Interaction of CPR5 with cell cycle regulators UVI4 and OSD1 in Arabidopsis

Abstract

The impact of cell cycle on plant immunity was indicated by the enhancement of disease resistance with overexpressing OSD1 and UVI4 genes that are negative regulators of cell cycle controller APC (anaphase promoting complex). CPR5 is another gene that is implicated in cell cycle regulation and plant immunity, but its mode of action is not known. Here we report the analysis of genetic requirement for the function of UVI4 and OSD1 in cell cycle progression control and in particular the involvement of CPR5 in this regulation. We show that the APC activator CCS52A1 partially mediates the function of OSD1 and UVI4 in female gametophyte development. We found that the cpr5 mutation suppresses the endoreduplication defect in the uvi4 single mutant and partially rescued the gametophyte development defect in the osd1 uvi4 double mutant while the uvi4 mutation enhances the cpr5 defects in trichome branching and plant disease resistance. In addition, cyclin B1 genes CYCB1;1, CYCB1;2, and CYCB1;4 are upregulated in cpr5. Therefore, CPR5 has a large role in cell cycle regulation and this role has a complex interaction with that of UVI4 and OSD1. This study further indicates an intrinsic link between plant defense responses and cell cycle progression.

Figure 1. Overexpression of OSD1 and UVI4 affect endoreduplication in leaves.

(A) Ploidy levels in the first pair of leaves from the wild-type Ler, uvi4-2, and osd1-2 grown under 12 hour light/day for 4 weeks. (B) Ploidy levels in the first pair of leaves from Ws, bon1-2, osd1-4 and bon1 osd1-4 grown under 12 hour light/day for 4 weeks. Error bars indicate standard deviations. The number above the column indicates the ploidy index. Averages of three replicas for each sample were shown in (A) and (B). Ploidy indices were used for statistical analysis, and the letter a indicates a statistical significance determined by student t-test. The representative data were shown from two independent measurements. (C) Frequencies of each class of trichome numbers in Col-0, uvi4 and UVI4-OE transgenic lines 5 and 17 in Col-0 and lines 3 and 5 in uvi4. Approximately 100 to 150 cells were examined for each genotype. Error bars indicate standard deviations. The difference between overexpression lines and wild-type Col-0 or uvi4was determined by chi-square test (UVI4-OE/Col-0 compared to Col-0, L5: P = 0.01<0.05, L17: P = 0.00<0.05; UVI4-OE/uvi4 compared to Col-0, L3: P = 0.02<0.05, L5: P = 0.21>0.05; UVI4-OE/uvi4 compared to uvi4, L3: P = 0.00<0.05, L5: P = 0.00<0.05).

Figure 2. CCS52A1 partially mediates the lethality of osd1 uvi4.

Shown are numbers of plants of each genotype in an analyzed population. The two numbers separated by a semicolon in parentheses are the observed ratios of that genotype relative to the top left genotype (left) and the expected ratios when there is no reduced transmission of the gametes or zygotes (right). (A) Analysis of progenies of osd1/OSD1 UVI4/uvi4. No osd1/osd1 uvi4/uvi4 plants (shaded) were found. The difference between observed and expected segregation ratio was determined by chi-square test (P = 0.004<0.05). (B) Analysis of progenies of osd1/OSD1 uvi4/uvi4. Notice that both OSD1/OSD1 uvi4/uvi4 and osd1/OSD1 uvi4/uvi4 (shaded) were about half of expected and there were no osd1/osd1uvi4/uvi4 (shaded) progenies produced. The significance was determined by chi-square test (P = 6.24E-12<0.05). (C) Analysis of gamete transmission inferred from reciprocal crosses between osd1/OSD1 uvi4/uvi4 and Col-0. The osd1 uvi4 genotype had a lower transmission rate through female gametes (shaded) but not male gametes. The significance was determined by chi-square test (male, P = 0.13>0.05; female, P = 2.53E-10<0.05). (D) Opened siliques from wild-type Col-0 (upper) and osd1/OSD1 uvi4/uvi4 (lower) plants. Aborted ovules and embryos can be seen in the mutant silique. (E) Analysis of progenies of osd1/OSD1 uvi4/uvi4 ccs52a1/ccs52a1. Notice that two osd1/osd1 uvi4/uvi4 ccs52a1/ccs52a1 plants (shaded) were found. (F) Morphology of the osd1/osd1 uvi4/uvi4 ccs52a1/ccs52a1 triple mutant plant.

Figure 3. Genetic interactions of uvi4 and osd1 with cpr5 in the regulation of endoreduplication and meiosis.

(A) Frequency of cells with different branching numbers in Col-0, cpr5, uvi4, and uvi4 cpr5 plants on the forth leaves. Approximately 100 to 150 cells were examined for each genotype. (B) DNA ploidy levels of Col-0, cpr5, uvi4, and uvi4 cpr5 shown as percentage of cells with 2C to 32C. (C) DNA ploidy levels of Col-0, cpr5, osd1 BC7F3, and cpr5 osd1 F3 plants shown as percentage of cells with 2C to 32C. Error bars indicate standard deviations. The number above the column indicates the ploidy index. Numbers in (B) and (C) are averages of three replicas. Ploidy indices were used for statistical analysis, and the letter a indicates a statistical significance determined by student t-test. The representative data were shown from two independent measurements.

Figure 4. The cpr5 mutation partially suppresses the lethality of osd1 uvi4 double mutant.

Shown are numbers of plants of each genotype in an analyzed population. The two numbers separated by a semicolon in parentheses are the observed ratios of that genotype relative to the top left genotype (left) and the expected ratios when there is no reduced transmission of the gametes or zygotes (right). (A) Analysis of gamete transmission inferred from of reciprocal crosses between CPR5/cpr5 and Col-0. The cpr5 had a higher transmission rate as male gamete (shaded) but not female gamete. The significance was determined by chi-square test (male, P = 0.011<0.05; female, P = 0.78>0.05). (B) Analysis of progenies from osd1/OSD1 uvi4/UVI4 cpr5/cpr5. Notice the presence of osd1/osd1 uvi4/uvi4 cpr5/cpr5 (shaded) while there was no osd1/osd1 uvi4/uvi4 in progenies of osd1/OSD1 UVI4/uvi4 (Figure 2A). Also notice that plants with osd1/OSD1 cpr5/cpr5 genotypes irrespective of the uvi4 genotypes (darker shaded) were fewer than expected, suggesting a lower transmission of osd1 cpr5 than OSD1 cpr5. The difference between observed segregation ratio and normal segregation ratio was determined by chi-square test (P = 1.07E-14<0.05). (C) Analysis of progenies from osd1/OSD1 uvi4/uvi4 cpr5/cpr5. (D) Morphology of Col-0, uvi4, osd1, cpr5, cpr5 uvi4, cpr5 osd1and cpr5 uvi4 osd1 grown for 6 weeks under 12 h light/12 h dark condition. (E) Analysis of gamete transmission inferred from of reciprocal crosses between osd1/OSD1 uvi4/uvi4 CPR5/cpr5 and Col-0 or uvi4. Notice a lower transmission (20 versus 50 with Col-0 and 15 versus 33 with uvi4) of osd1uvi4CPR5 (shaded) compared to OSD1uvi4CPR5 as female gametes but an increased transmission rate (29 versus 40 with Col-0 and 41 versus 38 with uvi4) of osd1uvi4cpr5 (darker shaded) compared to that of osd1uvi4CPR5. The difference of gamete transmission between osd1/OSD1 uvi4/uvi4 and osd1/OSD1 uvi4/uvi4 CPR5/cpr5 was determined by chi-square test (reciprocal cross with Col-0, P = 1.71E-36<0.05; reciprocal cross with uvi4, P = 4.20E-37<0.05).

Figure 5. Expression of cell cycle genes in cpr5 mutant.

Expression levels of AtCYCB1;1 in both the first pair of leaves and whole seedlings of 2-week-old plants, and AtCYCB1;2 and AtCYCB1;4 in whole seedlings analyzed by quantitative real time RT-PCR. Error bars indicate standard deviations.

Figure 6. Both uvi4 and osd1 mutations enhance defense responses in cpr5.

Bacterial growth assay in Col-0, uvi4, osd1, cpr5, cpr5 uvi4 and cpr5 osd1 (all diploid plants) inoculated by spray inoculation of Pst DC3000. Error bars indicate standard deviations. Letters a, b and c indicate the statistical significance determined by student t-test.

Figure 7. Roles of the OSD1, UVI4 and CPR5 genes.

Both UVI4 and OSD1 negatively regulate APC/C activities partially through their interaction with CCS52A1. APC/C inhibits activities of various CDK-cyclin complexes (indicated by shaded circles) each of which regulates processes including meiosis, female gametogenesis, endoreduplication and defense response. CPR5 negatively regulates the transcript levels of some cyclin genes and thus the activities some CDK-cyclin complexes (indicated by darker shades), and affects female gametogenesis, endoreduplication and defense responses. The exact action point of CPR5 is yet to be determined.