Abstract
The nucleotides guanosine tetraphosphate and pentaphosphate (together known as (p)ppGpp or magic spot) are produced in plant plastids from GDP/GTP and ATP by RelA-SpoT homologue (RSH) enzymes. In the model plant Arabidopsis (p)ppGpp regulates chloroplast transcription and translation to affect growth, and is also implicated in acclimation to stress. However, little is known about (p)ppGpp metabolism or its evolution in other photosynthetic eukaryotes. Here we studied (p)ppGpp metabolism in the marine diatom Phaeodactylum tricornutum. We identified three expressed RSH genes in the P. tricornutum genome, and determined the enzymatic activity of the corresponding enzymes by heterologous expression in bacteria. We showed that two P. tricornutum RSH are (p)ppGpp synthetases, despite substitution of a residue within the active site believed critical for activity, and that the third RSH is a bifunctional (p)ppGpp synthetase and hydrolase, the first of its kind demonstrated in a photosynthetic eukaryote. A broad phylogenetic analysis then showed that diatom RSH belong to novel algal RSH clades. Together our work significantly expands the horizons of (p)ppGpp signalling in the photosynthetic eukaryotes by demonstrating an unexpected functional, structural and evolutionary diversity in RSH enzymes from organisms with plastids derived from red algae.
Highlights
The nucleotides guanosine tetraphosphate and pentaphosphate (together known as (p)ppGpp or magic spot) are produced in plant plastids from GDP/GTP and ATP by RelA-SpoT homologue (RSH) enzymes
We showed that the genome of the diatom P. tricornutum encodes three RSH enzymes: PtRSH1, PtRSH4a and PtRSH4b
Using heterologous expression in E. coli (p)ppGpp mutants we showed that all three P. tricornutum RSH are (p)ppGpp synthetases, and that PtRSH1 is a (p)ppGpp hydrolase (Fig. 2)
Summary
The nucleotides guanosine tetraphosphate and pentaphosphate (together known as (p)ppGpp or magic spot) are produced in plant plastids from GDP/GTP and ATP by RelA-SpoT homologue (RSH) enzymes. Together our work significantly expands the horizons of (p)ppGpp signalling in the photosynthetic eukaryotes by demonstrating an unexpected functional, structural and evolutionary diversity in RSH enzymes from organisms with plastids derived from red algae. Bacteria-like regulatory systems that may be involved in acclimation to environmental perturbation are present in plastids[2,3] One of these regulatory-systems is mediated by the nucleotides guanosine tetraphosphate and pentaphosphate (referred to as (p)ppGpp hereafter) whose levels are controlled by the antagonistic action of RelA-SpoT homologues (RSH). Plastid-targeted RSH enzymes from the RSH1, RSH2/3 and RSH4 families have been identified in many green and red algae, yet the metabolism and functions of (p)ppGpp in these organisms have barely been investigated[3,12,16]. The inducible expression of CmRSH4b in C. merolae results in a reduction in plastid size and rRNA transcription in a similar manner to the expression of a (p)ppGpp synthetase in Arabidopsis[11]
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