Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue

After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen–stigma interactions that regulate pollen tube growth in Nicotiana

Pollination induces synthesis and release of NaStEP into the stigmatic exudate

Total protein from exudate of unpollinated and pollinated stigmas of cv Breakthrough were sequentially extracted, separated by SDS–PAGE, and blotted to nitrocellulose for immunostaining with anti-NaStEP (top) or silver nitrate stained (bottom). Equal volumes (30 μl) were loaded in each lane. (a) Total protein from exudate of unpollinated stigmas at anthesis and at 48 h old. (b) Total proteins from exudate after 5, 12, and 24 h of self-pollination. (c) Total proteins from exudate after 5, 12, and 24 h of pollination with pollen. (d), (e), and (f) Densitometric analysis of NaStEP expression and its presence in the stigmatic exudate of before and after pollination with self- and pollen. L, low-salt wash; H, high-salt wash; T, proteins remaining in the stigma after washes.<p><b>Copyright information:</b></p><p>Taken from "Pollination in stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue"</p><p></p><p>Journal of Experimental Botany 2008;59(11):3187-3201.</p><p>Published online Jan 2008</p><p>PMCID:PMC2504342.</p><p></p

NaStEP is discharged from the vacuole to the stigmatic exudate upon pollination

Unpollinated stigma from SC cv Breakthrough showing NaStEP localization in vacuoles and osmiophilic bodies. (a) Cross-section of mature papillae surrounded by copious exudate containing secretory droplets. Arrows show cell wall interruptions. (b) A papillary cell with a large vacuole and osmiophilic bodies. (c) Anti-NaStEP labelling of vacuolar electron-dense bodies. Arrows show immunogold secondary antibody labelling. cv Breakthrough stigma 10 h after pollination with pollen. (d) Degenerating papillary cells (arrow), showing abundant exudate and a disordered cytoplasm with abnormal organelles and disintegrated vacuoles. (e) Vacuole showing poor anti-NaStEP labelling of the osmiophilic bodies (arrows). (f) A close-up of the labelling shown in (e) (black arrows) showing anti-NaStEP labelling (white arrows) in smaller osmiophilic bodies. Stigmatic exudate of cv Breakthrough. (g) and (h) Diagrams of a stigma plus style of and a transverse section of a stigma. The arrow and square show zones where images were taken. (i) Unpollinated stigmas. (j) At 10 h after pollination with pollen. (k) At 10 h after pollination with pollen. (l) At 10 h after self-pollination. N, nucleus; m, mitochondria; c, chloroplast; v, vacuole; osb, osmiophilic bodies; ex, exudate. Bars, 500 nm for (a), 1 μm for (b) and (d), 240 nm for (c), 500 nm for (e), and 200 nm for (i)–(l).<p><b>Copyright information:</b></p><p>Taken from "Pollination in stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue"</p><p></p><p>Journal of Experimental Botany 2008;59(11):3187-3201.</p><p>Published online Jan 2008</p><p>PMCID:PMC2504342.</p><p></p

Alignment of the amino acid sequences of the cDNA encoding NaStEP and NaSoEP proteins

The black boxes under the alignment indicate the degree of sequence consensus. Arrowheads indicate the potential signal peptide cleavage sites. Cysteine residues are marked with an asterisk. Putative glycosylation sites are single underlined. Presumed vacuolar sorting signals are boxed. The antigenic region is double underlined. A dash in the sequence indicates a gap introduced to maintain a good alignment.<p><b>Copyright information:</b></p><p>Taken from "Pollination in stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue"</p><p></p><p>Journal of Experimental Botany 2008;59(11):3187-3201.</p><p>Published online Jan 2008</p><p>PMCID:PMC2504342.</p><p></p

NaStEP-like proteins are expressed in stigmas of SI species but no in SC species

(a) Total proteins (10 μg) from stigmas and styles of SI , SI , SI , SI , SI , SC , SC Breakthrough, SI Hybrid 1 (SC SI ), SI Hybrid 2 (SC SI ), SC , SC , SC , and SC were separated by SDS–PAGE and either Coomassie blue stained (left) or blotted to nitrocellulose for immunostaining with anti-NaStEP antibody (right). (b) Cross-sections of Breakthrough, SI SS, SI , SC , SC , SC , and SC stigmas immunostained with anti-NaStEP. I and II: diagrams of a stigma plus style of and a transverse section of a stigma. The arrow and square show zones where images were taken. UTT, upper transmitting tissue; SC, secretory cells; PC, papillae cell. Bars, 50 mm.<p><b>Copyright information:</b></p><p>Taken from "Pollination in stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue"</p><p></p><p>Journal of Experimental Botany 2008;59(11):3187-3201.</p><p>Published online Jan 2008</p><p>PMCID:PMC2504342.</p><p></p

Native NaStEP from stigmas and serine proteinase inhibitor activity assay

Purified HPLC fractions (29–36) of NaStEP were resolved by SDS–PAGE. (a) Proteins present in the HPLC fractions were transferred onto nitrocellulose and immunostained with the anti-NaStEP antibody. (b) Silver-stained proteins. (c) Inhibitory activity gel assay against trypsin. A 3 μg aliquot of the soybean trypsin inhibitor (STI) was run as a positive control. (d) A silver-stained replicate of the inhibitory activity assay gel.<p><b>Copyright information:</b></p><p>Taken from "Pollination in stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue"</p><p></p><p>Journal of Experimental Botany 2008;59(11):3187-3201.</p><p>Published online Jan 2008</p><p>PMCID:PMC2504342.</p><p></p