Influence of Human p53 on Plant Development

<div><p>Mammalian p53 is a super tumor suppressor and plays a key role in guarding genome from DNA damage. However, p53 has not been found in plants which do not bear cancer although they constantly expose to ionizing radiation of ultraviolet light. Here we introduced <i>p53</i> into the model plant Arabidopsis and examined p53-conferred phenotype in plant. Most strikingly, p53 caused early senescence and fasciation. In plants, fasciation has been shown as a result of the elevated homologous DNA recombination. Consistently, a reporter with overlapping segments of the <i>GUS</i> gene (1445) showed that the frequency of homologous recombination was highly induced in <i>p53</i>-transgenic plants. In contrast to p53, SUPPRESSOR OF NPR1-1 INDUCIBLE 1 (SNI1), as a negative regulator of homologous recombination in plants, is not present in mammals. Comet assay and clonogenic survival assay demonstrated that SNI1 inhibited DNA damage repair caused by either ionizing radiation or hydroxyurea in human osteosarcoma U2OS cancer cells. RAD51D is a recombinase in homologous recombination and functions downstream of SNI1 in plants. Interestingly, p53 rendered the <i>sni1</i> mutants madly branching of inflorescence, a phenotype of fasciation, whereas <i>rad51d</i> mutant fully suppressed the p53-induced phenotype, indicating that human p53 action in plant is mediated by the SNI1-RAD51D signaling pathway. The reciprocal species-swap tests of p53 and SNI1 in human and Arabidopsis manifest that these species-specific proteins play a common role in homologous recombination across kingdoms of animals and plants.</p></div

The reciprocal species-swap test of p53 and SNI1 between Arabidopsis and human.

<p>(A) Somatic recombination in wild type (WT) and <i>p53</i>-transgenic (<i>p53</i>) plants is shown in blue sectors by a reporter with overlapping segments of the <i>GUS</i> gene (1445). (B) Quantitative result of panel A. Experiments were performed in three <i>p53</i>-transgenic lines (n = 50 ~ 100) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162840#pone.0162840.s005" target="_blank">S1 Table</a>). The result of line 1 is shown. Error bars represent SEs. ***, p value < 0.001 compared to WT by binomial test. (C) Human osteosarcoma U2OS cancer cells transfected with empty vector (EV) or hemagglutinin (HA)-tagged SNI1 (SNI1). Proteins extracted from the transfected U2OS cancer cells were blotted with anti-HA antibody (abcam, ab1265). Anti-α-tubulin was used as an internal loading control. (D) The comet assay was carried out on the transfected U2OS cancer cells which were treated with 10 Gy of ionizing radiation (IR) and recovered with indicated time. The level of DNA break repair was visualized with the length of comet tail. (E) Images in panel B were analyzed using CometScore software (Tritek) to quantify the comet tail moment of at least 75 cells for each sample. Error bars represent SEs. ***, p value < 0.001, compared to EV by binomial test. Experiments were performed three times with similar results. (F) The transfected U2OS cancer cells were pulse-treated with hydroxyurea (HU) for 24 hours to introduce DNA damage and recovered in drug-free medium. (G) Quantitative results of panel D. After 14 days of culture, colonies were counted and normalized to untreated control. Error bars represent SEs. Experiments were carried out in triplicate.</p

Human p53 acts through the SNI1-RAD51D signaling pathway in plant.

<p>(A) Fascinated inflorescence of <i>sni1</i> mutant. Inset (box in red): part of the fascinated inflorescence is enlarged. (B) A single plant of five-week-old <i>sni1</i> mutant and four-month-old <i>p53</i>-transgenic <i>sni1</i> mutant (<i>sni1</i>/<i>p53</i>). (C) Three-week-old WT, <i>p53</i>-transgenic (<i>p53</i>), <i>rad51d</i> and <i>p53</i>-transgenic <i>rad51d</i> (<i>rad51d</i>/<i>p53</i>) plants. Arrows indicate cotyledons. (D) Number (#) of secondary inflorescences of WT, <i>p53</i>, <i>rad51d</i> and <i>rad51d</i>/<i>p53</i> plants was plotted. The letter above the bar indicates a statistically significant difference between groups at p value < 0.01. Experiments were conducted in triplicate (n > 30) with similar results.</p

Influence of p53 on plant transcriptome.

<p>(A) Gene Ontology (GO) analysis of microarray data. Ten-day-old wild type (WT) and <i>p53</i>-transgenic (<i>p53</i>) seedlings were used for microarray analysis (GEO accession number: GSE79678). The differential expressed genes (t test, p value < 0.05 and fold change > 2) were analyzed for enriched biological processes by Gene Ontology (GO: <a href="https://www.arabidopsis.org/tools/bulk/go/index.jsp" target="_blank">https://www.arabidopsis.org/tools/bulk/go/index.jsp</a>). Experiments were performed in triplicate. (B) The expressions of <i>SNI1</i>, 7 <i>SSN</i>s (<i>SUPPRESSORS OF SNI1</i>s) and 3 fascination-associated genes in <i>p53</i>-transgenic plants were compared to those in WT plants. The red line indicates the expression with no change. (C) Ten-day-old wild type (WT) and <i>p53</i>-transgenic (<i>p53</i>) seedlings were used for RNA extraction. <i>RAD51D</i> transcripts were quantified by qPCR. <i>UBQ5</i> was used as an internal control. Error bars represent SEs. Experiments were conducted in triplicate.</p

Human p53-conferred phenotype in plant.

<p>(A) Leaves of 3-week-old wild type (WT) and <i>p53</i>-transgenic plants (<i>p53</i>). Arrows indicate cotyledons. (B) Four-week-old WT and <i>p53</i> plants. Arrows indicate the first pair of true leaves. (C) Bolting WT and <i>p53</i> plants. (D) Left panel: inflorescences of WT and <i>p53</i>. Insets show enlarged stems (in yellow box). Right panel: number (#) of secondary inflorescences. Error bars represent standard errors (SEs). ***, p value < 0.001, compared to WT by binomial test. Experiments were carried out in triplicate (n > 30) with similar results. (E) Siliques of WT and <i>p53</i>. Arrow indicates clustered (fascinated) siliques.</p

Image_1_The Canonical E2Fs Are Required for Germline Development in Arabidopsis.pdf

<p>A number of cell fate determinations, including cell division, cell differentiation, and programmed cell death, intensely occur during plant germline development. How these cell fate determinations are regulated remains largely unclear. The transcription factor E2F is a core cell cycle regulator. Here we show that the Arabidopsis canonical E2Fs, including E2Fa, E2Fb, and E2Fc, play a redundant role in plant germline development. The e2fa e2fb e2fc (e2fabc) triple mutant is sterile, although its vegetative development appears normal. On the one hand, the e2fabc microspores undergo cell death during pollen mitosis. Microspores start to die at the bicellular stage. By the tricellular stage, the majority of the e2fabc microspores are degenerated. On the other hand, a wild type ovule often has one megaspore mother cell (MMC), whereas the majority of e2fabc ovules have two to three MMCs. The subsequent female gametogenesis of e2fabc mutant is aborted and the vacuole is severely impaired in the embryo sac. Analysis of transmission efficiency showed that the canonical E2Fs from both male and female gametophyte are essential for plant gametogenesis. Our study reveals that the canonical E2Fs are required for plant germline development, especially the pollen mitosis and the archesporial cell (AC)-MMC transition.</p

Identification of spinach SIAMESE and analysis of its function in plant immunity

<p>Diseases cause a significant loss in both yield and quality of spinach. The cell cycle signalling pathway plays a central role in balancing development and immunity of plants. Cyclin-Dependent Kinase Inhibitor (CKI) is a core cell cycle regulator. It has been found that two Arabidopsis CKIs, SIAMESE (SIM) and SMR1 (SIAMESE-Related 1), function redundantly not only as negative regulators of cell proliferation but also as positive regulators of plant immunity. In the spinach genome, we identified a homologue of Arabidopsis SIM, referred to as <i>Spinacia oleracea</i> SIAMESE (SoSIM). To investigate the function of SoSIM, we introduced the <i>35S</i> promoter-driven <i>SoSIM</i> (<i>35S:SoSIM</i>) into Arabidopsis <i>sim smr1</i> double mutants. Over-expression of <i>SoSIM</i> phenocopied RNA interference of <i>Cyclin-Dependent Kinase A1</i> (<i>CDKA1</i>) which exhibited dwarf and serrated leaves, confirming that SIM is a CDK inhibitor. Arabidopsis wild-type trichomes are single and unicellular, whereas <i>sim smr1</i> mutant trichomes are clustered and multicellular. SoSIM restored wild-type trichome phenotype of <i>sim smr1</i> mutant. The <i>sim smr1</i> mutants were susceptible to the avirulent pathogen <i>Pseudomonas syringae</i> pv. <i>maculicola</i> as compared with wild-type plants. Our data showed that the resistance of <i>35S:SoSIM</i>-transgenic <i>sim smr1</i> lines to the pathogen was fully restored, indicating that SoSIM activates plant immunity. These data verify that SoSIM functions as its Arabidopsis counterpart in inhibition of plant development and activation of plant immunity. Therefore, SoSIM can be explored to control the balance between development and immunity in spinach.</p