Evidence for a Role of ANAC082 as a Ribosomal Stress Response Mediator Leading to Growth Defects and Developmental Alterations in Arabidopsis

Abstract

Ribosome-related mutants in Arabidopsis thaliana share several notable characteristics regarding growth and development, which implies the existence of a common pathway that responds to disorders in ribosome biogenesis. As a first step to explore this pathway genetically, we screened a mutagenized population of root initiation defective2 (rid2), a temperature-sensitive mutant that is impaired in pre-rRNA processing, and isolated suppressor of root initiation defective two1 (sriw1), a suppressor mutant in which the defects of cell proliferation observed in rid2 at the restrictive temperature was markedly rescued. sriw1 was identified as a missense mutation of the NAC transcription factor gene ANAC082 The sriw1 mutation greatly alleviated the developmental abnormalities of rid2 and four other tested ribosome-related mutants, including rid3 However, the impaired pre-rRNA processing in rid2 and rid3 was not relieved by sriw1 Expression of ANAC082 was localized to regions where phenotypic effects of ribosome-related mutations are readily evident and was elevated in rid2 and rid3 compared with the wild type. These findings suggest that ANAC082 acts downstream of perturbation of biogenesis of the ribosome and may mediate a set of stress responses leading to developmental alterations and cell proliferation defects.

Figure 1.

Phenotypic Comparison between the sriw1 rid2 Double Mutant, the Wild Type, and rid2. (A) Seedlings of the wild type, rid2, and sriw1 rid2 grown for 14 d at 22°C or 28°C. Bar = 1 cm. (B) Adventitious root formation induced from hypocotyl explants of the wild type, rid2, and sriw1 rid2 by culture on root-inducing medium (RIM) for 16 d at 22°C or 28°C. Bar = 1 cm. (C) Callus formation induced from hypocotyl explants of the wild type, rid2, and sriw1 rid2 by culture on CIM for 21 d at 22°C or 28°C. Bar = 1 cm. (D) Differential interference contrast images of hypocotyl explants of the wild type, rid2, and sriw1 rid2 cultured on CIM for 5 d at 22°C or 28°C. Each yellow bar represents the size (long diameter) of the nucleolus. Bar = 20 μm. (E) Nucleolar size of stele-derived cells in hypocotyl explants of the wild type, rid2, and sriw1 rid2 cultured on CIM for 5 d at 22°C or 28°C. Mean values obtained from 20 nucleoli are shown, with standard deviations. Values with different letters are significantly different from each other at P < 0.05 (two-tailed Student’s t test; Supplemental Table 1). (F) Fluorescence micrographs showing nucleoplasms and nucleoli in the wild type, rid2, and sriw1 rid2. Sections were prepared from the hypocotyl explants cultured for 5 d on CIM at 28°C and double-stained with 4’,6-diamidino-2-phenylindole (blue, DNA) and SYTO RNASelect (green, RNA). Nu and No represent the nucleoplasm and nucleolus, respectively. Bar = 20 μm.

Figure 2.

Lack of Recovery from the Impaired Pre-rRNA Processing in the sriw1 rid2 Mutant. (A) Schematic representation of the pre-rRNA processing pathways in Arabidopsis according to Weis et al. (2015a, 2015b). Letters above the rDNA unit represent pre-rRNA processing sites. Red bars indicate the positions of hybridization probes used in RNA gel blot analysis. (B) RNA gel blot analysis of the accumulation of rRNA precursors in the wild type, rid2, and sriw1 rid2. RNA samples were prepared from the wild-type and mutant plants that had been grown for 12 d at 22°C or 28°C and subjected to RNA gel blot analysis with 5′-ETS-, ITS1-, and ITS2-specific probes (a, b, and c, respectively, in [A]) for hybridization. 25S rRNA bands visualized by staining with GelRed (Biotium) are shown as an index of the loaded amounts of total RNA (because 25S rRNA constitutes a large fraction of total RNA, any changes in the 25S rRNA content, which can be caused by RNA biosynthesis-defective mutations such as rid2, are expected to be reflected in changes of the total RNA content). This experiment was repeated three times using independently prepared materials to confirm the reproducibility of the result. (C) Ratios of 18S rRNA to 25S rRNA in the wild type, rid2, and sriw1 rid2. RNA samples were prepared from the wild-type and mutant plants that had been grown for 12 d at 22°C or 28°C and analyzed by capillary electrophoresis. Relative 18S/25S ratios were calculated with reference to the data of the wild type grown at 22°C. The mean values of the relative ratios obtained from four independently prepared material sets are shown, with standard deviations. Values with different letters are significantly different from each other at P < 0.05 (two-tailed Student’s t test; Supplemental Table 2).

Figure 3.

Positional Identification of ANAC082 as the SRIW1 Gene. (A) Chromosomal location of the sriw1 mutation and ANAC082. Numbers in parentheses indicate recombinant numbers in F2 plants derived from crossing sriw1 rid2 with the Col plants heterozygous for the T-DNA insertion mutation of RID2 (SALK_014062). (B) Gene structure of ANAC082 and mutant alleles. (C) Complementation test for the function of the ANAC082 transgene by adventitious rooting assay. Hypocotyl explants of the sriw1 rid2 double mutant without or with the ANAC082 transgene were cultured on RIM at 28°C for 16 d, and adventitious root formation was compared. Bar = 1 cm. (D) Suppression of the cell proliferation defect of rid2 by the anac082-6 mutation in tissue culture. Hypocotyl explants of the wild type, anac082-6, rid2, and anac082-6 rid2 were cultured on CIM at 28°C for 21 d, and callus growth was compared. Bar = 1 cm.

Figure 4.

Effects of the sriw1 Mutation on the Defects of Seedling Development of rid3 and rh10 and on Pre-rRNA Processing in rid3. (A) Seedlings of the Ler wild type, rid3, sriw1, and sriw1 rid3 grown for 14 d at 22°C or 28°C. Bar = 1 cm. (B) Seedlings of the Col wild type, rh10-1, and sriw1 rh10-1 grown for 14 d at 22°C or 28°C. Bar = 1 cm. (C) RNA gel blot analysis of the accumulation of rRNA precursors in the wild type, rid2, and sriw1 rid2. RNA samples were prepared from wild-type and mutant plants that had been grown for 14 d at 22°C or 28°C and subjected to RNA gel blot analysis with 5′-ETS-, ITS1-, and ITS2-specific probes (a′, b′, and c, respectively, in Figure 2A) for hybridization. This experiment was repeated twice using independently prepared materials to confirm the reproducibility of the result.

Figure 5.

Effects of the sriw1 Mutation on Adventitious Root Formation in Hypocotyl Explants of Ribosome-Unrelated, Temperature-Sensitive Mutants. (A) Hypocotyl explants of the wild type, sriw1, rid2, sriw1 rid2, rpd1-1, sriw1 rpd1-1, rpd1-2, and sriw1 rpd1-2 were cultured on RIM for 16 d at 28°C. Bar = 1 cm. (B) Hypocotyl explants of the wild type, sriw1, rid2, sriw1 rid2, rgd3, and sriw1 rgd3 were cultured on RIM for 16 d at 28°C. Bar = 1 cm.

Figure 6.

Effects of the sriw1 Mutation on Shoot Regeneration in Hypocotyl Explants of rid2 and rid3. (A) Hypocotyl explants of the wild type, rid2, rid3, sriw1 rid2, and sriw1 rid3 cultured on SIM at 19°C, 25°C, or 28°C for 14 d after 6 d of CIM culture at 19°C. Bar = 1 cm. (B) Relative quantities of CUC1 and STM mRNAs in hypocotyl explants of the wild type, rid2, rid3, sriw1 rid2, and sriw1 rid3. Explants were cultured on CIM at 19°C for 6 d and then cultured on SIM at 19°C, 25°C, or 28°C. Total RNA samples were isolated from the explants collected immediately after CIM culture (0 d) and after 5 d of SIM culture (5 d) and subjected to RT-qPCR analysis. mRNA relative quantities were calculated with reference to the data of the wild type at 0 d after normalization. The mean values of the relative quantities obtained from four independent cultures are shown, with standard deviations. Statistical analyses of these data are shown in Supplemental Tables 3 and 4.

Figure 7.

Effects of the sriw1 Mutation on Leaf Morphology in rid2, rid3, rh10, oli5, and rpl4d Seedlings. The first or second true leaves of seedlings of the wild type (Col and Ler), sriw1, rid2, rid3, rh10-1, oli5-1, rpl4d-3, sriw1 rid2, sriw1 rid3, sriw1 rh10-1, sriw1 oli5-1, and sriw1 rpl4d-3 grown at 22°C or 28°C on vermiculite for 16 d. After chlorophyll was removed, leaf samples were mounted with water and photographed. L, C, and M in parentheses indicate genetic backgrounds of Ler, Col, and mixture of Ler and Col, respectively. For each strain, more than 10 leaves were observed, and a representative one is shown. Bars = 2 mm.

Figure 8.

Comparison of Sensitivities of Callus Formation to Agents That Interfere with rRNA Biosynthesis or Ribosomal Function between the Wild Type and sriw1. (A) Hypocotyl explants of the wild type and sriw1 were cultured at 22°C for 28 d on CIM containing actinomycin D (Act D), 5-fluorouracil (5-FU), camptothecin (CPT), cycloheximide (CHX), puromycin (Puro), DTT, or 2,6-dichlorobenzonitrile (DCB) at various concentrations. For each agent, cultures were performed at least three times and a representative one is shown. Bars = 1 cm. (B) Fresh weights of hypocotyl explants cultured at 22°C for 28 d on CIM containing the indicated agent. For a solvent control, explants were cultured with 0.1% DMSO. Cultures were set up in six-well plates or small dishes. In one well or dish, eight explants in total, consisting of four wild type and four sriw1, were cultured. The four explants of each strain cultured in the same well or dish were gathered together and weighed. Mean values obtained from three sets of cultures are shown, with standard deviations. Asterisks and double asterisks represent significant differences at P < 0.05 and P < 0.01, respectively (two-tailed Student’s t test; Supplemental Table 5).

Figure 9.

Expression of ANAC082 as Influenced by the rid2 and rid3 Mutations. (A) Expression patterns of ANAC082pro:GUS in seedlings of the wild type, rid2, and sriw1 rid2 grown for 12 d at 22°C and then for an additional 4 d at 22°C or 28°C. Bar = 100 μm. (B) Effects of the rid2 and rid3 mutations on the level of ANAC082 expression. Total RNAs were isolated from wild-type, rid2, and rid3 seedlings that were grown for 12 d at 22°C and then for an additional 4 d at 22°C or 28°C, and subjected to RT-qPCR analysis. The relative quantities of the ANAC082 mRNA were calculated with reference to the data of the wild type at 22°C after normalization. The mean values of the relative quantities obtained from three independently prepared material sets are shown, with standard deviations. Values with different letters are significantly different from each other at P < 0.05 (two-tailed Student’s t test; Supplemental Table 6). (C) GUS activity of ANAC082pro:GUS in hypocotyl explants cultured on CIM at 22°C for the indicated times. Bar = 200 μm.

Figure 10.

GAL4 Transactivation Assay of ANAC082 in Budding Yeast. Constructs for expression of the GAL4 DNA binding domain alone (#1) and a chimeric protein consisting of the GAL4 DNA binding domain fused with the NAC domain of ANAC082 (#2) or with full-length ANAC082 (#3) were transformed into the PJ69-4A cells of budding yeast. The transformed cells were incubated for 3 d at 28°C with or without histidine. Growth in the absence of histidine indicated transactivation of the HIS3 reporter by the chimeric protein.

Figure 11.

Hypothetical Model for the Role of ANAC082 and Ribosomal Stress Response. (A) Activation of the ANAC082-dependent pathway and direct influence of the deficiency of functional ribosomes on global translation at different degrees of ribosomal disorders. This model assumes that the ANAC082-dependent pathway responds sensitively to mild disorders of ribosome biogenesis even when the number of functional ribosomes is still sufficiently large and direct influence on global translation is negligible. (B) Comparison of the framework of ribosomal stress signaling between animals and plants. The ribosomal stress signaling pathway of animals involves the transcription factor p53 as a key mediator. In plants, which have no p53 homologs, ANAC082, a member of the plant-specific transcription factor family, plays a role comparable to p53 in the ribosome stress signaling pathway.