John E. Fowler

An Exocyst Complex Functions in Plant Cell Growth in Arabidopsis and Tobacco

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.

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

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.

The Arabidopsis Exocyst Complex Is Involved in Cytokinesis and Cell Plate Maturation

The plant cell cytokinesis is driven from the onset by highly organized vesicle fusion resulting in cell plate and new cell wall formation separating daughter cells. The evolutionarily conserved exocyst complex regulating exocytic vesicle binding to the plasma membrane is involved in both the final separation of cells as in animals and also in the initiation of cell plate in plant cells. Cell reproduction is a complex process involving whole cell structures and machineries in space and time, resulting in regulated distribution of endomembranes, organelles, and genomes between daughter cells. Secretory pathways supported by the activity of the Golgi apparatus play a crucial role in cytokinesis in plants. From the onset of phragmoplast initiation to the maturation of the cell plate, delivery of secretory vesicles is necessary to sustain successful daughter cell separation. Tethering of secretory vesicles at the plasma membrane is mediated by the evolutionarily conserved octameric exocyst complex. Using proteomic and cytologic approaches, we show that EXO84b is a subunit of the plant exocyst. Arabidopsis thaliana mutants for EXO84b are severely dwarfed and have compromised leaf epidermal cell and guard cell division. During cytokinesis, green fluorescent protein–tagged exocyst subunits SEC6, SEC8, SEC15b, EXO70A1, and EXO84b exhibit distinctive localization maxima at cell plate initiation and cell plate maturation, stages with a high demand for vesicle fusion. Finally, we present data indicating a defect in cell plate assembly in the exo70A1 mutant. We conclude that the exocyst complex is involved in secretory processes during cytokinesis in Arabidopsis cells, notably in cell plate initiation, cell plate maturation, and formation of new primary cell wall.

Conserved Subgroups and Developmental Regulation in the Monocot rop Gene Family

Rop small GTPases are plant-specific signaling proteins with roles in pollen and vegetative cell growth, abscisic acid signal transduction, stress responses, and pathogen resistance. We have characterized the rop family in the monocots maize (Zea mays) and rice (Oryza sativa). The maize genome contains at least nine expressed rops, and the fully sequenced rice genome has seven. Based on phylogenetic analyses of all available Rops, the family can be subdivided into four groups that predate the divergence of monocots and dicots; at least three have been maintained in both lineages. However, the Rop family has evolved differently in the two lineages, with each exhibiting apparent expansion in different groups. These analyses, together with genetic mapping and identification of conserved non-coding sequences, predict orthology for specific rice and maize rops. We also identified consensus protein sequence elements specific to each Rop group. A survey of ROP-mRNA expression in maize, based on multiplex reverse transcriptase-polymerase chain reaction and a massively parallel signature sequencing database, showed significant spatial and temporal overlap of the nine transcripts, with high levels of all nine in tissues in which cells are actively dividing and expanding. However, only a subset of rops was highly expressed in mature leaves and pollen. Intriguingly, the grouping of maize rops based on hierarchical clustering of expression profiles was remarkably similar to that obtained by phylogenetic analysis. We hypothesize that the Rop groups represent classes with distinct functions, which are specified by the unique protein sequence elements in each group and by their distinct expression patterns.

The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis

In land plants polar auxin transport is one of the substantial processes guiding whole plant polarity and morphogenesis. Directional auxin fluxes are mediated by PIN auxin efflux carriers, polarly localized at the plasma membrane. The polarization of exocytosis in yeast and animals is assisted by the exocyst: an octameric vesicle-tethering complex and an effector of Rab and Rho GTPases. Here we show that rootward polar auxin transport is compromised in roots of Arabidopsis thaliana loss-of-function mutants in the EXO70A1 exocyst subunit. The recycling of PIN1 and PIN2 proteins from brefeldin-A compartments is delayed after the brefeldin-A washout in exo70A1 and sec8 exocyst mutants. Relocalization of PIN1 and PIN2 proteins after prolonged brefeldin-A treatment is largely impaired in these mutants. At the same time, however, plasma membrane localization of GFP:EXO70A1, and the other exocyst subunits studied (GFP:SEC8 and YFP:SEC10), is resistant to brefeldin-A treatment. In root cells of the exo70A1 mutant, a portion of PIN2 is internalized and retained in specific, abnormally enlarged, endomembrane compartments that are distinct from VHA-a1-labelled early endosomes or the trans-Golgi network, but are RAB-A5d positive. We conclude that the exocyst is involved in PIN1 and PIN2 recycling, and thus in polar auxin transport regulation.

Arabidopsis exocyst subunits SEC8 and EXO70A1 and exocyst interactor ROH1 are involved in the localized deposition of seed coat pectin

• Polarized deposition of cell wall pectins is a key process in Arabidopsis thaliana myxospermous seed coat development. The exocyst, an octameric secretory vesicle tethering complex, has recently been shown to be involved in the regulation of cell polarity in plants. Here, we used the Arabidopsis seed coat to study the participation of the exocyst complex in polarized pectin delivery. • We characterized the amount of pectinaceous mucilage and seed coat structure in sec8 and exo70A1 exocyst mutants. Using a yeast two-hybrid screen, we identified a new interactor of the exocyst subunit Exo70A1, termed Roh1, a member of the DUF793 protein family. • T-DNA insertions in SEC8, EXO70A1 caused considerable deviations from normal seed coat development, in particular reduced pectin deposition and defects in the formation of the central columella of seed epidermal cells. A gain-of-function mutation of ROH1 also caused reduced pectin deposition. Interestingly, we observed a systematic difference in seed coat development between primary and secondary inflorescences in wild-type plants: siliques from secondary branches produced seeds with thicker seed coats. • The participation of exocyst subunits in mucilage deposition provides direct evidence for the role of the exocyst in polarized cell wall morphogenesis.

ROP GTPases Act with the Receptor-Like Protein PAN1 to Polarize Asymmetric Cell Division in Maize

This study demonstrates a role for Rho family GTPases (ROPs) in asymmetric cell division in maize. Functional and localization studies together with analysis of physical interactions demonstrate that ROPs function cooperatively with the receptor-like protein PAN1 to promote the premitotic polarization of subsidiary mother cells during stomatal complex development. Plant Rho family GTPases (ROPs) have been investigated primarily for their functions in polarized cell growth. We previously showed that the maize (Zea mays) Leu-rich repeat receptor-like protein PANGLOSS1 (PAN1) promotes the polarization of asymmetric subsidiary mother cell (SMC) divisions during stomatal development. Here, we show that maize Type I ROPs 2 and 9 function together with PAN1 in this process. Partial loss of ROP2/9 function causes a weak SMC division polarity phenotype and strongly enhances this phenotype in pan1 mutants. Like PAN1, ROPs accumulate in an asymmetric manner in SMCs. Overexpression of yellow fluorescent protein-ROP2 is associated with its delocalization in SMCs and with aberrantly oriented SMC divisions. Polarized localization of ROPs depends on PAN1, but PAN1 localization is insensitive to depletion and depolarization of ROP. Membrane-associated Type I ROPs display increased nonionic detergent solubility in pan1 mutants, suggesting a role for PAN1 in membrane partitioning of ROPs. Finally, endogenous PAN1 and ROP proteins are physically associated with each other in maize tissue extracts, as demonstrated by reciprocal coimmunoprecipitation experiments. This study demonstrates that ROPs play a key role in polarization of plant cell division and cell growth and reveals a role for a receptor-like protein in spatial localization of ROPs.

Arabidopsis Myosin XI-K Localizes to the Motile Endomembrane Vesicles Associated with F-actin

Plant myosins XI were implicated in cell growth, F-actin organization, and organelle transport, with myosin XI-K being a critical contributor to each of these processes. However, subcellular localization of myosins and the identity of their principal cargoes remain poorly understood. Here, we generated a functionally competent, fluorescent protein-tagged, myosin XI-K, and investigated its spatial distribution within Arabidopsis cells. This myosin was found to associate primarily not with larger organelles (e.g., Golgi) as was broadly assumed, but with endomembrane vesicles trafficking along F-actin. Subcellular localization and fractionation experiments indicated that the nature of myosin-associated vesicles is organ- and cell type-specific. In leaves, a large proportion of these vesicles aligned and co-fractionated with a motile endoplasmic reticulum (ER) subdomain. In roots, non-ER vesicles were a dominant myosin cargo. Myosin XI-K showed a striking polar localization at the tips of growing, but not mature, root hairs. These results strongly suggest that a major mechanism whereby myosins contribute to plant cell physiology is vesicle transport, and that this activity can be regulated depending on the growth phase of a cell.

Discovery of novel transcripts and gametophytic functions via RNA-seq analysis of maize gametophytic transcriptomes

BackgroundPlant gametophytes play central roles in sexual reproduction. A hallmark of the plant life cycle is that gene expression is required in the haploid gametophytes. Consequently, many mutant phenotypes are expressed in this phase.ResultsWe perform a quantitative RNA-seq analysis of embryo sacs, comparator ovules with the embryo sacs removed, mature pollen, and seedlings to assist the identification of gametophyte functions in maize. Expression levels were determined for annotated genes in both gametophytes, and novel transcripts were identified from de novo assembly of RNA-seq reads. Transposon-related transcripts are present in high levels in both gametophytes, suggesting a connection between gamete production and transposon expression in maize not previously identified in any female gametophytes. Two classes of small signaling proteins and several transcription factor gene families are enriched in gametophyte transcriptomes. Expression patterns of maize genes with duplicates in subgenome 1 and subgenome 2 indicate that pollen-expressed genes in subgenome 2 are retained at a higher rate than subgenome 2 genes with other expression patterns. Analysis of available insertion mutant collections shows a statistically significant deficit in insertions in gametophyte-expressed genes.ConclusionsThis analysis, the first RNA-seq study to compare both gametophytes in a monocot, identifies maize gametophyte functions, gametophyte expression of transposon-related sequences, and unannotated, novel transcripts. Reduced recovery of mutations in gametophyte-expressed genes is supporting evidence for their function in the gametophytes. Expression patterns of extant, duplicated maize genes reveals that selective pressures based on male gametophytic function have likely had a disproportionate effect on plant genomes.

Localization to the rhizoid tip implicates a Fucus distichus Rho family GTPase in a conserved cell polarity pathway

Generation and expression of cell polarity in brown algal zygotes of the Fucales involve regulation of the actin cytoskeleton and localized secretion. We used degenerate PCR to isolate cDNAs that encode two small GTPases, FdRac1 and FdRab8, from zygotes of Fucus distichus (L.) Powell. Sequence analysis placed FdRac1 in the Rho family, which regulates actin, and FdRab8 in the Rab family, which regulates vesicle transport. As expected, bacterially expressed forms of both proteins bound GTP in vitro. When expressed in budding yeast, FdRac1 showed some functional overlap with CDC42, the Saccharomyces cerevisiae Rho family gene required for yeast cell polarity. Immunolocalization revealed an asymmetric distribution of FdRac1 in polarized zygotes and embryos, with FdRac1 concentrated at or near the growing tip of the algal rhizoid. Our data support the hypothesis that FdRac1 regulates algal cell polarity, possibly via the actin cytoskeleton. Because brown algae belong to the heterokont group, which diverged from other groups early in eukaryotic evolution, we argue that the Rho family function of regulating cell polarity is ancient and may extend throughout the eukaryotes.