GeBP/GPL transcription factors regulate a subset of CPR5-dependent processes

Abstract

The CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5 (CPR5) gene of Arabidopsis (Arabidopsis thaliana) encodes a putative membrane protein of unknown biochemical function and displays highly pleiotropic functions, particularly in pathogen responses, cell proliferation, cell expansion, and cell death. Here, we demonstrate a link between CPR5 and the GLABRA1 ENHANCER BINDING PROTEIN (GeBP) family of transcription factors. We investigated the primary role of the GeBP/GeBP-like (GPL) genes using transcriptomic analysis of the quadruple gebp gpl1,2,3 mutant and one overexpressing line that displays several cpr5-like phenotypes including dwarfism, spontaneous necrotic lesions, and increased pathogen resistance. We found that GeBP/GPLs regulate a set of genes that represents a subset of the CPR5 pathway. This subset includes genes involved in response to stress as well as cell wall metabolism. Analysis of the quintuple gebp gpl1,2,3 cpr5 mutant indicates that GeBP/GPLs are involved in the control of cell expansion in a CPR5-dependent manner but not in the control of cell proliferation. In addition, to our knowledge, we provide the first evidence that the CPR5 protein is localized in the nucleus of plant cells and that a truncated version of the protein with no transmembrane domain can trigger cpr5-like processes when fused to the VP16 constitutive transcriptional activation domain. Our results provide clues on how CPR5 and GeBP/GPLs play opposite roles in the control of cell expansion and suggest that the CPR5 protein is involved in transcription.

Figure 1.

Gene ontology of gebp gpl1,2,3 and VP16:GPL2 transcriptomic data and transcriptomic similarities with the cpr5 mutant series. A, Gene ontology of gebp gpl1,2,3 (left) and VP16:GPL2 (right) transcriptomic data. Distribution of gene sets among functional biological pathways using singular enrichment analysis of AgriGO are shown. Colors are as in AgriGO, and only the most significant pathways are shown. The ratio of genes involved in each pathway and P values are indicated within boxes together with the gene ontology accession number. The highest probabilities are for the stimuli/stress response (gebp gpl1,2,3 and VP16:GPL2) and cell wall process (VP16:GPL2) pathways. Several entries were not associated to GO terms. Hence 81 genes instead of 88 were used in this analysis for the gebp gpl1,2,3 data and 323 genes instead of 332 for the VP16:GPL2 data. B, Hierarchical clusterings of genes misregulated in the gebp gpl1,2,3 (left) or VP16:GPL2 (right) and cpr5 mutant series. Graphics were generated using Genevestigator and MultiExperiment viewer software.

Figure 2.

Phenotypic similarities between VP16:GPL2 and cpr5-2 mutants. A, Top row, rosettes of 3-week-old wild-type, cpr5-2, and VP16:GPL2 plants grown in soil. White arrows indicate early senescing leaves. Middle row, individual third leaves of 4-week-old plants. Control lines expressing the VP16 domain alone showed no visible phenotypes as previously described (Chevalier et al., 2008). Scale bars: 1 mm. Bottom row, sizes of trichomes. Scale bars: 100 μm. B, Trypan blue staining of wild-type, cpr5-2, gebp gpl1,2,3, and VP16:GPL2 leaves. Scale bars: 500 μm. C, Bacterial populations in wild-type, cpr5-2, VP16:GPL2, and gebp gpl1,2,3 plants. Inoculations with Pst DC3000 strain were performed on leaves without lesions with a bacterial suspension at 2 × 10⁵ cfu mL⁻¹. Bacterial populations were measured at 0 (white bars) and 3 d (dark bars) postinoculation. Mean bacterial densities are shown (three to five replicates with corresponding sds) for one representative experiment from two or three independent experiments. Asterisks denote significantly different values from bacterial number in the wild type according to the Student’s t test (P ≤ 0.05). D, Transcript levels of PR1 and PR5 genes in gebp gpl1,2,3 and VP16:GPL2 relative to the wild type. Real-time RT-PCR was performed on 3-week-old rosettes. [See online article for color version of this figure.]

Figure 3.

Vegetative growth of wild-type, cpr5-2, and gebp gpl1,2,3 cpr5 mutants. A, Rosettes of wild type, gebp gpl1,2,3 quadruple mutant, cpr5-2, and gebp gpl1,2,3 cpr5 quintuple mutant grown in soil for 3 weeks. Scale bar: 3 mm. B, Leaf area of the wild type, gebp gpl1,2,3 quadruple mutant, cpr5-2, and gebp gpl1,2,3 cpr5 quintuple mutant. C, Leaf elongation rate in wild type, cpr5-2, gebp/gpl quadruple mutant, and gebp/gpl cpr5-2 quintuple mutant. Plants were grown in soil, and measurements of the third leaf were taken at daily intervals. Initial growth rates were similar in all types of plant. [See online article for color version of this figure.]

Figure 4.

Aphidicolin sensitivity assay and DNA levels in wild type, gebp gpl1,2,3 quadruple mutant, cpr5-2, and gebp gpl1,2,3 cpr5 quintuple mutant. A, Aphidicolin sensitivity assay. Plants were grown in vitro for 3 weeks in the absence (−) or presence (+) of aphidicolin (12 μg mL⁻¹). Scale bars: 5 mm. B, Distribution of nuclei according to DNA content in cells of third rosette leaves. [See online article for color version of this figure.]

Figure 5.

Size and number of adaxial pavement cells in leaves of wild-type and mutant plants. Epidermal cell size and number in wild type, gebp gpl1,2,3 quadruple mutant, cpr5-2, and gebp gpl1,2,3 cpr5 quintuple mutant. A, Adaxial pavement cell size of third leaves of 3-week-old plants grown in soil. Scale bars: 200 μm. B, Class distribution of epidermal cell size (top histogram), epidermal cell density (bottom left), and estimated epidermal cell number per leaf (bottom right). Class distribution of cell size was performed by measuring cell area from third leaves of 3-week-old plants grown in soil (Columbia, n = 147; quadruple mutant, n = 149; cpr5, n = 294; quintuple mutant, n = 191) using ImageJ software. The total number of epidermal cells per leaf was estimated by dividing the leaf area by the average cell area.

Figure 6.

Intracellular localization and functional analysis of CPR5 and CPR5ΔTM proteins. A, Subcellular localization of GFP:CPR5 protein in tobacco cells under confocal microscopy. Scale bars: 10 μm. B, Trichome development in plants transformed with 35S:GFP:CPR5 on a cpr5-2 mutant background. Young rosette leaves are shown. Scale bars: 1 mm. C, Phenotype of plants transformed with 35S:HA:VP16:CPR5 or 35S:HA:VP16:CPR5ΔTM on a wild-type background. Young rosettes are shown. White arrows indicate early senescing cotyledons. Scale bars: 3 mm. D, Detection of VP16 fusion proteins in western blots (two top sections) and transcript levels of PR1 in wild-type and VP16 plants using semiquantitative RT-PCR (two bottom sections) in wild-type and VP16 transgenic lines. The star indicates a weak band in the VP16:CPR5 lane corresponding to a protein with a size similar to that of the VP16:CPR5ΔTM protein. Contrast has been increased to better visualize this band. VP16 fusions were detected with a monoclonal anti-HA antibody (Roche). The KARI protein used as a loading control was detected with a polyclonal anti-KARI antibody.