SEARCH

SEARCH BY CITATION

Keywords:

  • shade avoidance;
  • plant immunity;
  • phytochrome;
  • phosphorylation;
  • NPR1;
  • Arabidopsis thaliana

Summary

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

In dense stands of plants, such as agricultural monocultures, plants are exposed simultaneously to competition for light and other stresses such as pathogen infection. Here, we show that both salicylic acid (SA)-dependent and jasmonic acid (JA)-dependent disease resistance is inhibited by a simultaneously reduced red:far-red light ratio (R:FR), the early warning signal for plant competition. Conversely, SA- and JA-dependent induced defences did not affect shade-avoidance responses to low R:FR. Reduced pathogen resistance by low R:FR was accompanied by a strong reduction in the regulation of JA- and SA-responsive genes. The severe inhibition of SA-responsive transcription in low R:FR appeared to be brought about by the repression of SA-inducible kinases. Phosphorylation of the SA-responsive transcription co-activator NPR1, which is required for full induction of SA-responsive transcription, was indeed reduced and may thus play a role in the suppression of SA-mediated defences by low R:FR-mediated phytochrome inactivation. Our results indicate that foraging for light through the shade-avoidance response is prioritised over plant immune responses when plants are simultaneously challenged with competition and pathogen attack.


Introduction

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

At high density, either in nature or in agriculture, plants are at risk of becoming shaded by the surrounding vegetation. To avoid getting cut off from the light plants in dense vegetation try to outgrow neighbours through increased leaf angles, stem elongation, apical dominance and early flowering; an escape strategy known as the shade-avoidance syndrome (Vandenbussche et al., 2005; Franklin, 2008). The first signal known to announce the presence of surrounding plants is a decrease in the ratio red:far-red light (R:FR) (Morgan and Smith, 1976; Ballaré et al., 1990). This cue is specific for plants, as red light (R) is absorbed for photosynthesis, whilst far-red light (FR) is reflected by green tissue (Smith, 2000). Red and far-red levels are perceived through the family of phytochrome photoreceptors (phyA-E in Arabidopsis), which exist in two photo-convertible forms: the active, far-red light absorbing form Pfr, and the inactive, red light absorbing form Pr (Smith and Holmes, 1977). PhyB is the main phytochrome involved in shade-avoidance responses, with redundant roles for phyD and phyE (Clack et al., 1994; Franklin et al., 2003). Active phyB is thought to bind the elongation promoting phytochrome interacting factors (PIF) 4, 5 and 7, members of a subfamily of basic helix–loop–helix (bHLH) transcription factors, in the nucleus, thereby targeting them for degradation through the proteasome and thus preventing induction of the shade-avoidance signalling cascade (Lorrain et al., 2008; Li et al., 2012). Far-red light inactivates phytochrome, thus alleviating phytochrome-mediated PIF degradation. This situation leads to rapid induction of gene expression, including genes encoding other transcription factors such as PIF-like (PIL)1 and several homeodomain/leucine zipper (HD-zip) proteins (Salter et al., 2003; Sessa et al., 2005) and stimulates biosynthesis and signalling of various growth-promoting hormones including auxin (Tao et al., 2008; Keuskamp et al., 2010; Li et al., 2012), gibberellins (Peng and Harberd, 1997; Djakovic-Petrovic et al., 2007), brassinosteroids (Kozuka et al., 2010) and ethylene (ET) (Finlayson et al., 1999; Pierik et al., 2004) ultimately resulting in enhanced elongation growth.

Another threat to plant survival in dense stands is the spreading of infectious diseases from one plant to another. There are two major hormonal pathways involved in plant immune responses: the salicylic acid (SA) and the jasmonic acid (JA) pathway. The SA and JA defence pathways each induce a different set of response genes and are often mutually antagonistic, which enables plants to fine-tune their defence response to a specific pathogen (Spoel and Dong, 2008; Pieterse et al., 2012).

A key transcriptional regulator of SA-induced defence is the ankyrin-repeat and BTB/POZ protein–protein interaction domains containing protein NPR1 (Cao et al., 1997; Ryals et al., 1997). Under un-induced conditions NPR1 resides in the cytosol as an oligomer linked through intermolecular disulfide bridges between cysteine residues. Upon accumulation of SA an increase in cellular reducing capacity releases NPR1 monomers, which subsequently translocate to the nucleus (Kinkema et al., 2000; Mou et al., 2003; Tada et al., 2008). NPR1 interacts with TGA transcription factors to co-activate transcription of primary defence-associated genes (Després et al., 2000; Fan and Dong, 2002), and NPR1 may be part of an SA receptor complex (Fu et al., 2012; Wu et al., 2012). NPR1 phosphorylation and subsequent proteasome-mediated turnover of NPR1 are required for full induction of SA-induced transcription (Spoel et al., 2009).

Induction of the JA pathway depends on the alleviation of transcriptional repression by proteins of the jasmonate ZIM domain (JAZ) family. The biologically active JA conjugate (+)-7-iso-jasmonoyl-l-isoleicine (JA-Ile) binds to the JA receptor complex coronatine insensitve (COI)1/JAZ (Fonseca et al., 2009). COI1 is an F-box protein that is part of the E3 ubiquitin-ligase complex SCFCOI1 (Xu et al., 2002). Upon binding of JA-Ile, the SCFCOI1 complex targets the JAZ proteins for degradation via the 26S proteasome (Chini et al., 2007; Thines et al., 2007). This situation allows for JA-induced gene expression through the bHLH transcription factors MYC2, MYC3 and MYC4, which are associated with herbivore resistance (Lorenzo et al., 2004; Fernandez-Calvo et al., 2011). Resistance against necrotrophic pathogens is regulated by the combined action of JA and ET and depends on the Apetala/Ethylene response factor (AP2/ERF) transcription factors ORA59 and ERF1 (Lorenzo et al., 2003; Pré et al., 2008).

Shading and pathogen attack are studied elaborately as single stresses. There are, however, several reports that highlight interplay between plant light signalling and pathogen defence. Total light intensity and the light period after infection have been shown to be important for the level of induction of SA-dependent defence (Genoud et al., 2002; Griebel and Zeier, 2008). Light quality has also been shown to affect defence responses. Red light, but not light of other wavelengths, stimulates disease resistance against both biotrophic and necrotrophic pathogens in a variety of plant species (Islam et al., 1998, 2008; Rahman et al., 2010; Wang et al., 2010), suggesting that phytochrome signalling may play a role in pathogen defence. The constitutively shade-avoiding Arabidopsis phytochrome mutants phyAphyB and phyB and the constitutive shade avoidance (csa)1 mutant display reduced basal resistance against the hemi-biotrophic pathogen Pseudomonas syringae (Genoud et al., 2002; Faigón-Soverna et al., 2006), further suggesting a role for phytochromes in SA-dependent defence. The phyAphyB mutant furthermore had decreased SA-dependent systemic immunity in tissues away from the infection site (Griebel and Zeier, 2008).

Functional evidence for interaction between competition for light and JA signalling has been shown in studies combining shade with herbivore attack. Chenopodium album plants exposed to simulated canopy shade were less resistant against subsequent herbivore attack than plants in control light (Kurashige and Agrawal, 2005). Inactivation of phytochromes was further shown to increase susceptibility to herbivores, as both Arabidopsis and Nicotiana longiflora plants pre-treated with low R:FR, as well as phyB mutants of Arabidopsis and tomato (Solanum lycopersicum), supported higher caterpillar growth (Izaguirre et al., 2006; Moreno et al., 2009). Recently, it was shown that FR pre-treatment also enhances susceptibility to the necrotrophic pathogen Botrytis cinerea in Arabidopsis (Cerrudo et al., 2012).

In the current research we studied the interaction between shade avoidance and SA-dependent defence, on the one hand, and JA-dependent defence, on the other. We investigated how shade avoidance and pathogen defence interact when both stresses are induced simultaneously at the physiological and genome-wide transcriptional levels. We found enhanced disease susceptibility and reduced gene expression profiles for both the SA- and JA-dependent pathways under low R:FR. Interestingly, shade-avoidance traits were not affected by defence induction, implying dominance of phytochrome signalling over defence induction. The SA-dependent transcript profile was affected radically by addition of a low R:FR signal. We provide evidence that suggests that this suppression is brought about by a mechanism in which the low R:FR signalling pathway targets SA-inducible phosphorylation cascades and compromises the balance between monomerization and phosphorylation of the transcriptional co-activator NPR1.

Results

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

Shade avoidance increases susceptibility to both a biotrophic and a necrotrophic pathogen

The effect of phytochrome signalling on pathogen defence was tested both by using the phytochrome mutant phyB and through subjecting plants grown in control light to low R:FR, achieved by supplementing control white light with FR-emitting light emitting diodes (LEDs). Constitutively shade-avoiding phyB plants inoculated with the virulent hemi-biotrophic pathogen Pseudomonas syringae pv. tomato DC3000 (Pst) supported significantly higher bacterial proliferation than the Col-0 wild-type (Figure 1a). Mutant phyB plants inoculated with the necrotrophic pathogen Botrytis cinerea (Bc) were also more susceptible than Col-0 plants (Figure 1b), showing that both SA- and JA-dependent basal resistance are attenuated when constitutive expression of shade avoidance is genetically programmed. Resistance against Bc was also attenuated in plants that had been subjected to shading conditions through growth in high density (Figure S1). When Col-0 plants were placed in low R:FR immediately after inoculation with Pst or Bc they also showed reduced resistance as compared with inoculated plants kept in control light (Figure 1c,d). This finding indicates that defence against both a hemi-biotrophic and a necrotrophic pathogen is suppressed by the shade-avoidance response even when the two responses are induced simultaneously.

image

Figure 1. Disease incidence and petiole elongation of wild-type and phyB plants. (a, c) Bacterial growth quantification 3 days post infection (dpi) of Pst-inoculated leaves of phyB mutant and Col-0 wild-type plants subjected to control light or low red:far-red (R:FR). Cfu = colony-forming units. (b, d) Lesion diameters scored 3 dpi on leaves inoculated with a 5 μl droplet of Bc spore solution. I: lesion diameter <2 mm, II: lesion diameter ≥2 mm and ≤4 mm, III: lesion diameter >4 mm (n > 20 leaves). (e, f) Petiole elongation of plants inoculated with Pst or Bc. Plants were transferred to control light or low R:FR 1 dpi. Data represent means ± standard error (SE). Asterisks indicate significant difference for (a, c) Student's t-test; (b, d) chi-squared test; (e,f) two-way analysis of variance (anova); < 0.05). L, light treatment; P, pathogen treatment; L:P, interaction between light treatment and pathogen treatment; NS, not significant.

Download figure to PowerPoint

Pathogen defence does not affect the shade-avoidance response

To study the effect of induced defence on the shade-avoidance response, we subjected Col-0 plants inoculated with Pst or Bc to a low R:FR signal 1 day post-inoculation (dpi) and measured the petiole elongation in infected leaves over 24 h. Growth was inhibited in control light due to Pst infection, but the low R:FR-induced elongation response remained intact in Pst-inoculated plants (Figure 1e). Petiole growth in control light was not affected in leaves on which Bc spores had been placed (Figure 1f), although defence was induced in the petioles as measured by the induction of the marker gene PDF1.2 (Figure S2). Petiole elongation in response to low R:FR, however, was not affected by Bc inoculation (Figure 1f). These results demonstrate that an induced defence response against either a biotrophic or necrotrophic pathogen does not inhibit the low R:FR-induced elongation response.

The interaction between shade avoidance and defence is not a direct consequence of resource partitioning

It is possible that one response overrules the other through resource partitioning (Cipollini, 2004). Here, we measured the response of the defence overexpression mutants cpr1, which has constitutively induced SA-dependent defence, and cev1, which has constitutively activated JA and ET signalling, to low R:FR. As these two mutants constitutively express defence, induction of shade avoidance would indicate that there is no immediate energetic restraint for both responses to occur simultaneously. First, we verified that expression of the SA-responsive marker gene PR1 was unaffected by low R:FR in cpr1 (Figure 2a), indicating that the genetically programmed constitutive expression of SA-dependent defence in this mutant was not compromised by induction of a shade-avoidance response. Expression of the JA defence marker gene PDF1.2 was reduced in cev1 under low R:FR as compared with control light (Figure 2b). However, PDF1.2 expression was still over a 100-fold higher in cev1 than in its wild-type Col-5, showing that the JA pathway is still highly expressed in this mutant under low R:FR. Interestingly, although both cpr1 and cev1 were more disease resistant than wild-type, low R:FR increased susceptibility in both mutants (Figure S3). This result suggests that a response to phytochrome inactivation by low R:FR can affect pathogen defence even when the defence machinery is induced constitutively. We then measured whether the defence-overexpressing mutants were still able to respond to low R:FR. Both mutants displayed stunted growth under control conditions (Figure 2c,d), suggesting that the constitutively expressed defence response goes at the expense of general growth. However, in response to the low R:FR treatment, petiole elongation was still enhanced in cpr1 (Figure 2c), indicating that SA defence and shade avoidance can be expressed simultaneously. For cev1 an interaction effect was found between genotype and light treatment (Figure 2d), but when the effect of light treatment was tested in the subset of the cev1 genotype petiole elongation was increased significantly. Thus, constitutive activation of the JA-mediated defence pathway reduces low R:FR-induced petiole elongation, but does not inhibit fully the shade-avoidance response in the cev1 mutant. This situation may be facilitated by the constitutive activation of ET signalling, a positive regulator of the shade-avoidance response, in the cev1 mutant. Induction of the shade-avoidance marker gene PIL1 by low R:FR remained unchanged in both defence overexpressors (Figure 2e,f), indicating that shade-avoidance signalling was not compromised by constitutive overexpression of SA- or JA-dependent defence. The low R:FR-induced petiole elongation and PIL1 expression in combination with the overexpression of defence in cpr1 and cev1 shows that plants are able simultaneously to direct resources to shade avoidance and defence responses.

image

Figure 2. Marker gene expression and petiole elongation in the constitutive defence overexpressors cpr1 and cev1. (a) Defence marker gene expression in cpr1 (light grey bars) and (b) cev1 (black bars) and their wild-types Col-0 and Col-5, respectively (white bars) in control light and low red:far-red (R:FR) (= 4). (c, d) Petiole elongation after 24 h control light or low R:FR in cpr1 and cev1 (= 8). (e, f) Relative expression of the shade-avoidance marker gene PIL1 (n = 4). Data represent means ± standard error (SE). Asterisks indicate significant difference (two-way analysis of variance (anova), < 0.05).). L, light treatment; G, genotype; L:G, interaction between light treatment and genotype; NS, not significant.

Download figure to PowerPoint

Shade avoidance suppresses defence triggered by exogenous application of SA or MeJA

The increased susceptibility of shade-avoiding plants might be caused by conflicting responses of shade avoidance and pathogen defence. This outcome could be through antagonistic signal transduction steps or through downstream physiological consequences such as loosening cell walls for elongation versus cell wall fortification against pathogen penetration. Here, we investigated whether the interaction between pathogen defence and shade avoidance is also found when defence is induced by hormones. The SA pathway was induced with 0.5 mm SA, whereas the JA pathway was triggered with 0.1 mm methyl jasmonate (MeJA), and plants were then placed under low R:FR. SA-induced expression of PR1 was strongly suppressed by low R:FR treatment (Figure 3a). Likewise, induction of PDF1.2 was reduced in MeJA-treated plants placed under low R:FR (Figure 3b). Interestingly, basal levels of the defence genes were also reduced in mock-treated plants in low R:FR, as we found a significant effect of light treatment, but no interaction between light and hormone treatment (Figure 3a,b). Low R:FR-induced petiole elongation was not affected by application of SA, and neither was the expression of PIL1 (Figure 3c,e). Petiole elongation both in control light and low R:FR was reduced by application of MeJA as compared with mock-treated plants (Figure 3d). However, elongation was still enhanced when MeJA-treated plants were subjected to low R:FR (Figure 3d) and the induction of PIL1 was not affected by MeJA (Figure 3f). These results confirm that defence is inhibited by simultaneous induction of a shade-avoidance response, whereas the shade-avoidance response is not hindered by induction of defence responses.

image

Figure 3. Hormone-induced marker gene expression and petiole elongation under low red:far-red (R:FR). (a, b) Relative expression of defence marker genes in plants sprayed with 0.5 mm salicylic acid (SA) (light grey bars), 0.1 mm methyl jasmonate (MeJA) (black bars) or mock solution (white bars) and placed in control light and low red:far-red (R:FR) immediately after hormone treatment (= 4). (c, d) R:FR-induced petiole elongation after 24 h (= 8). (e, f) Relative expression of the shade-avoidance marker gene PIL1 (= 4). Data represent means ± SE. Asterisks indicate significant difference (two-way analysis of variance (anova), < 0.05). L, light treatment; H, hormone treatment; G, interaction between light treatment and hormone treatment; NS, not significant.

Download figure to PowerPoint

Low R:FR suppresses both SA- and JA-dependent genome-wide transcript profiles

It is striking that both the SA- and JA-dependent pathways are overruled by shade avoidance, whereas the SA and JA pathways can act antagonistically to each other (Kunkel and Brooks, 2002; Pieterse et al., 2012). As both shade avoidance and defence responses involve extensive transcriptional changes (Devlin et al., 2003; De Vos et al., 2005; Sessa et al., 2005; Wang et al., 2006), we used a genome-wide transcriptomics survey on plants simultaneously treated with low R:FR and SA or JA to study the interaction at the transcriptome level.

Two separate experiments were conducted for SA and MeJA treatments. In order to study events early in signalling while still having induction of the defence pathway, we harvested after 2 h of treatment. As expected, there was a large overlap in differentially expressed genes (DEGs) between the low R:FR treatment and the combined treatment of low R:FR and defence hormone (Figure 4). This situation was especially evident in the SA experiment, in which 78% of the genes regulated by low R:FR were also expressed differentially in the combined treatment with SA, as compared with 66% overlap in low R:FR-induced genes in the combined treatment with MeJA. The overlap in low R:FR-induced DEGs further confirms a minor effect of defence on the shade-avoidance response.

image

Figure 4. Microarray data analysis. (a) Differentially expressed genes in microarray experiment 2 h after treatment with 0.5 mm salicylic acid (SA); or (b) 0.1 mm methyl jasmonate (MeJA), low red:far-red (R:FR), or combination of defence-inducing hormone and low R:FR.3 replicate arrays were executed for each treatment.

Download figure to PowerPoint

Changes in gene expression as induced by SA single treatment were massively reduced by the addition of a low R:FR signal, as only 15% of all DEGs regulated by SA were also expressed in the combined treatment with low R:FR (Figure 4a). A substantial number of DEGs appeared exclusively in the combined treatment of SA and low R:FR, however gene ontology (GO) analysis of this subset showed no over-representation of genes linked directly to defence (Table S1). We therefore conclude that the SA-induced transcriptional defence response is reduced severely by simultaneous perception of low R:FR. 66% of the DEGs by MeJA treatment (including a number of defence-related genes) were still expressed in the combined treatment with low R:FR, although at a lower level in the combined treatment (Figure 5a,c and Table S2). Conversely, the expression levels of low R:FR-regulated genes that were also induced in the combined treatment of low R:FR and MeJA were highly similar in both treatments (Figure 5b,d and Table S2).

image

Figure 5. Linear regression of expression levels of two subsets of genes from the methyl jasmonate (MeJA) experiment in single and combined treatments. (a) Linear regression on log2 fold changes (log2 FC) of genes induced by MeJA alone or combined with low red:far-red (R:FR); and (b) of genes induced by low R:FR alone or in combination with MeJA. (c) Log2 FC of genes within GO categories belonging to jasmonic acid (JA) defence (GO IDs 9753, 9611, 51707, 19760, 9812 and 9695) as induced by MeJA alone or combined with low R:FR; and (d) of genes belonging to shade avoidance (GO IDs 9639, 9638, 10017, 10218 and 10202) as induced by low R:FR alone or in combination with MeJA. Genes plotted in (c) and (d) are specified in Table S2. Dotted line represents y = x. Asterisks indicate significant regression analysis (linear mixed model, P ≤ 0.001).

Download figure to PowerPoint

Surprisingly, there was hardly any functional over-representation in the genes that were significantly regulated exclusively in the combined treatments (Table S1; MeJA + FR only and SA + FR only). As both defence pathways may be suppressed by phytochrome signalling through a common mechanism, we compared the DEGs that were unique to the combined treatment of SA or MeJA with low R:FR. There was no overlap between the DEGs in the combined treatment of low R:FR with SA and those in the combination of low R:FR with MeJA (Figure S4), suggesting that low R:FR affects the transcriptome of the different defence pathways without inducing common transcriptional responses in the two combined treatments. Together, the microarray data indicate that low R:FR suppresses both the SA- and JA transcript profiles, whereby the MeJA-responsive transcript profile is more moderately reduced in expression level than the SA-responsive profile, which is almost completely abolished in low R:FR.

Low R:FR affects SA-dependent phosphorylation

The massive phytochrome-mediated reduction in DEGs of the SA-regulated transcript profile prompted us to investigate whether NPR1, the central activator of SA-induced transcription, is a target of low R:FR signalling. We compared the list of the 45 SA-responsive genes that were repressed by low R:FR with the microarray data from Wang et al. (2006), for which the NPR1-dependency of transcripts induced by the SA analogue benzothiadiazole S-methylester (BTH) was determined. This comparison revealed that the majority of low R:FR-inhibited DEGs was NPR1 dependent, suggesting that NPR1 may indeed (but not exclusively) be targeted by low R:FR signalling. This suggestion was confirmed by the reduction of the SA-induced expression of the direct targets of NPR1-mediated transcription WRKY18, WRKY38 and WRKY62 in low R:FR (Figure 6a). Additionally, GO analysis of the SA arrays indicated that the expression of a number of SA-inducible kinases was repressed by low R:FR (Table S1 and S3). Phosphorylation has been shown to be important for NPR1-dependent transcription (Spoel et al., 2009) and we hypothesized that low R:FR inhibits SA-dependent defence by targeting SA-induced phosphorylation cascades. We assessed whether induction of SA-responsive genes requires phosphorylation in a kinase inhibitor assay. SA-induced expression of both NPR1-dependent and -independent genes was inhibited by the global kinase inhibitor staurosporine (Figure 6b), which suggests that low R:FR signalling could indeed inhibit SA-induced transcription through suppression of SA-inducible kinases. To verify whether SA-induced phosphorylation is targeted by the shade-avoidance signalling pathway, we studied NPR1 phosphorylation in low R:FR. As NPR1 phosphorylation in the nucleus is preceded by NPR1 monomerization in the cytosol and translocation to the nucleus, we first studied whether SA-induced monomerization was affected by low R:FR. Surprisingly, NPR1 monomerization occurred in low R:FR even in the absence of SA and SA-induced monomerization was further enhanced by low R:FR than in control light (Figure 6c). The NPR1 monomer subsequently translocated to the nucleus as shown by confocal imaging (Figure 6d), showing that the SA pathway is inhibited by low R:FR despite NPR1 presence in the nucleus and an activating SA signal. Using an antibody specific against phosphorylated NPR1, we found that phosphorylation of NPR1 was clearly enhanced in plants treated with SA (Figure 6c). However, although plants in low R:FR contained increased amounts of monomer in the nucleus, phosphorylation was not enhanced proportionally in these plants, also not in the combined treatment with SA. These results indicate that NPR1 phosphorylation is compromised in low R:FR, confirming the hypothesis that SA-induced phosphorylation cascades could be important targets for the interaction between SA defence and shade avoidance.

image

Figure 6. Phosphorylation-dependent gene expression and NPR1 protein analysis. (a) WRKY gene expression in petioles from Col-0 plants sprayed with 0.5 mm SA after 24 h of control light or low red:far-red (R:FR). Data represent means ± standard error (SE) (= 3). Asterisks indicate significant difference. (b) Semi-quantitative salicylic acid (SA)-induced expression of NPR1-dependent (PR1, WRKY18, WRKY38,WRKY62) and NPR1-independent [NAC102 (Blanco et al., 2005), At4 g13180 (Wang et al., 2006)] genes in Col-0 and npr1-1 seedlings in liquid medium. Here, 4.2 μm staurosporine was added to inhibit kinase activity. (c) Total protein extracts from 35S::NPR1-GFP plants sprayed with mock solution or 0.5 mm SA in control light or low R:FR after 8 h of treatment. Protein separation by SDS-PAGE was done in the presence or absence of 50 mm DTT and analysed by western blotting using an anti-GFP antibody or an antibody specific against NPR1 phosphorylated at Ser11 and Ser15 (Spoel et al., 2009). O, NPR1 oligomer; M, NPR1 monomer; T, total NPR1. (d) Confocal imaging in petioles of 35S::NPR1–GFP plants sprayed with mock solution or 0.5 mm SA in control light or low R:FR after 8 h of treatment. Pictures represent a Z-stack from the epidermis inwards.

Download figure to PowerPoint

Discussion

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

Plants have developed a variety of plastic responses to survive adverse environmental conditions and our knowledge of the mechanisms underlying plant responses to single stresses is forever expanding. In the natural environment, however, especially combinations of stressful circumstances might occur. As crosstalk between inducible signalling pathways helps to fine tune the most suitable response in a complex environment, the understanding of a plant's response to its complex environment requires that stress responses be studied in combination (Mittler, 2006; Sultan, 2010). A few reports have described that plants that are shade avoiding are more susceptible to pathogens and, in particular, herbivores (Genoud et al., 2002; Izaguirre et al., 2006; Griebel and Zeier, 2008; Moreno et al., 2009; Cerrudo et al., 2012). Here, we extend these observed interactions and describe how the shade-avoidance pathway may affect defence signalling.

Shade avoidance is prioritised over pathogen defence

Whereas previous reports on interaction between light signalling and pathogen defence have focused mainly on the defence response, we have also measured the effect on shade avoidance. We found that when both stresses are induced simultaneously the shade-avoidance response to low R:FR is hardly affected, both at the levels of transcription and of the final elongation response (Figures 1-5). This finding implies that competition for light is prioritised over pathogen defence. Indeed, the shade-avoidance response is crucial for the plant's survival during competition for light, as a small difference in height with neighbouring vegetation results in an excessive difference in light capture (Ballaré et al., 1988; Pierik et al., 2003). Plants thus seem to have evolved a mechanism that ensures they can keep up with neighbours and maintain light interception for photosynthesis even when they are under attack by pathogens.

Pathogen defence is compromised by shade avoidance

In accordance with previous reports, we have found that the constitutive shade-avoidance mutant phyB has reduced SA-dependent resistance against Pst as well as reduced JA-dependent defence against Bc. Even when phytochrome signalling was modified through supplemental FR light simultaneously with pathogen inoculation, plants were more susceptible to both the SA- and JA-resisted pathogens (Figure 1). This result is important as it shows that the inhibition of the defence responses (which we observed in the phyB mutant, and others in phy mutants or prolonged FR pre-treatment) is not necessarily because shade avoidance was induced first – so the defence response could not be induced due to, for instance, resource partitioning. Indeed, even with constitutively induced defence, the cev1 and cpr1 mutants were still able to display enhanced elongation in low R:FR (Figure 2). Accordingly, C. album plants with induced defence due to previous herbivore attack were still able to elongate stems upon shading (Kurashige and Agrawal, 2005). These data show that under experimentally manipulated conditions plants can, in principle, express both a shade avoidance and a defence response. It remains to be investigated whether this situation also holds for a long-standing pathogen infection, in which the plant's resources might be more severely depleted or redirected compared with in our short-term experiments. Previously, it has been shown that the sav3-2 shade-avoidance mutant which does not display a shade-avoidance response to low R:FR still has reduced resistance against herbivores in low R:FR, despite the fact that this mutant does not direct resources towards elongation growth upon low R:FR (Moreno et al., 2009). This situation further hints towards a low R:FR-mediated reduction of defence that is not directly through resource partitioning.

The fact that both SA- and JA-induced pathogen defence responses are overruled by the shade-avoidance response is quite remarkable, as SA- and JA-dependent defence signalling have been shown often to be mutually antagonistic (Pieterse et al., 2012). The inhibition of both defence responses could be brought about through a common low R:FR-induced process or might be regulated through a specific mechanism for both defence pathways. The lack of any shared DEGs in the combined treatment of SA or MeJA with low R:FR that could indicate the induction of a similar downstream mechanism (Figure S4 and Table S1) implies that both defence pathways are each targeted specifically by the phytochrome pathway.

Low R:FR-induced repression of JA-dependent defence

The transcript profiles of SA- and JA-mediated defence were both suppressed by low R:FR, but in different ways. The numbers of genes in the JA profile were not reduced as rigorously as in the SA profile, but the expression levels of MeJA-dependent DEGs were generally decreased by low R:FR (Figure 5 and Table S2). A reduction in JA defence marker gene expression by low R:FR-induced phytochrome inactivation has been shown in Arabidopsis by Moreno et al. (2009) and in Lotus japonicus (Suzuki et al., 2011). JA-induced gene expression can also be inhibited by cytosolic NPR1 upon SA treatment (Spoel et al., 2003). Interestingly, this SA-dependent suppression of the JA response was shown to be dependent on SA-induced redox changes (Koornneef et al., 2008), which suggests that the enhanced monomerization of NPR1 that we observed in low R:FR might negatively affect the JA signalling pathway. However, decreased resistance to Bc after FR-pre-treatment was maintained in npr1 plants and in the SA biosynthesis mutant sid2 (Cerrudo et al., 2012), showing that suppression of JA-dependent pathogen defence by phytochrome inactivation is not dependent on SA and NPR1.

Reduced JA biosynthesis would be an obvious candidate for a reduced JA response by phytochrome signalling. JA levels in the phyAphyB Arabidopsis mutant, however, have been found to be comparable with wild-type controls, both in control conditions and after wounding (Zhai et al., 2007). From the five DEGs in our transcript profiling that are regulated by MeJA and fall into the GO category ‘JA biosynthesis’ (GO ID 9695) only ALLENE OXIDE CYCLASE (AOC)1, which encodes an enzyme that catalyzes an essential step in JA biosynthesis, is repressed. However, this situation could be due either to direct suppression of JA biosynthesis or a suppressed feed-forward loop in low R:FR.

The JAZ repressors of JA-induced transcription are likely targets for attenuation of the JA response. JAZs are degraded upon JA perception to allow JA-induced gene expression; the expression of the JAZ genes themselves is rapidly upregulated by a positive feedback loop to JA, herbivory and wounding (Thines et al., 2007; Chung et al., 2008). Moreno et al. (2009) found increased MeJA-induced expression of JAZ10 in low R:FR-pre-treated plants, although only at a relatively high MeJA concentration of 450 μm. Bc resistance was not found to be suppressed in two low R:FR-treated JAZ10 RNAi lines (Cerrudo et al., 2012), suggesting that JAZ10 indeed plays a role in the interaction. It will be interesting to see how phytochrome signalling affects protein stability of JAZ10 and other JAZs. In our microarrays without prolonged FR pre-treatment and a MeJA concentration of 100 μm, the expression of all MeJA-induced JAZs (including JAZ10) were somewhat reduced in the simultaneous treatment with low R:FR, except for JAZ1 (Figure S5).

Low R:FR-induced repression of SA-dependent defence

Transcription of SA-responsive genes was strikingly reduced in low R:FR (Figure 4). NPR1 has long been established as the central regulator of SA-induced defence, as npr1 mutants fail to raise an SA response. In the presence of SA, NPR1 oligomers in the cytosol are monomerized due to a change in the redox state of the cell and translocated to the nucleus. As the SA response is inhibited by low R:FR, it could be expected that NPR1 monomerization or translocation to the nucleus would be inhibited by phytochrome inactivation. However, we found the opposite: NPR1 monomerization was enhanced in low R:FR both with and without SA (Figure 6). This puzzling result implies that low R:FR affects the redox environment of the cell. It is well known that the two light harvesting complexes photosystems (PS) I and II have distinct pigment protein complexes, which preferentially absorb different wavelengths of the incident light. When the light environment becomes FR-enriched, this outcome will lead to enhanced excitation of the preferentially FR-absorbing PSI over PSII, which is more sensitive to R wavelengths. The imbalance of the relative number of photons captured by the two photosystems causes a shortage in electron supply from PSII to PSI. This outcome can lead eventually to adjustment of the stoichiometry of the photosystems to optimize photosynthesis quantum yield but may initially affect the redox state of the electron transport chain (Chow et al., 1990; Walters, 2005), which might lead to changes in the redox state of the cell and subsequently affect NPR1 monomerization.

Previously, it has been shown that two NPR1 cysteine mutants had constitutive NPR1 monomer accumulation and enhanced PR1 expression in the absence of elevated SA, indicating that monomeric NPR1 is sufficient to induce PR gene expression (Mou et al., 2003). Here, we show that, in low R:FR, NPR1 monomer accumulates and translocates to the nucleus but does not induce defence-associated genes (Figure 6). SA-induced transcription is inhibited even when SA is applied together with low R:FR, indicating that defence-associated transcription is repressed by low R:FR-induced phytochrome inactivation. NPR1 does not have a DBD and transcription of defence genes depends on its interaction with TGA transcription factors, whose binding activity to promoter elements requires interaction with NPR1 (Després et al., 2000; Fan and Dong, 2002). Whether in low R:FR the physical interaction between NPR1 and TGAs, the binding capacity of the TGAs to target genes, or recruitment of the initiation complex and RNA polymerase II are affected remains to be investigated.

Phosphorylation of NPR1 in the nucleus has been shown to be necessary for full expression of NPR1-mediated target genes (Spoel et al., 2009). We indeed found that phosphorylation of NPR1 did not increase proportionally to the increase in NPR1 monomers in low R:FR (Figure 6). If NPR1 is not phosphorylated and thus not cleared from the promoters of target genes, the transcription cycle might not be re-initiated and transcription could be stalled (Spoel et al., 2009). This situation would explain why NPR1-dependent transcription is inhibited by low R:FR, even though NPR1 monomer is present in the nucleus.

The repression of SA-induced transcription through repression of phosphorylation cascades appears not to be restricted to NPR1-dependent genes. GO analysis of the DEGs revealed that a number of SA-regulated genes whose products are involved in protein phosphorylation were no longer expressed in combination with low R:FR (Table S1). The fact that several SA-induced kinases were inhibited by low R:FR (Table S3) suggests that defence-related phosphorylation is a general target for the shade-avoidance pathway. Accordingly, we found that SA induction of NPR1-independent genes can also be repressed by inhibition of kinase activity (Figure 6b). Phosphorylation cascades play an important role in both SA and JA defence, as well as in abiotic stress signalling (Colcombet and Hirt, 2008) and are thus important for directing stress responses. Targeting of kinases specific for certain responses might be an important aspect of interactions between signalling pathways and thus in the regulation of plant multiple stress responses.

Experimental Procedures

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

Plant growth and treatments

Wild-type, phyB (Reed et al., 1993), cpr1 (Bowling et al., 1994) and 35S::NPR1-GFP (Kinkema et al., 2000) plants were in the A. thaliana Columbia (Col-0) background, cev1 was on the Col-5 background (Ellis and Turner, 2001). Seeds were sown on 1:2 potting soil:perlite substrate with additional nutrients (Millenaar et al., 2005) and stratified at 4°C for 3 days. Ten-day-old seedlings were transferred to individual pots of 70 ml soil. Plants were watered daily and grown in a 9-h light period of 180 μmol m−2 sec−1 at 20°C and 70% RH. 35S::NPR1-GFP plants were grown in a 16-h light period. Four-week-old rosettes were sprayed with 0.5 mm SA, 0.1 mm MeJA, or a mock solution containing 0.1% v/v ethanol such that the leaves were covered in a fine mist. Plants were left to dry before they were put in different light treatments. A red:far-red ratio (R:FR) of 0.2 was obtained through supplemental far-red LEDs (730 nm; Philips Green Power, http://www.philips.com) added to a control white light background (R:FR 2.2; Philips HPI-T Plus, 400 W), with PAR of 110 μmol m−2 sec−1. For elongation growth experiments one petiole per plant of approximately 5 mm in length was measured with a digital calliper at the start of the light treatment and 24 h later. Petioles of the same developmental age were used in microarray and real-time reverse transcription polymerase chain reaction (RT-PCR) assays.

Pathogen assays

The hemi-biotrophic bacterial pathogen Pseudomonas syringae pv tomato DC3000 was grown overnight in liquid King's B medium (King et al., 1954). Bacteria cells were collected and resuspended in 10 mm MgSO4. Three upper leaves of 4-week-old plants were pressure-infiltrated with bacterial suspension of OD600 = 6 × 10−4. Inoculated plants were transferred to the light conditions mentioned above and kept in 100% RH for 1 h to facilitate the infection. Two leaf discs (0.3 cm2 each) per plant were harvested in 10 mm MgSO4 3 days post-inoculation (dpi). Appropriate dilutions of homogenized samples were plated on King's B agar supplemented with 50 mg L−1 rifampicin, from which the number of colony-forming units per cm2 of infected leaf tissue could be determined after 48 h. For cpr1 bioassay and elongation growth measurements in Pst-inoculated leaves the plants were dipped in a bacterial suspension of OD600 = 0.02 and 0.015% Silwet L-77 and kept at high RH throughout the experiment.

The necrotrophic fungus Botrytis cinerea strain B0510 was grown on half-strength potato dextrose agar (PDA; Difco Laboratories) at 22°C. After 2 weeks, conidia were harvested in half-strength potato dextrose broth. A 5 μl droplet of 5 × 105 spores/ml suspension was placed on leaf laminas of 4-week-old plants, which were kept at 100% RH throughout the experiment. Lesion diameters were scored 3 dpi and divided into classes according to size.

RNA isolation and real-time RT-PCR

Petioles were harvested and snap frozen 2 h (microarray) or 24 h (real-time RT-PCR) after the start of light treatments. Each biological replicate consisted of three petioles pooled from three different plants. Total RNA was isolated from homogenized material using the RNeasy plant mini kit (Qiagen, http://www.qiagen.com) with on-column DNA digestion following the company's instructions. Total RNA was transcribed to cDNA by Superscript III reverse transcriptase (Invitrogen, http://www.invitrogen.com) using random hexamers at 50°C in a total volume of 20 μl containing 100 units of reverse transcriptase III, 4 μl of first-strand buffer, 40 units of RNase inhibitor (Qiagen) and 1 μl of 0.1 m dithiothreitol. Real-time RT-PCR was performed on a 20 μl reaction mixture containing 10 μl SYBR Green Supermix (Bio-Rad, http://www.bio-rad.com) in a Bio-Rad MyIQ single-colour real-time PCR detection system. Gene-specific primers were designed using the primer3plus software (Untergasser et al., 2007; sequences in Table S4). Gene expression was calculated with the 2−ΔΔCt method (Livak and Schmittgen, 2001) with UBQ5 as internal standard.

Transcriptional profiling

cDNA synthesis, cRNA synthesis and hybridization to ATH1 Affymetrix Arabidopsis Gene Chips were performed commercially by Service XS Leiden, The Netherlands (authorized service provider Affymetrix, http://www.affymetrix.com). Three biological replicates were used for each treatment. Microarray data were analysed using the Bioconductor packages in R (www.bioconductor.org). Data were normalized with the RMA algorithm and differential expression was assessed using the empirical Bayes method in the R LIMMA package (Smyth, 2004) and Benjamini–Hochberg multiple testing correction (Benjamini and Hochberg, 1995). As the separately performed JA and SA microarray experiments hybridized differently (Figure S6), we used different statistical cut-offs for both experiments. For the JA experiment (see Cerrudo et al., 2012), genes were considered differentially expressed when adjusted P-value < 0.003. For the SA experiment, an adjusted P-value <0.05 combined with a log2 fold change >1 was used to define DEGs. Gene ontology (GO) analysis was done with the BiNGO plugin of Cytoscape (Maere et al., 2005). Data are available at the NCBI gene expression and hybridization array data repository, Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo; accession no. GSE35700 and GSE45728).

Kinase inhibition assay

Col-0 and npr1-1 plants were grown on plates containing Murashige and Skoog (MS) medium (pH 5.7, supplemented with 20 g L−1 sucrose, 0.8% (w/v) agar, and 1 × Gamborg's vitamin solution). After 12 days, seedlings were submerged in solutions with or without 0.5 mm SA. To inhibit kinase activity, solutions were supplemented with 4.2 μm staurosporine (in 2% DMSO), while controls were treated with 2% DMSO alone. Seedlings were harvested after 2 h and 24 h and gene expression was analysed with semi-quantitative RT-PCR using gene-specific primers (Table S5).

Protein analysis

Frozen leaf tissue of 35S::NPR1-GFP plants was ground in extraction buffer (50 mm Tris–HCl (pH7.5), 150 mm NaCl, 5 mm EDTA, 0.1% Triton X-100, 0.2% Nonidet P-40) with inhibitors (40 μm MG132, 50 μg ml−1 TPCK, 50 μg ml−1 TLCK, 0.6 mm PMSF and 1% Sigma phosphatase inhibitor cocktail). Homogenized samples were then centrifuged (16 000 g) at 4°C for 20 min. SDS sample buffer was added to protein extracts, as well as 50 mm DTT for the reducing gel. After heating for 10 min at 70°C the protein samples were separated on SDS-PAGE gels and transferred to nitrocellulose membranes, which were probed with anti-GFP (Roche, https://www.roche-applied-science.com/) or an antibody specific against phosphorylated Ser11 and Ser15 residues of NPR1 (α-pS11/15) (Spoel et al., 2009).

Confocal imaging

NPR1–GFP fluorescence was visualised in 35S::NPR1–GFP plants using an inverted confocal laser scanning microscope (Zeiss LSM Pascal, ×40 C-apochromat objective) with an excitation wavelength of 488 nm, a 505–530 bandpath filter for GFP emission and a 560 nm long pass filter for red fluorescence visualisation by chloroplasts.

Statistical analysis

Data were analysed through analysis of variance (anova) or chi-squared test in the R statistical environment (R Development Core Team 2009).

Acknowledgements

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

We thank Saskia van Wees and Irene Vos for advice on the bioassays and Dr Xinnian Dong and Mindy Sponsel for providing the α-pS11/15 antibody. The feedback of two anonymous reviewers on a previous version of this manuscript was highly appreciated. Research was funded by The Netherlands Organization for Scientific Research (NWO, grant no. 021001030 to M.deW. and 86306001 to R.P.) and The Royal Society (grant Uf090321 to S.H.S.).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
  • Ballaré, C.L., Sanchez, R.A., Scopel, A.L. and Ghersa, C.M. (1988) Morphological responses of Datura ferox L seedlings to the presence of neighbors – their relationships with canopy microclimate. Oecologia, 76, 288293.
  • Ballaré, C.L., Scopel, A.L. and Sánchez, R.A. (1990) Far-red radiation reflected from adjacent leaves: an early signal of competition in plant canopies. Science, 247, 329332.
  • Benjamini, Y. and Hochberg, Y. (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Series B Methodol, 57, 289300.
  • Blanco, F., Garretón, V., Frey, N., Dominguez, C., Pérez-Acle, T., Van der Straeten, D., Jordana, X. and Holuigue, L. (2005) Identification of NPR1-dependent and independent genes early induced by salicylic acid treatment in Arabidopsis. Plant Mol. Biol. 59, 927944.
  • Bowling, S.A., Ailan, G., Hui, C., Gordon, A.S., Klessig, D.F. and Dong, X. (1994) A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance. Plant Cell, 6, 18451857.
  • Cao, H., Glazebrook, J., Clarke, J.D., Volko, S. and Dong, X. (1997) The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell, 88, 5763.
  • Cerrudo, I., Keller, M.M., Cargnel, M.D., Demkura, P.V., De Wit, M., Patitucci, M.S., Pierik, R., Pieterse, C.M.J. and Ballaré, C.L. (2012) Low Red:Far-Red ratios reduce Arabidopsis resistance to Botrytis cinerea and jasmonate responses via a COI1-JAZ10-dependent, salicylic acid-independent mechanism. Plant Physiol. 158, 20422052.
  • Chini, A., Fonseca, S., Fernández, G. et al. (2007) The JAZ family of repressors is the missing link in jasmonate signalling. Nature, 448, 666671.
  • Chow, W.S., Melis, A. and Anderson, J.M. (1990) Adjustments of photosystem stoichiometry in chloroplasts improve the quantum efficiency of photosynthesis. Proc. Natl Acad. Sci. USA 87, 75077511.
  • Chung, H.S., Koo, A.J., Gao, X., Jayanty, S., Thines, B., Jones, A.D. and Howe, G.A. (2008) Regulation and function of Arabidopsis JASMONATE ZIM-domain genes in response to wounding and herbivory. Plant Physiol. 146, 952964.
  • Cipollini, D. (2004) Stretching the limits of plasticity: can a plant defend against both competitors and herbivores? Ecology, 85, 2837.
  • Clack, T., Mathews, S. and Sharrock, R.A. (1994) The phytochrome apoprotein family in Arabidopsis is encoded by five genes: the sequences and expression of PHYD and PHYE. Plant Mol. Biol. 25, 413427.
  • Colcombet, J. and Hirt, H. (2008) Arabidopsis MAPKs: a complex signalling network involved in multiple biological processes. Biochem. J. 413, 217226.
  • De Vos, M., Van Oosten, V.R., Poecke, R.M.P.V. et al. (2005) Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack. Mol. Plant Microbe In. 18, 923937.
  • Després, C., DeLong, C., Glaze, S., Liu, E. and Fobert, P.R. (2000) The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors. Plant Cell, 12, 279290.
  • Devlin, P.F., Yanovsky, M.J. and Kay, S.A. (2003) A genomic analysis of the shade avoidance response in Arabidopsis. Plant Physiol. 133, 16171629.
  • Djakovic-Petrovic, T., De Wit, M., Voesenek, L.A.C.J. and Pierik, R. (2007) DELLA protein function in growth responses to canopy signals. Plant J. 51, 117126.
  • Ellis, C. and Turner, J.G. (2001) The Arabidopsis mutant cev1 has constitutively active jasmonate and ethylene signal pathways and enhanced resistance to pathogens. Plant Cell, 13, 10251033.
  • Faigón-Soverna, A., Harmon, F.G., Storani, L., Karayekov, E., Staneloni, R.J., Gassmann, W., Más, P., Casal, J.J., Kay, S. and Yanovsky, M.J. (2006) A constitutive shade-avoidance mutant implicates TIR-NBS-LRR proteins in Arabidopsis photomorphogenic development. Plant Cell, 18, 29192928.
  • Fan, W. and Dong, X. (2002) In vivo interaction between NPR1 and transcription factor TGA2 leads to salicylic acid-mediated gene activation in Arabidopsis. Plant Cell, 14, 13771389.
  • Fernandez-Calvo, P., Chini, A., Fernandez-Barbero, G. et al. (2011) The Arabidopsis bHLH transcription factors MYC3 and MYC4 are targets of JAZ repressors and act additively with MYC2 in the activation of jasmonate responses. Plant Cell, 23, 701715.
  • Finlayson, S.A., Lee, I.J., Mullet, J.E. and Morgan, P.W. (1999) The mechanism of rhythmic ethylene production in sorghum. The role of phytochrome B and simulated shading. Plant Physiol. 120, 10831089.
  • Fonseca, S., Chini, A., Hamberg, M., Adie, B., Porzel, A., Kramell, R., Miersch, O., Wasternack, C. and Solano, R. (2009) (+)-7-iso-Jasmonoyl-L-isoleucine is the endogenous bioactive jasmonate. Nat. Chem. Biol. 5, 344350.
  • Franklin, K.A. (2008) Shade avoidance. New Phytol. 179, 930944.
  • Franklin, K.A., Praekelt, U., Stoddart, W.M., Billingham, O.E., Halliday, K.J. and Whitelam, G.C. (2003) Phytochromes B, D, and E act redundantly to control multiple physiological responses in Arabidopsis. Plant Physiol. 131, 13401346.
  • Fu, Z.Q., Yan, S., Saleh, A. et al. (2012) NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants. Nature, 486, 228232.
  • Genoud, T., Buchala, A.J., Chua, N.-H. and Métraux, J.-P. (2002) Phytochrome signalling modulates the SA-perceptive pathway in Arabidopsis. Plant J. 31, 8795.
  • Griebel, T. and Zeier, J. (2008) Light regulation and daytime dependency of inducible plant defenses in Arabidopsis: phytochrome signaling controls systemic acquired resistance rather than local defense. Plant Physiol. 147, 790801.
  • Islam, S.Z., Honda, Y. and Arase, S. (1998) Light-induced resistance of broad bean against Botrytis cinerea. J. Phytopathol. 146, 479485.
  • Islam, S.Z., Babadoost, M., Bekal, S. and Lambert, K. (2008) Red light-induced systemic disease resistance against root-knot nematode Meloidogyne javanica and Pseudomonas syringae pv. tomato DC 3000. J. Phytopathol. 156, 708714.
  • Izaguirre, M.M., Mazza, C.A., Biondini, M., Baldwin, I.T. and Ballaré, C.L. (2006) Remote sensing of future competitors: impacts on plants defenses. Proc. Natl Acad. Sci. USA 103, 71707174.
  • Keuskamp, D.H., Pollmann, S., Voesenek, L.A.C.J., Peeters, A.J.M. and Pierik, R. (2010) Auxin transport through PIN-FORMED 3 (PIN3) controls shade avoidance and fitness during competition. Proc. Natl Acad. Sci. USA 107, 2274022744.
  • King, E.O., Ward, M.K. and Raney, D.E. (1954) Two simple media for the demonstration of pyocyanin and fluorescin. J. Lab. Clin. Med. 44, 301307.
  • Kinkema, M., Fan, W. and Dong, X. (2000) Nuclear localization of NPR1 is required for activation of PR gene expression. Plant Cell, 12, 23392350.
  • Koornneef, A., Leon-Reyes, A., Ritsema, T., Verhage, A., Den Otter, F.C., Van Loon, L.C. and Pieterse, C.M.J. (2008) Kinetics of salicylate-mediated suppression of jasmonate signaling reveal a role for redox modulation. Plant Physiol. 147, 13581368.
  • Kozuka, T., Kobayashi, J., Horiguchi, G., Demura, T., Sakakibara, H., Tsukaya, H. and Nagatani, A. (2010) Involvement of auxin and brassinosteroid in the regulation of petiole elongation under the shade. Plant Physiol. 153, 16081618.
  • Kunkel, B.N. and Brooks, D.M. (2002) Cross talk between signaling pathways in pathogen defense. Curr. Opin. Plant Biol. 5, 325331.
  • Kurashige, N.S. and Agrawal, A.A. (2005) Phenotypic plasticity to light competition and herbivory in Chenopodium album (Chenopodiaceae). Am. J. Bot. 92(1), 2126.
  • Li, L., Ljung, K., Breton, G. et al. (2012) Linking photoreceptor excitation to changes in plant architecture. Genes Dev. 26, 785790.
  • Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods, 25, 402408.
  • Lorenzo, O., Piqueras, R., Sanchez-Serrano, J.J. and Solano, R. (2003) ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense. Plant Cell, 15, 165178.
  • Lorenzo, O., Chico, J.M., Sanchez-Serrano, J.J. and Solano, R. (2004) JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis. Plant Cell, 16(7), 19381950.
  • Lorrain, S., Allen, T., Duek, P.D., Whitelam, G.C. and Fankhauser, C. (2008) Phytochrome-mediated inhibition of shade avoidance involves degradation of growth-promoting bHLH transcription factors. Plant J. 53, 312323.
  • Maere, S., Heymans, K. and Kuiper, M. (2005) BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics, 21, 34483449.
  • Millenaar, F.F., Cox, M.C.H., van Berkel-de Jong, Y.E.M., Welschen, R.A.M., Pierik, R., Voesenek, L.A.J.C. and Peeters, A.J.M. (2005) Ethylene-induced differential growth of petioles in Arabidopsis. Analyzing natural variation, response kinetics, and regulation. Plant Physiol. 137, 9981008.
  • Mittler, R. (2006) Abiotic stress, the field environment and stress combination. Trends Plant Sci. 11, 1519.
  • Moreno, J.E., Tao, Y., Chory, J. and Ballaré, C.L. (2009) Ecological modulation of plant defense via phytochrome control of jasmonate sensitivity. Proc. Natl Acad. Sci. USA 106, 49354940.
  • Morgan, D.C. and Smith, H. (1976) Linear relationship between phytochrome photoequilibrium and growth in plants under simulated natural radiation. Nature, 262, 210212.
  • Mou, Z., Fan, W. and Dong, X. (2003) Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes. Cell, 113, 935944.
  • Peng, J. and Harberd, N.P. (1997) Gibberellin deficiency and response mutations suppress the stem elongation phenotype of phytochrome-deficient mutants of Arabidopsis. Plant Physiol. 113, 10511058.
  • Pierik, R., Visser, E.J.W., De Kroon, H. and Voesenek, L.A.C.J. (2003) Ethylene is required in tobacco to successfully compete with proximate neighbours. Plant, Cell Environ. 26, 12291234.
  • Pierik, R., Whitelam, G.C., Voesenek, L.A.C.J., de Kroon, H. and Visser, E.J.W. (2004) Canopy studies on ethylene-insensitive tobacco identify ethylene as a novel element in blue light and plant-plant signaling. Plant J. 38, 310319.
  • Pieterse, C.M.J., Van der Does, D., Zamioudis, C., Leon-Reyes, A. and Van Wees, S.C.M. (2012) Hormonal modulation of plant immunity. Annu. Rev. Cell Dev. Biol. 28, 489521.
  • Pré, M., Atallah, M., Champion, A., De Vos, M., Pieterse, C.M.J. and Memelink, J. (2008) The AP2/ERF domain transcription factor ORA59 integrates jasmonic acid and ethylene signals in plant defense. Plant Physiol. 147, 13471357.
  • Rahman, M.Z., Khanam, H., Ueno, M., Kihara, J., Honda, Y. and Arase, S. (2010) Suppression by red light irradiation of Corynespora leaf spot of cucumber caused by Corynespora cassiicola. J. Phytopathol. 158, 378381.
  • R Development Core Team (2009) R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. ISBN 3-900051-07-0, URL http://www.R-project.org.
  • Reed, J.W., Nagpal, P., Poole, D.S., Furuya, M. and Chory, J. (1993) Mutations in the gene for the red/far-red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development. Plant Cell, 5, 147157.
  • Ryals, J., Weymann, K., Lawton, K. et al. (1997) The Arabidopsis NIM1 protein shows homology to the mammalian transcription factor inhibitor IκB. Plant Cell, 9, 425439.
  • Salter, M.G., Franklin, K.A. and Whitelam, G.C. (2003) Gating of the rapid shade-avoidance response by the circadian clock in plants. Nature, 426, 680683.
  • Sessa, G., Carabelli, M., Sassi, M., Ciolfi, A., Possenti, M., Mittempergher, F., Becker, J., Morelli, G. and Ruberti, I. (2005) A dynamic balance between gene activation and repression regulates the shade avoidance response in Arabidopsis. Genes Dev. 19, 28112815.
  • Smith, H. (2000) Phytochromes and light signal perception by plants - an emerging synthesis. Nature, 407, 585591.
  • Smith, H. and Holmes, M.G. (1977) The function of phytochrome in the natural environment? III. Measurement and calculation of phytochrome photoequilibria. Photochem. Photobiol. 25, 547550.
  • Smyth, G.K. (2004) Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article 3. doi: 10.2202/1544-6115.1027.
  • Spoel, S.H. and Dong, X. (2008) Making sense of hormone crosstalk during plant immune responses. Cell Host Microbe, 3, 348351.
  • Spoel, S.H., Koornneef, A., Claessens, S.M.C. et al. (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol. Plant Cell, 15, 760770.
  • Spoel, S.H., Mou, Z., Tada, Y., Spivey, N.W., Genschik, P. and Dong, X. (2009) Proteasome-mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity. Cell, 137, 860872.
  • Sultan, S.E. (2010) Plant developmental responses to the environment: eco-devo insights. Curr. Opin. Plant Biol. 13, 96101.
  • Suzuki, A., Suriyagoda, L., Shigeyama, T. et al. (2011) Lotus japonicus nodulation is photomorphogenetically controlled by sensing the red/far red (R/FR) ratio through jasmonic acid (JA) signaling. Proc. Natl Acad. Sci. USA 108, 1683716842.
  • Tada, Y., Spoel, S.H., Pajerowska-Mukhtar, K., Mou, Z., Song, J., Wang, C., Zuo, J. and Dong, X. (2008) Plant immunity requires conformational changes of NPR1 via S-nitrosylation and thioredoxins. Science, 321, 952956.
  • Tao, Y., Ferrer, J.-L., Ljung, K. et al. (2008) Rapid synthesis of auxin via a new tryptophan-dependent pathway is required for shade avoidance in plants. Cell, 133, 164176.
  • Thines, B., Katsir, L., Melotto, M., Niu, Y., Mandaokar, A., Liu, G., Nomura, K., He, S.Y., Howe, G.A. and Browse, J. (2007) JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling. Nature, 448, 661665.
  • Untergasser, A., Nijveen, H., Rao, X., Bisseling, T., Geurts, R. and Leunissen, J.A. (2007) Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res. 35, W71W74.
  • Vandenbussche, F., Pierik, R., Millenaar, F.F., Voesenek, L.A.C.J. and Van der Straeten, D. (2005) Reaching out of the shade. Curr. Opin. Plant Biol. 8, 462468.
  • Walters, R.G. (2005) Towards an understanding of photosynthetic acclimation. J. Exp. Bot. 56, 435447.
  • Wang, D., Amornsiripanitch, N. and Dong, X. (2006) A genomic approach to identify regulatory nodes in the transcriptional network of systemic acquired resistance in plants. PLoS Pathog. 2, 10421050.
  • Wang, H., Jiang, Y.P., Yu, H.J., Xia, X.J., Shi, K., Zhou, Y.H. and Yu, J.Q. (2010) Light quality affects incidence of powdery mildew, expression of defence-related genes and associated metabolism in cucumber plants. Eur. J. Plant Pathol. 127, 125135.
  • Wu, Y., Zhang, D., Chu, J.Y., Boyle, P., Wang, Y., Brindle, I.D., De Luca, V. and Després, C. (2012) The Arabidopsis NPR1 protein is a receptor for the plant defense hormone salicylic acid. Cell, 28, 639647.
  • Xu, L., Liu, F., Lechner, E., Genschik, P., Crosby, W.L., Ma, H., Peng, W., Huang, D. and Xie, D. (2002) The SCFCOI1 ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis. Plant Cell, 14, 19191935.
  • Zhai, Q., Li, C.-B., Zheng, W., Wu, X., Zhao, J., Zhou, G., Jiang, H., Sun, J., Lou, Y. and Li, C. (2007) Phytochrome chromophore deficiency leads to overproduction of jasmonic acid and elevated expression of jasmonate-responsive genes in Arabidopsis. Plant Cell Physiol. 48, 10611071.

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
tpj12203-sup-0001-FigureS1.epsimage/eps1356KFigure S1. Bc infection in plants grown at high density.
tpj12203-sup-0002-FigureS2.epsimage/eps441KFigure S2. Leaf response to Bc-inoculation.
tpj12203-sup-0003-FigureS3.epsimage/eps693KFigure S3. Disease incidence of the constitutive defence overexpressors cpr1 and cev1 in control light and low R:FR.
tpj12203-sup-0004-FigureS4.epsimage/eps477KFigure S4. Overlap in differentially expressed genes induced by combined treatment of SA and low R:FR and combined treatment of MeJA and low R:FR.
tpj12203-sup-0005-FigureS5.epsimage/eps305KFigure S5. Heatmap of log2 Fold Change expression values of JAZ genes in MeJA treatment and MeJA combined with low R:FR.
tpj12203-sup-0006-FigureS6.epsimage/eps1820KFigure S6. Volcano plots of JA and SA microarrays.
tpj12203-sup-0007-TableS1.pdfapplication/PDF87KTable S1. GO analysis of differentially expressed genes from SA- and MeJA microarrays.
tpj12203-sup-0008-TableS2.pdfapplication/PDF56KTable S2. Gene IDs corresponding to Figure 5c,d.
tpj12203-sup-0009-TableS3.pdfapplication/PDF175KTable S3. SA-induced kinases repressed by low R:FR.
tpj12203-sup-0010-TableS4.pdfapplication/PDF41KTable S4. Primer sequences (5′[RIGHTWARDS ARROW]3′) used for Real-Time RT-PCR.
tpj12203-sup-0011-TableS5.pdfapplication/PDF38KTable S5. Primer sequences (5′[RIGHTWARDS ARROW]3′) used for semi-quantitative RT-PCR.
tpj12203-sup-0012-Legends.docxWord document106K 

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.