Farhah F. Assaad

The Arabidopsis HINKEL Gene Encodes a Kinesin-Related Protein Involved in Cytokinesis and Is Expressed in a Cell Cycle-Dependent Manner

Plant cytokinesis starts in the center of the division plane, with vesicle fusion generating a new membrane compartment, the cell plate, that subsequently expands laterally by continuous fusion of newly arriving vesicles to its margin. Targeted delivery of vesicles is assisted by the dynamic reorganization of a plant-specific cytoskeletal array, the phragmoplast, from a solid cylinder into an expanding ring-shaped structure. This lateral translocation is brought about by depolymerization of microtubules in the center, giving way to the expanding cell plate, and polymerization of microtubules along the edge. Whereas several components are known to mediate cytokinetic vesicle fusion [8-10], no gene function involved in phragmoplast dynamics has been identified by mutation. Mutations in the Arabidopsis HINKEL gene cause cytokinesis defects, such as enlarged cells with incomplete cell walls and multiple nuclei. Proper targeting of the cytokinesis-specific syntaxin KNOLLE [8] and lateral expansion of the phragmoplast are not affected. However, the phragmoplast microtubules appear to persist in the center, where vesicle fusion should result in cell plate formation. Molecular analysis reveals that the HINKEL gene encodes a plant-specific kinesin-related protein with a putative N-terminal motor domain and is expressed in a cell cycle-dependent manner similar to the KNOLLE gene. Our results suggest that HINKEL plays a role in the reorganization of phragmoplast microtubules during cell plate formation.

The Arabidopsis KNOLLE and KEULE genes interact to promote vesicle fusion during cytokinesis

Partitioning of the cytoplasm during cytokinesis or cellularisation requires syntaxin-mediated membrane fusion [1-3]. Whereas in animals, membrane fusion promotes ingression of a cleavage furrow from the plasma membrane [4,5], somatic cells of higher plants form de novo a transient membrane compartment, the cell plate, which is initiated in the centre of the division plane and matures into a new cell wall and its flanking plasma membranes [6,7]. Cell plate formation results from the fusion of Golgi-derived vesicles delivered by a dynamic cytoskeletal array, the phragmoplast. Mutations in two Arabidopsis genes, KNOLLE (KN) and KEULE (KEU), cause abnormal seedlings with multinucleate cells and incomplete cell walls [1,8]. The KN gene encodes a cytokinesis-specific syntaxin which localises to the cell plate [9]. Here, we show that KN protein localisation is unaffected in keu mutant cells, which, like kn, display phragmoplast microtubules and accumulate ADL1 protein in the plane of cell division but vesicles fail to fuse with one another. Genetic interactions between KN and KEU were analysed in double mutant embryos. Whereas the haploid gametophytes gave rise to functional gametes, the embryos behaved like single cells displaying multiple, synchronously cycling nuclei, cell cycle-dependent microtubule arrays and ADL1 accumulation between pairs of daughter nuclei. This complete inhibition of cytokinesis from fertilisation indicates that KN and KEU, have partially redundant functions and interact specifically in vesicle fusion during cytokinesis of somatic cells.

Closely related receptor complexes differ in their ABA selectivity and sensitivity

The recent discovery of a variety of receptors has led to new models for hormone perception in plants. In the case of the hormone abscisic acid (ABA), which regulates plant responses to abiotic stress, perception seems to occur both at the plasma membrane and in the cytosol. The cytosolic receptors for ABA have recently been identified as complexes between protein phosphatases 2C (PP2C) and regulatory components (RCAR/PYR/PYL) that bind ABA. Binding of ABA to the receptor complexes inactivates the PP2Cs, thereby activating the large variety of physiological processes regulated by ABA. The Arabidopsis genome encodes 13 homologues of RCAR1 and approximately 80 PP2Cs, of which six in clade A have been identified as negative regulators of ABA responses. In this study we characterize a novel member of the RCAR family, RCAR3. RCAR3 was identified in a screen for interactors of the PP2Cs ABI1 and ABI2, which are key regulators of ABA responses. RCAR3 was shown to repress ABI1 and ABI2 in vitro, and to stimulate ABA signalling in protoplast cells. RCAR3 conferred greater ABA sensitivity to the PP2C regulation than RCAR1, whereas stereo-selectivity for (S)-ABA was less stringent with RCAR3 as compared with RCAR1. In addition, regulation of the protein phosphatase activity by RCAR1 and RCAR3 was more sensitive to ABA for ABI1 than for ABI2. Based on the differences we have observed in transcriptional regulation and biochemical properties, we propose a model whereby differential expression of the co-receptors and combinatorial assembly of the receptor complexes act in concert to modulate and fine-tune ABA responses.

The KEULE gene is involved in cytokinesis in Arabidopsis

Abstract We present evidence to show that the KEULE gene of Arabidopsis is involved in cytokinesis. Mutant keule embryos have large multinucleate cells with gapped or incomplete cross walls, as well as cell wall stubs that are very similar to those observed upon caffeine inhibition of cytokinesis in plants. These defects are observed in all populations of dividing cells in the mutant, including calli, but less frequently in mature cells. Cell division appears to be slowed down, and the planes of cell division are often misoriented. In late embryos and seedlings, cross-wall formation usually appears complete, suggesting that the requirement for KEULE during cytokinesis is not absolute. Nonetheless, keule mutants die as seedlings with large polyploid cells. The bloated surface layer of keule seedlings does not uniformly behave like wild-type epidermis, and patches of this layer assume characteristics of the underlying ground tissue. The cytokinesis defect of keule mutants may influence aspects of cellular differentiation.

The timely deposition of callose is essential for cytokinesis in Arabidopsis

The primary plant cell wall is laid down over a brief period of time during cytokinesis. Initially, a membrane network forms at the equator of a dividing cell. The cross-wall is then assembled and remodeled within this membrane compartment. Callose is the predominant luminal component of the nascent cross-wall or cell plate, but is not a component of intact mature cell walls, which are composed primarily of cellulose, pectins and xyloglucans. Widely accepted models postulate that callose comprises a transient, rapid spreading force for the expansion of membrane networks during cytokinesis. In this study, we clone and characterize an Arabidopsis gene, MASSUE/AtGSL8, which encodes a putative callose synthase. massue mutants are seedling-lethal and have a striking cytokinesis-defective phenotype. Callose deposition was delayed in the cell plates of massue mutants. Mutant cells were occasionally bi- or multi-nucleate, with cell-wall stubs, and we frequently observed gaps at the junction between cross-walls and parental cell walls. The results suggest that the timely deposition of callose is essential for the completion of plant cytokinesis. Surprisingly, confocal analysis revealed that the cell-plate membrane compartment forms and expands, seemingly as far as the parental wall, prior to the appearance of callose. We discuss the possibility that callose may be required to establish a lasting connection between the nascent cross-wall and the parental cell wall.

Cytokinesis-defective mutants of Arabidopsis.

We have identified mutations in six previously uncharacterized genes of Arabidopsis, named club,bublina, massue, rod,bloated, and bims, that are required for cytokinesis. The mutants are seedling lethal, have morphological abnormalities, and are characterized by cell wall stubs, gapped walls, and multinucleate cells. In these and other respects, the new mutants are phenotypically similar to knolle,keule, hinkel, and pleiademutants. The mutants display a gradient of stomatal phenotypes, correlating roughly with the severity of their cytokinesis defect. Similarly, the extent to which the different mutant lines were capable of growing in tissue culture correlated well with the severity of the cytokinesis defect. Phenotypic analysis of the novel and previously characterized loci indicated that the secondary consequences of a primary defect in cytokinesis include anomalies in body organization, organ number, and cellular differentiation, as well as organ fusions and perturbations of the nuclear cycle. Two of the 10 loci are required for both cytokinesis and root hair morphogenesis. The results have implications for the identification of novel cytokinesis genes and highlight the mechanistic similarity between cytokinesis and root hair morphogenesis, two processes that result in a rapid deposition of new cell walls via polarized secretion.

Plant Cytokinesis Is Orchestrated by the Sequential Action of the TRAPPII and Exocyst Tethering Complexes

Plant cytokinesis is initiated in a transient membrane compartment, the cell plate, and completed by a process of maturation during which the cell plate becomes a cross wall. How the transition from juvenile to adult stages occurs is poorly understood. In this study, we monitor the Arabidopsis transport protein particle II (TRAPPII) and exocyst tethering complexes throughout cytokinesis. We show that their appearance is predominantly sequential, with brief overlap at the onset and end of cytokinesis. The TRAPPII complex is required for cell plate biogenesis, and the exocyst is required for cell plate maturation. The TRAPPII complex sorts plasma membrane proteins, including exocyst subunits, at the cell plate throughout cytokinesis. We show that the two tethering complexes physically interact and propose that their coordinated action may orchestrate not only plant but also animal cytokinesis.

Tethering Factors Required for Cytokinesis in Arabidopsis

At the end of the cell cycle, the nascent cross wall is laid down within a transient membrane compartment referred to as the cell plate. Tethering factors, which act by capturing vesicles and holding them in the vicinity of their target membranes, are likely to play an important role in the first stages of cell plate assembly. Factors required for cell plate biogenesis, however, remain to be identified. In this study, we used a reverse genetic screen to isolate tethering factors required for cytokinesis in Arabidopsis (Arabidopsis thaliana). We focused on the TRAPPI and TRAPPII (for transport protein particle) tethering complexes, which are thought to be required for the flow of traffic through the Golgi and for trans-Golgi network function, as well as on the GARP complex, thought to be required for the tethering of endocytotic vesicles to the trans-Golgi network. We found weak cytokinesis defects in some TRAPPI mutants and strong cytokinesis defects in all the TRAPPII lines we surveyed. Indeed, four insertion lines at the TRAPPII locus AtTRS120 had canonical cytokinesis-defective seedling-lethal phenotypes, including cell wall stubs and incomplete cross walls. Confocal and electron microscopy showed that in trs120 mutants, vesicles accumulated at the equator of dividing cells yet failed to assemble into a cell plate. This shows that AtTRS120 is required for cell plate biogenesis. In contrast to the TRAPP complexes, we found no conclusive evidence for cytokinesis defects in seven GARP insertion lines. We discuss the implications of these findings for the origin and identity of cell plate membranes.

Of weeds and men : what genomes teach us about plant cell biology

It has generally been assumed that fundamental cellular processes are conserved at the molecular level. Genome comparisons, however, suggest that the molecular mechanisms underlying programmed cell death, defense, adaptation and development may differ considerably between the plant and animal kingdoms. Phylogenetic analyses have revealed a great deal of novelty in the plant genes that are implicated in conserved processes such as transcription, cytoskeletal dynamics and vesicle trafficking. The Arabidopsis genome highlights the highly dynamic and regulated nature of the plant cell, which is fine-tuned to light, water, nutrient availability, temperature, touch and wind.

Plant Cytokinesis: Terminology for Structures and Processes

Plant cytokinesis is orchestrated by a specialized structure, the phragmoplast. The phragmoplast first occurred in representatives of Charophyte algae and then became the main division apparatus in land plants. Major cellular activities, including cytoskeletal dynamics, vesicle trafficking, membrane assembly, and cell wall biosynthesis, cooperate in the phragmoplast under the guidance of a complex signaling network. Furthermore, the phragmoplast combines plant-specific features with the conserved cytokinetic processes of animals, fungi, and protists. As such, the phragmoplast represents a useful system for understanding both plant cell dynamics and the evolution of cytokinesis. We recognize that future research and knowledge transfer into other fields would benefit from standardized terminology. Here, we propose such a lexicon of terminology for specific structures and processes associated with plant cytokinesis.