Molecular Mechanism Of MADS-box Transcription Factor SIFYFL Regulating Senescence And Fruit Ripening In Tomato

Plant hormone ethylene plays important roles in many aspects of plant growth and development, including leaf senescence, fruit ripening, abscisic and other procedural senescence processes and signaling of stress resistance.MADS-box genes are a class of transcription factor family, widely distributed in eukaryotic. In higher plants, MADS-box proteins regulate the growth and development processes, including the formation of floral organs, flowering time, fruit ripening and abscisic and plant senescence. In2002, Vrebalov and others found a MADS-box transcription factor S1MADS-RIN through map-based cloning method, and proved it is an important tomato fruit ripening transcription factor, and regulate ethylene biosynthesis pathway. In order to further explore the functions of other unknown genes in tomato MADS-box family, we screened out and cloned a MADS-box protein that is highly homologous to S1MADS-RIN through bioinformatics analysis, and is an ortholog to Arabidopsis AtFYF(AtAGL42), so it is named for SIFYF-Like (S1MBP18), then we submitted it to the NCBI database (GenBank accession number:KF709444).In this study, the expression patterns of SIFYFL gene were analyzed using real-time PCR technology. The results show that the expression levels of SIFYFL gene are higher in tomato leaves, fruits, sepals and abscission zone, indicating the gene may be associated with leaf development, fruit ripening and abscission and sepal development. To further explore the function of this gene, both positive and negative aspects of transgenic technology were performed by Agrobacterium-mediated method. On the one hand, SIFYFL gene was over-expressed in wild-type tomato AC, on the other hand, SIFYFL gene was silenced in wild-type tomato AC.By detecting the related indicators of physiological and biochemical and molecular level of overexpression and silencing transgenic tomato lines of SIFYFL, we got the results that are as follows:First, SIFYFL participates in regulating the expression of ethylene-related genes, thereby regulate the content of chlorophyll in tomato leaves to regulate the degree of senescence of tomato leaves. On the one hand, over-expression of SIFYFL delays leaf senescence, increases chlorophyll content, the expression of photosynthesis genes are up-regulated and the expression of ethylene synthesis genes is reduced in over-expression transgenic leaves in the same period; On the other hand, RNAi silence transgenic lines exhibit the opposite situations. Second, SIFYFL associated with the ripening process of tomato fruits by regulating the expression of genes of ethylene synthesis. In over-expression transgenic lines, tomato fruit ripening are delayed, the accumulation of carotenoids is reduced, fruit storage time becomes longer, fruit firmness are increased, the amount of ethylene biosynthesis are reduced. Expression of fruit ripening-related genes, including carotenoid biosynthetic genes (SIPSY1, SIPDS and SIZDS), cell wall metabolism genes (SlPG, SlEXP1, SIXTH5and SITBG4) and fatty acid metabolism genes (SlTomLoxA, SITomLoxB and SlTomLoxC) in over-expression transgenic fruit are significantly inhibited. Ethylene biosynthetic genes (ACS2, ACO1and ACO3) and ethylene response genes (E4and E8) in over-expressing transgenic fruits are significantly inhibited. Ethylene synthesis in fruits of over-expression transgenic tomato is only40%-50%compared with that of wild-type, while in silence transgenic lines, the above-mentioned series of indicators all emerges opposite results. These results suggest that SIFYFL plays a role in the upstream of ethylene and affects fruit ripening in tomato. In addition, SIFYFL involves in the ethylene-related tomato abscission zone development. In over-expression transgenic lines, the abscission of tomato fruits is delayed, fruit stalk cell development of abscission zone is postponed and the break strength are increased. Real-time PCR results show that the expression of a set of abscission zone-related genes (SlMADS-MC, SIMADS-JOLNTLESS, BL, WUS, LS and GOB) are significantly reduced, the expression of ethylene biosynthesis genes (ACS4, ACS2, ACSlA, ACO1and ACO6) are significantly decreased in over-expression transgenic plants, and in the silence transgenic plants, these results are opposite. These results show that SIFYFL gene may through affects ethylene and thus regulate the expression of genes related to the development of abscission zone.Since the MADS-box proteins are usually formed homologous or heterodimers to play regulatory roles. Yeast two-hybrid results show that, SIFYFL can interact with itself and fruit ripen related S1MADS-RIN, S1MADS1, respectively, indicating SIFYFL possibly interacts with S1MADS-RIN and S1MADS1, thus affects the ethylene thereby regulates tomato fruit ripening. In addition, SIFYFL also can interact with tomato abscission zone development related protein JOINTLESS, and it is reported that JOINTLESS can interact with tomato abscission zone development related protein S1MADS-MC, indicating that SIFYFL protein might form complex with JOINTLESS and S1MADS-MC and regulate the formation of tomato abscission zone. In summary, this study focuses on exploring the functions of MADS-box transcription factor S1FYFL. These results show that SIFYFL may involve in the pathway of ethylene and participates in tomato ethylene-related developmental processes, such as senescence, ripening and abscission. This study provides a theoretical foundation to completely elucidate the functions of MADS-box genes in tomato, and also provides a practical foundation to improve the storability of tomato fruits.