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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.