Polyamines inhibit NADPH oxidase-mediated superoxide generation and putrescine prevents programmed cell death induced by polyamine oxidase-generated hydrogen peroxide

Our previous results indicate that during protoplast isolation an oxidative burst occurs [A.K. Papadakis and KA Roubelakis-Angelakis (1999) Plant Physiol 127:197–205] and that suppression of totipotency is correlated with reduced antioxidant activity and low redox state [A.K. Papadakis et al. (2001b) Plant Physiol 126:434–444]. Polyamines are known to affect cell development and to act as antioxidants. Polyamines applied during isolation of tobacco (Nicotiana tabacum L.) protoplasts reduced the accumulation of O₂^·– but not that of H₂O₂. This antioxidant effect is probably due to the inhibition of microsomal membrane NADPH oxidase, which occurred in a concentration-dependent manner, with spermine exerting the highest inhibitory effect. However, during protoplast culture, polyamine oxidase activity increased severalfold in spermidine- and spermine-treated protoplasts, concomitant with H₂O₂ titers. A cell death program was executed in untreated protoplasts, as documented by membrane malfunction, induced DNase activity, DNA fragmentation and a positive TUNEL reaction. Protoplast cell death was prevented in protoplasts treated with putrescine, but not by treatment with spermidine or spermine, which rather had the opposite effect. The data presented suggest that PAs may be implicated in the expression of plant protoplast totipotency.     

Spermidine exodus and oxidation in the apoplast induced by abiotic stress is responsible for H2O2 signatures that direct tolerance responses in tobacco

Polyamines (PAs) exert a protective effect against stress challenges, but their molecular role in this remains speculative. In order to detect the signaling role of apoplastic PA-derived hydrogen peroxide (H2O2) under abiotic stress, we developed a series of tobacco (Nicotiana tabacum cv Xanthi) transgenic plants overexpressing or downregulating apoplastic polyamine oxidase (PAO; S-pao and A-pao plants, respectively) or downregulating S-adenosyl-l-methionine decarboxylase (samdc plants). Upon salt stress, plants secreted spermidine (Spd) into the apoplast, where it was oxidized by the apoplastic PAO, generating H2O2. A-pao plants accumulated less H2O2 and exhibited less programmed cell death (PCD) than did wild-type plants, in contrast with S-pao and samdc downregulating plants. Induction of either stress-responsive genes or PCD was dependent on the level of Spd-derived apoplastic H2O2. Thus, in wild-type and A-pao plants, stress-responsive genes were efficiently induced, although in the latter at a lower rate, while S-pao plants, with higher H2O2 levels, failed to accumulate stress-responsive mRNAs, inducing PCD instead. Furthermore, decreasing intracellular PAs, while keeping normal apoplastic Spd oxidation, as in samdc downregulating transgenic plants, caused enhanced salinity-induced PCD. These results reveal that salinity induces the exodus of Spd into the apoplast, where it is catabolized by PAO, producing H2O2. The accumulated H2O2 results in the induction of either tolerance responses or PCD, depending also on the levels of intracellular PAs.     

A Serine Carboxypeptidase-Like Acyltransferase Is Required for Synthesis of Antimicrobial Compounds and Disease Resistance in Oats

Serine carboxypeptidase-like (SCPL) proteins have recently emerged as a new group of plant acyltransferases. These enzymes share homology with peptidases but lack protease activity and instead are able to acylate natural products. Several SCPL acyltransferases have been characterized to date from dicots, including an enzyme required for the synthesis of glucose polyesters that may contribute to insect resistance in wild tomato (Solanum pennellii) and enzymes required for the synthesis of sinapate esters associated with UV protection in Arabidopsis thaliana. In our earlier genetic analysis, we identified the Saponin-deficient 7 (Sad7) locus as being required for the synthesis of antimicrobial triterpene glycosides (avenacins) and for broad-spectrum disease resistance in diploid oat (Avena strigosa). Here, we report on the cloning of Sad7 and show that this gene encodes a functional SCPL acyltransferase, SCPL1, that is able to catalyze the synthesis of both N-methyl anthraniloyl- and benzoyl-derivatized forms of avenacin. Sad7 forms part of an operon-like gene cluster for avenacin synthesis. Oat SCPL1 (SAD7) is the founder member of a subfamily of monocot-specific SCPL proteins that includes predicted proteins from rice (Oryza sativa) and other grasses with potential roles in secondary metabolism and plant defense.