Methyl jasmonate activates the biosynthesis of many natural compounds, previously know as secondary metabolites, in plants. In this study, we used tobacco BY-2 cells and large-scale gene expression analysis to further investigate MJ-dependent metabolic changes in this model system. One of the main aims of our work was the characterization of novel MJ signal transduction components. Time-course expression data for various MJ-responsive putative regulators were aligned with the expression data for known metabolic genes, resulting in the identification of a novel MJ-regulated MYB gene in tobacco. This transcriptionally controlled NtMYBJS1 gene was shown to interact with several phenylpropanoid biosynthesis genes in an MJ-dependent fashion, leading to the enhanced accumulation of hydroxycinnamoyl–polyamine conjugates in the cells.
NtMYBJS1 is an MJ-dependent regulator of phenylpropanoid biosynthesis
A number of phenylpropanoid-related MJ-inducible genes were identified using current tobacco microarray (see Matsuoka and Galis, 2006, for further details). In addition, we found good agreement between metabolic data and classification of several of these genes in the constitutively MJ-induced group 1. Because the MJ responsiveness of the basic phenylpropanoid biosynthesis genes has already been documented (4CL: Lee and Douglas, 1996; Suzuki et al., 2005, and this report; PAL: Ellard-Ivey and Douglas, 1996; Sharan et al., 1998; Suzuki et al., 2005; Taguchi et al., 1998; Yang et al., 2001, and this report; CHS: Richard et al., 2000), we concentrated on the identification of potential MJ-signal transduction elements in this study.
Among the possible candidates, MYB proteins are known regulators of diverse metabolic and developmental processes in plants. For example, MYB proteins are involved in control of trichome differentiation (AtMYB0/GLABROUS 1; Oppenheimer et al., 1991), root hair development (AtMYB66/WEREWOLF; Lee and Schiefelbein, 1999), leaf patterning (AtMYB91/AS; Byrne et al., 2000) and regulation of cell cycle by MYB3R factors (Ito et al., 2001). However, the best established role of MYB proteins, and especially of their largest family, the R2R3-MYB proteins, is in the regulation of metabolic pathways in plants.
For example, the AtMYB34/ATR1 gene has been shown to regulate tryptophan biosynthesis (Bender and Fink, 1998) and AtMYB2 induced alcohol dehydrogenase gene expression during the response to low oxygen (Hoeren et al., 1998). In addition, a significant number of MYB transcription factors, including that encoded by the tobacco NtMYBJS1 gene described in this paper, have been implicated in the control of phenolic biosynthesis in plants (for reviews, see e.g. Endt et al., 2002; Jin and Martin, 1999; Weisshaar and Jenkins, 1998).
Among the best characterized examples of MYB-phenylpropanoid pathway regulators are the maize R- or B-cofactor-dependent regulators of anthocyanin biosynthesis C1 (Pazares et al., 1986, 1987) and Pl (Cone et al., 1993), and the P gene, which acts alone in activating gene transcription (Grotewold et al., 1991, 1994). The overexpression of another maize (Zea mays) MYB gene, ZmMYB-IF35, in a way similar to that of NtMYBJS1 in tobacco, induced ectopic accumulation of ferulic and chlorogenic acids and other related compounds in the cultured cells (Dias and Grotewold, 2003). The results presented in this paper suggest that the NtMYBJS1 protein also does not require a cofactor for its activity, as the overexpression of the MYB protein alone induced phenylpropanoid genes and accumulation of phenylpropanoid conjugates in tobacco cells. Other well-characterized flower-specific MYB regulators, AmMYB305, AmMYB340, AmMYB308 and AmMYB330 (Moyano et al., 1996; Sablowski et al., 1994; Tamagnone et al., 1998), were proposed to regulate accumulation of flavonoids in the flowers of Antirrhinummajus L. (snapdragon) plants. The overexpression of AmMYB308 (AmMYB330) in heterologous tobacco host plants resulted in suppressed expression of the C4H, 4CL and CAD genes (Tamagnone et al., 1998), suggesting a strong conservation of MYB DNA-binding motifs among plant species. In tobacco, an anther-specific NtmybAS1 gene has been shown to regulate two different PAL gene promoters when expressed in tobacco leaf protoplasts (Yang et al., 2001) and a lignin-related MYB protein, NtLim, which is mainly expressed in tobacco stem tissues, can control the expression of the PAL, 4CL, and CAD genes (Kawaoka et al., 2000). Another good example of MYB regulators may include the Arabidopsis AtMYB75/PAP1 gene which controls transcription levels of the PAL, chalcone synthase (CHS) and dihydroflavonol reductase (DFR) genes in anthocyanin biosynthesis (Borevitz et al., 2000; Tohge et al., 2005). Finally, the Arabidopsis AtMYB12 protein, with a high degree of functional and structural similarity to the P gene from maize, was recently identified as a flavonol-specific regulator of phenylpropanoid biosynthesis (Mehrtens et al., 2005). In the context of the many examples described in this paragraph, the NtMYBJS1 gene represents a novel member of the large family of MYB genes which specifically regulate phenylpropanoid biosynthesis in plants. Moreover, we have shown that the expression of this novel gene is controlled by MJ in a hormone-dependent manner.
Previously, several MYB genes, in a similar way to the NtMYBJS1 gene, were tentatively shown to be involved in abiotic stress and pathogen response. These include, for example, the UV-B-inducible AtMYB4 (Jin et al., 2000) and soybean (Glycine max L.) GmMYB29A1 (Shimizu et al., 2000) genes, the MJ-inducible rice (Oryza sativa L.) OsJAMYBgene (Lee et al., 2001), the ABA/dehydration-responsive AtMYB2 gene (Urao et al., 1993) and the wounding/elicitor-inducible NtMYBLBM1 gene (NtMYB2; Sugimoto et al., 2000; Takeda et al., 1999). It is noteworthy that the NtMYBLBM1, GmMYB29A1, NtMYBJS1 and Arabidopsis AtMYB13-15 proteins share certain common structural features (Figure 3b), suggesting the functional conservation of these genes in the stress/pathogen response. In support of this proposal, we found that the NtMYBJS1 gene was induced in tobacco leaves in response to wounding or direct exogenous application of MJ (IG and KM, unpublished results).
Binding of the MYB DNA-binding domain of NtMYBJS1 protein to PAL promoters (Figure 6) further strengthens the previous conclusion that NtMYBJS1 is an intrinsic transcriptional regulator of phenylpropanoid genes in tobacco. This protein preferably recognized the ACCAACCCC DNA motif in the PAL promoter sequence, which is consistent with the previously reported maize P-gene core binding sequence CC(T/A)ACC (Grotewold et al., 1994). Recently, a binding capacity of the elicitor and UV-B-inducible carrot (Daucus carota L.) DcMYB1 recombinant protein to the same core sequence tctcACCAACCCttg (box-L5) was reported (Maeda et al., 2005). In Arabidopsis, three corresponding MYB genes (AtMYB13, AtMYB14, and AtMYB15) encode proteins that have conserved motifs in common with NtMYBJS1 (Figure 3). Based on a clustalw-derived phylogenetic tree, these genes also have the most similar DNA-binding domain to the NtMYBJS1 gene (data not shown), suggesting that they could be involved in the control of similar genes in Arabidopsis. To find potential targets of these MYB genes in Arabidopsis, we carried out database searches for genes with the ACCAACCCC sequence in the promoter region, using the pattern-matching search site in The Arabidopsis Information Resource (http://www.arabidopsis.org/cgi-bin/patmatch/nph-patmatch.pl). Consequently, we found a possible linkage of this motif and phenylpropanoid genes. Among 27 184 genes, 65 contained the ACCAACCCC sequence motif within the 500-bp upstream sequence. Among these 65 genes, 12 (about 18%) encoded proteins for shikimate/phenylpropanoid or S-adenosylmethionine (SAM)/polyamine metabolism; these included two 4CL genes (At1g51680 and At3g21240), a caffeoyl-CoA 3-O-methyltransferase gene (At4g34050), a cinnamoyl-CoA reductase gene (At1g15950), two cinnamoyl-alcohol dehydrogenase genes (At4g34230 and At1g09510), two shikimate kinase genes (At4g39540 and At2g21940) and an S-adenosylmethionine synthetase gene (At2g36880). Furthermore, the wound-responsive Arabidopsis PAL1 (At2g37040) and PAL2 (At3g53260) genes contain a very similar motif (ACCAACCGC) in their promoter regions.
Taken together, these observations indicate that the NtMYBJS1 gene and its homologs are direct signaling components between MJ (hormone signal) and the accumulation of phenylpropanoid compounds in plants during stress.
The role of hydroxycinnamoyl–polyamine conjugates in plant defense
Our analysis of metabolites in MJ-treated BY-2 cells indicates that two major classes of compounds, nicotine alkaloids (Goossens et al., 2003; Hakkinen et al., 2004) and hydroxycinnamoyl–polyamine conjugates, accumulate in the cells. The latter were further identified as putrescine conjugates of caffeic acid and p-coumaric acid. One of these compounds, CP, is also the main differentially accumulated metabolite in MJ-treated Nicotiana attenuata leaves (Keinanen et al., 2001). The importance of polyamines and their conjugates in plant disease was recently reviewed by Walters (2003). In Nicotiana tabacum, hydroxycinnamoyl–polyamine conjugates accumulate in the reproductive organs, and the formation of putrescine conjugates is stimulated by viral or fungal infections (Wink, 1997). For example, hydroxycinnamic acid amides accumulate around or in necrotic lesions during the hypersensitive response to tobacco mosaic virus (TMV) infection (Martintanguy et al., 1976; Rabiti et al., 1998; Torrigiani et al., 1997). Torrigiani et al. (1997) suggested that high levels of polyamine conjugates may be required to limit virus movement and prevent systemic infection. Additionally, Martintanguy et al. (1976) showed that tobacco leaf discs treated with coumaroylputrescine and CP undergo a 90% reduction in local lesion formation following TMV inoculation.
Further to these observations, TMV infection causes a transient production of jasmonic acid (Seo et al., 2001) and induces patatin-like lipase (Dhondt et al., 2000), which is likely to be the enzyme involved in jasmonic acid biosynthesis. Thus, our result that MJ induces the production of hydroxycinnamoyl–polyamine conjugates through the action of NtMYBJS1 could explain the link between TMV infection and the accumulation of these polyamine conjugates. Future analysis of the effect of NtMYBJS1 on pathogen infection will clarify this possible linkage.
MJ-induced genes for hydroxycinnamoyl–polyamine conjugate formation
The accumulation of putrescine conjugates in BY-2 cells (Figure 1) includes two major biosynthetic pathways in tobacco: the PAL-driven phenylpropanoid pathway and the agrinine decarboxylase/ODC-controlled biosynthesis of polyamines. Interestingly, the two rate-limiting ODC genes on the BY-2 microarray showed differential induction during MJ treatments. One ODC gene, D89984, which is identical to the NtODC-2 gene (Xu et al., 2004), was classified in group 1 together with several key phenylpropanoid genes. This observation suggests that the NtODC-2 gene might be one of the main sources of putrescine for the synthesis of phenylpropanoid conjugates. The expression pattern of the NtODC-2 gene in NtMYBJS1ox cell lines lends further support to this hypothesis. This gene was induced in a 5-day culture of cell lines overexpressing NtMYBJS1 (Table S6), which overproduce phenylpropanoid substrates in the cells. In contrast, the alternative ODC gene on the array (BP526041) encodes a novel tobacco ODC protein, dissimilar to the proteins summarized by Xu et al. (2004), yet similar to the putative ODC from Solanum demissum. This ODC gene was classified into group 3 of the MJ-induced genes which typically show very low basal levels of gene expression in untreated cells and a strong increase in amplitude after MJ application. Significantly, the nicotine biosynthesis-controlling gene putrescine N-methyltransferase (PMT; BP532193; Xu and Timko, 2004) was co-classified with the BP526041 gene. Based on this co-expression data, we propose that the main function of this ODC enzyme may be the synthesis of putrescine required for alkaloid biosynthesis (Shoji et al., 2000). Additional experiments showing the proposed metabolic compartmentalization of ODC enzymes in plant cells are required to investigate this hypothesis.
In the previous paragraph, the role of two ODC genes in polyamine and alkaloid biosynthesis was discussed. However, the contribution of arginine decarboxylase (ADC), together with agmatine iminohydroalse and N-carbamoylputrescine amidohydrolase, in the formation of the putrescine pool in plants must also be considered. While ADC was not among the genes investigated on the current tobacco microarray, previous data suggest that this gene is also induced by MJ in BY-2 cells (Goossens et al., 2003). In addition, the EST BP528797, which is a close structural homolog of the Arabidopsis NLP1 gene with N-carbamoylputrescine amidohydrolase activity (Piotrowski et al., 2003), was upregulated in group 6 on the current microarray.
The enzymatic activity of putrescine hydroxycinnamoyl-CoA transferase required for CP and FP formation in tobacco has been previously characterized (Meurer-Grimes et al., 1989; Negrel et al., 1989, 1991, 1992). Additionally, the activity of this enzyme significantly increases in barley (Hordeum vulgare L.) during the hypersensitive reaction to powdery mildew fungus (Blumeria graminis f. sp hordei) (Cowley and Walters, 2002). Despite well-characterized enzymatic activity, the corresponding N-acetyltransferase gene, which encodes plant putrescine hydroxycinnamoyl-CoA transferase, has not been identified. Interestingly, reasonably good candidates for this gene could be found among our MJ-upregulated transcripts. For example, the BP135698 transcript, encoding a putative amino acid acetyltransferase gene, was co-regulated with the PAL, 4CL, and ODC (D89984) genes in group 1 during MJ elicitation. Additionally, the GNAT-like acetyltransferase genes, encoded by transcripts BP129058 and BP533687, not only showed induction in the presence of MJ but were also approximately twofold upregulated in cell lines overexpressing NtMYBJS1 (data not shown) with enhanced production of CP and FP (Figure 5c). Further characterization of these gene products will be required to identify the N-acetyltransferase gene.