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

Pollen tube growth in the pistils from wild-type and homozygous <i>siz1-2</i> plants.

<p>(A)–(G) decolorized aniline blue staining of pistils 1–2 days after pollination (DAP). (A) The whole scene of pollen tube growth within the <i>siz1-2</i> pistil. (B) The whole scene of pollen tube growth within the wild-type pistil. (C) The fertilized wild-type ovules showing pollen tubes (arrow) grew into the micropyle (arrowhead) and became bigger in volume. The pistil was harvested 2 days after pollination. (D) Mutant ovules without a pollen tube growing toward the funiculus, while many pollen tubes grew within the placenta. (E) The mutant ovule with pollen tube (arrow) growing around the funiculus, but turning away from the funiculus, without targeting the micropyle (arrowhead). (F) Two undeveloped ovules with pollen tubes (arrow) growing around the funiculus without targeting the micropyle. (G) Representative mutant ovule with pollen tube growing near the micropyle opening but failing to target the female gametophyte. (H)–(M) Scanning electron microscopy analysis of pistils 1–2 days after pollination. (H) Scanning electron micrograph of wild-type ovules showing that pollen tubes grew along the funiculus and then entered the micropyle (arrowhead). (I) Scanning electron micrograph of some <i>siz1-2</i> ovules showing that pollen tubes grew along the funiculus and then entered the micropyle (arrowhead), similar to those of the wild type. (J)–(M) Aberrant pollen tube guidance in <i>siz1-2</i> ovules. (L) A pollen tube stopped growing near the micropyle (arrowhead). (J) A pollen tube bypassing the micropyle and growing on the surface of the integument. (K) A pollen tube grew along the funiculus but failed to enter the micropyle and turned away. (M) An example showing that no pollen tube grew on the funiculus of the ovule. St, style; Tt, pollen tube transmitting tract. Arrows indicate pollen tubes and arrowheads show micropyle. Bar = 200 µm in (A) and (B), 40 µm in (C)–(M).</p

Ovule development from stage FG1 to FG7 in the wild type and <i>siz1-2</i> mutant.

<p>The upper panels show ovule development of the wild type, as revealed by laser scanning confocal microscope, while the lower panels show ovule development of the <i>siz1-2</i> mutant. Corresponding development stages of the ovules examined are indicated below. Bar = 20 µm.</p

Expression patterns of selected genes from <i>siz1-2</i> and wild-type siliques.

<p>The tubulin α-2 gene (AT1G04820) was used as the internal control, and its expression level was set arbitrarily as 1.</p

Final phenotypes of the female gametophyte in the wild type and <i>siz1-2</i> mutant.

<p>(A) LSCM images of an ovule derived from a wild-type flower; the pistil was harvested 2 day after emasculation. (B)–(D) LSCM images for ovules derived from <i>siz-1-2</i> flowers; the pistils were harvested 2 days after emasculation. Percentages of abnormal female gametophytes among the examined ovules are indicated below. Cn, central cell nucleus; En, egg cell nucleus; Sn, synergid cell nucleus. Bar = 40 µm.</p

Expression of the Pro_siz1::GUS–GFP gene.

<p>(A) Expression of Pro_siz1::GUS–GFP in the whole inflorescence. A GUS signal was detected in most of the flowers, except the latest ones, while the strongest GUS signal was found in sepals. The inflorescence in (A) was stained with 1 mM 5-bromo-4-chloro-3-indolyl-b-glucuronic acid (X-Gluc) for 12 h. (B) GUS signal in a flower after pollination. The style of the pistil was stained strongly by X-Gluc, and the upper part of the carpel and the stem of the stamen were also stained by X-Gluc. No GUS signal was seen in the anthers. (C) GUS signal in reproductive organs at the end of pollination. The style was strongly stained by X-Gluc, while no GUS signal was seen in the stigmatic cells or pollen. (D) No GUS signal was detected in the whole flower in the wild-type plants after staining with 1 mM X-Gluc for 12 h. (E) Expression of Pro_siz1::GUS–GFP could be detected in all cells within the ovule before fertilization after staining with 1 mM X-Gluc for 8 h. (F) GFP fluorescence of Pro_siz1::GUS–GFP can be seen in all cells of the ovules. (G) Wild-type ovule control. No fluorescence was detected in the wild-type ovule under LSCM. Pg, pollen grain; Sc, stigmatic cell; St, style.</p

Analysis of ovule development and <i>in vitro</i> germination of <i>siz1-2</i> pollen grains compared to the wild type by DIC microscopy.

<p>(A)–(C) Ovules in a <i>siz1-2</i> mutant under a DIC microscope. The fertilized ovule grew bigger and formed a quadrant embryo (Em) within the embryo sac (B); the unfertilized ovule stopped growing, with no proembryo appearing. (D) Wild-type pollen tubes cultured at 28°C <i>in vitro</i>. Pollen tubes with normal morphology are indicated by an arrow. (E) <i>siz1-2</i> pollen tubes incubated under the same condition as (A), showing no obvious difference compared to the wild-type pollen tube. Em, embryo. Pg, pollen grain. Pt, pollen tube. Bar = 50 µm in (A), 8 µm in (B) and (C), 200 µm in (D) and (E).</p

Silique development and seed-set of <i>siz1-2</i>, <i>nahG siz1-2</i>, wild-type, and the <i>Pro_siz1::SIZ1-GFP</i> construct-transformed <i>siz1-2</i> mutant plants (<i>SSG</i>).

<p>(A) Siliques of <i>siz1-2</i>, <i>nahG siz1-2</i>, wild-type, and <i>SSG</i> 8–10 days after pollination. (B) Dissected silique from <i>siz1-2</i> homozygous plants showing severely reduced seed-set and undeveloped ovules. Similar results were also found in line <i>siz1-3</i> (data not shown). De, defective embryo. (C) Dissected silique from <i>nahG siz1-2</i> plants showing severely reduced seed-set and undeveloped ovules, similar to <i>siz1-2</i>. De, defective embryo. (D) Dissected silique of a wild-type plant with a full seed-set. (E) Dissected silique of a <i>SSG</i> plant with full seed-set, similar to that of the wild-type plant. (F) Percentage of defective embryos in <i>siz1-2</i>, <i>nahG siz1-2</i>, Col-0, and <i>SSG</i> pistils. A mean value of three repeats, asterisks indicate no significant difference between percentage of defective embryos of <i>siz1-2</i> and <i>nahG siz1-2</i> (P<0.05).</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

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