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.
References
1) Stephens JC, Artz SW, and Ames BN, Proc. Natl. Acad. Sci.
USA, 72, 4389–4393 (1975).
2) Magnusson LU, Farewell A, and Nystrom T, Trends Microbiol.,
¨
13, 236–242 (2005).
3) Rhaese HJ, Dichtelmuller H, and Grade R, Eur. J. Biochem., 56,
¨
385–392 (1975).
4) Nascimento MM, Lemos JA, Abranches J, Lin VK, and Burne
RA, J. Bacteriol., 190, 28–36 (2008).
´
5) Friga GM, Borbely G, and Farkas GL, Arch. Microbiol., 129,
341–343 (1981).
6) Glass RE, Jones ST, and Ishihama A, Mol. Gen. Genet., 203,
265–268 (1986).
7) Tedin K and Bremer H, J. Biol. Chem., 267, 2337–2344 (1992).
8) Chatterji D, Fujita N, and Ishihama A, Genes Cells, 3, 279–287
(1998).
9) Wagner R, J. Mol. Microbiol. Biotechnol., 4, 331–340 (2002).
10) Sato M, Takahashi K, Ochiai Y, Hosaka T, Ochi K, and Nabeta
K, Chembiochem, 10, 1227–1233 (2009).
11) Cashel M, J. Biol. Chem., 244, 3133–3141 (1969).
12) Potrykus K and Cashel M, Annu. Rev. Microbiol., 62, 35–51
(2008).
Conclusion
13) Srivatsan A and Wang JD, Curr. Opin. Microbiol., 11, 100–105
(2008).
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.
14) Dalebroux ZD, Svensson SL, Gaynor EC, and Swanson MS,
Microbiol. Mol. Biol. Rev., 74, 171–199 (2010).
15) Takahashi K, Kasai K, and Ochi K, Proc. Natl. Acad. Sci. USA,
101, 4320–4324 (2004).
16) van der Biezen EA, Sun J, Coleman MJ, Bibb MJ, and Jones JD,
Proc. Natl. Acad. Sci. USA, 97, 3742–3752 (2000).
17) Tozawa Y, Nozawa A, Kanno T, Narisawa T, Masuda S, Kasai
K, and Nanamiya H, J. Biol. Chem., 282, 35536–35545 (2007).
18) Masuda S, Mizusawa K, Narisawa T, Tozawa Y, Ohta H, and
Takamiya K, Plant Cell Physiol., 49, 135–141 (2008).
19) Masuda S, Tozawa Y, and Ohta H, Plant Signal. Behav., 3,
1021–1023 (2008).
20) Givens RM, Lin MH, Taylor DJ, Mechold U, Berry JO, and
Hernandez VJ, J. Biol. Chem., 279, 7495–7504 (2004).
21) Kasai K, Kanno T, Endo Y, Wakasa K, and Tozawa Y, Nucleic
Acids Res., 32, 5732–5741 (2004).
22) Mizusawa K, Masuda S, and Ohta H, Planta, 228, 553–562
(2008).
23) Kasai K, Usami S, Yamada T, Endo Y, Ochi K, and Tozawa Y,
Nucleic Acids Res., 30, 4985–4992 (2002).
24) Braeken K, Moris M, Daniels R, Vanderleyden J, and Michiels
J, Trends Microbiol., 14, 45–54 (2006).
25) Bessman MJ, Frick DN, and O’handley SF, J. Biol. Chem., 271,
25059–25062 (1996).
26) Xu W, Dunn CA, O’handley SF, Smith DL, and Bessman MJ,
J. Biol. Chem., 281, 22794–22798 (2006).
27) McLennan AG, Cell. Mol. Life Sci., 63, 123–143 (2006).
28) Ogawa T, Ueda Y, Yoshimura K, and Shigeoka S, J. Biol.
Chem., 280, 25277–25283 (2005).
29) Ogawa T, Yoshimura K, Miyake H, Ishikawa K, Ito D, Tanabe
N, and Shigeoka S, Plant Physiol., 148, 1412–1424 (2008).
30) McLennan AG, Pharmacol. Ther., 87, 73–89 (2000).
31) Kraszewska E, Acta Biochim. Pol., 55, 663–671 (2008).
32) Gunawardana D, Likic VA, and Gayler KR, Comp. Funct.
Genomics, 2009, 1–13 (2009).
33) Fisher DI, Cartwright JL, Harashima H, Kamiya H, and
McLennan AG, BMC Biochem., 5, 7 (2004).