The Molecular Mechanism Study Of Regulation Floral Organ Development By JAG And TCP5 Genes In Arabidopsis Thaliana

The regulation of plant organ development is a basic developmental biology process controlled by a series of genetic mechanisms and environmental factors.Studies of the molecular mechanisms concerning organ development is a hotspot in plant science.In this study,we used floral petal of Arabidopsis thaliana as the model to dissect the function of JAGGED(JAG)and TEOSINTE BRANCHED 1,CYCLOIDEA AND PCF TRANSCRIPTION FACTOR 5(TCP5)in floral organ development,and genetic,molecular biology and bioinformatic methods were involved.The dissection of the genetic and molecular mechanisms of JAG and TCP5 provided a novel understanding of how these genes play crucial roles in plant organ development.The main results obtained from this study are summarized as follows:The phenotypic analysis of jag-1,tcp5 and the multi-gene mutant plants revealed that TCP5 regulate the development of petal negatively and functions redundantly with TCP13 and TCP17.tcp5 partially rescued the petal defection of jag-1 tcp5 double mutant,suggesting a putative downstream role of TCP5 in the JAG regulated petal developmental pathway.To explore the expression relationship between JAG and TCP5 in floral organ,we analyzed the gene expression of TCP5 in the jag-2 mutant and JAG over-expressing lines by qRT-PCR.The results of qRT-PCR analysis showed that the transcription factor JAG inhibited the expression of TCP5.The repression of TCP5 expression by JAG is likely to be direct.We also investigated the expression pattern of TCP5 using TCP5promoter-GUS line and histochemical staining analysis showed that compared with the wild-type Col-0,the TCP5 expression stage was advanced and expression duration was delayed in the jag-2 mutant petals.In addition,in the 11 th stage of petal development,the expression region range of TCP5 in the jag-2 mutant petals was wider than in the wild-type.In this study,we constructed an inducible expression vector JAGp:JAG:GR and overexpression vector 35S:JAGg by Gateway techniques.Furthermore,we also obtained a series of JAGp:JAG:GR and 35S:JAGg transgenic plants.MOCK/DEX treatment the inflorescence of JACp:JAG:GR(jag-2)transgenic plants revealed that compared with the MOCK treatment,the petal and petal blade surface area of JAGp:JAG:GR(jag-2)transgenic plants treated with DEX increased significantly(P < 0.05),suggesting that the JAG induced expression experiment is successful.To further explore the molecular mechanism of JAG and TCP5 in floral organ development,we analyzed their co-regulated downstream genes derived from the RNA-seq results obtained in our previous study and were focused on a set of gibberellin related genes,including JR-1,JR-2,JR-3,JP-1,TP-1,TP-2,TP-3,TP-4,TP-5 and TP-6.Furthermore,we analyzed the expression level of GA2ox1,GA3ox1 and GA20ox2,and conformed that the GA pathway is controlled by JAG and TCP5.In addition,the phenotypic analysis of Ler and jag-2 petals treated by a vary concentration of exogenous GA3 revealed that JAG had inhibitory effects on the inactivation of gibberellin acid.When the JAG gene was mutated,relatively high concentrations of exogenous GA3 could promote the growth.These results suggested that JAG and TCP5 genes regulate the development of floral organ by coordinating genes involved in the synthesis and metabolic pathway of gibberellin acid in Arabidopsis thaliana.To further dissect the role of JAG and analyze the downstream regulation network,we investigate the floral phenotypes of transgenic plants overexpressing an inducible form of JAG and observe the strong morphological defects in flower development when JAG is induced.Furthermore,JAG also affect chlorophyll biosynthesis.We detected a significant change of the chlorophyll a/b ratio in the JAG over-expressing floral tissues.Subsequent gene expression assay showed that the higher chlorophyll b in the JAG over-expression line is likely to be the consequence of increased expression of CAO,a key factor involved in chlorophyll a to b transition.The regulation of CAO by JAG appeared to be indirect.In addition,we also analyze the published Ch IP-seq and micro-array data,and revealed that 23 JAG direct targets are associated with chloroplast development.These results suggest the important role of JAG in controlling chloroplast functions.