| Sieve elements (SEs) in caryopsis of wheat (Triticum aestivum L.) are responsible for the transport of nutrients, ions, and water. Previou study have revealed that metaphloem sieve elements (MSEs) in the developing caryopsis of wheat undergo a unique type of programmed cell death (PCD) that called programmed cell semi-death (PCSD), main at0to7days after flowering (DAF). Cell inclusions (organelles, cytoplasm et al.) gradually degrade with the MSE differentiation while mature sieve elements keep active. However, the mechanism involved in PCSD are not reported in detail and still largely unknown. Such as, how did the definite degradation of nucleus during the MSEs differentiation? As the nuclear degradation associated and regulated with some proteases? How to regulate? And if them involved in ceased programmed cell death? In order to solve the problems above, this study take multiple plant micro-technology and ultramicro-technology, such as microstructural and ultrastructural immunohistochemistry, in situ hybridization and molecular techniques. The research mainly includes the following results:1. During the development of MSEs, transmission electron microscopy (TEM) showed that nuclei degraded main in2to5DAF. First, the degradation started at2～3DAF, mainly at3～4DAF; then the degraded fragments were swallowed by phagocytic vacuole at4DAF; Finally, nuclei almost completely degraded at5DAF. Mitochondria and amyloplast were detected at5DAF, which were close to cytomembrane and the membrane fusion, and there have a few cell contents wrapped by combig space.2. The results of the DNA agarose gel electrophoresis from abdominal tissue in wheat caryopsis showed the appearance of DNA ladder, which was the one of typical morphologic changes of PCD; Gene expression analysis showed the expression level of the two type-Ⅱ Metacaspase genes (TeaMCA II and TaMCA4) have the similar dynamic change, the higher expression were at1DAF and6DAF; Immunohistochemistry showed that caspase-3-like protein was localized in abdominal tissue of wheat caryopsis, the stronger brown positive signal appeared in MSEs at1DAF and4DAF at the microscopic level, no obvious signals were observed in3to7DAF.3. Gene expression analysis also showed the overexpression of BEN1-LIKE gene also in abdominal tissue of wheat caryopsis, the highest expression was at3DAF; We measured the BEN1-LIKE protein expression and focuses to location of BEN1-LIKE protein in differentiating MSEs of wheat. Immunohistochemistry showed that BEN1-LIKE protein was mainly localized in MSEs at3-4DAF at the microscopic level. In addition, in-situ hybridization showed that BEN1-LIKE mRNA was more obvious hybridization signal at3-4DAF. Furthermore, Immuno-electron microcoy studies further revealed that BEN1-LIKE protein was mainly localized in MSEs nuclei.4. MSEs differentiation tested using a TSQ Zn2+fluorescence probe showed that the dynamic change of Zn2+accumulation was similar as BEN1-LIKE protein expression, the blue fluorescence intensity was brighter at3and4DAF than at2and5DAF.In conclusion, the expression level of the two type-Ⅱ Metacaspase genes (TeaMCA Ⅱand TaMCA4) by gene expression analysis and the localization of Caspase3-like protein by immunohistochemistry assay showed that they may play a role in the early development of MSEs. Moreover, due to the dynamic change of BEN1-LIKE protein by microstructural and ultrastructural localization and the period of nucleus degradation, which suggested that nucleus degradation in wheat MSEs was associated with BEN1-LIKE protein. In addition, the dynamic change in Zn+accumulation is similar as BEN1-LIKE expression levels. The change of Zn2+accumulation may affect the expression of BEN1-LIKE protein, which may be associated with the cease of PCD in MSEs differentiation. Furthermore, a few incomplete degradation inclusion may be preserved due to the fusion of phagocytic vacuole membrane and plasma membrane, which was beneficial to maintaining the activity of MSEs. |
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