ECERIFERUM11/C-TERMINAL DOMAIN PHOSPHATASE-LIKE2 Affects Secretory Trafficking

Abstract

Secretory trafficking is highly conserved in all eukaryotic cells and is required for secretion of proteins as well as extracellular matrix components. In plants, the export of cuticular waxes and various cell wall components relies on secretory trafficking, but the molecular mechanisms underlying their secretion are not well understood. In this study, we characterize the Arabidopsis (Arabidopsis thaliana) dwarf eceriferum11 (cer11) mutant and we show that it exhibits reduced stem cuticular wax deposition, aberrant seed coat mucilage extrusion, and delayed secondary cell wall columella formation, as well as a block in secretory GFP trafficking. Cloning of the CER11 gene revealed that it encodes a C-TERMINAL DOMAIN PHOSPHATASE-LIKE2 (CPL2) protein. Thus, secretory trafficking in plant cells in general, and secretion of extracellular matrix constituents in developing epidermal cells in particular, involves a dephosphorylation step catalyzed by CER11/CPL2.

Figure 1.

Phenotypes of the cer11 mutant. A, Images of 5-week–old wild-type (Ler) and cer11 plants show that the cer11 plant is dwarf and bushy. Scale bar = 5 cm. B, Stem cuticular wax load determined by GC-FID. Wax analysis data are expressed as the mean percentage ± sd (n = 4; data shown is one of three independent experiments). Asterisk indicates that, statistically, this value was significantly different from wild type at P < 0.05 (Student’s t test). C, SEM images showing wax crystals deposited on the surface of wild-type and cer11 stems. Scale bar = 5 μm. D, Wild-type (Ler) and cer11 seeds after hydration with shaking in water or EDTA, and subsequent staining with ruthenium red. Lower magnification images show phenotypes of multiple seeds and higher magnification images show single representative seeds. Scale bar = 0.6 mm. E, Monosaccharide analysis of mucilage by HPAEC. The monosaccharide composition of mucilage extracted from seeds using or Na₂CO₃ or from AIR prepared from whole seeds was determined. Differences in rhamnose (Rha) and GalUA (GalA) content between wild type and cer11 are indicated by black arrows. Values are the mean ± sd (n = 4; independent seed lots for each genotype extracted and processed at the same time; data shown is one of three independent experiments) and are expressed as nmoles sugar normalized to milligrams of seed used for mucilage extraction or milligrams of AIR hydrolyzed (whole seed). Asterisks indicates that, statistically, this value was significantly different from wild type at P < 0.05 (Student’s t test). F, SEM images of wild-type and cer11 whole seeds (upper) and individual seed coat epidermal cells (lower). Columellae are indicated by white arrows. Scale bar = 20 µm. G, Light micrographs of cross sections through wild-type (upper) and cer11 (lower) seed coats stained with toluidine blue showing epidermal cells at 4, 7, 10, and 15 DPA. Development of the cer11 seed coat epidermal cells at 4, 7, and 10 DPA is indistinguishable from the wild type. However, at 15 DPA, cer11 columellae formation is not complete and the seed coat does not rupture upon hydration. Mucilage pockets are indicated by black arrowheads; a columella is indicated by a black arrow. Scale bar = 20 μm. WT, wild type.

Figure 2.

Secretion of secGFP and MUM2-YFP is disrupted in cer11. A, RNA blot analysis of secGFP transcript levels in wild-type and cer11 seedlings expressing secGFP. The RNA blot was performed using total RNA from seedlings, with ribosomal RNA used as a control. B, Confocal laser scanning microscopy images of wild-type and cer11 hypocotyl cells expressing secGFP show increased levels of GFP fluorescence in the ER network and fusiform bodies in cer11. Scale bar = 10 μm. C to M, MUM2-YFP localization by confocal microscopy in wild type (C–F) and cer11 (G–M) seed coat epidermal cells at the 4-DPA (C, G), 7-DPA (D, H), and 10-DPA (F, J) seed coat developmental stages in transverse section. YFP-MUM2 localization in wild type (E) and the cer11 (I) seed coat epidermal cells at 7 DPA in longitudinal section. Overlay (M) of MUM2-YFP (K) and the PM dye FM4-64 (L), enlarged from the white box in (I). The developmental stages of the embryos are shown in the insets. Abbreviations: mucilage pocket (m), cytoplasmic column (cc), primary cell wall (pcw), secondary cell wall (scw). Scale bars = 20 μm. WT, wild type.

Figure 3.

Phenotypes of the cpl2 mutants. A, The structure of the CER11/CPL2 gene (At5g01270) shows exons as black boxes, introns as solid lines, 5′-UTR as a white box, and 3′UTR as a black triangle. The locations of the T-DNA insertion of cpl mutants SALK_149234 (cpl2-1), SALK_059753 (cpl2-2), and GK-433F07 (cpl2-3) were mapped and are indicated by arrowheads. The location of the breakpoint in the cer11 mutant is indicated by a vertical black arrow. The locations of primers used for RT-PCR in (B) are indicated with short horizontal arrows. B, RT-PCR analysis of steady-state CER11/CPL2 transcript levels in wild type (Col-0) and mutant (cpl2-1, cpl2-2, and cpl2-3) leaves. RT-PCR was performed using total leaf RNA, and GAPC was used as a control. C, Images of 5-week–old wild-type (Col-0) and mutant (cpl2-1, cpl2-2, and cpl2-3) plants showing all the cpl2 mutant plants are dwarf and bushy compared to the wild type. Scale bar = 5 cm. D, Cuticular wax analysis for wild type and the mutant stems by GC-FID. Wax analysis data are expressed as the mean percentage ± sd (n = 4; data shown is one of three independent experiments). The asterisk indicates that, statistically, the mutant was significantly different from wild type (P < 0.05; Student’s t test). E, Mucilage extrusion from wild-type (Col-0) and cpl2-1, cpl2-2, and cpl2-3 seeds after hydration in water and staining with ruthenium red. Scale bar = 0.5 mm. F, SEM images of seed coat epidermis of seeds from wild type (Col-0) and mutants. Scale bar = 20 µm. WT, wild type.

Figure 4.

Expression pattern of the CER11/CPL2 gene and localization of the CER11/CER2-GFP protein and its colocalization with VHA-C-YFP. A, Expression levels of the CER11/CPL2 gene in different organs. Total RNAs from leaf, stem, flower, root, 10-d–old seedling, and developing seeds at 4 DPA, 7 DPA, and 10 DPA from wild type (Col-0) were analyzed for CER11/CPL2 and GAPC gene expression by RT-qPCR. CER11/CPL2 gene expression was normalized to GAPC expression values. Error bars represent sd (n = 4). B and C, Confocal microscopy of CER11/CPL2-GFP localization in stem epidermal cells (B) and seed coat epidermal cells at 7 DPA (C). A representative embryo for the developmental stage is shown in the inset. Scale bars = 20 µm. d and E, The dwarf phenotype of the VHA-C mutant det3-1 is complimented by VHA-C-YFP. In 6-week–old plants (D), the short stature of det3 is returned to wild-type height when VHA-C-YFP is introduced (E). Letters show statistical significance for n = 5, one-way ANOVA followed by Tukey's Honestly Significant Difference posthoc analysis performed using SPSS 25 software, P < 0.0001. Scale bar = 7 cm. F to K, Colocalization of CPL2-RFP and VHA-C-YFP in stem epidermal cells (F–H) and seed coat epidermal cells (I–K). Scale bar = 20 μm. WT, wild type.

Figure 5.

CPL2 interacts with VHA-C and can dephosphorylate VHA-C in vitro. A, Yeast-2-hybrid assay shows that the C-terminal domain of VHA-C (AD-VHA-C^176–375) can bind to CPL2 (BD-CPL2). AD, GAL4 activation domain fusions; BD, GAL4 DNA binding domain fusions. Serial 1:10 dilutions are shown. B, Yeast-2-hybrid assay shows that the N-terminal domain of CER11/CPL2 (CPL2^1–599) is sufficient for VHA-C binding. The domain structure of each of the CPL2 truncations tested are shown, with two major functional domains indicated. Catalytic FCP1 homology domain (blue); dsRNA binding motif (red). C, Split luciferase assay. Luciferase activity in ∼2-cm regions of N. benthamiana leaves coinfiltrated with Agrobacterium strains containing various nLuc and cLuc constructs. cLuc-VHA-C was tested for interaction with each of CER11/CPL2-nLuc (full length, FL), CER11/CPL2^1–392-nLuc (1–392), CER11/CPL2^1–599-nLuc (1–599), and nLuc, as well as between CER11/CPL2-nLuc (FL), and MPK6-cLuc or cLuc. Robust luciferase activity was detected when cLuc-VHA-C was coexpressed with CER11/CPL2-nLuc (FL) or CER11/CPL2^1–599-nLuc. Results are representative of 10 replicates (Reps), from two experiments. D, In vitro dephosphorylation assay. VHA-C-YFP was extracted from 10-d–old transgenic seedlings expressing VHACp::VHAC-YFP and incubated with buffer-only, 6×His-CER11/CPL2, or LPP. The total VHA-C-YFP and phosphorylated VHA-C-YFP (Phospho-VHA-C-YFP) were analyzed by western blotting with anti-GFP antibody and anti-Phospho-Ser/Thr antibody, respectively. E, Measurement of the band intensity of Phospho-VHA-C-YFP relative to Total VHA-C-YFP in (D) was used for quantitation.

Figure 6.

Wax and seed coat phenotypes of the det3-1 mutant. A, The structure of the VHA-C gene (At1g12840) shows exons as black boxes, introns as black lines, 5′-UTR as a white box, and 3′UTR as a black triangle. The location of the point mutation in det3-1 is indicated by a vertical arrow. The locations of primers used for RT-PCR in (B) are indicated with short horizontal arrows. B, RT-PCR analysis of steady-state VHA-C transcript levels in wild type (Col-0) and the det3-1 leaves. RT-PCR was performed using total leaf RNA, and the expression level of GAPC was used as a control. Two different sizes of VHA-C transcripts were detected in the det3-1, which are indicated by black arrows. C, A comparison of 5-week–old wild-type (Col-0) and the det3-1 plants shows that the det3-1 mutation causes a severe growth defect. Scale bar = 5 cm. D, Cuticular wax analysis for 5-week–old wild-type and the det3-1 stems by GC-FID. Wax analysis data are expressed as the mean percentage ± sd (n = 4; data shown is one of three independent experiments). E, Mucilage extrusion from wild-type and the det3-1 seeds after hydration in water or EDTA, and staining with ruthenium red. Scale bars = 0.5 mm. F, SEM images of seed coat epidermis of seeds from wild type and the mutant (det3-1). Scale bars = 20 μm. WT, wild type.