Serine esterases are required for pollen tube penetration of the stigma in Brassica

Abstract

We have investigated the diversity of serine esterases in pollen and stigma tissues of Brassica napus and the role of these enzymes in pollen germination and pollen tube penetration of the stigma. The serine esterase-specific inhibitor diisopropyl fluorophosphate was used as a probe in a tritiated form, [ 3 H]-DIPF, to determine the number and diversity of serine esterases in crude protein extracts from pollen and stigma. Seven serine esterases were identified in pollen and at least seven serine esterases were identified in stigma. The most abundant enzymes had molecular weights of 30-50 kDa. In the pollen extract a serine esterase was detected with the same molecular weight, 22 kDa, as an esterase previously shown to be a cutinase. Only one serine esterase (40 kDa) appeared to be shared between pollen and stigma extracts. Butyrate esterase activity in pollen and stigma extracts was assayed using p-nitrophenyl butyrate (PNB), an ester substrate frequently used in 'cutinase' assays. Total PNBase activity in pollen and stigma extracts was shown to be significantly reduced by the serine esterase inhibitors DIPF and ebelactone B. When DIPF and ebelactone B were applied to stigmas prior to pollination, pollen germination was not significantly affected but, at the highest inhibitor concentrations, up to 70% of germinating pollen tubes failed to penetrate the stigma surface. These data demonstrate that serine esterases, most probably cutinase(s), are required for pollen tube penetration of the dry cuticularised Brassica stigma.

AtVPS41-mediated endocytic pathway is essential for pollen tube–stigma interaction inArabidopsis

“…After germinating on the stigma, pollen tubes have to penetrate the cuticle covering the stigmatic surface (32)(33)(34). Pollen-held enzymes, such as pectin-degrading enzymes and enzymes that modify the cell wall, must be secreted to degrade the stigmatic cuticle and loosen the stigmatic cell wall.…”

Pollen and Stigma Structure and Function: The Role of Diversity in Pollination

“…Removing or disrupting the pellicle prevents compatible pollen tubes from entering the stigma, despite normal germination (Heslop-Harrison and HeslopHarrison, 1975;Heslop-Harrison, 1977;Heslop-Harrison and Shivanna, 1977). In addition, cutinase inhibitors significantly reduce the ability of pollen tubes to penetrate Brassica stigmas (Hiscock et al, 2002a). Hydrolysis of pectin in the stigma cell wall also is necessary, for which pollen expresses genes encoding pectin esterase and pectate lyase (Kim et al, 1996;Wu et al, 1996).…”

Genome-Wide Identification of Genes Expressed in Arabidopsis Pistils Specifically along the Path of Pollen Tube Growth

“…The stigma cuticle presents a barrier to pollen hydration and germination, and this barrier must be breached by the action of cutinases (esterases) from the stigma pellicle and pollen grain surface (Hiscock et al, 2002). Removal of the stigmatic pellicle or treatment of stigmas with a cutinase inhibitor inhibits pollen tube penetration through the stigma cell wall (HeslopHarrison and Heslop-Harrison, 1975;Hiscock et al, 2002).…”

“…Removal of the stigmatic pellicle or treatment of stigmas with a cutinase inhibitor inhibits pollen tube penetration through the stigma cell wall (HeslopHarrison and Heslop-Harrison, 1975;Hiscock et al, 2002). Furthermore, the heavily cutinated surface of leaf epidermal cells cannot support pollen tube growth, except in cuticle-defective mutants and cutinase-expressing transgenic Arabidopsis (Lolle and Cheung, 1993;Sieber et al, 2000).…”

“…In Arabidopsis (Arabidopsis thaliana), the stigma is capped by approximately 150 finger-like cells, called papillar cells (Bowman et al, 1999). Analysis of the dry stigmas of other crucifers (Heslop-Harrison and Shivanna, 1977) has indicated that the surface of each papillar cell is covered by an interrupted layer of cutin, called the cuticle, and a superficial proteinaceous pellicle layer of poorly defined molecular composition (Gaude and Dumas, 1986;Hiscock et al, 2002), which may function in the adhesion and hydration of pollen grains through interactions with components of the pollen surface (Stead et al, 1980;Gaude and Dumas, 1986;Zinkl et al, 1999). For their part, cells of the transmitting tract that line the path of pollen tube growth are thought to produce molecules that contribute to the rapid rate and directionality of tube elongation (Sanchez et al, 2004).…”