KCS7, KCS15 and KCS21 act downstream ofMS1
Dysfunctional tapetum1 (DYT1), Defective in tapetal development and function 1 (TDF1), Aborted microspore (AMS), MS188, and MS1 are essential tapetum regulators which form a genetic pathway (Sorensen et al., 2003; Wilson et al., 2001; W. Zhang et al., 2006; Z. B. Zhang et al., 2007; Zhu et al., 2008; Zhu, Lou, Xu, & Yang, 2011). Based on the microarray data, KCS7 , KCS15 and KS21 act downstream of these regulators (Fig. 2a) (Zhu et al., 2011). We collected the inflorescences of dyt1 , tdf1 , ams ,ms188 and ms1 for quantitative real-time PCR analysis. These results showed that the expressions of KCS7 , KCS15and KCS21 were downregulated while KCS20 was not affected in these mutants (Fig. 2b).
MS1 is the key regulator for late tapetum development in the genetic pathway (Zhu et al., 2011). The downregulation of KCS7 ,KCS15 and KCS21 in ms1 mutant suggested that these three KCS genes were regulated by MS1. To further analyze the molecular regulation between MS1 and KCS7 , KCS15and KCS21 , we introduced the pKCS::KCS-GFP construct into the ms1 plants and analyzed the distribution of KCS-GFP signals (Fig. 1a). The GFP signals could not be detected in ms1 mutant while they were clearly observed in the tapetal cells of wild type (Fig. 1a). These results were consistent with the microarray and qPCR results (Fig. 2a,b), which further showed that KCS7 , KCS15 andKCS21 act downstream of MS1 . MYB99 is a direct target of MS1 and its mutant displays a slight defect in pollen development (Alves-Ferreira et al., 2007). The expressions ofKCS7, KCS15 and KCS21 were not changed inmyb99 (Fig. 2c). MS188 is an upstream regulator of MS1 (Zhu et al., 2011). Our recent work showed that many PCPs were downregulated in both ms188 and ms1 , and expression ofMS1 driven by the MS188 promoter in ms188 mutant (ms188/pMS188::MS1 ) restored the expression of PCPs (Lu et al., 2020). We performed RT-qPCR in the same transgenic line (ms188/pMS188::MS1 ), and the expressions of KCS7 andKCS15 were fully restored in pMS188::MS1 transgenic plants (Fig. 2d). However, the expression of KCS21 did not show any recovery in pMS188::MS1 transgenic plants compared with that inms1 (Fig. 2d).