A gene encoding a putative guanosine 5′-diphosphate (or 5′-triphosphate) 3′-diphosphate ((p)ppGpp) synthetase, designated PepRSH (Pepper RelA/SpoT homologue), was isolated from hot peppers. PepRSH was found to contain five introns and six exons and a 2166-bp open reading frame encoding a protein of 721 amino acids; this protein displayed significant homology with other plant (p)ppGpp synthetases. A genomic DNA gel blot analysis revealed that the pepper genome has at least a single copy of PepRSH. PepRSH transcripts were highly accumulated in non-host resistance response-induced leaves and in leaves following induction with salicylic acid, methyl jasmonate, wounding, hydrogen peroxide, and ultraviolet-B. The expression of PepRSH was also influenced by abiotic stresses, such as flooding and high salinity. The deduced PepRSH protein has a putative chloroplast-targeting transit peptide at its N-terminus, and immunolocalization studies verified the translocation of PepRSH to the chloroplast. The predicted PepRSH protein is markedly similar to known plant and bacterial RSH proteins. Expression of a putative (p)ppGpp synthetase domain in an Escherichia coli single mutant (RelA-SpoT+) complemented growth of the mutant but not of an E. coli double mutant (RelA-SpoT-), demonstrating that PepRSH has (p)ppGpp synthetase activity only in the (p)ppGpp synthetase domain. Site-directed mutagenesis of the conserved histidine and aspartic acid (HD) site in the putative HD domain of PepRSH revealed that the histidine and aspartic acid dual sites were critical residues for the (p)ppGpp synthetase activity of PepRSH protein. Mutation of the HD site limited the tolerance of bacteria to both salt and osmotic stress. Our results indicate that pepper plants have a (p)ppGpp regulatory system that is similar to that of bacteria and which may transduce stress-related signals through the regulation of (p)ppGpp by PepRSH localized in chloroplasts. Crown
Bibliographical noteFunding Information:
This project was funded by a grant for Basic Research from the NIAB (National Institute of Agricultural Biotechnology) Research Program at the Rural Development Administration. This investigation was also supported by the Korea Research Foundation Grant funded by the Korean Government [KRF-2008-359-F00005]. We thank Dr. M. Cashel, National Institute of Health, USA, for providing E. coli mutants (CF1648, CF1652 and CF1693).
- (p)ppGpp synthetase
- Biotic and abiotic stress
ASJC Scopus subject areas
- Agronomy and Crop Science
- Plant Science