The microtubular preprophase band recruits Myosin XI to the cortical division site to guide phragmoplast expansion during plant cytokinesis.

In plant vegetative tissues, cell division employs a mitotic microtubule array called the preprophase band (PPB) that marks the cortical division site. This transient cytoskeletal array imprints the spatial information to be read by the cytokinetic phragmoplast at later stages of mitotic cell division. In Arabidopsis thaliana, we discovered that the PPB recruited the Myosin XI motor MYA1/Myo11F to the cortical division site, where it joined microtubule-associated proteins and motors to form a ring of prominent cytoskeletal assemblies that received the expanding phragmoplast. Such a myosin localization pattern at the cortical division site was dependent on the POK1/2 Kinesin-12 motors. This regulatory function of MYA1/Myo11F in phragmoplast guidance was dependent on intact actin filaments. The discovery of these cytoskeletal motor assemblies pinpoints a mechanism underlying how two dynamic cytoskeletal networks work in concert to govern PPB-dependent division plane orientation in flowering plants.

De novo TANGLED1 recruitment from the phragmoplast to aberrant cell plate fusion sites in maize.

Division plane positioning is crucial for proper growth and development in many organisms. In plants, the division plane is established before mitosis, by accumulation of a cytoskeletal structure called the preprophase band (PPB). The PPB is thought to be essential for recruitment of division site-localized proteins, which remain at the division site after the PPB disassembles. Here, we show that the division site-localized protein TANGLED1 (TAN1) is recruited independently of the PPB to the cell cortex by the plant cytokinetic machinery, the phragmoplast, from experiments using both the PPB-defective mutant discordia1 (dcd1) and chemical treatments that disrupt the phragmoplast in maize. TAN1 recruitment to de novo sites on the cortex is partially dependent on intact actin filaments and the myosin XI motor protein OPAQUE1 (O1). These data imply a yet unknown role for TAN1 and possibly other division site-localized proteins during the last stages of cell division when the phragmoplast touches the cell cortex to complete cytokinesis.

Spatiotemporal regulation of plant cell division.

Plant morphogenesis largely depends on the orientation and rate of cell division and elongation, and their coordination at all levels of organization. Despite recent progresses in the comprehension of pathways controlling division plane determination in plant cells, many pieces are missing to the puzzle. For example, we have a partial comprehension of formation, function and evolutionary significance of the preprophase band, a plant-specific cytoskeletal array involved in premitotic setup of the division plane, as well as the role of the nucleus and its connection to the preprophase band of microtubules. Likewise, several modeling studies point to a strong relationship between cell shape and division geometry, but the emergence of such geometric rules from the molecular and cellular pathways at play are still obscure. Yet, recent imaging technologies and genetic tools hold a lot of promise to tackle these challenges and to revisit old questions with unprecedented resolution in space and time.

De Novo TANGLED1 Recruitment to Aberrant Cell Plate Fusion Sites in Maize.

Division plane positioning is critical for proper growth and development in many organisms. In plants, the division plane is established before mitosis, by accumulation of a cytoskeletal structure called the preprophase band (PPB). The PPB is thought to be essential for recruitment of division site localized proteins, which remain at the division site after the PPB disassembles. Here, we show that a division site localized protein, TANGLED1 (TAN1), is recruited independently of the PPB to the cell cortex at sites, by the plant cytokinetic machinery, the phragmoplast. TAN1 recruitment to de novo sites on the cortex is partially dependent on intact actin filaments and the myosin XI motor protein OPAQUE1 (O1). These data imply a yet unknown role for TAN1 and possibly other division site localized proteins during the last stages of cell division when the phragmoplast touches the cell cortex to complete cytokinesis.

The making of a ring: Assembly and regulation of microtubule-associated proteins during preprophase band formation and division plane set-up.

The preprophase band (PPB) is a transient cytokinetic structure that marks the future division plane at the onset of mitosis. The PPB forms a dense cortical ring of mainly microtubules, actin filaments, endoplasmic reticulum, and associated proteins that encircles the nucleus of mitotic cells. After PPB disassembly, the positional information is preserved by the cortical division zone (CDZ). The formation of the PPB and its contribution to timely CDZ set-up involves activities of functionally distinct microtubule-associated proteins (MAPs) that interact physically and genetically to support robust division plane orientation in plants. Recent studies identified two types of plant-specific MAPs as key regulators of PPB formation, the TON1 RECRUITMENT MOTIF (TRM) and IQ67 DOMAIN (IQD) families. Both families share hallmarks of disordered scaffold proteins. Interactions of IQDs and TRMs with multiple binding partners, including the microtubule severing KATANIN1, may provide a molecular framework to coordinate PPB formation, maturation, and disassembly.

Natural variation of ZmLNG1 alters organ shapes in maize.

The development of a series of elite maize hybrids has greatly increased crop yield in the past decades. Parental lines of these hybrids usually come from different heterotic groups and contain many genetic differences. Identifications of important quantitative trait genes in the elite hybrids can extend our understanding of heterosis and also help to guide genetic improvement. Here, we mapped a major quantitative trait locus using a linkage population from an elite maize hybrid Zhengdan958 and identified ZmLNG1 as the causative gene controlling multiple morphologic traits in maize. A 6-kb deletion in one parental line of the hybrid leads to the fusion of ZmLNG1 with its nearby gene. The fusion event prevents the C-terminal of ZmLNG1 from interacting with ZmTON1, which resulted in the change of plant architecture. Further experiments demonstrated that ZmLNG1 could act as a mediator to connect ZmTON1 and ZmOFPs, which belong to another type of plant morphological regulatory proteins, thereby affecting the phosphorylation level of ZmOFPs. These results demonstrate the importance of ZmLNG1 in forming the TON1-TRM-PP2A complex and provide a model for the regulation of plant organ morphology by TON1-recruiting motifs (TRMs) and Ovate family proteins (OFPs).

ROP Interactive Partners are Involved in the Control of Cell Division Patterns in Arabidopsis Leaves.

Animal Rho GTP-binding proteins and their plant counterparts, Rho of plants (ROPs), regulate cell polarity, but they do so through different effector proteins. A class of ROP effectors, interactor of constitutive active ROPs (ICRs)/ROP interactive partners (RIPs), has been implicated in diverse biological processes; however, there are limited analyses of RIP loss-of-function mutants. Here, we report an analysis of the functions of the Arabidopsis thaliana RIPs in the leaf epidermis. Green Fluorescent Protein (GFP) fusion proteins of all the RIPs colocalized to cortical microtubules. RIP1, RIP3 and RIP4, but not RIP2 and RIP5, colocalized with the preprophase band (PPB), spindles and phragmoplasts. RIP2 and RIP5 did not colocalize with the PPB, spindles or phragmoplasts even when they were expressed under a promoter active in proliferative cells, indicating that there are differences among RIP protein properties. The overexpression of RIP1 or RIP4 resulted in the fragmentation of cortical microtubules, and the rip1 2 3 4 5 quintuple mutant showed increased growth rate of microtubules at their plus ends compared with the wild type. The rip1 2 3 4 5 mutant leaves and petals were narrow, which was explained by the decreased cell number along the transverse axis compared with that of the wild type. The rip1 2 3 4 5 mutant leaf epidermis possessed fewer PPBs oriented close to the long axis of the leaf compared with wild type, indicating the involvement of RIPs in cell division plane regulation and leaf shape determination.

De-Esterified Homogalacturonan Enrichment of the Cell Wall Region Adjoining the Preprophase Cortical Cytoplasmic Zone in Some Protodermal Cell Types of Three Land Plants.

The distribution of highly de-esterified homogalacturonans (HGs) in dividing protodermal cells of the monocotyledon Zea mays, the dicotyledon Vigna sinensis, and the fern Asplenium nidus was investigated in order to examine whether the cell wall region adjoining the preprophase band (PPB) is locally diversified. Application of immunofluorescence revealed that de-esterified HGs were accumulated selectively in the cell wall adjacent to the PPB in: (a) symmetrically dividing cells of stomatal rows of Z. mays, (b) the asymmetrically dividing protodermal cells of Z. mays, (c) the symmetrically dividing guard cell mother cells (GMCs) of Z. mays and V. sinensis, and (d) the symmetrically dividing protodermal cells of A. nidus. A common feature of the above cell types is that the cell division plane is defined by extrinsic cues. The presented data suggest that the PPB cortical zone-plasmalemma and the adjacent cell wall region function in a coordinated fashion in the determination/accomplishment of the cell division plane, behaving as a continuum. The de-esterified HGs, among other possible functions, might be involved in the perception and the transduction of the extrinsic cues determining cell division plane in the examined cells.

Division Plane Establishment and Cytokinesis.

Plant cells divide their cytoplasmic content by forming a new membrane compartment, the cell plate, via a rerouting of the secretory pathway toward the division plane aided by a dynamic cytoskeletal apparatus known as the phragmoplast. The phragmoplast expands centrifugally and directs the cell plate to the preselected division site at the plasma membrane to fuse with the parental wall. The division site is transiently decorated by the cytoskeletal preprophase band in preprophase and prophase, whereas a number of proteins discovered over the last decade reside continuously at the division site and provide a lasting spatial reference for phragmoplast guidance. Recent studies of membrane fusion at the cell plate have revealed the contribution of functionally conserved eukaryotic proteins to distinct stages of cell plate biogenesis and emphasize the coupling of cell plate formation with phragmoplast expansion. Together with novel findings concerning preprophase band function and the setup of the division site, cytokinesis and its spatial control remain an open-ended field with outstanding and challenging questions to resolve.

Preprophase-band positioning in isolated tobacco BY-2 cells: evidence for a principal role of nucleus-cell cortex interaction in default division-plane selection.

In some plant tissue types, new cross-walls tend to divide parental cells equally and to meet parental walls at right angles while tending to have minimal surface area. A previously proposed model that I call the reach model suggests that this feature originates from the tendency of premitotic division-plane selection or of the positioning of microtubule preprophase bands (PPBs) which predict the cortical division site, and that default division-plane selection involves nuclear centering and subsequent PPB microtubule assembly on the cell wall parts closest to the nucleus. In an initial effort to characterize truly default division-plane selection, the present study quantified division orientation and PPB positioning in protoplast-derived isolated elongate tobacco BY-2 cells. In this system, PPB-predicted and actual division planes were mostly oriented transversely, as predicted based on the reach model. Some sample elongate cells had asymmetric shapes that came from clear terminal-size differences and, in those cells, PPB-marked planes tended to be displaced from the centers of centrally located nuclei toward the narrower cell end, again as predicted based on the reach model. Such PPB positioning typically forecasted volumetrically asymmetric transverse division that would produce a smaller daughter cell from a parental cell part including the narrower cell end. These results provide experimental evidence that default division-plane selection tends to be close to or the same as the selection using the reach model's criterion, and that it does not use any criterion that specifically prioritizes the equality or verticality of division.

Dual localized kinesin-12 POK2 plays multiple roles during cell division and interacts with MAP65-3.

Kinesins are versatile nano-machines that utilize variable non-motor domains to tune specific motor microtubule encounters. During plant cytokinesis, the kinesin-12 orthologs, PHRAGMOPLAST ORIENTING KINESIN (POK)1 and POK2, are essential for rapid centrifugal expansion of the cytokinetic apparatus, the phragmoplast, toward a pre-selected cell plate fusion site at the cell cortex. Here, we report on the spatio-temporal localization pattern of POK2, mediated by distinct protein domains. Functional dissection of POK2 domains revealed the association of POK2 with the site of the future cell division plane and with the phragmoplast during cytokinesis. Accumulation of POK2 at the phragmoplast midzone depends on its functional POK2 motor domain and is fine-tuned by its carboxy-terminal region that also directs POK2 to the division site. Furthermore, POK2 likely stabilizes the phragmoplast midzone via interaction with the conserved microtubule-associated protein MAP65-3/PLEIADE, a well-established microtubule cross-linker. Collectively, our results suggest that dual localized POK2 plays multiple roles during plant cell division.

Advanced microscopy methods for bioimaging of mitotic microtubules in plants.

Mitotic cell division in plants is a dynamic process playing a key role in plant morphogenesis, growth, and development. Since progress of mitosis is highly sensitive to external stresses, documentation of mitotic cell division in living plants requires fast and gentle live-cell imaging microscopy methods and suitable sample preparation procedures. This chapter describes, both theoretically and practically, currently used advanced microscopy methods for the live-cell visualization of the entire process of plant mitosis. These methods include microscopy modalities based on spinning disk, Airyscan confocal laser scanning, structured illumination, and light-sheet bioimaging of tissues or whole plant organs with diverse spatiotemporal resolution. Examples are provided from studies of mitotic cell division using microtubule molecular markers in the model plant Arabidopsis thaliana, and from deep imaging of mitotic microtubules in robust plant samples, such as legume crop species Medicago sativa.

An overview of plant division-plane orientation.

Contents Summary 505 I. Introduction 505 II. Models of plant cell division 505 III. Establishing the division plane 506 IV. Maintaining the division plane during mitosis and cytokinesis 509 Acknowledgements 510 References 510 SUMMARY: Plants, a significant source of planet-wide biomass, have an unique type of cell division in which a new cell wall is constructed de novo inside the cell and guided towards the cell edge to complete division. The elegant control over positioning this new cell wall is essential for proper patterning and development. Plant cells, lacking migration, tightly coordinate division orientation and directed expansion to generate organized shapes. Several emerging lines of evidence suggest that the proteins required for division-plane establishment are distinct from those required for division-plane maintenance. We discuss recent shape-based computational models and mutant analyses that raise questions about, and identify unexpected connections between, the roles of well-known proteins and structures during division-plane orientation.

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.

Katanin Effects on Dynamics of Cortical Microtubules and Mitotic Arrays in Arabidopsis thaliana Revealed by Advanced Live-Cell Imaging.

Katanin is the only microtubule severing protein identified in plants so far. Previous studies have documented its role in regulating cortical microtubule organization during cell growth and morphogenesis. Although, some cell division defects are reported in KATANIN mutants, it is not clear whether or how katanin activity may affect microtubule dynamics in interphase cells, as well as the progression of mitosis and cytokinesis and the orientation of cell division plane (CDP). For this reason, we characterized microtubule organization and dynamics in growing and dividing cotyledon cells of Arabidopsis ktn1-2 mutant devoid of KATANIN 1 activity. In interphase epidermal cells of ktn1-2 cortical microtubules exhibited aberrant and largely isotropic organization, reduced bundling and showed excessive branched microtubule formation. End-wise microtubule dynamics were not much affected, although a significantly slower rate of microtubule growth was measured in the ktn1-2 mutant where microtubule severing was completely abolished. KATANIN 1 depletion also brought about significant changes in preprophase microtubule band (PPB) organization and dynamics. In this case, many PPBs exhibited unisided organization and splayed appearance while in most cases they were broader than those of wild type cells. By recording PPB maturation, it was observed that PPBs in the mutant narrowed at a much slower pace compared to those in Col-0. The form of the mitotic spindle and the phragmoplast was not much affected in ktn1-2, however, the dynamics of both processes showed significant differences compared to wild type. In general, both mitosis and cytokinesis were considerably delayed in the mutant. Additionally, the mitotic spindle and the phragmoplast exhibited extensive rotational motions with the equatorial plane of the spindle being essentially uncoupled from the division plane set by the PPB. However, at the onset of its formation the phragmoplast undergoes rotational motion rectifying the expansion of the cell plate to match the original cell division plane. Conclusively, KATANIN 1 contributes to microtubule dynamics during interphase, regulates PPB formation and maturation and is involved in the positioning of the mitotic spindle and the phragmoplast.

The Evolution of Cell Division: From Streptophyte Algae to Land Plants.

The mechanism of cell division has undergone significant alterations during the evolution from aquatic streptophyte algae to land plants. Two new structures evolved, the cytokinetic phragmoplast and the preprophase band (PPB) of microtubules, whereas the ancestral mechanism of cleavage and the centrosomes disappeared. We map cell biological data onto the recently emerged phylogenetic tree of streptophytes. The tree suggests that, after the establishment of the phragmoplast mechanism, several groups independently lost their centrosomes. Surprisingly, the phragmoplast shows reductions in the Zygnematophyceae (the sister to land plants), many of which returned to cleavage. The PPB by contrast evolved stepwise and, most likely, originated in the algae. The phragmoplast/PPB mechanism established in this way served as a basis for the 3D development of land plants.

Control of patterns of symmetric cell division in the epidermal and cortical tissues of the Arabidopsis root.

Controlled cell division is central to the growth and development of all multicellular organisms. Within the proliferating zone of the Arabidopsis root, regular symmetric divisions give rise to patterns of parallel files of cells, the genetic basis of which remains unclear. We found that genotypes impaired in the TONNEAU1a (TON1a) gene display misoriented symmetric divisions in the epidermis and have no division defects in the underlying cortical tissue. The TON1a gene encodes a microtubule-associated protein. We show that in the ton1a mutant, epidermal and cortical cells do not form narrow, ring-like preprophase bands (PPBs), which are plant-specific, cytoskeletal structures that predict the position of the division plane before mitosis. The results indicate that in the cortex but not in the epidermis, division plane positioning and patterning can proceed correctly in the absence of both a functional TON1a and PPB formation. Differences between tissues in how they respond to the signals that guide symmetric division orientation during patterning might provide the basis for organised organ growth in the absence of cell movements.

Plant cytokinesis-No ring, no constriction but centrifugal construction of the partitioning membrane.

Plants have evolved a unique way of partitioning the cytoplasm of dividing cells: Instead of forming a contractile ring that constricts the plasma membrane, plant cells target membrane vesicles to the plane of division where the vesicles fuse with one another to form the partitioning membrane. Plant cytokinesis starts in the centre and progresses towards the periphery, culminating in the fusion of the partitioning membrane with the parental plasma membrane. This membrane dynamics is orchestrated by a specific cytoskeletal array named phragmoplast that originates from interzone spindle remnants. Here we review the properties of the process as well as molecules that play specific roles in that process.

Microtubule dynamics of the centrosome-like polar organizers from the basal land plant Marchantia polymorpha.

The liverwort Marchantia employs both modern and ancestral devices during cell division: it forms preprophase bands and in addition it shows centrosome-like polar organizers. We investigated whether polar organizers and preprophase bands cooperate to set up the division plane. To this end, two novel green fluorescent protein-based microtubule markers for dividing cells of Marchantia were developed. Cells of the apical notch formed polar organizers first and subsequently assembled preprophase bands. Polar organizers were formed de novo from multiple mobile microtubule foci localizing to the nuclear envelope. The foci then became concentrated by bipolar aggregation. We determined the comet production rate of polar organizers and show that microtubule plus ends of astral microtubules polymerize faster than those found on cortical microtubules. Importantly, it was observed that conditions increasing polar organizer numbers interfere with preprophase band formation. The data show that polar organizers have much in common with centrosomes, but that they also have specialized features. The results suggest that polar organizers contribute to preprophase band formation and in this way are involved in controlling the division plane. Our analyses of the basal land plant Marchantia shed new light on the evolution of plant cell division.