A homeobox transcription factor HB34 suppresses jasmonic acid biosynthesis but promotes the expression of growth-related genes to balance plant immunity and growth in Arabidopsis

Abstract

Activation of plant immunity is commonly associated with the biosynthesis of defense-related metabolites, such as jasmonic acid (JA) and salicylic acid (SA). However, constitutive activation of the immune response or excessive accumulation of defense metabolites often negatively impacts plant growth. The regulatory mechanisms underlying the trade-off between plant immunity and growth remain elusive. In this study, we identified a homeobox transcription factor, HB34, as a key regulator that balances plant immunity and growth by differentially regulating the expression of JA biosynthetic and growth-related genes in Arabidopsis. Loss of HB34 enhances plant resistance to the necrotrophic pathogen Botrytis cinerea but impairs plant growth and development, accompanied by constitutive activation of JA-responsive genes. Mechanistically, HB34 negatively regulates the transcription of JA biosynthetic genes to suppress JA accumulation, and blocking JA biosynthesis attenuates the enhanced Botrytis cinerea resistance of hb34. Conversely, HB34 enhances the transcription of growth-related genes, whereas overexpression of these genes partially rescues growth defects, thereby decoupling the trade-off between enhanced defense and impaired plant growth. Our findings reveal a novel mechanism whereby a single transcription factor fine-tunes the trade-off between plant growth and immunity by differentially regulating JA biosynthetic and growth-related genes in Arabidopsis.

Keywords: Arabidopsis; HB34; growth-defense trade-off; jasmonic acid; transcriptional regulation.

Figure 1

Loss of HB34 enhances resistance to Botrytis cinerea (B. cinerea) but suppresses plant growth. (A) Representative leaves of WT and hb34 lines after B. cinerea infection. Drop-inoculation was performed on 4-week-old plants, and infected leaves were photographed at 2 days post-inoculation (dpi). Scale bar, 1 cm. (B) Lesion diameter of infected leaves. Lesion diameter was measured using ImageJ. Values represent means ± SD from 25 leaves per line. Statistical significance compared to WT was assessed using a two-tailed Student’s t-test (∗∗ p < 0.01). (C) Fungal biomass quantification by real-time PCR. DNA was extracted from infected leaves, and B. cinerea biomass was quantified using BcActin, normalized to AtActin2. Values represent means ± SD from three biological replicates. Statistical significance compared to WT was assessed by a two-tailed Student’s t-test (∗∗ p < 0.01). (D) Representative seedlings and hypocotyl length measurements. Values represent means ± SD from at least 25 seedlings per line. Scale bar, 3 mm. Statistical significance compared to WT was assessed by a two-tailed Student’s t-test (∗∗ p < 0.01). (E) Representative seedlings and leaf blade area of the indicated lines. Values represent means ± SD from at least 25 seedlings per line. Scale bar, 3 mm. Statistical significance compared to WT was assessed by a two-tailed Student’s t-test (∗∗ p < 0.01). (F) Representative seedlings and fresh weight measurements. Values represent means ± SD from at least 25 seedlings per line. Scale bar, 1 cm. Statistical significance compared to WT was assessed by a two-tailed Student’s t-test (∗∗ p < 0.01).

Figure 2

HB34 regulates a set of JA-responsive and growth-related genes in Arabidopsis. (A) Volcano plot showing differentially expressed genes (DEGs) in hb34-1 compared to WT from RNA-seq analysis. Red and blue dots represent up- and down-regulated genes, respectively. (B) GO enrichment analysis of up-regulated DEGs in hb34-1. (C) GO enrichment analysis of down-regulated DEGs in hb34-1. (D) RT-qPCR showing expression levels of representative genes in the indicated seedlings. Values represent means ± SD from three biological replicates. Different letters (a or b) indicate statistically significant differences determined by one-way ANOVA (p < 0.05). (E) Metagene plots showing average HB34 occupancy at transcription start sites (TSSs) and 2.0-kb flanking regions from ChIP-seq analysis. Input serves as a background control. RPGC, reads per genomic content. (F) Distribution of HB34-binding regions across genomic features. Regions corresponding to promoter TSS, exon, intron, intergenic, and transcription termination site (TTS) are shown. (G) GO enrichment analysis of genes bound by HB34. (H) Venn diagram showing the overlap between HB34-bound genes and up- or down-regulated DEGs in hb34-1.

Figure 3

Loss of HB34 results in constitutive activation of the JA response. (A) RT-qPCR showing HB34 expression levels in 12-day-old WT seedlings treated with 100 μM MeJA. Samples were harvested at the indicated time points. Values represent means ± SD from three biological replicates. Statistical significance compared to WT was assessed by two-tailed Student’s t-test (∗ p < 0.05; ∗∗ p  <  0.01). (B) RT-qPCR showing HB34 expression levels in WT seedlings after B. cinerea inoculation. Twenty-eight-day-old SD-grown plants were inoculated, and samples were collected at the indicated time points. Values represent means ± SD from three biological replicates. Statistical significance compared to WT was assessed by two-tailed Student’s t-test (∗ p < 0.05; ∗∗ p < 0.01). (C) RT-qPCR showing HB34 expression levels in 12-day-old seedlings of the indicated genotypes. Values represent means ± SD from three biological replicates. Statistical significance compared to WT was assessed by two-tailed Student’s t-test (∗ p < 0.05; ∗∗ p < 0.01). (D) Relative root growth inhibition in the indicated genotypes in response to MeJA treatment. Relative root growth was calculated as the ratio of root length under MeJA treatment to that without treatment. Different letters (a or b) indicate statistically significant differences determined by one-way ANOVA (p < 0.05). (E) Relative anthocyanin accumulation in the indicated genotypes in response to MeJA treatment. Relative anthocyanin accumulation was calculated as the ratio of anthocyanin levels with MeJA treatment to those without treatment. Different letters (a or b) indicate statistically significant differences determined by one-way ANOVA (p < 0.05). (F) RT-qPCR showing the expression levels of PDF1.2C, PDF1.3, PR3, AOC1, LOX2, and VSP2 in the indicated genotypes. Values represent means ± SD from three biological replicates. Different letters (a or b) indicate statistically significant differences determined by one-way ANOVA (p < 0.05).

Figure 4

HB34 negatively modulates the transcription of a set of JA biosynthetic genes. (A) Heatmap showing the expression levels of JA biosynthetic genes in WT and hb34-1 seedlings. (B) Integrated Genomics Viewer (IGV) snapshots and ChIP-qPCR results showing HB34 binding at the AOC1, LOX2, and OPR3 loci. IP #1 and IP #2 represent two biological replicates from ChIP-seq. Two independent ChIP-qPCR assays were performed in WT and p35S::HB34-GFP hb34-1 seedlings. Values represent means ± SD from three technical replicates. Statistical significance relative to WT was assessed by a two-tailed Student’s t-test (∗ p < 0.05; ∗∗ p < 0.01; n.s., no significance). (C) EMSA showing interactions between recombinant MBP-HB34 and the indicated DNA fragments. Competitive binding assays were performed using a 20-fold excess of unlabeled probes. (D) Transient dual-luciferase assays showing the effect of HB34 overexpression on the transcriptional activity of LOX2, AOC1, and OPR3 promoters. The promoters of LOX2, AOC1, and OPR3 were fused with luciferase (LUC) as reporters, while the full-length HB34 coding sequence under the 35S promoter served as the effector. p35S::GFP was used as a background control. Relative LUC activity (LUC:REN ratio) was normalized to REN activity, with the value for p35S::GFP set as 1. Statistical significance relative to the control was assessed by a two-tailed Student’s t-test (∗ p < 0.05; ∗∗ p < 0.01). (E) Expression levels of LOX2, AOC1, and OPR3 in HB34-ER seedlings under mock or estradiol treatment. Values represent means ± SD from three biological replicates, with Actin2 as the internal control. Statistical significance relative to the mock was assessed by a two-tailed Student’s t-test (∗ p  < 0.05; ∗∗ p < 0.01). (F) JA content in WT and hb34-1 seedlings. Values represent means ± SD from three biological replicates. Different letters (a or b) indicate statistically significant differences determined by one-way ANOVA (p < 0.05).

Figure 5

Blocking JA biosynthesis suppresses the increased resistance and growth defects in the hb34-1 mutant. (A and B) Representative leaves and lesion diameter of the indicated genotypes after B. cinerea infection. Values represent means ± SD from at least 25 plants per line. Scale bar, 1 cm. Different letters (a and b) indicate significant differences determined by one-way ANOVA (p < 0.05). (C) Quantification of fungal biomass by real-time PCR. DNA was extracted from infected leaves of the indicated plants. Values represent means ± SD from three biological replicates. Different letters (a and b) indicate significant differences determined by one-way ANOVA (p < 0.05). (D) RT-qPCR showing the expression levels of PDF1.2C, PDF1.3, LOX2, and AOC1 in the indicated genotypes. Values represent means ± SD from three biological replicates. Different letters (a and b) indicate significant differences determined by one-way ANOVA (p < 0.05). (E) Representative 5-day-old seedlings and hypocotyl length measurements of the indicated genotypes. Values represent means ± SD from at least 25 seedlings for each line. Scale bar, 3 mm. Different letters (a and b) indicate significant differences determined by one-way ANOVA (p < 0.05). (F) Representative 8-day-old seedlings and leaf blade area measurements of the indicated genotypes. Scale bar, 3 mm. Different letters (a and b) indicate significant differences determined by one-way ANOVA (p < 0.05). (G) Representative 18-day-old seedlings and fresh weight measurements of the indicated genotypes. Values represent means ± SD from at least 25 plants per line. Scale bar, 1 cm. Different letters (a and b) indicate significant differences determined by one-way ANOVA (p < 0.05).

Figure 6

HB34 promotes plant growth and development by enhancing the expression of a set of growth-related genes. (A) IGV and ChIP-qPCR results showing HB34 binding at the YUC8 and SAUR65 loci. IP #1 and IP #2 represent two biological replicates of ChIP-seq assays. Two independent ChIP-qPCR assays were performed in WT and p35S::HB34-GFP hb34-1 seedlings. Values represent means ± SD from three technical replicates. Statistical significance relative to WT was assessed by a two-tailed Student’s t-test (∗ p < 0.05; ∗∗ p < 0.01; n.s., no significance). (B) EMSA showing interactions between recombinant HB34 protein and the promoter fragments of YUC8 and SAUR65. Competitive binding was assessed by adding excess unlabeled probes. (C) Relative expression levels of YUC8 and SAUR65 in the indicated genotypes. Values represent means ± SD from three biological replicates, with Actin2 as the internal control. Different letters (a or b) indicate significant differences determined by one-way ANOVA (p < 0.05). (D and E) Transient dual-luciferase assays showing the effect of HB34 overexpression on the transcriptional activity of the YUC8 and SAUR65 promoters. Promoters of YUC8 and SAUR65 were fused to LUC as reporters. Relative LUC activity was normalized to that of REN, with p35S::GFP as the control. Statistical significance was assessed by a two-tailed Student’s t-test (∗ p < 0.05; ∗∗ p < 0.01). (F) Expression levels of YUC8 and SAUR65 in HB34-ER seedlings. Values represent means ± SD from three biological replicates. Statistical significance was assessed by a two-tailed Student’s t-test (∗ p < 0.05; ∗∗ p  < 0.01). (G) Representative 5-day-old seedlings and hypocotyl length measurements of the indicated genotypes. Values represent means ± SD from at least 25 seedlings per line. Scale bar, 3 mm. Different letters (a or b) indicate significant differences determined by one-way ANOVA (p < 0.05). (H) Representative 8-day-old seedlings and leaf blade area measurements of the indicated genotypes. Scale bar, 3 mm. Different letters (a or b) indicate significant differences determined by one-way ANOVA (p < 0.05). (I) Representative 18-day-old seedlings and fresh weight measurements of the indicated genotypes. Values represent means ± SD from at least 25 seedlings per line. Scale bar, 1 cm. Different letters (a or b) indicate significant differences determined by one-way ANOVA (p < 0.05). (J and K) Representative infected leaves and lesion diameter measurements of indicated lines after B. cinerea infection. Values represent means ± SD from at least 25 leaves per line. Scale bar, 1 cm. Different letters (a or b) indicate significant differences determined by one-way ANOVA (p < 0.05). (L) Fungal biomass of the indicated genotypes. Values represent means ± SD from three biological replicates. Different letters (a or b) indicate significant differences determined by one-way ANOVA (p < 0.05).

Figure 7

Proposed model for HB34 in regulating the trade-off between plant defense and growth. The homeobox transcription factor HB34 negatively regulates the expression of JA biosynthetic genes while positively modulating growth-related genes in Arabidopsis. Loss of HB34 enhances resistance to the necrotrophic pathogen B. cinerea but impairs plant growth and development. Under normal conditions, HB34 represses inappropriate activation of JA-responsive genes and prevents excessive JA accumulation, thereby promoting growth. Upon B. cinerea infection, HB34 transcription is downregulated, leading to de-repression of JA biosynthetic genes and rapid JA accumulation, which enhances immunity but compromises plant growth and development. These findings reveal that a single transcription factor fine-tunes the tradeoff between growth and immunity by differentially regulating JA biosynthesis and growth-related genes.