1. Volume 9, Issue 4, Pages 541-557 (April 2016)
Arabidopsis FHY3 and FAR1 Regulate Light-Induced myo-Inositol Biosynthesis and Oxidative Stress Responses by Transcriptional Activation of MIPS1 
Lin Ma, Tian Tian, Rongcheng Lin, Xing-Wang Deng, Haiyang Wang, Gang Li 
Molecular Plant 
Volume 9, Issue 4, Pages (April 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1
FHY3 and FAR1 Negatively Regulate Light-Induced Cell Death after Dark-Light Transition.
(A and B) Morphological phenotype of wild-type (No-0) and fhy3-4, far1-2, fhy3-4 far1-2 plants under long-day (LD, 16 h light/8 h dark) and short-day (SD, 8 h light/16 h dark) conditions. 3-week-old (LD) and 4-week-old (SD) plants were used to take photographs and measure the plant size (B, left panel, centimeters), fresh weight (B, middle panel, grams) and the percentage of leaves displayed with cell death (B, right panel) (Bars, 1 cm; n > 8, **p < 0.01; ***p < 0.001). Right panel of (A) is the magnified photograph of fhy3-4 far1-2 on the left.
(C) Morphological phenotype of transgenic plants of FHY3p:FHY3 and FAR1p:FAR1 in the fhy3-4 far1-2 mutant background. The photographs were taken when the plants were 4 weeks old under SD conditions. Bar, 1 cm.
(D) Morphological phenotype of fhy3-4 far1-2 mutants after plants were transferred from LD (3 weeks old) to SD conditions for 4 days with low light (left, 50 μmol m−2 s−1), or moderate high light conditions (right, 200 μmol m−2 s−1). Bars, 1 cm. Arrows indicate the regions of cell death under high light conditions and yellowing under low light conditions.
(E) RT–qPCR analysis of the transcript levels of FHY3 and FAR1 after plants were transferred from dark to light conditions with low (50 μmol m−2 s−1, indicated by L) or high light (200 μmol m−2 s−1, indicated by H). FHY3-H, FHY3-L, FAR1-H, FAR1-L indicated the transcript levels of FHY3 and FAR1 under high (H) or low (L) light intensity conditions, respectively. For RT–qPCR assays, UBQ1 was used as the internal control. Data are shown as means ± standard deviation; n = 3.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

3. Figure 2
Loss of FHY3 and FAR1 Causes Increased ROS Accumulation and Sensitivity to Oxidative Stress.
(A) Trypan blue, DAB, and NBT staining of No-0 and fhy3-4 far1-2 leaves. 4-week-old plants grown under SD conditions were used for histochemical staining assays. Bars, 1 mm.
(B) Quantification of hydrogen peroxide (H2O2) and superoxide (O2•−) in rosette leaves. 3-week-old (LD) and 4-week-old (SD) No-0 and fhy3-4 far1-2 plants were used. n ≥ 4; *p < 0.05; **p < 0.01; ***p <
(C and D) Increased sensitivity of fhy3-4 and fhy3-4 far1-2 mutants to exogenous MV treatment. 4-week-old plants grown in LD conditions were used for MV treatment, after being sprayed with 5 μM MV, plants were kept under LD (C) and continuous dark (D) conditions for 2 or 7 days. Bars, 1 cm.
(E) Morphological phenotype of various mutant or transgenic plants after 5 μM MV treatment. 4-week-old plants grown in LD conditions were used for MV treatment. FLAG-FHY3, 35Spro:3FLAG-FHY3-3HA; YFP-FHY3, FHY3pro:YFP-FHY3. FLAG-FHY3, and YFP-FHY3 are two kinds of transgenic plants of FHY3 in an fhy3-4 mutant background. Bars, 1 cm.
(F) Relative expression of ROS-responsive genes PR1, PR2, AOX1d, and ZAT12 in various plants after MV treatment. 4-week-old plants grown in LD conditions were sprayed with 5 μM MV and then incubated for various times. For RT–qPCR assays, UBQ1 was used as the internal control. Data are shown as means ± standard deviation; n = 3; p < 0.05.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

4. Figure 3
Light-Induced Cell Death after Dark-Light Transition Is Dependent on SA.
(A and B) Overexpression of salicylic acid 3-hydroxylase (S3H-OE) largely suppresses the cell death phenotype of fhy3-4 far1-2 mutants. The photographs were taken when the plants were 4 (SD) and 3 (LD) weeks old. OE3 and OE4 are two independent representative transgenic lines of S3H-OE in the fhy3-4 far1-2 mutant background. Arrows indicate the regions of cell death and leaf rugosity. Bars, 1 cm.
(C) Morphological phenotype of No-0, fhy3-4 far1-2, OE3, and OE4 after extended darkness treatment for 5 days (D5d) then transfer to LD for 2 days (L2d). The photograph of fhy3-4 far1-2 in (C) is the magnification of the same plant labeled with the red box in (B). Arrows indicate the regions of cell death after extended darkness. Bars, 1 cm.
(D) Expression of S3H, PR1, PR2, and ZAT12 in No-0, fhy3-4 far1-2, and transgenic lines of S3H-OE (line OE3). CK, the plants without dark treatment; L1d, L2d, 1 or 2 days under light conditions after 5-day dark treatment. For RT–qPCR assays, UBQ1 was used as the internal control. Data are shown as means ± standard deviation; n = 3. *p < 0.05.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

5. Figure 4 The Inositol Metabolic Pathway Is Regulated by FHY3 and FAR1.
(A) Comparison of ChIP-seq results of FHY3 under far-red and dark conditions with the microarray results of fhy3-4 far1-2 under SD conditions. 3-week-old plants grown under SD conditions were used for microarray analysis.
(B) A diagram showing the metabolic pathway of inositol. The gene names labeled with red indicate loci regulated by FHY3 and FAR1.
(C) Relative mRNA levels of inositol metabolic-related genes in fhy3-4 far1-2 mutants. One-week-old seedling plants grown on GM medium under LD conditions were harvested at ZT4 and used to perform the RT–qPCR assay. For RT–qPCR assays, UBQ1 was used as the internal control. Data are shown as means ± standard deviation; n = 3. **p < 0.01.
(D) ChIP-seq shows the promoter regions of MIPS1, MIPS2, MIOX2, and INT1 are specifically enriched in the DNA immunoprecipitated by FHY3. Data were extracted from our previous study of ChIP-seq of FHY3 (Ouyang et al., 2011) red arrows indicate the transcriptional start sites.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

6. Figure 5
Light Regulates the Synthesis of Inositol and the Transcript Levels of MIPS1/2.
(A) The content of inositol decreased in the dark and increased under light conditions. 3-week-old No-0 plants grown under LD conditions, followed by dark-light treatment for various times, were used to detect the content of inositol. D0, harvested after light on 4 h before dark treatment; D1, dark treatment for 1 day; L4, L24, light treatment for 4 and 24 h, respectively. **p < 0.01.
(B) The transcript levels of MIPS1 and MIPS2 under dark-light transition conditions. 3-week-old No-0 plants grown under LD conditions, followed by dark or light treatment for various times, were used to detect the relative expression of MIPS1/2. D0, harvested after light on 4 h before dark treatment; D1, D2, D3, darkness for 1, 2, or 3 days; L4-L5, light for 1 or 2 days.
(C) The transcript levels of MIPS1 and MIPS2 under high or low light intensity conditions. The No-0 plants were grown for 7 days under LD conditions (100 μmol m−2 s−1), followed by transfer to high (200 μmol m−2 s−1) or low (50 μmol m−2 s−1) light intensity conditions for 1 or 2 h after the end of the 7th night (CK).
(D) The relative expression of MIPS1 in phya, phyb, and fhy3 mutants. 4-day-old seedlings grown in continuous darkness and then transferred to red (left panel) or far-red light (right panel) conditions for various times (0, 1, 3, 6 h) were used to detect the expression of MIPS1.
(E) The relative expression of MIPS1 in wild-type and fhy3-4, far1-2 and fhy3-4 far1-2 mutants after white light treatment. 4-day-old etiolated seedlings (L0), followed by exposure to white light for 4 h (L4) were used to detect MIPS1 expression. No, Col, Ler, and C24 are wild-type plants of Arabidopsis in various ecotype backgrounds.
(F) The relative expression of MIPS1 in fhy3-4, and fhy3-4 far1-2 mutants after various light treatments. 4-day-old etiolated seedlings, followed by transferring to red (left), far-red (middle), or blue (right) light conditions for 1 or 3 h were used for RT–qPCR analysis. R0-R3, F0-F3, B0-B3, indicate red (R), far-red (FR), or blue (B) light treatment for 0, 1, 3 h, respectively.
For RT–qPCR assays in (B)–(F), UBQ1 was used as the internal control. Data are shown as means ± standard deviation; n = 3; *p < 0.05.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

7. Figure 6
FHY3 and FAR1 Directly Bind the MIPS1/2 Promoters and Activate Transcription.
(A) Comparison of nucleotide sequences in the promoter regions of MIPS1 and MIPS2. Conserved nucleotides are green, putative FHY3/FAR1 binding sites (FBS) are red, putative ‘ACGT’ cis-elements are blue. Dotted and dashed lines indicate omitted or inserted gaps for comparison.
(B and C) Quantitative yeast one-hybrid assays. M1Fw, M1Fm, wild-type, or mutated FBS1 cis-elements in the promoter region of MIPS1. M2F1w, F1m, F2w, wild-type, or mutated FBS1/2 cis-elements in the MIPS2 promoter. M3, the promoter region of MIPS3. AD, activation domain; FHY3, FAR1, AD-fusion protein of FHY3 or FAR1. *p < 0.05; **p < 0.01; n = 5.
(D) ChIP–qPCR assays showing that FHY3 binds to the promoter of MIPS1 (M1p) and MIPS2 (M2p), but not MIPS3 (M3p) or ACTIN. 4-day-old 3FLAG-FHY3-3HA/fhy3-4 transgenic plants grown in continuous darkness (D) or after exposure to white light for 4 h (D-L) were used to perform ChIP–qPCR assays. The promoter regions of MIPS3 and ACTIN were used as a negative control for the ChIP assay.
(E) Spatial expression patterns of FHY3pro::GUS transgenic plants.
(F) FHY3 directly activates the expression of MIPS1 and MIPS2. 7-day-old FHY3pro:FHY3 -GR/fhy3-4 transgenic plants grown in 12 h light/12 h dark conditions were treated with or without DEX (10 μM) for 1 or 2 h.
(G and H) Disruption of FHY3 and FAR1 suppresses light-induced high expression of MIPS1 and MIPS2 under both SD (G) and LD (H) conditions, compared with wild-type (No-0). 7-day-old seedlings were used to perform RT–qPCR. Gray, night period. For qPCR assays, UBQ was used as the internal control. Data are shown as means ± standard deviation; n = 3; p < 0.05.; p < *p < 0.05; **p < 0.01.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

8. Figure 7
MIPS1 and Inositol Are Involved in the FHY3- and FAR1-Mediated Oxidative Stress Response.
(A) RT–qPCR confirms the elevated expression level of MIPS1 in transgenic plants with MIPS1-OE/fhy3-4 far1-2 (MIPS1-OE). L27 and L45 are two independent transgenic lines. One-week old seedling plants grown in MS medium were used to detect the transcript level of MIPS1. **p < 0.01.
(B and C) Constitutive expression of MIPS1 in the fhy3-4 far1-2 mutants partially rescues the reduced inositol content (B) and suppresses the cellular concentration of hydrogen peroxide (C). 3-week-old plants grown under LD conditions were used to detect inositol and hydrogen peroxide in (B) and (C). Data are shown as means ± standard deviation; n = 3. **p < 0.01; ***p <
(D) Constitutive expression of MIPS1 in the fhy3-4 far1-2 mutants partially suppresses stress-induced precocious leaf senescence (LD conditions). 4-week-old plants were used to take photographs. Bars indicate 5 cm in the left panel and 1 cm in the right panel.
(E–G) Expression of genes related to senescence (E, PR1, PR2), ROS (F, ZAT7, ZAT10, ZAT12) and SA (G, EDS5, ICS1, ICS2) in fhy3-4 far1-2 and MIPS1-OE transgenic plants. 3-week-old plants grown in LD conditions were used to perform RT–qPCR assays. For RT–qPCR assays, UBQ1 was used as the internal control. Data are shown as means ± standard deviation; n = 3; *p < 0.05; **p < 0.01.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

9. Figure 8
A Model for the Role of FHY3 and FAR1 in Regulating Inositol Biosynthesis and Responding to Oxidative Stress.
Light signaling proteins FHY3 and FAR1 bind to the promoter and mediate light-induced transcription of MIPS1/2, with FHY3 playing a predominant role. FHY3 and FAR1 also activate the transcription of VTC4 indirectly. Meanwhile, light supplies energy for photosynthesis and thus provides the initial substrate for the biosynthesis of inositol. Therefore, FHY3 and FAR1 mediate light-induced inositol biosynthesis to suppress dark-light transition triggered oxidative stress. In addition, FHY3 and FAR1 negatively regulate oxidative stress in an SA-dependent manner, or other unknown mechanism. Green lines indicate the work presented in this study. Arrows, positive regulation; bar, negative regulation.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions