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- Materials and Methods
- Supporting Information
Plants adapt to environmental fluctuations by adjusting their physiology and morphology. Numerous genes are regulated during plant responses to biotic and abiotic stress conditions. With an aim of improving stress adaptability and productivity of crop plants, intensive works have been carried out to identity genes and molecular mechanisms underlying plant adaptation under various stress conditions (Ingram & Bartels, 1996; Schenk et al., 2000; Seki et al., 2002; Tao et al., 2003).
The stress genes function in a coordinate manner through a complex signaling network as well as through individual signaling pathways. While initial stimuli are obviously diverse, these signals are integrated into a unified scheme in many cases, resulting in common plant responses to different stress signals (Albrecht et al., 2003; Denekamp & Smeekens, 2003; Park et al., 2007). An example is a group of pathogenesis-related (PR) genes. They are well-known marker genes for plant pathogenesis that play primary roles in disease resistance response (Bol et al., 1990). Notably, it has been recently reported that the PR genes are also induced by abiotic stresses, such as cold, high salinity, and drought (Seo et al., 2008). Abiotic stresses are also known to confer disease resistance in Arabidopsis (Gaudet et al., 2003; Griffith & Yaish, 2004). An activation tagging allele of the Activated Disease Resistance 1 (ADR1) gene encoding the coiled-coil (CC) nucleotide-binding site (NBS) leucine-rich repeat (LRR) protein exhibits drought resistance as well as salicylic acid (SA)-mediated resistance to virulent pathogens (Grant et al., 2003; Chini et al., 2004), supporting a wide range of signaling crosstalks between biotic and abiotic stress signals.
Salicylic acid is an important growth hormone functioning in plant–microbe interactions. Impaired SA biosynthetic mutants, such as salicyclic acid induction deficient2 (sid2), and NahG transgenic plants overexpressing a salicylate hydroxylase that prevents accumulation of SA exhibit an increased susceptibility to pathogen infection by compromising the establishment of systemic acquired resistance (Gaffney et al., 1993; Wildermuth et al., 2001). SA also plays regulatory roles in plant response to various abiotic stresses. SA-deficient NahG transgenic plants are resistant to oxidative damage generated by osmotic stress (Borsani et al., 2001), although it is currently unclear whether the resistance is attributable to the reduced SA content or to the accumulated catechol in the transgenic plants (Borsani et al., 2001). In addition, the inhibitory effect of high salt and osmotic stress on seed germination is compromised by gibberellic acid (GA)-mediated induction of SA biosynthesis (Alonso-Ramírez et al., 2009).
Salicylic acid is mainly synthesized through the isochorismate pathway (Wildermuth et al., 2001; Garcion et al., 2008). Although the biochemical activity is not fully characterized, the SID2 gene plays a central role in the SA biosynthetic pathway (Wildermuth et al., 2001). SA is also synthesized from phenylalanine by phenylalanine ammonia lyase (PAL) activity (Lee et al., 1995), although its contribution to endogenous SA content is relatively lower than that of the isochorismate pathway.
While SA biosynthetic pathway genes have been relatively well characterized, regulatory mechanisms governing SA biosynthesis are poorly understood: only a few transcription factor genes have been reported to regulate SA biosynthesis. The Arabidopsis MYB30 transcription factor is related to the hypersensitive cell death program (Raffaele et al., 2006). The R2R3-type MYB transcription factor regulates hypersensitive response by modulating SA accumulation. Consequently, the MYB30-mediated cell death is abolished in SA biosynthetic mutants but is unaffected in SA signaling mutants, such as npr1 (Raffaele et al., 2006).
Anthocyanin accumulation is a prominent developmental appearance that is caused by diverse environmental stresses, such as ultraviolet light, nutrient deficiency, and abiotic stress conditions (Winkel-Shirley, 2001, 2002). It is also closely related to plant pathogenesis and frequently used as an easily visible marker for plant disease resistance response (Dixon, 2001). A protein complex composed of an MYB, a basic helix-loop-helix (bHLH), and WD40 proteins, thus designated an MBW complex, play a key role in anthocyanin biosynthesis (Broun, 2005; Koes et al., 2005). In Arabidopsis, it has been observed that the TRANSPARENT TESTA GLABRA1 (TTG1), which plays a central role in constituting the complex, interacts with GLABRA3 (GL3), ENHANCER OF GLABRA3 (EGL3), and TRANSPARENT TESTA8 (TT8) (Walker et al., 1999; Zhang et al., 2003). However, it is currently unclear whether the GL3 and EGL3 proteins are components of the MBW complex. In addition, two redundant MYB transcription factors, PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) and PAP2, also participate in the flavonoid biosynthetic pathway (Borevitz et al., 2000; Teng et al., 2005). Recently, some of these genes have been reported to be environmentally regulated and provide resistance to environmental stresses (Rowan et al., 2009).
The MYB transcription factors, one of the largest transcription factor families in plants, regulate diverse developmental processes and plant responses to environmental stimuli (Stracke et al., 2001), such as cell fate determination (Lee & Schiefelbein, 1999) and biotic and abiotic stresses (Mengiste et al., 2003; Jung et al., 2008). The MYB96 transcription factor, a R2R3-type MYB member, has recently been shown to serve as a positive regulator of drought resistance response. It enhances plant resistance to drought stress by inducing the RD22 gene (Seo et al., 2009). An activation tagging line myb96-1d exhibits an enhanced drought resistance with reduced lateral roots. By contrast, the drought resistance response is significantly reduced in the MYB96-deficient myb96-1 mutant. Interestingly, the MYB96 gene also mediates the auxin–ABA interactions during lateral root development. The MYB96 gene modulates abscisic acid (ABA)-mediated abiotic stress signals in inducing a small group of GH3 genes encoding IAA-conjugating enzymes and contributes to maintenance of endogenous IAA contents at an appropriate amount under drought conditions (Seo et al., 2009).
Here, we report that the MYB96 transcription factor links ABA-mediated abiotic stress signals with SA biosynthesis and pathogen resistance response. While the myb96-1d activation tagging line exhibited an enhanced disease resistance, the myb96-1 mutant was more susceptible to a virulent Pseudomonas syringae DC3000 strain. Consistent with this, the SID2 gene was markedly up-regulated, and endogenous concentrations of free SA and SA-β-glucoside (SAG) were elevated in myb96-1d. Interestingly, the myb96-1d phenotypes, including impaired leaf development and dwarfed growth, were suppressed in the myb96-1d X NahG genetic cross, indicating that SA is closely linked with MYB96-mediated ABA signaling. It is therefore proposed that the MYB96 gene serves as a molecular knot that integrates ABA- and SA-mediated signals under environmental stress conditions.
- Top of page
- Materials and Methods
- Supporting Information
Two major stress hormones functioning under biotic and abiotic stress conditions are ABA and SA. Accumulating evidence demonstrates that the two hormones act either individually or through intricate signaling crosstalks (Park et al., 2007; Flors et al., 2008; Yasuda et al., 2008), reflecting that a finely tuned hormone balance is critical for plant survival under stress conditions.
Abscisic acid is generally considered as a negative regulator of disease resistance. Exogenous application of ABA is correlated with an increased susceptibility to pathogen infection, and ABA-deficient mutants exhibit an enhanced pathogen resistance (Mauch-Mani & Mauch, 2005; Fan et al., 2009). In another case, while elevated concentrations of SA are required to build up an innate immune response, bacterial effectors rapidly activate ABA biosynthesis in plants to suppress defense responses (de Torres-Zabala et al., 2007). In this signaling scheme, ABA antagonizes SA-mediated defense responses, providing a mechanistic base for priming events during plant defense responses.
Although antagonistic interactions have been reported between ABA and SA, recent studies imply that positive interactions between the ABA signaling pathway and the biotic signaling network involving SA, jasmonic acid (JA) and ethylene (ET) enhance a tolerance response to abiotic and biotic stresses. It has been recently proven that plant pathogens take advantage of ABA signaling pathways to promote pathogenesis (Mengiste et al., 2003; Chini et al., 2004). The BOTRYTIS SUSCEPTIBLE1 (BOS1) gene controls both JA- and ABA-inducible genes. As a result, a loss-of-function bos1 mutant is susceptible to both necrotrophic pathogens and osmotic and oxidative stresses (Mengiste et al., 2003). The ADR1 gene is an another intriguing example supporting an intimate functional relationship between abiotic and biotic stress responses. While most of disease-resistant mutants do not exhibit an enhanced resistance to abiotic stresses, such as drought and high salinity (Chini et al., 2004), the activation-tagging mutant adr1 exhibits an enhanced resistance to both virulent pathogens and drought stress. We also observed similar phenotypes in the activation-tagging myb96-1d line. The ABA-mediated MYB96 regulation of SA biosynthesis might be another route for balancing plant responses to pathogen infection and abiotic stress condition.
In this work, we examined pathogen resistance responses of the activation-tagging myb96-1d line and the myb96-1 mutant. Expression levels of the SA biosynthetic and signaling genes were also examined. We found that the activation-tagging myb96-1d line, which has previously been shown to exhibit an enhanced resistance to drought (Seo et al., 2009), was also resistant to pathogen infection. By contrast, the T-DNA insertional myb96-1 mutant was susceptible to drought and pathogen infection. Interestingly, the SID2 gene was up-regulated, and the concentrations of endogenous SA were elevated in the activation-tagging myb96-1d line, indicating that the enhanced pathogen resistance of the mutant is derived from increased SA biosynthesis. ABA and abiotic stress conditions, such as drought, osmotic stress, and high salinity, also induced the SID2 gene. However, the inductive effects of ABA were reduced in the myb96-1 mutant, indicating that the MYB96 gene is, at least in part, required for the SID2 induction by ABA-mediated abiotic stress signals.
Our observations demonstrate that the MYB96 transcription factor acts as a signaling link that integrates ABA and SA signals and regulates a synergistic interaction between the two stress hormones. This scheme is also consistent with the improved disease resistance of plants exposed to abiotic stress conditions in Arabidopsis (Gaudet et al., 2003; Griffith & Yaish, 2004). The previous reports (Seo et al., 2009) and our data indicate that the MYB96 transcription factor plays diverse roles in plant responses to biotic and abiotic stresses. It regulates lateral root development under drought via the ABA-auxin crosstalk and shoot growth and disease resistance via the ABA–SA interaction. The ABA–SA interaction is particularly interesting, because ABA-mediated abiotic stress signals regulate SA biosynthesis by inducing a SA biosynthetic enzyme gene, SID2. It will be interesting whether the ABA–auxin and ABA–SA interactions are mutually independent or functionally interrelated. Phenotypic and molecular analysis of a series of higher-order mutants would provide insights into how the MYB96 transcription factor modulates the hormonal interactions.
Abiotic stress-mediated pathogenesis has been widely documented (Gaudet et al., 2003; Griffith & Yaish, 2004; Agarwal et al., 2006). A subset of PR genes (PR1, PR2, and PR5) is also induced by cold, high salt, and drought (Seo et al., 2008). The PR gene induction is correlated with enhanced disease resistance in many cases. We also found that the PR genes were up-regulated, and disease resistance is improved in the activation-tagging myb96-1d line. However, the data should be carefully interpreted, and more works are required to confirm the ABA–SA interaction. It has been reported that the PR3 gene plays a role in regulating seed germination in the presence of high salt (Seo et al., 2008). Other PR genes have also been implicated in various plant developmental processes (Doxey et al., 2007; Brininstool et al., 2008). It is therefore possible that the PR genes induced in the activation-tagging myb96-1d line may be related to a certain developmental process under abiotic stress conditions, and the altered disease resistance responses of the myb96-1d and myb96-1 plants would be an indirect effect.
Moreover, the activation-tagging myb96-1d line exhibited an array of phenotypic alterations, such as delayed growth and smaller leaves with an altered morphology. The phenotypic alterations may affect the pathogen resistance response, as previously reported (Calo et al., 2006; Tang et al., 2007). Cuticular lipids on the leaves, including cutin monomers and cuticular waxes, may be changed in the activation-tagging myb96-1d line. Delayed growth may also affect the defense responses. Bacterial cell infiltration assays on the myb96-1d and myb96-1 leaves showed that disturbed leaf morphology and structure, such as altered stomatal aperature, do not significantly affect the resistance responses (Fig. 2c). However, some doubt still remains, and further studies are required to resolve the issue.
Additional evidence supporting the role of the MYB96 gene in ABA–SA interaction was provided by the high accumulation of anthocyanin in the activation-tagging myb96-1d line and in wild-type plants grown in the presence of SA and NaCl. Anthocyanins accumulate in plants exposed to diverse biotic and abiotic stress conditions (Winkel-Shirley, 2001, 2002). We observed a high accumulation of anthocyanins in the activation-tagging myb96-1d line that exhibits enhanced resistance responses to both drought and pathogen infection (Seo et al., 2009; this work). Plant growth was severely delayed in the presence of either SA or NaCl. When wild-type plants were treated with SA and NaCl, anthocyanins accumulate to a high concentration in addition to growth retardation, indicating that anthocyanin accumulation requires both ABA and SA signals. Alternatively, the ABA and SA signals governing anthocyanin accumulation might be interconnected.
A notable observation was that while plant growth was delayed to a similar degree in both the abi1-1 mutant and the NahG transgenic plants in the presence of high salt and SA, anthocyanin accumulation was significantly reduced only in the abi1-1 mutant (Fig. 7b). By contrast, anthocyanins still accumulated to a high concentration in the NahG transgenic plants. This may be the result of the high accumulation of catechol in the NahG transgenic plants. Nevertheless, it is evident that ABA-mediated abiotic stress signaling plays a primary role in inducing anthocyanin accumulation.