The role and behavior of Arabidopsis thaliana lipid transfer proteins during cuticular wax deposition

The primary aerial surfaces of terrestrial plants are coated with a protective hydrophobic layer comprising insoluble and soluble lipids. The lipids are known collectively as cuticular wax. To generate the waxy cuticle during elongative growth, plants dedicate half of the fatty acid metabolism of their epidermal cells. It is unknown how cuticular wax is exported from the plasma membrane into the cell wall, and eventually, to the cuticle at the cell surface. I hypothesized that lipid transfer proteins (LTPs) were responsible for plasma membrane to cell wall transport of cuticular lipids. Using an epidermis-specific microarray, I identified five candidate Arabidopsis LTPs. I discovered that mutations in gene At1g27950 result in a stem wax phenotype: reduced cuticular lipid nonacosane resulting in reduced total wax compared to wildtype. This gene encodes a glycosylphosphatidylinositol (GPI)-linked LTP and thus was named LTPG. In contrast, to LTPG, no detectable wax phenotype was found in mutants for classical LTPs. In phylogenetic analyses, these LTPs clustered into a weakly related group that I named LTPAs. In an attempt to overcome genetic redundancy I made double and triple mutants from the candidate LTPAs. None of these mutants displayed detectable changes in wax compared with wildtype. Using live cell imaging, I showed that LTPG is localized to the epidermal cell plasma membrane and the cell wall and accumulates non-uniformly on the plant surface. I employed fluorescence recovery after photobleaching to demonstrate that, in the plasma membrane, LTPG is relatively immobile and exhibits a complicated recovery, the latter appears linked to the flux of cuticular lipids through the plasma membrane. LTPG accumulates over the long cell walls of stem epidermal cells and this protein moves when observed over 1 min intervals. I created a GPIlinked LTPA and demonstrated that it can rescue the ltpg-1 mutation. I demonstrate that LTPG is required for wax export by associating with the plant cell wall. This is the first experimental evidence linking the lipid transfer function of a plant LTP to a biological role, which in this case is lipid movement through the cell wall to the cuticle.Science, Faculty ofBotany, Department ofGraduat

Cell Geometry Guides the Dynamic Targeting of Apoplastic GPI-Linked Lipid Transfer Protein to Cell Wall Elements and Cell Borders in <i>Arabidopsis thaliana</i>

<div><p>During cellular morphogenesis, changes in cell shape and cell junction topology are fundamental to normal tissue and organ development. Here we show that apoplastic Glycophosphatidylinositol (GPI)-anchored Lipid Transfer Protein (LTPG) is excluded from cell junctions and flat wall regions, and passively accumulates around their borders in the epidermal cells of <i>Arabidopsis thaliana</i>. Beginning with intense accumulation beneath highly curved cell junction borders, this enrichment is gradually lost as cells become more bulbous during their differentiation. In fully mature epidermal cells, YFP-LTPG often shows a fibrous cellulose microfibril-like pattern within the bulging outer faces. Physical contact between a flat glass surface and bulbous cell surface induces rapid and reversible evacuation from contact sites and accumulation to the curved wall regions surrounding the contact borders. Thus, LTPG distribution is dynamic, responding to changes in cell shape and wall curvature during cell growth and differentiation. We hypothesize that this geometry-based mechanism guides wax-carrying LTPG to functional sites, where it may act to “seal” the vulnerable border surrounding cell-cell junctions and assist in cell wall fortification and cuticular wax deposition.</p> </div

YFP-LTPG is apoplastic.

<div><p>A, B Apoplastic marker SEC-YFP accumulates over anticlinal walls, but shows no filamentous patterning.</p> <p><b>A</b> Mature cotyledon epidermal cells (arrowheads show anticlinal enrichment). </p> <p><b>B</b> Immature unexpanded cotyledon epidermal cells (arrowheads show anticlinal enrichment; arrow shows lack of enrichment at recently formed cell wall).</p> <p><b>C</b> YFP-LTPG localization in leaf epidermal cells prior to treatment with mannitol, after 20 min in 500µM mannitol, and after 20 min rinsing in distilled water. YFP-LTPG fills in the apoplastic space between cell wall and plasma membrane in plasmolysed cells. Plasmolysis was complete in under 10 minutes, and rinsing in water restored initial pattern over anticlinal walls Inset shows striated YFP-LTPG fluorescence at the outer cell surface prior to plasmolysis. Arrows show striations.</p> <p><b>D</b> GFP-PIP2a localization in leaf epidermal cells prior to treatment with mannitol, after 20 min in 500µM mannitol, and after 20 min rinsing in distilled water. GFP-PIP2a localizes to the retraced plasma membrane and accompanying Hechtian strands. </p> <p>YFP-LTPG does not localize to the plasma membrane, and becomes concentrated within the concave side of epidermal cell lobes as the protoplast expands during deplasmolysis. Co-staining of YFP-LTPG and FM4-64.</p> <p><b>E</b> Loss of outer anticlinal enrichment and apoplastic filling of YFP-LTPG during plasmolysis. Arrowheads indicate anticlinal enrichment regions before and after plasmolysis. Shown are orthogonal views from cotyledon epidermal cells.</p> <p><b>F</b> Plasmolysis results in rapid and reversible loss of outer anticlinal YFP-LTPG fluorescence (arrowheads).</p> <p>Bars, 10 µm.</p></div

Hypothetical balloon in a box model to predict LTPG distribution patterns.

<div><p><b>A</b> Three different scenarios for inflating a balloon in a box with differentially curved walls. As the balloon inflates, LTPG accumulates passively to spaces, which appear below sharply curved regions of the cell. The box 1 is analogous to a young boxy epidermal cell. Box 2 is analogous to a bulbous mature epidermal cells. Box 3 represents the hypothetical scenario of a perfectly round/walled sphere, which results in perfectly uniform LTPG distribution. Box 4 is analogous to our cover glass flattening scenario, wherein induced flattening of a rounded surface generates small regions of curvature bordering the flattened region. </p> <p><b>B</b> Balloon in a leaky box model, wherein the naked internal walls of the epidermal cells created by the separation of underlying mesophyll cells leak apoplastic LTPG.</p></div

YFP-LTPG is excluded from cell-cell contacts and coverslip contact sites.

<div><p><b>A</b> Cotyledon epidermal cells showing inner face (yellow) and midplane (blue), and overlay of these two planes. Arrowheads show ridges of fluorescence surrounding fluorescence free zones that are in contact with underlying mesophyll cells. </p> <p><b>B</b> Contact with coverslip copies cell-cell contact clearing. 3D reconstruction tilted image from cotyledon petiole epidermal cells. Spaces occupy a plane on the outer periclinal face. Orthogonal views illustrate the planar nature of clearing (brackets), and show ridges as well. Arrow indicates normal anticlinal accumulation. Images in right panel show the top slice from the Z-series in C (top), and a maximum Z-projection of the series (bottom).</p> <p><b>C</b> Clear zones at cell-cell (left panel) and cell-coverslip (right panel) contact sites. In both cases, ridges (arrowheads) surround clear zones. </p> <p><b>D</b> Coverslip contact results in clear zones within the filamentous patterning (brackets) in mature epidermal cells.</p> <p><b>E</b> YFP-LTPG contact clear zones form and enlarge as the coverslip is appressed over time. Images are ~1 minute apart. Yellow arrowheads track leading edge of tip clearing as it spreads to the lateral walls. Red arrowheads show where two clearing from adjacent cells meet and cross over their shared anticlinal wall. See also movie S1.</p> <p>Scale bars, 5 µm.</p></div

Cell geometry-dependent distribution of apoplastic YFP-LTPG.

<div><p><b>A</b> Orthogonal views showing variable anticlinal and periclinal polarization. 1 - unexpanded leaf; 2 - young hypocotyl; 3 - mature hypocotyl; 4 - Loss of anticlinal enrichment in very bulged cells.</p> <p><b>B</b> Loss of anticlinal enrichment in the bulbous cotyledon epidermal cells of <i>mor1-1</i> and <i>clasp-1</i> mutants.</p> <p><b>C</b> Flattening of the outer periclinal face of bulging epidermal cells with coverslip generates increase in fluorescence over anticlinal walls (arrowheads).</p> <p><b>D</b> Surface view showing increase in external anticlinal fluorescence (arrowhead) upon flattening of cell.</p> <p><b>E</b> Formation of radial strations (arrowheads) along anticlinal walls in periclinally flattened epidermal cells. Left panel shows mid-stage cotyledon petiole cells, right panel shows mature hypocotyl cells. Bar, 5 µm.</p></div

YFP-LTPG shows filamentous patterning and accumulation over anticlinal walls.

<div><p><b>A</b> Mature cotyledon epidermal cell showing filamentous YFP-LTPG patterning (arrows).</p> <p><b>B</b> 3D rotation of mature cotyledon epidermal cell shows filaments extending down anticlinal walls (arrows).</p> <p><b>C</b> Schematic diagram showing nomenclature used in this article. </p> <p><b>D</b> Tilted image from z-series 3D reconstruction showing anticlinal YFP-LTPG accumulation in unexpanded leaf petiole cells (arrowheads).</p> <p><b>E</b> Radial striations (arrowheads) along anticlinal walls of unexpanded leaf petiole cells. Tilted image from 3D rotation.</p> <p><b>F</b> Orthogonal slice from confocal Z-series illustrates YFP-LTPG fluorescence accumulation over anticlinal walls (arrowheads).</p> <p><b>G</b> Optical midplane image of epidermal cells showing accumulation of YFP-LTPG over anticlinal walls (arrowheads). </p> <p><b>H</b> Tilted image from 3D reconstruction of YFP-LTPG expressed in unexpanded leaf. Recently-formed cell walls (brackets) contain faint, homogeneous fluorescence. As the anticlinal wall matures, YFP-LTPG accumulates above it non-uniformly (green highlight), showing a gradual increase in fluorescence with increasing distance from three-way junctions (arrows). Green highlight shows outer edge enrichment; pink shows anticlinal walls. Arrowheads indicate example of outer enrichment site. Bottom panel shows orthogonal slice. </p> <p><b>I</b> Mid-stage lobed leaf epidermal cell showing non-uniform accumulation of YFP-LTPG over anticlinal walls. Arrowheads indicate accumulation of YFP-LTPG within concave sides of cell lobes. Bars, 10 µm.</p></div

YFP-LTPG accumulates in intercellular spaces below epidermis.

<div><p><b>A</b>-<b>C</b>. Intercellular localization of YFP-LTPG (A, B) and control plasma membrane marker GFP-PIP2A (C) in young cotyledon cells. B shows subepidermal intercellular fluorescence in a stem. Mesophyll cells visualized with chloroplast fluorescence (red). For A and C, the left panels show a single optical plane through the mesophyll layer below the epidermis. The blue/green panels show merged images with the inner slices pseudo-colored blue, and the optical midplane of the epidermal cells shown in green. M = mesophyll cell; S = intercellular space.</p> <p><b>D</b> Intercellular YFP-LTPG distribution in roots. Fluorescence is limited specifically to atrichoblasts of the elongation zone (EZ). Left panel shows 3D reconstruction (arrowheads indicate subepidermal fluorescence lining inner borders of atrichoblasts). Right panel shows dual plane overlay, wherein subepidermal fluorescence appears as blue strips behind the green cell files in the overlay, and is indicated by arrowheads in orthogonal views. Arrow indicates subepidermal intercellular fluorescence beneath non-expressing cell file.</p> <p><b>E</b> YFP-LTPG fluorescence is not due to free YFP. Western blot using anti-GFP shows full length YFP-LTPG band at 47 KDa. Bars, 5 µm.</p></div