An exocyst complex functions in plant cell growth in Arabidopsis and tobacco

Abstract

The exocyst, an octameric tethering complex and effector of Rho and Rab GTPases, facilitates polarized secretion in yeast and animals. Recent evidence implicates three plant homologs of exocyst subunits (SEC3, SEC8, and EXO70A1) in plant cell morphogenesis. Here, we provide genetic, cell biological, and biochemical evidence that these and other predicted subunits function together in vivo in Arabidopsis thaliana. Double mutants in exocyst subunits (sec5 exo70A1 and sec8 exo70A1) show a synergistic defect in etiolated hypocotyl elongation. Mutants in exocyst subunits SEC5, SEC6, SEC8, and SEC15a show defective pollen germination and pollen tube growth phenotypes. Using antibodies directed against SEC6, SEC8, and EXO70A1, we demonstrate colocalization of these proteins at the apex of growing tobacco pollen tubes. The SEC3, SEC5, SEC6, SEC8, SEC10, SEC15a, and EXO70 subunits copurify in a high molecular mass fraction of 900 kD after chromatographic fractionation of an Arabidopsis cell suspension extract. Blue native electrophoresis confirmed the presence of SEC3, SEC6, SEC8, and EXO70 in high molecular mass complexes. Finally, use of the yeast two-hybrid system revealed interaction of Arabidopsis SEC3a with EXO70A1, SEC10 with SEC15b, and SEC6 with SEC8. We conclude that the exocyst functions as a complex in plant cells, where it plays important roles in morphogenesis.

Figure 1.

Mutations in Two Different Exocyst Components Show a Synergistic Effect on Hypocotyl Elongation in 5-d-Old Etiolated Seedlings. (A) Comparison of hypocotyl lengths of siblings that differ with respect to SEC5a and EXO70A1 genotypes. WT denotes plants that are homozygous wild-type or heterozygous for the gene of interest; mm denotes plants that are homozygous mutants. Shown are data for wild-type plants (WT WT, n = 76), plants homozygous for the sec5a-1 mutation alone (mm WT, n = 25), plants homozygous for the exo70A1-1 mutation alone (WT mm, n = 53), and plants homozygous for both the sec5a-1 and exo70A1-1 mutations (mm mm, n = 8). (B) Analogous results for sec8-4 and exo70A1-2 mutations (WT WT, n = 22; mm WT, n = 16; WT mm, n = 33; mm mm, n = 10). Error bars indicate se. Inset are photos of representative dark-grown hypocotyls arranged in the same order of genotypes as in the graphs. Bar = 5 mm.

Figure 2.

Exocyst Mutants Share a Similar Aberrant Pollen Tube Phenotype. (A) to (F) Germinated qrt1 pollen from wild-type plants compared with the qrt1 pollen from sibling plants mutated in putative exocyst component genes. Each panel is identified by the genotype of the parent. Bar = 50 μm. (A) Homozygous wild type with respect to exocyst genes. (B) Homozygous for sec5a-1 and heterozygous sec5b-1. (C) Heterozygous for sec6-1. (D) Heterozygous for sec8-3. (E) Heterozygous for sec15a-1. (F) Heterozygous for sec8-4. (G) to (I) Dimensions of aberrant pollen tubes in qrt1 pollen from plants heterozygous for an exocyst mutation compared with pollen tubes in the qrt1 pollen from wild-type siblings. Error bars indicate se. (G) Widths of aberrant pollen tubes from exocyst mutants (gray) compared with widths of shortest tubes in quartets of wild-type siblings (black). (H) Lengths of aberrant pollen tubes from exocyst mutants (gray) compared with the lengths of the shortest tubes in quartets of wild-type siblings (black). (I) Length of aberrant pollen tubes (gray) and nonaberrant pollen tubes (black) in quartet pollen from plants heterozygous for an exocyst mutation, relative to the length of all pollen tubes measured in wild-type siblings (n = 76 to199).

Figure 3.

Localization of EXO70, SEC6, and SEC8 to the Tip in Tobacco Pollen Tubes by Indirect Immunofluorescence. (A) and (B) Projections of confocal sections labeled by the EXO70A1 antibody. Transverse sections through the pollen tube made by three-dimensional reconstruction are displayed. (C) and (D) Projections of confocal sections labeled by the SEC6 antibody. (E) and (F) Projections of confocal sections labeled by the SEC8 antibody. (G) Double labeling by mouse EXO70A1 antibody and rabbit SEC8 antibody shows colocalization of these subunits at the tip of pollen tubes. (H) Double labeling by mouse SEC6 antibody and rabbit SEC8 shows colocalization of these subunits at the tip of pollen tubes.

Figure 4.

Accumulation of SEC6 and SEC8 in Germinating Tobacco Pollen. N. tabacum (cv Samsun) pollen was incubated in 10% sucrose and 0.01% boric acid for the time periods described. Cytosolic and membrane fractions (100,000g pellet) were loaded on 10% SDS-PAGE, 50 μg of total protein per lane, and assayed by protein gel blot analysis using anti-SEC6 and anti-SEC8 antibodies.

Figure 5.

Cofractionation of Exocyst Subunits in Column Chromatography. (A) SEGC of total protein extract from Arabidopsis suspension culture. Shown is the cofractionation of exocyst subunits in SEGC fractions as detected with exocyst-specific antibodies. (B) SEGC as the third purification step of the same extract, following hydroxyapatite affinity chromatography and ion exchange chromatography. Shown is the cofractionation of exocyst subunits in high molecular fractions of SEGC as detected with exocyst-specific antibodies. Top panels in (A) and (B) show protein gel blot analyses. Columns in bottom panels of (A) and (B) show relative total protein concentration measured with a UV detector at 254 nm. MM indicates approximate middle molecular mass calculated for each 5-mL fraction.

Figure 6.

Yeast Two-Hybrid Analysis of Pairwise Interactions of Exocyst Subunits. (A) Pairwise interaction of all exocyst subunits. Lines and columns describe fusion forms of exocyst subunits used for transformation. Single colonies were resuspended in 150 μL of sterile water and dropped on selective plates, and each drop was 10 μL. Yeast strain AH109 was grown on -ADE-HIS-LEU-TRP plates at 28°C. Combinations boxed by white squares also showed positive β-galactosidase activity. BD, the DNA binding domain; AD, the activation domain. (B) Strength of interaction compared with a positive control (see Methods). Single colonies were resuspended in 150 μL of sterile water. Serial dilutions 1:30, 1:900, and 1:2700 in sterile water were prepared and dropped on -ADE-HIS-LEU-TRP plates at 28°C, and each drop was 10 μL.