The bHLH Subgroup IIId Factors Negatively Regulate Jasmonate-Mediated Plant Defense and Development

<div><p>Plants have evolved sophisticated systems for adaptation to their natural habitat. In response to developmental and environmental cues, plants produce and perceive jasmonate (JA) signals, which induce degradation of JASMONATE-ZIM-Domain (JAZ) proteins and derepress the JAZ-repressed transcription factors to regulate diverse aspects of defense responses and developmental processes. Here, we identified the bHLH subgroup IIId transcription factors (bHLH3, bHLH13, bHLH14 and bHLH17) as novel targets of JAZs. These bHLH subgroup IIId transcription factors act as transcription repressors and function redundantly to negatively regulate JA responses. The quadruple mutant <i>bhlh3 bhlh13 bhlh14 bhlh17</i> showed severe sensitivity to JA-inhibited root growth and JA-induced anthocyanin accumulation, and exhibited obvious increase in JA-regulated plant defense against pathogen infection and insect attack. Transgenic plants overexpressing <i>bHLH13</i> or <i>bHLH17</i> displayed reduced JA responses. Furthermore, these bHLH factors functioned as transcription repressors to antagonize the transcription activators, such as MYC2 and the WD-repeat/bHLH/MYB complex, through binding to their target sequences. Coordinated regulation of JA responses by transcription activators and repressors would benefit plants by allowing fine regulation of defense and development, and survival in their frequently changing environment.</p></div

Expression patterns and subcellular localizations of bHLH3, bHLH13, bHLH14 and bHLH17.

<p>(A) <i>GUS</i> reporter gene was fused with the promoters of the four bHLH factors respectively to generate <i>Arabidopsis</i> transgenic plants (<i>P_bHLH3</i>-<i>GUS</i>, <i>P_bHLH13</i>-<i>GUS</i>, <i>P_bHLH14</i>-<i>GUS</i> and <i>P_bHLH17</i>-<i>GUS</i>). Histochemical GUS activity was detected in various tissues of transgenic seedlings. (B) Quantitative real-time PCR analysis of relative expression levels of <i>bHLH3</i>, <i>bHLH13</i>, <i>bHLH14</i> and <i>bHLH17</i> in root (R), stem (S), rosette leaf (RL), stem leaf (SL) and flower (F). <i>ACTIN8</i> was used as the internal control. Error bars represent SE (n = 3). (C) Subcellular localization of bHLH3, bHLH13, bHLH14 and bHLH17 in epidermal cells of <i>N. benthamiana</i> leaves. Constructs indicated on the left were infiltrated in leaves of <i>N. benthamiana</i>. GFP fluorescence was detected 50 hours after infiltration. The nuclei were indicated by DAPI staining. (D) Quantitative real-time PCR analysis of <i>bHLH3</i>, <i>bHLH13</i>, <i>bHLH14</i> and <i>bHLH17</i> in 11-day-old WT and <i>coi1-1</i> seedlings treated with 100 µM methyl-jasmonate (MeJA) for 0, 2, 6, and 10 hours. <i>ACTIN8</i> was used as the internal control. Error bars represent SE (n = 3).</p

bHLH3, bHLH13, bHLH14 and bHLH17 act as transcription repressors.

<p>(A) The schematic diagram shows the constructs used in the transient expression assays. (B) Transient expression assays show that the bHLH17 (indicated by H17) acts as a transcription repressor, while JAZ1 attenuates the bHLH17 repression function in a dosage-dependent manner. The MYC2 functions as a transcription activator, while JAZ1 represses the MYC2 activation activity. The GUS reporter and the internal control luciferase were cotransformed with the Control, bHLH17 (H17) and MYC2 effectors. The 0.2, 1, 5 or 10 µg JAZ1 plasmid DNA were respectively used for co-transformation with bHLH17 (H17). 10 µg JAZ1 plasmid DNA was used for co-transformation with MYC2 and Control. When JAZ1 was used for co-transformation with the Control vector only, the GUS activity was not affected, which is consistent with the previous observations that the negative regulator JAZ1 is not a transcription factor and cannot repress the promoter sequences (JAZ1 interacts with and represses its targeted proteins/transcription factors). Numbers on the brackets indicate the relative values of the GUS/LUC ratio of bHLH17 with JAZ1 to that of bHLH17. The GUS/LUC ratio represents the GUS activity relative to the internal control LUC. Error bars represent SE (n = 3). Asterisks represent Student's t-test significance between pairs indicated with brackets (*, P<0.05; **, P<0.01). (C) Transient expression assays show that bHLH3, bHLH13 and bHLH14 act as transcription repressors and their repression function can be attenuated by JAZ1. The GUS reporter and the luciferase (LUC) internal control were cotransformed with the indicated constructs (10 µg for each construct). Error bars represent SE (n = 3).</p

The <i>bhlh3 bhlh13 bhlh14 bhlh17</i> quadruple mutant exhibited enhanced JA responses.

<p>(A) Anthocyanin contents in the 11-day-old seedlings of WT, <i>bhlh3 bhlh17</i>, <i>bhlh3 bhlh13 bhlh17</i>, <i>bhlh3 bhlh13 bhlh14 bhlh17</i> (<i>Q4</i>) and <i>coi1-1</i> grown on MS medium containing indicated concentrations of MeJA. FW, fresh weight. Error bars represent SE (n = 3). (B) Quantitative real-time PCR analysis of <i>DFR</i>, <i>LDOX</i> and <i>UF3GT</i> expression levels in 11-day-old seedlings of indicated plants grown on MS medium supplied with 0 or 5 µM MeJA. <i>ACTIN8</i> was used as the internal control. (C) Phenotypes of 7-day-old (the upper panel, the middle panel that showed enlarged seedlings) and 11-day-old seedlings (the bottom panel) of WT, the quadruple mutant (<i>Q4</i>) and <i>coi1-1</i> grown on MS medium supplied without (CK) or with indicated concentrations (µM) of MeJA. (D) Root length of 11-day-old seedlings grown on MS medium containing indicated concentrations of MeJA. Error bars represent SE (n = 15). Asterisks denote Student's t-test significance compared with WT plants: **, P<0.01. (E) Quantitative real-time PCR analysis of <i>VSP1</i> expression level in WT, <i>Q4</i> and <i>coi1-1</i> seedlings grown for 11 days on MS medium supplied without (CK) or with 5 µM MeJA (JA). <i>ACTIN8</i> was used as the internal control. (F) Flowering phenotypes of five-week-old WT, <i>Q4</i> and <i>coi1-1</i> plants grown in a long-day growth chamber (16L/8D, 23°C). (G) Flowering time of WT, <i>Q4</i> and <i>coi1-1</i>. Data shown are the means from 24 plants. Error bars represent SE. Asterisks denote Student's t-test significance compared with WT plants: **, P<0.01. (H) Root phenotypes of 7-day-old WT, <i>Q4</i> and <i>coi1-1</i> seedlings grown on MS medium supplied without (CK) or with indicated concentrations (µM) of MeJA.</p

The bHLH3 and bHLH17 antagonize TT8/MYB75 and MYC2 to negatively regulate their downstream target genes.

<p>(A) The schematic diagram shows the constructs used in the transient transcriptional activity assays of (B) and (C). (B) Transient transcriptional activity assays show that activation of <i>DFR</i> promoter by TT8/MYB75 is repressed by bHLH17 in dosage-dependent manner. The <i>P_DFR-LUC</i> reporter was cotransformed with the indicated constructs. The 0.2, 1, 5 or 10 µg bHLH17 (H17) plasmid DNA were respectively used for co-transformation with TT8 (T8) and MYB75 (M75). Numbers on the brackets indicate the relative values of the LUC/REN ratio of TT8/MYB75 with bHLH17 to that of TT8/MYB75. The LUC/REN ratio represents the <i>P_DFR-LUC</i> activity relative to the internal control (<i>REN</i> driven by 35S promoter). Error bars represent SE (n = 3). Asterisks represent Student's t-test significance between pairs indicated with brackets (*, P<0.05; **, P<0.01). (C) Transient transcriptional activity assays show that activation of <i>DFR</i> promoter by TT8/MYB75 is repressed by bHLH3 and bHLH17. The <i>P_DFR-LUC</i> reporter was cotransformed with the indicated constructs (10 µg for each construct). Error bars represent SE (n = 3). Asterisks represent Student's t-test significance (**, P<0.01). (D) The schematic diagrams show the constructs used in the transcriptional activity assays of (E). (E) Transient transcriptional activity assays show that activation of <i>TAT1</i> promoter by MYC2 is repressed by bHLH3 and bHLH17. The <i>P_TAT1-LUC</i> reporter was cotransformed with the indicated constructs (10 µg for each construct). Error bars represent SE (n = 3). Asterisks represent Student's t-test significance (*, P<0.05; **, P<0.01).</p

JA responses were not obviously altered in the <i>bhlh3</i>, <i>bhlh13</i>, <i>bhlh14</i> and <i>bhlh17</i> single mutants.

<p>(A) Schematic diagrams of T-DNA insertion sites in <i>bHLH3</i>, <i>bHLH13</i>, <i>bHLH14</i> and <i>bHLH17</i>. White box, UTR; black box, exon; black line, intron; white triangle, T-DNA insertion site. Pair1 and Pair2, indicated by arrows, are the primer pairs for analyzing gene expression in (B). (B) Quantitative real-time PCR analysis of <i>bHLH3</i>, <i>bHLH13</i>, <i>bHLH14</i> and <i>bHLH17</i> in the respective T-DNA insertion mutants using primer pairs indicated by arrow pairs in (A). <i>ACTIN8</i> was used as the internal control. (C) Seedling phenotypes of 7-day-old (upper panel) and 11-day-old (bottom panel) Col-0 wild type (WT), <i>bhlh3</i>, <i>bhlh13</i>, <i>bhlh14</i> and <i>bhlh17</i> grown on MS medium supplied without (CK) or with indicated concentrations (µM) of MeJA. (D) Anthocyanin contents of the 11-day-old seedlings in WT, <i>bhlh3</i>, <i>bhlh13</i>, <i>bhlh14</i> and <i>bhlh17</i> single mutants grown on MS medium containing indicated concentrations of MeJA. FW, fresh weight. Error bars represent SE (n = 3). (E) Root phenotypes of 7-day-old seedlings of WT and single mutants of <i>bhlh3</i>, <i>bhlh13</i>, <i>bhlh14</i> and <i>bhlh17</i> grown on MS medium supplied without (CK) or with indicated concentrations (µM) of MeJA. (F) Root length of 11-day-old seedlings grown on MS medium containing indicated concentrations of MeJA. Error bars represent SE (n = 15). (G) Flowering time of WT, <i>bhlh3</i>, <i>bhlh13</i>, <i>bhlh14</i> and <i>bhlh17</i> single mutants. Data shown are the means from 24 plants. Error bars represent SE.</p

The bHLH subgroup IIId factors negatively regulate JA-mediated plant defense.

<p>(A) Disease symptoms of WT, the quadruple mutant (<i>Q4</i>) and the <i>coi1-1</i> mutant five days after spray inoculation with <i>Botrytis cinerea</i> or mock (CK) in the growth condition with ∼40% humidity. The enlarged leaves indicate obvious disease symptoms in WT, and severe disease symptoms <i>in coi1-1</i>. (B) Disease symptoms of WT, the quadruple mutant (<i>Q4</i>) and <i>coi1-1</i> mutant seven days after spray inoculation with <i>B. cinerea</i> or mock (CK) in the growth condition with high humidity (>90%). (C) Disease severity of the plants in (A) seven days after spray inoculation with <i>B. cinerea</i>. Disease rating was represented as no visible (0, white), weak (1, light grey), severe symptoms (2, grey), and completely dead plants (3, dark). Experiments were repeated three times with similar results. (D) Survival ratio of the plants in (B) nine days after spray inoculation. Error bars represents SE. Asterisks denote Student's t-test significance compared with WT plants: **, P<0.01. (E) Quantitative real-time PCR analysis of expression levels of <i>PDF1.2</i>, <i>Thi2.1</i>, <i>ERF1</i> and <i>LOX2</i> in WT and <i>Q4</i> germinated and grown on MS medium supplied without (CK) or with 5 µM MeJA (JA) for 11 days. <i>ACTIN8</i> was used as the internal control. (F) Disease symptoms on leaves of WT, <i>Q4</i> and <i>coi1-1</i> mutant three days after spray inoculation with <i>Pseudomonas syringae</i> pv. <i>tomato</i> (<i>Pst</i>) DC3000. (G) Growth of <i>Pst</i> DC3000 on WT, <i>Q4</i> and <i>coi1-1</i> mutant plants three days after spray inoculation as in (F). Bacterial counts are expressed as log (cfu/cm²). Error bars represent SE. The results are representative of three independent biological experiments. Asterisks represent Student's t-test significance compared with WT plants: **, P<0.01.</p

JAZ proteins interact with bHLH3, bHLH13, bHLH14 and bHLH17.

<p>(A) Yeast two-hybrid (Y2H) assay to detect interactions of JAZs with bHLH3, bHLH13, bHLH14 and bHLH17 factors. Twelve <i>Arabidopsis</i> JAZs were fused with the LexA DNA binding domain (BD) in pLexA respectively. The bHLH3, bHLH13, bHLH14 and bHLH17 were fused with the activation domain (AD) in pB42AD respectively. Interactions (represented by blue color) were assessed on 2% Gal/1% raffinose/SD/-Ura/-His/-Trp/-Leu/X-β-Gal medium. (B) Bimolecular fluorescence complementation (BiFC) assay to detect interactions of JAZ1 and JAZ10 (fused with nYFP) with bHLH3, bHLH13, bHLH14 and bHLH17 (fused with cYFP). Construct pairs were coexpressed in leaves of <i>N. benthamiana</i>. YFP fluorescence was detected 50 hours after infiltration. The nuclei were indicated by DAPI (4,6-diamidino-2-phenylindole dihydrochloride) staining. (C) Y2H assay to test interactions of bHLH13 domain constructs with twelve JAZs. The schematic diagram shows the bHLH13 domain constructs. The conserved JID domain and bHLH domain were shown with blue and gray box respectively. The numbers indicate the positions of amino acid. Different bHLH13 domains were fused with AD in pB42AD, and JAZs were fused with BD in pLexA. (D) Y2H assay to test interactions between different domains of JAZ8 and JAZ11 with bHLH3, bHLH13, bHLH14 and bHLH17 respectively. The schematic diagram shows the JAZ8 and JAZ11 domain constructs. The conserved ZIM and Jas domains are indicated by gray and green boxes respectively. Different domains of JAZ8 and JAZ11 were fused with BD, and these bHLH factors were fused with AD individually.</p

bHLH3 directly binds to promoter sequences of <i>TAT1</i> and <i>DFR</i> in ChIP-PCR assay.

<p>(A) ChIP-PCR analysis of the <i>in vivo</i> binding of myc-bHLH3 to the promoter of <i>DFR</i>. Chromatin from wild type plants (CK) and the transgenic plants constitutively expressing myc-bHLH3 (myc-bHLH3) was immunoprecipitated without (−) or with anti-myc antibody (+). Levels of the <i>DFR</i> promoter sequence (a 267-bp fragment containing two G-box motifs (CACGTG)) in the indicated chromatin were quantified by quantitative real-time PCR assay. PCR-amplification of a 77-bp 3′-UTR fragment of <i>DFR</i> was used as a negative control. The experiment was repeated three biological times with similar results. Error bars represent SE (n = 3). (B) ChIP-PCR analysis of the <i>in vivo</i> binding of myc-bHLH3 to the promoter of <i>TAT1</i>. Levels of the <i>TAT1</i> promoter sequence (a 111-bp fragment containing one G-box motif) in the indicated chromatin were quantified by quantitative real-time PCR assay. PCR-amplification of a 82-bp 3′-UTR fragment of <i>TAT1</i> was used as a negative control. The experiment was repeated three biological times with similar results. Error bars represent SE (n = 3).</p

A simplified model for coordinated regulation of JA responses by JAZ-targeted transcription activators and transcription repressors.

<p>(A) JAZ proteins interact with transcription activators (such as MYC2, TT8 and MYB75) and transcription repressors (including bHLH3, bHLH13, bHLH14 and bHLH17) to attenuate their transcriptional function. Upon perception of JA, SCF^COI1 recruits JAZs for ubiquitination and degradation via 26S proteasome. The JAZ-targeted transcription activators and repressors are then released to antagonistically and coordinately regulate their target genes (such as <i>TAT1</i> and <i>DFR</i>), which may further regulate JA responsive genes essential for various JA responses. (B) In wild type plant, both transcription activators and repressors, in response to JA signal, are released to regulate expression of their mutual target genes (such as <i>TAT1</i> and <i>DFR</i>). The balance between repression and activation would lead to an appropriate output of JA-responsive genes, resulting in an appropriate level of JA responses such as plant defense and anthocyanin accumulation. A, transcription activator; R, transcription repressor. (C) In the <i>bhlh3 bhlh13 bhlh14 bhlh1</i>7 quadruple mutant, the transcription repressors were severely abolished. The balance between repression and activation would lead to enhanced output of JA-responsive genes, resulting in increased JA responses. (D) In the plants overexpressing the bHLH subgroup IIId factors (OE), high level of the transcription repressors would exhibit strongest repression. The balance between repression and activation would lead to decreased output of JA-responsive genes, resulting in reduced JA responses.</p