The stringent response is one of the most important regulatory systems in bacteria for adaptation to environmental stresses such as amino acid starvation. This response is mediated by an unusual nucleotide, guanosine 3',5'-bis(pyrophosphate) (ppGpp), the levels of which are controlled by two enzymes, RelA and SpoT. ppGpp dramatically influences a broad range of physiological activities in bacterial cells, including transcription and translation as well as the enzymatic activities of several metabolic pathways. Recently, a growing number of RelA/SpoT homologues, designated RSH, have been identified in plants. Arabidopsis has four RSHs (AtRSH1, AtRSH2, AtRSH3 and AtCRSH). In order to reveal the specific roles of the individual Arabidopsis RSHs, we have analyzed the ppGpp synthase activities of the four RSHs and characterized the expression pattern of the RSH genes. Results from the present and previous studies indicate that all four RSH proteins are targeted into plastids. Complementation analysis of E. coli mutants revealed that AtRSH2, AtRSH3 and AtCRSH, but not AtRSH1, have ppGpp synthetase activity. Promoter analysis using reporter gene fusions indicated that the four Arabidopsis RSH genes are expressed in green tissues and flowers, suggesting the involvement of RSH functions in chloroplast development and reproduction. We also observed that all RSH transcripts exhibit a diurnal rhythm, and that induction of AtRSH1 and AtRSH2 transcripts are responsive to several environmental stresses. These results suggest that expression of the four RSH genes is coordinately regulated and that the ppGpp-dependent plastid stringent response has certain roles in the physiology of higher plants.