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

MMS21/HPY2 and SIZ1, Two Arabidopsis SUMO E3 Ligases, Have Distinct Functions in Development

<div><p>The small ubiquitin related modifier (SUMO)-mediated posttranslational protein modification is widely conserved among eukaryotes. Similar to ubiquitination, SUMO modifications are attached to the substrate protein through three reaction steps by the E1, E2 and E3 enzymes. To date, multiple families of SUMO E3 ligases have been reported in yeast and animals, but only two types of E3 ligases have been identified in Arabidopsis: <u>S</u>AP and M<u>iz</u> 1 (SIZ1) and <u>M</u>ethyl <u>M</u>ethanesulfonate-<u>S</u>ensitivity protein <u>21</u> (MMS21)/<u>H</u>IGH <u>P</u>LOID<u>Y</u> 2 (HPY2), hereafter referred to as HPY2. Both proteins possess characteristic motifs termed <u>S</u>iz/<u>P</u>IAS RING (SP-RING) domains, and these motifs are conserved throughout the plant kingdom. Previous studies have shown that loss-of-function mutations in HPY2 or SIZ1 cause dwarf phenotypes and that the phenotype of <em>siz1-2</em> is caused by the accumulation of salicylic acid (SA). However, we demonstrate here that the phenotype of <em>hpy2-1</em> does not depend on SA accumulation. Consistently, the expression of <em>SIZ1</em> driven by the <em>HPY2</em> promoter does not complement the <em>hpy2-1</em> phenotypes, indicating that they are not functional homologs. Lastly, we show that the <em>siz1-2</em> and <em>hpy2-1</em> double mutant results in embryonic lethality, supporting the hypothesis that they have non-overlapping roles during embryogenesis. Together, these results suggest that SIZ1 and HPY2 function independently and that their combined SUMOylation is essential for plant development.</p> </div

Double mutants of <i>hpy2-1</i> and <i>siz1-2</i> are embryonic lethal.

<p>Maturing seeds in the siliques of <i>hpy2-1</i>/+ (a), <i>hpy2-1</i>/+ <i>siz1-2</i>/− (b) and <i>siz1-2</i>/− (c). White arrows in (b) mark collapsed seeds. Numbers on the right represent the percentage of aborted seeds.</p

The SP-RING domain is conserved in plant SUMO E3 ligases.

<p>(a) The plant SUMO E3 ligases, HPY2/MMS21 and SIZ1. Characteristic domains are shown by grey boxes. SIZ1 possesses several domains, such as SAP, PHD, PINIT, SP-RING, SXS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046897#pone.0046897-Miura1" target="_blank">[4]</a>, while HPY2 possesses only SP-RING domain. (b) A schematic model of the SP-RING domain. The open circles indicate conserved amino acid residues, and the grey box marks the stabilising motif. A Zn ion coordinated in the SP-RING domain is indicated by a closed circle. (c) Sequence alignment of the SP-RING domain in the MMS21 homologues. The sequence data for the plant MMS21 homologues were obtained from the NCBI protein database and aligned based on Miura et al. (2007). <i>Vitis vinifera</i>, VvMMS21 (XP_002282690.1); <i>Populus trichocarpa</i>, PtMMS21 (XP_002317010.1); <i>Glycine max</i>, GmMMS21 (ACU20283.1); <i>Oryza sativa</i>, OsMMS21 (EEE64695.1); <i>Zea mays</i>, ZmMMS21 (ACF80287.1); <i>Physcomitrella patens</i>, PpMMS21 (XP_001767320.1). (d) Sequence alignment of the SP-RING domain in the SIZ1 homologues. The sequence data for the plant SIZ1 homologues were obtained from the NCBI protein database and aligned based on Miura et al. (2007). <i>Medicago truncatula</i>, MtSIZ1 (XP_003606454.1) and MtSIZ2 (ABD33066.1); <i>Vitis vinifera</i>, VvSIZ1 (XP_002282690.1); <i>Oryza sativa</i>, OsSIZ2 (NP_001051092.1) and OsSIZ1 (NP_001054517.1); <i>Sorghum bicolor</i>, SbSIZ1 (XP_002439205.1); <i>Hordeum vulgare</i>, HvSIZ1 (BAJ98904.1); <i>Physcomitrella patens</i>, PpSIZ1 (P_001767531.1).</p

The protein localisation of HPY2 and SIZ1 in roots.

<p>Confocal microscopy of <i>HPY2pro:HPY2-GFP</i> (a–c), <i>SIZ1pro:SIZ1-GFP</i> (d–f) and <i>HPY2pro:SIZ1-GFP</i> (g, h) roots at the tip, transition zone and differentiated region. Bar = 50 µm.</p

Introduction of <i>nahG</i> in <i>hpy2-1</i> does not restore the dwarf phenotype.

<p>(a) Seven-day-old WT and <i>hpy2-1</i> seedlings carrying <i>nahG</i>. (b) Relative root length of WT, <i>siz1-2</i> and <i>hpy2-1</i> plants with or without <i>nahG</i>.</p

The expression pattern of <i>HPY2</i> and <i>SIZ1 in planta</i>.

<p>The promoter activity of <i>HPY2</i> and <i>SIZ1</i> in aerial tissues as visualised by the GUS activity in <i>HPY2pro:GUS</i> and <i>SIZpro:GUS</i> plants. The <i>HPY2</i> promoter activity is detected in the anther, leaf vein and hypocotyl cells while the <i>SIZ1</i> promoter activity is detected in stigma, leaf vein, young seeds and hypocotyl cells. The images for true leaves (upper panel) are magnified by ∼4-fold (lower panel) to show clear GUS signals in leaf veins.</p

Ectopic expression of <i>SIZ1</i> by the <i>HPY2</i> promoter does not rescue the <i>hpy2-1</i> phenotype.

<p>(a) Seven-day-old WT and <i>hpy2-1</i> seedlings carrying <i>HPY2pro:HPY2-GFP</i> or <i>HPY2pro:SIZ1-GFP</i>. (b) Microscopic observation of <i>HPY2pro:SIZ1-GFP</i> in <i>hpy2-1</i> root tips. Despite the expression of SIZ1-GFP fusion proteins, as indicated by white arrows, the meristem defects are not restored in <i>hpy2-1</i>. (c) Relative root length of WT, <i>hpy2-1</i>, HPY2p:HPY2-GFP in WT, HPY2p:HPY2-GFP in <i>hpy2-1</i>, HPY2p:SIZ1-GFP in WT and HPY2p:SIZ1-GFP in <i>hpy2-1</i>.Bar = 50 µm in (b).</p

The dwarf phenotypes of SUMO E3 ligase mutants.

<p>(a) Seven-day-old wild-type (WT), <i>siz1-2</i>, <i>hpy2-1</i> and <i>hpy2-2</i> seedlings. (b) Quantification of the root length of seven-day-old seedlings. (c) Twenty five-day-old WT, <i>siz1-2</i> and <i>hpy2-2</i> plants. Most of the <i>hpy2-1</i> plants are no longer viable after bolting. (d) Quantification of the true leaf number of fifteen-day-old seedlings. (e) Quantification of the rosette diameter of fifteen-day-old seedlings. Bar = 1 cm in (a).</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