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

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 NH2 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 Ca2+ 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 Ca2+-activated (p)ppGpp synthetases in plant chloroplasts, implicating both Ca2+ and (p)ppGpp signaling in regulation of the genetic system of these organelles. 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 NH2 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 Ca2+ 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 Ca2+-activated (p)ppGpp synthetases in plant chloroplasts, implicating both Ca2+ and (p)ppGpp signaling in regulation of the genetic system of these organelles. Guanosine 5′-triphosphate (or 5′-diphosphate) 3′-diphosphate ((p)ppGpp) 2The abbreviations used are: (p)ppGpp, guanosine 5′-triphosphate (or 5′-diphosphate) 3′-diphosphate; GST, glutathione S-transferase; BAPTA, 1,2-bis(o-aminophenoxy) ethane-N, N, N′, N′-tetraacetic acid; ORF, open reading frame; RT, reverse transcription; IPTG, isopropyl β-d-thiogalactopyranoside; MOPS, 4-morpholinepropanesulfonic acid; Tricine, N-tris(hydroxymethyl) methylglycine. is an effector molecule responsible for the stringent response, a global regulatory system in bacteria (1Cashel, M., Gentry, D. R., Hernandez, V. J., and Vinella, D. (1996) Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd Ed., pp. 1458–1496, ASM Press, Washington, D. CGoogle Scholar, 2Magnusson L.U. Farewell A. Nyström T. Trends Microbiol. 2005; 13: 236-242Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar). 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 (1Cashel, M., Gentry, D. R., Hernandez, V. J., and Vinella, D. (1996) Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd Ed., pp. 1458–1496, ASM Press, Washington, D. CGoogle Scholar). Subsequently, carbon, fatty acid, or iron limitation was also found to trigger the accumulation of (p)ppGpp in E. coli (2Magnusson L.U. Farewell A. Nyström T. Trends Microbiol. 2005; 13: 236-242Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar, 3Xiao H. Kalma M. Ikehara K. Zemel S. Glaser G. Cashel M. J. Biol. Chem. 1991; 266: 5980-5990Abstract Full Text PDF PubMed Google Scholar, 4Seyfzadeh M. Keener J. Nomura M. Proc. Natl. Acad. Sci. U. 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Cell. 2007; 128: 865-875Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Two enzymes, RelA and SpoT, are responsible for fluctuations in (p)ppGpp concentration in E. coli. RelA is a ribosome-associated protein and synthesizes (p)ppGpp in response to the increase in the proportion of uncharged tRNAs that results from amino acid starvation (10Wendrich T.M. Blaha G. Wilson D.N. Marahiel M.A. Nierhaus K.H. Mol. Cell. 2002; 10: 779-788Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). In contrast, SpoT is a bifunctional (p)ppGpp synthetase and hydrolase and regulates (p)ppGpp levels in response to carbon source or fatty acid limitation (3Xiao H. Kalma M. Ikehara K. Zemel S. Glaser G. Cashel M. J. Biol. Chem. 1991; 266: 5980-5990Abstract Full Text PDF PubMed Google Scholar, 4Seyfzadeh M. Keener J. Nomura M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11004-11008Crossref PubMed Scopus (133) Google Scholar, 11Battesti A. Bouveret E. Mol. Microbiol. 2006; 62: 1048-1063Crossref PubMed Scopus (221) Google Scholar). Many other bacteria possess only a single bifunctional (p)ppGpp synthetase and hydrolase, designated Rel (12Mittenhuber G. J. Mol. Microbiol. Biotechnol. 2001; 3: 585-600PubMed Google Scholar). Rel proteins have been shown to be essential for the long term survival or virulence of pathogenic bacteria (13Gogfrey H.P. Burgrysheva J.V. Cabello F.C. Trends Microbiol. 2002; 10: 349-351Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Stringent factor and RelA-SpoT-like (p)ppGpp synthetases have also been identified in chloroplasts (14van der Biezen E.A. Sun J. Coleman M.J. Bibb M.J. Jones J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3747-3752Crossref PubMed Scopus (145) Google Scholar, 15Takahashi K. Kasai K. Ochi K. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4320-4324Crossref PubMed Scopus (117) Google Scholar, 16Kasai K. Usami S. Yamada T. Endo Y. Ochi K. Tozawa Y. Nucleic Acids Res. 2002; 30: 4985-4992Crossref PubMed Google Scholar, 17Givens R.M. Lin M.-H. Taylor D.J. Mechold U. Berry J.O. Hernandez V.J. J. Biol. Chem. 2004; 279: 7495-7504Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and we previously demonstrated the presence of intrinsic (p)ppGpp synthetase activity in pea chloroplasts (18Kasai K. Kanno T. Endo Y. Wakasa T. Tozawa Y. Nucleic Acids Res. 2004; 32: 5732-5741Crossref PubMed Scopus (60) Google Scholar). Three RelA-SpoT homolog genes (RSH1, RSH2, and RSH3) have been identified in plants (14van der Biezen E.A. Sun J. Coleman M.J. Bibb M.J. Jones J.D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3747-3752Crossref PubMed Scopus (145) Google Scholar, 17Givens R.M. Lin M.-H. Taylor D.J. Mechold U. Berry J.O. Hernandez V.J. J. Biol. Chem. 2004; 279: 7495-7504Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and one such gene (RSH) has been identified in the alga Chlamydomonas reinhardtii (16Kasai K. Usami S. Yamada T. Endo Y. Ochi K. Tozawa Y. Nucleic Acids Res. 2002; 30: 4985-4992Crossref PubMed Google Scholar). Enzymatic activity and specific localization to chloroplasts have been demonstrated for plant RSH2 and RSH3 and for algal RSH (16Kasai K. Usami S. Yamada T. Endo Y. Ochi K. Tozawa Y. Nucleic Acids Res. 2002; 30: 4985-4992Crossref PubMed Google Scholar, 17Givens R.M. Lin M.-H. Taylor D.J. Mechold U. Berry J.O. Hernandez V.J. J. Biol. Chem. 2004; 279: 7495-7504Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Chloroplasts of land plants and algae are thought to originate from ancient cyanobacteria-like prokaryotes. Indeed, the genetic system of chloroplasts, including the apparatuses for transcription, translation, and DNA replication, is highly similar to that of bacteria. A stringent control system similar to that of bacteria has therefore been suggested to operate in chloroplasts of photosynthetic eukaryotes (19Braeken K. Moris M. Daniels R. Vanderleyden J. Michiels J. Trends Microbiol. 2006; 14: 45-54Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). In a search for plant genes that encode RelA-SpoT-like proteins, we have now identified a previously unknown type of RSH gene for putative chloroplast-localizing proteins in rice (Oryza sativa). The deduced amino acid sequences of the novel RSH genes contain motifs similar to the EF-hand, a Ca2+-binding domain, at COOH We that the (p)ppGpp synthetase activity of the gene is by Ca2+ the EF-hand and we therefore CRSH RelA-SpoT to of regulation from that of other RSH thus have for Ca2+ signaling and (p)ppGpp accumulation in plant and plants were as previously Y. K. H. Wakasa K. Nucleic Acids Res. PubMed Scopus (69) Google Scholar). plants were at a with E. coli and were used for genetic complementation analysis (16Kasai K. Usami S. Yamada T. Endo Y. Ochi K. Tozawa Y. Nucleic Acids Res. 2002; 30: 4985-4992Crossref PubMed Google Scholar). and of CRSH recombinant DNA were as T. J. Molecular A Scholar). The was with a rice Y. K. H. Wakasa K. Nucleic Acids Res. PubMed Scopus (69) Google Scholar) and on a rice that similarity to and The were into the and DNA were also and used to with an system of a in for OsCRSH1 and of such were as previously Y. K. H. Wakasa K. Nucleic Acids Res. PubMed Scopus (69) Google Scholar). DNA for was as The which an of was from the of The of the open reading was from the of rice The of the in was by with the and the reverse The of the for the of was by with a Y. K. H. Wakasa K. Nucleic Acids Res. PubMed Scopus (69) Google Scholar) from the and the reverse The were to a with the and The were with from and to with the and The the was with and and into the of the T. T. R. Endo Y. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar), generating of RNA from and were as previously Y. K. H. Wakasa K. Nucleic Acids Res. PubMed Scopus (69) Google Scholar). of an OsCRSH1 a of OsCRSH1 was into the of and a was from the by in transcription with RNA specific for rice and were also and as Y. K. H. Wakasa K. Nucleic Acids Res. PubMed Scopus (69) Google Scholar). was from RNA of by as previously Y. K. H. Wakasa K. Nucleic Acids Res. PubMed Scopus (69) Google Scholar). was with the and for OsCRSH1 for or for a was also with OsCRSH2, or as and with the The of of of and of DNA was in a system with an of for by or of at for at for and at for A of the was by a and of DNA with of encoding or of OsCRSH1 was by with a the as a for the for a protein the amino for a protein the and the reverse The were with and and into the of The were designated and A for an OsCRSH1 mutant both the and was by with the encoding the the and the reverse The was designated of the the for the proteins were used as for in transcription with RNA as K. T. T. Endo Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar). the synthesis of glutathione proteins of or a DNA encoding by the amino acid which is the of was by with the T. M. R. A. Endo Y. Tozawa Y. 2007; PubMed Scopus Google Scholar) as the and the reverse The was into the and and the was into the of to OsCRSH1 were by with or as the the and the reverse or and the were with and into the of The were designated and of expression for and to the putative of protein were by with a the for for the for or for and the reverse for or for The were with and and into the of generating and protein synthesis was with to two the synthesis of proteins, the was as K. T. T. Endo Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar) in the presence of and of with the of a in of protein for the T. T. R. Endo Y. Proc. Natl. Acad. Sci. U. S. A. 2002; PubMed Scopus Google Scholar) was proteins by the were on a glutathione and with on the to the OsCRSH1 The of proteins in the presence of was by both of the and of the of the at for at The was and to a and was with a and with of OsCRSH1 was by in as T. A. Kasai K. M. Y. Wakasa K. Tozawa Y. 2005; PubMed Scopus Google Scholar). The gene was that in of Ca2+ was with Two were to the two of OsCRSH1 with the of The was with a for the Ca2+ with the nucleotide for the Ca2+ to the of the the was with a for the Ca2+ for the Ca2+ to the The were with of as the in a of of of DNA and of The of at for at for and at for by a at for The DNA sequences by the two were and to of at for at for and at for The were by with the and the the of at for at for and at for by a at for The were with and into the of The for proteins of with and Ca2+-binding were designated and of OsCRSH1 in E. E. coli and were as of by of into DNA to gene expression from the (16Kasai K. Usami S. Yamada T. Endo Y. Ochi K. Tozawa Y. Nucleic Acids Res. 2002; 30: 4985-4992Crossref PubMed Google Scholar). of an expression for the OsCRSH1 was to with the and The was with and and for the of The were with or to and to with (16Kasai K. Usami S. Yamada T. Endo Y. Ochi K. Tozawa Y. Nucleic Acids Res. 2002; 30: 4985-4992Crossref PubMed Google Scholar) with or IPTG, and at for of (p)ppGpp were in a of at and protein in and in the or presence of The was for at by the of of the of the was and at for at The was to and a was a was used as the as (18Kasai K. Kanno T. Endo Y. Wakasa T. Tozawa Y. Nucleic Acids Res. 2004; 32: 5732-5741Crossref PubMed Scopus (60) Google Scholar). analysis was as previously (18Kasai K. Kanno T. Endo Y. Wakasa T. Tozawa Y. Nucleic Acids Res. 2004; 32: 5732-5741Crossref PubMed Scopus (60) Google Scholar) with for the and by of of and of in of for the was to a and the was with a and with of (p)ppGpp pppGpp was in by in the presence of as in the for (p)ppGpp synthetase of the pppGpp was as (18Kasai K. Kanno T. Endo Y. Wakasa T. Tozawa Y. Nucleic Acids Res. 2004; 32: 5732-5741Crossref PubMed Scopus (60) Google Scholar). of and of the pppGpp with or the of The were at for and the was by the of of the of of the was and at for at The was to and a was to the and of SpoT DNA of E. coli gene for the amino of the SpoT protein was by and The DNA was with and and into the of E. coli with was in of at with the was at was to the at a concentration of and at for The were by at for at and in A The were by and the was by at for at The was to for at and the was to a and with The protein with at NH2 was from the with The was by and the was at the of of and of OsCRSH a search for (p)ppGpp synthetase genes in land we found a rice that of a putative with similarity to RelA and SpoT of E. and we a the from a rice in Y. K. H. Wakasa K. Nucleic Acids Res. PubMed Scopus (69) Google Scholar). we the of in the in the rice data base A data base search identified a similar in Arabidopsis The proteins encoded by the rice and Arabidopsis amino and similarity to RelA-SpoT proteins the deduced amino acid sequences of the plant proteins with of other RelA-SpoT we the in (p)ppGpp hydrolase and (p)ppGpp synthetase The plant sequences also an to contain a as as two EF-hand motifs in the A and the of these and characterization of the encoded we designated the rice gene as OsCRSH1 Ca2+-activated RelA-SpoT homolog A search in the identification of two genes and in the rice data a ORF, the of was as a in the reverse with the DNA to the of the encoded protein that the of rice the genes to and we the for both genes by from a from RNA of rice The deduced amino acid sequences of both genes a putative in the a central RelA-SpoT-like domain, and EF-hand motifs in the two genes were thus designated and The of nucleotide OsCRSH1 and or is and the for amino acid are and genes are on rice gene is OsCRSH1 and The of A. to contain only a single on that is to the OsCRSH is therefore likely that the OsCRSH genes as a of of the gene during rice and of the function of the of the putative OsCRSH1 we an in chloroplast as previously K. Kanno T. M. Y. Wakasa K. Tozawa Y. 2005; PubMed Scopus Google Scholar). The protein was with a protein synthesis system in the presence of and of the protein with pea chloroplasts in to a that was to with the The of protein to that OsCRSH1 protein was into results thus that OsCRSH1 was imported into chloroplasts, likely as a of putative We the of the of OsCRSH1 with that of also with the translation The of the of OsCRSH1 was similar to that of an OsCRSH1 mutant the amino that the is the in the The of the mutant is also similar to that of the of the Arabidopsis protein and the of these we used the of OsCRSH1 as the of the protein in of an E. coli by OsCRSH1 as a RelA-SpoT we the encoding the putative of the protein into the expression to of E. coli and with or as (16Kasai K. Usami S. Yamada T. Endo Y. Ochi K. Tozawa Y. Nucleic Acids Res. 2002; 30: 4985-4992Crossref PubMed Google Scholar) revealed that the rice protein the of the mutant on and In contrast, with the phenotype of the single mutant results therefore suggested that OsCRSH1 only a of (p)ppGpp synthetase activity similar to that of E. coli of OsCRSH the of OsCRSH1 expression in rice plants by The of which RNA and specific expression in Y. K. H. Wakasa K. Nucleic Acids Res. PubMed Scopus (69) Google Scholar), and which is in Y. K. H. Wakasa K. Nucleic Acids Res. PubMed Scopus (69) Google Scholar), were as A of was in and with the of in the in the that the similarity in the nucleotide sequences of OsCRSH2, and in of the OsCRSH1 in the we the expression of genes by The of and in both and was that of OsCRSH1 that OsCRSH1 is the highly gene among the in the rice of (p)ppGpp of OsCRSH1 in the function of OsCRSH1 as a (p)ppGpp we the for the of the protein into the to The protein encoded by was by in transcription and translation and with a glutathione The of the protein was on the by and the was at and for (p)ppGpp synthetase activity in the or presence of The protein pppGpp synthetase activity only in the presence of Ca2+ at of The was by and as pppGpp (18Kasai K. Kanno T. Endo Y. Wakasa T. Tozawa Y. Nucleic Acids Res. 2004; 32: 5732-5741Crossref PubMed Scopus (60) Google Scholar). The levels of pppGpp accumulation that were in the synthetase Ca2+ concentration be to the pppGpp hydrolase activity of the we the hydrolase activity of shown in the pppGpp hydrolase activity was by E. coli SpoT, which was as a control for pppGpp hydrolase, the hydrolase activity was in the of results thus that OsCRSH1 Ca2+-activated pppGpp synthetase the Ca2+ of activity is to the EF-hand motifs of we in which Ca2+ to the be to be M. A. A. M. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The and thus contain of at the of the Ca2+-binding mutant (p)ppGpp synthetase activity indicating that both EF-hand motifs are for the the two EF-hand motifs is for the pppGpp synthetase activity of we a mutant of the protein that the amino both The mutant protein (p)ppGpp synthetase activity in the or presence of Ca2+ that the is for of the of we that the of was that of the two We therefore the synthesis of and in the presence of of the Ca2+ 1,2-bis(o-aminophenoxy) N′-tetraacetic acid to The of the for synthesis was in the presence of In contrast, the of the was by of that of and are to pppGpp in the these results suggested that is in the for protein in of the source and for protein synthesis and a protein (p)ppGpp of OsCRSH the function of the other two rice CRSH and we the (p)ppGpp synthetase with protein The putative of the rice CRSH proteins were thus and in the presence of and in the or presence of synthesis was with the that was with rice proteins showed (p)ppGpp synthetase activity in the of BAPTA, and the activity of protein was by of Ca2+ We also that rice CRSH proteins shown to BAPTA, of EF-hand the synthesis of protein was by the presence of results thus that the rice CRSH proteins possess similar Ca2+-activated (p)ppGpp synthetase in have demonstrated the of ppGpp in plants K. Kasai K. Ochi K. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4320-4324Crossref PubMed Scopus (117) Google Scholar), the presence of (p)ppGpp synthetase activity in chloroplasts (18Kasai K. Kanno T. Endo Y. Wakasa T. Tozawa Y. Nucleic Acids Res. 2004; 32: 5732-5741Crossref PubMed Scopus (60) Google Scholar), and the encoding of genes for chloroplastic (p)ppGpp synthetase (16Kasai K. Usami S. Yamada T. Endo Y. Ochi K. Tozawa Y. Nucleic Acids Res. 2002; 30: 4985-4992Crossref PubMed Google Scholar, 17Givens R.M. Lin M.-H. Taylor D.J. Mechold U. Berry J.O. Hernandez V.J. J. Biol. Chem. 2004; 279: 7495-7504Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). We have now identified plant genes for a novel type of RelA-SpoT designated that to chloroplasts and (p)ppGpp synthetase The of CRSH proteins two Ca2+-binding motifs Z. J. Mol. 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PubMed Scopus Google Scholar) have been The of was the and a in the Ca2+ concentration of the from to and T. P. J. A. A. PubMed Scopus Google Scholar). In the we found that of the (p)ppGpp synthetase activity of OsCRSH1 a Ca2+ concentration of in We previously showed that the (p)ppGpp synthetase activity of pea chloroplasts the (18Kasai K. Kanno T. Endo Y. Wakasa T. Tozawa Y. Nucleic Acids Res. 2004; 32: 5732-5741Crossref PubMed Scopus (60) Google Scholar), that such as the increase the activity of OsCRSH1 or to Ca2+ at increase in Ca2+ concentration in the chloroplast thus the (p)ppGpp synthetase activity of CRSH proteins and the genetic system of the chloroplast in a similar to that in the stringent The and of of chloroplastic RelA-SpoT including to and to that (p)ppGpp signaling an in regulation of the genetic system of in land plants. RSH genes are conserved among land and CRSH genes to be only in land plants. We that Ca2+-activated (p)ppGpp signaling have with the of Ca2+ signaling in the chloroplasts of land plants. have land plants to to such as and We and for