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