ABSTRACT
Class III peroxidases (Prxs) are enzymes involved in a multitude of physiological and stress-related processes in plants. Here, we report on the characterization of a putative peroxidase-encoding gene from Coffea arabica (CaPrx) that is expressed in early stages of root-knot nematode (RKN) infection. CaPrx showed enhanced expression in coffee roots inoculated with RKN (at 12 h post-inoculation), but no significant difference in expression was observed between susceptible and resistant plants. Assays using transgenic tobacco plants harboring a promoter-ß-glucuronidase (GUS) fusion revealed that the CaPrx promoter was exclusively active in the galls induced by RKN. In cross sections of galls, GUS staining was predominantly localized in giant cells. Up-regulation of GUS expression in roots of transgenic plants following RKN inoculation was observed within 16 h. Moreover, no increase in GUS expression after treatment with jasmonic acid was detected. Altogether, these results point to a putative role of this peroxidase in the general coffee response to RKN infection.
Subject(s)
Coffea/enzymology , Coffea/genetics , Genes, Plant/genetics , Peroxidases/genetics , Plant Diseases/parasitology , Plant Roots/parasitology , Tylenchoidea/physiology , Animals , Base Sequence , Coffea/immunology , Coffea/parasitology , Cyclopentanes/pharmacology , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Disease Resistance/drug effects , Disease Resistance/genetics , Expressed Sequence Tags , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Plant/drug effects , Genes, Reporter/genetics , Glucuronidase/metabolism , Molecular Sequence Data , Organ Specificity/drug effects , Organ Specificity/genetics , Oxylipins/pharmacology , Peroxidases/metabolism , Phylogeny , Plant Diseases/genetics , Plant Diseases/immunology , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Roots/drug effects , Plant Roots/genetics , Plants, Genetically Modified , Promoter Regions, Genetic/genetics , Reproducibility of Results , Nicotiana/drug effects , Nicotiana/genetics , Tylenchoidea/drug effectsABSTRACT
A cDNA clone (designated CaIRL) encoding an isoflavone reductase-like protein from coffee (Coffea arabica) was retrieved during a search for genes showing organ/tissue-specific expression among the expressed sequence tags (EST) of the Brazilian coffee EST database. The CaIRL cDNA contains a single open reading frame of 946 nucleotides (nt) encoding 314 amino acids (predicted molecular weight of 34 kDa). Several features identified the predicted CaIRL protein as a new member of the PIP family of NADPH-dependent reductases. Expression studies demonstrated that CaIRL is expressed exclusively in coffee leaves and its transcript level is markedly increased in response to fungal infection and mechanical injury. Analysis of transgenic tobacco plants harboring a CaIRL 5'-flanking region (862 nt) fused to uidA reporter gene (GUS) confirmed the responsiveness of the putative promoter to abiotic stress in wounded leaves. In turn, a 5' deletion to -404 completely abolished promoter activation by abiotic stimulus in transgenic plants. The lack of GUS expression in non-wounded leaf tissues in transgenic tobacco was in contrast to the basal level of CaIRL expression observed in non-stressed healthy coffee leaves.
Subject(s)
Coffee/enzymology , Gene Expression Regulation, Plant , Oxidoreductases Acting on CH-CH Group Donors/genetics , Promoter Regions, Genetic , Stress, Physiological , 5' Flanking Region , Amino Acid Sequence , Coffee/genetics , Coffee/microbiology , Molecular Sequence Data , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Phylogeny , Plant Diseases/microbiology , Plant Leaves/enzymology , Plant Leaves/microbiology , Plants, Genetically Modified , Nicotiana/genetics , Nicotiana/metabolism , Nicotiana/microbiologyABSTRACT
An Arabidopsis thaliana cDNA clone encoding a plant uncoupling mitochondrial protein (AtPUMP1) was overexpressed in transgenic tobacco plants. Analysis of the AtPUMP1 mRNA content in the transgenic lines, determined by Northern blot, revealed variable levels of transgene expression. Antibody probing of Western blots of mitochondrial proteins from three independent transgenic lines showed significant accumulation of AtPUMP1 in this organelle. Overproduction of AtPUMP1 in transgenic tobacco plants led to a significant increase in tolerance to oxidative stress promoted by exogenous hydrogen peroxide as compared to wild-type control plants. These results provide the first biological evidence for a role of PUMP in protection of plant cells against oxidative stress damage.