Mining the enzymes involved in the detoxification of reactive oxygen species (ROS) in sugarcane

Adopting the sequencing of expressed sequence tags (ESTs) of a sugarcane database derived from libraries induced and not induced by pathogens, we identified EST clusters homologous to genes corresponding to enzymes involved in the detoxification of reactive oxygen species. The predicted amino acids of these enzymes are superoxide dismutases (SODs), glutathione-5-transferase (GST), glutathione peroxidase (GPX), and catalases. Three MnSOD mitochondrial precursors and 10 CuZnSOD were identified in sugarcane: the MnSOD mitochondrial precursor is 96% similar to the maize MnSOD mitochondrial precursor and, of the 10 Cl identified, seven were 98% identical to maize cytosolic CuZnSOD4 and one was 67% identical to putative peroxisomal CuZnSOD from Arabidopsis. Three homologues to class Phi GST were 87-88% identical to GST III from maize. Five GPX homologues were identified: three were homologous to cytosolic GPX from barley, one was 88% identical to phospholipid hydroperoxide glutathione peroxidase (PHGPX) from rice, and the last was 71% identical to GPX from A. thaliana. Three enzymes similar to maize catalase were identified in sugarcane: two were similar to catalase isozyme 3 and catalase chain 3 from maize, which are mitochondrial, and one was similar to catalase isozyme 1 from maize, whose location is peroxisomal subcellular. All enzymes were induced in all sugarcane libraries (flower, seed, root, callus, leaves) and also in the pathogen-induced libraries, except for Cl whose cDNA was detected in none of the libraries induced by pathogens (Acetobacter diazotroficans and Herbaspirillum rubrisubalbicans). The expression of the enzymes SOD, GST, GPX, and catalases involved in the detoxification was examined using reverse transcriptase-polymerase chain reaction in cDNA from leaves of sugarcane under biotic stress conditions, inoculated with Puccinia melanocephala, the causal agent of sugarcane rust disease.