Oxidative burst and expression of germin/oxo genes during wounding of ryegrass leaf blades: comparison with senescence of leaf sheaths

Abstract

Two bursts of H₂O₂ production have been detected by in situ 3,3′‐diaminobenzidine (DAB) staining after cutting of Lolium perenne L. leaf blades. The first burst, which occurred immediately after wounding was inhibited by Na‐diethydithiocarbamate (DIECA), a Cu/Zn–superoxide dismutase (SOD) inhibitor. The second burst, which was initiated several hours later, coincided with the induction of oxalate oxidase (G‐OXO) activity detected in vitro or visualized in situ by the α‐chloronaphtol assay. Four genes encoding G‐OXO have been identified from cDNA obtained from wounded L. perenne L. leaf blades. Comparison of protein sequences revealed more than 91% homology in the coding region between G‐OXOs of the true cereals and G‐OXOs of ryegrass, which is a Gramineae belonging to the tribe of Festucaceae. The wound‐dependent increase of G‐OXO activity in floated cut leaf blades was the result of differential induction of the four g‐oxo genes. The involvement of G‐OXOs in wound‐induced H₂O₂ production coincided with the presence in leaf tissues of oxalate throughout the period of increase of G‐OXO synthesis. Moreover, expression of g‐oxo genes was enhanced by an exogenous supply of H₂O₂ or methyljasmonate (MeJa). Expression of the four g‐oxo genes was also induced after in planta stinging of leaf blades. The pattern of their expression in planta was identical to that occuring in senescing leaf sheaths. These results emphasize the importance of G‐OXOs in H₂O₂ production in oxalate‐producing plant species such as ryegrass. G‐OXOs might be crucial during critical events in the life of plants such as cutting and senescence by initiating H₂O₂‐mediated defences against pathogens and foraging animals.