Calcium Oxalate Degradation Is Involved In Aerenchyma Formation In Typha Angustifolia Leaves

Calcium oxalate?CaOx?crystals are ubiquitous in all photosynthetic organisms,including algae,lower vascular plants,gymnosperms,and angiosperms.The main functions of calcium oxalate crystals in plants include high volume calcium?Ca?regulation and self-protection.Ultrastructuralanddevelopmentalanalyzesindicatethatthis biomineralization is not a simple and random process of endogenous oxalic acid synthesis and physicochemical precipitation of calcium from the environment.Instead,the specific shape and size of crystals are regulated by genetic regulators.The specific of crystal idioblast is characterized by the constancy of the internal morphology of the crystal,crystal growth and cell expansion.Researchers have used a variety of methods to begin to reveal the fine control mechanisms that are available to the different types of crystal idioblast,including Ca uptake and accumulation mechanisms,oxalic acid biosynthesis pathways,and crystal growth regulation.Typha angustifolia L.?Typhaceae?is a perennial aquatic or marsh plant,and aerenchyma contributes to the transportation of gas forms in its roots,rhizomes and leaves.The degradation of specific cells in the T.angustifolia leaves leads to the formation of lysigenous aerenchyma,and PCD is associated with the process of aerenchyma formation.The developing aerenchyma of T.angustifolia accompanied by changes in the formation and degradation of CaOx crystals?raphides?.Oxalate oxidase?OXO??oxalate:oxygen oxidoreductase,EC1.2.3.4?degrades calcium oxalate to carbon dioxide and hydrogen peroxide?H₂O₂?.H₂O₂ not only acts as a key inducer for different types of developmentally and environmentally programmed cell death?PCD?but also promotes the formation of aerenchyma.Therefore,the objective of this study was to describe the relationship between the degradation of CaOx crystals and aerenchyma formation.The results of this study are as follows:?1?By observing the development process of aerenchyma of T.angustifolia leaves,in the early stage of cavity formation,crystal idioblasts began to appear near the PCD-susceptible zone.The crystal idioblasts contained more crystal units,and the shape of the crystal was regular.During the formation of the cavity,the crystal idioblasts were distributed around the cavity,and the number of crystals was reduced and the morphology becomes irregular.When the cavity matures through the lysis of a large number of cells,the crystals are simultaneously degraded and the idioblasts dissociate.?2?H₂O₂ was observed by histochemical staining with DAB.It was found that strong reddish brown?H₂O₂?coloring was found in the crystal cells.The results of TBO staining showed that in the early stage of cavity formation,the crystal idioblasts stained more deeply than other cells.In the third stage,with the development of aerenchyma,the staining of the crystal idioblasts becomes more intense.The localization of cerium chloride cells showed that a large amount of H₂O₂ accumulated in the cell wall and intercellular space during the early cavity formation and the cavity formation phase.It does not exist in the mature phase of the cavity.?3?Quantitative real-time PCR was used to analyse the expression of the oxalate oxidase gene?TpOXO?and H₂O₂-related genes at different developmental stages during the formation of aerenchyma in T.angustifolia.During the degradation of CaOx crystal,TpOXO was highly expressed and the activity was highest,and the H₂O₂ concentration was highest at this stage.Moreover,H₂O₂ affected the formation of aerenchyma in T.angustifolia.?4?The oxalate oxidase gene of T.angustifolia was successfully cloned and named as TpCOXO?accession number:MF407410?.The full-length cDNA of this gene was 903 bp and contained 669 bp open reading frame,63 bp 5'untranslated region region and 167 bp3'untranslated region.The gene encoded TpOXO protein contains 223 amino acids,the predicted molecular weight and isoelectric point were 23.545 kDa and 5.74,respectively,which belongs to the hydrophobic protein.The protein is a member of the oxalate decarboxylase family and phylogenetic tree analysis shows that TpOXO was most closely related to Ananas comosus OXO.The shape and location of calcium oxalate crystals in specific tissues or cell types constitute an important classification basis.Calcium oxalate crystals have different functions in different plants.CaOx crystals occurred between PCD-susceptible cells in the early phase of aerenchyma formation in T.angustifolia leaves.When a large number of cells degrade to form a aerenchyma,high expression of oxalate oxidase and degradation of the crystal were accompanied.The degradation of CaOx crystals causes high levels of H₂O₂,which is considered a key signalling molecule that triggers PCD.This study was the first time to reveal the new function of calcium oxalate crystal degradation to produce H₂O₂ and to participate in aerenchyma PCD.