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J Plant Res (2017) 130:625634

DOI 10.1007/s10265-017-0922-8


Phylogenetic analysis ofproteins involved inthestringent

response inplant cells
DoshunIto1 YutaIhara1 HidenoriNishihara1 ShinjiMasuda2,3

Received: 9 June 2016 / Accepted: 7 February 2017 / Published online: 16 March 2017
The Botanical Society of Japan and Springer Japan 2017

Abstract The nucleotide (p)ppGpp is a second messen- cyanobacterium followed by gene loss of an ancestral RSH
ger that controls the stringent response in bacteria. The gene in the cyanobacterial linage. Phylogenetic analysis of
stringent response modifies expression of a large num- gppA/ppx families indicated that plant gppA/ppx homologs
ber of genes and metabolic processes and allows bacteria form an individual cluster in the phylogenetic tree, and
to survive under fluctuating environmental conditions. show a sister relationship with some bacterial gppA/ppx
Recent genome sequencing analyses have revealed that homologs. Although RSHs contain a plastidial transit pep-
genes responsible for the stringent response are also found tide at the N terminus, GppA/Ppx homologs do not, sug-
in plants. These include (p)ppGpp synthases and hydro- gesting that plant GppA/Ppx homologs function in the
lases, RelA/SpoT homologs (RSHs), and the pppGpp-spe- cytosol. These results reveal that a proto-plant cell obtained
cific phosphatase GppA/Ppx. However, phylogenetic rela- genes for the stringent response by lateral gene transfer
tionship between enzymes involved in bacterial and plant events from different bacterial phyla and have utilized them
stringent responses is as yet generally unclear. Here, we to control metabolism in plastids and the cytosol.
investigated the origin and evolution of genes involved in
the stringent response in plants. Phylogenetic analysis and Keywords Chloroplast GppA ppGpp pppGpp Ppx
primary structures of RSH homologs from different plant RelA/SpoT homolog
phyla (including Embryophyta, Charophyta, Chlorophyta,
Rhodophyta and Glaucophyta) indicate that RSH gene
families were introduced into plant cells by at least two Introduction
independent lateral gene transfers from the bacterial Deino-
coccus-Thermus phylum and an unidentified bacterial phy- The stringent response is a fundamental regulatory sys-
lum; alternatively, they were introduced into a proto-plant tem that enables bacterial cells to survive in fluctuating
cell by a lateral gene transfer from the endosymbiotic environments. This response is mediated by the synthe-
sis and hydrolysis of the second messengers guanosine
5-triphosphate 3-diphosphate (pppGpp) and guanosine
Electronic supplementary material The online version of this
article (doi:10.1007/s10265-017-0922-8) contains supplementary
5-diphosphate 3-diphosphate (ppGpp), the levels of which
material, which is available to authorized users. are maintained in Escherichia coli by two enzymes, RelA
and SpoT (Cashel et al. 1996; Dalebroux and Swanson
* Shinji Masuda 2012; Hauryliuk et al. 2015; Potrykus and Cashel 2008).
Both enzymes synthesize (p)ppGpp by phosphorylating
Graduate School ofBioscience andBiotechnology, Tokyo GTP or GDP using ATP in response to different environ-
Institute ofTechnology, Yokohama2268501, Japan mental changes such as starvation for amino acids, iron,
Center forBiological Resources andInformatics, Tokyo nitrogen, phosphate or carbon. When GTP is the sub-
Institute ofTechnology, Yokohama2268501, Japan strate, pppGpp is initially produced but can be converted
EarthLife Science Institute, Tokyo Institute ofTechnology, to ppGpp by a specific phosphatase called guanosine
Tokyo1528551, Japan 5-triphosphate 3-diphosphate pyrophosphatase (GppA)


626 J Plant Res (2017) 130:625634

or exopolyphosphatase (Ppx) (Hara and Sy 1983; Keasling 2012). Albi and Serrano (2014) characterized two GppA/
et al. 1993). In Escherichia coli, (p)ppGpp controls tran- Ppx homologs belonging to the HD-domain polyPases
scription of a large number of genes through direct inter- from the green-sulfur bacterium Chlorobium tepidum,
action with RNA polymerase and also controls translation, and showed that they are able to hydrolyze polyP, and one
DNA replication, GTP-binding proteins, proteases and of the two also has the nucleotide triphosphatase activ-
enzymes for purine biosynthesis, which is necessary for ity (Albi and Serrano 2014). These results suggested that
adaptation to environmental stimuli (Potrykus and Cashel GppA/Ppx-like proteins have remarkable functional plastic-
2008). pppGpp and ppGpp have differing affinities for the ity and some of them do not mediate the (p)ppGpp-depend-
target proteins (Mechold et al. 2013), suggesting that the ent stringent response; they mediate only inorganic polyP
ratio of the two nucleotides is physiologically important. metabolism.
SpoT, but not RelA, can also hydrolyze (p)ppGpp under Recent genome sequence data have indicated that RelA/
certain conditions (Cashel etal. 1996; Potrykus and Cashel SpoT-like proteins are conserved in algae and land plants
2008). Thus, SpoT is a bifunctional protein that catalyzes (Masuda 2012; Tozawa and Nomura 2011; van der Biezen
both (p)ppGpp synthesis and hydrolysis, whereas RelA has et al. 2000). In Arabidopsis, four genes encoding RelA/
only the (p)ppGpp synthase activity. SpoT and RelA pro- SpoT homologs (RSHs) have been identified: RSH1,
teins are only found in - and -proteobacteria (Mitten- RSH2, RSH3 and Ca2+-activated RSH (CRSH) (Tozawa
huber 2001), although SpoT homologs, having (p)ppGpp and Nomura 2011). All Arabidopsis RSHs are localized in
synthase and hydrolase domains, are widely distributed plastids (Maekawa etal. 2015; Masuda etal. 2008; Mizu-
among the bacterial kingdom, which are called Rel or Rsh sawa etal. 2008), indicating that they control plastidial (p)
(Atkinson etal. 2011); we refer the bacterial homologs as ppGpp level. Arabidopsis RSH2 and RSH3 are paralogs
Rel hereafter. with high (~80%) amino acid sequence identity (Atkinson
The enzymatic activities of E. coli RelA and SpoT are et al. 2011; Givens et al. 2004; Masuda 2012), and thus
also differentially regulated. RelA associates with ribo- Arabidopsis RSHs are classified into three groups, namely
somes and is activated when the ribosome A site is occu- RSH1, RSH2/RSH3 and CRSH, that differ in primary
pied by uncharged tRNA (Cashel etal. 1996; Potrykus and structure. Specifically, RSH1 does not have a conserved
Cashel 2008). This allows bacteria to rapidly accumulate Gly residue that is critical for (p)ppGpp synthase activ-
(p)ppGpp upon amino acid starvation. RelA, SpoT and Rel ity in Bacillus subtilis Rel (Wendrich and Marahiel 1997),
have a threonyl-tRNA synthetase, GTPase, SpoT (TGS) indicating that RSH1 does not have (p)ppGpp synthase
domain and an aspartate kinase chorismate mutase TyrA activity and has only (p)ppGpp hydrolase activity (Masuda
(ACT) domain in its C terminus, where acyl-carrier pro- et al. 2008). CRSH does not have the conserved His-Asp
tein, ribosomal tRNA, and other regulatory proteins are (HD) domain that is responsible for catalyzing (p)ppGpp
associated to control their (p)ppGpp synthesis and hydroly- hydrolysis (Masuda etal. 2008; Tozawa etal. 2007). RSH2/
sis activities (Brown etal. 2016; Loveland etal. 2016; Pot- RSH3 contains both the conserved Gly residue and HD
rykus and Cashel 2008). This allows bacteria to control (p) domain (Givens et al. 2004). This suggests that plastidial
ppGpp levels in response to environmental changes. (p)ppGpp levels in Arabidopsis are balanced through dif-
GppA/Ppx protein families are widely distributed ferent activities of the three RSH family enzymes. We pre-
among prokaryotes (bacteria and archaea), fungi, protists viously showed that over-accumulation of ppGpp in Arabi-
and metazoa; they are classified into six groups: GPPases, dopsis chloroplasts induces pleiotropic phenotypes, such as
GPPase-like polyPases, high molecular-weight polyPases, reduced chloroplast size and photosynthetic activities, indi-
low molecular-weight polyPases, HD-domain polyPases, cating that (p)ppGpp levels must be maintained within spe-
and RTG2 proteins (Albi and Serrano 2014). E. coli GppA cific concentration to ensure robust control of plastid activi-
and Ppx are classified into GPPases and GPPase-like poly- ties in plant cells (Maekawa etal. 2015). We also showed
Pases, respectively. Previous studies showed that enzy- that artificial over-accumulation of ppGpp in the cytosol
matic specificities for each GppA/Ppx family seem to dif- results in reduced plant growth (Ihara and Masuda 2016),
fer. Specifically, E. coli GppA and Ppx liberate Pi not only suggesting that (p)ppGpp produced in chloroplasts is trans-
by hydrolysis of pppGpp to generate ppGpp, but also by ferred to the cytosol to optimize metabolic processes.
progressive hydrolysis of the terminal phosphoanhydride Although the mechanisms of the plastidial stringent
bonds of the inorganic polyphosphate (polyP) (Hara and response are beginning to appear, it is unclear how RSH
Sy 1983; Keasling etal. 1993). Two GppA/Ppx homologs genes were introduced into plant cells during evolution.
belonging to the low molecular-weight polyPases from the Previously, Givens et al. (2004) performed a phylogenetic
pathogenic bacterium Mycobacterium tuberculosis show analysis of plant RSHs and reported that they originated
polyP phosphatase (polyPase) and ATPase activity, but do from an endosymbiotic cyanobacterium (Givens et al.
not catalyze hydrolysis of pppGpp to ppGpp (Choi et al. 2004). However, several other groups reported different

J Plant Res (2017) 130:625634 627

models indicating that plant RSHs were introduced into Ppx were separately applied for BLAST analysis against
plant cells by lateral gene transfer from pathogenic bacteria the E. coli database in NCBI, and if the top hit success-
and/or the Deinococcus-Thermus bacterial phylum (Atkin- fully identified the original E. coli GppA or Ppx sequence,
son et al. 2011; Masuda 2012; Suzuki and Miyagishima it was taken as an ortholog. For constructing phylogenetic
2010; van der Biezen etal. 2000). It is also unknown how trees, bacterial GppA/Ppx were searched via BLAST analy-
the three plant RSH families diverged and were established sis on the NCBI website using the amino acid sequence of
during plant evolution. the identified Arabidopsis GppA/Ppx homolog as a query
To clarify the origin and evolution of RSHs involved in sequence. Obtained sequences were cutoff by an E value
the land plant stringent response, we compared the primary of 11024, and then GppA/Ppx homologs were selected
structures of RSHs conserved in five plant phyla. We then from all bacterial phyla found in the list. Accession num-
performed a phylogenetic analysis of the plant RSHs iden- bers for the sequences and identifiers were as described in
tified. Our results suggest that plant RSHs originated from TableS2.
the Deinococcus-Thermus phylum and an unidentified bac- Domain structures of the identified proteins were ana-
terial phylum by independent lateral gene transfer events. lyzed using InterProScan (Mitchell et al. 2014). Based on
We also found that genes encoding GppA/Ppx homologs multiple protein sequence alignments, constructed below,
are conserved in plant cells, which are in the same branch critical amino acid residues for enzymatic activities (e.g.,
with those of several bacterial species. These results indi- ppGpp synthesis and hydrolysis) were analyzed for each
cate that the plant stringent response was established with protein sequence based on previous studies (Aravind and
enzymes from different origins. Koonin 1998; Hogg et al. 2004; Wendrich and Marahiel
Multiple amino-acid sequence alignments of RSH and
Materials andmethods GppA/Ppx homologs were constructed using MAFFT ver.
7 (Katoh and Standley 2013), followed by manual adjust-
To identify plant orthologs of RSHs, BLAST searches ment based on structural considerations. The region used
were performed with amino acid sequences of Arabidop- to construct the RSH phylogenetic trees corresponds to
sis RSH1, RSH2 and/or CRSH against nucleotide and residues 42342 of E. coli SpoT. The region used to con-
amino acid sequence databases of the National Center for struct the GppA/Ppx phylogenetic tree corresponds to resi-
Biotechnology Information (NCBI), Phytozome database dues 18323 of Arabidopsis GppA/Ppx homolog. After
(https://phytozome.jgi.doe.gov/pz/portal.html), Cyan- constructing the original alignments, we excluded gap-rich
ophora Genome Project (Price et al. 2012), and Klebsor- regions from each alignment by a home-made Perl script,
midium flaccidum genome project (Hori etal. 2014). Iden- which removes sites where gaps were found in over 95% of
tified plant RSHs were then separately applied for BLAST the sequences. All alignments used for phylogenetic analy-
analysis against the Arabidopsis database in NCBI, and if sis are shown in Supplementary Dataset. Substitution mod-
the top hit successfully identified the original Arabidop- els were tested for each of the alignments with ProtTest ver.
sis sequence (RSH1, RSH2 or CRSH), it was taken as an 3.4.2 (Darriba etal. 2011) under consideration of empirical
ortholog. Bacterial Rel proteins were searched via BLAST amino acid frequencies (+F) and a discrete gamma distri-
analysis using the amino acid sequence of E. coli SpoT. For bution (+G). The LG substitution model (Le and Gascuel
phylogenetic analysis of plant RSHs, amino-acid sequences 2008) with +G was chosen as the best-fit model based
of bacterial Rel were selected based on datasets used in on Bayesian information criterion (BIC) for all the dataset.
previous phylogenetic studies (Givens et al. 2004; Mas- Bayesian inference was performed using MrBayes 3.2.3
uda 2012; Suzuki and Miyagishima 2010; van der Biezen (Ronquist et al. 2012) under the LG+G model. Two par-
et al. 2000), which include those from Cyanobacteria, allel runs of 1107 generations with twelve chains were
Deinococcus-Thermus, Proteobacteria, Firmicutes, Ther- carried out with tree sampling every 100 generations. The
momicrobium, and Chlamydiae. Accession numbers for the first 2.5106 generations were discarded as burn-in. The
sequences and identifiers were as described in TableS1. run parameter convergence was assessed by average stand-
To identify plant orthologs of GppA/Ppx, BLAST ard deviation of split frequencies (<0.01) and by monitor-
searches were performed with the amino acid sequence of ing the trace plots and effective sample size (>200) using
E. coli GppA against the same nucleotide and amino acid Tracer v.1.6 (Rambaut etal. 2013). The trees were visual-
sequence databases of the National Center for Biotechnol- ized by FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/
ogy Information (NCBI), Phytozome database (https://phy- figtree/). Phylogenetic trees were also constructed by the
tozome.jgi.doe.gov/pz/portal.html), Cyanophora Genome maximum likelihood (ML) method using RAxML ver. 8
Project (Price et al. 2012), and Klebsormidium flaccidum (Stamatakis 2014) under the LG+G model. Bootstrap (BP)
genome project (Hori et al. 2014). Identified plant GppA/ values were obtained by 1,000 replications.


628 J Plant Res (2017) 130:625634

To assess the possibility for the sister-relationship RSH family with bacterial SpoT. We found that RSH1 fam-
between the cyanobacterial Rel and RSH1, RSH2 or CRSH, ily is in the same clade comprising Deinococcus-Thermus
the approximately unbiased (AU) test was carried out by Rel (Fig. S3), but RSH2 family is not clearly resolved from
using each sequence data shown in Supplementary Data- the clade comprising bacterial Rel (Fig. S1). CRSH family
set. For each of the RSH2 and CRSH data, the approximate is clearly in the same clade comprising Deinococcus-Ther-
likelihoods were calculated using the ProtML program in mus Rel with a BIPP value of 1.00 (Fig. S2).
MOLPHY 2.3b (Adachi and Hesegawa 1996) or all the We carried out the AU test to assess the possibility of
945 possible trees under the constraints of RSH2/CRSH, the cyanobacterial origin for each of the RSH1, RSH2 and
Cyanobacteria, Proteobacteria, Firmicutes+Chlamydiae, CRSH families by using CONSEL (for detail, see Materi-
Thermomicrobium, Dictyoglomus, and Deinococcus-Ther- als and methods section). The sister relationships of the
mus according to the results in Figs. S1, S2, respectively. cyanobacterial Rel with RSH1 or CRSH were significantly
For RSH1, the best 3,200 trees with the highest approxi- rejected by the AU test (p=0.00005 and p=0.012, respec-
mate likelihood were selected for this evaluation from the tively). On the other hand, the sister-relationship between
10,395 combinations because Meiothermus Rel and other RSH2 and cyanobacterial Rel was not significantly rejected
Deinococcus-Thermus clade were not constrained (see Fig. by the AU test (p=0.144).
S3). Per-site log-likelihoods were calculated with RAxML Amino-acid sequences of RSHs from each phylum were
under the LG+G model and were subsequently used for then applied for analysis by InterProScan (Mitchell et al.
the AU test with CONSEL (Shimodaira and Hasegawa 2014) to identify domain structures. As shown in Fig. 2a,
2001). InterProScan revealed that all RSH1 sequences had simi-
Chloroplast transit peptide prediction was performed larity to (p)ppGpp hydrolase and synthase domains. How-
with the ChloroP 1.1 server (http://www.cbs.dtu.dk/ ever, the Gly residue that is critical for (p)ppGpp synthase
services/ChloroP/). activity in B. subtilis Rel was not conserved in all RSH1
homologs (Fig. 3a), suggesting that they do not have (p)
ppGpp synthase activity. All RSH1-like proteins had the
Results TGS domain found in bacterial Rel (e.g., Synechocystis and
Meiothermus Rel) (Fig. 2a). In addition, the ACT domain
We searched for RSH family proteins in genomic sequences was conserved among RSH1-like proteins from all plant
from five plant phyla (Embryophyta, Charophyta, Chloro- phyla except Embryophyta (Arabidopsis and Physcom-
phyta, Rhodophyta, Glaucophyta) using Arabidopsis RSH1, itrella) (Fig. 2a) . The lack of ACT domain in Embryo-
RSH2 and CRSH as query sequences. To find evolutional phyta RSH1 may not be due to alternative splicing of the
trails for RSHs in land plants, we mainly focused on the gene since the primary structures were revealed from their
green plant linage, but not other lineages including those of genomic sequences, but not cDNA sequences.
secondary symbionts. Figure1 shows the phylogenetic tree Recently, the complex structure of the ribosome and E.
based on amino acid sequence alignment of the (p)ppGpp coli RelA was solved by cryo-electron microscopy (Brown
hydrolase and synthase domains of bacterial Rel and plant et al. 2016; Loveland et al. 2016). In the structure, the
RSHs. Strikingly, plant RSHs are classified into three indi- TGS domain of RelA binds the CCA tail to orient the free
vidual clusters, which are separated from each other. The 3 hydroxyl group of the terminal adenosine, which leads
classified families were termed as RSH1, RSH2 and CRSH steric preclusion of an aminoacylated tRNA at this posi-
families, since Arabidopsis RSH1, RSH2 and CRSH were tion (Brown etal. 2016), indicating the importance of the
grouped in the clusters, respectively. RSH1 homologs were interaction for sensing ribosome activity. More specifically,
found in species from three plant phyla, other than Embry- Pro411, Lys412, His432 and Arg438 of E. coli RelA inter-
ophyta, including K. flaccidum (Charophyta), Ostreococcus act with the 3 CCA tail of the A-site tRNA (Fig. S4, red
tauri (Chlorophyta) and Cyanidioschyzon merolae (Rhodo- boxes), and Arg487, Arg489, Lys491, His493, Arg497,
phyta). No gene encoding an RSH1-like protein was found and Lys498 interact with the tRNA phosphate backbone
in the genome sequence of Cyanophora paradoxa (Glauco- (Fig. S4, blue boxes). Among the amino-acid residues,
phyta). RSH2-like proteins were found in four plant-phyla positively-charged amino acids (e.g., Lys, Arg, and His)
Embryophyta, Charophyta, Chlorophyta and Glaucophyta, were particularly important for the interaction (Brown etal.
but not found in Rhodophyta (Fig.1). CRSH-like proteins 2016). The amino-acid sequence alignment of the TGS
were found in all plant phyla except Glaucophyta (C. para- domain of bacterial Rel and plant RSH1 reveals that at least
doxa; Fig. 1). Deinococcus-Thermus Rel is grouped out half of the positively-charged amino-acids were conserved
from other bacterial Rel, and form an individual cluster in both bacterial Rel, and plant RSH1 (Fig. S4), suggest-
with plant RSH1- and CRSH-family proteins (Fig.1). We ing that RSH1 homologs interact with the ribosomes in
also constructed phylogenetic trees based on each plant chloroplasts.

J Plant Res (2017) 130:625634 629

Fig.1Phylogenetic tree based on (p)ppGpp synthase and hydrolase tutions per site, as indicated by the scale bar. Gain and loss of the
domains of plant RSHs and bacterial Rel. The trees were constructed critical Gly residue, aspartate kinase chorismate mutase TyrA domain
based on Bayesian inference and ML using the LG+G model in (ACT), Ca2+-binding helix E and F motif (EF hand), and tetratrico-
MrBayes and RAxML, respectively. Support values are Bayesian peptide repeat motif (TPR) are expressed by solid and dotted arrows,
posterior probabilities (left) and bootstrap values of ML (right), and respectively
branch lengths are proportional to the expected number of substi-

Primary structures of RSH2-like proteins found in to other RSH2-like proteins in these regions. The TGS
four plant-phyla Embryophyta, Charophyta, Chlorophyta domain is only found in RSH2-like proteins of C. para-
and Glaucophyta were characterized (Fig. 2b). One of doxa (Glaucophyta). The lack of TGS domain in other
the two RSH2-like proteins in O. tauri (RSH2b) lacks phyla is not due to alternative splicing of the gene since
Gly residue, although the sequences are general similar we obtained primary structures of RSH2 homologs form


630 J Plant Res (2017) 130:625634

Fig.2Schematic depiction
of the primary structures of
RSH1 (a), RSH2 (b) and CRSH
(c) family proteins. Hyd: (p)
ppGpp hydrolase domain; hyd:
(p)ppGpp hydrolase domain
lacking some critical amino-
acids; Syn: (p)ppGpp synthase
domain; syn: (p)ppGpp synthase
domain lacking the critical
Gly residue; ACT: aspartate
kinase chorismate mutase TyrA
domain; EF hand: C a2+-binding
helix E and F motif; TGS:
threonyl-tRNA synthetase,
GTPase, SpoT domain; TPR:
tetratricopeptide repeat motif;
AA: amino-acids

genomic sequences of K. flaccidum, Arabidopsis and Rhodophyta (C. merolae; Fig. 2c). CRSH-like proteins
Physcomitrella. from O. tauri and K. flaccidum have C-terminal tetratrico-
CRSH-like proteins were found in all plant phyla except peptide repeat motifs (Fig.2c), which mediate proteinpro-
Glaucophyta (C. paradoxa; Fig. 2c). These CRSH-like tein interactions.
proteins lack certain critical residues in the HD domain A computer-aided similarity search showed that
(Fig. 3b), suggesting that they lack (p)ppGpp hydro- GppA/Ppx homologs have been conserved in the plant
lase activity. The CRSH-like proteins from Chlorophyta phyla Embryophyta (e.g. Arabidopsis and Populus) and
(O. tauri and Volvox carteri) lack the conserved Gly Charophyta Klebsormidium; these contain both a GppA/
residue in the (p)ppGpp synthase domain (Fig. 3a), sug- Ppx phosphatase domain and an HD domain or an HD-
gesting that they have lost (p)ppGpp synthase activity. like domain, which resembles to the bacterial HD-domain
The Ca2+-binding EF hand motifs have been conserved polyPases, including E. coli GppA and Ppx (Fig. 4).
in CRSH-like proteins from most plant phyla, except

J Plant Res (2017) 130:625634 631

Fig.3Alignment of partial a Arabidopsis RSH2 419 GI - - - - - - -

amino acid sequences for the Arabidopsis RSH3 423 GI - - - - - - -
(p)ppGpp synthase (a) and Physcomitrella RSH2a 256 GV - - - - - - -
- Q F VD L S GRP K N L Y S I Y K K RSH2 family
Klebsormidium RSH2 517 GI - - - - - - -
hydrolase (b) domains. The Gly Ostreococcus RSH2a 278 NA - - - - - - -
residue that is critical for syn- Cyanidioschyzon RSH 642 PQL - HS Y I -
thase activity and the His-Asp ** * ** *
Arabidopsis CRSH 296 L V L - A EMV - - NDV Y I K GRY K S RY SMMK K
(HD) critical for hydrolysis are Physcomitrella CRSH 239 P EM - QS L I - - E SV K I S GRCK S K Y S TMK K
indicated in red Klebsormidium CRSH 552 L L I T QP Y VPGMEV T L S GR L K S L Y S AHCK CRSH family
Ostreococcus CRSHa 157 P TM - NA L I GR GGV RV L ARRK S RY S TMK K
Cyanidioschyzon CRSH 305 P E L - RR - - - - YRY E I T GRA K N L F S T F K K
* * * *
Synechocystis Rel 235 G I - - - - - - - - E H F E L QGRP K H L Y G I Y Y K
Bacterial Rel
Meiothermus Rel 245 S V L - GA S I - - QGY E V T GR T K H L Y S I WK K
* * *
Arabidopsis RSH1 363 QF L - D L V T - - V N T DV RS V CK E T Y S I Y K A
Physcomitrella RSH1 327 QF L - GYMT - - A D I K V A T L S K E L Y S I Y K K
Klebsormidium RSH1 356 A C L - - C L T - - HS F RV E T L CK E A Y S I Y K K RSH1 family
Ostreococcus RSH1 304 D F L - RGHA - - V A V S L GA K QK E P Y GV HRS
Cyanidioschyzon RSH1 413 S I L - K P L V - - RE I C T K V A L K N L F S I Y QR
b Arabidopsis RSH1 176 P V A V A R I L GE L E L DWE S I V A GL L HD
Physcomitrella RSH1 135 P V E V A R I L GE L EMDWE T V V A GL L HD
Klebsormidium RSH1 169 P V A V A E I L GQMQMDCE T I I A GL L HD RSH1 family
Ostreococcus RSH1 104 P V A V A A I L A D L E L DHE S L I A GL L HD
Cyanidioschyzon RSH1 139 P V A V A L L L A E L RMD VD T L I A GL L HD
** ** * * * * ** *
Synechocystis Rel 56 P V A V A GL L RD L GGDE AM I A A GF L HD
Meiothermus Rel 45 P V A V A E I L A E L GL DA E T L MA GL L HD Bacterial Rel
** ** * * * * ***
Arabidopsis RSH2 237 CV E T AML L A N I GA NS T V V V A GL L HD
Arabidopsis RSH3 241 CV E T AML L A D I GA NS T V V V A G I L HD
Physcomitrella RSH2 a 44 CV E T A L I L A A T GV GN T V V A A GL L HD RSH2 family
Klebsormidium RSH2 334 C L E A A K I L A E S T RDRVMV A A GL L HD
Ostreococcus RSH2a 99 CV E T A V T L A K L GMD T K T V A V GL L HD
* * * **
Arabidopsis CRSH 116 A L S L S I I L AD L QMDA E V I S A S I L S E
Physcomitrella CRSH 59 A L S V A F I L AD L RMDA E V I A A GL L RE CRSH family
Klebsormidium CRSH 373 - - - - - - - VWGV K A E L E D L C F A V L Q -
Cyanidioschyzon CRSH 123 S V RNA A F L AR L GV DA D T I CA A L V - D

Fig.4Schematic representa-
tion of the primary structures of
GppA/Ppx homologs. Domain
structures were analyzed using
InterProScan. AA amino-acids

When we performed BLAST search, with Arabidopsis Discussion

GppA/Ppx homolog used as a query sequence, to find
genes showing similarity more than a 1104 E value, It has been shown that Arabidopsis RSH1 does not func-
only three bacterial GppA/Ppx families were found in tionally complement E. coli relA or the relA-spoT double
addition to plant GppA/Ppx, which belong to HD-domain mutation, although Arabidopsis RSH2, RSH3 and CRSH
polyPases, GPPases and GPPase-like polyPases. By the do complement these mutations (Mizusawa et al. 2008).
analysis, high molecular-weight polyPases, low molecu- These results indicate that Arabidopsis RSH1 does not have
lar-weight polyPases, and RTG2 proteins including fun- (p)ppGpp synthase activity. Here, we confirmed that Arabi-
gal, protistan and/or metazoan GppA homologs were dopsis RSH1 does not have the critical Gly residue in the
not found. Phylogenetic analysis revealed that the plant (p)ppGpp synthase domain (Fig. 3a). Indeed, RSH1-like
GppA/Ppx homologs are in the sister clade comprising proteins were found in Embryophyta, Charophyta, Chloro-
HD-domain polyPases from bacterial phyla Spirochetes, phyta and Rhodophyta (Figs.1, 2a), none of which have the
Actinobacteria and Chlorobi (Fig.5). Gly residue (Fig.3a), suggesting that they had lost the (p)
ppGpp synthase activity. The RSH1 family proteins from


632 J Plant Res (2017) 130:625634

J Plant Res (2017) 130:625634 633

Fig.5Phylogenetic tree based on GppA/Ppx domains of GppA/Ppx clearly show that the origin of RSH2 differs from that of
homologs. The trees were constructed based on Bayesian inference RSH1 and CRSH (Fig. 1). Specifically, the sister rela-
and ML using the LG+G model in MrBayes and RAxML, respec-
tively. Support values are Bayesian posterior probabilities (left), boot-
tionships of the cyanobacterial Rel with RSH1 or CRSH
strap values of ML (right), and branch lengths are proportional to the were significantly rejected by the AU test (p=0.00005
expected number of substitutions per site, as indicated by the scale and p=0.012, respectively); on the other hand, the sister-
bar. Organisms whose GppA/Ppx primary structures are shown in relationship between RSH2 and cyanobacterial Rel was
Fig.4 are highlighted by red squares
not significantly rejected by the AU test (p=0.144) (see
Results section). From these observations, we propose
the plant phyla have retained the conserved TGS domain, that plant RSHs were introduced into proto-plant cells by at
like bacterial Rel (Fig. 2a), which may associate with the least two different horizontal gene transfer events: one from
ribosome in chloroplasts (Fig. S4). Furthermore, the ACT Deinococcus-Thermus (for RSH1 and CRSH families) and
domain has been conserved in the RSH1 homologs from the other from an undefined bacterial species (for RSH2
Charophyta, Chlorophyta and Rhodophyta, but not in family). Although the RSH2 origin could not be resolved
Embryophyta. These results indicate that RSH1 is structur- by this phylogenetic analysis because of low BIPP or BP
ally similar to bacterial Rel proteins. Phylogenetic analysis values between RSH2 and bacterial Rel, it is still possible
indicates that RSH1 proteins are clustered with Deinococ- that RSH2 was derived from an endosymbiotic cyanobac-
cus-Thermus Rel proteins (Fig.1, S3). It has been reported terium. If this is the case, one could assume that the RSH
that nuclear-encoded plastidial proteins are derived from homologs (RSH1, CRSH and RSH2 families) were intro-
the endosymbiotic cyanobacterium as well as other bacte- duced into a proto-plant cell by the endosymbiosis of the
rial species (Suzuki and Miyagishima 2010). These results ancestral cyanobacterium and the RSH1 and CRSH gene
suggest that the plant RSH1-like protein was introduced families were then selectively lost in the cyanobacterial
into a proto-plant cell by lateral gene transfer at least before linage. A close evolutionary relationship of Cyanobacteria
the separation of Chlorophyta and Rhodophyta, followed and Deinococcus-Thermus phyla was reported (Gupta and
by loss of the functional ACT domain after separation of Johari 1998), supporting the idea. In any case, plant RSH
Embryophyta and Charophyta (Fig.1). families were sub-functionalized during plant evolution;
CRSH-like proteins were found in Embryophyta, Cha- e.g., RSH1 may have been arranged to associate with ribo-
rophyta, Chlorophyta and Rhodophyta (Figs. 1, 2c). Phy- some through the TGS domain, but others have not (Fig.2).
logenetic analysis clearly showed that CRSH and RSH1 This is similar to the bacterial (p)ppGpp synthases/hydro-
family proteins are in the same cluster but different line- lases; RelA and SpoT were sub-functionalized as the major
ages; CRSH was separated from RSH1 and Deinococcus- (p)ppGpp synthesis and the hydrolysis, respectively, in the
Thermus Rel with high BIPP and BP values of 0.83 and 63, - and -proteobacterial linage (Potrykus and Cashel 2008).
respectively (Fig. 1). This suggests that CRSH and RSH1 We found that GppA/Ppx-like proteins, having both
were introduced into a proto-plant cell by lateral gene trans- GppA/Ppx and HD (or HD-like) domains, are conserved
fer from Deinococcus-Thermus prior to the separation of in Embryophyta and Charophyta, and the protein family
Chlorophyta and Rhodophyta, and the introduced gene(s) is in the clade comprising bacterial HD-domain polyPases
was functionally and genetically divided at an early step and the GPPases/GPPase-like polyPases (Figs. 4, 5). This
after gene transfer. Arabidopsis CRSH does not have (p) suggests that GppA/Ppx-like protein was introduced into a
ppGpp hydrolase activity owing to loss of the HD domain, proto-plant cell by lateral gene transfer, although the donor
and this property is conserved in all CRSHs from different bacterial species could not be defined by the phylogenetic
plant phyla (Fig.2c). Thus, CRSH and RSH1 have adopted analysis. The HD-domain (or HD-like domain) polyPases
separate (p)ppGpp synthase and hydrolase roles, respec- are structurally similar to the GPPases and GPPase-like
tively, during the evolution of plant cells. polyPases including E. coli GppA and Ppx that convert
Complementation analysis of the E. coli relA mutant pppGpp to ppGpp to mediate the stringent response (Hara
and relA-spoT double mutant suggests that Arabidopsis and Sy 1983; Keasling et al. 1993), suggesting that plant
RSH2 and RSH3 have both (p)ppGpp synthase and hydro- GppA/Ppx homologs may have such an enzymatic activity.
lase activities (Mizusawa et al. 2008). RSH2-like proteins It should be noted that Arabidopsis GppA/Ppx do not have
were found in four plant phyla including Embryophyta, a predicted chloroplast transit peptide at their N terminus
Charophyta, Chlorophyta and Glaucophyta, and most have (Fig. S5); no transit peptide is also predicted for the GppA/
the conserved (p)ppGpp synthase and hydrolase domains Ppx homolog from K. flaccidum (Charophyta), although it
(Fig.2b). Notably, two RSH2-like proteins in C. paradoxa has a long N-terminal sequence (Fig.4). These results sug-
have the TGS domain, like bacterial Rel (Fig. 2b), sug- gest that plant GppA/Ppx homologs localize in the cytosol.
gesting that RSH2 was introduced into an ancestral Glau- We previously showed that over-accumulation of ppGpp in
cophyta by lateral gene transfer. The phylogenetic analyses the cytosol results in reduced plant growth, indicating that


634 J Plant Res (2017) 130:625634

ppGpp is utilized in the cytosol (Ihara and Masuda 2016). Ihara Y, Masuda S (2016) Cytosolic ppGpp accumulation induces
If plant GppA/Ppx-like proteins could convert pppGpp to retarded plant growth and development. Plant Signal Behav
ppGpp, plant-type GppA/Ppx-like proteins may control Katoh K, Standley DM (2013) MAFFT multiple sequence alignment
the ratio of pppGpp and ppGpp in the cytosol during the software version 7: improvements in performance and usability.
stringent response in plant cells. Genetic and biochemical Mol Biol Evol 30:772780
analyses of plant GppA/Ppx-like proteins will help clarify Keasling JD, Bertsch L, Kornberg A (1993) Guanosine pentaphosphate
phosphohydrolase of Escherichia coli is a long-chain exopolyphos-
the exact function of (p)ppGpp in the cytosol, and such a phatase. Proc Natl Acad Sci USA 90:70297033
study is currently under way in our laboratory. Le SQ, Gascuel O (2008) An improved general amino acid replacement
matrix. Mol Biol Evol 25:13071320
Acknowledgements This work was supported in part by JSPS Loveland AB, Bah E, Madireddy R, etal (2016) Ribosome-RelA struc-
KAKENHI Grant Nos. 16H03280 and 16K14694 to SM. tures reveal the mechanism of stringent response activation. Elife
Maekawa M, Honoki R, Ihara Y, et al (2015) Impact of the plastidial
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