The Arabidopsis Transcription Factor ANAC032 Represses Anthocyanin Biosynthesis in Response to High Sucrose and Oxidative and Abiotic Stresses

Abstract

Production of anthocyanins is one of the adaptive responses employed by plants during stress conditions. During stress, anthocyanin biosynthesis is mainly regulated at the transcriptional level via a complex interplay between activators and repressors of anthocyanin biosynthesis genes. In this study, we investigated the role of a NAC transcription factor, ANAC032, in the regulation of anthocyanin biosynthesis during stress conditions. ANAC032 expression was found to be induced by exogenous sucrose as well as high light (HL) stress. Using biochemical, molecular and transgenic approaches, we show that ANAC032 represses anthocyanin biosynthesis in response to sucrose treatment, HL and oxidative stress. ANAC032 was found to negatively affect anthocyanin accumulation and the expression of anthocyanin biosynthesis (DFR, ANS/LDOX) and positive regulatory (TT8) genes as demonstrated in overexpression line (35S:ANAC032) compared to wild-type under HL stress. The chimeric repressor line (35S:ANAC032-SRDX) exhibited the opposite expression patterns for these genes. The negative impact of ANAC032 on the expression of DFR, ANS/LDOX and TT8 was found to be correlated with the altered expression of negative regulators of anthocyanin biosynthesis, AtMYBL2 and SPL9. In addition to this, ANAC032 also repressed the MeJA- and ABA-induced anthocyanin biosynthesis. As a result, transgenic lines overexpressing ANAC032 (35S:ANAC032) produced drastically reduced levels of anthocyanin pigment compared to wild-type when challenged with salinity stress. However, transgenic chimeric repressor lines (35S:ANAC032-SRDX) exhibited the opposite phenotype. Our results suggest that ANAC032 functions as a negative regulator of anthocyanin biosynthesis in Arabidopsis thaliana during stress conditions.

Keywords: ANAC032; Arabidopsis thaliana; anthocyanin biosynthesis; high light; oxidative stress; salinity.

FIGURE 1

Regulation of sucrose–induced anthocyanin by ANAC032 in Arabidopsis. (A) qRT-PCR analysis of ANAC032 expression in response to sucrose treatment. Five-day-old seedlings were transferred to fresh half-strength MS plates supplemented with 1% or 6% sucrose for 24 h under continuous light conditions. Data represent mean values (±SD). UBC21 was used as internal control. Data were analyzed using Student’s t-test (^∗∗∗P< 0.01). (B,C) β-glucuronidase activity of P_ANAC032: GUS line in response to high sucrose treatment. Five-day-old seedlings of P_ANAC032: GUS line, grown on half-strength MS plates with 0% (B) and 1% (C) sucrose were transferred to MS plates supplemented with 6% sucrose for 24 h, followed by incubation in GUS-staining solution overnight in dark at 37°C. (D) Phenotypic response of wild-type (WT) and ANAC032 transgenic lines (OX32; overexpressors and SRDX32; chimeric repressors) after 5 days of growth on half-strength agar plates containing 0 and 6% sucrose. (E) Anthocyanin content in the seedlings of ANAC032 transgenic lines, grown on half-strength MS agar plates containing 0, 1.5, 3, and 6% sucrose, after 5 days of growth. Data represent mean values (±SD; n = 3). (F) qRT-PCR analysis of DFR (dihydroflavanol reductase) expression in response to high sucrose treatment. Five-day-old seedlings of WT and ANAC032 transgenic lines were transferred to fresh half strength MS plates containing 1 and 6% sucrose for 24 h. UBC21 was used as internal control. Within each treatment, bars with different letters in (E,F) are statistically not similar to each other according to one way ANOVA LSD test (P < 0.05).

FIGURE 2

Regulation of high light-induced anthocyanin biosynthesis. (A) Expression analysis of ANAC032 in response to high light stress using qRT-PCR assay. Data represent mean values (±SD). ACT7 was used as internal control. Data were analyzed statistically using Student’s t-test (^∗∗∗P < 0.01). (B) β-glucuronidase activity of P_ANAC032: GUS line in response to high light treatment. Twenty-day-old plants of P_ANAC032 : GUS line were transferred to control/optimal (150 μmol m^-2 s^-1) and high light (∼1000 μmol m^-2 s^-1) for 24 h and then incubated in GUS-staining solution overnight (scale bar = 1 cm). (C) Phenotype of WT and ANAC032 transgenic lines grown under optimal (control) and high light. (D) Biochemical analysis of anthocyanin content in WT and ANAC032 transgenic lines in response to high light stress. (E) Analysis of total soluble sugars in response to HL stress. 17-day-old plants of WT and ANAC032 overexpression and SRDX lines were grown under high light (∼450 μmol m^-2 s^-1) for 4 days. Plants grown under optimal light conditions (∼150 μmol m^-2 s^-1) were considered as control. Data represent values from three biological replicates. Bars with different letters are not statistically similar to each other according to one way ANOVA LSD test (P < 0.05).

FIGURE 3

Effect of 3-AT on anthocyanin biosynthesis in WT and ANAC032 transgenic lines. (A) Phenotypic response of WT and ANAC032 overexpression and SRDX lines for anthocyanin biosynthesis in response to 3-AT treatment after 17 days of growth under long-day conditions. (B) Biochemical analysis of anthocyanin content in WT and ANAC032 transgenic lines after 17 days on 0 and 7.5 μM of 3-AT. (C) Template showing the position of the genotypes shown in (A). Data represent mean values from three biological replicates. Within each treatment, bars with different letters are not statistically similar to each other according to one way ANOVA LSD test (P < 0.05).

FIGURE 4

Expression analysis of anthocyanin biosynthesis and regulatory genes in response to high light stress. (A) qRT-PCR analysis of anthocyanin biosynthesis genes (ABGs). (B) qRT-PCR analysis of positive regulators (transcriptional activators) of anthocyanin biosynthesis. (C) qRT-PCR analysis of negative regulators (transcriptional repressors) of anthocyanin biosynthesis. Seventeen-day-old WT and ANAC032 transgenic lines were treated with optimal/control (150 μmol m^-2 s^-1) and high light (∼450 μmol m^-2 s^-1) for 4 days. Data represent mean relative expression values (±SD). ACT7 was used as internal reference gene. Within each treatment, bars with different letters are not statistically similar to each other according one way ANOVA LSD test (P < 0.05).

FIGURE 5

Hormonal regulation of anthocyanin accumulation. (A) Phenotypic response of WT and ANAC032 transgenic lines for anthocyanin biosynthesis in response to MeJA treatment after 15 days of growth under long-day conditions. (B) Anthocyanin content in response to MeJA treatment. Seeds were germinated and grown on half-strength MS agar plates (1% sucrose) supplemented with 0, 25, and 50 μM MeJA for 12 days under long-day conditions. Twenty seedlings were pooled in each replicate for each genotype to analyze anthocyanin content. (C) Phenotypic response of WT and ANAC032 transgenic lines for anthocyanin biosynthesis in response to ABA treatment. (D) Anthocyanin content in response to ABA treatment. Eight-day-old seedlings of wild-type and ANAC032 transgenic lines were exposed to 0, 10, and 20 μM ABA and were grown under long-day condition for 4 days. Eight seedlings were pooled in each replicate for each genotype. Data represent mean values (±SD, n = 3). Within each treatment, bars with different letters in (B,D) are not similar to each other statistically according to one way ANOVA LSD test (P < 0.05).

FIGURE 6

Salinity-induced anthocyanin biosynthesis in ANAC032 transgenic lines. (A) Anthocyanin content in response to salinity stress. Three-week-old plants of WT and ANAC032 transgenic lines were treated with 200 mM NaCl for 2 weeks. Data represent values from three biological replicates (±SD). Bars with same letters are not significantly different from each other according to one way ANOVA LSD test (P < 0.05). (B) qRT-PCR analysis of ABGs in leaves of plants subjected to salinity stress. (C) qRT-PCR analysis of transcription factors that positively regulate the expression of ABGs plants subjected to salinity stress. ACT7 was used as internal control. For each gene, bars with same letters are not significantly different from each other according to one way ANOVA LSD test (P < 0.05).

FIGURE 7

(A) Phenotype of 10-day-old seedlings grown on half-strength MS agar plates supplemented with 1% sucrose under long-day condition. (B) Primary root lengths of 7-day-old seedlings of wild-type and ANAC032 transgenic lines. Data represent values from three biological replicates. Each replicate included 20 roots. Bars with same letters are not statistically different from each other according to one way ANOVA-LSD test (P < 0.05). (C) Analysis of lignification pattern in WT and ANAC032 transgenic lines. Phloroglucinol-HCl staining of lignin in roots and hypocotyl regions of 7-day-old seedlings. Roots were analyzed under a light microscope (10 X magnification). (R-H, root-hypocotyl junction).