Two SEPALLATA MADS-Box Genes, SlMBP21 and SlMADS1, Have Cooperative Functions Required for Sepal Development in Tomato.

MADS-box transcription factors have crucial functions in numerous physiological and biochemical processes during plant growth and development. Previous studies have reported that two MADS-box genes, SlMBP21 and SlMADS1, play important regulatory roles in the sepal development of tomato, respectively. However, the functional relationships between these two genes are still unknown. In order to investigate this, we simultaneously studied these two genes in tomato. Phylogenetic analysis showed that they were classified into the same branch of the SEPALLATA (SEP) clade. qRT-PCR displayed that both SlMBP21 and SlMADS1 transcripts are preferentially accumulated in sepals, and are increased with flower development. During sepal development, SlMBP21 is increased but SlMADS1 is decreased. Using the RNAi, tomato plants with reduced SlMBP21 mRNA generated enlarged and fused sepals, while simultaneous inhibition of SlMBP21 and SlMADS1 led to larger (longer and wider) and fused sepals than that in SlMBP21-RNAi lines. qRT-PCR results exhibited that the transcripts of genes relating to sepal development, ethylene, auxin and cell expansion were dramatically changed in SlMBP21-RNAi sepals, especially in SlMBP21-SlMADS1-RNAi sepals. Yeast two-hybrid assay displayed that SlMBP21 can interact with SlMBP21, SlAP2a, TAGL1 and RIN, and SlMADS1 can interact with SlAP2a and RIN, respectively. In conclusion, SlMBP21 and SlMADS1 cooperatively regulate sepal development in tomato by impacting the expression or activities of other related regulators or via interactions with other regulatory proteins.

A MADS-box transcription factor, SlMADS1, interacts with SlMACROCALYX to regulate tomato sepal growth.

In flowering plants, sepals play important roles in the development of flowers and fruit, and both processes are regulated by MADS-box (MADS) transcription factors (TFs). SlMADS1 was previously reported to act as a negative regulator of fruit ripening. In this study, expression analysis shown that its transcripts were very highly expressed during the development of sepals. To test the role of SlMADS1, we generated KO-SlMADS1 (knock-out) tomato mutants by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) technology and over-expression of SlMADS1 (OE-SlMADS1). The sepals and individual cells of KO-SlMADS1 mutants were significantly elongated, compared with the wild type (WT), whereas the sepals of OE-SlMADS1 tomatoes were significantly shorter and their cells were wider. RNA-seq (RNA-sequencing) of sepal samples showed that ethylene-, gibberellin-, auxin-, cytokinin- and cell wall metabolism-related genes were significantly affected in both KO-SlMADS1 and OE-SlMADS1 plants with altered sepal size. Since SlMACROCALYX (MC) is known to regulate the development of tomato sepals, we also studied the relationship between SlMC and SlMADS1 and the result showed that SlMADS1 interacts directly with SlMC. In addition, we also found that manipulating SlMADS1 expression alters the development of tomato plant leaves, roots and plant height. These results enrich our understanding of sepal development and the function of SlMADS1 throughout the plant.