Research On Aerenchyma Formation Pattern And Dynamic Changes Of Reactive Oxygen Species Induced By Waterlogging In Wheat Seminal Roots

Aerenchyma formation and ROL barrier development has been two important mophorlogical adaptions to flooding.Wheat is an upland crop, which develops aerenchyma under waterlogging conditions, ROS has been reported as a signal that mediate the cell death in many species under adverse conditions.The aim of this study is to characterize the pattern of cortical aerenchyma formation, along with the development of ROL barrier and its regulation by ROS. Plants were harvested after the flooding treatment at the time indicated. The development pattern of aerenchyma and charateristics of pre-aerenchymal cells were viewed with a light, fluoresencent or transmision electron microscope. Genes regulating the homestatis of H2O2were studied by the q-PCR method. The main results were as follows:Evans blue staining indicated that some cortical cells started to be stained at4h waterlogging. By8h and12h waterlogging, the staining became stronger in cortex, and the number of stained cells increased. By24h and48h waterlogging, most cortical cells were stained. Cell acidification staining showed that no cells were stained under0h waterlgging treatment indicating that no cell acidification detected under normal growth conditions. By4h waterlogging, the staining became stronger in cortex, and the number of stained cells increased indicating cell adicification became stronger. By24h waterlogging, some stained cortical cells started to lyse indicating the beginning of aerenchyma formation. By48h waterlogging, aerenchyma had developed, and the staining were only detected in the cells surrounding gas spaces.Phloroglucinol staining indicated that lignin was observed in endodermis, but not in the exodermis under normal growth conditions. Exposing roots to anaerobic conditions resulted in heavy lignin deposition in endodermis and exodermis.Casparian bands staining showed that in wheat seedling roots, grown in aerated or stagnant conditions, Casparian bands were observed in endodermis in both treatment, but only in the exodermis of roots under waterlogging conditions, the development of Casparian bands was detected.Cross-section of wheat roots under0-72h waterlogging treatment indicated that first cortical cells death first started at the mid-cortex cells. With the continuing of waterlogging treatment, the cell death from the mid-cortex spread to its neighboring cells; by72h, well developed aerenchyma had formed. Analysis of aerenchyma along the wheat root revealed that aerenchyma started to form at10mm behind the root tip, and significantly increased towards the center of the roots, then decreased toward the basal regions of the root.In-situ detection of ROS showed that ROS accumulation was first detected in the mid cortex cells coinciding with the results of first cells death position. Further waterlogging treatment resulted in the accumulation of ROS in the cortical cells which were the zone where aerenchyma developed, at the final stage of waterlogging; ROS accumulation decreased with the formation of aerenchyma and was only observed in the cells surrounding the gas sapces. On the other hand, H2O2ultracytochemical localization revealed the accumulation and distribution of H2O2at the subcellular level which further indicated the involvement of ROS in the cortical cell death process (i.e. aerenchyma formation). Subcellular localization of ROS indicated that:waterlogging induced H2O2production is mainly divided into three stages according to the H2O2accumulation locations. The first stage starts shortly after the onset of waterlogging treatment (6h), the H2O2accumulation is mainly on the cell membrane but is not observed in other subcellular locations. The second phase (12h～24h) starts with the intercellular and intracellular accumulation of H2O2production. At this stage cell degradation is observed, especially the rupture of mitochondria which has been recognized as central players in ROS-dependent apoptotic cell death characterized by the release of cytochrome C which is necessary for capaspase activation triggered cell death In the third stage (48h), H2O2accumulation is only observed in the degrading debris vesicles.Besides, gene expression analysis showed that the expression level of genes controlling the ROS production were up-regulated while genes controlling ROS elimination were down-regulated. These results indicated that ROS production might be the result of up-regulation of ROS producing genes and down-regulation of ROS detoxifying genes.These results suggest that the aerenchyma development in wheat roots starts from the mid-cortex cells and its formation is regulated by ROS.