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

Yuzuru Tozawa,Akira Nozawa,Takuya Kanno,Takakuni Narisawa,Shinji Masuda,Koji Kasai,Hideaki Nanamiya

doi:10.1074/jbc.m703820200

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.

Highlights

The hyperphosphorylated guanosine nucleotides ppGpp and pppGpp were initially identified as “magic spots” that accumulate during amino acid deprivation in Escherichia coli and induce rapid down-regulation of stable RNA synthesis [1]
Whereas AK058438 contains a full-length open reading frame (ORF), the sequence of AK110850 was annotated as a cDNA in the reverse direction, with the DNA sequence corresponding to the COOH-terminal region of the encoded protein being missing
Given that the similarity in the nucleotide sequences of OsCRSH1, OsCRSH2, and OsCRSH3 might result in cross-hybridization of the OsCRSH1 probe in Northern analysis, even under the high stringency conditions used, we examined the expression of all three genes by RT-PCR analysis

Summary

Introduction

The hyperphosphorylated guanosine nucleotides ppGpp and pppGpp were initially identified as “magic spots” that accumulate during amino acid deprivation in Escherichia coli and induce rapid down-regulation of stable RNA synthesis [1]. For construction of cell-free expression vectors for OsCRSH2 and OsCRSH3, cDNA fragments corresponding to the putative mature form of each protein were amplified by PCR with a plasmid containing the full-length ORF (clone AK058438 for OsCRSH2; pECRSH3 for OsCRSH3), the forward primer CR2DN14F (5Ј-ATAACTAGTATGGCGGCCGCGGTGGCGCCTGA-3Ј) for OsCRSH2 or CR3DN12F (5ЈATAACTAGTATGGCCGTCGCCATTGACGCACC-3Ј) for OsCRSH3, and the reverse primer CR2R (5Ј-ATAAGATCTTCAGCCGGAGACGAGCTTGT-3Ј) for OsCRSH2 or CR3R for OsCRSH3.

Results

Conclusion