Enzymatic and molecular characterization of arabidopsis ppGpp pyrophosphohydrolase, AtNUDX26

Article abstract of DOI:10.1271/bbb.120523

Not only in bacteria but also in plant cells, guanosine- 3',5'-tetraphosphate (ppGpp) is an important signaling molecule, that affects various cellular processes. In this study, we identified nucleoside diphosphates linked to some moiety X (Nudix) hydrola

Full text of DOI:10.1271/bbb.120523

Biosci. Biotechnol. Biochem., 76 (12), 2236–2241, 2012
Enzymatic and Molecular Characterization of
Arabidopsis ppGpp Pyrophosphohydrolase, AtNUDX26
Daisuke ITO,1 Takahiro KATO,1 Takanori MARUTA,1;2 Masahiro TAMOI,1
y
1;
Kazuya YOSHIMURA,3 and Shigeru SHIGEOKA
1Department of Advanced Bioscience, Faculty of Agriculture, Kinki University,
3327-204 Nakamachi, Nara 631-8505, Japan
2Faculty of Life and Environmental Science, Shimane University,
1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan
3Department of Food and Nutritional Science, College of Bioscience and Biotechnology, Chubu University,
1200 Matsumoto-cho, Kasugai, Aichi 487-8501, Japan
Received July 5, 2012; Accepted August 26, 2012; Online Publication, December 7, 2012
[doi:10.1271/bbb.120523]
Not only in bacteria but also in plant cells, guanosine-
30,50-tetraphosphate (ppGpp) is an important signaling
molecule, that affects various cellular processes. In this
study, we identified nucleoside diphosphates linked to
some moiety X (Nudix) hydrolases, AtNUDX11, 15, 25,
and 26, having ppGpp pyrophosphohydrolase activity
from Arabidopsis plants. Among these, AtNUDX26
localized in chloroplasts had the highest Vmax and kcat
values, leading to high catalytic efficiency, kcat/Km. The
activity of AtNUDX26 required Mg2þ or Mn2þ ions as
cofactor and was optimal at pH 9.0 and 50 ꢀC. The
expression of AtNUDX26 and of ppGpp metabolism-
associated genes was regulated by various types of
stress, suggesting that AtNUDX26 regulates cellular
ppGpp levels in response to stress and impacts gene
expression in chloroplasts. This is the first report on the
molecular properties of ppGpp pyrophosphohydrolases
in plants.
The biosynthesis of ppGpp in E. coli is catalyzed by
two homologous enzymes, RelA and SpoT.2,11) In
E. coli cells grown under conditions of amino acid
depletion, pppGpp is synthesized from ATP and GTP by
ribosome-associated RelA, and subsequently converted
to ppGpp by 50-nucleotidase. Thereafter, ppGpp is
degraded to GDP and pyrophosphate by SpoT, which
has reciprocal activities for ppGpp degradation and
synthesis. Van der Biezen et al.10) isolated three RelA/
SpoT homologs, designated RSH genes (AtRSH1,
AtRSH2, and AtRSH3), from Arabidopsis thaliana. In
addition, Arabidopsis had a single CRSH gene, a Ca2þ
-
activated RelA-SpoT homolog, for an enzyme having
ppGpp synthase activity.17–19) It has been found that
ppGpp and the ppGpp synthetase occur in chloroplasts
along with all RSH and CRSH proteins in Arabidopsis
plants.15,20–22) The chloroplast is a semi-autonomous
organelle that originates not only from an ancestral host
cell but also from a cyanobacterium-like photosynthetic
prokaryote. Therefore, it is plausible that a stringent
control system due to ppGpp similar to that of bacteria
exists in plant chloroplasts.20,23,24)
Key words: Arabidopsis; ganosine 30,50-bispyrophos-
phate (ppGpp); nucleoside diphosphates
linked to some moiety X (Nudix) hydrolase
Nudix hydrolases, distributed in all living organisms,
constitute a large family of proteins that share a highly
conserved amino acid sequence, GX5EX7REXEEUXGU,
where U is an aliphatic, hydrophobic residue, although
several interesting examples exist with altered consensus
sequences.25–27) Nudix hydrolases hydrolyze a wide
variety of substrates that contain a nucleoside diphos-
phate linked to some other moiety, X.25,27–32) Large
numbers of these substrates, including nucleoside di- and
tri-phosphates and their oxidized forms, dinucleoside
polyphosphates, nucleotide sugars, coenzymes, and
capped RNAs, have been identified.33–37) These sub-
strates are either potentially toxic compounds, cell-
signaling molecules, regulators of cellular metabolism,
or metabolic intermediates. Hence it has been suggested
that Nudix hydrolases play protective, regulatory, and
signaling roles in metabolism by hydrolytically remov-
ing such compounds.25,38) Arabidopsis thaliana has 28
A hyperphosphorylated guanosine nucleotide, ppGpp,
termed an alarmone, is a low-molecular-weight effector
that helps bacteria survive in limited environments.1,2)
The level of ppGpp in Escherichia coli (E. coli)
increases 10 to 100-fold during amino acid starvation
as compared to the level in exponentially growing
cells.3,4) ppGpp also accumulates in cyanobacteria under
nitrogen starvation.5) It binds to the ꢀ-subunit of RNA
polymerase and induces structural change.6–10) The
ppGpp that accumulates in the cells leads to an increase
in the synthesis of many enzymes required for producing
amino acids, and to changes in the pattern of expression
of various other products.2,11–14) Notably, it has been
reported that the level of ppGpp in plants is increased by
environmental stress, including drought, salinity, and
UV irradiation.15) In addition, ppGpp has been found to
act as a regulator of chloroplast RNA polymerase.16)
y
To whom correspondence should be addressed. Tel/Fax: +81-742-43-8083; E-mail: shigeoka@nara.kindai.ac.jp
Abbreviations: CRSH, Ca2þ-activated RelA-SpoT homolog; Nudix, nucleoside diphosphates linked to some moiety X; NUDX, nudix hydrolase;
pGp, ganosine 30,50-bisphosphate; ppGpp, ganosine 30,50-bispyrophosphate; pppGpp, ganosine 30-diphosphate,50-triphosphate; RSH, RelA-SpoT
homolog

Enzymatic and Molecular Characterization of AtNUDX26
2237
(ppGpp) and the reaction product (pGp) were detected by their UV
absorbances at 252 nm.
genes (AtNUDX1–27 and AtDCP2) encoding Nudix
hydrolase homologs located in the cytosol, mitochondria,
and chloroplasts.28,29,39) There are various AtNUDXs
having pyrophosphohydrolase activity, with a wide range
of substrate specificities: 8-oxo-7,8-dihydro-20-(deoxy)
guanosine 50-triphosphate (AtNUDX1), ADP-ribose
(AtNUDX2 and 7), ADP-glucose (AtNUDX14), long-
chain diadenosine polyphosphates (ApnA)(AtNUDX13,
25, 26, and 27), NAD(P)H (AtNUDX6, 7, and 19),
coenzyme A (AtNUDX11 and 15), FAD (AtNUDX23),
and mRNA caps (AtDCP2).28,29,39,40) Notably, there is
increasing evidence of regulatory roles of the respective
Nudix hydrolases in various physiological processes in
plants, biotic and abiotic stress responses and the
metabolism of various molecules.39,41–46) However, there
are numerous AtNUDXs (AtNUDX3–5, 8, 9, 12, 16–18,
20–22, and 24) that show no activity toward any
substrate.
Recently, it was reported that ppGpp is recognized as
a substrate for a Nudix hydrolase, Ndx8, in Thermus
thermophilus,37) suggesting that Nudix hydrolases have
the potential to act as transcriptional regulators through
hydrolysis of ppGpp. In fact, intracellular ppGpp levels
in the ndx8 mutant briefly increased at the end of the
logarithmic growth phase and then dropped during the
stationary phase, indicating that Ndx8 is involved in the
regulation of ppGpp levels during growth-phase tran-
sition by acting on the degradation of ppGpp.37) In this
study, we identified the ppGpp pyrophosphohydrolases
(AtNUDX11, 15, 25, and 26) in Arabidopsis. Among
these, judging from its subcellular localization, enzy-
matic properties (enzymatic ability and kinetic param-
eters) toward ppGpp, and gene expression in response to
environmental stress, AtNUDX26 is involved in the
metabolism of ppGpp in chloroplasts.
Plant materials and stress treatments. Arabidopsis thaliana wild-
type (Col-0) was grown on Murashige and Skoog’s medium under a
light intensity of 100 mmol photons/m2/s. Two-week-old Arabidopsis
plants were subjected to various types of stress: salinity, paraquat,
drought, and heat. Paraquat at 50 mM was sprayed on the plants.
Salinity stress was imposed by transferring the plants to MS medium
containing 250 mM NaCl for 0 to 9 h. Drought stress was imposed by
subjecting them to dehydration on paper towels for 0 to 6 h. Heat stress
was imposed by incubating the cultures for 0 to 3 h at 37 ꢀC.
Quantitative Real-Time PCR experiments. Quantitative Real-Time
PCR (RT–PCR) experiments were performed following Nishizawa
et al.49) The primer sequences were as follows:
NUDX26-primerF (50-ATCCGATTGGAAGGGTCAAGCAC-30),
NUDX26-primerR (50-CTCAGGTTTCTCAGTACCATCGC-30),
RSH1-primerF (50-TTCTTGCGTGGCCGAGATTGACA-30),
RSH1-primerR (50-CACCTTAGCGCAAACACACTAACCAAACT-30),
RSH2-primerF (50-CTGAAGATGGGTGATGTGGTGG-30),
RSH2-primerR (50-CGCGAGTCCTCGATCATACATG-30),
RSH3-primerF (50-ACACCTGTAAGTGATCTGAAATGCAAG-30),
RSH3-primerR (50-CGCAAGCCCTCGATCATACATC-30),
CRSH-primerF (50-CAGACGAATTCGATACGTTTCAGAAAC-30),
CRSH-primerR (50-TCATGAAGCTTCTCGTCTAAAAGACTC-30),
Actin2-QF (50-GGTGGTTCCATTCTTGCTTCCC-30),
Actin2-QR (50-TCATACTCGGCCTTGGAGATCC-30).
Data analysis. Significance of differences between data sets was
evaluated by t-test. Calculations were carried out with Microsoft Excel
software.
Accession numbers. The Arabidopsis Genome Initiative locus
identifiers for the genes mentioned here are as follows: AtNUDX1
(At1g68760), AtNUDX2 (At5g47650), AtNUDX3 (At1g79690),
AtNUDX4 (At1g18300), AtNUDX5 (At2g04430), AtNUDX6
(At2g04450), AtNUDX7 (At4g12720), AtNUDX8 (At5g47240),
AtNUDX9 (At3g46200), AtNUDX10 (At4g25434), AtNUDX11
(At5g45940), AtNUDX12 (At1g12880), AtNUDX13 (At3g26690),
AtNUDX14 (At4g11980), AtNUDX15 (At1g28960), AtNUDX16
(At3g12600), AtNUDX17 (At2g01670), AtNUDX18 (At1g14860),
AtNUDX19 (At5g20070), AtNUDX20 (At5g19460), AtNUDX21
(At1g73540), AtNUDX22 (At2g33980), AtNUDX23 (At2g42070),
AtNUDX24 (At5g19470), AtNUDX25 (At1g30110), AtNUDX26
(At3g10620), AtNUDX27 (At5g06340), RSH1 (At4g02260), RSH2
(At3g14050), RSH3 (At1g54130), CRSH (At3g17470), and Actin2
(At3g18780).
Materials and Methods
Expression and purification of the recombinant AtNUDX proteins.
Recombinant forms of AtNUDXs (AtNUDX1–27) were produced
using E. coli strain BL21 (DE3) pLysS cells, and were purified from
the extract using a HiTrap chelating HP column (GE Healthcare, Little
Chalfont, UK) following previous reports.28,29,40) The protein content
was determined by the Bradford method, using bovine serum albumin
as standard.47) The molecular masses of the recombinant AtNUDX
proteins agreed with the predicted values, calculated from the amino
acid sequence of the mature protein plus the hexahistidine-tag (data
not shown).
Results and Discussion
Identification of AtNUDXs having pyrophosphohy-
drolase activity toward ppGpp
To identify AtNUDXs having pyrophosphohydrolase
activity toward ppGpp, we prepared His-tagged
recombinant AtNUDX1–27 proteins in the absence of
the predicted transit peptide expressed in E. coli
cells. Production and purification of the recombinant
AtNUDXs were carried out by a method reported
previously.28,29,40) The hydrolase activity of AtNUDXs
toward ppGpp in the presence of 5 mM Mg2þ as
cofactor was examined by HPLC analysis. Recombi-
nant AtNUDX26 hydrolyzed ppGpp with relatively
high activity (0:19 ꢂ 0:05 mmol/min/mg protein).
AtNUDX11 (0:06 ꢂ 0:01 mmol/min/mg protein), 15
(0:02 ꢂ 0:01 mmol/min/mg protein), and 25 (0:06 ꢂ
0:01 mmol/min/mg protein) exhibited barely detectable
activity. No ppGpp hydrolase activity was detected in
the other AtNUDXs (AtNUDX1–10, 12–14, 16–24,
and 27).
Enzyme assay and HPLC. ppGpp and ganosine 30,50-bisphosphate
(pGp) were purchased from TriLink Biotechnologies (San Diego, CA).
The hydrolytic activities of recombinant forms of AtNUDXs toward
ppGpp were assayed by a method described previously.28,29) The
reaction mixture (60 mL), containing 50 mM Tris–HCl (pH 8.0), 5 mM
MgCl2, 25 mM ppGpp, and 0.2–1.0 mg of the purified recombinant
protein, was incubated at 37 ꢀC for 10 min. For the assay of Km, ppGpp
at 5 mM to 800 mM was added to the reaction. For the assay of divalent
cation-dependency, Mg2þ was replaced with Ni2þ, Zn2þ, Ca2þ, or
Mn2þ (5 mM each). For the assay of pH-dependency, the Tris–HCl
buffer was replaced with Glycine-NaOH buffer (100 mM each) at basic
pH (pH 8.5–10.0). Temperature dependency was assayed at 20–60 ꢀC.
The reaction was terminated by adding 10 mL of 100 mM EDTA. In
addition, the reaction was carried out under reducing conditions with
1 mM DTT following Olejnik et al.48) The mixture was then analyzed
by HPLC using a COSMOSIL C18 column (4:6 ꢁ 250 mm, Nacalai
Tesque, Kyoto, Japan) at a flow rate of 0.6 mL/min for the mobile
phase buffer, which contained 73 mM KH2PO4, 5 mM tetrabutylammo-
nium dihydrogenphosphate, and 21% methanol. The substrates

2238
D. ITO et al.
pGp
A
None
Ni2+
GDP
GTP
Zn2+
Ca2+
Mn2+
Mg2+
ppGpp
0
20
40 60 80 100
Activity (% of maximum)
B
C
100
80
60
40
20
0
Tris-HCl
Glycine
-NaOH
0
5
10
15
20
Retention time (min)
Fig. 1. Identification of the Products of the Degradation of ppGpp by
AtNUDX26.
6.0
7.0
8.0
pH
9.0
10.0
Reaction mixtures containing 25 mM (1.5 nmol/60 mL) ppGpp were
incubated with 5 mM Mg2þ in the absence and the presence of the
purified recombinant AtNUDX26 protein at 37 ꢀC for 10 min and
then subjected to HPLC with a COSMOSIL C18 column, as
described in ‘‘Materials and Methods.’’ The elution profiles of the
reaction mixture without the enzyme (gray line) and with the enzyme
(black line) is shown. Positions of standards (0.9 nmol of GDP,
1.5 nmol of GTP, 1.5 nmol of ppGpp, and 1.5 nmol of pGp) are
shown in the upper panel. Asterisks indicate either pGpp or ppGp.
100
80
60
40
20
0
20
30
40
°C
50
60
Fig. 2. Enzymatic Characterization of AtNUDX26.
It has been reported that the activity of some Nudix
hydrolases, such as AtNUDX13 and 26, which have
ApnA pyrophosphohydrolase activity, increased under
reducing conditions.29,48) However, in the presence of
the reducing agent, dithiothreitol (DTT: 1 mM), in the
reaction solution, the activities of AtNUDX11 (0:05 ꢂ
0:02 mmol/min/mg protein), 25 (0:05 ꢂ 0:03 mmol/
min/mg protein), and 26 (0:12 ꢂ 0:04 mmol/min/mg
protein) were similar to those in the absence of the
agent. No ppGpp hydrolase activity was detected for the
other AtNUDXs under reducing conditions (data not
shown).
A, The requirement of divalent cations for ppGpp pyrophospho-
hydrolase activity was determined in the presence and the absence of
divalent cations (5 mM) at 37 ꢀC for 10 min, as described in
‘‘Materials and Methods.’’ B, The pH-dependency of ppGpp
pyrophosphohydrolase activity was determined under the assay
conditions described in ‘‘Materials and Methods,’’ except that Tris–
HCl buffer was replaced with Glycine-NaOH buffer (100 mM each)
at the indicated pH. C, The temperature dependency of ppGpp
pyrophosphohydrolase activity was determined as described in
‘‘Materials and Methods,’’ at 20–60 ꢀC.
Table 1. Analysis of the Enzymatic Properties of AtNUDX11, 15,
25, and 26
The reaction products generated by the activity of
AtNUDX26 toward ppGpp were determined by HPLC
analysis (Fig. 1). SpoT and its homolog, RSH, degrade
ppGpp to GDP and pyrophosphate.2,10,11) Typical Nudix
hydrolase cleaves the 50-pyrophosphate linkage of its
substrate. On the other hand, Thermus thermophilus
Ndx8 degrades ppGpp to pGp and two phosphates by
hydrolysis not only of the 50-diphosphate linkage but
also of 30-diphosphate linkage of ppGpp.37) 30-Diphos-
phatase activity is an unique property of Ndx8. In
addition to the peak of ppGpp as substrate, three peaks
with absorbance at 252 nm were detected in the reaction
solution. The peak that eluted first (approximately
9.50 min) corresponded to that of standard pGp, while
the others were estimated to be either pGpp or ppGp.
These findings indicate that like Thermus thermophilus
Ndx8, AtNUDX26 has pyrophosphohydrolase activity
toward both diphosphate linkages in ppGpp, and gen-
erates pGp as final product. At present, the fates of pGp
and pGpp/ppGp are unknown, even in bacterial cells.37)
The standard assay was used with concentrations of 5 to 800 mM
for ppGpp at 37 ꢀC with 5 mM Mg2þ, as described in ‘‘Materials and
Methods.’’ Data are means of three independent determinations ꢂ SD.
Km
Vmax
kcat
kcat/Km
1/s/M
Protein
mM
mmol/min/mg
1/s
AtNUDX11
AtNUDX15 145:3 ꢂ 9:8
29:1 ꢂ 2:5
0:15 ꢂ 0:03
0:05 ꢂ 0:01
0:35 ꢂ 0:07
1:64 ꢂ 0:30
0.07 2:3 ꢁ 103
0.03 2:0 ꢁ 102
0.12 1:9 ꢁ 103
0.49 3:6 ꢁ 103
AtNUDX25 63:7 ꢂ 3:6
AtNUDX26 135:1 ꢂ 9:6
and Mn2þ. No activity was detected in the absence of
these metal ions, or in the presence of the other metal
ions tested.
The optimum pH for activity was determined using
Tris–HCl (pH 6.5–9.0) and glycine–NaOH (pH 8.5–
10.0) buffers. As shown in Fig. 2B, AtNUDX26 was
most active at pH 9.0, with 50% activity at pH 7.0 and
pH 9.5. The optimum temperature for the enzyme was
50 ꢀC (Fig. 2C).
Kinetic parameters for ppGpp were measured
(Table 1). The Km values of AtNUDX11, 15, 25, and
26 were 29:1 ꢂ 2:5, 145:3 ꢂ 9:8, 63:7 ꢂ 3:6, and
135:1 ꢂ 9:6 mM respectively. The Vmax of AtNUDX26
was 1:64 ꢂ 0:30 mmol/min/mg, the highest value ob-
tained. Thus AtNUDX26 had the highest catalytic
efficiency (kcat/Km) for ppGpp among these AtNUDXs.
It has been reported that AtNUDX26, with a chloro-
Enzymatic properties and kinetic parameters of
AtNUDX26 as a ppGpp pyrophosphohydrolase
Most previously characterized Nudix hydrolases
require bivalent metal ions for their activity. Hence we
studied the effects of a series of bivalent metal ions on
the activity of AtNUDX26 (Fig. 2A). The activity of
AtNUDX26 was detected only in the presence of Mg2þ

Enzymatic and Molecular Characterization of AtNUDX26
2239
plastic transit peptide, is distributed in the chloroplasts
of Arabidopsis cells, whereas AtNUDX11 and 25 are in
the cytosol and AtNUDX15 is in the mitocondria.28,29) In
addition, AtNUDX11 and 15 have higher hydrolysis
activity toward CoA and its derivatives than ppGpp, and
thus appear to act on the regulation of various CoA-
related metabolisms.39) On the other hand, AtNUDX25
and 26 have highest activity toward ApnA among
various substrates,40) but the substantial physiological
role of ApnA remains unclear. These findings and the
results obtained here suggest that AtNUDX26 contrib-
utes to the regulation of ppGpp levels via hydrolysis in
the chloroplasts.
conserved across several species (PROSITE: http://
prosite.expasy.org/).27,31,39,50–53) Previous studies have
found that AtNUDX11 and 15 have the UPF0035 motif
conserved in CoA pyrophosphohydrolases.39) AtNUDX25
and 26 have been identified as ApnA pyrophosphohy-
drolases with a unique conserved amino acid sequence
(GGGX5EX7REUXEEXGUX2GX6G).29,31,40) This sug-
gests that the mechanism of the recognition of similar
to that CoA and ApnA.
Next, we compared the amino acid sequences of
AtNUDXs with T. thermophilus Ndx8, known to be a
ppGpp pyrophosphohydrolase.37) Ndx8 did not share
significant sequence homology to any of the AtNUDXs
(3–24%), including AtNUDX11 (19%), 15 (17%), 25
(24%), and 26 (18%), and no region other than the
Nudix motif was conserved among them (data not
shown). These findings suggest that plant Nudix hydro-
lases having ppGpp pyrophosphohydrolase activity
undergo their own evolutionary process. Alternatively,
there might be unidentified amino acid residues essential
for ppGpp pyrophosphohydrolase activity, since several
residues have been reported to be essential for activity or
substrate recognition in certain NUDXs, including
AtNUDX1 and E. coli NudG.31)
Comparison of amino acid sequences between
AtNUDXs and T. thermophilus Ndx8
Nudix hydrolases contain the highly conserved Nudix
box GX5EX7REXEEUXGU, where U is a bulky hydro-
phobic amino acid such as Ile, Leu, or Val.25–27) In
addition, some Nudix hydrolases, with hydrolytic activ-
ity toward ADP-ribose (Proline 15 or 16 aa downstream
of the Nudix box), ApnA (GGGX5EX7REUXEEX-
GUX2GX6G), coenzyme A (UPF0035; LLTXR[SA]X3-
RX3GX3FPGG), NADH (SQPWPFPQS), or UTP
(L[VL]VRK and AANE), contain additional regions
A
Time(h)
Time(h)
Time(h)
Time(h)
Time(h)
Time(h)
B
Time(h)
Time(h)
Fig. 3. Expression Profiles of AtNUDX26, AtRSH1, AtRSH2, AtRSH3, and CRSH under Various Stressful Conditions.
Two-week-old Arabidopsis plants were subjected to various forms of stress: treatment with salinity (250 mM NaCl), PQ (50 mM), drought
(dehydration on a paper towel), and heat (37 ꢀC under dark conditions). Total RNA extracted from Arabidopsis leaves was converted into first-
strand cDNA with the oligo (dt)20 primer. A quantitative PCR analysis was carried out to determine the expression levels of AtNUDX26 (A) and
AtRSH1, AtRSH2, AtRSH3, and CRSH (B). The details of the procedures are described in ‘‘Materials and Methods.’’ Relative amounts were
normalized to Actin2 mRNA. Data are mean values ꢂ SD for three individual experiments (n ¼ 3). Asterisks indicate values significantly
different from the control treatment (p < 0:05).

2240
D. ITO et al.
Expression of AtNUDX26 in response to various types
of stress
It has been reported that ppGpp levels in plant cells
Acknowledgment
This work was supported by a Grant-in-Aid for
Scientific Research (A) (S. S: 22248042) from the
Ministry of Education, Culture, Sports, Science, and
Technology (MEXT) of Japan, and by the Strategic
Project to Support the Formation of Research Bases at
Private Universities: A Matching Fund Subsidy from
MEXT, 2011–2015 (S1101035).
increased in response to various types of environmental
stress.15) We analyzed changes in the expression of
AtNUDX26 and the genes encoding enzymes involved in
ppGpp metabolism, AtRSH1, AtRSH2, AtRSH3, and
CRSH, under various stressful conditions. RT–PCR
analysis revealed that the transcript levels of AtNUDX26
increased approximately 4-fold at 6 h after drought stress
(Fig. 3). A significant increase in expression under
drought stress was observed for all the genes tested
here. In particular, the levels of AtRSH2 and AtRSH3
increased approximately 19- and 24-fold respectively.
Conversely, expression of AtNUDX26 decreased under
oxidative stress caused by treatment with paraquat (PQ,
a generator of reactive oxygen species) and under
salinity caused by treatment with NaCl (Fig. 3). Sim-
ilarly, the levels of CRSH decreased in response to both
PQ and NaCl treatments. In contrast, the levels of
AtRSH2 increased in response to these treatments. The
expression of AtNUDX26 was not significantly changed
by treatment with heat, although the expression levels of
some AtRSHs were affected (Fig. 3). These results
suggest that the metabolism of ppGpp is regulated
depending on the type of stress, and that AtNUDX26
might contribute to regulation.
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In plant cells, ppGpp is an important signaling
molecule, that affects various cellular processes, includ-
ing transcription, translation, DNA replication, amino
acid and nucleotide metabolism, secondary metabolism,
and infectivity.2,11–14,54) For the first time, we charac-
terized the molecular and enzymatic properties of
ppGpp pyrophosphohydrolases in plants, AtNUDX11,
15, 25, and 26. It has been found that in plant
chloroplasts ppGpp is synthesized and degraded by
RSHs and directly affects the translation sys-
tem.15,20–22,54,55) In addition to the importance of ppGpp
metabolism in plant chloroplasts, the finding that among
AtNUDXs having ppGpp pyrophosphohydrolase activ-
ity, chloroplastic AtNUDX26 has the highest catalytic
efficiency and that its expression is regulated in response
to various types of environmental stress suggests that
AtNUDX26 impacts the metabolism of ppGpp in
chloroplasts through the hydrolysis of ppGpp under
stress. Since ppGpp is a substrate for the hydrolysis
reaction by AtNUDX26, it might play a role in the fine
tuning of ppGpp signaling in combination with AtRSHs
in the chloroplasts. In addition, it is possible that not
only ppGpp but also pGp, the final product of the
reaction of AtNUDX26, have signaling roles in cellular
responses, since its analog, 30-phosphoadenosine 50-
phosphate, has an inhibitory effect on certain enzymes,
such as 30-phosphoadenosine 50-phosphosulfate, nucleo-
side diphosphate kinase, and poly (ADP-ribose) poly-
merase, which are involved in various biological
processes.56–61) To determine the importance of the
degradation of ppGpp by AtNUDX26, we are analyzing
the effect of the knockout and overexpression of
AtNUDX26 on levels of ppGpp and the response to
various types of stress.
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