关键词: 玉米（Zea mays L.）, 细胞质雄性不育, TdT介导的dUTP DNA末端标记, 细胞色素C, 细胞程序性死亡

Abstract:

The normal gametophyte development of S-type cytoplasmic male sterility（CMS-S）of maize is disrupted, and then microspores are aborted which fails to pollination. A set of near isogenic lines of CMS, S-Mo17^Rf3Rf3 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) process 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 apoptosis of pollens . Results from TUNEL assay showed that nuclear DNA cleavage was happened from microspore 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 microspores 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 tha development of microspores, such as providing nutrition, excreting b-1,3-glucanase to make microspores apart from tetrad. The release of pollen depends on the programmed death of anther celll too. The entire process of pollen development involves a series of fine modulation on molecular and cytological level. Each misregulation, such as abnormal excreting b-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 preestablished programmer was a potential reason of pollen abortion in maize CMS-S.

Key words: Maize (Zea mays L.), Cytoplasmic male sterility, TdT-mediated dUTP nick-end labeling, Cytochrome C, Programmed cell death