1. Repression of MYBL2 by Both microRNA858a and HY5 Leads to the Activation of Anthocyanin Biosynthetic Pathway in Arabidopsis 
Yulong Wang, Yiqing Wang, Zhaoqing Song, Huiyong Zhang 
Molecular Plant 
Volume 9, Issue 10, Pages (October 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1 Anthocyanin Accumulation in MIR858a-OX and STTM858 Seedlings.
(A) 6-day-old seedlings of wild-type (WT), mybl2-2, MIR858a-OX, and STTM858 grown under standard long-day (16 h of light/8 h of dark) conditions showed similar morphological phenotypes.
(B) Phenotypic characterization of the indicated genotypic seedlings grown on norflurazon-containing medium, showing the overaccumulation of purple anthocyanins in the MIR858a-OX plants compared with WT.
(C) Quantitative measurement of anthocyanins in 6-day-old seedlings grown under long-day conditions. Values are means ± SD of five biological experiments. The same letters indicate no statistical difference, while different letters denote groups with significant differences (ANOVA, p < 0.01).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

3. Figure 2
Expression of Anthocyanin Biosynthetic and Regulatory Genes in Arabidopsis Seedlings.
(A and B) Quantitative RT–PCR analysis of mRNA levels for biosynthetic genes (A) and regulatory genes (B) of the anthocyanin biosynthetic pathway in 6-day-old seedlings. Genes directly for anthocyanin biosynthesis are labeled in red. Actin2 was used as an internal control. Expression level in WT was set to 1. Data are means ± SD of three biological experiments. The same letters indicate no statistical difference, while different letters denote groups with significant differences (ANOVA, p < 0.01).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

4. Figure 3 Translational Repression of MYBL2 by miR858a.
(A) Transcripts level of MYBL2 in the seedlings of different genotypes by semi-quantitative RT–PCR. Positions of the regions used to monitor MYBL2 transcript levels are also indicated.1, WT; 2, MYBL2pro:MYBL2-HA; 3, MIR858a-OX; 4, 5, the progeny of MYBL2pro:MYBL2-HA and MIR858-OX or STTM858, respectively. Two biological replicates were performed and generated similar results (Supplemental Figure 4).
(B) RT–qPCR analysis of MYBL2 expression levels by determining the different regions across the full-length cDNA sequence of MYBL2 gene as indicated in (A). Data are transcript levels relative to Actin2, set to one for WT. Values are means ± SD of three biological experiments. There was no significant difference among the various genotypes by ANOVA analysis.
(C) Immunoblot analysis of HA-fused MYBL2 protein levels in seedlings. Values below the blots represent HY5 levels normalized against the loading control RPT5 using ImageJ and set to one for MYBL2pro:MYBL2-HA.
(D) Quantification of anthocyanins in the seedlings of different genotypes. Values are means ± SD of five biological experiments. The same letters indicate no statistical difference, while different letters denote groups with significant differences (ANOVA, p < 0.01).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

5. Figure 4 MYBL2 Is a Direct Target of HY5.
(A) HY5 occupancy at the MYBL2 locus based on global ChIP data, which were mapped onto the Arabidopsis genome coordinates and visualized using the Affymetrix Integrated Genome Browser.
(B) Confirmation of HY5 binding to the MYBL2 locus by ChIP-qPCR analysis. Specific primers were used for various fragments of the MYBL2 coding or promoter region. Data are means ± SD of three biological experiments. The asterisks above the columns indicate a significant difference in percent input between hy5 and WT (t test, *p < 0.05, **p < 0.01).
(C) EMSA analysis of HY5 binding to the MYBL2 locus (probes as shown in A). Lanes 1, 5, and 9, labeled probe alone; lanes 2 and 6, labeled probe incubated with recombinant HY5; lanes 3, 4, 7, and 8, excessive unlabeled probe as a competitor was added into the reaction system; lane 10, labeled probe incubated with recombinant HY5 as in lanes 2 and 6; lane 11, five times more recombinant HY5. FP, free probe; SB, shift band.
(D) Expression of MYBL2 in WT, hy5, and mybl2-2 seedlings by RT–qPCR. Data are transcript levels relative to Actin2 and set to 1 for WT. The letters above the columns indicate significant differences among various genotypes (ANOVA, p < 0.01).
(E) HY5 represses MYBL2 transcription via chromatin modifications. Specific primers were used for various regions as shown in (B). Specific primers were used for various regions as in (B). Data are means ± SD of three biological experiments. The same letters indicate no statistical difference, while different letters denote groups with significant differences (ANOVA, p < 0.01).
(F) Immunoblot analysis of HA-tagged MYBL2 protein levels in seedlings. Values below the blots represent MYBL2 levels normalized against the loading control RPT5 using ImageJ and set to 1 for MYBL2pro:MYBL2-HA.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

6. Figure 5 HY5 Directly Binds and Activates MIR858a Expression.
(A) A cluster of four consensus motifs for HY5 binding is found in the promoter of MIR858a, including G-box (CACGTG), CG-box (CACGTC), Z-box (CACGTA), and A-box (TACGTA). ChIP-qPCR analysis confirmed the binding of HY5 to the MIR858a promoter, and the precursor of miR858a was used as control. Specific primers were used for various fragments across the MIR858a promoter. Data are means ± SD of three biological experiments. The asterisks above the columns indicate significant differences in percent input between hy5 and WT (t test, p < 0.01).
(B) EMSA analysis of HY5 binding to the MIR858 promoter. Four promoter regions as shown in (A) were used as probes. Lanes 1, 5, 9, and 13, labeled probe alone; lanes 2, 6, 10, and 14, labeled probe incubated with recombinant HY5; lanes 3, 4, 7, 8, 15, and 16, excessive unlabeled probe as a competitor was added into the reaction system, respectively. FP, free probe; SB, shift band.
(C) RT–qPCR analysis of pri-miR858a and miR858a in wild-type, hy5, and 35S:HA-HY5 seedlings. Data are transcript levels relative to Actin2 and set to 1 for wild-type. Values are means ± SD of three biological experiments. The same letters indicate no statistical difference, while different letters denote groups with significant differences (ANOVA, p < 0.01).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

7. Figure 6
Light-Responsive Expression of MIR858a in Arabidopsis Seedlings.
(A) Transcript accumulation of MIR858a was analyzed as well as MYBL2 and HY5 by RT–qPCR using seedling samples of wild-type under low light (LL, 50 μmol m−2 s−1) or high light (HL, 400 μmol m−2 s−1) conditions. Data are transcript levels relative to Actin2 and set to 1 for LL conditions. Values are means ± SD of three biological experiments. The asterisks above the columns indicate significant differences between LL and HL conditions for different genes (t test, **p < 0.01).
(B) Quantitative analysis of MIR858a transcript levels in wild-type and hy5 seedlings. Seedlings were grown for 4 days under dark conditions, transferred to HL at time 0, and assayed by RT–qPCR at the indicated time points thereafter. Values are means ± SD of three biological experiments.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

8. Figure 7
Simplified Model for MIR858a-HY5-Regulated MYBL2 Repression in Anthocyanin Biosynthesis.
The anthocyanin biosynthetic pathway is affected by multiple environmental factors, including biotic and abiotic stresses (such as light, low temperature, drought, and pathogen infection). The biosynthetic genes are regulated directly by HY5 in response to light signaling or indirectly by other signals through modulating the expression of MYBL2, which interferes with the MBW complexes. In the present study, HY5 directly represses MYBL2 transcription and activates MIR858a expression, while miR858a represses MYBL2 expression at the translation step. The reduction of MYBL2 levels enhances the stabilization of the MBW complexes, thereby promoting expression of the late biosynthetic genes and thus leading to increased anthocyanin accumulation.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions