Atypical E2F activity coordinates PHR1 photolyase gene transcription with endoreduplication onset

Abstract

Because of their sessile life style, plants have evolved the ability to adjust to environmentally harsh conditions. An important aspect of stress adaptation involves the reprogramming of the cell cycle to ensure optimal growth. The atypical E2F transcription factor DP-E2F-like 1 (E2Fe/DEL1) had been found previously to be an important regulator of the endocycle onset. Here, a novel role for E2Fe/DEL1 was identified as a transcriptional repressor of the type-II cyclobutane pyrimidine dimer-photolyase DNA repair gene PHR1. Upon ultraviolet-B (UV-B) treatment, plants knocked out for E2Fe/DEL1 had improved DNA repair abilities when compared with control plants, whereas those overexpressing it performed less well. Better DNA repair allowed E2Fe/DEL1 knockout plants to resume endoreduplication faster than control plants, contributing in this manner to UV-B radiation resistance by compensating the stress-induced reduction in cell number by ploidy-dependent cell growth. As E2Fe/DEL1 levels decreased upon UV-B treatment, we hypothesize that the coordinated transcriptional induction of PHR1 with the endoreduplication onset contributes to the adaptation of plants exposed to UV-B stress.

Figure 1

Regulation of PHR1 transcription by E2Fe/DEL1. (A) PHR1 levels in the first leaf pair of 14-day-old seedlings. All values were normalized to the housekeeping gene ACT2. Measurements were made relative to Col-0 wild-type (WT) and are represented as mean±s.d. (n=3, P-value Student's t-test, ^*<0.01; ^**<0.05). (B) ChIP analysis showing binding of E2Fe/DEL1 to the PHR1 promoter. (C) ChIP scanning of the PHR1 promoter showing the strongest association of E2Fe/DEL1 around the putative E2F cis-acting element. Numbers in the x-axis correspond to the start and stop position of the promoter fragment tested. Data show the fold enrichment, representing the ratio between primer fragment amplification in control and E2Fe/DEL1^KO plants.

Figure 2

Efficient CPD repair of E2Fe/DEL1^KO plants. CPD concentrations were measured by ELISA in the fifth leaves irradiated with 116 kJ/m² UV-B, immediately harvested after irradiation (dark) or after recovery for 5 h in white light (grey). The CPD amounts in the Col-0 wild-type (WT) plants harvested immediately after treatment was arbitrarily set to 1. The CPD repair was statistically better in E2Fe/DEL1^KO leaves than in control (n=3, P-value Student's t-test, ^*<0.01).

Figure 3

Growth dose response of Col-0 wild-type (WT), E2Fe/DEL1^KO, and E2Fe/DEL1^OE plants towards UV-B. (A) Eighteen-day-old plants irradiated in a sun simulator with the indicated doses. Plants were photographed 9 days after treatment. (B) Fresh weight measurements of untreated and UV-B irradiated plants. Data are represented as mean±s.e. (n>3).

Figure 4

Leaf growth analysis of Col-0 wild-type (WT) and E2Fe/DEL1^KO plants under UV-B treatment. (A–C) Eighteen-day-old plants were not treated (white) or irradiated with 35 kJ/m² UV-B in a sun simulator (dark). The fifth leaves were harvested 9 days after irradiation for leaf size (A), epidermal cell number (B), and epidermal cell size (C). (D) Endoreduplication kinetics. Eighteen-day-old wild-type (black) and E2Fe/DEL1^KO (grey) plants were not treated (full lines) or irradiated with 35 kJ/m² UV-B (dashed lines). Data are represented as mean±s.e. (n=5).

Figure 5

Leaf age-dependent recovery in growth and endoreduplication after UV-B irradiation. (A) E2Fe/DEL1 transcript levels in the third (L3) and fifth (L5) leaf of Col-0 wild-type (WT) and E2Fe/DEL1^KO plants. (B) Eighteen-day-old plants irradiated with 59 kJ/m² UV-B. Radiation recovery is plotted as the percentage reduction in leaf size of the UV-B treated versus untreated plants. (C, D) E2Fe/DEL1^KO plants harvested at a 3-day interval to measure the endoreduplication kinetics in third (C) and fifth (D) untreated (grey) and irradiated (dark) leaves. Data are represented as mean±s.e. (n>10).

Figure 6

Requirement of PHR1 activity for efficient endoreduplication. (A–D) Representative DNA ploidy distributions of leaf 5 of 25-day-old Ler wild-type (WT) (A, B) and PHR1^KO (C, D) plants, either untreated (A, C) or irradiated with 34 kJ/m² UV-B 15 days after sowing (B, D). (E) Endoreduplication index. Data are represented as mean±s.e. (n=10).

Figure 7

Effect of UV-B on E2Fe/DEL1 and PHR1 transcript levels. (A) PHR1 transcript levels in 18-day-old Col-0 wild-type (WT) and E2Fe/DEL1^KO control (white) versus UV-B-irradiated seedlings (59 kJ/m²) (black). Plants were harvested for RNA extraction immediately after treatment. (B) E2Fe/DEL1 transcript levels in Col-0 wild-type (WT) plants 14 days after sowing of whole seedlings either untreated (−UV) or treated with 35 kJ/m² of UV-B (+UV). Plants were harvested immediately after treatment. Data are represented as mean±s.e. (n=3).