Diplomirani studenti na Odsjeku za informacijske znanosti Filozofskog fakulteta Sveučilišta u Osijeku za razdoblje 2014.-2016.

<p><b>Copyright information:</b></p><p>Taken from "The SUMO E3 ligase, , regulates flowering by controlling a salicylic acid-mediated floral promotion pathway and through affects on chromatin structure"</p><p></p><p>The Plant Journal 2008;53(3):530-540.</p><p>Published online Jan 2008</p><p>PMCID:PMC2254019.</p><p>© 2007 The Authors Journal compilation 2007 Blackwell Publishing Ltd</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

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

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

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

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

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

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

Presentation_1_The High-Affinity Potassium Transporter EpHKT1;2 From the Extremophile Eutrema parvula Mediates Salt Tolerance.pdf

<p>To survive salt stress, plants must maintain a balance between sodium and potassium ions. High-affinity potassium transporters (HKTs) play a key role in reducing Na⁺ toxicity through K⁺ uptake. Eutrema parvula (formerly known as Thellungiella parvula), a halophyte closely related to Arabidopsis, has two HKT1 genes that encode EpHKT1;1 and EpHKT1;2. In response to high salinity, the EpHKT1;2 transcript level increased rapidly; by contrast, the EpHKT1;1 transcript increased more slowly in response to salt treatment. Yeast cells expressing EpHKT1;2 were able to tolerate high concentrations of NaCl, whereas EpHKT1;1-expressing yeast cells remained sensitive to NaCl. Amino acid sequence alignment with other plant HKTs showed that EpHKT1;1 contains an asparagine residue (Asn-213) in the second pore-loop domain, but EpHKT1;2 contains an aspartic acid residue (Asp-205) at the same position. Yeast cells expressing EpHKT1;1, in which Asn-213 was substituted with Asp, were able to tolerate high concentrations of NaCl. In contrast, substitution of Asp-205 by Asn in EpHKT1;2 did not enhance salt tolerance and rather resulted in a similar function to that of AtHKT1 (Na⁺ influx but no K⁺ influx), indicating that the presence of Asn or Asp determines the mode of cation selectivity of the HKT1-type transporters. Moreover, Arabidopsis plants (Col-gl) overexpressing EpHKT1;2 showed significantly higher tolerance to salt stress and accumulated less Na⁺ and more K⁺ compared to those overexpressing EpHKT1;1 or AtHKT1. Taken together, these results suggest that EpHKT1;2 mediates tolerance to Na⁺ ion toxicity in E. parvula and is a major contributor to its halophytic nature.</p

AdipoRs are expressed on the plasma membrane in <i>S. cerevisiae</i>.

<p>(A) Subcellular localization of AdipoRs by confocal microscopy. <i>S. cerevisiae</i> strain BY4741 carrying plasmid pYES-EGFP (GFP), pYES-EGFP-AdipoR1 (GFP-AdipoR1) and pYES-EGFP-AdipoR2 (GFP-AdipoR2) were cultured in selective minimal medium containing 2% galactose. Shown are images of cells that were in the early log phase of growth. (B) Western blot analysis of total membrane protein extracts that were fractionated by 10% SDS-PAGE. The predicted molecular weights of GFP-AdipoR1 and GFP-AdipoR2 are around 65 kDa.</p