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Keywords:

  • abscisic acid;
  • Arabidopsis;
  • disease resistance;
  • MYB96;
  • salicylic acid;
  • SID2

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • The Arabidopsis MYB96 transcription factor plays a role in abscisic acid (ABA)-mediated drought response. Notably, anthocyanins accumulate in the activation-tagging myb96-1d line, suggesting a role of MYB96 in biotic and abiotic stress responses in plants. Here, we investigate the role of MYB96 in salicylic acid (SA) biosynthesis and plant defense and explore the mechanisms underlying the ABA–SA interaction.
  • myb96-1d and myb96-1 were subject to pathogen infection assays, and expression of SA biosynthetic and defense genes was examined. myb96-1d was crossed with the NahG transgenic plants to investigate the role of MYB96 in ABA regulation of SA biosynthesis.
  • Whereas myb96-1d exhibited an enhanced disease resistance, myb96-1 was susceptible to pathogen infection. A subset of pathogenesis-related (PR) genes was up-regulated in myb96-1d. However, PR transcript abundances were reduced in myb96-1d X NahG. Interestingly, a SA biosynthetic gene SALICYLIC ACID INDUCTION DEFICIENT2 (SID2) was up-regulated, and concentrations of SA and SA-β-glucoside (SAG) were elevated in myb96-1d. In addition, the inductive effects of abiotic stresses on SID2 were reduced in aba3-1.
  • Our observations indicate that MYB96-mediated ABA signals enhance plant disease resistance by inducing SA biosynthesis. It is envisioned that MYB96 is a molecular link that mediates ABA-SA crosstalks.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. 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.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant materials and growth conditions

All Arabidopsis thaliana (L.) Heynh. lines used were in the Columbia background (Col-0), unless otherwise specified. Plants were grown in a controlled culture room set at 22°C with a relative humidity of 60% under long-day conditions (16 h light and 8 h dark), with white light illumination (120 μmol photons m−2 s−1) provided by fluorescent FLR40D/A tubes (Osram, Seoul, Korea). The activation-tagging line myb96-1d and the myb96-1 knockout mutant have been previously described (Seo et al., 2009).

Transcript abundance analysis

Quantitative real-time RT-PCR (qRT-PCR) was employed for measuring transcript abundances. Total RNA sample preparation, reverse transcription, and qRT-PCR were carried out based on the rules that have recently been proposed by Udvardi et al. (2008) to ensure reproducible and accurate measurements. Extraction of total RNA samples from appropriate plant materials and qRT-PCR conditions have been previously described (Kim et al., 2006). The RNA samples were extensively pretreated with an RNAse-free DNAse to eliminate any contaminating genomic DNA before use. The PCR primers used are listed in Supporting Information, Table S1.

Quantitative real-time RT-PCR was carried out in 96-well blocks with an Applied Biosystems 7500 Real-Time PCR System (Foster City, CA, USA) using the SYBR Green I master mix in a volume of 25 μl. The PCR primers were designed using the Primer Express Software installed into the system. The two-step thermal cycling profile used was 15 s at 94°C and 1 min at 68°C. An eIF4A gene (At3g13920) was included in the assays as an internal control for normalizing the variations in cDNA amounts used (Gutierrez et al., 2008). The qRT-PCR reactions were carried out in biological triplicates and technical duplicates using RNA samples extracted from three independent plant materials grown under identical growth conditions. The comparative ΔΔCT method was used to evaluate the relative quantities of each amplified product in the samples. The threshold cycle (CT) was automatically determined for each reaction by the system set with default parameters. The specificity of the PCR was determined by melt curve analysis of the amplified products using the standard method installed in the system.

Treatments with growth hormones and abiotic stresses

Two-week-old plants grown on MS-agar plates were transferred to MS liquid cultures supplemented with various growth hormones, including methyl jasmonate (mJA) or 1-aminocyclopropane-1-carboxylic acid (ACC) (20 μM each, unless otherwise specified), for the indicated time periods, and plant materials were harvested for total RNA extraction. ABA was used at a final concentration of either 1 or 5 μM for MS-agar plates or 20 μM for MS liquid cultures.

For the assays on the effects of drought on gene expression, 2-wk-old plants grown on MS-agar plates were put on a dry 3MM paper and incubated at room temperature for the indicated time periods. For the assays on the effects of high salinity on gene expression, 2-wk-old plants grown on MS-agar plates were soaked in MS liquid cultures containing 200 mM NaCl and incubated with gentle shaking under constant light for the indicated time periods.

Assays on pathogen infection

Bacterial cells of P. syringae pv. tomato strain DC3000 were cultured for 2 d at 28°C in King’s B medium supplemented with rifampicin (50 μg l−1) (Park et al., 2007). A bacterial cell suspension was prepared at 107 cfu ml−1 in 10 mM MgCl2 supplemented with 250 ppm TWEEN 80 and sprayed directly on to the leaf surface. After incubation for 16 h at 25°C and 100% relative humidity in complete darkness, the inoculated plants were transferred to a growth chamber set at 23°C and 80% relative humidity and grown further under long days. Measurements of bacterial cell growth were carried out as previously described (Park et al., 2007) using whole leaves of 4-wk-old plants grown in soil.

For the direct infiltration assays, bacterial cells of P. syringae pv. tomato strain DC3000 were prepared as described (Park et al., 2007). Bacterial cells were collected and resuspended in resuspension buffer containing 10 mM MgCl2. The sixth leaves of 4-wk-old plants grown in soil were infiltrated with the bacterial cell suspensions by injecting into the abaxial side of the leaves using 1 ml needleless syringes.

Analysis of anthocyanin concentrations

Extraction and quantification of anthocyanins from the leaf tissues were carried out as described previously (Rabino & Mancinelli, 1986). Plant leaves were homogenized in liquid nitrogen, and anthocyanins were extracted using methanol that contained 1% HCl (v/v). Extraction steps were conducted at 4°C. After centrifugation, the supernatant was used for measurements of absorbance at 530 and 657 nm. The formula A530 – 0.25 × A657 was used to calculate the amounts of anthocyanins.

Measurements of endogenous SA contents

Extraction and quantification of endogenous SA and SAG from the leaf tissues of 2-wk-old plants grown on MS-agar plates were carried out as described previously (Bowling et al., 1994). Three independent measurements were averaged. Statistical significance was determined using Student’s t-test.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Anthocyanins accumulate in the myb96-1d leaves

We have recently reported that the MYB96 transcription factor is intimately related with ABA-mediated drought stress responses, particularly during lateral root development (Seo et al., 2009). The activation-tagging myb96-1d line is characterized by having reduced growth with disturbed leaf morphology and by exhibiting an enhanced resistance to drought (Fig. 1a; Seo et al., 2009). Notably, we also found that anthocyanins accumulate to a high concentration in the leaves of older myb96-1d plants (Fig. 1a, right panels). There were no discernible differences in the amounts of anthocyanin during the seedling growth stage. However, it was significantly elevated in the later growth stages, evidently 32 d after germination. Anthocyanin accumulation was initiated in the leaf margin of the myb96-1d line and later spread throughout the whole leaf area (Fig. 1a, right panels). Anthocyanin content was higher by c. 80-fold in the myb96-1d leaves than in wild-type leaves (Fig. 1b). By contrast, chlorophyll content was not discernibly changed in the leaves of the activation-tagging myb96-1d line (Fig. 1c). The MYB96-deficient mutant, myb96-1, had indistinguishable phenotypes compared with wild-type plants under normal growth conditions (Seo et al., 2009).

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Figure 1.  Anthocyanins accumulate to a high concentration in the leaves of the MYB96-overexpressing myb96-1d activation-tagging line. (a) High accumulation of anthocyanins in the adult leaves of the activation-tagging myb96-1d line and 35S:MYB96 transgenic Arabidopsis plants. Five-week-old plants grown in soil were photographed (left panel). Anthocyanin accumulation is evident, particularly in the rosette leaves of older mutant plants (right, upper panel). Two representative rosette leaves with visible anthocyanin accumulation are shown (right, bottom panel). Bar, 1 cm. (b) Measurement of anthocyanins in the mutant leaves. The normalized value of wild-type plants was used as a reference value of (1) for the relative anthocyanin contents. (c) Measurements of chlorophyll in the mutant leaves. Total chlorophyll levels were measured. The normalized value of wild-type plants was used as a reference (100%) for the comparison of relative chlorophyll contents. In (b) and (c), the leaves of 5-wk-old plants grown in soil were used for extraction of anthocyanins and chlorophylls, and five measurements were averaged and statistically treated using Student’s t-test (*< 0.01). Bars indicate standard error of the mean. (d) Transcript abundances of several anthocyanin biosynthetic enzyme genes in the mutants (PAL1, DFR, PAP1 and PAP2). Whole plants grown on MS-agar plates for 2 wk were used for extraction of total RNAs. Transcript abundances were compared by quantitative real-time RT-PCR (qRT-PCR). Biological triplicates and technical duplicates were averaged and statistically treated (t-test, *< 0.01). Bars indicate standard error of the mean. A MYB96-deficient mutant myb96-1 was also included in the assays of (b)–(d).

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We examined expression patterns of the genes encoding anthocyanin biosynthetic enzymes and constituting the MBW complex by qRT-PCR. We found that the PAP1 and PAP2 genes were significantly up-regulated in the activation-tagging myb96-1d line (Fig. 1d; Fig. S1). The SA biosynthetic enzyme gene PAL1 and the DIHYDROFLAVONOL 4-REDUCTASE (DFR) gene that catalyzes the conversion of dihydroquercetin to leucocyanidin in the anthocyanin biosynthesis were also induced by c. two- to threefold in the activation-tagging myb96-1d plant. These observations indicate that the MYB96-mediated signaling induces a subset of anthocyanin biosynthetic genes, resulting in a high accumulation of anthocyanins in the myb96-1d plants.

The activation-tagging myb96-1d line and the myb96-1 mutant exhibit altered resistance responses to pathogen infection

Anthocyanin accumulation in plant tissues is closely related to abiotic and biotic stress responses (Dixon & Paiva, 1995; Chalker-Scott, 1999). It is sometimes used as a physiological marker for disease resistance response in some plant species (He & Dixon, 2000; Dixon, 2001). In addition, we found that a SA biosynthetic enzyme gene, PAL1, is up-regulated in the activation-tagging myb96-1d line. Based on previous observations as well as our own, it was hypothesized that the MYB96 gene might be related to pathogen resistance response in addition to its role in drought resistance.

To examine the hypothesis, we infected the myb96-1d and myb96-1 plants with a virulent P. syringae DC3000 strain. As expected, counting of bacterial cell growth in the infected mutants revealed that, whereas the MYB96-overexpressing myb96-1d line exhibited an enhanced disease resistance to pathogen infection, the myb96-1 mutant was relatively more susceptible to pathogen infection (Fig. 2a). Transgenic plants overexpressing the MYB96 gene under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter were also resistant to pathogen infection similar to the activation-tagging myb96-1d line.

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Figure 2.  The activation-tagging myb96-1d line exhibits an enhanced resistance to Pseudomonas syringae infection. (a) Pathogen infection assays on the myb96-1d and myb96-1 plants. Arabidopsis plants were infected with a virulent P. syringae strain by spray inoculation, and the numbers of bacterial cells were counted. Four-week-old plants grown in soil were used for infection assays. Five independent countings were averaged and statistically treated (t-test, *< 0.01). Bars indicate standard error of the mean. (b) Expression of the pathogenesis-related (PR) genes in the activation-tagging myb96-1d line and the myb96-1 mutant. Whole plants grown on MS-agar plates for 2 wk were used for extraction of total RNAs. Transcript abundances were compared by quantitative real-time RT-PCR (qRT-PCR). Biological triplicates and technical duplicates were averaged and statistically treated (t-test, *< 0.01). Bars indicate standard error of the mean. The y-axis was presented on a logarithmic scale for better comparison of fold changes. (c) Pathogen infection assays on the myb96-1d and myb96-1 leaves. The sixth leaves of 4-wk-old plants grown in soil were used for infiltration assays. Three independent experiments were averaged and statistically treated (t-test, *< 0.01). Bars indicate standard error of the mean. Open bars, 0 d postinfection (dpi); closed bars, 4 dpi.

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Consistent with the altered resistance responses to pathogen infection in the mutants, expression of several PR genes were changed in the mutants. We observed that while the transcript abundances of PR1, PR2, and PR5, which participate in SA signaling (Seo et al., 2008), were significantly higher in the activation-tagging myb96-1d line, they were slightly but reproducibly lower in the myb96-1 mutant (Fig. 2b).

We have recently reported that MYB96 regulates drought resistance by modulating stomatal opening. It has been known that stomatal closure is triggered in response to bacterial infection and pathogen-associated molecular pattern (PAMP), which serves as part of the plant defense mechanisms to restrict bacterial invasion (Melotto et al., 2006). It was therefore suspected that the reduced stomatal aperture in the activation-tagging myb96-1d line would be related to the observed disease resistance. To answer the question, we infiltrated P. syringae pv. tomato DC3000 directly into the leaves of the myb96-1d and myb96-1 plants. While the activation-tagging myb96-1d line exhibited an enhanced resistance, the myb96-1 mutant showed a higher susceptibility (Fig. 2c), indicating that the reduced or increased stomatal closure does not contribute significantly to the altered resistance responses in the myb96-1d and myb96-1 leaves. Together, these observations indicate that the MYB96 gene is intimately related with disease resistance response in plants.

The SID2 gene is induced in the activation-tagging myb96-1d line

To investigate the molecular cause underlying the altered disease resistance responses of the activation-tagging myb96-1d line and the myb96-1 mutant, we examined the expression patterns of SA biosynthetic and signaling genes. The PAL1 gene was induced moderately (2.3-fold) in the activation-tagging myb96-1d line (Fig. 1d). In particular, the SID2 gene was significantly up-regulated in the activation-tagging myb96-1d line but slightly suppressed in the myb96-1 mutant (Fig. 3a). By contrast, the expression patterns of other SA biosynthetic genes, such as PAL2, PAL3, and SID1/ICS2, and of SA signaling genes, including GST6, NPR1, and TGA2, were unaffected to a noticeable degree by the myb96 mutations (Fig. S2). These observations support the proposition that the MYB96 gene positively regulates the SID2 gene, which would result in SA accumulation in the activation-tagging myb96-1d line. The MYB96 gene was uninfluenced by exogenous SA application (Fig. 3b), indicating that the MYB96 gene acts upstream of SID2 expression and thus SA biosynthesis.

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Figure 3.  The SALICYLIC ACID INDUCTION DEFICIENT2 (SID2) gene is up-regulated in the activation-tagging myb96-1d line. Two-week-old, whole Arabidopsis plants grown on MS-agar plates were used for extraction of total RNAs or for subsequent treatment with salicylic acid (SA) or flagellin22 (Flg22). Transcript abundances were compared by quantitative real-time RT-PCR (qRT-PCR). Biological triplicates and technical duplicates were averaged and statistically treated (t-test, *< 0.01). Bars indicate standard error of the mean. (a) Up-regulation of the SID2 gene in the activation-tagging myb96-1d line. (b) Effects of SA on MYB96 expression. Plants were incubated with gentle shaking in liquid MS cultures supplemented with 0.1 mM SA for the indicted time periods before extraction of total RNAs. Mo, mock. (c) Expression kinetics of the MYB96, SID2, and pathogenesis-related1 (PR1) genes after application of 1 μM Flg22. Peaks of the transcript abundances for individual genes are indicated by arrows. The relative expression levels were fold changes relative to the transcript abundances at 0 h time points for each gene.

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Our data suggest that the MYB96 gene might be induced by pathogen infection and the MYB96 induction would occur earlier than induction of SA biosynthesis and PR gene induction. To examine this possibility, we investigated expression kinetics of the MYB96, SID2, and PR1 genes after treatment with the bacterial flagellin peptide elicitor (Flg22), which efficiently elicits plant defense responses (Takai et al., 2008). The MYB96 gene was rapidly induced by the Flg22 treatment, and the transcript abundance reached the peak within 1 h (Fig. 3c). The SID2 induction was initiated 2 h after treatments and reached the peak 6 h after Flg22 application. The PR1 induction was initiated 12 h after treatments, supporting the notion that the MYB96 induction is an early event in plant disease resistance response, occurring ahead of the promotion of SA biosynthesis and PR gene induction.

Endogenous concentrations of SA and SAG are elevated in the activation-tagging myb96-1d line

Enhanced disease resistance and up-regulation of the SA biosynthetic enzyme gene SID2 in the activation-tagging myb96-1d line suggested that SA biosynthesis would be elevated in the mutant. We therefore measured the endogenous concentrations of SA and SAG in the myb96-1d and myb96-1 plants. As expected, the endogenous concentrations of free SA and conjugated SA (SAG) were c. sevenfold and 10-fold higher, respectively, in the activation-tagging myb96-1d line (Fig. 4a). The concentrations of SA and SAG in the myb96-1 mutant were not detectably different from those in wild-type plants. This might be the result of functional redundancy among the multiple MYB transcription factors functioning in pathogen resistance response.

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Figure 4.  Endogenous concentrations of salicylic acid (SA) and its conjugated form SA-β-glucoside (SAG) are elevated in the activation-tagging myb96-1d line. (a) Endogenous contents of SA and SAG in the activation-tagging myb96-1d line and the myb96-1 mutant. The leaves of 2-wk-old Arabidopsis plants grown on MS-agar plates were used for extraction of SA and SAG. Three measurements were averaged and statistically treated (t-test, *< 0.01). Bars indicate standard error of the mean. (b) Phenotypic comparison of the myb96-1d X Col-0 and myb96-1d X NahG genetic crosses. The activation-tagging myb96-1d line was crossed with the SA-deficient NahG transgenic plants. Four-week-old plants grown in soil were photographed. (c) Transcript abundances of the pathogenesis-related (PR) genes in the genetic crosses. Two-week-old plants grown on MS-agar plates were used for extraction of total RNAs. Transcript abundances were compared by quantitative real-time RT-PCR (qRT-PCR). Biological triplicates and technical duplicates were averaged and statistically treated (t-test, *< 0.01). Bars indicate standard error of the mean. The y-axis was presented on a logarithmic scale for better comparison of fold changes. (d) Pathogen infection assays on the myb96-1d X Col-0 and myb96-1d X NahG genetic crosses. Four-week-old plants grown in soil were used for the spray inoculation assays. The third and fourth leaves were photographed (upper panel). Three independent experiments were averaged and statistically treated (t-test, *< 0.01). Bars indicate standard error of the mean (bottom panel). Open bars, 0 d postinfection (dpi); closed bars, 5 dpi.

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Our data indicate that the increased SA biosynthesis contributes to the myb96-1d phenotypes, such as enhanced disease resistance and altered leaf morphology. To confirm the intimate relationship between SA and phenotypic alterations in the activation-tagging myb96-1d line, we genetically crossed the mutant with SA-deficient NahG transgenic plants that express a salicylate hydroxylase (Friedrich et al., 1995). Strikingly, the myb96-1d phenotypes, such as impaired leaf morphology and growth retardation, disappeared in the myb96-1d X NahG cross (Fig. 4b) as well as in the myb96-1d X sid2 cross (Fig. S3). In addition, the transcript abundances of the PR1, PR2, and PR5 genes were reduced in the myb96-1d X NahG cross to an amount comparable to or even lower than those observed in wild-type plants (Col-0) (Fig. 4c). By contrast, the transcript abundance of the MYB96 gene was unaffected in the genetic cross, which is consistent with the lack of SA effects on the MYB96 expression (Fig. 3b).

To further confirm the role of MYB96 in disease resistance, we infected the myb96-1d X Col-0 and myb96-1d X NahG crosses with P. syringae pv. tomato DC3000 cells by spray inoculation. Consistent with the expression patterns of the PR genes (Fig. 4c), whereas the myb96-1d X Col-0 cross showed an enhanced resistance, the disease resistance of the myb96-1d X NahG cross was compromised to a degree comparable to that observed in wild-type plants (Fig. 4d). It is therefore evident that the myb96-1d phenotypes are, at least in part, attributable to elevated SA biosynthesis in the mutant.

The MYB96-mediated ABA signals confer an enhanced drought resistance via a RD22-mediated pathway (Seo et al., 2009). However, expression of the RD22 gene was unaltered in the myb96-1d X NahG cross (Fig. S4), indicating that the MYB96-mediated regulation of the RD22 gene during drought resistance response is functionally separated from the MYB96-mediated regulation of SA biosynthesis and thus of pathogen resistance response.

MYB96 plays a role in pathogen induction of PR genes

The activation-tagging myb96-1d line showed an enhanced pathogen resistance, and a small group of PR genes are up-regulated in the mutant. The concentration of endogenous SA content was also elevated in the activation-tagging myb96-1d line. A critical question was therefore whether the MYB96 gene contributes to the PR induction in infected plants.

To examine this, wild-type, myb96-1d, and myb96-1 plants were infected with P. syringae cells by spray inoculation, and the PR1 expression kinetics were investigated by qRT-PCR after infection. The MYB96 gene was rapidly induced after pathogen infection, and the transcript abundance decreased gradually after the peak at 12 h (Fig. 5a). Under the same infection conditions, the PR1 gene induction was initiated 48 h after infection, and the transcript abundance was further elevated throughput the time course. The PR1 gene was induced in a similar kinetics in the infected myb96-1 mutant. However, the PR1 transcript abundance was lower by c. 40% in the myb96-1 mutant than in wild-type plants (Fig. 5b). It is well known that the PR1 gene is induced after pathogen infection in a SA-dependent manner (Bol et al., 1990). It was therefore concluded that at least a portion of the PR1 induction after pathogen infection depends on a functional MYB96 activity.

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Figure 5.  The MYB96 gene contributes to the pathogenesis-related (PR) induction by pathogen infection. Four-week-old Arabidopsis plants grown in soil were infected with a virulent Pseudomonas syringae strain. Transcript abundances of the MYB96 gene (a) and of the PR1 gene (b) were compared by quantitative real-time RT-PCR (qRT-PCR) at the indicated time points after infection. Biological triplicates and technical duplicates were averaged. Bars indicate standard error of the mean.

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MYB96-mediated abiotic stress signals induce the SID2 gene in an ABA-dependent manner

The MYB96 gene is a component of ABA signaling in drought stress response. Therefore, related questions were whether ABA and abiotic stresses affect the SID2 expression and, if so, whether the effects of abiotic stresses on the SID2 expression depend on ABA.

Wild-type plants were treated with ABA and mannitol that confers osmotic stress on plants, and the transcript abundances of the SID2 gene were examined by qRT-PCR. The SID2 expression was induced by more than fourfold in the presence of ABA (Fig. 6a, left panel). Mannitol treatment also exhibited a similar effect on the SID2 expression (Fig. 6a, right panel). However, the inductive effect of ABA on the SID2 expression was reduced by more than 30% in the myb96-1 mutant (Fig. 6b), indicating that a functional MYB96 activity is required for the ABA induction of the SID2 gene.

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Figure 6.  The MYB96 gene modulates abscisic acid (ABA)-mediated abiotic stress signals in inducing the SALICYLIC ACID INDUCTION DEFICIENT2 (SID2) gene. Two-week-old Arabidopsis plants grown on MS-agar plates were used for treatments with ABA, salicylic acid (SA), mannose (Man), NaCl, or drought (DR). Whole plants were used for extraction of total RNAs. Transcript abundances were compared by quantitative real-time RT-PCR (qRT-PCR). Biological triplicates and technical duplicates were averaged and statistically treated (t-test, *< 0.01). Bars indicate standard error of the mean. (a) Effects of ABA and SA (left panel) and mannitol (right panel) on the SID2 expression. (b) Effects of ABA on SID2 expression in the myb96-1 mutant. (c) Effects of drought (left panel) and NaCl (right panel) on SID2 expression. (d) Effects of NaCl on SID2 expression in the aba3-1 and sid2 mutants.

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Drought and high salinity also showed inductive effects on the SID2 expression (Fig. 6c). In addition, the effects of high salinity on the SID2 expression were reduced by c. 40% in the ABA-deficient aba3-1 mutant (Fig. 6d), indicating that abiotic stresses induce the SID2 gene, at least in part, via the ABA-dependent pathway. Together, our observations indicate that ABA-mediated abiotic stress signals regulate the SID2 gene and that the MYB96 gene plays a role in these signaling cascades.

MYB96 serves as a molecular link that integrates ABA and SA signals

We found that the activation-tagging myb96-1d line phenotypes, such as dwarfed growth with altered leaf morphology, are efficiently rescued in the myb96-1d X NahG cross (Fig. 4b). The transcript abundances of the PR genes and disease resistance were also compromised in the cross. Furthermore, the activation-tagging myb96-1d line exhibits an enhanced resistance to drought (Seo et al., 2009) as well as to pathogen infection (Fig. 2a). These observations strongly support the proposition that the ABA and SA signals are closely linked, and the myb96-1d phenotypes, such as anthocyanin accumulation, would be caused by simultaneous stimulation of both ABA and SA responses in the mutant.

To examine this, wild-type plants were treated with SA (0.1 mM) or NaCl (150 mM), or both, and plant phenotypes were analyzed. Plant growth was delayed in the presence of either SA or NaCl with a more severe retardation in the NaCl-treated plants (Fig. 7a, left panel). When the plants were treated with both SA and NaCl, a remarkable amount of anthocyanins was accumulated in the leaves, similar to that observed in the myb96-1d leaves (Fig. 7a, right panel), supporting the notion that the myb96-1d phenotypes are caused by an additive effect of ABA and SA signals.

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Figure 7.  The MYB96 gene integrates abscisic acid (ABA) and salicylic acid (SA) signals during plant stress responses. (a) Anthocyanin accumulation in wild-type Arabidopsis plants treated with SA and NaCl. In the presence of SA and NaCl, plant growth is significantly reduced (left panel), and anthocyanins accumulate to a high concentration (right panel). (b) Anthocyanin accumulation in the abi1-1 mutant and NahG transgenic plants treated with SA and NaCl. Whereas plant growth is reduced to a similar degree in both plants (left panel), anthocyanin accumulation is reduced only in the abi1-1 mutant (right panel).

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We next treated an ABA signaling mutant, abi1-1, and the NahG transgenic plants with both SA and NaCl. Plant growth was severely delayed in all the treated plants (Fig. 7b). However, anthocyanins accumulated to a high concentration only in the NahG transgenic plants, but the accumulation was greatly reduced in the abi1-1 mutant, indicating that anthocyanin accumulation is regulated primarily by ABA-mediated signaling (see the following section).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. 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.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Jae-Yong Ryu for growing plants. This work was supported by the Brain Korea 21, Biogreen 21 (20080401034001), and National Research Laboratory programs and by grants from the Plant Signaling Network Research Center (2009-0079297), the Korea Science and Engineering Foundation (2007-03415), and from the Agricultural R&D Promotion Center (309017-5), Korea Ministry for Food, Agriculture, Forestry and Fisheries.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information