Flying saucer1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed mucilage

Abstract

Pectins are complex polysaccharides that form the gel matrix of the primary cell wall and are abundant in the middle lamella that holds plant cells together. Their degree of methylesterification (DM) impacts wall strength and cell adhesion since unesterified pectin regions can cross-link via Ca(2+) ions to form stronger gels. Here, we characterize flying saucer1 (fly1), a novel Arabidopsis thaliana seed coat mutant, which displays primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence. These defects appear to result from a lower DM in mucilage and are enhanced by the addition of Ca(2+) or completely rescued using alkaline Ca(2+) chelators. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. FLY1 is orthologous to TRANSMEMBRANE UBIQUITIN LIGASE1, a Golgi-localized E3 ligase involved in the quality control of membrane proteins in yeast. However, FLY1-yellow fluorescent protein (YFP) fusions are localized in punctae that are predominantly distinct from the Golgi and the trans-Golgi network/early endosome in the seed coat epidermis. Wortmannin treatment, which induces the fusion of late endosomes in plants, resulted in enlarged FLY1-YFP bodies. We propose that FLY1 regulates the DM of pectin in mucilage, potentially by recycling pectin methylesterase enzymes in the endomembrane system of seed coat epidermal cells.

Figure 1.

fly1-1 Seeds Display Primary Wall Detachment and Reduced Mucilage Extrusion. (A) and (B) Mature seeds shaken in water for 2 h and stained with RR. The mucilage capsule of fly1-1 is smaller than the wild type (WT) and is surrounded by disc-like structures. (C) and (D) Mature seeds shaken in water for 2 h. The fly1-1 discs, unlike mucilage, can be clearly seen without staining. (E) to (H) Mature seeds shaken in water for 2 h and stained with S4B. (E) and (F) S4B signal from multiple optical slices. (G) and (H) Optical slices through the middle of seeds. Arrowheads indicate S4B-labeled primary cell walls attached to columellae. The loss of primary wall attachment (asterisk) correlates with the position of fly1-1 discs (arrow). (I) to (L) The development of fly1-1 seed coat epidermal cells is indistinguishable from the wild type at 4 DPA ([I] and [J]) and 7 DPA ([K] and [L]). M, mucilage pockets. A, amyloplasts. Bars = 200 µm in (A) to (D), 100 µm in (E) to (H), and 15 µm in (I) to (L).

Figure 2.

Analysis of Dry and Hydrated Seeds by Scanning Electron Microscopy and Cryo-Scanning Electron Microscopy. (A) and (B) The surface morphology of dry mature fly1-1 seeds viewed with scanning electron microscopy is indistinguishable from the wild type (WT). (C) to (F) Cryo-scanning electron microscopy of mucilage extruded from mature seeds hydrated in water. Wild-type seeds showed an irregular mucilage matrix before (C) and after sputter coating (E). fly1-1 seeds have discs on top of the mucilage matrix before (D) and after sputter coating (F). Bars = 200 µm in (A) to (D) and 50 µm in (E) and (F).

Figure 3.

Effects of Ca²⁺ and EDTA on fly1-1 Mucilage Extrusion. (A) to (F) Mature seeds shaken for 45 min in water ([A] and [B]), 50 mM CaCl₂ ([C] and [D]), or 50 mM EDTA ([E] and [F]) and then stained with RR. Only a few CaCl₂-treated fly1-1 cells release mucilage, but these appear to have discs atop compact mucilage columns ([D], inset). EDTA-treated wild-type (WT) and fly1-1 seeds have equally large mucilage capsules. (G) and (H) Unstained wild-type and fly1-1 seeds shaken in EDTA have primary walls attached to all columellae and do not display discs. (I) and (J) S4B staining confirms that the columellae of EDTA-hydrated seeds have primary cell walls attached to them. Note the absence of fly1-1 discs ([H] and [J]). Bars = 200 µm in (A) to (F), 150 µm in (G) and (H), and 75 µm in (I) and (J).

Figure 4.

Immunolabeling and Biochemical Analysis of Pectin DM in Seeds and Mucilage. (A) to (D) 2F4 immunolabeling (green) of unesterified HG and seed intrinsic fluorescence (magenta). (A) and (B) Maximum intensity signals from multiple optical stacks. (C) and (D) Optical slices through the middle of seeds. In the wild type (WT), 2F4 labels attached primary walls (arrowheads). For fly1-1, 2F4 labels both the detached primary walls (arrows) and the underlying mucilage. Bars = 50 µm. (F) Biochemical determination of pectin DM in mucilage and whole seeds. Wild-type and fly1-1 whole seeds have a similar DM, but fly1-1 mucilage has a significantly lower DM than wild-type mucilage. Values represent the mean ± se of four technical replicates. Two additional biological replicates showed the same trend.

Figure 5.

Characterization of the FLY1 Gene and Its Encoded Protein. (A) FLY1 gene structure and mutations. T-DNA insertions (fly1-2 to fly1-6) are indicated with arrows. Boxes and connecting lines represent exons and introns. (B) and (C) Genomic complementation of fly1-1 with At4g28370. (B) fly1-1 plants transformed with FLY1_pro:FLY1-YFP produce seeds that do not release discs. (C) fly1-1 plants transformed with an empty pGreenII0229 vector produced seeds that still released discs. (D) RT-PCR analysis of FLY1 and FLY2 transcripts in Col-2 tissues. Embryo and seed coat RNA was isolated from seeds at 7 DPA. GAPC was used as a loading control. (E) Predicted architecture of the FLY1 protein. FLY1 has a signal peptide (SP), seven α-helices (Schwacke et al., 2003), and a RING-H2 domain. Bars = 300 bp in (A), 150 µm in (B) and (C), and 100 amino acids in (E). (F) The FLY1 RING domain has E3 ubiquitin ligase activity in vitro. Complete (C) ubiquitination assays contained E1 enzyme 6xHis-At-UBA1, E2 enzyme 6xHis-At-UBC8, E3 enzyme GST-Fly1RING (only includes the soluble RING domain), and Ubiquitin (Ub). Top: Immunoblot using anti-Ub. The ladder and smear represent poly-Ub species of the enzymes present in the reaction. No poly-Ub is detected when omitting 6xHis-At-UBC8 (-E2 lane), GST-Fly1RING (-E3 lane), or ubiquitin (-Ub lane) from the assay. The asterisk marks anti-Ub cross-reacting with the E3 enzyme. Center: anti-His to detect E1 and E2 enzymes. Bottom: anti-GST to detect GST-Fly1RING.

Figure 6.

Localization of FLY1-YFP, ST-RFP, and VHAa1-RFP in Seed Coat Epidermal Cells. Maximum intensity signals from multiple stacks. Composite images (right panels) of FLY1-YFP (left panels) and RFP-tagged markers (middle panels). FLY1-YFP is localized in punctae at 4 DPA ([A] and [G]) and in punctae and larger bodies (arrows) at 7 DPA ([D] and [J]). FLY1-YFP punctae are associated with (arrowheads) but predominantly distinct from the Golgi marker ST-RFP ([C] and [F]) and the TGN/EE marker VHAa1-RFP ([I] and [L]). A, amyloplasts. M, mucilage pockets. Bars = 10 µm.