Superoxide and its metabolism during germination and axis growth ofVigna radiata(L.) Wilczek seeds

Abstract

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.

Role of peroxidase activity and Ca2+ in axis growth during seed germination

“…3a). Localization of such Prx activity coincided with the region where reduction of NBT stain specific for O 2 Á-was also observed earlier with the same system (Singh et al 2014). In absence of external supply of H 2 O 2 , the blue oxidation product of TMB indicates the accumulation of H 2 O 2 in vivo.…”

“…In our earlier works with germinating seeds of V. radiata involvement of ROS during germination, axis growth has been clearly demonstrated by studying the activities of NADPH oxidase, O 2 Á-producing enzyme, and SOD, further metabolizing O 2 Á-to H 2 O 2 in the apoplastic space (Singh et al 2014). Moreover, a direct role of ROS in the form of OH Á through cell wall relaxation in connection with axis extension growth during germination was also indicated.…”

“…Earlier studies have revealed the existence and involvement of a major producer of ROS, the plasma membrane-localized NADPH oxidases (called Respiratory burst oxidase homologue, Rboh, in plants) during germination of V. radiata (Singh et al 2014). The generation of O 2 Á-radical by Rboh in the apoplast and its consequent dismutation into H 2 O 2 either spontaneously or through the action of superoxide dismutases (SOD) appeared to have an important role in axis growth during seed germination (Bailly 2004;Müller et al 2009;Singh et al 2014). H 2 O 2 is reported to be involved in different developmental and physiological processes including root development (Dunand et al 2007), growth of root hairs (Foreman et al 2003 (Passardi et al 2005;Cosio and Dunand 2009).…”

ROS Signaling: Relevance with Site of Production and Metabolism of ROS

“…Ca 2+ ions may also have some role in this process, but less is known about this mechanism. However, a direct involvement of ROS (OH • ) resulting from further metabolism of O 2 •À and H 2 O 2 in the cell wall by cleaving polysaccharides (Müller et al 2009) has also been implicated for seed germination and secondary root growth (Kranner et al 2010;Roach and Kranner 2011;Singh et al 2014). Apparently, apoplastic space is an ideal site for ROS signaling since this compartment has low redox buffering capacity (Noctor et al 2002), thus less affecting signal strength.…”

“…•À in the apoplast (Singh et al 2014). In fact, plant NADPH oxidases, known as respiratory burst oxidase homologues (rbohs), are homologues of catalytic domain of mammalian gp91…”