Research To grow or defend? Low red : far-red ratios reduce jasmonate sensitivity in Arabidopsis seedlings by promoting DELLA degradation and increasing JAZ10 stability Melisa Leone1, Mercedes M. Keller1, Ignacio Cerrudo1,2 and Carlos L. Ballar
e1,2 1 IFEVA, Consejo Nacional de Investigaciones Cient
ıficas y T
ecnicas–Universidad de Buenos Aires, C1417DSE Buenos Aires, Argentina; 2IIB-INTECH, Consejo Nacional de Investigaciones Cient
ıficas y T
ecnicas–Universidad Nacional de San Mart
ın, B1650HMP Buenos Aires, Argentina Summary Author for correspondence: Carlos L. Ballar e Tel: +54 11 4524 8070 Email: ballare@ifeva.edu.ar Received: 8 May 2014 Accepted: 2 July 2014 New Phytologist (2014) 204: 355–367 doi: 10.1111/nph.12971 Key words: DELLA, GAI1, gibberellin, immunity, jasmonate, JAZ10, photoreceptor, phytochrome.
How plants balance resource allocation between growth and defense under conditions of competitive stress is a key question in plant biology. Low red : far-red (R : FR) ratios, which signal a high risk of competition in plant canopies, repress jasmonate-induced defense responses. The mechanism of this repression is not well understood. We addressed this problem in Arabidopsis by investigating the role of DELLA and JASMONATE ZIM domain (JAZ) proteins.
We showed that a quintuple della mutant and a phyB mutant were insensitive to jasmonate for several physiological readouts. Inactivation of the photoreceptor phyB by low R : FR ratios rapidly reduced DELLA protein abundance, and the inhibitory effect of FR on jasmonate signaling was missing in the gai-1 mutant, which encodes a stable version of the GAI DELLA protein.
We also demonstrated that low R : FR ratios and the phyB mutation stabilized the protein JAZ10. Furthermore, we demonstrated that JAZ10 was required for the inhibitory effect of low R : FR on jasmonate responses, and that the jaz10 mutation restored jasmonate sensitivity to the phyB mutant.
We conclude that, under conditions of competition for light, plants redirect resource allocation from defense to rapid elongation by promoting DELLA degradation and enhancing JAZ10 stability. Introduction Under natural conditions, plants must strike a balance in the allocation of limited resources to different physiological activities, including growth and defense. For plants, growth is essential not only to accumulate resources that can be transferred to offspring, but also to position new resource-harvesting structures in places within the canopy or the soil in which resources are less contested by competitors. In turn, investment of resources in defense is critical to fend off attacks from a wide variety of consumer organisms that feed on plants. The fact that resources are limited, but their uses are multiple, creates resource allocation tradeoffs. The ‘growth vs defense’ allocation dilemma has received considerable attention in the ecological literature (Herms & Mattson, 1992; Agrawal, 2000; Cipollini, 2004; Izaguirre et al., 2006; Ballar
e, 2009). However, the molecular mechanisms used by plants to solve this dilemma and to produce allocation decisions that are adaptive and appropriate for each particular environment are not well understood. Recent work has demonstrated that part of the plant’s solution to this dilemma is based on the regulation of jasmonate (JA) signaling by the photoreceptor phytochrome (Ballar
e, 2011). It has Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust been shown that low red to far-red (R : FR) ratios, which indicate a high risk of competition in plant canopies, antagonize JA responses (Moreno et al., 2009; Cerrudo et al., 2012; De Wit et al., 2013; Izaguirre et al., 2013; Kegge et al., 2013; Cargnel et al., 2014; Chico et al., 2014). A down-regulation of JA responses reduces the expenditure of resources in defense, which presumably helps the plant to focus its fitness strategy on growth and morphological responses to overtop other plants. JAs are the principal hormones involved in the orchestration of plant defense against herbivores and necrotrophic pathogens (Howe & Jander, 2008; Browse, 2009; Wasternack & Hause, 2013). In turn, phytochrome, particularly phytochrome B (phyB), is the principal photoreceptor used by plants to detect the proximity of competitors (Ballar
e, 1999; Smith, 2000; Casal, 2012). phyB has two inter-convertible forms: Pfr, which is biologically active, and Pr, which is inactive. Pfr has an absorption maximum in the FR region of the spectrum, whereas Pr absorbs maximally in the R region. Light absorption leads to photoconversion between Pfr and Pr; therefore, under natural light conditions, the relative fraction of phyB molecules that are in the active Pfr form depends on the ratio of R to FR radiation (the R : FR ratio) (Smith, 1995). Green leaves strongly absorb R but New Phytologist (2014) 204: 355–367 355 www.newphytologist.com 356 Research not FR radiation; therefore, in plant canopies, a reduction in R : FR from the characteristic value of 1.2 to values < 1 is a good signal of a heightened risk of competition (Ballar
e et al., 1990). Plants of shade-intolerant species respond to this signal with increased apical dominance and a rapid acceleration of stem and petiole elongation, which allows them to position new leaves in well-illuminated, upper canopy strata. This reconfiguration of plant morphology, triggered by low R : FR and other competition signals, is often referred to as the shade-avoidance syndrome (SAS) (Smith, 1995). At the molecular level, SAS involves increased activity of several growth-promoting hormones, including auxin (Morelli & Ruberti, 2000; Roig-Villanova et al., 2007; Tao et al., 2008; Keuskamp et al., 2010; Hornitschek et al., 2012; Li et al., 2012) and gibberellins (GAs) (Garcia-Martinez & Gil, 2001; Djakovic-Petrovic et al., 2007; Kurepin et al., 2007). GAs act by promoting the proteasomal degradation of a family of growth-repressing proteins, known as DELLA proteins (Harberd et al., 2009). The JA pathway is activated in response to tissue damage caused by chewing insects and necrotrophic pathogens. This pathway begins with the release of a-linolenic acid from chloroplast membrane lipids, leading to the production of jasmonic acid (Wasternack & Hause, 2013). Jasmonic acid can then be conjugated to amino acids, such as isoleucine, to form the bioactive hormone, JA-Ile (Browse, 2009). Perception of the hormone is achieved by a co-receptor formed by the ubiquitin E3 ligase complex Skp1-Cul1-F-box protein CORONATINE INSENSITIVE 1 (SCFCOI1) and JA ZIM DOMAIN (JAZ) proteins. JA-Ile stimulates the specific binding of COI1 and JAZ proteins, which leads to ubiquitination of JAZs by SCFCOI1 and their subsequent proteasome-mediated degradation (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007, 2009; Melotto et al., 2008; Pauwels et al., 2010; Sheard et al., 2010). JAZ proteins block JA responses by repressing the activity of: critical transcription factors that regulate resistance to insects (Fern
andez-Calvo et al., 2011); anthocyanin biosynthesis and trichome initiation (Qi et al., 2011); and JA–ethylene interactions and resistance to necrotrophic pathogens (Zhu et al., 2011). Therefore, the degradation of JAZs triggers the activation of JA-induced defenses (Pauwels & Goossens, 2011; Kazan & Manners, 2012). Repression of JA responses by phyB inactivation has been shown in Arabidopsis and other species and for several defenses that include phenolic compounds, volatile terpenes and extrafloral nectar (reviewed in Ballar
e, 2014). This down-regulation is not a simple by-product of resource diversion to SAS (Moreno et al., 2009; Cerrudo et al., 2012; De Wit et al., 2013), and the available genetic (Cerrudo et al., 2012) and physiological (De Wit et al., 2013) evidence demonstrates that it is not caused by increased salicylic acid (SA) signaling. In contrast with the effect of SA as a repressor of JA responses, the effect of low R : FR ratios appears to occur at the level of the COI1–JAZ co-receptor module (Cerrudo et al., 2012). The mechanisms by which low R : FR ratios antagonize JA responses are unclear. One possibility is that phyB inactivation leads to a shift in the balance between JAZ and DELLA proteins (Ballar
e, 2014). Low R : FR ratios may result in increased GA New Phytologist (2014) 204: 355–367 www.newphytologist.com New Phytologist signaling (Garcia-Martinez & Gil, 2001; Kurepin et al., 2007), and prolonged treatments of low R : FR ratio have been shown to reduce the abundance of DELLA fusion proteins (RGA-GFP) in rapidly elongating Arabidopsis hypocotyls and petioles (Djakovic-Petrovic et al., 2007). DELLAs are positive regulators of JA responses (Navarro et al., 2008), because DELLAs bind to JAZs and prevent them from repressing their target transcription factors, such as MYCs (Hou et al., 2010; Yang et al., 2012). Therefore, increased DELLA turnover could make more JAZ proteins available for interaction with MYCs, and hence for the repression of JA-mediated defense responses (Hou et al., 2010; Yang et al., 2012; Huot et al., 2014). Although this model is attractive because of its simplicity, it is unclear whether DELLA turnover induced by low R : FR is sufficiently fast to explain the rapid effects of light quality on JA-induced gene expression (Moreno et al., 2009; Cerrudo et al., 2012; De Wit et al., 2013), and whether the degradation patterns detected in hypocotyls and petioles are representative of those that take place in organs in which the bulk of the defense responses are expressed (e.g. leaf laminas). Recent work has also indicated that supplemental FR radiation can increase the stability of JAZ proteins (Chico et al., 2014), but the evidence that this stabilization plays a functional role in dampening JA signaling is limited to the observation that the JAZ10 gene appears to be necessary for the effect of low R : FR ratios increasing plant susceptibility to the necrotrophic fungus Botrytis cinerea (Cerrudo et al., 2012). Supplemental FR radiation has been shown recently to accelerate the degradation of the transcription factor MYC2 (Chico et al., 2014). However, clear effects of low R : FR ratios reducing JA responses have also been demonstrated for genes that are not targets of MYCs (such as PDF1.2) (Moreno et al., 2009; Cerrudo et al., 2012; De Wit et al., 2013), suggesting that multiple mechanisms could connect phyB with JA signaling (Moreno & Ballar
e, 2014). In the experiments reported here, we investigate the mechanisms of repression of JA signaling by low R : FR ratios in Arabidopsis seedlings using two sets of readouts for the JA response, one connected with growth inhibition (hypocotyl elongation and biomass accumulation) and one related to defense (accumulation of leaf phenolics). We found that a quintuple della mutant and a phyB mutant are insensitive to JA for the physiological responses characterized in our study. We show that phyB inactivation triggers rapid DELLA degradation, and that this effect is required to suppress JA responses, because the inhibitory effect of FR is missing in the gai-1 mutant, which produces a version of the GAI1 DELLA protein that is resistant to GA-induced degradation. We further demonstrate that low R : FR ratios and the phyB mutation reduce the turnover of the JAZ10 protein, and that the JAZ10 gene is required for the inhibitory effect of low R : FR ratios or the phyB mutation on JA signaling. We conclude that, under conditions of high competition for light, shade-intolerant species, such as Arabidopsis, rapidly change the balance between DELLA and JAZ proteins, in favor of the latter, and that this change plays an important role in the reconfiguration of their resource allocation strategy, from defense to rapid elongation. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust New Phytologist Materials and Methods Plant material and growth conditions Surface-sterilized seeds of Arabidopsis (Arabidopsis thaliana (L.) Heynh) were germinated on 0.7% agar, 1.5% sucrose and Murashige and Skoog (MS) medium in glass jars with filtered vents that allowed gas exchange but prevented contamination at 22°C in a growth chamber (Supporting Information Fig. S1). White light (WL; photosynthetic photon flux density, 150 lmol m2 s1; R : FR ratio = 4.5) was provided by fluorescent lamps. Two photoperiods were used in the experiments, as indicated in the relevant figure legends: long days (LD), 14 h WL : 10 h darkness; short days (SD), 8 h WL : 16 h darkness. The phyB-9 (Reed et al., 1994), sav3-2 (Tao et al., 2008), RNAi7 (Yan et al., 2007), jaz8 (CS849856) (Jiang et al., 2014), jaz9-1 (SALK_004872C) (Yang et al., 2012), jaz10.1 (SAIL_92_D08), 35S::JAZ1-GUS (Thines et al., 2007), and the 35S::JAZ10-GUS (Chico et al., 2014) lines were all in the Col-0 background. The GA-insensitive gain-offunction gai-1 mutant (Koorneef et al., 1985) and the gai-t6 rga-t2 rgl1-1 rgl3-1 rgl2-1 quintuple della (59della, CS 16298-ABRC) (Keller et al., 2011) and gai-t6 rga-t2 rgl1-1 rgl2-1 quadruple della (49della) (Achard et al., 2006) knockout mutants were all in the Ler background. In most experiments, 12-d-old plants were used for measurements of hypocotyl length, fresh weight, phenolic compounds and protein stability. The double jaz10phyB mutant was obtained by crossing the phyB-9 and jaz10.1 mutants. Long-hypocotyl seedlings from the F2 generation were selected, and tested for resistance to glufosinate-ammonium (Basta, Bayer) and for JAZ10 genomic sequence integrity by PCR using the primers 50 -ATGTCGAAAGCTACCATAGAA-30 and 50 -TTAGGCCGATGT CGGATAGTA-30 (Chung et al., 2008). PCR products were resolved on a 2.5% agarose gel and plants without a product for this reaction were selfed until the F4 generation. The presence of the phyB-9 mutation was tested in the successive generations based on the long-hypocotyl phenotype and photomorphogenic behavior under R light. The transgenic line 35S::JAZ10-GUS was crossed with the phyB-9 mutant and long-hypocotyl F2 seedlings selected for hygromycin resistance and positive GUS staining. Selected plants were selfed and phenotypically tested until the F4, which used to perform the experiments. Light treatments Arabidopsis plants receiving 150 lmol m2 s1 WL were placed in front of banks of water-cooled incandescent lamps covered with either opaque screens (ambient ‘Amb’ light treatment) or FR-transmitting filters (‘FR’ treatment), as described by Moreno et al. (2009) (Fig. S1). The FR treatment reduced the R : FR ratio of the integrated horizontal radiation to c. 0.55. Previous studies in canopies of mustard (Sinapis alba) and chamico (Datura ferox) seedlings have indicated that this R : FR ratio in the horizontal light flux corresponds to a leaf area index of c. 0.5, in which mutual shading among neighboring plants is negligible (Ballar
e et al., 1991). Neither air temperature nor the level of WL received by the plants was affected by the FR treatment. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust Research 357 JA treatments Hormone treatments were performed by adding solutions of methyl-JA (MeJA) (Sigma) at the indicated concentrations on top of the medium in which plants were grown, following the protocols described in the relevant figures. Plants not assigned to the hormone treatment were treated with distilled water, which was supplemented with ethanol in the same proportion as that used to dissolve MeJA. Wounding treatments were carried out by applying pressure with a small forceps for 5 s on two leaves of the rosette. Measurements of growth, secondary metabolites and gene expression Hypocotyl growth was measured at the end of the experiment with a caliper; fresh weight was determined with an analytical balance. For measurement of soluble phenolic compounds, the seedlings were weighed and placed in 400 ll of acidified methanol (99 : 1, v/v) at 4°C for 48 h (Rabino & Mancinelli, 1986; Demkura et al., 2010) After the addition of 300 ll of deionized water, soluble phenolics were separated from chlorophylls by the addition of 700 ll of chloroform. The absorbance of the extracts was determined using a spectrophotometer (UV1700; Shimadzu, Kyoto, Japan), and A320 values were referred to fresh weight. These absorbance values correlated well with anthocyanin content and were less variable than the absorbance data at 530 nm. Total RNA was extracted from 100 mg of frozen tissue using the LiCl–phenol/chloroform method (Izaguirre et al., 2003). Purified fractions of total RNA were subjected to RQ1 (RNase-free) DNase treatment (Promega) to avoid contamination with genomic DNA. For cDNA synthesis, fractions of 2 lg of RNA were reverse transcribed using oligo(dT) as primer and M-MLV reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Quantitative real-time PCR (qPCR) was performed in a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s standard method for absolute quantification using FastStart Universal SYBR Green Master Mix (Roche Applied Science, Indianapolis, IN, USA) and primers at a final concentration of 500 nM. The A. thaliana UBIQUITIN (UBC) gene was used to normalize for differences in the concentrations of cDNA samples. Primer sequences were as follows: UBC, CTGCGACTCAGGGAATCTTCTAA (forward primer); TTGTGCCATTGAATTGAACCC (reverse primer); PDF1.2, TTGCTGCTTTCGACGCA (forward primer), TGTCCCACTTGGCTTCTCG (reverse primer); ERF1, CCT CGGCGATTCTCAATTTTT (forward primer), CCGAAAGC GACTCTTGAACTCT (reverse primer). b-Glucuronidase (GUS) histochemical staining and activity Seedlings were incubated in ice-cold 90% acetone for 20 min. The solution was changed to GUS staining solution (1 mM X-Gluc (5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid), 100 mM NaPi (sodium phosphate) buffer, pH 7.0, 10 mM EDTA, 0.1% (v/v) Triton X-100). The seedlings were incubated overnight at 37°C in the dark, followed by de-staining and New Phytologist (2014) 204: 355–367 www.newphytologist.com New Phytologist 358 Research clearing by several changes of 70% ethanol. Seedlings were inspected under a dissection microscope for staining intensity, and photographed. For the quantification of GUS activity, 12-dold seedlings were harvested, frozen in liquid nitrogen and ground in 50 ll of extraction buffer (phosphate buffer 50 mM, pH 7, Na2EDTA 10 mM, pH 8, sodium dodecylsulfate (SDS) 0.1%, Triton X100 0.1% and b-Me (2-mercaptoethanol) 4.32 mM). The tissue was stored at 80°C. GUS activity was measured by monitoring the cleavage of the b-glucuronidase 4methylumbelliferyl b-D-glucuronide (MUG) substrate (Jefferson et al., 1987) using a Beckman Coulter DTX 800/880 fluorometer (Pasadena, CA, USA). Total protein content (Lowry et al., 1951) was used to normalize GUS activity. Immunoblots Total proteins were extracted using plant protein extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% NP-40, 1 mM phenylmethylsulfonylfluoride (PMSF) and 1 9 protease inhibitor cocktail (Roche)). Protein content was quantified by the Bradford assay (Bradford, 1976). Equal amounts of protein were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting analysis. Immunodetection of RGA was performed using anti-RGA (Agrisera, AS11 1630, V€ann€as, Sweden). Rabbit antigoat immunoglobulin G horseradish peroxidase (IgG HRP) conjugate secondary antibody (BioRad) was used for detection. Statistics and data analysis Statistical analyses were carried out using INFOSTAT software (InfoStat/Professional version 1.1, Universidad Nacional de C
ordoba, C
ordoba, Argentina). Data were analyzed using factorial ANOVA, with light treatment, MeJA or genotype as factors. Differences between means were tested using a post-hoc Tukey test only when the relevant interaction terms were significant in the ANOVA. Appropriate transformations of the primary data were used when needed to meet the assumptions of the analysis. The number of independent experiments (n) used to calculate the treatment means are indicated in the relevant figure legends. In each experiment, 20 (growth and pigment analyses) or four to five (protein stability) individual seedlings were pooled to obtain the response value for each treatment/genotype combination. Therefore, the physiological results are based on measurements of between 100 and 200 seedlings per treatment/genotype combination, and the protein stability data (Western blot and GUS activity data) on tissue obtained from 15–25 seedlings per treatment/genotype combination. growth (biomass accumulation), and induced the accumulation of soluble leaf phenolics (putative defenses). These responses to JA required a functional COI1–JAZ co-receptor module (Fig. S2). For all growth and defense readouts, the JA effect was absent in plants grown under low R : FR (WL + FR) or in phyB mutants (Fig. 1b–d). The hypocotyl elongation results are in contrast with those reported in another study, where increased JA-induced growth inhibition was apparently stronger in phyB than in Col-0 (see Fig. S5 in Hou et al., 2010), but they agree well with those reported by Chen et al. (2013), and are consistent with the majority of the evidence indicating that phyB Pfr is a positive regulator of JA sensitivity (Ballar
e, 2011). The interactive effects of the R : FR ratio and MeJA treatments on growth and metabolite readouts (Fig. 1) were qualitatively similar to those obtained for responses at the gene expression level under our in vitro system (Fig. S3), and were therefore used throughout this study to evaluate phyB-induced changes in JA sensitivity. Growth and phenolic responses to JA require DELLA DELLA proteins are positive regulators of JA responses (Navarro et al., 2008; Hou et al., 2010; Yang et al., 2012). We tested the role of DELLAs in our system by comparing JA responses between wild-type (Ler-0) and quintuple della seedlings (59della).The Ler-0 wild-type responded to JA and low R : FR ratio in the same way as the Col-0 seedlings used as controls in previous experiments: treatment with 50 lM MeJA inhibited growth and promoted the accumulation of soluble phenolics, and low R : FR canceled the effect of JA on growth and defense (Fig 2a,c,e). Under the conditions used in our experiments, 59della mutant seedlings were not taller than Ler-0 seedlings. This is probably an effect of the LD photoperiod, because, under SD, 59della hypocotyls were c. 25% longer than those of Ler-0 seedlings (Fig. 2b). Regardless of photoperiod, MeJA failed to inhibit hypocotyl elongation (Fig. 2a,b) and biomass accumulation (Fig. 2c,d) in the 59della mutant. The mutant also failed to respond to MeJA with increased accumulation of leaf phenolics (Fig. 2e,f). The lack of growth inhibition in 59della is consistent with previous observations (Yang et al., 2012).The lack of a phenolic response to JA in 59della has not been reported previously. A quadruple della mutant (49della: gai-t6 rga-t2 rgl1-1 rgl2-1), showed a similar lack of sensitivity to JA for the physiological responses measured in this study (Fig. S4), suggesting that this lack of response to JA in 59della is not exclusively caused by the lack of RGA-LIKE3 (RGL3), which is known to be a positive modulator of JA signaling (Wild et al., 2012). Low R : FR ratios trigger rapid DELLA turnover Results JA inhibits seedling growth and promotes the accumulation of phenolic compounds, and these effects are eliminated by low R : FR ratios acting through phyB In de-etiolating 7-d-old Arabidopsis seedlings, JA treatment (50 lM MeJA) inhibited hypocotyl elongation and seedling New Phytologist (2014) 204: 355–367 www.newphytologist.com Previous work using pRGA::GFP–RGA (Djakovic-Petrovic et al., 2007) and pUBQ10::mCITRINE-RGA or pUBQ10::mCITRINEGAI (Y. Jaillais & J. Chory, unpublished) Arabidopsis plants demonstrated that low R : FR ratios reduce DELLA abundance in petioles and hypocotyls, presumably as a consequence of increased GA signaling. These previous experiments generally used prolonged irradiation treatments, and it is unclear whether Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust New Phytologist Research 359 (a) MeJA or mock 0 7d 0 +5 d +2 d WL (Amb) WL (Amb) WL + FR (FR) Harvest Germination L = 0.8647 JA = 0.8438 L x JA P = 0.9896 (b) Hypocotyl length (cm) 1.0 L x JA < 0.0001 0.8 0.6 0.4 c c b a 0.2 0.0 Amb FR Amb FR Col-0 (c) Fresh weight (mg) 25 phyB L x JA = 0.0065 b b 20 L = 0.8968 JA = 0.3650 L x JA = 0.4599 b 15 The effect of low R : FR ratios repressing JA-mediated responses is missing in a DELLA gain-of-function mutant, gai-1 Having determined that phyB inactivation causes a rapid decline in DELLA levels, we reasoned that DELLA degradation could mediate the effect of low R : FR canceling JA responses, as DELLAs can interfere with the ability of JAZ proteins to interact with their target transcription factors (Hou et al., 2010). We tested this hypothesis with the GA-insensitive, gain-of-function gai-1 mutant (Koorneef et al., 1985), which encodes a version (gai) of the GAI DELLA protein that is resistant to GA-induced degradation (Peng & Harberd, 1997). The effects of low R : FR ratio, canceling JA-induced growth inhibition (Fig. 4a,b) and the accumulation of leaf phenolics (Fig. 4c), were clearly missing in gai-1. 10 a JAZ10 links phyB inactivation with repression of JA sensitivity 5 0 Amb FR Amb Col-0 25 15 L = 0.6640 JA = 0.8681 L x JA = 0.2424 L x JA = 0.0187 b 20 a 10 FR phyB (d) Phenolics (Abs320 g–1 FW) the effects of low R : FR on DELLA degradation are sufficiently fast to account for the rapid effects of phyB manipulations on JA sensitivity. We used an anti-RGA antibody to monitor DELLA stability in whole seedlings under the experimental conditions described in Fig. 1a. Immunoblot analysis demonstrated that the low R : FR ratios caused a rapid (within minutes) reduction in RGA protein abundance, and that the phyB mutant had constitutively low RGA levels (Fig. 3). a a 5 0 Amb FR Col-0 Amb FR phyB Fig. 1 Experimental protocol and interactive effects of jasmonate (JA) and phytochrome B (phyB) inactivation on Arabidopsis growth and defense. (a) Schematic representation of the experimental protocol. Seedlings were germinated and grown for 7 d under white light (WL), and then transferred to the test light conditions: WL (Amb) or far-red (FR) (WL supplemented with FR radiation, WL + FR). Two days after transfer, seedlings were treated with methyl jasmonate (MeJA) (50 lM) or a mock solution, and left for three additional days under the light treatments before being harvested for the quantification of growth and defense responses. (b) Hypocotyl length. (c) Seedling biomass (fresh weight). (d) Accumulation of soluble phenolic compounds. Seedlings were grown under long days (14 h WL: 10 h darkness) unless indicated otherwise. Open bars, control; closed bars, JA. Error bars, +1 SE; n = 8–10. Significant terms in the factorial analysis of variance are indicated for each response variable with their associated P value: L, light treatment (Amb vs FR); JA (MeJA vs mock). When the L 9 JA interaction term was statistically significant, means were separated using the Tukey test, and different letters indicate significant differences between treatment means. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust It has been shown recently that the effect of simulated shadelight decreasing Arabidopsis resistance to the necrotrophic pathogen Botrytis cinerea is missing in lines disrupted in the expression of the JAZ10 gene (Cerrudo et al., 2012; see also Cargnel et al., 2014). Because plant defenses against necrotrophs are orchestrated by JA, we investigated the role of JAZ10 in the cross-talk between phyB and JA signaling. Previous work in de-etiolating Arabidopsis seedlings has demonstrated a requirement for phyA, a member of the phy family not involved in R : FR responses, in the degradation of another JAZ protein: JAZ1 (Robson et al., 2010). More recently, FR supplementation has been shown to reduce the turnover of several JAZ proteins (including JAZ1 and JAZ10) (Chico et al., 2014). We tested the effects of the R : FR treatment used in our physiological experiments on JAZ10 turnover in a transgenic line in which JAZ10–GUS was expressed under the 35S constitutive promoter (35S::JAZ10-GUS) (Chico et al., 2014). In preliminary experiments, we found that JAZ10– GUS levels were significantly higher in plants receiving light with a low R : FR ratio than in plants grown under WL. The effect of low R : FR increasing JAZ10 levels persisted even after 30 min of leaf wounding (Fig. 5). Seedlings treated with MeJA (20 lM) displayed a rapid turnover of JAZ10–GUS, as expected (Fig. 6). When the kinetics of JAZ10–GUS levels were measured after transferring Arabidopsis seedlings from high to low R : FR ratio (ambient light ? ambient light supplemented with FR), we found that GUS activity tended to accumulate if the seedlings were not exposed to MeJA. Moreover, FR supplementation counteracted the negative effect of MeJA (20 lM) on JAZ10–GUS New Phytologist (2014) 204: 355–367 www.newphytologist.com New Phytologist 360 Research LD 1.2 L x JA = 0.0195 1.0 b b b 0.8 L = 0.2348 JA = 0.5970 L x JA = 0.5369 a 0.6 0.4 0.2 0.0 Amb FR Amb 1.0 0.8 0.6 0.4 0.2 0.0 FR Amb FR Amb Ler-0 (c) FR 5xdella (d) Fresh weight (mg) L x JA = 0.0398 20 15 35 L = 0.7620 JA = 0.9916 L x JA = 0.6782 25 Fresh weight (mg) L = 0.1108 JA = 0.2457 L x JA = 0.4340 L = 0.0003 JA = 0.0631 L x JA = 0.3607 1.2 5xdella Ler-0 bc c 10 b a 5 L = 0.8874 JA = 0.2388 L x JA = 0.7714 L = 0.0001 JA = 0.0040 L x JA = 0.1393 30 25 20 15 10 5 0 0 Amb FR Amb Ler-0 FR Amb FR Amb Ler-0 5xdella (e) FR 5xdella (f) L x JA = 0.0065 15 L = 0.6185 JA = 0.4365 L x JA = 0.7466 Phenolics (Abs320 g–1 FW) c 15 Phenolics (Abs320 g–1 FW) SD (b) Hypocotyl length (cm) Hypocotyl length (cm) (a) b 10 ab a 5 0 b L x JA = 0.0011 L x JA = 0.0076 10 a a a ab ab b a 5 0 Amb FR Amb Ler Amb 5xdella Ler-0 (a) FR Ler PAC 5xdella FR Ler-0 Col-0 Col-0 FR 1 h Col-0 FR 6 h RGA (b) RGA protein 0.15 0.10 0.05 0.00 Col-0 Col-0 FR 30´ Col-0 FR 1 h Col-0 FR 6 h phyB Fig. 3 Far-red (FR) supplementation of white light triggers DELLA (RGA) protein degradation. (a) Turnover of the RGA protein in 12-d-old Arabidopsis seedlings treated with supplemental FR radiation for 1 or 6 h. The 59della mutant (Ler background) was used as a negative control; Ler + paclobutrazol (PAC), which is a gibberellin (GA) biosynthesis inhibitor, was used as a positive control. RGA protein was detected with an anti-RGA antibody. Image is from a representative experiment; Coomassie blue staining of the gel was used as loading control. (b) Quantification of RGA levels relative to the relevant loading controls at different times after treatment with supplemental FR and in the phyB mutant. Error bars, +1SE; values are means of three independent experiments (n = 3). New Phytologist (2014) 204: 355–367 www.newphytologist.com Amb 5xdella FR Fig. 2 DELLAs are required for Arabidopsis growth and defense responses to jasmonate (JA) under long day (LD; 14 h white light (WL) : 10 h darkness) and short day (SD; 8 h WL : 16 h darkness) conditions. Open bars, control; closed bars, JA. (a, b) Hypocotyl length. (c, d) Seedling biomass (fresh weight). (e, f) Accumulation of soluble phenolic compounds. Irradiation and methyl jasmonate (MeJA) treatment protocol as in Fig. 1(a). Error bars, +1 SE; n = 8. Significant terms in the factorial analysis of variance are indicated for each response variable with their associated P value: L, light treatment (Amb vs far-red (FR)); JA (MeJA vs mock). When the L 9 JA interaction term was statistically significant, means were separated using the Tukey test, and different letters indicate significant differences between treatment means. stability (Fig. 6). Finally, JA-induced degradation of JAZ10– GUS was slower in the phyB background that in the Col-0 background (Fig. 7). We found no evidence of an effect of alteration of R : FR on JAZ1-GUS protein stability (Fig. S5), suggesting that JAZ1 turnover is not affected by reductions in R : FR ratio that simulate the proximity of non-shading neighbors. We next used a genetic approach to test whether JAZ10 is required for the effect of low R : FR ratios decreasing JA sensitivity. We used a jaz10 mutant and an independent line in which JAZ10 expression was down-regulated by RNAi (Yan et al., 2007). In both genotypes (jaz10 and RNAi7), the effect of low R : FR ratio depressing JA responses was significantly attenuated or missing, and this was true for JA-induced growth inhibition and accumulation of phenolic compounds (Figs 8, S6). Interestingly, exploration of the phenotype of other jaz single mutants (jaz8, jaz9, see the Materials and Methods section) did not reveal FR-insensitive phenotypes (Figs S7, S8). These experiments demonstrate that the effect of low R : FR ratios reducing JA sensitivity requires JAZ10, which could provide a functional explanation for the bioassay results reported by Cerrudo et al. (2012). To further test the role of JAZ10, we introgressed the jaz10 mutation in the phyB background and tested the double mutant for JA response phenotypes. The phyB jaz10 double mutant displayed an elongated (SAS) phenotype that was essentially Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust New Phytologist Research 361 (a) L x JA P = 0.0382 Hypocotyl length (cm) 1.0 (a) L = 0.0358 JA < 0.0001 L x JA = 0.1024 b b 35s::JAZ10-GUS b 0.8 a 0.6 Amb 0.4 0.2 0.0 Amb FR Amb Ler-0 L x JA = 0.0003 Fresh weight (mg) 25 L = 0.3950 JA = 0.0214 L x JA = 0.6296 b b b 20 FR 15 10 a 5 0 Amb FR Phenolics (Abs320 g–1 FW) (c) 25 gai-1 1.0 0.8 0.6 0.4 0.2 0.0 0 L x JA P = 0.0007 b 15 a a a 5 0 Amb FR Ler-0 Amb FR gai-1 Fig. 4 DELLA (GAI) turnover is required for the inhibitory effect of low red : far-red (R : FR) ratios on jasmonate (JA) response in Arabidopsis seedlings. Open bars, control; closed bars, JA. (a) Hypocotyl length. (b) Seedling biomass (fresh weight). (c) Accumulation of soluble phenolic compounds. Irradiation and methyl jasmonate (MeJA) treatment protocol as in Fig. 1(a). Error bars, +1 SE; n = 6–8. Significant terms in the factorial analysis of variance are indicated for each response variable with their associated P value: L, light treatment (Amb vs FR); JA (MeJA vs mock). When the L 9 JA interaction term was statistically significant, means were separated using the Tukey test, and different letters indicate significant differences between treatment means. identical to that of the phyB simple mutant (Fig. 9a). Remarkably, however, the double mutant regained sensitivity towards JA for the markers analyzed (hypocotyl, growth inhibition and accumulation of soluble phenolics) (Fig. 9). These results demonstrate that jaz10 is epistatic to phyB for attenuation of JA sensitivity. Discussion Down-regulation of JA responses by low R : FR ratios is a central mechanism by which shade-intolerant plants redirect resources Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust 15 30 60 Time after wounding (min) L = 0.7999 JA = 0.0001 L x JA = 0.9217 20 10 FR Amb Ler-0 (b) GUS activity (nmol mg–1 min–1) (b) FR gai-1 Fig. 5 Low red : far-red (R : FR) ratios increase the levels of JAZ10-GUS and retard JAZ10-GUS degradation triggered by leaf wounding in Arabidopsis seedlings. (a) Photographs of 35S::JAZ10-GUS 2-wk-old plants taken 4 h after transfer to the white light (WL) + FR treatment (FR); seedlings that remained under WL (Amb) are shown for comparison. Plants were cultivated in Murashige and Skoog (MS) medium with 1% sucrose in clear plastic boxes under WL and short-day (SD: 8 h WL : 16 h darkness) conditions. Entire plants were carefully harvested and subjected to histochemical b-glucuronidase (GUS) staining (see the Materials and Methods section) and photographed. (b) Kinetics of JAZ10-GUS degradation after wounding seedlings of the FR (red) and Amb (black) treatments with a small forceps, as indicated in the Materials and Methods section. Bars, 1SE; n = 11–14. from defense into rapid growth when they face an increased risk of competition, which has important implications for crop health in modern agricultural settings (Ballar
e et al., 2012). Our experiments suggest that the molecular basis of this reduced JA sensitivity in plants exposed to low R : FR ratios involves a shift in the balance between DELLA and JAZ proteins, which results in repression of the JA pathway. The principal lines of experimental evidence supporting this model (Fig. 10) are discussed below. The rapid turnover of DELLA proteins, as demonstrated in this study for RGA (Fig. 3), could provide a mechanism by which phyB inactivation at high canopy density suppresses JA-induced defenses. Our data support this mechanism by showing that: (1) for several physiological outputs connected with growth (inhibition of hypocotyl elongation and fresh weight accumulation) and putative defense (accumulation of phenolic compounds), a 59della mutant was indistinguishable from the phyB mutant in terms of its lack of responses to MeJA (Fig. 2); and (2), the gai-1 New Phytologist (2014) 204: 355–367 www.newphytologist.com New Phytologist 362 Research (a) 0 12 d 30' (a) 1.5 WL (Amb) WL (Amb) Germination FR Amb (b) Harvest JA or mock Relative GUS activity WL+ FR (FR) 1.0 * * 0.5 0.0 0 (c) 15 30 Time after MeJA (min) 2.0 Relative GUS activity (b) 1.5 FR 1.0 35S::JAZ10GUS PHYB Amb FR JA 0.5 Amb JA 35S::JAZ10GUS phyB 0.0 0 30 Time after JA and/or FR (min) Fig. 6 Low red : far-red (R : FR) ratios increase JAZ10 protein stability in Arabidopsis seedlings. (a) Protocol used to evaluate the effects of light quality on JAZ10 kinetics. Seedlings were germinated and grown for 12 d under white light (WL) (14 h WL : 10 h darkness). Seedlings were treated with methyl jasmonate (MeJA; 20 lM) or a mock solution and transferred to the test light conditions: WL (Amb) or WL + FR (FR). After 30 min, the seedlings were harvested for measurement of b-glucuronidase (GUS) activity. (b) Representative photographs of 35S::JAZ10-GUS seedlings after 4 h under Amb or FR light conditions (no JA treatment). (c) Kinetics of JAZ10-GUS under the four combinations of light and hormone treatment. Values are given relative to the GUS activity value at time zero. Bars, 1 SE; n = 7. mutant, which encodes a stable version of the GAI DELLA protein (Peng & Harberd, 1997), failed to attenuate JA responses when exposed to low R : FR ratios (Fig. 4). The latter result suggests that, at least for the physiological outputs measured in this study, stabilization of GAI is sufficient to eliminate the low R : FR effect. It should be noted that, in older plants (4-wk-old), grown in soil, MeJA has measurable inhibitory effects on the growth of both phyB and 59della mutants (I. Cerrudo et al., unpublished). These results suggest that ontogeny or stress conditions may change the relative importance of DELLAs in the model proposed in Fig. 10 for the modulation of JA signaling. DELLA proteins positively regulate JA defense signaling (Navarro et al., 2008) by interfering with the ability of JAZ proteins to interact with their target transcription factors (Hou New Phytologist (2014) 204: 355–367 www.newphytologist.com Fig. 7 JAZ10 protein stability increases in the phyB background. (a) Dynamics of b-glucuronidase (GUS) activity in response to methyl jasmonate (MeJA). Arabidopsis seedlings were germinated and grown for 2 wk on Murashige and Skoog (MS) medium with 1% sucrose under white light (WL) (8 h WL : 16 h darkness). Seedlings were treated with MeJA (20 lM) or a mock solution and harvested at the indicated times for measurement of enzymatic activity. Activity values are given relative to the initial value. Black circles, 35S::JAZ10-GUS PHYB; red squares 35S::JAZ10GUS phyB. Bars, 1 SE; asterisks indicate significant differences between treatment means (P < 0.05; n = 14–18). (b) Representative images of 35S:: JAZ10-GUS PHYB (top) and 35S::JAZ10-GUS phyB (bottom) seedlings (note the elongated phenotype of the 35S::JAZ10-GUS phyB line). et al., 2010). Therefore, the rapid DELLA turnover triggered by low R : FR ratios documented here (Fig. 3) is predicted to release JAZ proteins from JAZ–DELLA complexes, thereby facilitating the repression of JA responses (Fig. 10). In addition to reducing DELLA abundance, low R : FR ratios will increase the availability of (growth-promoting) bHLH transcription factors, known as PHYTOCHROME INTERACTING FACTORS (PIF) (Lorrain et al., 2008; Hornitschek et al., 2012; Li et al., 2012). As PIFs interact with (and are repressed by) DELLAs (de Lucas et al., 2008; Feng et al., 2008), an increase in PIF protein levels under low R : FR ratios is predicted to decrease the number of DELLA molecules available for Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust New Phytologist Research 363 (a) 0.6 L x JA = 0.0132 c c 0.4 L < 0.0001 JA < 0.0001 L x JA = 0.8293 b a 0.2 Col-0 Amb FR FR Amb Col-0 jaz10 (b) Fresh weight (mg) 15 L x JA = 0.0244 L = 0.0338 JA = 0.0497 L x JA = 0.4048 b 10 b b 5 (b) a 1.0 G x JA ≤ 0.0001 d d d c 0.6 0.4 b b a a 0.2 0.0 Col-0 FR Amb Col-0 20 (c) jaz10 L = 0.0454 JA = 0.0028 L x JA = 0.9215 L x JA = 0.0043 b 15 a a a phyB jaz10 jaz10phyB FR Fresh weight (mg) Amb Phenolics (Abs320 g–1 FW) jaz10phyB 0.8 0 (c) phyB 0.0 Hypocotyl length (cm) Hypocotyl length (cm) (a) 10 20 c G X JA = 0.0155 bc 15 10 a a ab a a 5 a 5 0 Col-0 0 Amb FR Col-0 Amb FR jaz10 (d) phyB jaz10 jaz10phyB 50 c interaction with JAZ, which will result in further suppression of JA responses (Fig. 10). The repression of JA responses by low R : FR ratios is unlikely to be solely dependent on changes in DELLA availability. We found that the low R : FR treatment that is effective in suppressing JA responses (e.g. Fig. 1) produces an increase in JAZ10– GUS protein levels (Figs 5, 6), which is consistent with a recent report (Chico et al., 2014), and we further demonstrated that low R : FR ratios reduce the rate of JAZ10–GUS degradation elicited by wounding and MeJA treatment (Figs 5, 6). In the light of our physiological experiments (Figs 8, 9), this increased JAZ stability is predicted to have a significant effect, attenuating JA responses. It is unclear why we did not find stabilization of JAZ1-GUS by low R : FR, as reported by Chico et al. (2014), but the difference Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust Phenolics (Abs320 g–1 FW) G x JA = 0.0010 Fig. 8 Low red : far-red (R : FR) ratios fail to inhibit jasmonate (JA) responses in a jaz10 mutant. (a) Hypocotyl length. (b) Seedling biomass (fresh weight). (c) Accumulation of soluble phenolic compounds. Irradiation of Arabidopsis seedlings and methyl jasmonate (MeJA) treatment protocol as in Fig. 1(a). Open bars, control; closed bars, JA. Error bars, +1 SE; n = 6–8. Significant terms in the factorial analysis of variance are indicated for each response variable with their associated P value. L, light treatment (Amb vs FR); JA (MeJA vs mock). When the L 9 JA interaction term was statistically significant, means were separated using the Tukey test, and different letters indicate significant differences between treatment means. 40 b 30 20 b ab ab a b a 10 0 Col-0 phyB jaz10 jaz10phyB Fig. 9 The jaz10 mutation rescues the jasmonate (JA)-insensitive phenotype of a phyB mutant. (a) Photographs of 10-d-old Arabidopsis seedlings showing that the constitutive expression of the shade-avoidance syndrome (SAS) phenotype is similar in the simple phyB mutant and in the double jaz10 phyB mutant. Bar, 1 cm. (b) Hypocotyl length. (c) Seedling biomass (fresh weight). (d) Accumulation of soluble phenolic compounds. Irradiation and methyl jasmonate (MeJA) treatment protocol as in Fig. 1(a). Open bars, control; closed bars, JA. Error bars, +1 SE; n = 5. Significant terms in the factorial analysis of variance are indicated for each response variable with their associated P value: G, genotype; JA (MeJA vs mock). Primary data for fresh weight were log-transformed to meet the assumptions of the ANOVA. When the G 9 JA interaction term was statistically significant, means were separated using the Tukey test, and different letters indicate significant differences between treatment means. could be related to the use of different 35S:JAZ1-GUS lines, or to the fact that the FR treatment used by Chico et al. (2014) resulted in a much lower R : FR ratio (0.2) than that used in our New Phytologist (2014) 204: 355–367 www.newphytologist.com New Phytologist 364 Research (a) High R : FR (b) Low R : FR JA-Ile DELLA PIFs JAZ DELLA MYCs & other TFs PIFs Defense Growth, SAS JAZ10 MYC Fig. 10 A regulatory hub for growth and defense? Current model of the mechanism by which low red : far-red (R : FR) ratios affect the activity of key jasmonate (JA) signaling players and antagonize JA responses in Arabidopsis seedlings. (a) Under high R : FR, phyB Pfr levels are high, which reduce the levels and activity of PHYTOCHROME INTERACTING FACTORS (PIFs) (Lorrain et al., 2008; Hornitschek et al., 2012; Li et al., 2012; Park et al., 2012). PIFs are also inhibited by abundant DELLA proteins (Feng et al., 2008; de Lucas et al., 2008), which, in addition, keep JAZ proteins from repressing their target transcription factors, such as MYCs (Hou et al., 2010). Under these conditions, elongation growth is repressed and JA-mediated defense responses can be readily activated following JA-induced JAZ protein degradation. (b) Under low R : FR ratios, PIF levels and activity increase, and DELLAs are rapidly degraded (Fig. 3), thereby promoting the shade-avoidance syndrome (SAS). DELLA degradation frees up JAZ proteins, which are also stabilized (Fig. 6, see also Chico et al., 2014) and are therefore present and available to repress defense-activating transcription factors. Under these conditions, MYC levels can also be reduced (Chico et al., 2014), which further suppress defense responses. TF, transcription factor. experiments (0.55 in the radiation coming from the sides of the plant). Importantly, an additional layer of regulation uncovered by Chico et al. (2014) is the de-stabilization of MYC transcription factors under their low R : FR treatment, which should further contribute to the attenuation of JA responses (Fig. 10). There are 12 JAZ genes in the Arabidopsis genome, and genetic studies have revealed a great deal of functional redundancy among the various JAZ proteins. Single knockouts in the JAZ1, JAZ2, JAZ5, JAZ7 and JAZ9 loci failed to produce JA-related phenotypes in Arabidopsis (Thines et al., 2007; Demianski et al., 2012), although, in Populus, stabilization of a single JAZ protein (PtJAZ6) appeared to be sufficient to block JA signaling during the interaction between the plant and the mutualistic fungus Laccaria bicolor (Plett et al., 2014). In terms of interactions with DELLAs, several Arabidopsis JAZ proteins, including JAZ1, JAZ3, JAZ4, JAZ9, JAZ10 and JAZ11, have been shown to interact with different strengths with the conserved GRAS domain of DELLA proteins (Hou et al., 2010; Yang et al., 2012). We focused on JAZ10 because previous work had shown that the FR-induced increase in Arabidopsis sensitivity to the necrotrophic fungal pathogen B. cinerea was missing in New Phytologist (2014) 204: 355–367 www.newphytologist.com jaz10 and two RNAi lines disrupted for the expression of JAZ10 (Cerrudo et al., 2012; see also Cargnel et al., 2014). The present data clearly demonstrate that JAZ10 is required to mediate the repressive action of low R : FR on several JA responses (Figs 8, S6), and further show that the low sensitivity of phyB mutant seedlings to MeJA can be rescued by the jaz10 mutation, although the presence of the jaz10 mutation does not reverse the constitutive SAS phenotype of the phyB mutant (Fig. 9). Other jaz mutations tested in our experiments (jaz8 and jaz9) failed to produce an FR-insensitive phenotype (Figs S7, S8). Collectively the genetic evidence obtained in this study indicates that the protein JAZ10 plays a key role linking phyB inactivation with the attenuation of JA signaling in Arabidopsis seedlings. We do not know whether the apparently distinct function of JAZ10 in linking phyB and JA signaling is related to the fact that alternative splicing within the Jas domain results in two JAZ10 isoforms (JAZ10.3 and JAZ10.4) that are relatively stable in the presence of bioactive JA (Chung & Howe, 2009; Chung et al., 2010). If supplemental FR results in increased levels of these isoforms, this could hint at a plausible mechanism for JA de-sensitization. The availability of Arabidopsis lines in which the various splice variants of JAZ10 are expressed from the native JAZ10 promoter in the jaz10 mutant background (Moreno et al., 2013) provides tools to understand the potential role of these proteins in attenuating JA signaling under low R : FR ratios. Conclusion Down-regulation of JA signaling under light conditions that indicate a high risk of competition has been documented for a wide range of physiological responses in several species. Using a highly simplified setup to test for JA responses in young Arabidopsis seedlings, we conclude that DELLA turnover and increased JAZ10 stability play an important role in the molecular mechanism by which JA signaling is repressed in seedlings exposed to low R : FR ratios. The evidence obtained in this and other recent studies suggests that the regulation of JA responses by competition signals has multiple levels of control, which include: a reduced DELLA pool available for interactions with JAZ proteins; increased JAZ stability; and increased turnover of MYC transcription factors (Fig. 10). In addition, the effects of phyB inactivation have been shown to be local (i.e. restricted to the plant parts that experience a low R : FR ratio; Izaguirre et al., 2013). This regulation at multiple molecular levels and at a (local) modular scale may provide a powerful mechanism to fine tune the strength of the JA-mediated defense response as a function of the intensity and spatial distribution of the light signals that indicate a threat of competition. Acknowledgements This research was financially supported by grants from CONICET (Consejo Nacional de Investigaciones Cientı́ficas y T
ecnicas), ANPCyT (Agencia Nacional de Promoci
on Cientı́fica y Tecnol
ogica) and UBACyT (Universidad de Buenos Aires Ciencia y T
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Yang D-L, Yao J, Mei C-S, Tong X-H, Zeng L-J, Li Q, Xiao L-T, Sun T-P, Li J, Deng X-W et al. 2012. Plant hormone jasmonate prioritizes defense over growth by interfering with gibberellin signaling cascade. Proceedings of the National Academy of Sciences, USA 109: E1192–E1200. Zhu Z, An F, Feng Y, Li P, Xue L, Mu A, Jiang Z, Kim J-M, To TK, Li W et al. 2011. Derepression of ethylene-stabilized transcription factors (EIN3/EIL1) mediates jasmonate and ethylene signaling synergy in Arabidopsis. Proceedings of the National Academy of Sciences, USA 108: 12539–12544. Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Experimental setup used to test the interactive effects of the red : far-red (R : FR) ratio and jasmonate (JA) on growth and defense in Arabidopsis seedlings. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust New Phytologist Fig. S2 Methyl jasmonate (MeJA)-induced growth inhibition and accumulation of soluble phenolic compounds are missing in the coi1-1 mutant and in a transgenic line that constitutively expresses the JAZ10.4 splice variant of the JAZ10 protein (Chung & Howe, 2009). Fig. S3 Interactive effects of red : far-red (R : FR) ratio and jasmonate (JA) treatment on expression of JA marker genes in the experimental setup depicted in Fig. S1. Fig. S4 A quadruple della mutant (49della, gai-t6 rga-t2 rgl1-1 rgl2-1) has a jasmonate (JA)-insensitive phenotype that resembles that of the 59della (gai-t6 rga-t2 rgl1-1 rgl3-1 rgl2-1) mutant. Research 367 Fig. S6 Low red : far-red (R : FR) ratios fail to inhibit jasmonate (JA) responses in a RNAi line disrupted for the expression of the JAZ10 gene. Fig. S7 The effect of low red : far-red (R : FR) ratios antagonizing jasmonate (JA) responses was fully conserved in a jaz8 mutant. Fig. S8 The effect of low red : far-red (R : FR) ratios antagonizing jasmonate (JA) responses was fully conserved in the jaz9-1 mutant. Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Fig. S5 Low red : far-red (R : FR) ratios do not increase JAZ1 protein stability. New Phytologist is an electronic (online-only) journal owned by the New Phytologist Trust, a not-for-profit organization dedicated to the promotion of plant science, facilitating projects from symposia to free access for our Tansley reviews. Regular papers, Letters, Research reviews, Rapid reports and both Modelling/Theory and Methods papers are encouraged. We are committed to rapid processing, from online submission through to publication ‘as ready’ via Early View – our average time to decision is <25 days. 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