Functional Study Of A Transcription Factor Squamosa Promoter Binding Protein Like 3 In Tomato Flower Abscission Zone Development

Abscission is a physiological process that plant cells, tissues or organs shedding from the plant in response to external environment and self-regulation. The region where abscission occurs is called abscission zone (AZ). Tomato (Solanum lycopersicum) is a model plant for flower abscission zone study because of its unique structure. A number of tomato abscission zone related genes such as JOINTLESS (J), JOINTLESS2 (J2) and LATERAL SUPPRESSOR (LS) have been isolated. J belongs to the plant specific transcription factor MADS-box gene family which involved in many plant growth processes. It has been known that SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors regulate many MADS-box genes in plant development. SPLs are involved in several important and divergent biological processes such as flower and fruit development and architecture formation. In this project, the yeast one-hybrid system was used to screen genes that directly bind to J promoters. Forward genetic analysis was used to study their roles in abscission zone development. The key findings are:1. A SPL gene LeSPL3 was shown to bind directly to J promoters. Yeast one hybrid system was used to find the regulator genes of J. The results show that 5 transcription factors belong to the known gene families. Four out of five transcription factors are plant specific, which are named by the Arabidopsis homologous genes, LeSCL3, LeSPL3, LeREV and LePHA. All of these transcription factors show in vitro binding activity to J promoter by yeast one hybrid. Electrophoretic Mobility Shift Assay(EMSA)and Chromatin Immunoprecipitation(ChIP)were used for further analysis of LeSPL3 because of possible cis-element in J promoter region. All the binding results indicate that the LeSPL3 can bind to the J upstream cis-element TCTGTACAA. Therefore, LeSPL3 is the direct upstream regulating gene of J.2. The functions of LeSPL3 were studied by generating Arabidopsis and tobacco (Nicotiana tabacum) transgenic lines. The genomic sequence was obtained by yeast one hybrid sequence result. LeSPL3 and AtSPL3 have 69.1% similarity in amino acid sequence, and the gene has 2 exons and 1 intron, and the 3'UTR region has recognition sites of microRNA miR156/157. Tissue-specific analysis showed that LeSPL3 expression is much higher in tomato inflorescence and pedicel than other tissues. In transgenic tobacco plants overexpressing LeSPL3 the abscission zone cell layers increase. In addition the tobacco transgenic lines also showed thinner flower pedicels and smaller pedicel cell size. These results indicate that LeSPL3 may affect the pedicel and pedicel AZ development. Meanwhile overexpression of LeSPL3 in Arabidopsis and tobacco all have curly leaves and flower earlier which are similar as AtSPL3 overexpression lines. These results indicate that LeSPL3 has the same function as AtSPL3 in regulating the leaf development and flowering time.3. A comparative study of NtSPL3 and tomato LeSPL3 was performed. Tobacco is a close species of tomato. Amino acid sequences analysis showed that NtSPL3 has 94% similarity with LeSPL3. NtSPL3 affects the pedicel AZ development. In transgenic tobacco plants overexpressing NtSPL3 showed increased abscission zone cell layers, but knock down of NtSPL3 caused the absence of AZ. In the other hand NtSPL3 also affect the leaf development and flowering time in tobacco. The guard cells number changed in transgenic lines more in overexpression lines and less in gene knock down lines compare to the wild type. The results showed that NtSPL3 affects the guard cell development which was not seen in LeSPL3 overexpression plants.In summary, we showed that LeSPL3 is involved in pedicel and pedicel AZ development by directly regulate MADS-box gene JOINTLESS. These discoveries extended the molecular mechanism of pedicel AZ development. In addition, NtSPL3 may confer a wider function in plant development, especially in guard cell development. This result indicates the functional diversification between LeSPL3 and NtSPL3. NtSPL3 may play an important role in plant drought tolerance. The study revealed the molecular mechanism of abscission AZ development that can be potentially applied in agriculture to increase the yield by reducing fruit loss.