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Plant Physiology and Biochemistry 41 (2003) 207–213 www.elsevier.com/locate/plaphy

Original article

Molecular cloning and characterization of a novel starvation inducible MAP kinase gene in rice Shih-Feng Fu a, Wuan-Pin Lin a, Shin-Lon Ho b,1, Wan-Chi Chou a, Dinq-Ding Huang a, Su-May Yu b, Hao-Jen Huang a,* a

Department of Biology, National Cheng Kung University, No. 1 University Rd., Tainan 701, Taiwan b Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan Received 2 July 2002; accepted 18 October 2002

Abstract Signaling pathways, specifically mitogen-activated protein kinase (MAPK) cascades, have been implicated in the regulation of stress and developmental signals in plants. Here, we report the characterization of a rice (Oryza sativa L. cv. TN-67) MAPK gene, OsMAPK3, isolated by screening a sugar-starved treated rice cell cDNA library. The OsMAPK3 gene was abundantly expressed in mature leaves and panicles of 3-month-old plants, but poorly in young leaves. Its developmental regulation was suggested by the fact that OsMAPK3 mRNA levels changed during leaf differentiation and root development. Furthermore, the amount of this transcript increased markedly in sugar-starved or mannose-fed cells. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Environmental stresses; Mitogen-activated protein kinase; Oryza sativa; Phosphorylation; Sugar starvation

1. Introduction Plants have developed systems that quickly and accurately transmit stimuli from the external environment and adjust metabolic pathways by modulating the expression of genes. Activation and/or inactivation of appropriate genes in response to particular stimuli is mediated through well-tuned signal transduction systems in which protein phosphorylation cascades play pivotal roles. One of the major pathways by which environmental stimuli are transduced intracellularly is the mitogen-activated protein kinases (MAPKs) cascade. It plays a critical role in the regulation of physiological and biochemical responses associated with extracellular stimuli. MAPKs are a family of conserved serine/threonine protein kinases that have been adapted throughout evolution

Abbreviations: Man-6-P, mannose-6-phosphate; MAPK, mitogenactivated protein kinase. * Corresponding author. E-mail address: haojen@mail.ncku.edu.tw (H.J. Huang). 1 Present address: Department of Agronomy, National Chia-Yi University, Chia-Yi 600, Taiwan.

© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. DOI: 1 0 . 1 0 1 6 / S 0 9 8 1 - 9 4 2 8 ( 0 3 ) 0 0 0 1 2 - 3

to form parts of diverse signal-transduction pathways in all eukaryotes [32]. MAPKs are activated by a highly conserved mechanism of sequential phosphorylation events in which a MAP kinase kinase kinase (MAPKKK) activates a MAP kinase kinase (MAPKK), which in turn activates a MAP kinase by phosphorylation of both tyrosine and threonine residues in the TXY motif [7]. Considering the conserved MAPK cascade present in many organisms, it is likely that the activation of the plant MAPK is achieved posttranslationally by MAPK kinases [21]. Interestingly, increased transcript levels of genes encoding a MAPK in plants have been observed in response to environmental stresses [19]. For instance, drought and cold induce mRNA accumulation of MsSAMK in alfalfa [11] and AtMPK3 in Arabidopsis [19]. Similarly, the NtWIPK transcript encoding MAPK in tobacco accumulates after wounding [28]. The increase in expression of these MAPK genes may contribute to the stress response by increasing the amount of proteins available for activation [20]. Therefore, it can be concluded that transcriptional regulation is an important control mechanism in MAPK signaling cascades in plants.

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Fig. 1. Alignment of the predicted OsMAPK3 protein with MAPKs from rice, Arabidopsis, and Petunia (A) and the relationship tree of OsMAPK3 to the cloned plant MAPKs (B). Roman numerals indicate the 12 major conserved subdomains of protein kinases identified previously [5]. Tyrosine and threonine residues, which must be phosphorylated for MAP kinase activation, are indicated by diamonds. The black boxes indicate identical residues, and gray boxes indicate conservative substitutions. The protein sequences shown in these diagrams are listed in the GenBank database under the following accession numbers: NtNTF4 (X83880), CaMK2 (AF247136), NtSIPK (U94192), EeMPK (AF242308), MsSIMK (X66469), PsD5 (X70703), AtMPK6 (D21842), ZmMPK5 (AB016802), MsSAMK (X82270), PsMAPK3 (AF153061), AtMPK3 (D21839), NtWIPK (D61377), CaMK1 (AF247135), IbMPK (AF149424), PcMAPKI (Y12785), OsMAPK2 (AJ250311), ZmMPK4 (AB016801), AsMAP1 (X79993), TaWCK-1 (AF079318), AtMPK10 (CAB75798), NtNRK1 (AB055515), NtNTF6 (X83879), MsMMK3 (AJ224336), AtMPK13 (AAF75067), AtMPK5 (D21841), AtMPK12 (AAC62906), MsMMK2 (X82268), AtMPK4 (D21840), AtMPK11 (AAF81314), AtMPK7 (D21843), AtMPK14 (CAB16812), OsMAPK3 (AF241166), OsMAPK4 (AJ251330), AtMPK1 (D14713), AtMPK2 (D14714), PaMAPK (AF134730), PsMAPK2 (AF154329), NtNTF3 (X69971), PhMEK1 (X83440), OsMEK1 (AF216314). Similarity searches of nucleotide sequence were performed at the National Center Biotechnology Information using the BLAST network service [1]. The alignments were generated by the BOXSHADE web site (http://www.ch.embnet.org/software/BOX_form.html). Relationship tree was performed using MEGA 1.02 software [15].


More than 50 cDNAs encoding putative MAPKs have been cloned from various plants [3,9,10,14,18,35]. A large number of MAPKs have been described in plants and can be divided into at least four subgroups [10]. The available data suggest that each subgroup is responsive for particular types of stimuli or is involved in particular cellular processes [2,10,31,40]. However, knowledge about the functions of subgroup C is just emerging [3,27,36]. In this report, we describe the cloning and characterization of a plant MAPK from rice, OsMAPK3. OsMAPK3 protein was most closely related to rice OsMAPK4 and Arabidopsis AtMPK1, and all belonged to subgroup C. This study proposes the developmental regulation of the OsMAPK3 gene and abiotic stimuli, mannose exposure and sugar-starvation, leading to MAPK activation at the transcriptional level. The possible roles of rice subgroup C MAPKs in signal transduction under environmental stresses are also compared and discussed.

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2. Results 2.1. Isolation of a new MAP kinase, OsMAPK3, from rice Screening of the rice cDNA library constructed from sugar-starved cells resulted in the isolation of a full-length cDNA of 1544 bp, called OsMAPK3. Sequence analysis showed clear similarity to MAPKs, but also revealed distinction from the other rice MAPKs isolated so far [8]. The nucleotide sequence of the OsMAPK3 cDNA contained an open reading frame that would encode a protein of 370 amino acids, with an ATG codon at the 158th nucleotide, and the poly(A)+ tail starting at the 1527th nucleotide downstream from the stop codon. The molecular mass of the predicted polypeptide is 42.4 kDa. An alignment of the predicted amino acid sequences of OsMAPK3 with the cloned MAPKs from various plants is shown in Fig. 1A. The OsMAPK3 protein contained all 12 conserved amino acids and peptide motifs characteristic of 12 subdomains of protein kinases with serine/threonine specificity. The TEY motif, which includes the threonine and tyrosine residues whose phosphorylation is necessary for MAP kinase activation and is a characteristic feature of MAP kinase, was also conserved in the OsMAPK3 protein sequence (Fig. 1A). It has been proposed from an analysis of sequence homology of the predicted amino acid sequences that plant MAPKs can be grouped into at least four distinct groups [10]. Based on the relationship tree of cloned plant MAPKs, OsMAPK3 can be grouped into subgroup C (Fig. 1B). Comparison of the predicted protein sequences of the OsMAPK3 with MAP kinases of other plants shows that OsMAPK3 is most homologous to the Oryza sativa OsMAPK4 (GenBank accession number AJ251330) and Arabidopsis AtMPK1, which also encode subgroup C MAP kinases [18]. 2.2. Genomic analysis of OsMAPK3 gene The genomic organization of OsMAPK3 genes was analyzed by Southern blot hybridization. Knowing the sequence conservation and size similarity of the mRNA encoded by OsMAPK family members, it was important to choose small probes to avoid cross-hybridization between family members. Genomic DNA was isolated from rice suspensioncultured cells, digested with BamHI, BglII, KpnI, XhoI, ClaI, EcoRI and HindIII, and hybridized with the PCR fragments containing mostly 3’-untranslated regions of OsMAPK3. Under high-stringency conditions, the probe of OsMAPK3 hybridized to one main restriction band (Fig. 2). The results indicated that OsMAPK3 was a single-copy gene and that the probe was gene-specific. 2.3. Expression of OsMAPK3 in different tissues of rice plant

Fig. 1 (continued)

The spatial expression of OsMAPK3 gene in rice plant was analyzed by Northern hybridization. OsMAPK3 was

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To determine whether OsMAPK3 plays a role in root development, Northern blots were used to follow the accumulation of its transcript during root development. Different stages of root development were selected as follows: roots of 10-d-old (10 d), 20-d-old (20 d), 30-d-old (30 d) and 40-d-old (40 d) rice. Only a trace of OsMAPK3 transcript was detected in 10 d roots. The transcript level gradually rose at 20 d, and peaked at 30 d, then decreased sharply at 40 d (Fig. 3B). 2.4. Kinetics of mRNA expression in relation to OsMAPK3 and OsMAPK4 in sugar-starved and mannose-fed cells

Fig. 2. Southern blot analysis of genomic sequences that correspond to OsMAPK3. Rice genomic DNA (10 µg) was digested by BamHI (B1), BglII (B2), KpnI (K), XhoI (X), ClaI (C), EcoRI (E) and HindIII (H), respectively. Hybridization was performed with the gene-specific cDNA probes amplified by PCR using primers corresponding to the 3’ end fragments of OsMAPK3.

Fig. 3. Developmental regulation of rice OsMAPK3 transcripts in various organs (A), roots (B) and leaf segments (C). Each lane was loaded with 10 µg of total RNA extracted from young leaves (YL) of 10-d-old seedlings, mature leaves (ML) of 3-month-old plants, roots (R) of 10-d-old seedlings, young panicles (YP) at the stage before heading, and mature panicles (MP) at the heading stage. The RNA was isolated from roots at four different stages of 10-d-old (10 d), 20-d-old (20 d), 30-d-old (30 d) and 40-d-old (40 d) rice. The apical (A), middle (M) and basal (B) segments of leaves were harvested from 10-d-old seedlings. The transcripts are shown by arrows on the right. The rRNA level served as the internal loading control.

expressed predominantly in basal segments of the leaf, but not in middle and apical segments (Fig. 3C). This gene was also distributed abundantly in mature leaves of 3-month-old plants and panicles, but poorly in young leaves and roots of 10-d-old seedlings (Fig. 3A).

As shown in Fig. 4B, the level of OsMAPK3 transcript increased within 3 h in response to starvation. The preferential induction by starvation was consistent with the cloning of OsMAPK3 gene by screening the cDNA library constructed from sugar-deprived cells. Furthermore, the level of both OsMAPK3 and OsMAPK4 transcript increased in a similar manner in response to starvation (Fig. 4B). In a continuing effort to access the stress-responsive signal transduction pathways in rice, the OsMAPK3 gene was analyzed in suspension cells subjected to mannose treatment, resulting in toxicity. It should be noted that the same membranes used to monitor the OsMAPK4 expression were stripped and rehybridized with the OsMAPK3 probe to have an expression profile which could be, as far as possible, used for a comparative analysis. Interestingly, different expression patterns between OsMAPK3 and OsMAPK4 genes were observed in response to the mannose treatment (Fig. 4A). The steady state level of OsMAPK3 mRNA in suspensioncultured cells was first induced within 3 h. This increase was transient, since transcript levels began to decrease after 12 h. In addition, an increase of OsMAPK4 transcript level was already detectable at 3 h after mannose treatment, reaching maximal levels after 12 h. OsMAPK4 mRNA levels were also elevated by mannose, but the kinetics of induction were slower than that of OsMAPK3 (Fig. 4B). These results suggested that the expression of OsMAPK3 and OsMAPK4 genes were differentially regulated in response to mannose treatment. 3. Discussion Given the large number of plant MAPK genes, it is pertinent to address the question of their tissue-specific expression and the implications with respect to MAPK in plant development and physiology. In tobacco, the NtNTF3 gene is apparently expressed constitutively during pollen development [36], while NtNTF4 expression is confined to pollen and seeds [34,37]. A Petunia × hybrida PhMEK1 gene was shown to accumulate preferentially in female reproductive organs [3]. It has also been found that gibberellins and cytokinins regulate PsMAPK3 mRNA levels in pea ovary, shortly after fruit set [16]. Similarly, organ-specific transcription patterns were observed for MsSIMK in alfalfa [13,22] and for CaMK2 in pepper [29]. In our study, OsMAPK3 mRNA was


Fig. 4. The effect of mannose treatment (A) and sucrose starvation (B) on the gene expression of OsMAPK3 and OsMAPK4 in rice suspension cells. RNA samples were prepared from mannose or starvation-treated rice suspension cells, which were sampled at indicated times. The rRNA transcript level served as an internal loading control. Hybridization signals were quantitated with an AlphaImager™ (Alpha Innotech Corporation, CA, USA). The values were normalized with respect to ribosomal RNA content. The signal at 0 h was defined as 1.0 (arbitrary units), and other abundances are expressed relative to that value. (—"—), OsMAPK3 (mannose treatment); (-●-), OsMAPK4 (mannose treatment); (--"--), OsMAPK3 (sugar starvation); (- -●- -), OsMAPK4 (sugar starvation).

expressed strongly in mature leaves but only very weakly in young leaves. The observation suggests that the gene is predominantly expressed in leaves at a late developmental stage. The monocot leaf displays spatial separation between cell proliferation and differentiation. Leaf growth takes place by continued cell divisions at a basal meristem. Thus, a developing monocot leaf possesses the most immature cells at its base, and the mature and differentiated cells at its tip [4]. The patterns of the transcription of OsMAPK3 in the rice leaf confirmed the notion that this gene is regulated by leaf differentiation programming. Our study also showed that the accumulation of the OsMAPK3 transcript is regulated by root development. Therefore, it is concluded that the OsMAPK3

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system described here may perform unique signaling roles or developmental programs in leaf and root. In many plants, mannose is actively transported into cells and can lead to intracellular disturbances. The metabolic perturbations are initiated by the phosphorylation of the sugar analog by hexokinase to mannose-6-phosphate (Man6-P). However, Man-6-P is not further metabolized, due to the absence in plants of mannose-6-phosphate isomerase (D-mannose-6-phosphate ketol-isomerase; EC 5.3.1.8). Its continued accumulation may deplete intracellular Pi and reduce synthesis of ATP [6]. Thus, mannose has been described to be toxic [6]. At the plant as well as cell level, mannose has been shown to inhibit growth of shoots, roots, stems, and pollen tubes [6] or to cause an imbalance in glucose and ion uptake [6]. D-mannose has also been reported to induce wilting of spinach beet [6] and inhibit germination of Arabidopsis seeds [24]. Stein and Hansen [30] developed a system for studying apoptosis of Arabidopsis roots and maize cell culture. The system is based on the toxicity of D-mannose. We used the toxicity of mannose as a stress-related stimulus to investigate the MAPK signaling pathway in rice cells. Interestingly, the OsMAPK3 and OsMAPK4 genes are differently regulated. When cells are starved of sugar, no significantly different expression pattern is observed between the two genes. Nevertheless, the kinetics of mRNA induction of OsMAPK3 in response to mannose is faster than that of OsMAPK4. The differences in kinetics of mRNA induction may reflect distinct functions of OsMAPK3 and OsMAPK4. They may involve different regulatory elements such as transcriptional factors and signal transduction systems. Distinct subgroups of MAPKs have evolved in plants for transmitting different types of signals, e.g. stress factors in members of subgroup A and mitogenic stimuli in subgroup B [12]. Very little is known about the functions of members of subgroup C, which includes NtNTF3 of tobacco [36], AtMPK-1, -2, -7 of Arabidopsis [18] and PhMEK1 of Petunia [3]. The NtNTF3 of tobacco is reportedly expressed during anther development [36]. In Arabidopsis, the mRNA level of AtMPK1 has been observed to increase slightly under high-salinity stress [19]. The circadian clock controls abundance of AtMPK7 [27]. Mizoguchi et al. [17] suggested that auxin might be an activator of AtMPK2. The PhMEK1 gene expression is clearly induced by auxin treatment and may be involved in regulating the cell cycle [33]. In this study, we showed that mannose treatment and sugar starvation induce transcriptional regulation of OsMAPK3 gene in rice. Relationship tree reveals that two closely related isozymes of MAPKs within the same subgroup can exist in a single plant species [12]. In tobacco, NtNTF4 and NtSIPK have been identified as belonging to subgroup A of plant MAPKs [35,39], while NtNTF6 and NtNRK1 are homologues of subgroup B [23,35]. In Arabidopsis, AtMPK1 and AtMPK2 fall into subgroup C; both showing similar spatial expression patterns [17]. Family members tend to share highly similar sequences and spatial expression patterns. Whether the MAPKs cited above are functional homologues or possess

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different functions remains to be clarified. In this investigation, OsMAPK3 showed similarities with OsMAPK4, when compared for sequence conservation. The levels of two subgroup C MAPK, OsMAPK3 and OsMAPK4, transcripts were differentially affected by mannose treatment. To our knowledge, a comparative study of the stress-induced expression of two different MAPK genes in the same subgroup has not been reported previously. Future studies of OsMAPK3 and OsMAPK4 genes in rice will have to address OsMAPK protein levels under environmental stresses.

4. Methods 4.1. Plant materials and stress treatments Rice plants (O. sativa L. cv. TN-67) were grown as described by Sauter [26], with some modifications. The plants were grown in a growth chamber under a 12-h photoperiod and at 28 °C and 70% relative humidity. Plants were watered daily with tap water. Suspension-cultured cells of rice (O. sativa L. cv. TN-67) were prepared as described by Yu et al. [38]. The suspension cells were cultured on Murashige and Skoog (MS) medium (Sigma, St. Louis, MO, USA) supplemented with 0.99 µM 2,4-D, 30 g l–1 sucrose. The cells were kept on a rotary shaker at 125 rpm in darkness at 26 °C and subcultured weekly. Three-day-old suspension-cultured cells were exposed to different stresses before total RNA extraction. To cause sucrose starvation, suspension cells were cultured in the absence of sucrose. For mannose treatment, 80 mM D-mannose (Sigma) was added to fresh medium, devoid of sucrose. 4.2. Cloning of MAPK genes from rice A cDNA library was constructed in kgt22A vector with poly(A)+ RNA from rice suspension cells that had been exposed to 4 h of sugar starvation. One of two reverse degenerate primers corresponding to conserved sequences, either DIWTAGVI or DKDGSGFIT, was used for amplification by PCR of a rice genomic DNA template. The forward degenerate sequence used in both cases was HRDLKPEN. The amplified fragment was 32P-labeled and used to probe the cDNA library, generated from sugar-starved cells. Positive clones were obtained and their DNA sequences were determined. One clone was identified as a novel member of plant subgroup C and given the identity OsMAPK3 (previously denoted as O. sativa MAP kinase 2; accession number AF241166).

transferred onto a Hybond-XL nylon membrane (Amersham, Buckinghamshire, UK) by capillary transfer, using 20 × SSC (1 × SSC = 0.15 M NaCl and 15 mM sodium citrate, pH 7.0). The blots were prehybridized for 2 h at 65 °C in 5 × SSPE (1 × SSPE = 0.18 M NaCl, 10 mM NaH2PO4 and 1 mM EDTA, pH 7.0), 5 × Denhard’s solution, and 0.5% (w/v) SDS, then hybridized overnight at 65 °C using a PCR probe labeled with [a-32P]dCTP (Amersham) by Rediprime Labeling System (Amersham). As the OsMAPK3-specific probe (GenBank accession number AF241166), a 250-bp fragment was amplified by PCR using these primers: forward, 5’-TGC CCG ATG ATC TTC AAC TG-3’ and reverse, 5’-GCA CTT CTT AAT GGT CCA TA-3’. As the OsMAPK4-specific probe (GenBank accession number AJ251330), a 286-bp fragment was amplified by PCR using these primers: forward, 5’-GAG TGA ATA TGT GAC AGG CA-3’ and reverse, 5’-AGC ATC TAA CAT TAC AAG CC-3’. The blots were washed twice in 2 × SSPE, 0.1% SDS at room temperature for 15 min and once in 0.1 × SSPE, 0.1% SDS at 65 °C for 10 min. The membranes were exposed to BioMax MR Film (Kodak, New York, USA).

4.4. Northern blot Northern blots were also performed according to Sambrook et al. [25]. Total RNA was extracted from leaves, roots, panicles, seedlings and suspension-cultured cells by using the RNeasy kit (QIAGEN, Hilden, Germany). The denatured RNA was loaded onto gels containing 1% agarose and 10 mM sodium phosphate buffer and subjected to electrophoresis with 10 mM sodium phosphate buffer as running buffer. The RNA was transferred by the TransVac vacuum blotting unit (Amersham) to nylon membrane (MSI, MA, USA), using 25 mM sodium phosphate buffer. Prehybridization was conducted in a solution containing 51.5% (v/v) formamide, 0.75 M NaCl, 1 × TPSE (0.1 M Tris pH 7.5, 2% Na4P2O7, 2.5% SDS and 0.05 M EDTA) and 10% dextran sulfate at 42 °C for 2 h in a HB-1D Hybridiser (TECHNE, Cambridge, UK). The specific [a-32P]dCTP-labeled DNA probe, prepared according to the procedure above, was added to the hybridization mixture, then hybridized at 42 °C for 24 h. Filters were washed twice with 2 × SSC and 0.1% SDS at room temperature for 15 min, and once with 0.1 × SSC and 0.1% SDS at 42 °C for 15 min. Exposures were made at – 70 °C on BioMax MR (Kodak) X-ray Film.

4.3. Southern blot

Acknowledgements

Southern blots were performed by the method of Sambrook et al. [25]. Genomic DNA (10 µg) of rice suspensioncultured cells was digested with BamHI, BglII, KpnI, XhoI, ClaI, EcoRI and HindIII (Promega, Madison, WI, USA) and

We are deeply grateful to Professor Toshio Murashige for critical reading of the manuscript. This work was supported by the grant from National Science Council (NSC 89-2311B-006-005) of the Republic of China.


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