Song Liping, Zhou Zhengfu, Tang Shan, Zhang Zhiqiang, Xia Shengqian, Qin Maomao, Li Bao, Wen Jing, Yi Bin, Shen Jinxiong, Ma Chaozhi, Fu Tingdong, Tu Jinxing
National Key Laboratory of Crop Genetic Improvement, National Center of Rapeseed Improvement in Wuhan, Huazhong Agricultural University, Wuhan 430070, China.
Wheat Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China.
Plant Cell Physiol. 2016 Sep;57(9):1972-84. doi: 10.1093/pcp/pcw119. Epub 2016 Jul 7.
Tapetal programmed cell death (PCD) is essential in pollen grain development, and cysteine proteases are ubiquitous enzymes participating in plant PCD. Although the major papain-like cysteine proteases (PLCPs) have been investigated, the exact functions of many PLCPs are still poorly understood in PCD. Here, we identified a PLCP gene, BnaC.CP20.1, which was closely related to XP_013596648.1 from Brassica oleracea. Quantitative real-time PCR analysis revealed that BnaC.CP20.1 expression was down-regulated in male-sterile lines in oilseed rape, suggesting a connection between this gene and male sterility. BnaC.CP20.1 is especially active in the tapetum and microspores in Brassica napus from the uninucleate stage until formation of mature pollen grains during anther development. On expression of BnaC.CP20.1 prior to the tetrad stage, BnA9::BnaC.CP20.1 transgenic lines in Arabidopsis thaliana showed a male-sterile phenotype with shortened siliques containing fewer or no seeds by self-crossing. Scanning electron microscopy indicated that the reticulate exine was defective in aborted microspores. Callose degradation was delayed and microspores were not released from the tetrad in a timely fashion. Additionally, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay indicated that BnaC.CP20.1 ectopic expression led to premature tapetal PCD. Transmission electron microscopy analyses further demonstrated that the pollen abortion was due to the absence of tectum connections to the bacula in the transgenic anthers. These findings suggest that timely expression of BnaC.CP20.1 is necessary for tapetal degeneration and pollen wall formation.
绒毡层程序性细胞死亡（PCD）在花粉粒发育过程中至关重要，而半胱氨酸蛋白酶是参与植物PCD的普遍存在的酶。尽管主要的木瓜蛋白酶样半胱氨酸蛋白酶（PLCPs）已被研究，但许多PLCPs在PCD中的具体功能仍知之甚少。在此，我们鉴定了一个PLCP基因BnaC.CP20.1，它与来自甘蓝的XP_013596648.1密切相关。定量实时PCR分析表明，油菜雄性不育系中BnaC.CP20.1的表达下调，表明该基因与雄性不育之间存在联系。在甘蓝型油菜的花药发育过程中，从单核期到成熟花粉粒形成，BnaC.CP20.1在绒毡层和小孢子中特别活跃。在四分体阶段之前表达BnaC.CP20.1时，拟南芥中的BnA9::BnaC.CP20.1转基因系表现出雄性不育表型，自交后角果缩短，种子较少或没有种子。扫描电子显微镜表明， aborted小孢子中的网状外壁有缺陷。胼胝质降解延迟，小孢子没有及时从四分体中释放出来。此外，末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）分析表明，BnaC.CP20.1异位表达导致绒毡层过早发生PCD。透射电子显微镜分析进一步证明，转基因花药中的花粉败育是由于外壁与基粒棒之间缺乏连接。这些发现表明，BnaC.CP20.1的及时表达对于绒毡层退化和花粉壁形成是必要的。
