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. 2014 Apr 10;9:e28745. doi: 10.4161/psb.28745

The interplay of transcription factors in suppression of UV-B induced flavonol accumulation by flg22

Dirk Schenke 1,*, Daguang Cai 1
PMCID: PMC4091570  PMID: 24721804

Abstract

Biotic stress can be mimicked by application of elicitors, which comprise of microbe-associated molecular patterns (MAMPs). Treatment of plant cell cultures with MAMPs such as flg22 suppressed the expression of UV-B-induced flavonol pathway genes (FPGs) in parsley, carrot and Arabidopsis. This is thought to allow the plant focusing its secondary metabolism on the pathogen defense during MAMP-triggered immunity (MTI). Recently we reported that this suppression also depends on prevention of histone 3 acetylation at lysine 9 (H3K9ac), a hallmark for gene activation. Here we describe a possible regulation between UV-B and flg22 signaling cascades, and the interplay of MYB and WRKY transcription factors in regulating the expression of the FPGs.

Keywords: crosstalk, UV-B, flg22, ChIP, H3K9ac, MYB, WRKY, HY5, histone modification

Introduction

Suppression of UV-B-induced flavonol pathway genes (FPGs) after activation of plant innate immunity is known for more than two decades and observed in both dicots and monocots. This is thought to benefit the plant by directing more phenylalanine precursor into metabolic branches for the production of phytoalexins and lignins.1,2 The production of phytoalexins and lignins, known to hinder the spread of the pathogen within the host tissue is important for basal resistance during MAMP (microbe-associated molecular pattern) induced immunity (MTI). Initial experiments (all supplemented with white light, VIS) using Arabidopsis cell cultures treated with water (control), flg22 (an 22 amino acid peptide from bacterial flagella as an elicitor to induce MTI / mimic pathogen infection), UV-B (to induce the FPGs) and co-treated with UV-B/flg22 (suppressing the UV-B induced FPGs), showed that UV-B protective metabolites, such as quercetin- and kaempferol-derivatives, were strongly reduced by the co-treatment when compared with the increased accumulation in response to UV-B treatment.2 Two MYB transcription factors (TFs), the positive regulator MYB12 (UV-B upregulated and flg22 suppressed) and MYB4, a negative regulator (activated by UV-B, but much faster by flg22) have been implicated in the regulation of the FPGs.2 Four genes (MYB12; chalcone synthase, CHS; chalcone-flavone isomerase, CFI and flavanone 3-hydroxylase, F3H) involved in flavonol biosynthesis have been investigated by chromatin immunoprecipitation (ChIP) and showed identical H3K9 acetylation (H3K9ac) pattern in response to the treatments described above, being all increased by UV-B and suppressed by flg22.3 Thus, our data point to the TFs MYB4 and MYB12 as well as H3K9ac playing a crucial role in regulation of the FPGs.2,3

UV-B Signaling

A breakthrough in the dissection of UV-B signaling was the identification of the UV-B receptor UVR8 in 2011.4 In its inactive state, the UVR8 forms a homodimer, which monomerizes upon UV-B stress and then translocates from the cytosol into the nucleus to interact with COP1, an E3 ubiquitin ligase.5,6,7 COP1, however, is regulated by white light, which explains why UV-B light alone is not effective to activate FPGs.6 In the nucleus the UVR8-COP1 complex formation involves also SPA1 (Suppressor of PHY A-105 1) and this stabilizes the bZIP transcription factor (TF) HY5 by preventing its degradation.6,7,8 HY5 is the master-regulator of the photomorphogenic UV-B responses, comprising the activation of the FPGs.7,9,10 In addition, the FPGs proved to be highly co-regulated with MYB12 during crosstalk between UV-B and MAMP-signaling, being strongly upregulated by UV-B irradiation and suppressed by co-treatment with flg22.2 This activation by UV-B depends on the main positive regulator, MYB12, which promoter is targeted by HY5 via binding to the ACE/G-box [CACGTG].10,11 MYB12 itself binds then to the MYB-recognition element or MRE-box [ACC(T/A)A(C/A)C] within the FPG promoters12 and both positive regulators, HY5 and MYB12, are thought to act in concert to activate the FPGs.2 Promoter analysis showed that MYB12 might be involved in activation of MYB4 expression, which in turn has a negative impact on MYB12, the FPGs and its own expression.2 To unravel the regulation of the FPGs and MYB12 in more detail, ChIP experiments were deployed to investigate changes in posttranslational histone modifications at four independent genomic loci (MYB12, CHS, CFI, and F3H).3 The UV-B treatment induced H3K9 acetylation at both promoter and open reading frame, while this hallmark of gene activation was suppressed by the co-treatment with flg22.3 Charron et al. (2009) reported, that H3K9ac increased at the HY5 locus after transition from dark to light,13 while this mark was suppressed at some light-regulated loci in the hy5–215 mutant.14 Thus, it is reasonable to believe that HY5 and/or its down-stream target MYB12 are responsible for H3K9 acetylation and that MYB TFs might indeed interact with histone acetyltransferases (HATs) or histone deacetylases (HDACs).3 Such histone modifications have an impact on the chromatin structure and since UVR8 was found to associate with chromatin at the HY5 and MYB12 loci, also UVR8 may influence the chromatin state.7,15 It is interesting to note, that CHS gene expression in response to UV-B required calcium ions, calmodulin, and protein phosphorylation as demonstrated by inhibitor studies with cell suspension cultures.9 Intriguingly, UV-B stress can induce a pathway distinct from UVR8 signaling, but depending on MAP kinase (MPK) phosphatase 1 that interacts with MPK3 and MPK6.16 Thus, both MPKs are activated by UV-B stress16 as well as during MTI17 as described below. Calcium and/or kinase activity might have a direct effect on the activity of HY5 or other TFs.18 However, as described in animal systems, calcium and phosphorylation can influence also the chromatin structure or DNA conformation,19,20 and thus they might serve as a prerequisite step in gene activation. This means that besides rendering chromatin generally accessible, a parallel induced signaling pathway is required for specific gene activation, e.g., by TFs binding to their corresponding cis responsive elements.

flg22-Signaling

The pattern recognition receptor (PRR) for the bacterial MAMP flg22 has been identified in 2000.21 FLS2 is a receptor like kinase (RLK) located in the plasmamembrane with an extracellular leucine-rich repeat (LRR) domain, necessary for flg22 binding and an intracellular serine/threonine kinase domain for downstream signaling.22 Also FLS2 needs complex formation to initiate MTI. It interacts with the Brassinosteroid-Insensitive1 associated receptor kinase1 (BAK1)23 and ligand-induced heteromerization with members of this kinase family appears to be generally required for MAMP induced defense responses.24 Another player in this complex formation is BIK1 (Botrytis-induced kinase1). After Fls2-BAK1 dimerization, the associated BIK1 kinase becomes phosphorylated, dissociates from the complex and contributes to initiation of a signal transduction cascade.25 This involves a Ca2+ burst that might activate Ca2+-dependent protein kinases (CDPKs) and the production of reactive oxygen species (ROS) by the activation of NADPH-oxidase AtRbohD, which is also required to amplify the Ca2+ signal in planta.26 Though there is no direct link of these events to mitogen-activated protein kinase (MAPK) cascade signaling, two MAPK cascades (MAP3K–MKK4/MKK5–MPK3/MPK6 and MEKK1–MKK1/MKK2–MPK4) have been identified as downstream components in flg22-signaling.17,27,28 Some WRKY TFs, such as WRKY33 could be directly linked to MAPK signaling via the interaction of MPK4 with the VQ protein MKS1,29 but probably WRKYs can be generally recognized by VQ motif containing proteins.30,31 It is generally believed that these WRKY TFs are activated to regulate plant stress responsive genes.32,33 Interestingly AtWRKY38 and AtWRKY62 have been reported to interact with HDAC19 as negative regulators of basal defense and might therefore also affect histone acetylation.34

Concluding Remarks

As reported in our previous study,2 the FPGs are highly co-regulated, including the 4CL3. All these genes contain two conserved cis elements in their promoters, a MRE and a G-box, being targets for MYB TFs (e.g., MYB4, MYB12) or bZIP TFs, such as HY5, respectively. Though there are single W-boxes in most of the FPG promoters, they might not be important targets for MTI-induced WRKY TFs, since the 4CL3 promoter sequence does not contain a W-box.2 Figure 1 shows a working model elucidating how UV-B-induced FPGs can be negatively affected during MTI. Both MYB TFs, the positive regulator MYB12 and the negative regulator MYB4 are conversely regulated by flg22 treatment in which MYB12 is strongly suppressed, while MYB4 is fast and strongly upregulated. Thus, flg22 signaling appears to co-opt the negative feedback mechanism of FPG regulation.2 Two scenarios are possible: 1) H3K9ac is prevented by fast activation of the negative regulator MYB4, suppressing the positive regulator MYB12 (assuming that MYB12 mainly recruits HAT activity to the FPG loci) or 2) H3K9ac is removed by HDAC activity recruited by e.g., MYB4. Perhaps, both scenarios take place in concert to fine-tune H3K9ac levels. The fact that MYB4 and MYB12 contain highly conserved W-boxes in their promoters suggests that both might be a target for flg22-induced WRKY TFs as shown in Figure 2. Since WRKYs comprise positive (activating) and negative (repressing) regulators,35 they could target the promoters of MYB4 (with 4 W-boxes) or MYB12 (containing 2 W-boxes) with different effects, respectively. If negatively acting WRKYs recruit HDAC activity to the MYB12 (and possibly other FPG loci), they might also contribute to the reduction of the activation mark H3K9ac in response to flg22. Though a model of the WRKY–DNA-binding interface already exists,36 it cannot be predicted in how far the arrangement and location of W-boxes within a given promoter has an impact on binding of positively or negatively acting WRKYs (Imre Somssich, MPIPZ Cologne, personal communication). To evidence this, CHIP-qPCR experiments with anti-all WRKY antibodies should be conducted to prove WRKY binding at the promoters of these two MYB TFs. Finally, molecular identification and functional characterization of these WRKYs, e.g., in Arabidopsis knockout mutants displaying impaired crosstalk will help to shed light on the signal crosstalk in plant responses to biotic and abiotic stresses.

graphic file with name psb-9-e28745-g1.jpg

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Figure 1. Working model displaying the interaction of transcription factors in regulation of the flavonol pathway genes (FPGs). The suppressive effect of flg22-induced signaling (MTI) on UV-B-activated FPG expression may be mediated by WRKY TFs via downregulating the positive regulator MYB12 and/or upregulating the negative regulator MYB4. The two MYB TFs are good candidates to recruit HAT or HDAC activity, respectively, thereby being responsible for changes in H3K9 acetylation at the FPG loci.

graphic file with name psb-9-e28745-g2.jpg

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Figure 2. In silico promoter analysis of the negative regulator MYB4 and the positive regulator MYB12. The arrows within the boxes, which represent cis responsive elements, indicate the orientation (forward or reverse complement).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Glossary

Abbreviations:

4CL

4-coumarate:CoA ligase

CFI

chalcone-flavone isomerase

CHS

chalcone synthase

ChIP

chromatin immunoprecipitation

F3H

flavanone 3-hydroxylase

flavonol pathway genes (FPGs)

H3, histone 3

H3K9ac

histone 3 lysine 9 acetylation

MAMP

microbe-associated molecular pattern

MTI

MAMP triggered immunity

UV

ultraviolet

VIS

visible (white) light

Schenke D, Cai D, Scheel D. Suppression of UV-B stress responses by flg22 is regulated at the chromatin level via histone modification. Plant Cell Environ. 2014:n/a–n/a. doi: 10.1111/pce.12283.

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