SUMO E3 Ligase SIZ1 stabilizes MYB75 to regulate anthocyanin accumulation under high light conditions in Arabidopsis

Journal Pre-proof SUMO E3 Ligase SIZ1 stabilizes MYB75 to regulate anthocyanin accumulation under high light conditions in Arabidopsis Ting Zheng, Yanling Li, Wei Lei, Kang Qiao, Baohui Liu, Dawei Zhang, Honghui Lin

PII:

S0168-9452(19)31528-6

DOI:

https://doi.org/10.1016/j.plantsci.2019.110355

Reference:

PSL 110355

To appear in:

Plant Science

Received Date:

12 September 2019

Revised Date:

19 November 2019

Accepted Date:

21 November 2019

Please cite this article as: Zheng T, Li Y, Lei W, Qiao K, Liu B, Zhang D, Lin H, SUMO E3 Ligase SIZ1 stabilizes MYB75 to regulate anthocyanin accumulation under high light conditions in Arabidopsis, Plant Science (2019), doi: https://doi.org/10.1016/j.plantsci.2019.110355

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SUMO E3 Ligase SIZ1 stabilizes MYB75 to regulate anthocyanin accumulation under high light conditions in Arabidopsis Ting Zheng1, Yanling Li1, Wei Lei1, Kang Qiao1, Baohui Liu2, Dawei Zhang1,* and Honghui Lin1,* 1

Ministry of Education Key Laboratory for Bio-Resource and Eco-Environment,

College of Life Science, State Key Laboratory of Hydraulics and Mountain River

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Engineering, Sichuan University, Chengdu, 610064, China School of Life Sciences, Guangzhou University, Guangzhou, 510006, China

*Author for correspondence: Dawei Zhang

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E-mail: [email protected] Honghui Lin

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E-mail: [email protected].

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SUMO E3 Ligase SIZ1 regulates anthocyanin accumulation via sumoylation of

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MYB75 and thereby increases its stability under high light conditions in Arabidopsis.

Abstract Sumoylation is one of post-translational modification (PTM) in which SUMO (small ubiquitin-like modifier) are covalently conjugated to protein substrates through a range of biochemical steps. This paper presents evidence that SUMO E3 ligase SIZ1

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positively regulates anthocyanin accumulation. Loss-of-function siz1 mutant seedlings exhibit anthocyanin accumulation-reduced phenotype under high light conditions.

Moreover, SIZ1 interacts and sumoylates MYB75/PAP1, a key transcription factor in anthocyanin accumulation. Loss-of-function siz1 or K246R substitution in MYB75

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blocked SIZ1-mediated sumoylation in vitro and in vivo. Anthocyanin accumulation in

mutant myb75-c can not be rescued by expressing MYB75K246R, but expression of wild-

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type MYB75WT complements the mutant phenotype. It suggested that sumoylation is important for MYB75 function. We further prove that sumoylation is essential for

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MYB75 protein stability. And SIZ1 is involved in the light-induced accumulation of anthocyanins. Our findings reveal an important role for sumoylation of MYB in

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regulation of anthocyanin accumulation in plants. Key words: AtSIZ1, anthocyanin accumulation, high light, sumoylation, MYB75,

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post-translational modification

Introduction In eukaryotic cells, post-translational modification of protein by small or large molecules such as phosphate, carbohydrate, and small proteins is crucial for plant growth and development (Geiss-Friedlander and Melchior 2007). Sumoylation is one

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of post-translational modification (PTM) in which SUMO (small ubiquitin-like modifier) are covalently conjugated to cellular target proteins through a range of biochemical steps (Augustine and Vierstra 2018; Miura, et al. 2007). Firstly, proteolysis

of SUMO precursors exposes the C-terminal double glycine (Gly-Gly) via SUMO-

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specific proteases (Miura and Hasegawa 2010). Subsequently, the mature SUMO is

activated by SUMO-activating E1 enzyme and transferred to a C residue in SUMO E2

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conjugating enzyme (Miura and Hasegawa 2010). Finally, SUMO is conjugated to target proteins via isopeptide bond formed between the Gly-Gly of SUMO and the

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lysine residue within SUMO consensus motif (ΨKxE/D; Ψ, a large hydrophobic residue; K, the acceptor lysine; x, any amino acid; E/D, glutamate or aspartate) in the substrate,

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a process that requires the SUMO E3 ligase in vivo (Augustine and Vierstra 2018; Miura and Hasegawa 2010). There are four SUMO isoforms (SUMO1, SUMO2, SUMO3, and SUMO5) acting as functional PTM in Arabidopsis thaliana, with the highly related

2010).

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SUMO1/SUMO2 subfamily (83% similarity) being dominant (van den Burg, et al.

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Increasing evidence have characterized several SUMO E3 ligase and demonstrated their crucial impact on cellular function in plants. The largest group of SUMO E3 ligases is identified by the presence of an SP-RING motif, which is necessary for their function (Geiss-Friedlander and Melchior 2007; Wang and Dasso 2009). METHYL METHANESULFONATE SENSITIVITY GENE21 (MMS21), the subgroup of SPRING ligase, is involved in DNA repair and cell cycle regulation via dissociating the E2Fa/DPa complex in Arabidopsis (Liu, et al. 2016). Other SP-RING ligase include

SAP and Miz1 (SIZ1) protein, which participated in nutrient homeostasis, abiotic stress and hormone signaling in Arabidopsis (Kim, et al. 2015; Miura, et al. 2007; Miura, et al. 2005; Miuraa, et al. 2009; Park, et al. 2011; Saleh, et al. 2015; Zheng, et al. 2012). Recent studies revealed that protein stability is modulated by sumoylation in plants. Sumoylation antagonizes ubiquitination by contesting with acceptor K residues. Sumoylation of INDUCER OF CBF/DREB1 EXPRESSION 1 (ICE1) by SIZ1 regulated cold stress through inhibiting ubiquitination in Arabidopsis (Miura, et al. 2007). SIZ1 affected nitrate assimilation by increasing stability of NITRATE

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REDUCTASES (NRs) (Park, et al. 2011). On the other hand, ubiquitination is facilitated by sumoylation via recruiting SUMO-targeted ubiquitin ligases (STUbLs) to

sumoylate substrates. Sumoylation of Snf1-related Protein Kinase 1 (SnRK1) by SIZ1 resulted in ubiquitination and proteasomal degradation (Crozet, et al. 2016).

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Sumoylation triggers NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1

(NPR1) degradation upon immune induction (Saleh, et al. 2015). Sumoylation may

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implicate in other metabolic and developmental process through tight regulation between sumoylation and ubiquitination, however, it remains to be illustrated.

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As sessile organisms, plants generate various secondary metabolites which play an essential role in development and stress tolerance (Dixon and Paiva 1995). Anthocyanin,

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a class of flavonoids, are amidst the crack investigative such secondary metabolites (Jaakola 2013). They confer plants with purple to blue colors and enhance plants stress tolerance via their antioxidant capability to scavenge reactive oxygen species (ROS)

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(Nakabayashi, et al. 2014; Winkel-Shirley 2001). Thus, plants have evolved subtle molecular mechanisms to regulate anthocyanin biosynthesis during growth and

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development. In addition, anthocyanin biosynthesis is stimulated by hormone signaling and environmental factors such as jasmonic acid (JA) signaling (Qi, et al. 2011), high light (Li, et al. 2016), drought (Nakabayashi, et al. 2014), and cold stress (Perea-Resa, et al. 2017). Mounting research obviously expound the anthocyanin biosynthetic pathway and its modulatory mechanisms at the transcriptional and post-translational level in plants. Anthocyanin biosynthesis derives from flavonoid biosynthetic pathway and three anthocyanin-specific genes encoding dihydroflavonol 4-reductasae (DFR),

leucoanthocyanidin dioxygenase (LDOX), UDP-glucose: flavonoid 3-oglucosyl transferase (UF3GT) have been identified (Jaakola 2013; Stracke, et al. 2007). In Arabidopsis, expression of these genes is controlled by a ternary MBW protein complex, which is composed of R2R3-MYB transcription factors (TFs), basic helix-loop-helix (bHLH) TFs, and WD40-repeat proteins (Gonzalez, et al. 2008; Zimmermann, et al. 2004). Among the regulatory factors, it seems that R2R3-MYB TFs are central factors to regulate anthocyanin accumulation because overexpressing R2R3-MYB TFs enhance anthocyanin accumulation in Arabidopsis (Gonzalez, et al. 2008). MYB75,

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which is also defined as PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1), plays a key role in anthocyanin accumulation (Borevitz, et al. 2000; Gonzalez, et al. 2008).

Prior

research

confirms

PHOTOMORPHOGENIC1/SUPPRESSOR

that

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PHYA-105

CONSTITUTIVELY (COP1)

restrained

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anthocyanin accumulation by degrading MYB75 in darkness (Maier, et al. 2013). Oppositely, phosphorylation of MYB75 by MAP KINASE4 (MPK4) increases its

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stability (Li, et al. 2016). However, overexpressing phosphodeficient MYB75 still raise anthocyanin accumulation in Arabidopsis (Li, et al. 2016), demonstrating an additional

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regulatory mechanism by which unknown factors maintain the MYB75 stability. A number of research have thoroughly proven that SIZ1 is involved in abscisic acid

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(ABA) signaling, cold stress, nitrate assimilation, and flowering time. Here, we show that SIZ1 positively regulates anthocyanin accumulation and that sumoylation of MYB75 at amino acids K246 by SIZ1 is crucial for its function in anthocyanin

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accumulation. Sumoylation of MYB75 by SIZ1 increases MYB75 stability, which is essential for anthocyanin accumulation. Our results proffer the molecular, biochemical,

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and genetic evidence that sumoylation is a crucial PTM required for the regulation of anthocyanin accumulation in Arabidopsis. Materials and methods Plant materials and growth conditions The Arabidopsis thaliana pap1-D (Borevitz, et al. 2000), myb75-c (Li, et al. 2016), siz12 (Miura, et al. 2005), SSG (Jin, et al. 2008), and sum1-1 amiR-SUM2 (van den Burg, et al. 2010) were described as previously. The half-strength Murashige and Skoog

medium with Arabidopsis seeds were placed at 4 ℃ for 2 d before moving to 22 ℃ under diverse light conditions. For high light-induced research, plates under 40 μmol m-2 s-1 condition were estimated control or 180 μmol m-2 s-1 conditions were estimated high light (Li, et al. 2016). Nicotiana benthamiana grown in soil at 22 ℃ under 16-h-light/8h-dark conditions was used for the BiFC assays. Anthocyanin measurement Anthocyanin content were estimated as previous study (Rabino and Mancinelli 1986). Briefly, anthocyanin were extracted with extraction buffer (methanol containing 1%

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HCl) and incubated at 4 ℃ overlight. After centrifuging, the supernatants were used for absorbance calculated at 530 and 657 nm. (A530-0.25×A657) per gram fresh weight was quantified for relative anthocyanin content. Plasmid construction and plant transformation

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To generate MYB75 wild type or variable plasmids, the 1500 bp genomic sequence of MYB75 contained the coding area was obtained and cloned into the pCM1307 vector

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to create 35S:HA-MYB75 (Zhou, et al. 2014). These constructs were transformed to Arabidopsis (Columbia) by using Agrobacterium tumefaciens (strain GV3101)-

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mediated floral-dip method (Zhang, et al. 2006).

Transcriptional inhibition assays in protoplast

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For transcriptional activity assays in Arabidopsis protoplast, a 512-bp DFR promoter was obtained by PCR amplification and fused with pGreenII 0800-LUC (Li and He 2016). The PEG/CaCl2-mediated transfection of different combinations of effectors,

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reporter and internal control were performed in Arabidopsis protoplasts (Yoo, et al. 2007). The Promega dual-luciferase reporter assay system and a GloMax 20-20

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luminometer (Promega, http://www.promega.com) were used for luciferase assays. The firefly LUC activity was normalised to Renilla LUC activity. Yeast two-hybrid For yeast two-hybrid assays, the coding sequence of MYB75 were amplified and fused with pGADT7 (Clontech). The coding sequence of SIZ1 were amplified and fused with pGBKT7 (Clontech). The pairs of plasmids were transformed into yeast strain (AH109). After grown on supplement (SD-Leu/-Trp) for 3 days, the co-transformants were shifted

onto selective supplement (SD-Leu/-Trp/-Ade/-His) to test for protein-protein interactions. It has been reported that TRANSPARENT TESTA 8 (TT8) could interact with MYB75, so we used this interaction for positive control (Gonzalez, et al. 2008; Zimmermann, et al. 2004). Bimolecular fluorescence complementation (BiFC) assays For BiFC assays, the coding sequence of MYB75 was obtained and fused with the pXY103-nYFP vector (Yu, et al. 2008). The the coding sequence of of SIZ1 was obtained and fused with pXY104-cYFP vector (Yu, et al. 2008). The constructs were

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transformed into Agrobacterium tumefaciens strain (GV3101) respectively, and the different combinations were injected with the lower epidermis of Nicotiana benthamiana plants. Scanning microsystem (Leica) was used for observing fluorescent signals 48 hours later.

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In vitro pull-down assays

The coding sequence of MYB75 were fused with the pMAL-C2X vectors with MBP

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tag. The coding sequence of SIZ1 were fused with the pET28a vectors with 6×HIS tag. In vitro pull-down assays were performed as previous study (Yin, et al. 2005). Ni-NTA

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beads containing 3 μg HIS-SIZ1 proteins were incubated with 3 μg MBP-MYB75 in pull-down buffer (20 mM Tris, 150 mM NaCl, 0.2% Triton X-100, 1 mM PMSF, 1%

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protease inhibitor cocktail [pH 8.0]) for 2 h at 4℃. After being rinsed four times with the pull-down buffer, the beads were boiled in 95℃ with 30 μL SDS-PAGE loading buffer and then subjected to SDS-PAGE. The anti-MBP antibies and anti-6×HIS

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antibies were used for immunoblotting. Co-IP assay

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Nicotiana benthamiana plants expressing SIZ1-GFP and HA, SIZ1-GFP and HAMYB75 were extracted with and thawed in extraction buffer containing 50 mM TrisHCl (pH 7.5), 150 mM NaCl, 10% (v/v) glycerol, 0.1% (v/v) NonidetP-40, and 1×complete protease inhibitor cocktail (Roche) (Wang, et al. 2013). The mixture was centrifuged at 12,000 g at 4 ℃ for 10 min, and the supernatant was incubated with agarose conjugated Anti-HA antibody at 4 ℃ for 3 h. Then the mmunoprecipitates were washed with extraction buffer and then separated by SDS-PAGE. Anti-HA and Anti-

GFP antibodies were used for immunoblotting analysis. In vitro Sumoylation Assay pMAL-C2X-MYB75 was used as template with primer pairs listed in Supplemental Table 1 to generate pMAL-C2X-MYB75K62R, pMAL-C2X-MYB75K122R, pMAL-C2XMYB75K160R, pMAL-C2X-MYB75K187R, pMAL-C2X-MYB75K225R, and pMAL-C2XMYB75K246R, respectively. In vitro sumoylation assay was performed as described previously with minor modifications (Park, et al. 2011). Briefly, 50 ng of HIS AtSAE1b, 50 ng of HIS-AtSAE2, 50 ng of HIS-AtSCE1, 8 μg of HIS-AtSUMO1-GG,

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and 100 ng of wild-type or variable MBP-MYB75 were incubated with 30 μl reaction buffer (5 mM MgCl2, 20 mM HEPES pH7.5, 2 mM ATP) at 30°C for 3 h. Sumoylated MBP-MYB75 was analyzed by anti-MBP antibodies. In vivo Sumoylation Assay

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To determine the sumoylation status of MYB75, The 35S:HA-MYB75WT, 35S:HA-

MYB75K187R, 35S:HA-MYB75K246R and 35S:MYC-SUMO1 were coexpressed in wild-

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type or siz1-2 protoplasts by PEG/CaCl2-mediated transformation (Yoo, et al. 2007). The protoplasts were thawed in 1 mL extraction buffer containing 50mM Tris-HCl pH

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7.5, 150mM NaCl, 10% (v/v) glycerol, 0.1% (v/v) NonidetP-40, and 1×complete protease inhibitor cocktail (Roche) after a 20-h incubation at 23 °C (Wang, et al. 2013).

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10 μL agarose conjugated Anti-HA antibody (Sigma) were incubated with the extract supernatant at 4 °C for 2 h. Then the mmunoprecipitates were washed with extraction buffer and then separated by SDS-PAGE. HA-MYB75-MYC-SUMO1 conjugation was

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detected by Anti-MYC antibodies. The abundance of HA-MYB75 as a control was detected by anti-HA antibodies.

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Protein Degradation Assays Total proteins were thawed in degradation buffer containing 10 mM NaCl, 25 mM TrisHCl pH 7.5, 4 mM PMSF, 10 mM MgCl2, 5 mM DTT, and 10 mM ATP (Naidoo, et al. 1999; Osterlund, et al. 2000). The supernatant was collected and Bio-Rad protein assay was used for determining protein concentration. The total protein extracts prepared from wild type and siz1-2 mutants were adjusted to equal concentration in the degradation buffer for each assay. Then, in vitro degradation assays as indicated were

selectively treated with MG132. For plant derived HA-MYB75 and HA-MYB75K246R protein degradation, 35S:HA-MYB75WT, 35S:HA-MYB75WT siz1-2, and 35S:HAMYB75K246R transgenic plants were treated with 100 μM translational inhibitor cycloheximide (CHX) and 50 μM MG132 for the indicated periods of time and sampled simultaneously to detect HA-MYB75 and HA-MYB75K246R protein abundance using anti-HA antibody. In Vivo Ubiquitination Assay In vivo ubiquitination assay was performed as previously described (Miura, et al. 2007).

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Briefly, 35S:HA-MYB75, 35S:HA-MYB75K246R plasmids were transiently expressed overnight in wild type or siz1-2 Arabidopsis protoplasts respectively, which were then

treated with 50 μM MG132 for 2 h. The protoplasts were thawed in 500 μL extraction buffer containing 300 mM NaCl, 100 mM Tris-HCl pH 7.5, 1% Triton X-100, 2 mM

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EDTA pH 8.0, 10% glycerol, 50 μM MG132, and protease inhibitor . The supernatant

was incubated with agarose conjugated Anti-HA antibody (Sigma) at 4 ℃ for 2 h. After

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being rinsed four times with the pull-down buffer, the beads were boiled in 95℃ with 50 μL SDS-PAGE loading buffer and then subjected to SDS-PAGE. Ubn-MYB75

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conjugation was detected by anti-Ub antibodies. The abundance of HA-MYB75 as a control was detected by anti-HA antibodies.

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Real-Time PCR Analysis

RNA was extracted as described previously, and cDNA synthesis was performed by one microgram (Zhang, et al. 2010). qRT-PCR analysis was conducted by using SYBR

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Green PCR Master Mix. Three separate experiments and technical triplicates of each experiment were implemented. Gene expression was standardized to the ACTIN 8

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transcript levels.

Statistical Analysis Samples were analyzed in three individual biological replicates, and the data are indicated as the mean ± SD. Two-way ANOVA (LSD’s multiple-range test) or Student’s t-test were performed at a significance level of P<0.05. Results The SUMO E3 Ligase SIZ1 is involved in high light-induced anthocyanin

accumulation Previous research evidenced that SIZ1 regulated anthocyanin accumulation under drought stress in Arabidopsis (Catala, et al. 2007), thus we further investigated how SIZ1 participated in regulation of anthocyanin accumulation. As prior reported, high light is an efficient factor to induce anthocyanin accumulation (Albert, et al. 2014; Li, et al. 2016), therefore, Arabidopsis seedlings were maintained under weak light of 40 μmol m-2 s-1 (hereafter called Control), a control light intensity would not generate high levels of anthocyanin accumulation in wild-type plants (Maier, et al. 2013). Seedlings

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were then moved to moderate high light (180 μmol m-2 s-1, hereafter called high light) to induce anthocyanin biosynthesis. As shown in Fig. 1A, compared with wild type,

pap1-D seedlings, the activation tag mutant constitutively overexpresses MYB75/PAP1, showed more anthocyanin accumulation under high light conditions. By comparison,

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myb75-c (MYB75 knockout mutants were generated in the Col-0 ecotype using the

CRISPR-Cas9 system) and siz1-2 displayed less anthocyanin accumulation (Fig. 1A).

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Quantification of anthocyanin also verified these results (Fig. 1B). We next detected the expression of anthocyanin-specific biosynthetic genes, DFR, LDOX, and UF3GT.

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Consistently, the transcript levels of these genes were also dramatically reduced in siz12 under high light conditions (Fig. 1C-1E). We further investigated whether SUMO

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conjugates accumulated in response to high light. As shown in Fig. 1F, SUMO conjugates levels were elevated in wild type after exposure to high light, while high light-induced accumulation of SUMO conjugates were significantly reduced in siz1-2

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mutant. Summarily, these results confirm that SIZ1 is involved in anthocyanin accumulation and mediates high light-induced accumulation of SUMO conjugates.

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SIZ1 positively regulates anthocyanin accumulation and impaired SUMO1/2 modification decreases anthocyanin accumulation Next we checked whether SIZ1 positively regulates anthocyanin accumulation. Expression of ProSIZ1:SIZ1-GFP in siz1-2 plants (complemented lines referred to as SSG) (Jin, et al. 2008) rescued the siz1-2 phenotype under high light conditions, indicating SIZ1 positively regulates anthocyanin accumulation (Fig. 2A and 2B). To determine whether SIZ1-mediated SUMO1/2 modification is involved in the

regulation of anthocyanin accumulation, we determined the anthocyanin levels of sum1 and sum2 double mutant under control and high light conditions. SUMO1 and 2 have redundant functions, and the sum1 sum2 double knockout mutant is embryo lethal (Saracco, et al. 2007). Therefore, we used a viable weak allele, sum1-1 amiR-SUM2, in which SUMO2 expression is down-regulated by RNAi in the sum1-1 knockout mutant background (van den Burg, et al. 2010). Similar to siz1-2, sum1-1 amiR-SUM2 seedlings exhibited minimal anthocyanin accumulation under high light conditions compared with wild type, suggesting that SUMO1/2 modification regulates

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anthocyanin accumulation (Fig. 2C and 2D). Taken together, these data suggest that SUMO1/2 modification is essential for anthocyanin accumulation. SIZ1 interacts with MYB75

To elucidate the molecular mechanism by which SIZ1 modulates anthocyanin

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accumulation, we aimed to identify interacted TFs of SIZ1 by performing yeast twohybrid screening. MYB75 (At1g56650), an MYB transcription factor, was ascertained

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in this screening. A further pairwise experiment evidenced the interaction between SIZ1 and MYB75 in the yeast two-hybrid assay (Fig. 3A). In vitro pull-down assays also

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validated that HIS-SIZ1 directly interacted with MBP-MYB75, but not with MBP alone (Fig. 3B).

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Next, we examined the interaction between SIZ1 and MYB75 in vivo using bimolecular fluorescence complementation (BiFC) assays on Nicotiana benthamiana leaves. Coexpression of MYB75 fused with the amino terminus of the yellow fluorescent protein

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(nYFP) and SIZ1 fused with the carboxy terminus of YFP (cYFP) in N. benthamiana obviously showed that MYB75 interacted with SIZ1 in epidermal cells of N.

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benthamiana leaves, however, no fluorescence was observed in epidermal cells transformed with nYFP and SIZ1-cYFP or MYB75-nYFP with cYFP construct (Fig. 3C). We further perform a co-immunoprecipitation (co-IP) experiment in Arabidopsis protoplasts transiently expressing HA-MYB75 or HA and SIZ1-GFP respectively. The co-IP assays suggested that SIZ1-GFP was immunoprecipitated by HA-MYB75 rather than HA (Fig. 3D). In conclusion, SIZ1 interacts with MYB75 in vitro and in vivo. SIZ1 mediates sumoylation of MYB75

On the basis of the interaction between SIZ1 and MYB75 in vitro and in vivo, we further investigate whether SIZ1 functions as an E3 SUMO ligase for MYB75. To test this possibility, we performed an in vitro sumoylation assay to determine sumoylation of MYB75 as described previously (Lin, et al. 2016; Park, et al. 2011). As shown in Fig. 4A, MYB75 was sumoylated in the presence of SUMO E1 (HIS6-SAE1b and HIS6SAE2), SUMO E2 (HIS6-SCE1), SUMO E3 (HIS6-SIZ1) and HIS6-SUMO1-GG (a conjugation-normal wild type) instead of HIS6-SUMO1-AA (a conjugation-deficient mutant), suggesting that MYB75 could be a substrate for SIZ1. To test this hypothesis,

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we transferred 35S:MYC-SUMO1 and 35S:HA-MYB75 constructs into either wild type or siz1-2 protoplasts, and monitored the sumoylation of MYB75. Sumoylation of MYB75 was detected in wild type but not in the siz1-2 mutant, demonstrating that SIZ1

modulates the sumoylation of MYB75 in vivo (Fig. 4B). Next we determine whether

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high light conditions affected sumoylation of MYB75. As shown in Fig. 4C, sumoylation of MYB75 was enhanced by high light conditions. Bioinformatics analysis

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showed that MYB75 has seven potential sumoylation sites (K62, K111, K122, K160, K187, K225, and K246) (Fig. 4D). To map the sumoylation site(s) in MYB75, we

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generated MYB75 mutants in which the lysine residue was mutated into arginine individually. The sumoylation of MYB75 was abolished in K246R mutant, but was

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unaffected in other single mutant (Fig. 4E), suggesting that sumoylation of MYB75 probably occurs at residue K246. Previous studies demonstrated that SUMO interacted with substrate through SUMO-interaction motifs (SIMs), independent of an E3 ligase

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(Kerscher 2007; Wang and Dasso 2009). Based on this hypothesis, we searched for the SIM sequence [VIL]-x-[VIL]-[VIL] or [VIL]-[VIL]-x-[VIL] in MYB75 and found two

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putative SIMs (Supplementary Fig. S1A). However, mutagenesis showed that these SIMs (SIM1 and SIM2) mutant didn’t block the sumoylation of MYB75 (Supplementary Fig. S1B), indicating SUMO1 does not covalently interact with MYB75 through these SIMs. To determine whether MYB75 is sumoylated and the sumoylation site in plants, we generated 35S:MYC-SUMO1, 35S:HA-MYB75, 35S:HA-MYB75K187R, and 35S:HAMYB75K246R constructs. MYC-SUMO1 plasmid was cotransformed with these HA-

MYB75 plasmids individually into wild type protoplasts. SUMO1 conjugation of MYB75 was detected in protein extracts isolated from MYB75WT and MYB75K187R, but greatly reduced in MYB75K246R (Fig. 4F). These results indicate that K246 is the principal site of SUMO conjugation on MYB75 in Arabidopsis. Sumoylation of MYB75 by SIZ1 is critical for anthocyanin accumulation To further confirm that sumoylation of MYB75 by SIZ1 is required for anthocyanin accumulation, we crossed pap1-D mutant with siz1-2 to generate the pap1-D siz1-2 double mutant. The pap1-D siz1-2 double mutant accumulated remarkably lower

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anthocyanin than that of pap1-D and exhibited almost identical anthocyanin pigment compared with siz1-2 mutant (Fig. 5A and 5B). Consistent with this, expression of the

anthocyanin-specific biosynthetic genes DFR, LDOX, and UF3GT in the pap1-D siz12 double mutant were also lower than that in the pap1-D mutant, suggesting that

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MYB75 function is inhibited in siz1-2 (Fig. 5C-5E). Collectively, these results demonstrated that sumoylation of MYB75 by SIZ1 is critical for MYB75-mediated

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anthocyanin accumulation.

MYB75 sumoylation at residue K246 is necessary for anthocyanin accumulation

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Next, we expressed 35S:HA-MYB75WT or 35S:HA-MYB75K246R in myb75-c plants. Two transgenic lines showed almost equivalent expression of MYB75WT or

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MYB75K246R, but the anthocyanin content of 35S:MYB75K246R/myb75-c was much lower than that of 35S:MYB75WT/myb75-c plants (Fig. 6A-6C). Consistent with the phenotype, expression of anthocyanin-specific biosynthetic genes DFR and LDOX

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were also lower in 35S:MYB75K246R/myb75-c compared with 35S:MYB75WT/myb75-c plants (Fig. 6D and 6E), suggesting that sumoylation of K246 is essential for MYB75

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function in anthocyanin accumulation. Sumoylation by SIZ1 increases the stability of MYB75 Previous research described that sumoylation affects protein stability, subcellular localization, and functional activity. The results that anthocyanin accumulation was minimal in pap1-D siz1-2 mutant and 35S:MYB75K246R/myb75-c prompt us to determine the functions of sumoylation on MYB75. Both MYB75WT-GFP and MYB75K246R-GFP localized to the nucleus in Arabidopsis protoplasts, indicating that

sumoylation of MYB75 did not affect protein localization (Supplementary Fig. S2A). Additionally, MYB75K246R mutation did not drastically affect MYB75 transcriptional activity in myb75-c protoplasts treated with MG132 (Supplementary Fig. S2B). We next compared MYB75 protein levels in wild type and siz1-2 plants using a cell-free degradation assay. When incubated with total proteins from siz1-2 plants, the abundance of MBP-MYB75 was rapidly reduced, whereas MBP-MYB75 was more resistant to degradation in the total proteins from wild type (Fig. 7A). Notably, MG132 resulted in stabilization of MBP-MYB75, suggesting that MYB75 degradation requires

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proteasome activity (Fig. 7A and 7B). Moreover, the protein level of MYB75 was analyzed among the 35S:HA-MYB75WT, 35S:HA-MYB75WT siz1-2, and 35S:HA-MYB75K246R transgenic plants. Total proteins were extracted from these transgenic plants and used for western blotting assays with

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an anti-HA antibody. As shown in Figure 7B, the degradation of MYB75 was expedited

in 35S:HA-MYB75WT siz1-2 and 35S:HA-MYB75K246R extracts compared with that in

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35S:HA-MYB75WT extracts. In addition, the degradation of MYB75 was effectively blocked by MG132 (Figure 7C and 7D). It is well established that light regulated the

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stability of MYB75, we examined the abundance of MYB75 proteins in dark-adapted seedlings to further explained whether MYB75 sumoylation affects its stability. As

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shown in Fig. 7E, MYB75 were almost undetectable in dark-adapted wild type, 35SMYB75 siz1-2 or 35S:MYB75K246R seedlings. Notably, the levels of MYB75 in wild type seedlings increased greatly, whereas that of MYB75 in siz1-2 and MYB75K246R

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was only slightly increased when we treated dark-adapted seedlings with 50 μM MG132. In addition, we conducted an in vivo ubiquitination assay using the wild type

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and siz1-2 protoplasts treated with 50 μM MG132. The results indicated that the ubiquitination levels of HA-MYB75 in the siz1-2 protoplasts were much higher than that in the wild type protoplasts (Fig. 7F). Meanwhile, the ubiquitination levels of MYB75 in the wild type protoplasts were much fewer than MYB75K246R in the wild type protoplasts (Fig. 7F). Taken together, our results indicate that sumoylation of MYB75 by SIZ1 opposes degradation and thus stabilizing MYB75. Discussion

In this study, we established that SIZ1 participates in anthocyanin accumulation by sumoylating MYB75 (Fig. 1). MYB75 is a R2R3-MYB transcription factor that has been identified to be a key factor in anthocyanin accumulation (Borevitz, et al. 2000). It is well studied that COP1 and MPK4 oppositely modulates MYB75 stability. Our research revealed that sumoylation of MYB75 at K246 by SIZ1 enhanced its stability (Fig. 7), thus inspiring a new post-translational modification for MYB75 function in anthocyanin accumulation under high light conditions. SIZ1 coordinates stress conditions and anthocyanin accumulation

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We ascertained that high light conditions enhanced SIZ1 expression (Supplementary Fig. S3) and induced SIZ1-dependent SUMO1 conjugate accumulation (Fig. 1D). Additionally, sumoylation of substrate by SIZ1 is involved in numerous biological

processes related to stress conditions including freezing, cold, drought and disease

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(Catala, et al. 2007; Miura, et al. 2007; Saleh, et al. 2015). The extreme stresses result in reactive oxygen species (ROS) accumulation in plant cell, which disturb cellular

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metabolism and reduce yield (Choudhury, et al. 2017; Dietz, et al. 2016; Mittler 2017). Recent study evidenced that hydrogen peroxide (H2O2) induced SUMO-conjugate

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accumulation in Arabidopsis (Rytz, et al. 2018). Notably, anthocyanin accumulation induced by stress conditions protect the plants against cellular damage through

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scavenging ROS. Therefore, we speculate that on the one hand sumoylation of proteins which are involved in signal transduction by SIZ1 could elevate the expression of stress-related genes, and on the other sumoylation of MYB75 by SIZ1 endow plants

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with anthocyanin accumulation to scavenge ROS. Plants evolved this dual mechanism to cope with and adapt to adverse environmental conditions.

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Sumoylation of MYB75 by SIZ1 regulates anthocyanin accumulation during plant senescence

Anthocyanin accumulation is not simply regulated by environmental stimuli, the expression of anthocyanin biosynthetic and regulatory genes accordingly alters during plant development (Bhargava, et al. 2010; Kubasek, et al. 1992; Nesi, et al. 2000; Walker, et al. 1999). We noticed that SIZ1 and MYB75 displayed high expression in senescent leaves (Supplementary Fig. S4), identical conclusion was obtained in gene

expression analysis using the Arabidopsis microarray data displayed in the eFP browser where presented very similar expression patterns of SIZ1 and MYB75 (Supplementary Fig. S5), suggesting they may function together to regulate anthocyanin accumulation during plant senescence. An interesting study observed that senescence dramatically induced MYB90 expression in Arabidopsis, indicating that MYB90 is likely involved in SIZ1-regulated anthocyanin accumulation (Wingler, et al. 2012). Nevertheless, we didn’t observe the interaction between SIZ1 and MYB90 in yeast (Supplementary Fig. S6).

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The sugar-sensing model proposed that high sugar content triggered senescence through suppressing protein kinase SnRK1 (Thomas 2013). It should be pointed out that sugar levels induced transcript levels of MYB75 and SUMO1 conjugate

accumulation in a SIZ1-dependent manner (Castro, et al. 2015; Solfanelli, et al. 2006).

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Hence, we suppose that sumoylation of MYB75 induced by SUMO-conjugate

accumulation play an important role in anthocyanin accumulation in senescent leaves.

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Meanwhile, the SIZ1-regulated anthocyanin accumulation derived from sugar enrichment constitute a feedback to extend leaf longevity in senescent leaves.

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Diverse post-translational modification of MYB75 fines tune anthocyanin accumulation

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Our previous study demonstrated that HAT1, the transcription factor of class II homeodomain-leucine zipper family, inhibits anthocyanin accumulation via interaction with MYB75 at post-translational level (Zheng, et al. 2019). Additionally, sumoylation

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and ubiquitination can interact competitively or cooperatively on the same substrate to regulate protein stability and function (Miura, et al. 2007; Zheng, et al. 2018). For

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example, phosphorylation of the human Flap Endonuclease 1 (FEN1) induces its sumoylation, which in turn leads to ubiquitination and subsequent proteasomal degradation (Guo, et al. 2012). In contrast, ICE1, which is a bHLH transcription factor that functions as a positive regulator in cold stress, is sumoylated at K393. Sumoylation of K393 reduces ubiquitination of ICE1 thus could increase ICE1 stability (Miura, et al. 2007). Recent study reveal that sumoylation and ubiquitination act antagonistically in the ABA response to regulate the stability of MYB30 by occupying the same residue

(Zheng, et al. 2018). Our results suggested that sumoylation of MYB75 decreased ubiquitination of MYB75. Additionally, degradation of MYB75 is mediated by COP1 in the dark (Maier, et al. 2013). It remains challenging to clarify the relationship between ubiquitination and sumoylation of MYB75. Much work has focused on protein phosphorylation and sumoylation for stress responses and development in plants (Tomanov, et al. 2018). Sumoylation of NPR1 is restrained by phosphorylation at Ser55/Ser59 (Saleh, et al. 2015). Phosphorylation of CESTA, a transcription factor participated in brassinosteroid response, likely prevents

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its sumoylation (Khan, et al. 2014). In contrast to phosphorylation, ABSCISIC ACID INSENSITIVE5 (ABI5) activity is weaken by sumoylation (Miuraa, et al. 2009). Unlike the antagonistic effects of sumoylation and phosphorylation on these proteins,

SIZ1 and MPK4 positively modulate MYB75 stability. Further effort is required to

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understand how the interplay between sumoylation and phosphorylation of MYB75 regulates anthocyanin accumulation.

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In conclusion, our results suggested that sumoylation of MYB75 at K246 by SIZ1 increases protein stability. High light elevates transcript levels of SIZ1 and thus

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enhancing sumoylation of MYB75. Consequently, sumoylated MYB75 facilitates anthocyanin-specific biosynthetic gene (DFR, LDOX, and UF3GT) expression to

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promote anthocyanin accumulation under high light conditions (Fig. 8). Supplementary data

Supplementary data are available at Plant Science online.

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Figure S1. Mutant of SIMs didn’t block the sumoylation of MYB75 Figure S2. Localization and transcriptional activity analysis of MYB75K246R

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Figure S3. High light induces SIZ1 expression Figure S4. Expression of SIZ1 and MYB75 in senescent leaves Figure S5. Expression pattern of SIZ1 and MYB75 in plants Figure S6. SIZ1 does not interact with MYB90 Figure S7. Confirmation of MYB75WT and MYB75K246R in different genotype Figure S8. Full scan images of Western blots present in this study Figure S9. Full scan images of Western blots present in this study

Author contributions H.H.L and D.W.Z conceived and supervised the study. T.Z and D.W.Z designed experiments and analyzed data. T.Z, Y.L.L, W.L, K.Q and B.H.L performed experiments. T.Z and D.W.Z wrote the manuscript.

Conflict of Interest

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The authors have declared that no competing interests exist. Acknowledgements

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We thank Prof. Jingbo Jin for providing SSG, sum1-1 amiR-SUM2, pMAL-C2-MBPSIZ1-Myc and HIS6-SUMO1AA. The pap1-D and myb75-c were kindly provided by Prof. Daoxin Xie and Prof. Jinlong Qiu respectively. We acknowledge Prof. Junxian He for providing the vector of pGreenII 0800-LUC. This work was supported by grants from the National Natural Science Foundation of China (http://www.nsfc.gov.cn/) [31570237 to DWZ ; 31670235 to HHL]; the Development Project of Transgenic Crops of China (http://program.most.gov.cn/) (2016ZX08009003-002 to HHL); the National Basic Research Program of China (http://program.most.gov.cn/) [973 Program (2015CB150100) to HHL]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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Fig. 1. SIZ1 is involved in high light-induced anthocyanin accumulation (A) Anthocyanin accumulation in seedlings of the indicated genotypes grown on plates under different conditions for 14 days. Bars = 0.5 cm.

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(B) Anthocyanin levels in extracts from seedlings in (A). The experiments were performed in biological triplicate (representing anthocyanin content measured from 15 plants of each genotype and treatment were pooled for one replicate). FW, fresh weight. Error bars denote ± SD (n=3). Different letters represented statistically significant differences (two-way ANOVA, p<0.05). (C-E) qPCR analysis of DFR, LDOX, and UF3GT expression levels in 14-day-old seedlings in (A) grown on plates under different conditions for 9 h, respectively. Expression levels were standardized to ACTIN 8, the results of wild type under control conditions were set at 1. Error bars denote ± SD (n=3). Different letters represented statistically significant differences (two-way ANOVA, p<0.05).

(F) Accumulation of SUMO conjugates levels is induced by high light. Protein extracts

were analyzed by protein gel blots using anti-SUMO1 antibodies to detect SUMO

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conjugates. The plant β-ACTIN was used as a loading control.

Fig. 2. SIZ1 positively regulates anthocyanin accumulation and impaired SUMO1/2 modification decreases anthocyanin accumulation (A) Anthocyanin accumulation in seedlings of the indicated genotypes grown on plates under different conditions for 14 days. Bars = 0.5 cm. (B) Anthocyanin levels in extracts from seedlings in (A). The experiments were performed in biological triplicate (representing anthocyanin content measured from 15 plants of each genotype and treatment were pooled for one replicate). FW, fresh weight. Error bars denote ± SD (n=3). Different letters represented statistically significant differences (two-way ANOVA, p<0.05). (C) Anthocyanin accumulation in seedlings of the indicated genotypes grown on plates under different conditions for 14 days. Bars = 0.5 cm.

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(D) Anthocyanin levels in extracts from seedlings in (C). The experiments were performed in biological triplicate (representing anthocyanin content measured from

15 plants of each genotype and treatment were pooled for one replicate). FW, fresh

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weight. Error bars denote ± SD (n=3). Different letters represented statistically

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significant differences (two-way ANOVA, p<0.05).

Fig. 3. SIZ1 interacts with MYB75 (A) The interaction of SIZ1 and MYB75 in yeast. The ability of cells to grow on Quadruple DO supplement lacking Leu, Trp, His, and Ade (-LWHA) suggested the interaction. Interaction between MYB75 and TT8 served as a positive control. AD, GAL4 activation domain. BD, GAL4 DNA binding domain. (B) Pull-down assay showing the interaction of SIZ1 and MYB75. Purified HIS-SIZ1 proteins were immunoprecipitated with HIS beads. Immunoprecipitated proteins were

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incubated with MBP or MBP-MYB75 proteins, and anti-MBP antibody was used to detect MBP-MYB75. (C) BiFC analysis of the interaction between SIZ1 and MYB75 in N. benthamiana. Interaction between MYB75 and TT8 served as a positive control. Bars = 50 μm. (D) Co-IP assay showing the interaction of MYB75 with SIZ1 in vivo. The construct combinations were expressed in N. benthamiana leaves. Total proteins were extracted and immunoprecipitated with anti-HA agarose beads. The proteins were detected with antiHA and anti-GFP antibodie.

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Fig. 4. SIZ1 mediates sumoylation of MYB75 at K246 (A) In vitro sumoylation of MYB75. Sumoylated MYB75 was detected with anti-MBP antibodies. SUMO1GG and SUMO1AA respectively represent a conjugation-normal wild type and a conjugation-deficient mutant.Arrowheads indicate possible sumoylated MYB75. (B) SIZ1 mediates the sumoylation of MYB75 in vivo. Combinations of MYC-SUMO1 and HA-MYB75 were coexpressed in Arabidopsis protoplasts. Soluble extracts from protoplasts were immunoprecipitated with anti-HA antibody (IP:HA) and the immunoprecipitated products were detected with anti-MYC antibody to monitor SUMOMYB75 conjugates. Protein loading was detected with anti-HA antibody. (C) Sumoylation of MYB75 was enhanced by high light conditions. Combinations of MYC-SUMO1 and HA-MYB75 were coexpressed in Arabidopsis protoplasts. For the high light treatment, the protoplasts were incubated under high light conditions for 1 h. Sumoylation was detected as described in Fig 4B. (D) Amino acids marked with red indicate predicted sumoylation sites in MYB75. (E) In vitro identification of the sumoylation site on MYB75. Sumoylated MYB75 was detected with anti-MBP antibodies. Arrowheads indicate possible sumoylated MYB75.

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(F) In vivoidentification of the sumoylation site on MYB75. Combinations of MYCSUMO1 and HA-MYB75WT, HA-MYB75K187R, or HA-MYB75K246R were coexpressed in Arabidopsis protoplasts. Soluble extracts from protoplasts were immunoprecipitated with anti-HA antibody (IP:HA) and the immunoprecipitated products were detected with antiMYC antibody to monitor SUMO-MYB75 conjugates. Protein loading was detected with anti-HA antibody.

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Fig. 5. SIZ1 is essential for MYB75-mediated anthocyanin accumulation (A) Anthocyanin accumulation in seedlings of the indicated genotypes grown on plates under different conditions for 14 days. Bars = 0.5 cm. (B) Anthocyanin levels in extracts from seedlings in (A). The experiments were performed in biological triplicate (representing anthocyanin content measured from 15 plants of each genotype and treatment were pooled for one replicate). FW, fresh weight. Error bars denote ± SD (n=3). Different letters represented statistically significant differences (two-way ANOVA, p<0.05). (C-E) qPCR analysis of DFR, LDOX, and UF3GT expression levels in 14-day-old seedlings in (A) grown on plates under different conditions for 9 h, respectively. Expression levels were standardized to ACTIN 8, the results of wild type under control conditions were set at 1. Error bars denote ± SD (n=3). Different letters represented statistically significant differences (two-way ANOVA, p<0.05).

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Fig. 6. Sumoylation of MYB75 is required for its function in anthocyanin accumulation (A) Anthocyanin accumulation in seedlings of the indicated genotypes grown on plates under different conditions for 14 days. Bars = 0.5 cm. (B) Anthocyanin levels in extracts from seedlings in (A). The experiments were performed in biological triplicate (representing anthocyanin content measured from 15 plants of each genotype and treatment were pooled for one replicate). FW, fresh weight. Error bars denote ± SD (n=3). Different letters represented statistically significant differences (two-way ANOVA, p<0.05). (C) qPCR analysis of MYB75 transcript levels in 14-day-old seedlings in (A) grown on plates under control conditions, respectively. Expression levels were standardized to ACTIN 8, and results in the wild type under control conditions were set at 1. Error bars denote ± SD (n=3). (D-E) qPCR analysis of DFR, and LDOX expression levels in 14-day-old seedlings in (A) grown on plates under different conditions for 9 h, respectively. Expression levels were standardized to ACTIN 8, the results of wild type under control conditions were set at 1. Error bars denote ± SD (n=3). Different letters represented statistically significant differences (two-way ANOVA, p<0.05).

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Fig. 7. MYB75 protein stability is suppressed by blocking the sumoylation of MYB75 (A) Cell-free degradation assay. Total protein extracts from wild type (WT) and siz1-2 mutant were incubated with equal quantities of recombinant MBP-MYB75 for different durations, which were then treated with DMSO (-MG132) or 50 μM MG132 for the indicated periods of time and sampled simultaneously to detect and sampled simultaneously to detect MBP-MYB75 protein abundance using anti-MBP antibody. βACTIN in total protein extracts was used as a loading control. (B) The MBP-MYB75 protein levels in (A) were quantified. All protein levels were normalized to β-ACTIN and their initial value. Three independent biological replicates were analyzed. Data presented means ± SD. (C) HA-MYB75 protein degradation assay in vivo. Indicated transgenic seedlings were treated with translational inhibitor cycloheximide (CHX) for the indicated periods of

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time and sampled simultaneously to detect HA-MYB75 protein abundance using anti-HA antibody. β-ACTIN in total protein extracts was used as a loading control. DMSO (MG132) was used as a negative control of the 26S proteasome inhibitor MG132. (D) The HA-MYB75 protein levels in (C) were quantified. All protein levels were normalized to β-ACTIN and their initial value. Three independent biological replicates were analyzed. Data presented means ± SD. (E) Sumoylation of MYB75 is vital for MYB75 protein stability. Dark-adapted, 35S:MYB75WT, 35S:MYB75 siz1-2, and 35S:MYB75K246R seedlings were treated with MG132 or DMSO for 12 h in darkness. The levels of MYB75 were determined by using anti-HA antibody. β-ACTIN was used as loading control. (F) Ubiquitination assay of HA-MYB75 in the wild type and siz1-2 protoplasts treated with 50 μM MG132 in vivo. The HA-MYB75 and HA-MYB75-Ub proteins were immunoprecipitated with anti-HA agarose and were detected by western blotting with anti-Ub antibodies (upper gels) and anti-HA antibodies (lower gels).

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Fig. 8. A proposed model depicting how SIZ1 regulates anthocyanin accumulation under high light conditions High light elevates transcript levels of SUMO E3 ligase SIZ1 thus enhancing sumoylation of MYB75. Sumoylation of MYB75 by SIZ1 increase its protein stability. Consequently, sumoylated MYB75 facilitates anthocyanin-specific biosynthetic gene (DFR, LDOX, and UF3GT) expression to promote anthocyanin accumulation under high light conditions.