Alternative splicing variants of IiSEP3 in Isatis indigotica are involved in floral transition and flower development

The SEPALLATA3 genes regulate several aspects of plant development. This study identified four distinct splicing isoforms of the SEPALLATA3 gene in Isatis indigotica (I. indigotica). IiSEP3-1 and IiSEP3-2 have eight exons and were named as IiSEP3-2/1. However, IiSEP3-3 and IiSEP3-4 with the missing sixth exon were labeled IiSEP3ΔK3. Furthermore, the IiSEP3-1 and IiSEP3-4 amino acids sequences lack the V90. IiSEP3 splicing variants were primarily expressed in floral organs, with petals showing the highest expression. Ectopic expression of IiSEP3-2 or IiSEP3-3 may cause early flowering and reduce the number of sepals, petals, and stamens. The ectopic expression of IiSEP3-2 resulted in cauline leaves and sepals converting to carpelloid structures. In contrast, the four floral whorls prematurely wilted, and the entire flower displayed an abortive state when IiSEP3-3 was expressed ectopically. Silencing the IiSEP3 gene of I. indigotica employing VIGS (tobacco rattle virus-mediated virus-induced gene silencing) technology using the TRV-IiSEP3-2/1 vector delayed flowering time and reduced the number of petals and stamens. Plants silenced with TRV-IiSEP3ΔK3 also exhibited similar phenotypes, including fewer sepals. The transcriptome analysis of silenced plants (TRV-IiSEP3-2/1 treatment group) indicated significant alterations in 1861 genes, with 1035 upregulated and 826 downregulated. TRV-IiSEP3ΔK3 treatment altered the expression of 2063 genes in plants, with 1289 genes upregulated and 774 genes transcription inhibited. Y2H and BIFC experiments revealed that IiSEP3-2 and IiSEP3-3 had distinct interacting proteins. Thus, we can conclude that IiSEP3-2 and IiSEP3-3 interact with different proteins, affecting floral transition and organ development in I. indigotica.