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. 2016 Sep 20;11(9):e0162840.
doi: 10.1371/journal.pone.0162840. eCollection 2016.

Influence of Human p53 on Plant Development

Huimin Ma  1 , Teng Song  1 , Tianhua Wang  1 , Shui Wang  1
Affiliations

Influence of Human p53 on Plant Development

Huimin Ma et al. PLoS One. .

Abstract

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 p53 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 GUS gene (1445) showed that the frequency of homologous recombination was highly induced in p53-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 sni1 mutants madly branching of inflorescence, a phenotype of fasciation, whereas rad51d 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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Human p53-conferred phenotype in plant.
(A) Leaves of 3-week-old wild type (WT) and p53-transgenic plants (p53). Arrows indicate cotyledons. (B) Four-week-old WT and p53 plants. Arrows indicate the first pair of true leaves. (C) Bolting WT and p53 plants. (D) Left panel: inflorescences of WT and p53. 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 p53. Arrow indicates clustered (fascinated) siliques.
Fig 2
Fig 2. The reciprocal species-swap test of p53 and SNI1 between Arabidopsis and human.
(A) Somatic recombination in wild type (WT) and p53-transgenic (p53) plants is shown in blue sectors by a reporter with overlapping segments of the GUS gene (1445). (B) Quantitative result of panel A. Experiments were performed in three p53-transgenic lines (n = 50 ~ 100) (S1 Table). 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.
Fig 3
Fig 3. Human p53 acts through the SNI1-RAD51D signaling pathway in plant.
(A) Fascinated inflorescence of sni1 mutant. Inset (box in red): part of the fascinated inflorescence is enlarged. (B) A single plant of five-week-old sni1 mutant and four-month-old p53-transgenic sni1 mutant (sni1/p53). (C) Three-week-old WT, p53-transgenic (p53), rad51d and p53-transgenic rad51d (rad51d/p53) plants. Arrows indicate cotyledons. (D) Number (#) of secondary inflorescences of WT, p53, rad51d and rad51d/p53 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.
Fig 4
Fig 4. Influence of p53 on plant transcriptome.
(A) Gene Ontology (GO) analysis of microarray data. Ten-day-old wild type (WT) and p53-transgenic (p53) 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: https://www.arabidopsis.org/tools/bulk/go/index.jsp). Experiments were performed in triplicate. (B) The expressions of SNI1, 7 SSNs (SUPPRESSORS OF SNI1s) and 3 fascination-associated genes in p53-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 p53-transgenic (p53) seedlings were used for RNA extraction. RAD51D transcripts were quantified by qPCR. UBQ5 was used as an internal control. Error bars represent SEs. Experiments were conducted in triplicate.
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Grants and funding

This work was supported by grant 31571254 from National Natural Science Foundation of China (http://www.nsfc.gov.cn).