An Arabidopsis SUMO E3 Ligase, SIZ1, Negatively Regulates Photomorphogenesis by Promoting COP1 Activity

<div><p>COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1), a ubiquitin E3 ligase, is a central negative regulator of photomorphogenesis. However, how COP1 activity is regulated by post-translational modifications remains largely unknown. Here we show that SUMO (small ubiquitin-like modifier) modification enhances COP1 activity. Loss-of-function <i>siz1</i> mutant seedlings exhibit a weak constitutive photomorphogenic phenotype. SIZ1 physically interacts with COP1 and mediates the sumoylation of COP1. A K193R substitution in COP1 blocks its SUMO modification and reduces COP1 activity <i>in vitro</i> and <i>in planta</i>. Consistently, COP1 activity is reduced in <i>siz1</i> and the level of HY5, a COP1 target protein, is increased in <i>siz1</i>. Sumoylated COP1 may exhibits higher transubiquitination activity than does non-sumoylated COP1, but SIZ1-mediated SUMO modification does not affect COP1 dimerization, COP1-HY5 interaction, and nuclear accumulation of COP1. Interestingly, prolonged light exposure reduces the sumoylation level of COP1, and COP1 mediates the ubiquitination and degradation of SIZ1. These regulatory mechanisms may maintain the homeostasis of COP1 activity, ensuing proper photomorphogenic development in changing light environment. Our genetic and biochemical studies identify a function for SIZ1 in photomorphogenesis and reveal a novel SUMO-regulated ubiquitin ligase, COP1, in plants.</p></div

Sumoylation may enhances the transubiquitination activity of COP1.

<p>(<b>A</b>) qRT-PCR analysis of <i>COP1</i> expression in Col-0 and <i>siz1-2</i>. Five-day-old dark-grown seedlings were transferred to white light for the indicated time periods. The relative expression level of <i>COP1</i> was normalized to that of <i>UBC</i>, and data represent the mean ± SE (n = 3). (<b>B</b>) COP1 levels in Col-0 and <i>siz1-2</i> under dark and light conditions. Five-day-old dark-grown seedlings (Dark) were exposed to light for 12 h (Light 12 h). COP1 was detected with anti-COP1 antibody. Tubulin was detected with anti-Tubulin antibody as a loading control. Numbers above the blot indicate the relative level of COP1 normalized to that of Tubulin. (<b>C</b>) Analysis of the effect of SUMO modification on COP1 dimerization. FLAG-COP1 and FLAG-SUMO1 were transiently co-expressed with Myc-COP1 or Myc-COP1^K193R in <i>N</i>. <i>benthamiana</i> leaves. Myc-COP1 or Myc-COP1^K193R was immunoprecipitated (IP) with anti-Myc antibody, and FLAG-COP1 in the precipitates was detected with anti-FLAG antibody. Sumoylated COP1 was detected with anti-SUMO1 and anti-FLAG antibody (longer exposure). The level of immunoprecipitated Myc-COP1 or Myc-COP1^K193R was detected with anti-Myc antibody. (<b>D</b>) <i>In vitro</i> immunoprecipitation analysis of the COP1-HY5 interaction. GST-HY5 was incubated with total protein extract isolated from <i>N</i>. <i>benthamiana</i> co-expressing Myc-COP1 with FLAG-SUMO1 or FLAG-SUMO1^AA under dark (0 h) or light (12 h) conditions in the presence of 50 μM MG132. After 1.5 h incubation at 4°C, Myc-COP1 was immunoprecipitated with anti-Myc antibody, and co-immunoprecipitated GST-HY5 was detected with anti-GST antibody. Immunoprecipitated Myc-COP1 was quantified with anti-Myc antibody and sumoylated COP1 was detected with anti-FLAG antibody. (<b>E</b>) Nuclear fractions were isolated from <i>COP1 OE</i> and <i>COP1 OE siz1-2</i> under dark (0 h) and light (12 h white light exposure) conditions, and level of nuclear COP1 was detected with anti-COP1 antibody. Nuclear fraction of Col-0 and total protein extract of <i>cop1-4</i> was used as a control. Histone 3 and tubulin were used as nuclear and cytosol marker proteins, respectively. (<b>F</b>) <i>In vitro</i> sumoylated and non-sumoylated MBP-COP1-FLAG were used as E3 ligases to perform an <i>in vitro</i> HY5 ubiquitination assay. Bead-conjugated GST-HY5 was used as substrate. After reaction, the beads were washed and ubiquitinated GST-HY5 were eluted for immunoblot analysis with anti-GST and anti-ubiquitin antibodies. MBP-FLAG was used as a negative control. The vertical line indicates ubiquitinated HY5. Input E3s were detected with anti-FLAG antibody. Asterisks indicate non-specific bands.</p

SUMO E3 ligase-mediated SUMO1/2 modification, but not SA, regulates hypocotyl elongation in response to light.

<p>(<b>A</b>) <i>siz1-2</i>, <i>sum1-1 amiR-SUM2</i>, and <i>NahG siz1-2</i> seedlings display shorter hypocotyls than the control plants Col-0 and <i>NahG</i> under red (R; 10 μmol m^-2 s^-1), blue (BL; 14 μmol m^-2 s^-1), and far-red (FR; 12 μmol m^-2 s^-1) light conditions. Bar = 2 mm. (<b>B</b>) Hypocotyl length of five-day-old Col-0, <i>siz1-2</i>, <i>sum1-1 amiR-SUM2</i>, <i>NahG</i>, and <i>NahG siz1-2</i> seedlings under darkness and the indicated light conditions. Data are the mean ± SE of 30 seedlings.</p

COP1 promotes the ubiquitination and degradation of SIZ1.

<p>(<b>A</b>) COP1 enhances the degradation of SIZ1. Total proteins extracted from Myc-COP1 expressing or non-transformed (control) <i>N</i>. <i>benthamiana</i> leaves were mixed with total proteins extracted from SIZ1-GFP expressing <i>N</i>. <i>benthamiana</i> leaves, and incubated for the indicated periods at 4°C under dark condition. SIZ1-GFP and Myc-COP1 protein levels were analyzed with the anti-GFP and anti-Myc antibody, respectively. Actin was used as a loading control and detected with anti-Actin antibody. Numbers indicate the relative protein levels of SIZ1. (<b>B</b>) MG132 inhibits the degradation of SIZ1-GFP. Total proteins extracted from Myc-COP1 or SIZ1-GFP expressing leaves were mixed together, and treated with or without 50 μM MG132 for an additional 4 h at 4°C under dark condition. The levels of SIZ1-GFP and Myc-COP1 were analyzed as describe above. Actin was used as a loading control and detected with anti-Actin antibody. Numbers indicate the relative protein levels of SIZ1 and COP1. (<b>C</b>) qRT-PCR analysis of the transcription level of <i>SIZ1</i> in Col-0 and <i>cop1-4</i>. Seedlings were grown under white light for 5 days. Relative expression was normalized to that of <i>UBC</i>. Data indicate the mean ± SE (n = 3). (<b>D</b>) <i>In vitro</i> ubiquitination of SIZ1 by COP1. MBP-COP1-FLAG and bead-conjugated MBP-SIZ1-Myc were used as E3 ligases and substrate, respectively, to perform an <i>in vitro</i> ubiquitination assay. MBP-FLAG was used as a negative control. Input E3s were detected with anti-FLAG antibody. After reaction, the beads were washed and ubiquitinated MBP-SIZ1-Myc was eluted for immunoblot analyses with anti-Myc and anti-ubiquitin antibodies. The vertical line indicates ubiquitinated SIZ1 proteins.</p

SIZ1-mediated sumoylation enhances COP1 activity.

<p>(<b>A</b>) The hypocotyls of <i>35S-Myc-COP1</i> seedlings, but not <i>35S-Myc-COP1</i>^<i>K193R</i>, are longer than those of Col-0 seedlings under 50 μmol m^-2 s^-1 fluence rate of white light. Bar = 2 mm. (<b>B</b>) Hypocotyl length of five-day-old white light-grown seedlings at the indicated fluence rates. Data represent the mean ± SE (n = 30). (<b>C</b>) Total protein extracts from five-day-old dark-grown <i>35S-Myc-COP1</i>, <i>35S-Myc-COP1</i>^<i>K193R</i>, Col-0 and <i>cop1-4</i> seedlings, and bead-conjugated 6xHis-HY5 were used as E3 ligases and substrate, respectively, to perform an <i>in vitro</i> ubiquitination assay. After reaction, the beads were washed and ubiquitinated 6xHis-HY5 were eluted for immunoblot analysis with anti-His and anti-ubiquitin antibodies. Arrowhead indicates ubiquitinated HY5. Asterisk indicates non-specific band. COP1 was detected with anti-COP1 antibody. (<b>D</b>) <i>siz1-2</i> suppresses the long-hypocotyl phenotype of <i>COP1 OE</i>. Col-0, <i>siz1-2</i>, <i>COP1 OE siz1-2</i>, and <i>COP1 OE</i> seedlings were grown under white light for 5 days. Bar = 2 mm. (<b>E</b>) Hypocotyl length of five-day-old light-grown seedlings. Data represent the mean ± SE (n = 30). ** Student’s <i>t</i>-test indicates significant differences between the <i>COP1 OE siz1-2</i> and <i>COP1 OE</i> (P ≤ 0.01). (<b>F</b>) Total protein extracts from five-day-old dark-grown Col-0 and <i>siz1-2</i> seedlings, and bead-conjugated His-HY5 were used as E3s and substrate, respectively, to perform an <i>in vitro</i> ubiquitination assay. <i>cop1-4</i> was used as a negative control. Ubiquitinated HY5 was determined using anti-His and anti-ubiquitin antibody. Arrowhead indicates ubiquitinated HY5. Asterisk indicates non-specific bands. COP1 was detected with anti-COP1 antibody. (<b>G</b>) Genetic interaction between <i>COP1</i> and <i>SIZ1</i>. Col-0, <i>siz1-2</i>, <i>cop1-4 siz1-2</i>, and <i>cop1-4</i> seedlings were grown under darkness for 5 days. Bar = 2 mm. (<b>H</b>) Hypocotyl length of five-day-old dark-grown seedlings. Data represent the mean ± SE (n = 30). ** Student’s <i>t</i>-test indicates significant differences between the <i>siz1-2</i> and <i>cop1-4 siz1-2</i> (P ≤ 0.01).</p

Protein abundance of HY5 is increased in <i>siz1-2</i>.

<p>(<b>A</b>) Immunoblot analysis of HY5 in Col-0, <i>siz1-2</i>, <i>cop1-4</i>, <i>cop1-4 siz1-2</i> and <i>hy5-215</i> seedlings. Total proteins were extracted from five-day-old continuous white light-grown seedlings. HY5 was detected with anti-HY5. Actin was used as a loading control and detected with anti-Actin antibody. Numbers indicate the relative protein levels of HY5. (<b>B</b>) qRT-PCR analysis of the <i>HY5</i> expression level in five-day-old continuous white light-grown Col-0 and <i>siz1-2</i> seedlings. Relative expression was normalized to that of <i>UBC</i>. Data represent the mean ± SE (n = 3). (<b>C</b>) qRT-PCR assay showing the decreased expression level of cell elongation-related genes in five-day-old continuous white light-grown <i>siz1-2</i> seedlings compared to that of Col-0. Relative expression was normalized to that of <i>UBC</i>, and data represent the mean ± SE (n = 3). (<b>D</b>) <i>hy5-215</i> suppresses the short-hypocotyl phenotype of <i>siz1-2</i> under red (R; 10 μmol m^-2 s^-1), blue (BL; 14 μmol m^-2 s^-1), and far-red (FR; 12 μmol m^-2 s^-1) light conditions. Bar = 2 mm. (<b>E</b>) Hypocotyl length of five-day-old seedlings under darkness and red (R), blue (BL), and far-red (FR) light conditions at the indicated fluence rates. Data represent the mean ± SE (n = 30).</p

The <i>siz1</i> mutant seedlings display a short-hypocotyl phenotype.

<p>(<b>A</b>) <i>siz1-2</i> seedlings exhibit a short-hypocotyl phenotype under red (R: 10 μmol m^-2 s^-1), blue (BL: 14 μmol m^-2 s^-1), and far-red (FR: 12 μmol m^-2 s^-1) light conditions, and this phenotype is rescued by complementation with <i>SIZ1</i> driven by its own promoter (SSG). Bar = 2 mm. (<b>B</b>) Hypocotyl length of five-day-old Col-0, <i>siz1-2</i>, and SSG under darkness and red (R), blue (BL), and far-red (FR) light conditions at the indicated fluence rates. Data are the mean ± SE of 30 seedlings. (<b>C</b>) qRT-PCR analysis showing the enhanced responsiveness of light-responsive genes in <i>siz1-2</i> seedlings compared to those in the wild type under the dark to light transition. Five-day-old dark-grown seedlings were transferred to white light for an additional 6 h. Relative expression was normalized to that of <i>UBC</i>. Error bars indicate ± SE (n = 3). (<b>D</b>) <i>siz1-2</i> seedlings exhibit unfolded apical hooks under dark conditions (DK). Bar = 0.5 mm. (<b>E</b>) <i>siz1-2</i> seedlings show more opened cotyledon compared to Col-0 under dark (DK), red (R; 10 μmol m^-2 s^-1), blue (BL; 14 μmol m^-2 s^-1), and far-red (FR; 12 μmol m^-2 s^-1) light conditions. ** Student’s <i>t</i>-test indicates significant differences between the Col-0 and <i>siz1-2</i> (P ≤ 0.01).</p

SIZ1 physically interacts with COP1, and mediates SUMO modification of COP1 at K193.

<p>(<b>A</b>) BiFC assay indicating that SIZ1-YFP^C interacts with COP1-YFP^N (left panel) in the nucleus of <i>N</i>. <i>benthamiana</i> leaf cells in the light (1 h white-light). <i>N</i>. <i>benthamiana</i> cells co-expressing SIZ1-YFP^C and YFP^N (middle panel) and YFP^C and COP1-YFP^N (right panel) were used as negative controls. Bar = 10 μm. (<b>B</b>) Co-immunoprecipitation analysis showing that SIZ1-GFP is associated with Myc-COP1. SIZ1-GFP and Myc-COP1 were transiently co-expressed in Col-0 protoplasts. Co-immunoprecipitated SIZ1-GFP was detected with anti-GFP antibody. Empty Myc vector (Vector) was used as a negative control. (<b>C</b>) <i>In vitro</i> sumoylation of COP1. Sumoylated COP1 was detected with anti-FLAG and anti-SUMO1 antibodies. Arrowheads indicate possible sumoylated COP1. (<b>D</b>) <i>In vivo</i> sumoylation of COP1. Myc-COP1 and FLAG-SUMO1 were transiently co-expressed in Col-0 protoplasts. Myc-COP1 was immunoprecipitated with anti-Myc antibody and sumoylated COP1 (SUMO1-COP1) was detected with anti-FLAG antibody. FLAG-SUMO1^AA was co-transformed with Myc-COP1 as a negative control. Input Myc-COP1 was detected with anti-Myc antibody. Input FLAG-SUMO1 and FLAG-SUMO1 ^AA were detected with anti-FLAG antibody in a separate blot, shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006016#pgen.1006016.s005" target="_blank">S5A Fig</a>. (<b>E</b>) Sumoylation of COP1 <i>in planta</i>. Total proteins were extracted from five-day-old dark-grown <i>35S-Myc-COP1</i> and Col-0 (control) seedlings, and anti-Myc antibody was used to immunoprecipitate Myc-COP1. Anti-SUMO1 antibody was used to determine sumoylated COP1. Input and immunoprecipitated Myc-COP1 were detected with anti-Myc antibody. Arrowhead indicates non-sumoylated COP1 band. Asterisks indicate sumoylated COP1 bands. (<b>F</b>) Light exposure reduces sumoylation levels of COP1. Myc-COP1 and FLAG-SUMO1 co-expressing <i>N</i>. <i>benthamiana</i> leaves were incubated under darkness for 12 h, and then exposed to white light (150 μmol m^-2 s^-1) for 12 h. The nuclear proteins were isolated at the end-of-dark (0 h) and the end-of-light (12 h) period, and the sumoylation level of COP1 was analyzed as described in (D). Input FLAG-SUMO1 and FLAG-SUMO1 ^AA were detected with anti-FLAG antibody in a separate blot, shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006016#pgen.1006016.s005" target="_blank">S5B Fig</a>. (<b>G</b>) The level of COP1 sumoylation was substantially lower in <i>siz1-2</i> than in Col-0. Myc-COP1 and FLAG-SUMO1 were transiently co-expressed in Col-0 or <i>siz1-2</i> protoplasts, and the sumoylation level of COP1 was analyzed as described in (D). (<b>H</b>) K193 is a primary sumoylation site in COP1. FLAG-SUMO1 was transiently co-expressed with Myc-COP1, Myc-COP1^K14R, Myc-COP1^K193R, or Myc-COP1^K653R in Col-0 protoplasts, and immunoprecipitation was performed as described in (D).</p

Proposed model of how SIZ1 and COP1 regulate photomorphogenesis.

<p>Under darkness, COP1 mediates the ubiquitination and degradation of HY5 (thinner T-bar). SIZ1-mediated SUMO modification enhances COP1 ubiquitin E3 ligase activity toward HY5 (thicker T-bar), but COP1 in turn promotes ubiquitination and 26S proteasome-mediated SIZ1 degradation. Under light, COP1 activity is repressed by photoreceptors and nuclear exclusion. Light exposure reduces the sumoylation level of COP1, which may also contribute to the repression of COP1 activity. SUMO protease possibly mediates the desumoylation of COP1 in response to light. T-bars indicate negative regulation and arrows indicate positive regulation. Dotted line indicates hypothetical regulation. S and Ub indicate SUMO and Ubiquitin, respectively.</p