Ulrike Mayer

Cytokinesis in the Arabidopsis Embryo Involves the Syntaxin-Related KNOLLE Gene Product

The embryo of the flowering plant Arabidopsis develops by a regular pattern of cell divisions and cell shape changes. Mutations in the KNOLLE (KN) gene affect the rate and plane of cell divisions as well as cell morphology, resulting in mutant seedlings with a disturbed radial organization of tissue layers. At the cellular level, mutant embryos are characterized by incomplete cross walls and enlarged cells with polyploid nuclei. The KN gene was isolated by positional cloning. The predicted KN protein has similarity to syntaxins, a protein family involved in vesicular trafficking. During embryogenesis, KN transcripts are detected in patches of single cells or small cell groups. Our results suggest a function for KN in cytokinesis.

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.

Plant Cytokinesis Requires De Novo Secretory Trafficking but Not Endocytosis

During plant cytokinesis membrane vesicles are efficiently delivered to the cell-division plane, where they fuse with one another to form a laterally expanding cell plate. These membrane vesicles were generally believed to originate from Golgi stacks. Recently, however, it was proposed that endocytosis contributes substantially to cell-plate formation. To determine the relative contributions of secretory and endocytic traffic to cytokinesis, we specifically inhibited either or both trafficking pathways in Arabidopsis. Blocking traffic to the division plane after the two pathways had converged at the trans-Golgi network disrupted cytokinesis and resulted in binucleate cells, whereas impairment of endocytosis alone did not interfere with cytokinesis. By contrast, inhibiting ER-Golgi traffic by eliminating the relevant BFA-resistant ARF-GEF caused retention of newly synthesized proteins, such as the cytokinesis-specific syntaxin KNOLLE in the ER, and prevented the formation of the partitioning membrane. Our results suggest that during plant cytokinesis, unlike animal cytokinesis, protein secretion is absolutely essential, whereas endocytosis is not.

Molecular analysis of the Arabidopsis pattern formation of gene GNOM : gene structure and intragenic complementation

TheGNOM gene is required for pattern formation along the main body axis of the embryo in the flowering plantArabidopsis thaliana. Mutations in theGNOM gene alter the asymmetric division of the zygote and interfere with the formation of distinct apical-basal regions in the developing embryo. We have isolated theGNOM gene by positional cloning, characterised its structure and determined the molecular lesions in mutant alleles. Although the predicted 163 kDa GNOM protein has a conserved domain in common with the yeast secretory protein Sec7p, it is most closely related in size and overall similarity to the product of the yeastYEC2 gene, which is not essential for cell viability. Four fully complementinggnom alleles carry missense mutations in conserved regions, seven partially complementing alleles have premature stop codon mutations and two non-complementing alleles have splice-site lesions. Our results suggest that the GNOM protein acts as a complex of identical subunits and that partial complementation may involve low levels of full-length protein generated by inefficient translational read-through.

Embryogenesis - the humble beginnings of plant life.

Each plant starts life from the zygote formed by the fusion of an egg and a sperm cell. The zygote gives rise to a multicellular embryo that displays a basic plant body organization and is surrounded by nutritive endosperm and maternal tissue. How the body organization is generated had already been studied before the genome sequence of Arabidopsis thaliana was completed 10 years ago, but several regulatory mechanisms of embryo development have since been discovered or analysed in more detail. Although this progress did not strictly depend on the availability of the genome sequence itself, several advances were considerably facilitated. In this review, we mainly address early embryo development, highlighting general mechanisms and crucial regulators, including phytohormones, that are involved in patterning the embryo and were mainly analysed in the post-genome decade. We also highlight some unsolved problems, provide a brief outlook on the future of Arabidopsis embryo research, and discuss how the knowledge gained from Arabidopsis could be translated to crop species.

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.

Heat Shock Protein Cognate 70-4 and an E3 Ubiquitin Ligase, CHIP, Mediate Plastid-Destined Precursor Degradation through the Ubiquitin-26S Proteasome System in Arabidopsis

Plastid-targeted proteins pass through the cytosol as unfolded precursors. If proteins accumulate in the cytosol, they can form nonspecific aggregates that cause severe cellular damage. Here, we demonstrate that high levels of plastid precursors are degraded through the ubiquitin-proteasome system (UPS) in Arabidopsis thaliana cells. The cytosolic heat shock protein cognate 70-4 (Hsc70-4) and E3 ligase carboxy terminus of Hsc70-interacting protein (CHIP) were highly induced in plastid protein import2 plants, which had a T-DNA insertion at Toc159 and showed an albino phenotype and a severe defect in protein import into chloroplasts. Hsc70-4 and CHIP together mediated plastid precursor degradation when import-defective chloroplast-targeted reporter proteins were transiently expressed in protoplasts. Hsc70-4 recognized specific sequence motifs in transit peptides and thereby led to precursor degradation through the UPS. CHIP, which interacted with Hsc70-4, functioned as an E3 ligase in the Hsc70-4–mediated protein degradation. The physiological role of Hsc70-4 was confirmed by analyzing Hsc70-4 RNA interfernce plants in an hsc70-1 mutant background. Plants with lower Hsc70 levels exhibited abnormal embryogenesis, resulting in defective seedlings that displayed high levels of reactive oxygen species and monoubiquitinated Lhcb4 precursors. We propose that Hsc70-4 and CHIP mediate plastid-destined precursor degradation to prevent cytosolic precursor accumulation and thereby play a critical role in embryogenesis.

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.

Syntaxin specificity of cytokinesis in Arabidopsis

Syntaxins interact with other SNAREs (soluble NSF-attachment protein receptors) to form structurally related complexes that mediate membrane fusion in diverse intracellular trafficking pathways. The original SNARE hypothesis postulated that each type of transport vesicle has its own distinct vesicle-SNARE that pairs up with a unique target-SNARE, or syntaxin, on the target membrane. However, recent evidence suggests that small G-proteins of the Rab family and their effectors mediate the initial contact between donor and acceptor membranes, providing complementary specificity to SNARE pairing at a later step towards membrane fusion. To assess the role of syntaxin specificity in membrane recognition requires a biological assay in which one syntaxin is replaced by other family members that do not normally function in that trafficking pathway. Here, we examine whether membrane fusion in Arabidopsis thaliana cytokinesis, which involves a plant-specific syntaxin, the cell-cycle-regulated KNOLLE (KN) protein, can be mediated by other syntaxins if expressed under the control of KN cis-regulatory sequences. Only a non-essential syntaxin was targeted to the plane of cell division and sufficiently related to KN to perform its function, thus revealing syntaxin specificity of cytokinesis.

Microtubule cytoskeleton: a track record

The plant microtubule cytoskeleton forms unique arrays during cell division and morphogenesis. Recent studies have addressed the biogenesis, turnover, spatio-temporal organisation and cellular function of microtubules. The results suggest that both conserved eukaryotic mechanisms and plant-specific modifications determine microtubule dynamics and function.