The pollen coat is reduced in kcs7/15/21 triple mutant
To investigate the functions of the KCS7 , KCS15 andKCS21 in anther development, T-DNA insertion lines ofKCS7 , KCS15 and KCS21 were identified (Fig. S2). These mutants exhibited drastically reduced expression of the relevant genes (Fig. S2). No obvious vegetative or reproductive morphological abnormalities were observed in any of the single (kcs7, kcs15, kcs21 ), double (kcs7/15, kcs7/21, kcs15/kcs21 ) and triple mutants of KCS7 , KCS15 and KCS21(kcs7/15/21, Fig. S3). Aborted pollen grains were occasionally observed in kcs15 single mutant (70 of 1030), and the percentage of aborted pollen grains was increased to 13% inkcs7/kcs15/kcs21 triple mutant (110 of 853, Fig. S3).
Lipids were deposited in pollen coat during pollen development, and KCS proteins were responsible for lipid synthesis. To analysis whether mutation in KCS7 , KCS15 and KCS21 affect the pollen coat lipids, the scanning electron microscopy (SEM) and transmission electron microscope (TEM) observations were used. WT pollen grains displayed a well-organized exine and a smooth pollen coat. However, the well-smoothed pollen coat was disturbed, with many particles in the cavity of triple mutant pollen exine (Fig. 3). In addition, the severely defective pollen of kcs7/kcs15/kcs21 was small and displayed dis-organized exine covered with additional materials (Fig. 3). The diethyloxadicarbocyanine iodide (DiOC2) was used to stain the fatty acid content of pollen wall and fatty acid exhibit a red fluorescence of DiOC2 staining (Gu et al., 2014). A strong red fluorescence of DiOC2 staining was observed in exine and pollen coat of the WT mature pollen (Fig. 3b,c). Whereas,kcs7/kcs15/kcs21 mature pollen showed weaker red signals with little or no signals observed in the pollen coat (Fig. 3b,c). Further TEM analysis showed kcs7/kcs15/kcs21 pollen coat is disordered and thinner than the wild type (Fig. 3d,e,f,g). Therefore, these results indicate that lipid content may be reduced in the pollen coat of