Calcium-activated (p)ppGpp Synthetase in Chloroplasts of Land Plants

Abstract

The genetic system of chloroplasts, including the machinery for transcription, translation, and DNA replication, exhibits substantial similarity to that of eubacteria. Chloroplasts are also thought to possess a system for generating guanosine 5'-triphosphate ((p)ppGpp), which triggers the stringent response in eubacteria, with genes encoding chloroplastic (p)ppGpp synthetase having been identified. We now describe the identification and characterization of genes (OsCRSH1, OsCRSH2, and OsCRSH3) for a novel type of (p)ppGpp synthetase in rice. The proteins encoded by these genes contain a putative chloroplast transit peptide at the NH(2) terminus, a central RelA-SpoT-like domain, and two EF-hand motifs at the COOH terminus. The recombinant OsCRSH1 protein was imported into chloroplasts in vitro, and genetic complementation analysis revealed that expression of OsCRSH1 suppressed the phenotype of an Escherichia coli mutant deficient in the RelA and SpoT enzymes. Biochemical analysis showed that the OsCRSH proteins possess (p)ppGpp synthetase activity that is dependent both on Ca(2+) and on the EF-hand motifs. A data base search identified a CRSH homolog in the dicotyledon Arabidopsis thaliana, indicating that such genes are conserved among both monocotyledonous and dicotyledonous land plants. CRSH proteins thus likely function as Ca(2+)-activated (p)ppGpp synthetases in plant chloroplasts, implicating both Ca(2+) and (p)ppGpp signaling in regulation of the genetic system of these organelles.

Chloroplast-mediated activation of plant immune signalling in Arabidopsis

“…1d -f ), suggesting that stromal Ca 2 + signatures may be involved in stressspecifi c responses, including rapid inhibition of photosynthesis and accumulation of JA 39,40 . Interestingly, chloroplasts contain several EF-hand Ca 2 + sensor proteins and other types of Ca 2 + -binding proteins 41 .…”

Diversity in Guanosine 3′,5′-Bisdiphosphate (ppGpp) Sensitivity among Guanylate Kinases of Bacteria and Plants

“…Primers for PCR are listed in Table 2. For construction of expression vectors for ND45 and ND73 forms of OsGKpm, the corresponding DNA fragments were amplified by PCR from cDNA of O. sativa (8) with the primers OsGKpm-⌬1-forward and OsGKpm-reverse for ND45, and OsGKpm-⌬2-forward and OsGKpm-reverse for ND73. The PCR products were cloned directly into the pTA2 vector with the use of a Target Clone Kit (Toyobo, Osaka, Japan).…”

Protein Synthesis and the Stress Response

“…RelA/SpoT homologous proteins have also been found in chloroplast probably with functions similar as in bacteria. Another ppGpp synthetase was found in chloroplasts of land plants that is sensitive to Ca ++ [90]. Also in metazoan, another SpoT related protein, Mesh1, was recently identified [91].…”