玉米CMS-S小孢子败育过程中的细胞程序性死亡

Programmed Cell Death during Abortion of Microspore in S-type Cytoplasmically Male-sterile Maize

以玉米(Zea maysL.)S型细胞质雄性不育系(CMS-S)的1对近等基因系S-Mo17Rf3Rf3和S-Mo17rf3rf3为材料,采用TdT介导的dUTP DNA末端标记(TUNEL)、细胞色素C免疫原位杂交和DNA寡聚核小体片段电泳等方法,分别在细胞学水平和DNA水平上研究了玉米CMS-S小孢子败育的细胞程序性死亡(PCD)过程。结果表明,在花粉母细胞减数分裂后的四分体解离时期,不育花药的绒粘层细胞较可育花药提前裂解;在不育系S-Mo17rf3rf3花药和花粉S-rf3中均明显出现PCD过程的DNA片段化以及线粒体细胞色素C外渗的现象,证明玉米CMS-S的花粉败育与花药绒粘层细胞的提前凋亡和小孢子细胞的程序性死亡有关。

The normal gametophyte development of S-type cytoplasmic male sterility（ CMS-S） of maize is disrupted, and then microsperes are aborted which fail to pollination. A set of near isogenic lines of CMS, S-Mo17^Rrf3Rrf3 and S-Mo17^rf3rf3 , was used, and TdT-mediated dUTP nick-end labeling （TUNEL）, in situ immunolocalization of cytochrome C and DNA ladder assay were employed to confirm that programmed cell death （PCD） processes at cytology and DNA level. It is evident that the tapetal cells of fertile anthers kept intact until microspores, and then were disintegrated on later developmental stage. Whereas the tapetal cells of sertile anthers were earlier disintegrated when tetrad cells were disjoined. DNA ladder assay revealed that the cleavage of nuclear DNA into Oligonucleosome-sized fragments took place in pre-emergent anther, as well as in pollen cells from tasseled anther of S-Mo17^rf3rf3 . These results indicated that the abortion of sterile pollens was from not only prematuration of sterile anthers but also apeptosis of pollens . Results from TUNEL assay showed that nuclear DNA cleavage happened from microspere stage to the stage of pollen aborting in sterile anther cell, but not in fertile anther cell. These results also supported the conclusion based on DNA electrophoresis. In addition, the release of cytochrome C from mitochondria to cytoplasm is a pivotal signal of launching PCD. The analysis of immunolocalization of cytochrome C showed that cytochrome C was not released before microsperes stage in S-（Rf3） , but began to release when tetrad was disjoined in S-（rf3）. During starch filling of pollen grain, DNA cleavage and the release of cytochrome C of tapetal cells were found in sterile and fertile anthers, however, these behaviors took place earlier in sterile anthr cell than in fertile anther cell. Tapetal cell of anther plays important functions during the development of microsperes, such as providing nutrition, excreting β3-1,3-glucanase to make microspores apart from tetrad. The release of pollen depends on the programmed death of anther cell too. The entire process of pollen development involves a series of fine modulation on molecular and cytological level. Each misregulation, such as abnormal excreting β-1,3-glucanase, prematuration of tapetal cells and abnormal trigger of PCD of anther cells, is likely to cause male sterility. The release of cytochrome C and the cleavage of DNA were observed at tetrad stage in sterile anthers but not in fertile anthers, implying that PCD which was launched earlier than pre-established programmer was a potential reason of pollen abortion in maize CMS-S.