Journal of Experimental Botany, Vol. 51, No. 351, pp. 1763-1764,
October 2000
© 2000 Oxford University Press
Gene Note |
Oxidative stress, heat shock and drought differentially affect expression of a tobacco protein phosphatase 2C1
2 Vakgroep Moleculaire Genetica and Departement Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium
3 GSF-Forschungszentrum für Umwelt und Gesundheit, Institut für Biochemische Pflanzenpathologie, D-85764 Neuherberg, Germany
Received 2 May 2000; Accepted 16 May 2000
Abstract
A protein phosphatase 2C (PP2C)-homologous cDNA was isolated from Nicotiana tabacum (NtPP2C1). The deduced protein sequence of 416 amino acids showed the highest degree of similarity to the PP2C of Arabidopsis thaliana (AtPP2CA) implicated in abscisic acid signalling. The expression of NtPP2C1 was strongly induced by drought, but repressed by oxidative stress and heat shock. It is suggested that NtPP2C1 operates at the junction of drought, heat shock and oxidative stress.
Key words: Drought, heat shock, Nicotiana, oxidative stress, PP2C.
Protein phosphatases 2C (PP2Cs) are a class of evolutionarily conserved serine/threonine protein phosphatases primarily involved in stress responses. Structurally, plant PP2Cs consist of a catalytic core domain and an N-terminal extension that is believed to regulate the catalytic activity of the enzyme (Rodriguez, 1998
). In the past few years, several plant genes encoding PP2Cs have been identified (Rodriguez, 1998; Kapranov et al., 1999; Miyazaki et al., 1999). Sequence analysis of PP2C family members indicates the existence of at least seven distinct classes of plant PP2Cs (Miyazaki et al., 1999). Most of the isolated PP2Cs have been implicated in abscisic acid (ABA) signal transduction. The Arabidopsis thaliana genes ABI1, ABI2, and AtPP2C-HA are transcriptionally upregulated by ABA (Leung et al., 1997; Rodriguez et al., 1998). Moreover, ectopic expression of wild-type and mutated forms of ABI1 and AtPP2CA as well as analysis of ABI1 loss-of-function mutants demonstrated that these phosphatases function as negative regulators of ABA responses (Sheen, 1998; Gosti et al., 1999).
A 3' cDNA fragment (383 bp) with homology to plant PP2Cs was identified in a differential screen (RNAmapTM; GenHunter) for Nicotiana tabacum genes of which the mRNA levels were altered upon oxidative stress (E Vranová, unpublished results). 5' rapid amplification of cDNA ends (RACE; MarathonTM cDNA Amplification Kit; Clontech) using total leaf RNA and a gene-specific 3' primer generated two types of 5' RACE products, of which only one was complementary to the 3' cDNA sequence. The full-length sequence reconstituted from 5' and 3' cDNA fragments was designated NtPP2C1 (accession number aj277086). The second 5' RACE product lacking the 3' part of the coding sequence was named NtPP2C2 (accession number aj277087). Both sequences share 94% identity at the amino acid level (Fig. 1B), and probably represent the PP2C orthologues from the two ancestor tobacco species (N. sylvestris and N. tomentosiformis) that constitute the amphidiploid N. tabacum genome. Therefore, only NtPP2C1 was analysed further. The NtPP2C1 cDNA is 1447 bp long and encodes a protein of 416 amino acids with a predicted molecular mass of 46 kDa. NtPP2C1 is most similar to AtPP2CA of A. thaliana (Kuromori and Yamamoto, 1994) with 64% amino acid identity and 69% amino acid similarity (Fig. 1). The N-terminal extensions of the two proteins are more divergent (32% identity) than their catalytic domains (73% identity). NtPP2C1 possesses two conserved G residues (G155 and G161) flanking the DGH158–160 active site uniquely conserved in Arabidopsis PP2Cs implicated in ABA signalling (Sheen, 1998), suggesting that NtPP2C1 is a functional homologue of the Arabidopsis PP2Cs involved in ABA signalling. Strong and sustained induction of NtPP2C1 during drought stress (Fig. 2) supports this assumption.
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In contrast to its transcriptional response to drought stress, the expression of NtPP2C1 was down-regulated by oxidative stress. Exposure of tobacco leaf discs to methyl viologen (MV) resulted in a sustained decrease of NtPP2C1 transcript levels (Fig. 2
). This result prompted the analysis of the NtPP2C1 expression under various stress conditions (Fig. 2). NtPP2C1 transcript levels decreased in catalase 1-deficient tobacco plants under conditions that promote H2O2 production in these plants (Chamnongpol et al., 1996). However, down-regulation of NtPP2C1 expression was only transient and less pronounced than in MV-treated leaf discs. A similar response was observed when tobacco plants were exposed to UV-B radiation or ozone. This result indicates that down-regulation of the NtPP2C1 expression is a common response to various oxidative stress stimuli. The reason for the more sustained response to MV in leaf discs, when compared to H2O2, ozone, and UV-B responses in plants is unknown. Expression of NtPP2C1 was also analysed during heat shock. Similarly to oxidative stress, exposure of tobacco plants to elevated temperatures was followed by a rapid decrease of the NtPP2C1 transcript level. Down-regulation induced by heat shock was readily reverted during the recovery period at 24 °C.
The expression data show that heat shock and oxidative stress have opposite effects on NtPP2C1 expression as compared to drought. If NtPP2C1 functions as a negative regulator in ABA signalling, like its homologues in Arabidopsis (Sheen, 1998; Gosti et al., 1999), down-regulation of NtPP2C1 expression by oxidative stress or heat shock would likely sensitize the plant to ABA, thus enhancing and/or prolonging ABA downstream responses. Synergistic effects of ABA and heat shock on gene expression have indeed been observed for the RD29A gene in A. thaliana (Xiong et al., 1999). The results presented here suggest that NtPP2C1 (and probably its functional homologues in other species) may constitute a point of convergence between heat shock, oxidative stress, and drought/ABA signalling pathways.
Acknowledgments
The authors thank F Seidlitz (GSF) for co-operation in UV-B experiments, F De Winter and S Morsa for help with RNA gel blot analysis, the sequencing group for technical assistance with DNA sequencing, F Van Breusegem, J Dat, and E Belles-Boix for critical reading of the manuscript, and M De Cock and S Debruyne for help in preparing it. This work was supported by a grant from the European Union (BIOTECH Program ERB-BIO4-CT96-0101).
Notes
1 The sequence of the mRNAs reported here has been deposited in the DDBJ/EMBL/GenBank Databases under accession nos aj277086 and aj277087. 
4 Permanent address: Department of Experimental Botany and Genetics, P.J. 
afárik University, Mánesova 23, 04154 Ko
ice, Slovakia.
5 Present address: CropDesign N.V., Technologiepark 3, B-9052 Zwijnaarde, Belgium.
6 To whom correspondence should be addressed. Fax: +32 9 264 5349. E-mail: diinz{at}gengenp.rug.ac.be
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