Calcium Oxalate Degradation Involved In The Programmed Death Of Tobacco Anther Crack Tissue And Ring Cell Cluster

Calcium oxalate crystals in tobacco anthers exist in the heterologous cells and ring cell clusters in the drug septum.The process of calcium oxalate crystal accumulation to degradation is closely related to not only the ring cell cluster but also the development,maturity and death of the drug-resistant tissue.The paraffin sections of tobacco anthers in different development periods were observed.It was found that calcium oxalate crystal presented obvious accumulation and degradation with the development of tobacco anthers.NBT and DAB staining demonstrated that when calcium oxalate crystal degradation,a large amount of H₂O₂ was produced in tobacco anthers.It is concluded that when calcium oxalate crystal is degraded and oxalic acid is released into cells,oxalic acid is decomposed into CO₂ and H₂O₂ by the action of oxalate oxidase.In this paper,the anthers of model plant tobacco were treated with ascorbic acid and H₂O₂ respectively.The results indicated that the anthers in H₂O₂ treatment group displayed a significant tendency of early cracking compared with the control group,while the anthers of ascorbic acid treatment group showed slight effect of delaying cracking compared with the control group.The results revealed that when the calcium oxalate crystal in the septum of tobacco anther was degraded in advance,the ring cell cluster also entered PCD process in advance,forming a clear cavity.The results exhibited that H₂O₂ had a significant effect on the development and cracking process of tobacco anthers.In order to further explore the relationship between the degradation of calcium oxalate crystal and anther cracking,GLP0806 group in Germin-like proteins family was selected as the research object.The Germin-Like proteins family was considered to have oxalate oxidase activity.In this paper,GLP0806 gene was successfully cloned and proved to be expressed in tobacco anthers.The GLP0806 protein encoded by this gene contains 226 amino acid residues,with a relative molecular weight of 23884.42,and the predicted isoelectric point（PI）of 6.17,which is shown as Homo hexamer possessing instrong ion ligands.The results of Agrobacterium transient transformation and eukaryotic expression system of Saccharomyces cerevisiae presented that GLP0806 could express the protein with stable oxalate oxidase activity in plant and yeast cells.The above experiments represent that the H₂O₂ produced by the degradation of calcium oxalate crystal under the action of oxalate oxidase is involved in the programmed death process of tobacco anther crack tissue and ring cell cluster.The results revealed that the relationship between the degradation of calcium oxalate crystal and PCD in tobacco anther cracking was studied.