College of Horticulture, Shenyang Agricultural University, Shenyang 110866, Liaoning, China; Institute of Horticulture Crops, Xinjiang Academy of Agricultural Sciences (Key Laboratory of Genome Research and Genetic Improvement of Xinjiang Characteristic Fruits and Vegetables), Urumqi 830091, China.
Liaoning Institute of Pomology, Xiongyue 115009, Liaoning, China.
J Proteomics. 2022 Mar 30;256:104505. doi: 10.1016/j.jprot.2022.104505. Epub 2022 Feb 2.
The self-incompatibility recognition mechanism determines whether the gametophyte is successfully fertilized between pollen tube SCF (SKP1-CUL1-F-box-RBX1) protein and pistil S-RNase protein during fertilization is unclear. In this study, the pistils of two almond cultivars 'Wanfeng' and 'Nonpareil' were used as the experimental materials after self- and nonself/cross-pollination, and pistils from the stamen-removed flowers were used as controls. We used fluorescence microscopy to observe the development of pollen tubes after pollination and 4D-LFQ to detect the protein expression profiles of 'Wanfeng' and 'Nonpareil' pistils and in controls. The results showed that it took 24-36 h for the development of the pollen tube to 1/3 of the pistil, and a total of 7684 differentially accumulated proteins (DAPs) were identified in the pistil after pollinating for 36 h, of which 7022 were quantifiable. Bioinformatics analysis based on the function of DAPs, identified RNA polymerases (4 DAPs), autophagy (3 DAPs), oxidative phosphorylation (3 DAPs), and homologous recombination (2 DAPs) pathways associated with the self-incompatibility process. These results were confirmed by parallel reaction monitoring (PRM), protein interaction and bioinformatics analysis. Taken together, these results provide the involvement of serine/threonine kinase protein in the reaction of pollen tube recognition the nonself- and the self-S-RNase protein. SIGNIFICANCE: Gametophytic self-incompatibility (GSI) is controlled by the highly polymorphic S locus or S haplotype, with two linked self-incompatibility genes, one encoding the S-RNase protein of the pistil S-determinant and the other encoding the F-box/SLF/SFB (S haplotype-specific F-box protein) protein of the pollen S-determinant. The recognition mechanism between pollen tube SCF protein and pistil S-RNase protein is divided into nonself- and self-recognition hypothesis mechanisms. At present, two hypothetical mechanisms cannot explain the recognition between pollen and pistil well, so the mechanism of gametophytic self-incompatibility recognition is still not fully revealed. In this experiment, we investigated the molecular mechanism of pollen-pistil recognition in self-incompatibility using self- and nonself-pollinated pistils of almond cultivars 'Wanfeng' and 'Nonpareil'. Based on our results, we proposed a potential involvement of the MARK2 (serine/threonine kinase) protein in the reaction of pollen tube recognition of the nonself- and the self-S-RNase protein. It provides a new way to reveal how almond pollen tubes recognize the self and nonself S-RNase enzyme protein.
自交不亲和识别机制决定了花粉管 SCF（SKP1-CUL1-F-box-RBX1）蛋白与雌蕊 S-RNase 蛋白之间的配子体是否在受精过程中成功受精，目前尚不清楚。本研究以两个巴旦木品种‘万丰’和‘非那利’的雌蕊为实验材料，进行自交和异交授粉后，以去雄花的雌蕊作为对照。我们使用荧光显微镜观察授粉后花粉管的发育情况，并使用 4D-LFQ 检测‘万丰’和‘非那利’雌蕊及对照中雌蕊的蛋白质表达谱。结果表明，花粉管发育到雌蕊的 1/3 处需要 24-36 小时，授粉 36 小时后共鉴定出 7684 个差异积累蛋白（DAP），其中 7022 个是可定量的。基于 DAP 功能的生物信息学分析，鉴定出与自交不亲和过程相关的 RNA 聚合酶（4 个 DAP）、自噬（3 个 DAP）、氧化磷酸化（3 个 DAP）和同源重组（2 个 DAP）途径。这些结果通过平行反应监测（PRM）、蛋白质相互作用和生物信息学分析得到了验证。综上所述，这些结果表明丝氨酸/苏氨酸激酶蛋白参与了花粉管识别非自身和自身 S-RNase 蛋白的反应。
配子体自交不亲和性（GSI）由高度多态性的 S 座位或 S 单倍型控制，包含两个连锁的自交不亲和基因，一个编码雌蕊 S 决定簇的 S-RNase 蛋白，另一个编码花粉 S 决定簇的 F-box/SLF/SFB（S 单倍型特异性 F-box 蛋白）蛋白。花粉管 SCF 蛋白与雌蕊 S-RNase 蛋白之间的识别机制分为非自身和自身识别假说机制。目前，两种假设机制都不能很好地解释花粉与雌蕊之间的识别，因此配子体自交不亲和性的识别机制仍未完全揭示。在这项实验中，我们使用‘万丰’和‘非那利’巴旦木品种的自交和异交雌蕊，研究了自交不亲和中花粉-雌蕊识别的分子机制。基于我们的结果，我们提出了 MARK2（丝氨酸/苏氨酸激酶）蛋白可能参与了花粉管识别非自身和自身 S-RNase 蛋白的反应。这为揭示巴旦木花粉管如何识别自身和非自身 S-RNase 酶蛋白提供了一种新方法。
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