High Ambient Temperature Represses Anthocyanin Biosynthesis through Degradation of HY5

Abstract

Anthocyanins are flavonoid compounds that protect plant tissues from many environmental stresses including high light irradiance, freezing temperatures, and pathogen infection. Regulation of anthocyanin biosynthesis is intimately associated with environmental changes to enhance plant survival under stressful environmental conditions. Various factors, such as UV, visible light, cold, osmotic stress, and pathogen infection, can induce anthocyanin biosynthesis. In contrast, high temperatures are known to reduce anthocyanin accumulation in many plant species, even drastically in the skin of fruits such as grape berries and apples. However, the mechanisms by which high temperatures regulate anthocyanin biosynthesis in Arabidopsis thaliana remain largely unknown. Here, we show that high ambient temperatures repress anthocyanin biosynthesis through the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) and the positive regulator of anthocyanin biosynthesis ELONGATED HYPOCOTYL5 (HY5). We show that an increase in ambient temperature decreases expression of genes required in both the early and late steps of the anthocyanin biosynthesis pathway in Arabidopsis seedlings. As a result, seedlings grown at a high temperature (28°C) accumulate less anthocyanin pigment than those grown at a low temperature (17°C). We further show that high temperature induces the degradation of the HY5 protein in a COP1 activity-dependent manner. In agreement with this finding, anthocyanin biosynthesis and accumulation do not respond to ambient temperature changes in cop1 and hy5 mutant plants. The degradation of HY5 derepresses the expression of MYBL2, which partially mediates the high temperature repression of anthocyanin biosynthesis. Overall, our study demonstrates that high ambient temperatures repress anthocyanin biosynthesis through a COP1-HY5 signaling module.

Keywords: Arabidopsis; HY5; anthocyanin; flavonoid; gene expression; high temperature stress.

FIGURE 1

Schematic diagram of the anthocyanin biosynthetic pathway in Arabidopsis thaliana. PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumaroyl: CoA-ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; DFR, dihydroflavonol reductase; LDOX, leucoanthocyanidin dioxygenase; UF3GT, UDP-glucose flavonoid 3-O-glucosyltransferase; AAT, anthocyanin acyltransferase.

FIGURE 2

High temperature represses the accumulation of anthocyanin pigments. (A) Gene Ontology (GO) analyses showed that genes in the ANTHOCYANIN_CONTAINING_COMPOUND_BIOSYNTHETIC_PROCESS are highly enriched among high temperature (27°C)-repressed genes identified in a previous RNA-seq experiment (Jung et al., 2016). Other molecular processes enriched in the high temperature-repressed genes are shown in Supplementary Table 1. (B) Heat map of the anthocyanin biosynthetic genes at Zeitgeber Time 0 (ZT0) in previous RNA-seq experiment (Jung et al., 2016). Red color indicates genes that were down-regulated by high temperatures and green color indicates genes that were up-regulated by high temperatures. (C) Growth conditions in experiments (D,E). Seedlings were grown on MS medium containing 3% sucrose at different temperatures (17, 20, or 28°C) for 5 days under continuous white light, and then harvested for anthocyanin extraction. (D,E) A lower level of anthocyanin accumulation was found in seedlings grown at the higher temperatures. Representative seedlings are shown in (D) and quantification of anthocyanin content is shown in (E). Error bars in (E) indicate s.d. (n = 3). ^∗P < 0.05 (Student’s t-test). (F) Growth conditions in experiments (G,H). Seedlings were either grown at 17°C for 4 days and then at 28°C for 24 h before harvesting, or were kept at 17°C as a control. (G,H) Anthocyanin accumulation was reduced by the high temperature treatment. Representative seedlings are shown in (G) and quantification of anthocyanin content is shown in (H). Error bars in (H) indicate s.d. (n = 3). ^∗∗P < 0.01 (Student’s t-test).

FIGURE 3

High temperature represses expression of anthocyanin biosynthetic genes. The qRT-PCR analyses showed that expression of anthocyanin biosynthetic genes was decreased at 28°C. Wild type seedlings were grown at 17°C under continuous white light for 4 days and then either transferred to 28°C for 24 h (17°C → 28°C) or kept at 17°C before harvesting for total RNA extraction. Gene expression levels were normalized to PP2A (AT1G13320) and are presented as values relative to those of seedlings kept at 17°C. Error bars indicate s.d. (n = 3). ^∗∗P < 0.01 (Student’s t-test).

FIGURE 4

Anthocyanin biosynthesis is insensitive to high temperature in hy5 mutant. (A) Anthocyanin accumulation was repressed by high temperature treatment in pif4;pif5 double mutant seedlings. The pif4;pif5 seedlings were grown at 17°C under continuous white light for 4 days and then either transferred to 28°C for 24 h (17°C → 28°C) or kept at 17°C before harvesting for anthocyanin extraction. (B) Anthocyanin accumulation in the hy5 mutant was not significantly affected by an increase in ambient temperature. Seedlings of wild type and hy5 mutant were grown under the same conditions as (A). Error bars in (A,B) indicate s.d. (n = 3). ^∗∗P < 0.01 (Student’s t-test); ns, not significant (P ≥ 0.05). (C–F) The qRT-PCR analyses showed that expression of both early and late anthocyanin biosynthetic genes were insensitive to high temperature in the hy5 mutant. Seedlings were grown at 17°C for 4 days and then transferred to 28°C for 24 h (17°C → 28°C) or kept at 17°C before harvesting for total RNA extraction. Gene expression levels were normalized to PP2A (AT1G13320) and are presented as values relative to those of wild type seedlings kept at 17°C. Error bars indicate s.d. (n = 3). ^∗∗P < 0.01 (Student’s t-test) and ns, not significant (Student’s t-test P ≥ 0.05).

FIGURE 5

High temperature induces the degradation of HY5 protein. (A) Growth conditions in experiments (B–F). Seedlings were grown on MS medium containing 3% sucrose at 17°C for 4 days and then transferred to 28°C for 6 or 24 h (17°C → 28°C) or kept at 17°C. The seedlings were then harvested for extraction of total RNA, protein, and anthocyanin. (B,C) The expression of HY5 was not significantly affected by the 6- or 24-h high temperature treatment. Gene expression levels were normalized to PP2A (AT1G13320) and are presented as values relative to that of seedlings kept at 17°C. Error bars indicate s.d. (n = 3). (D,E) Western blotting with anti-GFP antibody shows that the stability of HY5 protein is reduced at high temperature in wild type, but not in cop1-4 mutant. Total protein was extracted from 35S::HY5-GFP (D) or 35S::HY5-GFP;cop1-4 (E) mutant plants. Equal loading of samples is shown by Ponceau S staining. (F) Anthocyanin pigment accumulation was not affected by high temperature (24 h) in cop1-4 mutant seedlings. Error bars indicate s.d. (n = 3). ^∗∗P < 0.01 (Student’s t-test) and ns, not significant (Student’s t-test P ≥ 0.05).

FIGURE 6

MYBL2 partially mediates the high temperature repression of anthocyanin biosynthesis. (A) Expression of MYBL2 in wild type and hy5 mutant seedlings grown at 17°C for 4 days and then transferred to 28°C for 24 h (17°C → 28°C) or kept at 17°C. MYBL2 expression levels were normalized to PP2A (AT1G13320) and are presented as values relative to that of wild type seedlings kept at 17°C. Error bars indicate s.d. (n = 3) and ns, not significant (Student’s t-test P ≥ 0.05). (B) Anthocyanin levels of wild type and mybl2 mutant seedlings grown at two different temperatures. The wild type and mybl2-1 mutant seedlings were grown at 17°C under continuous white light for 4 days and then either transferred to 28°C for 24 h (17°C → 28°C) or kept at 17°C before harvesting for anthocyanin extraction. ^∗∗P < 0.01 (Student’s t-test) and numbers indicate the ratio of anthocyanin levels (17°C → 28°C/17°C).

FIGURE 7

Hypothetical model of the regulation of anthocyanin biosynthesis by high temperature in Arabidopsis. At low ambient temperatures, HY5 directly activates the expression of both early (CHS and CHI) and late (DFR, LDOX, and UF3GT) anthocyanin biosynthetic genes. HY5 also indirectly represses the expression of the late genes by repression of MYBL2, which encodes a repressor of DFR and LDOX. In contrast, at high ambient temperatures, increased COP1 activity induces the degradation of HY5 protein, which results in low expression of both the early and late anthocyanin biosynthetic genes. The HY5 degradation also transcriptionally activates MYBL2, which also contributes the repression of the late genes at high temperatures.