Pollen hydration was delayed in kcs7/15/21 triple mutant
Pollen coat is generally considered to involve in pollen-stigma interaction. A pollen hydration assay was carried out to analyze if the reduced pollen coat in kcs7/kcs15/kcs21 affect the pollen-stigma interaction. Ms188 is a male sterile line without any pollen inside anther while its stigmas are not affected (Z. B. Zhang et al., 2007). The pollen hydration assay was carried out by pollinating stigmas of ms188 with pollen grains derived from WT and thekcs7/15/21 triple mutant (Fig. 4). After landing on stigma, the wild-type pollen began to absorb water and became spherical (pollen hydration) in about 5 min (Fig. 4a). However, the hydration of thekcs7/15/21 triple mutant pollen occurred in 10 min after pollination. Accordingly, pollen germination of the triple mutant is also delayed (Fig. 4a). To determine if the hydration delay is derived from a defect in water absorption, an in vitro hydration of WT and kcs7/15/21 triple mutant pollen in PEG 3350 series was carried out (Fig. S4). The in vitro hydration was not significantly different between WT and kcs7/15/21 triple mutant pollen in PEG 3350 series, indicating that the absence of KCS7, KCS15, and KCS21 proteins did not impair the ability of pollen to absorb water. To test whether the subsequent steps of pollination were also affected in kcs7/15/21 triple mutant, we monitored pollen tube initiation and growth as previously described (Mayfield & Preuss, 2000) (Fig. S5,4b). 20% of WT pollen grains showed a pollen-tube emergence within 20 min of pollination, while, kcs7/15/21 triple mutant pollen-tube emergence was postponed to 30 min (Fig. 4c,S5). After 2 h, WT pollen produced significantly longer tubes (470 um) and penetrated into the style, while pollen tubes of kcs7/15/21 triple mutant were much shorter (266 um) (Fig. S5). Therefore, the elongation of pollen tube was postponed in kcs7/15/21 triple mutant (Fig. 4b,c,S5). Despite the observed defects in pollen hydration and pollen tube elongation in kcs7/15/21 triple mutant, there was no significant difference in seed set in kcs7/15/21 triple mutant compared with WT plants.