Possible targets of “magic spots” in plant signalling

Abstract

The prokaryotic signalling molecules (p)ppGpp, also called “magic spots”, regulate a wide variety of physiological activities in bacteria, including transcription, translation, and replication as well as some enzymatic activities such as those of some GTP-binding proteins, which are necessary for bacterial cells to adapt their physiology to different environmental stimuli. This response is called the stringent response. Recently, (p)ppGpp molecules and (p)ppGpp synthetase homologues, designated RSHs, have been identified in plants. At least some of the RSHs are targeted to chloroplasts. A knockdown mutation in one of the RSHs results in unusual flower development in Arabidopsis, suggesting that the plastid stringent response has important roles in the physiology of higher plants. Possible (p)ppGpp target proteins are investigated.

Introduction

The stringent response, one of the most important regulatory systems in bacteria, is mediated by the unusual nucleotides guanosine 5′-triphosphate, 3′-diphosphate and guanosine 5′-diphosphate, 3′-diphosphate, known collectively as (p)ppGpp, which are also referred to as “magic spots” because they were identified over 40 years ago as spots on thin-layer chromatograms when nucleotides were extracted from bacterial cells grown under starvation conditions.¹ The level of (p)ppGpp is maintained in Escherichia coli cells by two enzymes, RelA and SpoT. Both enzymes synthesize (p)ppGpp by phosphorylating GTP using ATP, and the synthesized (p)ppGpp is degraded by SpoT.¹ Recent genome sequence data have indicated that RelA/SpoT-homologues (RSHs) are conserved in plants.^2–8 The model plant Arabidopsis has four RSHs (RSH1, RSH2, RSH3 and CRSH). All of the RSHs are targeted to chloroplasts,^5,7–9 although the possibility that they also function in other cellular components such as in mitochondria cannot be excluded. The chloroplast localization of RSHs suggests that they were introduced into host cells through symbiosis of a photosynthetic cyanobacterium. Recent biochemical and genetic analyses have indicated that the RSHs are functionally differentiated in plant cells, showing different primary structures, enzyme activities, subcellular localizations (present in soluble or membrane fractions), and organ specificity as well as diurnal rhythms in expression.^2–9 These observations suggest that the RSHs maintain plastid ppGpp levels in response to different environmental stimuli, and these levels may have important roles in the physiology of higher plants. However, the target proteins of (p)ppGpp in plant cells are still unknown.

Possible Targets of (p)ppGpp in Plants

In bacteria, (p)ppGpp regulates transcription by altering RNA polymerase (RNAP) activity, although the role of (p)ppGpp in the regulatory mechanism is not the same in all organisms. Specifically, in E. coli, (p)ppGpp binds to the β and/or β' subunit of RNAP, which may result in a decrease in the lifetime of open complexes formed by promoter DNA and RNAP to inhibit transcription of rRNA.^10–14 In this case, another RNAP-associated protein, DksA, is necessary for full regulation of transcription.^15,16 On the other hand, in Bacillus subtilis, (p)ppGpp inhibits RNAP activity indirectly, such that an increase in the (p)ppGpp level reduces the amount of NTP that is required for full activity of RNAP.¹⁷ In plant cells, transcription of the plastid genome depends on two types of RNAP, nuclear-encoded plastid RNA polymerase (NEP) and plastid-encoded plastid RNA polymerase (PEP), which are a T3/T7 phage type RNAP and a bacterial type RNAP, respectively.¹⁸ Thus, PEP, but not NEP, may be one of the targets of (p)ppGpp in chloroplasts. Given that a dksA-like gene has not been found in the sequenced genome of Arabidopsis, (p)ppGpp may indirectly inhibit RNAP activity in vivo as in B. subtilis, although exogenous addition of (p)ppGpp slightly inhibits RNA synthesis in isolated chloroplasts in vitro.⁶

(p)ppGpp also controls prokaryotic translation. Specifically, translation initiation factor 2 (IF2), which requires GTP for activity, binds ppGpp at the same nucleotide-binding site as GTP and with similar affinity, which results in a conformational change of the protein that interferes with translation initiation under stringent conditions.¹⁹ ppGpp also binds to the GTP-binding site of Obg, which is involved in sporulation control of B. subtilis.²⁰ Thus, (p)ppGpp work as regulatory analogues of GDP and GTP and control activities of many other GTP-binding proteins in an allosteric manner. In Arabidopsis, there are two IF2 homologues, cpIF2 (FUG1) and mtIF2, which are targeted to chloroplasts and mitochondria, respectively,²¹ suggesting that activity of cpIF2 is controlled by (p)ppGpp. We searched for prokaryotic families of GTP-binding proteins in the Arabidopsis genome (Table 1). Surprisingly, there are a number of prokaryotic GTP-binding proteins that may function in chloroplasts (or mitochondria), including the Obg, Era, EngA, YchF, YihA and HflX protein families,²² suggesting that the activities of these protein families may be modulated by (p)ppGpp. Perhaps the GTP-binding proteins have functionally co-evolved with RSHs in plant cells. The GTP-binding proteins control pleiotropic phenotypes in bacterial cells including the regulation of carbon metabolism and the cell cycle, some aspects of which are essential for cell viability.²³ In the future, it should be interesting to characterize the protein families with special attention to the RSH-dependent plastidic stringent response.

Table 1.

Families of prokaryotic GTP-binding proteins in Arabidopsis

Familya	Locus	Target Pb
Obg	At1g07615	m
	At5g18570	c
Era	At1g30960	m
	At5g66470	c
EngA	At3g12080	c
	At5g39960	m
DRG	At4g39520	o
	At1g72660	o
	At1g17470	o
ThdF	At1g78010	m
YchF	At1g56050	c
	At1g30580	o
YihA	At2g22870	m
	At5g11480	c
	At5g58370	m
HflX	At5g57960	c

^aProtein families are defined previously²² except for HflX family.

^bPossible localization of proteins estimated by Target P program (http://www.cbs.dtu.dk/services/TargetP/). c, chloroplast; m, mitochondria; o, others.

Recently, (p)ppGpp was shown to directly inhibit DNA primase (DnaG) in B. subtilis, which prolongs DNA replication elongation under starved conditions.²⁴ However, we found no DnaG homologue in Arabidopsis, suggesting that this mechanism is not involved in the control of chloroplast genome replication, although the replication mechanisms and even the components of the DNA replication machinery of chloroplasts are not well understood.

To summarize, (p)ppGpp production under stringent conditions contributes to regulation of bacterial physiology in two ways: direct inhibition of enzymatic activities by (p)ppGpp and reduction of available NTP pools by substrate consumption during (p)ppGpp synthesis. Chloroplast NTP concentrations may also influence basal levels of cytokinins, because they are synthesized from adenine nucleotides derived from plastids.²⁵ We recently showed that a knockdown mutant of one of the Arabidopsis RSHs (CRSH) showed abnormal flower development,⁸ as similarly observed in cytokinin signal mutants,^26–30 supporting this idea. Cleary, further biochemical and genetic analysis of RSHs, together with GTP-binding proteins, will be necessary for our understanding of the functional roles of “magic spots” in the physiology of higher plants; such studies are currently underway.

Addendum to: Mizusawa K, Masuda S, Ohta H. Expression profiling of four RelA/SpoT-like proteins, homologues of bacterial stringent factors, in Arabidopsis thaliana. Planta. 2008;228:553–562. doi: 10.1007/s00425-008-0758-5.