Bioinformatics Analysis Of S40 Family And Functional Analysis Of Members In Plant Senescence And Development

Caragana intermedia and C.korshinskii are species of the Caragana Fabr.,Leguminosae,they are rich in protein and are taken as an important natural livestock forage plants in the northwest of China.Both of them have good resistances to abiotic stresses and senescence.The leaves of C.intermedia and C.korshinskii maintain a long green period until the late autumn and thus provide sufficient forage for livestock.The senescence regulator(Sn R)family,also known as S40 family,is a unique family in plants,and is deeply associated with plant leaf senescence.There are few reports on leaf senescence in C.intermedia and C.korshinskii so far,and the mechanism of S40 family regulating leaf senescence remains elusive.In this study,the causes of leaf anti-senescence ability in C.korshinskii and C.intermedia,as well as the mechanism of S40 family members senescence regulation were investigated,through S40 family bioinformatics analysis,plant phenotype observation and the signaling pathways related.Meanwhile,this study also found that S40 family members are involved in the process of plant growth and development of Arabidopsis thaliana.And the results are as follows:1.Totally 2820 protein sequences of S40 family were collected from 219 species in different databases and were selected.The relevant protein domain sequences of S40 family were used for phylogenetic analysis,and the S40 family members were classified into seven subfamilies from A to G.In addition,there were 4 conserved amino acid Motifs that were identified in the S40 family protein domain sequences.2.The anti-senescence ability in C.korshinskii leaves was significantly stronger than that A.thaliana.Based on the genome data of A.thaliana and C.korshinskii,the number of S40 family members had not amplified in C.korshinskii compared to A.thaliana.However,the basal level expression of most S40 family genes in C.korshinskii is strikingly higher than that in A.thaliana.Perhaps this is one of the key reasons contributing to the senescence resistance ability of C.korshinskii leaves.3.The function of Ci S40-11 in C.intermedia,which is the ortholog of evm.model.Chr6.3333 that had the highest constitutive expression level in C.korshinskii,were investigated.The results showed that Ci S40-11 enhanced anti-senescence ability of leaves,no matter when it was transiently expressed in C.intermedia or was stably expressed in A.thaliana.4.Through analysis of transcriptome data of Ci S40-11 overexpressed A.thaliana,we found that Ci S40-11 inhibited the expression of MYB2 and released its negative regulation on the expression of IPTs to promote the synthesis of cytokinins in transgenic A.thaliana.The cytokinins content detection results showed that two active cytokinins,i PR and tz R,increased obviously in the Ci S40-11 overexpression lines.5.The overexpression lines of S40 family members of A.thaliana,AtS40-5,AtS40-6 and AtS40-11,which are orthologs of Ci S40-11,exhibited dwarf phenotype in transgenic A.thaliana.The leaf cell diameters of AtS40-5(AT1G11700),AtS40-6(AT1G61930)and AtS40-11(AT4G21930)overexpression plants were significantly smaller than that of wildtype based on the microscopic structure observation.And the AtS40-11 overexpression lines showed the most dramatic phenotype.Correspondingly,the mutants ats40-5a,ats40-6a and ats40-11 a showed a taller phenotype,and the leaf cell diameters increased significantly,compared with those in wild-type.6.Expression pattern analysis showed that AtS40-5,AtS40-6 and AtS40-11 responded to gibberellic acid induction.AtS40-11 was used as an example to investigate the potential mechanism of S40 family.It was proved that the content of active gibberellins increased in the AtS40-11 overexpression lines,while no significant change was detected in the ats40-11 a mutant.Yeast two-hybrid experiments showed that AtS40-11 can interact with GID1 B,the gibberellin receptor.In addition,AtS40-11 gene did not affect the expression of genes related to gibberellin synthesis,metabolism and signal transduction.In summary,this study presents a systematic classification and thorough bioinformatics analysis of the S40 family.It also reveals that the Ci S40-11 gene promotes the synthesis of cytokinins to inhibit the process of leaf senescence.Additionally,the study finds that the AtS40-5,AtS40-6,and AtS40-11 genes can inhibit the transduction of gibberellin signaling which affects plant development,and this finding has not been reported previously.