Ca^(2+)和Ca^(2+)-ATPase在小麦颖果筛分子分化中的动态变化

Dynamic Changes of Ca^(2+) and Ca^(2+)-ATPase in Sieve Elements in the Developing Caryopsis of Triticum aestivum L.

【目的】探讨Ca2+和Ca2+-ATPase在小麦颖果筛分子(sieve elements,SEs)分化过程中的动态变化及其在SEs的细胞程序性死亡(programmed cell death,PCD)中的作用。【方法】用透射电子显微术观察小麦颖果韧皮部分化过程中的超微结构变化;用Ca2+特异性荧光染色法和焦锑酸钾沉淀法,对小麦颖果韧皮部分化过程中的Ca2+进行组织和亚细胞水平的定位;同时用铅盐沉淀法对Ca2+-ATPase进行定位。【结果】超微结构观察发现,在SEs发育初期,细胞壁逐渐加厚,且内壁呈突起状,随着分化的进行,SEs细胞壁较以前明显变薄且平滑。Ca2+荧光试验表明,花后6～10d,SEs细胞壁中有Ca2+的积累,其中花后9d,SEs细胞壁Ca2+浓度最高;到花后14d,细胞壁Ca2+浓度下降至对照水平。Ca2+亚细胞定位表明,在SEs中,花后1～2dCa2+主要分布在细胞膜上和细胞核中;花后4d,SEs细胞质中Ca2+浓度增加,并且线粒体中也出现Ca2+颗粒;但到花后5～8d,Ca2+主要分布在SEs细胞壁中,此时线粒体中未发现Ca2+颗粒;在花后10～18d,Ca2+再次从细胞壁转移到胞内;花后20d,SEs中Ca2+消失。在中间细胞(intermediarycells,ICs)中,花后1～18d始终都有Ca2+颗粒,主要分布在细胞内壁上和液泡中。在SEs发育过程中,Ca2+-ATPase的活性发生显著变化。花后3d时,SEs中的Ca2+-ATPase活性最弱;花后4～14dSEs有较强的Ca2+-ATPase活性,且主要分布在SEs的细胞壁、细胞膜、胞间连丝等部位和线粒体、细胞核等细胞器上。【结论】Ca2+和Ca2+-ATPase在小麦颖果SEs的分化过程中呈动态变化,Ca2+可能参与介导了SEs的PCD过程。此外,Ca2+和Ca2+-ATPase可能对SEs细胞壁的加厚和SEs的功能实施有一定调控作用。

【Objective】 Previous study revealed that sieve elements（SEs） in the developing caryopsis of Triticum aestivum L.underwent a unique type of programmed cell death（PCD） .In this paper,the dynamic changes and the roles of Ca^2＋ and Ca^2＋-ATPase in SEs during the PCD were studied.【Method】 The ultrastructural aspects of phloem cells in wheat caryopsis were examined by transmission electron microscopy（TEM）.Using specific fluorescence staining and potassium pyroantimonate precipitation method,Ca^2＋ was localized at histological and sub-cellular levels in SEs in the developing wheat caryopsis.TEM and lead nitrate were used to locate Ca^2＋-ATPase in SEs.【Result】 TEM studies showed that the cell walls of SEs thickened at the beginning of differentiation,and then became thinner and smoother.Fluorescence staining showed that the fluorescence due to Ca^2＋ appeared in cell walls of SEs from 6 to 10 d after flowering.The fluorescence due to Ca^2＋ in cell walls of SEs was most notable on 9 d after flowering and disappeared on 14 d after flowering.Sub-celluar localization of Ca^2＋ showed that Ca^2＋ was localized on plasma membrane and in nuclei from 1 to 2 d after flowering.On 4 d after flowering,Ca^2＋ was localized in cytoplasm and mitochondria of SEs.From 5 to 8 d after flowering,Ca^2＋ was transported to the cell walls of SEs and no Ca^2＋ precipitates were observed in mitochondria.From 10 to 18 d after flowering,Ca^2＋ was transported into the cytoplasm again from cell walls and no Ca^2＋ precipitates were observed on 20 d after flowering.In intermediary cells（ICs）,Ca^2＋ precipitates were observed from 1 to 18 d after flowering,and Ca^2＋ mainly distributed on intine and tonoplast.The activity of Ca^2＋-ATPase changed obviously during the SEs differentiation.There was lowest activity of Ca^2＋-ATPase on 3 d after flowering in SEs.High levels of Ca^2＋-ATPase activity were found from 4 to 14 d after flowering in SEs,and the enzyme was mainly localized in cell walls,cytoplasm,plasmodesmata,mitochondria and nuclei.【Conclusion】 These results showed the dynamic changes of Ca^2＋ and Ca^2＋-ATPase in SEs differentiation.Ca^2＋ might play important roles in SEs during the PCD.In addition,Ca^2＋ and Ca^2＋-ATPase might be also related to cell wall thickening and functions of SEs.