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

摘要

[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,Ca2+ 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.%[目的]探讨Ca~(2+)和Ca~(2+)-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Ca~(2+)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Ca~(2+)进行组织和亚细胞水平的定位;同时用铅盐沉淀法对Ca~(2+)-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.Ca~(2+)荧光试验表明,花后6～10 d,SEs细胞壁中有Ca~(2+)的积累,其中花后9 d,SEs细胞壁Ca~(2+)浓度最高;到花后14 d,细胞壁Ca~(2+)浓度下降至对照水平.Ca~(2+)亚细胞定位表明,在SEs中,花后1-2 d Ca~(2+)主要分布在细胞膜上和细胞核中;花后4 d,SEs细胞质中Ca2+浓度增加,并且线粒体中也出现Ca~(2+)颗粒;但到花后5-8 d,Ca~(2+)主要分布在SEs细胞壁中,此时线粒体中未发现Ca~(2+)颗粒;在花后10～18 d,Ca~(2+)再次从细胞壁转移到胞内;花后20 d,SEs中Ca~(2+)消失.在中间细胞(intermediary cells,ICs)中,花后1～18 d始终都有Ca~(2+)颗粒,主要分布在细胞内壁上和液泡中.在SEs发育过程中,Ca~(2+)-ATPase的活性发生显著变化.花后3 d时,SBs中的Ca~(2+)-ATPase活性最弱;花后4-14 d SEs有较强的Ca~(2+)-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]Ca~(2+)和Ca~(2+)-ATPase在小麦颖果SEs的分化过程中呈动态变化,Ca~(2+)可能参与介导了SEs的PCD过程.此外,Ca~(2+)和Ca~(2+)-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.