The SEPLLATA-like MADS-box Gene SLMBP21Is Required For Tomato(Solanum Lycopersicum L.) Flower Abscission Zone Development

Organ abscission is a key step in plant life cycle and an important agronomic trait for crops. For most plants, abscission occurs at a specific region named abscission zone (AZ). Tomato has proven to be an excellent model for AZ study because of its distinctive joint-like AZ structure in the middle of flower (fruit) pedicels. Control of organ abscission is of commercial significance for most crops because fruit dropping (abscission) is one of the major factors for yield loss under adverse growth conditions. In tomato, two MADS-box genes JOINTLESS (J) and MACROCALYX (MC) have been reported to be required for AZ formation. However, the detailed mechanism underlying AZ development is still not well understood. In this work, based on previous yeast two hybridization data, we identified that the SEPALLATA group MADS-box gene SLMBP21was required for tomato pedicel AZ development and then studied its mode of action both on protein-protein interaction level and transcriptome level. The main results are as follows:1. We generated antisense and overexpression transgenic lines of SLMBP21and found that loss of function of SLMBP21caused the elimination of AZ at tomato pedicels. The observation was confirmed by additional RNAi transgenic plants, indicating that SLMBP21is required for tomato AZ development. Overexpression of SLMBP21generated more AZ-like small cells at the proximal section of the pedicel as well as the peduncle. Exogenous ethylene treatment showed that these cells conferred partial AZ functions.2. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays showed that pair-wise interactions exsisted among SLMBP21, J, and MC, and both SLMBP21and J could form homodimers. We then generated fusion proteins of J-Flag, MC-GFP, and SLMBP21-Myc, and conducted co-immunoprecipitation (Co-IP) on protein extracts from tobacco leaves. The results showed that J, MC, and SLMBP21could redily form a protein complex in vivo when expressed together, indicating that they may function as protein complexes.3. In situ expression analyses showed that SLMBP21, J, and MC were expressed with distinct patterns but overlapped in the incipient AZ vasculature which corresponds to the outmost L3layer during early stages of flower development, providing an explanation for the important role that the outmost L3taking in AZ development. Co-expression of the three genes also provides needed spatiotemporal conditions for their products to interact with each other.4. Further transactivation analysis using yeast cells showed that among SLMBP21, J, and MC, only SLMBP21had a strong transcription activity. Therefore, SLMBP21has a unique role in the SLMBP21/J/MC protein complex as the sole transcription activator.5. We then carried out a RNA-seq analysis on pedicels from the wild-type and antisense transgenic plants. Through comparison with published data for J and MC, we found seven AZ-specific genes that were down-regulated in any of the loss-of-function plant of SLMBP21, J, and MC, including a subset of meristem-related genes such as LeWUS, Blind (Bl), and Lateral suppressor (Ls). These results are in line with the notion that AZ cells may confer meristematic nature. These seven genes can be good candidate downstream genes of SLMBP21/J/MC protein complex for AZ development and should be studied further. We also found that AZ development was a complicated process that required multiple pathways and signals.6. Phylogenetic analysis showed that SLMBP21belonged to the FBP9/23group of the LOFSEP supclade in SEP clade. Apart from SLMBP21, we also found proteins from other species of Solanaceae, Malus, and Pyrus in the FBP9/23group. Since the FBP9/23group is unique to Solanaceae and absent from Arabidopsis, the SLMBP21-mediated protein complex may represent a distinct molecular mechanism for plant AZ development.Overall, this work identifies a novel function for the MADS-box gene SLMBP21in tomato AZ development which can be potentially applied in tomato abscission manipulation. In light of the absence of SLMBP21-like genes in Arabidopsis, the mode of action of these genes in tomato may represent a distinguished mechanism for plant AZ development.