Functional Analysis Of The SEVEN IN ABSENTIA(SINA)Ubiquitin Ligase Gene Family And Its Impact On Fruit Development

Biotechnology is the most potential technology in the development of food science nowadays.As a kind of biotechnology,genetic engineering,has been successfully applied to improve the quality and yield of fruits.So,understanding the molecular mechanism under plants growth and development will great contribute to taking full advantage of genetic engineering in food science.Seven in absentia(SINA)protein family is one subgroup of ubiquitin ligases that are characterized by a conserved N-terminal cysteine-rich C3H4 RING domain,two zinc finger motifs and a C-terminal domain responsible for substrate-binding and dimerization.We recently identified six SINA genes in the tomato(Solanum Lycopersicum,Sl)genome.One of them,termed SlSINA3,encodes an E3 ubiquitin ligase that specifically ubiquitinates the defense-related transcription factor SlNAC1 promoting its degradation.In this study,we characterized all six SlSINA genes and the gene products biochemically and investigated their roles in diverse physiologic processes,including defense response and development.The bioinformatic assay indicates the difference of SlSINA1 in amino acid.We conducted real-time PCR analysis,it shows that SlSINA genes are differentially regulated in leaf,bud,stem,flower and root.All SlSINA proteins possess RING-dependent E3 ubiquitin ligase activity,which exhibits similar specificity towards the E2 ubiquitin-conjugating enzyme,as presented in the in vitro ubiquitination assay.Among those E2s from tomato plant,only Group ? can work well with SlSINAs,that may indicate the characteristic of SISINAs in the plant.We also tested the subcellular localization of SlSINAs by fused with GFP,SlSINA1,3,4,5,and 6 are localized in both cytoplasm and nucleus,whereas SlSINA2 is exclusively localized in the nucleus.In addition,it is confirmed through yeast wo-hybrid assay and Co-immunoprecipitation assay that all SlSINAs can interact with each other for homo-and hetero-dimerization.The functionality of SlSINA proteins has been investigated.SlSINA4 plays a positive role in defense signaling,as manifested by elicitation of E3-dependent hypersensitive response(HR)-like cell death;The other SISINAs are negative regulator of defense signaling,as indicated by suppression of HR cell death.In addition,we get several transgenic plants for most of the SISINA genes through Agrobacterium-mediated transformation.In the transgenic tomato plants overexpressing SISINA2 exhibit pale-green leaf phenotype,suggesting SlSINA2 regulates chlorophyll biosynthesis;whereas transgenic tomato plants overexpressing SlSINA5 have altered floral structure with exserted stigma,implicating its role in flower development.As E3 ligases,SlSINAs may participate in tomato life by controlling the stability of some proteins.And the stress responsive transcription factor SINAC4 may be targeted by SlSINA4 for degration in plant.Overexpressing SlNAC4 resulted in smaller fruits and dwarf plants,which seriously impaired the yield.