The Mechanism Research Programmed Cell Semi-Death Of Sieve Elements In Wheat

The main function of sieve elements (SEs) in the ventral vascular bundle of wheat caryopsis transported the photoassimilate to endosperm of the caryopsis. The differentiation of SEs went through the nucleus and the cytoplasm degradation, including ribosomes, endoplasmic reticulums, mitochondria, Golgi and vacuole, and kept cell membrane and a little cytoplasm, such as a small amount of intact mitochondria organelles. The study had confirmed that SEs differentiation experienced programmed cell death (PCD) process, but it was different from PCD, so calling programmed cell semi-death (PCSD). The mechanism of PCSD is not so clear that many problems would be needed to be done. For example, although many studies had confirmed that the cytoplasm was degraded, how cytoplasm degraded was without relevant reports? How did intracellular pH change during SEs differentiation? Why did programmed cell death process cease in SE differentiation, whether did a protease inhibitor which plant made by evolution play a role? For solving problems above, we took advantage of various technologies to carry out the research, including plant microscopy methods, immunohistochemical and ultrastructural localization, and molecular biology technology. The results obtained were as follows:1. It was found that the characteristics of SEs structure were as follows:(1) The degradation of chromatin happened in the nucleus, and the nucleus did not split. The nuclear membrane remained intact after chromatin was completely degradation. The nuclear pore enlarged. The vacuole was intact after the nucleus was degraded.(2) The cytoplasm was degraded in the vacuole, which occured after the nucleus degradation. At4.5DAF a mount of cytoplasm began to be degraded. A lot of the cytoplasm bulged inward the vacuole, and then be degraded by hydrolase in the vacuole, which is called microautophagy.(3) At5and6DAF, it appeared a special change that a part of tonoplast and plasma membrane merged, and some vacuole membrane ruptured after much of the cytoplasm were degraded in the vacuole So the portion of cytoplasm could be kept in space which was consisted of tonoplast and plasma membrane. The rupture made that vacuolar enzymes and surplus cytoplasm are mixed with each other.(4) At0and1DAF, endoplasmic reticulum(ER) broke into short fragments which appeared frequently around small vacuoles. The cavity of short ER fragments inflated and the expansion of the structures also were arranged around small vacuoles. The end of ER connected with rupture of tonoplasm.2. We detected acid changes in different developmental stages of SEs by pH fluorescent probe. The results noted SEs showed yellow-green fluorescence extremely weak only at5DAF. At the same time, bright yellow-green fluorescence was found in the perycarp and TE of1DAF. Compared with the acid change of perycarp and TE, SEs showed weak acid at5DAF according to fluorescence color.3. Immunohistochemical localization of WAP1displayed that WAP1was located in the development of SEs. The following was showed the expression of WAP1from2DAF to6DAF. The little expression of WAP1was first present in SEs and companion cells at2DAF. The labeling intensity increased slightly at3d,4d and5d after flowering. However, WAP1expression was the most obvious at5DAF (indicated by the arrow); there was barely intensity at6DAF. We used immune-electron microscopy technique to firstly confirm that WAP1were found in the vacuole of SEs at4DAF. Some of gold particles were connected to the electron dense. At5DAF, the label evenly distributed in the cytoplasm of SEs, but gold particles were not existed in organelles and cytoplasm close to cell wall. In situ hybridization results of WAP1mRNA showed that WAP1mRNA was transcripted from2DAF to6DAF. But the transcription quantity reached a maximum in the ventral vascular bundle at2DAF, and then decreased gradually. But WAP1mRNA was only transcripted in part of the cells of ventral vascular bundle, the volume of which was generally bigger than peripheral cells.4. In situ hybridization results of wheat cystatins mRNA showed that a small amount of WCs mRNA was only transcriped in ventral vascular bundle at4DAF and5DAF, much WCs mRNA was transcripted in parenchyma cells near TEs. In addition, cystatins mRNA was transcriped in SEs of wheat seedling root. 5. We used in situ detection of cathepsin B activity in tissues. The results showed cathepsin B activity was in SEs of ventral vascular bundle. According to the fluorescence intensity, cathepsin B activity was strongest in SEs after4DAF. At2DAF, cathepsin B activity began to appear. When cathepsin B activity in SEs was strongest, cathepsin B activity reduced gradually. Cathepsin B activity also appeared in SEs of development root tip.The experimental results confirmed that the degradation of cytoplasm was mediated by microautophagy of the vacuole, and then tonoplast selectively fused with the plasma membrane after cytoplasm in the vacuoles basically degraded completely. Combing space contained a small amount of cytoplasm, so that fraction of the cytoplasm was preserved. The cytosol appeared weak acid during membranes fusion. We also demonstrated that wheat aspartic protease (WAP1) and proteases including cathepsin B activity (PICA) were involved in programmed cell semi-death of SEs. PICA possessed the strongest activity before mass of cytoplasm was degraded, which might be kept sieve element stability, while WAP1mainly degraded the cytoplasm. Therefore, programmed cell semi-death of SEs might be vacuoles and multiple proteases the result of joint action. Vacuoles played an important role in the development of SEs.