GNOM-LIKE1/ERMO1 and SEC24a/ERMO2 are required for maintenance of endoplasmic reticulum morphology in Arabidopsis thaliana

Abstract

The endoplasmic reticulum (ER) is composed of tubules, sheets, and three-way junctions, resulting in a highly conserved polygonal network in all eukaryotes. The molecular mechanisms responsible for the organization of these structures are obscure. To identify novel factors responsible for ER morphology, we employed a forward genetic approach using a transgenic Arabidopsis thaliana plant (GFP-h) with fluorescently labeled ER. We isolated two mutants with defects in ER morphology and designated them endoplasmic reticulum morphology1 (ermo1) and ermo2. The cells of both mutants developed a number of ER-derived spherical bodies, approximately 1 microm in diameter, in addition to the typical polygonal network of ER. The spherical bodies were distributed throughout the ermo1 cells, while they formed a large aggregate in ermo2 cells. We identified the responsible gene for ermo1 to be GNOM-LIKE1 (GNL1) and the gene for ermo2 to be SEC24a. Homologs of both GNL1 and SEC24a are involved in membrane trafficking between the ER and Golgi in yeast and animal cells. Our findings, however, suggest that GNL1/ERMO1 and SEC24a/ERMO2 have a novel function in ER morphology in higher plants.

Figure 1.

Abnormal Development of a Large Number of ∼1-μm-Diameter Spherical Bodies in Both ermo1-1 and ermo2 Cells. Two mutants that showed defects in ER morphology (ermo1-1 and ermo2) were isolated from an ethyl methanesulfonate–mutagenized GFP-h pool. Both ermo1-1 and ermo2 developed a number of abnormal spherical bodies within the cells. Cotyledons ([A], [B], [E], and [F]), petioles (D), and hypocotyls (C) of ermo1-1, ermo2, and GFP-h plants were observed using a confocal laser scanning microscope. The inset in (D) shows the enlarged view of the boxed area. Bars = 10 μm in (A) to (F) and 1 μm in inset in (D).

Figure 2.

Spherical Bodies of ermo1-1 and ermo2 Are ER-Derived Structures. Seven-day-old plants were subjected to ultrastructural analysis. Sb, spherical body; Eb, ER body; rER, rough ER; G, Golgi body; V, vacuole; N, nucleus; Mt, mitochondrion; Pt, plastid; Per, Peroxisome; Cw, cell wall. (A) and (B) Cotyledon epidermal cells of a GFP-h plant. (C) to (F) Cotyledon epidermal cells of a ermo1-1 plant. (D) and (F) are enlarged views of (C) and (E), respectively. Note that the spherical bodies were surrounded by ribosomes (arrowheads). Arrows indicate that spherical bodies were connected to the tubular rER. (G) to (K) Cotyledon epidermal cells of ermo2. (H) and (I) are enlarged views of (G), and (K) is an enlarged view of (J). The spherical bodies were found both in the aggregate ([G] to [I]; indicated by red asterisks) and the periphery of the cells ([J] and [K]). The spherical bodies in ermo2 were also surrounded by ribosomes (arrowheads) and connected to the tubular rER (arrows). Bars = 1 μm in (A) to (C), (E), (G), (H), and (J) and 100 nm in (D), (F), (I), and (K).

Figure 3.

Maintenance of Spherical Bodies in ermo1-1 and ermo2 Is Independent of Actin Filaments. GFP-h and ermo mutants were transformed with tdTomato-ABD2 to visualize actin filaments (magenta). Bars = 10 μm. (A) to (F') In GFP-h cells ([A] and [B]), a large fraction of the ER (green) colocalized with actin filaments, shown as white signals. Typical networks of actin filaments were seen in ermo1-1 ([C] and [D]) and ermo2 ([E] and [F]) cells, and the spherical bodies were distributed along thick actin bundles ([C] to [E]). The aggregation of spherical bodies in ermo2 was surrounded by actin filaments (F). A three-dimensional reconstruction of (F) is shown in (F'). The single-channel SP-GFP-HDEL (left), tdTomato-ABD2 (center), and merged (right) images are shown. (G) to (L) Seedlings of the transformants were treated with 100 μM Lat B for 2 h. In GFP-h cells ([G] and [H]), the ER network collapsed and formed aggregations (arrowheads). The spherical bodies in ermo1-1 ([I] and [J]) and ermo2 (K) cells were maintained in the presence of Lat B, and typical Lat B–induced aggregates were formed (arrowheads). The formation of spherical body aggregates in ermo2 was not affected ([L], arrow). (M) and (N) Time-lapse image sequences of cotyledon epidermal cells of ermo1-1 (M) and ermo2 (N). The spherical bodies in each mutant moved along the actin bundle (arrowheads).

Figure 4.

The Phenotype of the ermo1-1 ermo2 Double Mutant. Confocal images of the ermo1-1 ermo2 double mutant. Seven-day-old seedlings were observed using a confocal microscope. Bars = 10 μm. (A), (C), and (D) Epidermal cells of the hypocotyl. (B) Epidermal cells of the petiole. Note that the double mutant did not develop any aggregations of spherical bodies.

Figure 5.

Identification of the ERMO1/GNL1 Gene as Being Responsible for the ermo1 Mutation. (A) Positional cloning and DNA sequencing identified a mutation in the GNL1 locus of ermo1-1. Mutation sites of T-DNA–inserted ermo1 alleles and previously reported gnl1 mutants are also shown. Black boxes and black lines indicate exons and introns, respectively. ERMO1/GNL1 has several characteristic domains: DCB, dimerization/cyclophilin binding domain; HUS, homology upstream of Sec7 domain; Sec7, Sec7 domain; HDS, homology downstream of Sec7 domain. (B) to (E) SP-GFP-HDEL was transiently expressed in T-DNA insertion lines. Confocal images of the ER in GFP-h (B), ermo1-2 (C), ermo1-3 (D), and ermo1-4 (E) cells are shown. Bars = 10 μm. (F) and (G) Confocal images of F1 plants generated by crossing ermo1-1 with either ermo1-3 (F) or ermo1-4 (G). All the T-DNA insertion lines and the F1 lines showed abnormal ER morphology identical to the phenotype of ermo1-1. Bars = 10 μm. (H) RT-PCR analysis revealed that GNL1 mRNA was not transcribed in T-DNA insertion mutant alleles, with the exception of ermo1-1. Amplified regions are indicated by blue lines in (A). Two biological replicates were performed.

Figure 6.

A High Concentration of BFA Induces Abnormal Structures Similar to the Spherical Bodies of ermo1-1 and ermo2 Plants. (A) to (D) Seedlings of GFP-h ([A] and [B]) and ST-GFP ([C] and [D]) plants were treated with either 25 μg/mL BFA ([B] and [D]) or solvent control ([A] and [C]). BFA (25 μg/mL) induced BFA compartments, which were visualized with ST-GFP (D), but did not affect ER morphology (B). (E) to (J) Seedlings of GFP-h ([E] to [H]) or ST-GFP ([I] and [J]) plants were treated with 100 μg/mL BFA. Bars = 10 μm. (E) and (F) The treatment induced GFP fluorescent spherical structures that were similar to the spherical bodies in ermo1 mutants (arrowheads). (G) The spherical structures occasionally formed several aggregations within a cell. (H) The aggregations were associated with the BFA compartments that were visualized by FM4-64 (magenta). (I) and (J) The BFA treatment caused a redistribution of ST-GFP to both the ER and the BFA compartments. Note that ST-GFP also showed GFP fluorescent spherical structures (arrowheads in [I]).

Figure 7.

Identification of the SEC24a/ERMO2 Gene as Being Responsible for the ermo2 Mutation. (A) Positional cloning and DNA sequencing identified a mutation in the SEC24a locus of ermo2. Black boxes and black lines indicate exons and introns, respectively. SEC24a/ERMO2 has several characteristic domains: Zn, zinc finger domain; Trunk, Sec23/24 trunk domain; β, Sec23/24 β-sandwich domain; helical, Sec23/24 helical domain; Gelsolin, Gelsolin repeat domain. (B) and (C) Independent ermo2 lines transformed with tdTomato-SEC24a were observed using a confocal microscope. No visible defects in ER morphology were observed in the transformant lines, indicating that SEC24a was responsible for the ermo2 phenotype. Bars = 10 μm. (D) The amino acid (Arg) at the ermo2 mutation site is completely conserved in SEC24 proteins from Arabidopsis (At), Oryza sativa (Os), Physcomitrella patens (Pp), Homo sapiens (Hs), Drosophila melanogaster (Dm), and S. cerevisiae (Sc). The last amino acid positions are indicated after each sequence. The residues corresponding to Arg-693 in SEC24a/ERMO2 are indicated in red.

Figure 8.

Abnormal Formation of the Spherical Bodies Is Not Caused by Defects in Protein Export from the ER. (A) to (D) GFP-h (A), ermo1-1 (B), and ermo2 ([C] and [D]) cells were treated with 50 μM CHX for 2 h. Spherical bodies and aggregations were formed and maintained in the presence of CHX. The insets in (B) and (C) are enlarged views of the boxed area in each panel. Bars = 10 μm in (A) to (D) and 1 μm in insets in (B) and (C). (E) SP-GFP-HDEL was stably introduced into a mag2-1 mal double mutant. No defects in ER morphology were detected. (F) Total protein from dry seeds was extracted and subjected to immunoblot analysis using anti-12S globulin antibodies. The ermo1-1 and ermo2 mutants did not accumulate precursors of 12S globulin (p12S), while mag2-1 with a defect in ER-Golgi transport showed abnormal accumulation of precursor forms (arrowhead). (G) Suborganeller fractionation using 3-week-old GFP-h, ermo1-1, ermo2, and mag2-1 mal plants followed by immunoblotting using anti-RD21 antibodies was performed. P1, P8, P100, and S100 fractions are rich in nuclei, plastids, microsomes, and soluble proteins, respectively. Both intermediate (i) and mature (m) forms of RD21 were detected in the vacuole-rich fraction (S100) in each mutant.