Wang M, Hoekstra S, van Bergen S, Lamers G E, Oppedijk B J, van der Heijden M W, de Priester W, Schilperoort R A
Center for Phytotechnology RUL/TNO, TNO Department of Plant Biotechnology, Leiden, The Netherlands.
Plant Mol Biol. 1999 Feb;39(3):489-501. doi: 10.1023/a:1006198431596.
Intra-nucleosomal cleavage of DNA into fragments of about 200 bp was demonstrated to occur in developing anthers, in which microspores had developed into the mid-late to late uni-nucleate stage in situ, i.e. at the verge of mitosis. The same was observed, but to a much larger extent, if these anthers were pre-treated by a hyper-osmotic shock. Pretreatment of anthers before the actual culture of microspores was required for optimal androgenesis of microspores. The use of the TUNEL reaction, which specifically labels 3' ends of DNA breaks, after intra-nucleosomal cleavage of DNA, revealed that DNA fragmentation mainly occurred in the loculus wall cells, tapetum cells and filament cells. TUNEL staining was absent or infrequently observed in the microspores of developing anthers in situ. Electron microscopy studies showed condensed chromatin in nuclei of loculus wall cells in the developing anthers. These observations at the chromatin and DNA level are known characteristics of programmed cell death, also known as apoptosis. Features of apoptosis were infrequently found in microspores from freshly isolated mature anthers. However, most tapetum cells had disappeared in these anthers and the remaining cell structures showed loss of cellular content. The viability of microspores in pre-treated anthers was comparable to those in freshly isolated anthers and almost four times higher than in anthers from control experiments. This observation was correlated with three to four times less microspores showing TUNEL staining and a two times higher level of ABA in the anther plus medium samples than in controls. Addition of ABA to the controls enhanced the viability and lowered the occurrence of apoptosis linked characteristics in the microspores. These data suggest that pre-treatment is effective in stimulating androgenesis because it leads to an increase in ABA levels which protects microspores from dying by apoptosis.
已证明在发育中的花药中会发生核小体内DNA切割成约200 bp的片段，其中小孢子在原位发育到单核中期至晚期，即处于有丝分裂边缘。如果对这些花药进行高渗休克预处理，也会观察到同样的现象，只是程度要大得多。在实际培养小孢子之前对花药进行预处理是小孢子最佳雄核发育所必需的。在DNA核小体内切割后使用TUNEL反应（该反应特异性标记DNA断裂的3'末端），结果显示DNA片段化主要发生在药室壁细胞、绒毡层细胞和花丝细胞中。在原位发育的花药小孢子中未观察到或很少观察到TUNEL染色。电子显微镜研究显示发育中的花药药室壁细胞核内染色质浓缩。这些在染色质和DNA水平的观察结果是程序性细胞死亡（也称为细胞凋亡）的已知特征。在新鲜分离的成熟花药的小孢子中很少发现细胞凋亡特征。然而，这些花药中的大多数绒毡层细胞已经消失，其余细胞结构显示细胞内容物丧失。预处理花药中小孢子的活力与新鲜分离花药中的小孢子相当，几乎是对照实验花药中小孢子活力的四倍。这一观察结果与显示TUNEL染色的小孢子数量减少三到四倍以及花药加培养基样品中的ABA水平比对照高两倍相关。向对照中添加ABA可提高小孢子的活力并降低与细胞凋亡相关特征的发生率。这些数据表明预处理在刺激雄核发育方面是有效的，因为它导致ABA水平升高，从而保护小孢子免于因细胞凋亡而死亡。
