Transcription factors BBX11 and HY5 interdependently regulate the molecular and metabolic responses to UV-B

Abstract

UV-B radiation acts as a developmental cue and a stress factor for plants, depending on dose. Activation of the transcription factor ELONGATED HYPOCOTYL 5 (HY5) in a UV RESISTANCE LOCUS 8 (UVR8)-dependent manner leads to the induction of a broad set of genes under UV-B. However, the underlying molecular mechanisms regulating this process are less understood. Here, we use molecular, biochemical, genetic, and metabolomic tools to identify the B-BOX transcription factor B-BOX PROTEIN 11 (BBX11) as a component of the molecular response to UV-B in Arabidopsis (Arabidopsis thaliana). BBX11 expression is induced by UV-B in a dose-dependent manner. Under low UV-B, BBX11 regulates hypocotyl growth suppression, whereas it protects plants exposed to high UV-B radiation by promoting the accumulation of photo-protective phenolics and antioxidants, and inducing DNA repair genes. Our genetic studies indicate that BBX11 regulates hypocotyl elongation under UV-B partially dependent on HY5. Overexpression of BBX11 can partially rescue the high UV-B sensitivity of hy5, suggesting that HY5-mediated UV-B stress tolerance is partially dependent on BBX11. HY5 regulates the UV-B-mediated induction of BBX11 by directly binding to its promoter. BBX11 reciprocally regulates the mRNA and protein levels of HY5. We report here the role of a BBX11-HY5 feedback loop in regulating photomorphogenesis and stress tolerance under UV-B.

Figure 1

BBX11 expression is induced by UV-B. A, RT-qPCR analysis of BBX11 expression in 5-day-old Col-0 seedlings grown in constant white light and exposed to supplemental UV-B 2 (2.2 W/m²) (+UV-B) or not (−UV-B) for the indicated time period. GAPDH was used as internal control. Error bars represent sem, n = 3 (B) Histochemical staining of 5-day-old pBBX11:GUS seedlings grown in constant white light and exposed to supplemental UV-B (+UV-B) or not (−UV-B) for the indicated durations, Scale bar- 500 μm. In (A and B) fluence of UV-B used was 2.2 W/m² (C) RT-qPCR analysis of BBX11 expression in 5-day-old Col-0 seedlings grown in constant white light and exposed to supplemental UV-B of different doses UV-B 1 (0.91 W/m²), UV-B 2 (2.2 W/m²), UV-B 3 (3.9 W/m²), or not (−UV) for 6 h. GAPDH was used as internal control. Error bar represents sem, n = 3, Statistical groups indicated by letters were determined by one-way ANOVA, with Tukey’s test P ≤ 0.05. D, Histochemical staining of 5-day-old pBBX11:GUS seedlings grown in constant white light and exposed to supplemental UV-B 1, UV-B 2, UV-B 3, or not (−UV) for 10 h before staining. Scale bar: 500 μm.

Figure 2

BBX11 regulates UV-B mediated hypocotyl growth inhibition. A–C, Representative images (A), quantification of hypocotyl lengths (B) and relative hypocotyl lengths (C) of 5-day-old seedlings of the indicated genotypes grown in constant white light supplemented with UV-B 1 (+) or not (−). Scale bar in (A) indicates 2 mm. In (B) and (C), error bars represent sem, n = 3. Statistical groups indicated by letters were determined by two-way ANOVA, with Sidaks’s multiple comparison test, P ≤ 0.05.

Figure 3

BBX11 protects plants from UV-B radiation. A, 4-day-old Col-0, bbx11-1, and OE3 seedlings grown on plates under constant white light were treated with supplemental UV-B 3 (+UV-B) or not (−UV-B) for 30 h and allowed to recover under white light for 1 day before imaging. B, quantification of seedling survival percentage, calculated as mentioned in methods. Error bars represent sem, n = 4, statistical groups indicated by letters were determined by one-way ANOVA, with Tukey’s test P ≤ 0.05. C, Plants of the indicated genotypes were grown in soil under long-day conditions (16/8 h) in white light for 18 days and exposed to supplemental UV-B 3 (+UV-B) or not (−UV-B) for 3 days and allowed to recover under white light for 5 days before being photographed.

Figure 4

BBX11 promotes accumulation of photo-protective metabolites. A, Heat map showing the relative accumulation of central and secondary metabolites and (B) relative accumulation of phenolic acids, in 18-day-old seedlings of the indicated genotypes grown in constant white light and exposed to 0, 24, and 96 h of supplemental UV-B 3 before harvesting. In (A) heat map scale (+3 to −3) indicates abundance of metabolite. Four biological replicates were taken for the analyses from each of the treatments. In (B) error bars represent sem, n = 4. Statistical groups indicated by letters were determined by one-way ANOVA, with Tukey’s test P ≤ 0.05.

Figure 5

HY5 regulates the UV-B-mediated induction of BBX11 by directly binding to its promoter. A, RT-qPCR analysis of BBX11 expression in 5-day-old Col-0 and hy5-215 seedlings grown in constant white light and exposed to supplemental UV-B 2 (+) or not (−) for 6 h. GAPDH was used as internal control. Error bars represent sem, n = 3. Statistical groups indicated by letters were determined by one-way ANOVA, with Tukey’s test, P ≤ 0.05 (B) ChIP-qPCR analyses of HY5 binding to the BBX11 promoter in vivo. Fourteen-day-old Col-0 and hy5-215 seedlings grown in constant white light and exposed to supplemental UV-B 3 (+) or not (−) for 3 h before crosslinking. DNA–protein complexes were immunoprecipitated using antibodies against HY5 (anti-HY5) and rabbit IgG as negative control (IgG). ChIP DNA was quantified by RT-qPCR with primers specific to the previously known HY5-binding site. Error bars represent sd, n = 2.

Figure 6

BBX11 acts partially independent of HY5 to regulate photomorphogenesis and stress tolerance under UV-B. A–C, Representative images (A) quantification of hypocotyl lengths (B) and relative hypocotyl lengths (C) of 5-day-old seedlings of the indicated genotypes grown in constant white light supplemented with UV-B 1 (+) or not (−). Scale bars in (A) indicate 2 mm. In (B) and (C), error bars represent sem, n = 3. Statistical groups indicated by letters were determined by one-way ANOVA, with Tukey’s test (multiple comparison for (B), P ≤  0.05 (D, E) 5-day-old seedlings of the indicated genotypes as shown in (E) grown in constant white light were exposed to supplemental UV-B 3 (+UV-B) or not (−UV-B) for 20 h and allowed to recover under white light for 10 days before imaging. F, quantification of seedling survival percentage, calculated as mentioned in methods. Error bars represent sem, n = 3, Statistical groups indicated by letters were determined by one-way ANOVA, with Tukey’s test, P ≤ 0.05.

Figure 7

BBX11 induces photoprotection and DNA damage repair genes under UV-B. A–F, RT-qPCR analysis of ELIP1 (A) ELIP2 (B), CHS (C), CHI (D), UVR2 (E), and UVR3 (F) in 5-day-old seedlings grown in constant white light and exposed to supplemental UV-B 2 (+) or not (−) for 4 h. GAPDH was used as internal control. Error bars represent sem, n = 3. Statistical groups indicated by letters were determined by one-way ANOVA, with Tukey’s test, P ≤ 0.05.

Figure 8

HY5-BBX11 feedback loop regulates photomorphogenesis and stress tolerance under UV-B. A, qRT-PCR analysis of HY5 expression in 5-day-old seedlings of the indicated genotypes grown in constant white light and exposed to supplemental UV-B 2 (+) or not (−) for the 4 h. GAPDH was used as internal control. Error bars represent sem, n = 3. Statistical groups indicated by letters were determined by one-way ANOVA, with Tukey’s test, P ≤ 0.05. B, Immunoblot analysis of HY5 and actin (loading control) levels in Col-0, bbx11-1, and OE3 seedlings exposed to 6 h of supplemental UV-B 2 (+UV-B) or not (−UV-B). C, Working model indicating the role of an HY5-BBX11 feedback loop in regulating UV-B photomorphogenesis and stress tolerance. “X” indicates an unknown factor that might regulate BBX11 transcription in addition to HY5 under UV-B.