Interaction between a coating-borne peptide of the Brassica pollen grain and stigmatic S (self-incompatibility)-locus-specific glycoproteins.

Methods are described for the removal of the sporophytic pollen grain coating of Brassica oleracea and for the isolation of coat polypeptides. The coat contains a small number of proteins ranging from 6 to 45 kDa. Many of the larger proteins are glycosylated, while all carry high positive charges resulting in pI values from 8.5 to 11. Polypeptides with pI values of 9.5, 9.0, and 8.5 possess strong esterase activity. No major differences could be detected in either pI values or molecular masses of pollen-coating polypeptides from grains carrying different sporophytically expressed S (self-incompatibility) alleles. Mixing pollen coat proteins with stigmatic extracts results in a conspicuous binding interaction involving female S-locus-specific and perhaps S-locus-related glycoproteins. This interaction, which is reversed by heating in the presence of SDS, results in an apparent charge shift of the female glycoprotein(s) of up to 2 pI units. The male participant in this interaction has been isolated by using a combination of fast protein liquid chromatography and reverse-phase HPLC and was shown to be a 7-kDa nonglycosylated peptide. Experiments with whole pollen cultured in vitro show challenge with stigmatic extracts to stimulate the release of gametophytic and sporophytic polypeptides and to result in the formation of a conspicuous interaction product, demonstrating the 7-kDa peptide to be freely available within the coating of pollen in vivo. IMAGES:

本研究建立了去除甘蓝（Brassica oleracea）花粉粒的孢子体包被并分离其包被多肽的实验方法。该包被所含蛋白质的分子量介于6至45千道尔顿（kDa），且蛋白种类较少。其中多数分子量较大的蛋白为糖基化修饰蛋白，所有蛋白均携带大量正电荷，等电点（pI）处于8.5至11之间。等电点为9.5、9.0和8.5的多肽具备较强的酯酶活性。针对携带不同孢子体表达的S（自交不亲和，self-incompatibility）等位基因的花粉粒，其花粉包被多肽的等电点与分子量均未检测到显著差异。将花粉包被蛋白与柱头提取物混合后，可观察到显著的结合相互作用，该作用涉及雌性S位点特异性糖蛋白，以及可能存在的S位点相关糖蛋白。该相互作用可在十二烷基硫酸钠（SDS）存在下通过加热逆转，并会使雌性糖蛋白的表观电荷发生最多达2个等电点单位的偏移。本研究通过快速蛋白液相色谱（fast protein liquid chromatography, FPLC）与反相高效液相色谱（reverse-phase HPLC, RP-HPLC）联用的方法，分离得到了该相互作用中的雄性结合组分，经鉴定其为分子量7 kDa的非糖基化多肽。体外培养完整花粉的实验显示，用柱头提取物刺激可诱导配子体与孢子体多肽的释放，并形成显著的相互作用产物，由此证明该7 kDa多肽在体内的花粉包被中可自由获取。图像集：