Figure legend
Figure 1. Expression analysis of KCS proteins during anther
development. a, The expression of KCS7-GFP, KCS15-GFP and KCS21-GFP
fusion proteins were initially detected in tapetum at stage 8 and
subsequently accumulated in tapetum and anther locule at stages 9 and
10. After stage 10, the GFP signal was disappeared. The expression of
KCS20-GFP fusion protein was initially detected in tapetum at stage 9
and subsequently accumulated in anther locule and pollen at stages 10
and 11. GFP signal in tapetum was shown in enlarged view. No GFP signals
of KCS7-GFP, KCS15-GFP and KCS21-GFP were identified in ms1background. T, tapetum. Bars= 20 μm. b, The expression of KCS10-GFP
fusion protein was detected in epidermis and endothecium at stage 10 and
stage 11, while the GFP signal of KCS5 was only detected in epidermis
from anther stage 10 to 12. Bars= 10 μm.
Figure 2. MS1 regulates the expression of KCS7, KCS15 and
KCS21. a, Heatmap showing the expression of KCS7, KCS15, andKCS21 genes were downregulated in dyt1, tdf1, ams, ms188,and ms1 mutants, while the expression of KCS20 was not
affected in these mutants. b, qPCR analysis of KCS genes indyt1, tdf1, ams, ms188, and ms1 mutants. c, RT-PCR
analysis of KCS7, KCS15, and KCS21 genes in ms1,and myb99 mutants. d, Expression of KCS7 , KCS15 andKCS21 genes was analyzed in pMS188::MS1 transgenic plants
in the ms188 homozygous background (rescue).
Figure 3. Pollen coat was affected inkcs7/15/21 triple mutant. a, Scanning electron
microscopic (SEM) analysis of exine and pollen coat morphology in WT andkcs7 /15 /21 triple mutant plants. Red arrows and
blue arrows indicated irregular pollen coat and particles, respectively.
b and c, Lipid staining of semi-thin sections of WT andkcs7 /15 /21 triple mutant plants at anther stage 12.
The red fluorescence indicates the lipid staining on mature pollen
grains (white arrow). Bars= 10 μm. d, e, f and g, Analysis of pollen
wall structure by transmission electron microscopy (TEM). The
ultrastructure of the pollen coat of kcs7 /15 /21triple mutant showed decreased and disordered compared with the wild
type. Bars= 5 μm in d, e, and bars= 1 μm in f, g.
Figure 4. In vivo pollen hydration and pollen tube growth
analysis in kcs7/15/21 triple mutant. a, Diagram
of pollen hydration in WT and kcs7 /15 /21 triple
mutant. b, Diagram of pollen germination on stigma. c, In vivopollen tube emergence after manual pollination at 10 min, 20 min, 30
min, 45 min, 1 h and 2 h post-pollination. Pollen tubes were emerged in
WT pollen after manual pollination at 20 min, while pollen tubes were
identified in kcs7 /15 /21 triple mutant at 30 min.
Pollen grains were applied to stigmas of ms188 . Bars= 10 μm.
Figure 5. A proposal model for anther derived lipids in pollen
wall development. a, Expression pattern of KCS6, KCS15, CER2, and
CER2L2 proteins during anther development. The expression of KCS15-GFP
was detected in tapetum, while the expression of KCS6-GFP, CER2-GFP and
CER2L2-GFP fusion proteins were detected in endothecium. Bars= 20 μm. b,
A proposed model for tapetum and endothecium in biosynthesis of lipids
for pollen wall development. In tapetum, MS1 regulates the expression ofKCS7 , KCS15 and KSC21 , and the KCS-mediated lipids
were deposited in pollen coat. In endothecium, CER2 and CER2L2 interact
with KCS6 to modulate the biosynthesis of pollen coat VLCFAs for pollen
hydration.