Cell Wall Degradation And The Dynamic Changes Of Ca²⁺ In The Developing Aerenchyma Of Wheat (Triticum Aestivum L.) Roots Under Waterlogging

Waterlogging is a serious limitation to winter wheat grown in the Yangtze River valley of China. It has rarely been reported that programmed cell death (PCD) is involved in the formation of lysigenous aerenchyma in wheat especially the booting stage of wheat roots under waterlogging. This study use the booting stage of wheat (Triticum aestivum L. Huamai 8) as the material. This study determines the characteristics of cortical PCD in wheat roots by TUNEL assay and DNA agarose gel electrophoresis; Fluorescence microscopy was used to detect the accumulation of Ca2+in wheat roots; Detect the degradation of Cell wall in the formation of lysigenous aerenchyma in wheat roots by transmission electron microscopy (TEM); Detect the dynamic changes of Ca2+ Ca2+-ATPase and cellulose in wheat roots by Ultracytochemical localization. The results were as follows:1. The first detectable degradation site of cell wall in middle cortical cell was primary wall, and then the intercellular space. The structure of cell wall in middle cortical remained intact after 12 h of waterlogging. After 24 h of waterlogging, middle cortical cell wall turned thiner apparently. After 48 h of waterlogging, the aerenchyma has been formed, and the middle cortical present radial. This research displayed subcellular localization of cellulase indicates that the cellulase in cortical cells showed high activity after 8 h of waterlogging. After 24 h of waterlogging, cellulose accumulated and formed many degradation sites in cell wall of middle cortical. After 48 h of waterlogging, the cellulase activity decreased.2. Cortical cells in wheat roots have been degraded and form aerenchyma while some exist as the support of aerenchyma. In the formation of aerenchyma TUNEL-positive nuclei was observed. At the early stage the percentage of TUNEL-positive nuclei in wheat roots was low, very high at the middle stage, and the percentage of TUNEL-positive nuclei began to reducein at the later stage. The gel electrophoresis showed that nuclear DNA had been fragmented in response to flooding for 24 h. The smallest DNA fragment was about 180 bp which are characteristic of PCD. After flooding for 96 h and 120 h the laddering became less distinct. Chromatin condensation and cell wall became electron transparent were also observed by TEM. All of this are characteristic of PCD.3. In normal cortical cells, Ca2+ fluorescence evenly distributed in cell wall. At the later stage, Ca2+ fluorescence was observed in cell wall of endodermis, stele and which will be exist as the support of aerenchyma. Subcellular localization of Ca2+showed the speedy increased of Ca2+reaction deposits in plasma membrane, tonoplast and some vesicles in middle cortical after 4 h of waterlogging. A large number of Ca2+reaction deposits were observed in middle cortical after 12 h of waterlogging. After 24h of waterlogging, the number of Ca2+ reaction deposits began to decrease. Ca2+ reaction deposits had significant discrepancy between adjacent cells in PM and some small vesicle of cortical in wheat roots. Subcellular localization of Ca2+-ATPase showed its activity enhances markedly in intercellular space, plasma membrane and tonoplast of some middle cortical cells after 8 h of waterlogging. After 24 h of waterlogging the activity of Ca2+-ATPase still remained high, while it decreased after 48 h of waterlogging. The activity of Ca2+-ATPase had significant discrepancy between adjacent cells of cortical in wheat roots.All of the above suggests that PCD is involved in the formation of lysigenous aerenchyma in the booting stage of wheat roots under waterlogging. TUNEL assay and DNA agarose gel electrophoresis which are classic characteristic of PCD. The Ca2+plays an important role in the procedure of plant PCD as signal. Ca2+is an important regulator of plasmodesma apertures. Dense Ca2+can cause the plasmodesmata to close up, so through the closing of plasmodesmata, the cortical cells will shape symplasmic domains. It can be seen that Ca2+plays a vital role to the optional dying of cortical cells. Cellulose, Ca2+and Ca2+-ATPase show a dynamic distribution which associated with the cell wall degradation of middle cortical. Moreover, It is a feedback regulation between Ca2+and Ca2+-ATPase.