Involvement of reactive oxygen species in regulation of plant growth and development is recently being demonstrated with various results depending on the experimental system and plant species. Role of superoxide and its metabolism in germination and axis growth was investigated in case of Vigna radiata seeds, a non-endospermous leguminous species having epigeal germination, by studying the effect of different reactive oxygen species (ROS) inhibitors, distribution of O2(•)- and H2O2 and ROS enzyme profile in axes. Germination percentage and axis growth were determined under treatment with ROS inhibitors and scavengers. Localization of O2(•)- and H2O2 was done using nitroblue tetrazolium (NBT) and 3,3',5,5'-tetramethyl benzidine dihydrochloride hydrate (TMB), respectively. Apoplastic level of O2(•)- was monitored by spectrophotometric analysis of bathing medium of axes. Profiles of NADPH oxidase and superoxide dismutase (SOD) were studied by in-gel assay. Germination was retarded by treatments affecting ROS level except H2O2 scavengers, while axis growth was retarded by all. Superoxide synthesis inhibitor and scavenger prevented H2O2 accumulation in axes in later phase as revealed from TMB staining. Activity of Cu/Zn SOD1 was initially high and declined thereafter. Superoxide being produced in apoplast possibly by NADPH oxidase activity is further metabolized to (•)OH via H2O2. Germination process depends possibly on (•)OH production in the axes. Post-germinative axis growth requires O2(•)- while the differentiating zone of axis (radicle) requires H2O2 for cell wall stiffening.

Axis growth during seed germination is mediated by reactive oxygen species and apoplastic peroxidase plays a role by producing OH(·) from H2O2. Ca (2+) activates both apoplastic peroxidase and NADPH oxidase. Role of reactive oxygen species (ROS) in seed germination and axis growth has been demonstrated in our earlier works with Vigna radiata seeds by studying superoxide generation and its metabolism in axes (Singh et al. in Plant Signal Behav doi: 10.4161/psb.29278 , 2014). In the present study, the participation of apoplastic peroxidase along with the involvement of Ca(2+) in axis growth during germination and post-germination stage has been investigated. Pharmacological studies using peroxidase (POX) inhibitors (salicylhydroxamic acid, SHAM; sodium azide, NaN3) and OH(·) scavenger (sodium benzoate, NaBz) indicated that seed germination and early axis growth (phase I) depend much on POX activity. Subapical region of axes corresponding to radicle that elongated much particularly in phase II suggested high POX activity as well as high NADPH oxidase (Respiratory burst oxidase homologue, Rboh, in plants) activity as indicated from localization by staining with TMB (3,3',5,5'-tetramethyl benzidine dihydrochloride hydrate) and NBT (nitroblue tetrazolium chloride), respectively. Apoplastic class III peroxidase (Prx) and also cellular POX activity reached maximum at the time of radicle emergence as revealed by TMB staining, spectrophotometric and in-gel assay for POX activity. Treatment with Ca(2+) antagonists (La(3+), plasma membrane-located Ca(2+) channel blocker and EGTA, Ca(2+) chelator in apoplast) retarded seed germination and strongly inhibited axis growth, while Li(+) (blocks endosomal Ca(2+) release) was effective only in retarding phase II axis growth suggesting an involvement of Ca(2+) influx during early axis growth. From the effect of Ca(2+) antagonists on the localization of activities of POX and Rboh using stains, it appears that Ca(2+) plays a dual role by activating Prx activity in apoplast while activating Rboh by entering into cytosol.

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