenced the candidate gene DWF5(DWARF5),and the result indicated that there was a G-to-A substitution at the 1848th base in the ORF(OPEN READING FRAME),leading to the premature termination of DWF5 translation.The causal mutation was further confirmed by the restored phenotype exhibited by the plants transformed with a wild-type DWF5 genomic copy.Therefore,del2 was renamed dwf5.2.It is known that DWF5 encodes the Δ7-sterol reductase in the BR(brassinosteroid)biosynthetic pathway.The content of BL in dwf5 was significantly lower than that in WT.Moreover,BL(brassinolide)treatment could rescue the delayed vegetative phase change phenotype of dwf5.3.Quantitative determination of the relative expression of some key genes in the miR156-SPL pathway indicated that miR172 and MIR172B were both reduced remarkably in dwf5,whereas the expression of SPL3,SPL9,SPL13,TOE1(TARGET OFEAT1),and TOE2 remained unchanged compared with that in WT.Western blotting showed that the SPL9 protein in dwf5 was dramatically reduced.The function of SPL9 to activate MIR172B transcription in dwf5 was proved to be impaired by using the pSPL9::GR-rSPL9 dwf5 transgenic line treated with DEX(Dexamethathone).Western blotting of SPL9 in pSPL9::3×FLAG-rSPL9 dwf5 plants treated with MG132 and CHX indicated that SPL9 was less stable in dwf5,and was degraded through a proteasomal pathway.4.Genetic analysis indicated that overexpression of MIR172B or introduction of toeltoe2 could significantly rescue the delayed phase change phenotype of dwf5 except for the rounder leaf phenotype.These results suggest that miR172 and TOE1/2 functions downstream of DWF5,and the reduction of miR172 in dwf5 is partially responsible for the late abaxial trichome phenotype of dwf5,while leaf shape in dwf5 is regulated by other pathways.Overexpress pSPL9::rSPL9 in dwf5 impaired the function of SPL9 to promote vegetative phase change.5.BIN2(BR-INSENSITIVE2)was shown to interact with SPL9 and TOE1 in vitro and in vivo by yeast two-hybrid,BiFC(Bimolecular fluorescence complementation),pull down,and Co-IP(Coimmunoprecipitation)analyses.SPL9 and TOE1 have a classical BIN2 phosphorylation motif of TPVKT and TKLVT,respectively.IP-MS/MS(Immunoprecipitation-mass spectrometry)further indicated that the 102th amino acid of SPL9 and the 124th of TOE1 were phosphorylated inWT,but not in bin2-3 bill bill.In vitro kinase assay demonstrated that BIN2 could phosphorylate the above sites in SPL9 and TOE1,respectively.6.We introduced mutations of non-phosphorylation form(T-A)and constitutive phosphorylation form(T-D)in TPVKT of SPL9 and TKLVT of TOE1,respectively,and overexpressed the miR156/172-insensitive form of pSPL9::3×FLAG-rSPL9 and UBI10::3×FLAG-rTOEl with or without these mutations in WT.Phenotypic characterization of transgenic plants with above three forms showed that the T-A form of both pSPL9::3×FLAG-rSPL9 and UBI10::3×FLAG-rTOEl exhibited the strongest phenotype;the T-T form with no mutation showed an intermediate phenotype;while the T-D form exhibited the weakest phenotype.Western blotting suggested that the TA form accumulated more SPL9 and TOE1 proteins,and the T-T form had the intermediate levels of the SPL9 and TOE1 proteins,whereas the T-D form accumulated the lowest levels of the SPL9 and TOE1 proteins.These results imply that the BIN2 phosphorylation sites in SPL9 and TOE1 were critical for protein stability and their function to regulate vegetative phase change.7.The level of the miR1 72-insensitive form of TOE1(rTOEl)was reduced,while the miR172-sensitive form of TOE1(TOE1)was elevated in dwf5 compared with that in WT.This result suggests that plants employ both the BIN2-SPL9-miR172-TOE1 and the BIN2-TOE1 pathway to regulate the level of TOE1 antagonistically to maintain a homeostatic level of TOE1 with the function of the BIN2-SPL9-miR172-TOE1 pathway outweighing that of the BIN2-TOE1 pathway.Based on the above results,we proposed a model of how BRs participate in the miR156-SPL pathway to regulate vegetative phase change.In WT,the presence of normal levels of BRs suppresses the activity of BIN2 to phosphorylate and degrade SPL9 and TOE1 simultaneously to maintain a homeostatic level of TOE1 to modulate JAT;in dwf5,the reduced content of BRs leads to an elevated level of BIN2,thus increasing the phosphorylated form of SPL9 and TOE1 for subsequent proteolytic degradation.Under the co-regulation of both the BIN2-SPL9-miR172-TOE1 and the BIN2-TOE1 pathway,the TOE1 protein is elevated in dwf5,and it results in a delayed vegetative phase change phenotype in dwf5.This study uncovered that BRs as an important plant hormone play a novel role in regulation of vegetative phase change.BRs intergrate integrate into the miR156-SPL pathway through the direct interaction of BIN2 with SPL9 and TOE1.Therefore,this study reveals a sophisticated gene network for BRs in vegetative phase change in plants,and provides a novel model for future study of plant development. |
