SEC8, a subunit of the putative Arabidopsis exocyst complex, facilitates pollen germination and competitive pollen tube growth - PubMed (original) (raw)

SEC8, a subunit of the putative Arabidopsis exocyst complex, facilitates pollen germination and competitive pollen tube growth

Rex A Cole et al. Plant Physiol. 2005 Aug.

Abstract

The exocyst, a complex of eight proteins, contributes to the morphogenesis of polarized cells in a broad range of eukaryotes. In these organisms, the exocyst appears to facilitate vesicle docking at the plasma membrane during exocytosis. Although we had identified orthologs for each of the eight exocyst components in Arabidopsis (Arabidopsis thaliana), no function has been demonstrated for any of them in plants. The gene encoding one exocyst component ortholog, AtSEC8, is expressed in pollen and vegetative tissues of Arabidopsis. Genetic studies utilizing an allelic series of six independent T-DNA mutations reveal a role for SEC8 in male gametophyte function. Three T-DNA insertions in SEC8 cause an absolute, male-specific transmission defect that can be complemented by expression of SEC8 from the LAT52 pollen promoter. Microscopic analysis shows no obvious abnormalities in the microgametogenesis of the SEC8 mutants, and the mutant pollen grains appear to respond to the signals that initiate germination. However, in vivo assays indicate that these mutant pollen grains are unable to germinate a pollen tube. The other three T-DNA insertions are associated with a partial transmission defect, such that the mutant allele is transmitted through the pollen at a reduced frequency. The partial transmission defect is only evident when mutant gametophytes must compete with wild-type gametophytes, and arises in part from a reduced pollen tube growth rate. These data support the hypothesis that one function of the putative plant exocyst is to facilitate the initiation and maintenance of the polarized growth of pollen tubes.

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Figures

Figure 1.

Figure 1.

Microarray data for putative exocyst components in Arabidopsis indicate that transcripts for all eight subunits are expressed widely. In particular, the experiments of Honys and Twell (2004) show that At_SEC8_, as well as other putative exocyst components, are expressed in developing pollen. Pollen sources of RNA for the expression analysis included uninucleate microspores (UNM), bicellular pollen (BCP), immature tricellular pollen (TCP), and mature pollen grains (MPG). Additional sources of RNA for the analysis included cotyledons (COT), leaves (LEF), petioles (PET), stems (STM), roots (ROT), root hairs (RHR), and cell suspensions (SUS). White (no expression) indicates that expression was not consistently detected among replicates. For brevity, the data shown for At_EXO70_ represent only three of the 23 homologs of this exocyst component in Arabidopsis; these are the homologs highly expressed in the pollen. Nomenclature is based upon that of Elias et al. (2003).

Figure 2.

Figure 2.

T-DNA insertional mutations interrupt the coding region of SEC8 (At3g10380) at multiple sites. A, Six independent insertions (designated m1 through m6) interrupt the approximately 8.5-kb gene. Based on the sequence of a full-length 3.7-kb cDNA (accession no. AY059763), the gene contains 27 exons (boxed) and is predicted to encode a 1,053-residue protein. B, Stretches of primary sequence at the C-terminal end of plant SEC8 orthologs are conserved in widely divergent species, as shown by alignment of predicted protein sequences. Identical residues are shaded black; similar residues, gray. Insertion sites for four of the mutant alleles are designated by a black triangle. Arabidopsis (A.t.) and rice (O.s.) sequences are based on genomic data; all others are based on a consensus of assembled ESTs. Other species are as follows: M.d., Malus domestica (apple); Z.m., Zea mays (maize); P.t., Pinus taeda (loblolly pine); P.p., Physcomitrella patens (moss). Helx, Regions of predicted _α_-helical structure (gray boxes).

Figure 3.

Figure 3.

Insertional mutations affect the SEC8 transcript and transmission through the male gametophyte. A, The SEC8 transcript is detectable in several wild-type tissues, including mature pollen, by RT-PCR. Primers against ACTIN2 (sporophyte) and ACTIN3 (gametophyte) were included as internal controls. B, Schematic of the SEC8 transcript and the strategy to test for aberrant transcripts generated by the -m4, -m5, and -m6 alleles. RT-PCR product A is located 5′ to the insertion sites, whereas product B spans all three insertion sites. C and D, In RNA made from immature floral tissue, product A is detectable in the wild type and all three mutant homozygotes (C), whereas product B is only detectable in the wild type (D). E and F, Twenty outcross progeny were genotyped by PCR using a set of three primers (see “Materials and Methods”) to produce distinct wild-type (WT) and sec8-m3 heterozygote banding patterns. E, No heterozygous progeny were produced in an outcross to a wild-type homozygote using pollen from a sec8-m3 heterozygote. F, Heterozygous progeny were produced in an outcross using pollen from a sec8-m3 heterozygote also carrying a LAT52::SEC8 construct, demonstrating male transmission of the mutant allele, and complementation.

Figure 4.

Figure 4.

Alexander staining reveals that a SEC8 allele with an absolute transmission defect is associated with a pollen germination defect. A to E, Staining phenotypes of wild-type pollen grains in vivo, illustrating (from left to right, ungerminated to empty grain) a likely progression of cytoplasm (stained purple) out of the grain and into the growing pollen tube. F and G, Representative fields of stained pollen grains from a wild-type homozygote (F) and a sibling sec8-m3 heterozygote (G). White arrows, Ungerminated grains; stigma cells stain green. H, Quantitation of in vivo germination rates from three different pairs of lines, assayed blindly 4 h after pollination. Hatched bars, Mutants; white bars, wild-type siblings.

Figure 5.

Figure 5.

TEM images indicate that ungerminated pollen from sec8-m3 heterozygotes responds to signals to germinate. A, D, G, and H, Wild-type pollen grains in vivo, illustrating (respectively) a likely progression of intracellular morphologies during germination. D and G, Vacuolar space generated adjacent to the pollen cell wall (black arrow). H, An apparently emptied coat. Larger, apparently coalescing, vacuoles (examples marked by asterisks) are also characteristic of germinating grains and are not present in mature pollen (I). B, Detail of A, adjacent to the pollen cell wall. Mitochondria (white arrowhead) and small membrane-bound structures of unknown identity (black arrows) are apparent. E, Detail of a pollen grain similar to D, showing vacuoles (asterisks) apparently fusing with the coat-associated vacuolar space and a few of the apparent small membrane-bound structures seen in B. C and F, Representative ungerminated grains (i.e. no separation of the cytoplasm from the coat) from a sec8-m3 heterozygote of class I and II, respectively (see text). Class I and II grains show certain characteristics similar to A and D, respectively. Both contain larger vacuoles (examples marked by asterisks); class I grains (C) contain small membrane-bound structures, whereas class II grains do not. Bar = 2 _μ_m. White arrows, Sperm cells (A and I); black arrowhead, vegetative nucleus (I).

Figure 6.

Figure 6.

Transmission of the sec8-m4 allele increases in incompletely filled siliques. Pollinations (using sec8-m4/+ as the male) were done with either sparse (black squares) or excess (white circles) pollen placed on each wild-type stigma. Genotypes of progeny were determined by PCR.

Figure 7.

Figure 7.

The sec8-m4 mutation decreases pollen tube growth rate in culture. A histogram of the rates for individual pollen tubes (n = 81 mutant, 100 wild type) from five different homozygous mutant plants (cross-hatched bars) and five different sibling wild-type plants (white bars) shows a significantly different distribution in the two genotypes. Rates were determined by imaging germinated, growing pollen tubes at 15- to 30-min intervals.

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