Functional Analysis Of Grape Transcription Factor VvMADS39 And VvMADS28 And Its Regulation Mechanism On Ovule Development

Grapevine(Vitis vinifera L.)is one of the economic fruit trees widely cultivated in the world.Seedless grape has become one of hot-spot directions of market consumption and breeding due to its commercial advantages such as convenient use,great taste and simple processing,compare with seeded grape.At present,the outstanding problem are that the seedless varieties are single,and the molecular mechanism of seedless traits in grape is still unclear.So,it is of great significance to elucidating the formation of seedless grape and to create new seedless grape germplasm by using molecular biology techniques to excavate ovule development genes.Differentially expressed genes were obtained according to the seed transcriptomes of progeny derived from the V.vinifera seeded maternal parent ‘Red Globe’ and the seedless paternal parent ‘Centennial seedless’.Moreover,Vv MADS39 and Vv MADS28 related to ovule development were preliminarily screened based on transcriptome analysis and quantitative expression.In this study,we further verified the function of these two MADS-box family genes and characterized their developmental regulation mechanism.The main conclusions were listed as follows:1.Expression pattern,function of grape transcription factor Vv MADS39,and regulation on seedless fruit formation.Expression patterns of Vv MADS39 in different ovule development stages of seeded grape ’Red Globe’ and seedless grape ’Thompson Seedless’ found that,during ovule abortion(30-40 d),the expression of Vv MADS39 remarkable decreased in ’Red Globe’,but remained active in ’Thompson Seedless’,these results suggests that maintenance of Vv MADS39 actively expression in ’Thompson Seedless’ ovules may participate in the abortive process of ovule.In contrast,reduced expression of Vv MADS39 was associated with increased histone H3 lysine 27 trimethylation(H3K27me3)in the promoter region of Vv MADS39 in ’Red Globe’,allowing normal ovule development.Heterologous overexpression of Vv MADS39 in tomato(Micro-Tom)conferred a reduction in fruit,seed size and seed number.Targeted mutagenesis of the homologous Sl MADS39 in tomato conferred various floral and fruit defects,these results suggest that the MADS39 plays an important role in maintaining floral meristem characteristics,fruit and seed development.Yeast hybridization(Y1H/Y2H/Y3H),Co-immunoprecipitation(Co-IP),Bimolecular Fluorescence Complementation(Bi FC)and Electrophoretic Mobility Shift Assay(EMSA)found that Vv MADS39 interacts with Vv AGAMOUS(Vv AG)protein to form a stable dimer that specifically binds to the target gene Vv INO promoter to positively regulate its expression.Taken together,interaction of proteins and the regulation of inter-gene expression synergically control grape floral organ morphogenesis and seedless fruit formation.2.Expression pattern,function of grape transcription factor Vv MADS28,and regulation seed development.The real-time polymerase chain reaction results showed that Vv MADS28 expression level was highly expressed in the ovule of seeded variety during the whole period and stronger hybridization signals were localized to the integument of ’Red Globe’,these results suggesting that Vv MADS28 was required for formation of seed coat process.The observed stronger expression in ovules of ’Red Globe’ but weaker in ’Thompson Seedless’,was associated with increased H3K27me3 repressive marks in the promoter region of Vv MADS28 in ’Thompson Seedless’.Vv MADS28-RNAi in seeded grapevine cultivar ’Red Globe’ shown that inhibition of episperm and endosperm cells development correlate with reduced seed size.The most striking phenotype caused by 35S::Vv MADS28 in transgenic tomatoes were the unique sepals and shrunken fruits,however,there was no obvious morphological changes were observed in seed size.Vv MADS28 is positively regulated by upstream Vv ERF98 through Y1 H and dual-luciferase,moreover,both Vv ERF98 and Vv MADS28 were more highly upregulated in seed after treatment with ethephon,these results indicate that Vv ERF98 specifically responds to ethylene treatment and participates in the regulatory pathway of Vv MADS28 in seed development.Split luciferase assay and Co-IP demonstrated Vv MADS28 could interact with a Type I Mβ protein Vv MADS5.Combined with DNA affinity purification sequencing(DAP-seq),Y1 H,EMSA and dualluciferase assays,it was found that Vv MADS28 inhibited the expression of grapevine Vv WUS by binding to the CAr G site of Vv WUS promoter.In conclusion,this study preliminarily indicates the molecular mechanism of Vv MADS28 and Vv MADS39 involve in ovule development.Vv MADS39 causes parthenogenesis by regulating pollen grain development.Vv MADS39 can inhibit ovule development and contribute to the formation of seedless fruit;As an active regulator of ovule development,Vv MADS28 is involved in seed coat formation.Meanwhile,the maintenance of Vv MADS28 and Vv WUS expression homeostasis is important for seed and plant development.