1. Arogenate Dehydratase Isoforms Differentially Regulate Anthocyanin Biosynthesis in Arabidopsis thaliana 
Qingbo Chen, Cong Man, Danning Li, Huijuan Tan, Ye Xie, Jirong Huang 
Molecular Plant 
Volume 9, Issue 12, Pages (December 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1
ADT Isoforms Regulate Anthocyanin Biosynthesis in a Redundant and Differential Manner.
(A) Anthocyanin accumulation in 4-day-old seedlings of the representative mutants. Seeds were germinated in 1/2 MS liquid medium without sucrose for 2 days and then transferred to the anthocyanin induction medium containing 3% sucrose for an additional 2 days. Bars, 1 mm.
(B–F) Anthocyanin content of WT and various adt mutant seedlings grown under the same condition as in (A). Error bars indicate SD values for the means of three independent replicates. The means that do not share a letter are significantly different (P < 0.05).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

3. Figure 2 ADT Mutations Affect Cold-Induced Anthocyanin Accumulation.
Anthocyanin levels were measured from WT, adt1/3, and adt4/5 seedlings that were grown on 1/2 MS sucrose-free plates for 3 days at 22°C, and then transferred to ½ MS plates with 1% sucrose for an additional 3 days at 22°C or 7°C. Data are means ± SD from three independent replicates. The means that do not share a letter are significantly different (P < 0.05).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

4. Figure 3 Anthocyanin Profile Is Not Altered in adt Mutants.
(A) HPLC analysis of anthocyanin components of WT, adt1/3, and adt4/5 seedlings grown in the sucrose-induced anthocyanin system. The molecules of each labeled component are: a1, cyanidin3-O-[2-O-(xylosyl)-6-O-(4-O-(glucosyl)-p-coumaroyl) glucoside]-5-O-[6-O–(malonyl)glucoside]; a2, cyanidin3-O-[2-O-(2-O-(sinapoyl)xylosyl)-6-O-(4-O-(glucosyl)-p-coumaroyl)glucoside]-5-O-glucoside; a3, cyanidin3-O-[2-O–(2-O–(sinapoyl)xylosyl)-6-O–(4-O–(glucosyl)-p-coumaroyl)glucoside]-5-O-[6-O-(malonyl)glucoside]; a4, cyanidin3-O-[2-O–(2-O–(sinapoyl)xylosyl)-6-O–(p-coumaroyl) glucoside]-5-O-glucoside. HPLC chromatogram profiles of anthocyanins were detected at 520 nm. mAU, milliabsorbance unit.
(B) Quantification of the four main anthocyanin components based on peak areas. Cyanindin 3-O-glucoside was used as an internal standard. Data are means ± SD from three independent replicates. The means that do not share a letter are significantly different (P < 0.01).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

5. Figure 4 ADT2 Plays a Dominant Role in Anthocyanin Biosynthesis.
(A) qPCR analysis of transcriptional levels of ADT1–ADT6 in WT and two adt2-amiR transgenic lines. Transcript levels were normalized to ACTIN2, and then expression of each gene in WT was set to 1. Data are means ± SD from three independent replicates. *P < 0.05 and **P < 0.01 (Student t-test).
(B) Anthocyanin content of WT and two adt2-amiR transgenic plants. Seedlings were grown in the sucrose-induced anthocyanin system. Data are means ± SD from three independent replicates. The means that do not share a letter are significantly different (P < 0.01).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

6. Figure 5
Expression of ADT1–ADT6 Genes Are Differentially Induced by Sucrose.
Gene expression was analyzed by qPCR using total RNA extracted from WT seedlings grown in sucrose-free ½ MS solution for 2 days and then treated with 3% sucrose for indicated times. Transcript levels were normalized to ACTIN2, and then the value for the transcript of each gene at 0 h was set to 1. Data are means ± SD from three independent replicates.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

7. Figure 6
Reduced Anthocyanin Accumulation in adt1/3 Is Not due to Low Expression Levels of Anthocyanin Biosynthetic Genes.
Gene expression was analyzed by qPCR using total RNAs extracted from WT, adt1/3, and adt4/5 seedlings that were grown in sucrose-free ½ MS solution for 2 days and subsequently treated with 3% sucrose for indicated times. Transcript levels were normalized to ACTIN2, and then the value of the transcript of each gene at 0 h was set to 1. Data are means ± SD from three independent replicates.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

8. Figure 7 Anthocyanin Accumulation Is Regulated by Phe Levels In Vivo.
(A) UPLC analysis of Phe levels in WT, adt1/3, and adt4/5 seedlings grown in the sucrose-induced anthocyanin system. The seedlings were sampled at 24 h after addition of 3% sucrose. DW, dry weight. The means labeled with an asterisk are significantly different between WT and mutant at P < 0.05.
(B) Anthocyanin content of WT, adt1/3, and adt4/5 seedlings. Seeds were germinated in ½ MS liquid medium without sucrose for 2 days and then grown in anthocyanin induction medium with or without 0.4 mM Phe for an additional 2 days. Data are means ± SD from three independent replicates. The means labeled with double asterisks are significantly different between WT and mutants at P < 0.01.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

9. Figure 8
Effect of ADT Overexpression on Anthocyanin Biosynthesis and Phe Feedback Regulation of ADT2 and ADT4 Activity.
(A) Phenotypes of 35-day-old WT and transgenic plants overexpressing the ADT2, ADT4, or ADT5 gene. Bars, 1 cm.
(B) qPCR analysis of the level of ADTs expression in WT and transgenic lines. Total RNAs were extracted from the seedlings grown in the sucrose-induced anthocyanin synthesis system. Expression levels were normalized to ACTIN2, and then the value for the transcript of each gene in WT was set to 1. Data are means ± SD from three independent replicates.
(C) Anthocyanin content of WT and overexpression lines. Anthocyanin was extracted from the same seedlings as in (B). Data are means ± SD from three independent replicates. The means that do not share a letter are significantly different (P < 0.05).
(D and E) In vitro assays of ADT2 and ADT4 activity in the absence or presence of 100 μM Phe. Data are means ± SD from three independent replicates.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions