SUPERMAN Targets PHRAGMOPLAST ORIENTING KINESIN to Negatively Regulate Leaf Cell Division in Poplar.

Plant organs achieve their specific size and shape through the coordination of cell division and cell expansion, processes that are profoundly influenced by environmental cues. Cytokinesis during cell division depends on the position of the cytokinetic wall, but how this process responses to environment fluctuations remains underexplored. Here, we investigated a regulatory module involving C2H2-type zinc finger protein (C2H2-ZFP) in leaf morphology during drought stress. A total of 123 C2H2-ZFP members were identified through a comparative genome survey in Populus alba × P. glandulosa '84K'. Among them, PagSUPa, an orthologous gene of Arabidopsis SUPERMAN, was selected due to its responsiveness to drought stress and was further confirmed to play a role in leaf development. Phenotypic characterization and cellular analysis revealed that PagSUPa fine-tunes the duration of cell proliferation in the adaxial epidermis, thereby influencing leaf morphology by modulating leaf adaxial-abaxial polarity. Additionally, we found that PagSUPa directly suppresses the expression of PHRAGMOPLAST ORIENTING KINESIN1 (PagPOK1) and PagPOK2, genes encoding proteins involved in phragmoplast orientation and position, which results in impaired cytokinesis and cell wall organization. This study provides novel insights into the regulatory network governed by the SUP gene during leaf development, specifically in relation to cell division.

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.

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.

A possible pattern in the evolution of male meiotic cytokinesis in angiosperms.

Evolution of cellular characteristics is a fundamental aspect of evolutionary biology, but knowledge about evolution at the cellular level is very limited. In particular, whether a certain intracellular characteristic evolved in angiosperms, and what significance of such evolution is to angiosperms, if it exists, are important and yet unanswered questions. We have found that bidirectional cytokinesis occurs or likely occurs in male meiosis in extant basal and near-basal angiosperm lineages, which differs from the unidirectional cytokinesis in male meiosis in monocots and eudicots. This pattern of cytokinesis in angiosperms seems to align with the distribution pattern of angiosperms with the lineages basal to monocots and eudicots living in tropical, subtropical or temperate environments and monocots and eudicots in an expanded range of environments including tropical, subtropical, temperate, subarctic and arctic environments. These two cytokinetic modes seem to result from two phragmoplast types, respectively. A phragmoplast in the bidirectional cytokinesis dynamically associates with the leading edge of a growing cell plate whereas a phragmoplast in the unidirectional cytokinesis is localized to an entire division plane. The large assembly of microtubules in the phragmoplast in unidirectional cytokinesis may be indicative of increased microtubule stability compared with that of the small microtubule assembly in the phragmoplast in bidirectional cytokinesis. Microtubules could conceivably increase their stability from evolutionary changes in tubulins and/or microtubule-associated proteins. Microtubules are very sensitive to low temperatures, which should be a reason for plants to be sensitive to low temperatures. If monocots and eudicots have more stable microtubules than other angiosperms, they will be expected to deal with low temperatures better than other angiosperms. Future investigations into the male meiotic cytokinetic directions, microtubule stability at low temperatures, and proteins affecting microtubule stability in more species may shed light on how plants evolved to inhabit cold environments.

Spatially resolved proteomics of the Arabidopsis stomatal lineage identifies polarity complexes for cell divisions and stomatal pores.

Cell polarity is used to guide asymmetric divisions and create morphologically diverse cells. We find that two oppositely oriented cortical polarity domains present during the asymmetric divisions in the Arabidopsis stomatal lineage are reconfigured into polar domains marking ventral (pore-forming) and outward-facing domains of maturing stomatal guard cells. Proteins that define these opposing polarity domains were used as baits in miniTurboID-based proximity labeling. Among differentially enriched proteins, we find kinases, putative microtubule-interacting proteins, and polar SOSEKIs with their effector ANGUSTIFOLIA. Using AI-facilitated protein structure prediction models, we identify potential protein-protein interaction interfaces among them. Functional and localization analyses of the polarity protein OPL2 and its putative interaction partners suggest a positive interaction with mitotic microtubules and a role in cytokinesis. This combination of proteomics and structural modeling with live-cell imaging provides insights into how polarity is rewired in different cell types and cell-cycle stages.

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.

Localized calcium transients in phragmoplast regulate cytokinesis of tobacco BY-2 cells.

KEY MESSAGE: Plants exhibit a unique pattern of cytosolic Ca2+ dynamics to correlate with microtubules to regulate cytokinesis, which significantly differs from those observed in animal and yeast cells. Calcium (Ca2+) transients mediated signaling is known to be essential in cytokinesis across eukaryotic cells. However, the detailed spatiotemporal dynamics of Ca2+ during plant cytokinesis remain largely unexplored. In this study, we employed GCaMP5, a genetically encoded Ca2+ sensor, to investigate cytokinetic Ca2+ transients during cytokinesis in Nicotiana tabacum Bright Yellow-2 (BY-2) cells. We validated the effectiveness of GCaMP5 to capture fluctuations in intracellular free Ca2+ in transgenic BY-2 cells. Our results reveal that Ca2+ dynamics during BY-2 cell cytokinesis are distinctly different from those observed in embryonic and yeast cells. It is characterized by an initial significant Ca2+ spike within the phragmoplast region. This spike is followed by a decrease in Ca2+ concentration at the onset of cytokinesis in phragmoplast, which then remains elevated in comparison to the cytosolic Ca2+ until the completion of cell plate formation. At the end of cytokinesis, Ca2+ becomes uniformly distributed in the cytosol. This pattern contrasts with the typical dual waves of Ca2+ spikes observed during cytokinesis in animal embryonic cells and fission yeasts. Furthermore, applications of pharmaceutical inhibitors for either Ca2+ or microtubules revealed a close correlation between Ca2+ transients and microtubule organization in the regulation of cytokinesis. Collectively, our findings highlight the unique dynamics and crucial role of Ca2+ transients during plant cell cytokinesis, and provides new insights into plant cell division mechanisms.

Subcellular positioning during cell division and cell plate formation in maize.

Introduction: During proliferative plant cell division, the new cell wall, called the cell plate, is first built in the middle of the cell and then expands outward to complete cytokinesis. This dynamic process requires coordinated movement and arrangement of the cytoskeleton and organelles. Methods: Here we use live-cell markers to track the dynamic reorganization of microtubules, nuclei, endoplasmic reticulum, and endomembrane compartments during division and the formation of the cell plate in maize leaf epidermal cells. Results: The microtubule plus-end localized protein END BINDING1 (EB1) highlighted increasing microtubule dynamicity during mitosis to support rapid changes in microtubule structures. The localization of the cell-plate specific syntaxin KNOLLE, several RAB-GTPases, as well as two plasma membrane localized proteins was assessed after treatment with the cytokinesis-specific callose-deposition inhibitor Endosidin7 (ES7) and the microtubule-disrupting herbicide chlorpropham (CIPC). While ES7 caused cell plate defects in Arabidopsis thaliana, it did not alter callose accumulation, or disrupt cell plate formation in maize. In contrast, CIPC treatment of maize epidermal cells occasionally produced irregular cell plates that split or fragmented, but did not otherwise disrupt the accumulation of cell-plate localized proteins. Discussion: Together, these markers provide a robust suite of tools to examine subcellular trafficking and organellar organization during mitosis and cell plate formation in maize.

AtMAC stabilizes the phragmoplast by crosslinking microtubules and actin filaments during cytokinesis.

The phragmoplast, a structure crucial for the completion of cytokinesis in plant cells, is composed of antiparallel microtubules (MTs) and actin filaments (AFs). However, how the parallel structure of phragmoplast MTs and AFs is maintained, especially during centrifugal phragmoplast expansion, remains elusive. Here, we analyzed a new Arabidopsis thaliana MT and AF crosslinking protein (AtMAC). When AtMAC was deleted, the phragmoplast showed disintegrity during centrifugal expansion, and the resulting phragmoplast fragmentation led to incomplete cell plates. Overexpression of AtMAC increased the resistance of phragmoplasts to depolymerization and caused the formation of additional phragmoplasts during cytokinesis. Biochemical experiments showed that AtMAC crosslinked MTs and AFs in vitro, and the truncated AtMAC protein, N-CC1, was the key domain controlling the ability of AtMAC. Further analysis showed that N-CC1(51-154) is the key domain for binding MTs, and N-CC1(51-125) for binding AFs. In conclusion, AtMAC is the novel MT and AF crosslinking protein found to be involved in regulation of phragmoplast organization during centrifugal phragmoplast expansion, which is required for complete cytokinesis.

Rice kinesin-related protein STD1 and microtubule-associated protein MAP65-5 cooperatively control microtubule bundling.

MAIN CONCLUSIONS: STD1 specifically interacts with MAP65-5 in rice and they cooperatively control microtubule bundles in phragmoplast expansion during cell division. Microtubules play critical roles during the cell cycle progression in the plant cell. We previously reported that STEMLESS DWARF 1 (STD1), a kinesin-related protein, was localized specifically to the phragmoplast midzone during telophase to regulate the lateral expansion of phragmoplast in rice (Oryza sativa). However, how STD1 regulates microtubule organization remains unknown. Here, we found that STD1 interacted directly with MAP65-5, a member of the microtubule-associated proteins (MAPs). Both STD1 and MAP65-5 could form homodimers and bundle microtubules individually. Compared with MAP65-5, the microtubules bundled by STD1 were disassembled completely into single microtubules after adding ATP. Conversely, the interaction of STD1 with MAP65-5 enhanced the microtubule bundling. These results suggest STD1 and MAP65-5 might cooperatively regulate microtubule organization in the phragmoplast at telophase.

Redundant mechanisms in division plane positioning.

Redundancies in plant cell division contribute to the maintenance of proper division plane orientation. Here we highlight three types of redundancy: 1) Temporal redundancy, or correction of earlier defects that results in proper final positioning, 2) Genetic redundancy, or functional compensation by homologous genes, and 3) Synthetic redundancy, or redundancy within or between pathways that contribute to proper division plane orientation. Understanding the types of redundant mechanisms involved provides insight into current models of division plane orientation and opens up new avenues for exploration.

Rice microtubule-associated protein OsMAP65-3.1, but not OsMAP65-3.2, plays a critical role in phragmoplast microtubule organization in cytokinesis.

In plants, MAP65 preferentially cross-links the anti-parallel microtubules (MTs) and plays an important role for cytokinesis. However, the functions of MAP65 isoforms in rice (Oryza sativa. L) are largely unknown. Here, we identified two MAP65-3 homologs in rice, OsMAP65-3.1 and OsMAP65-3.2. We found that both OsMAP65-3.1 and OsMAP65-3.2 were similar in dimerization and location to AtMAP65-3, and the expression of either rice genes driven by the AtMAP65-3 promoter suppressed the cytokinesis failure and growth defect of atmap65-3. However, OsMAP65-3.1 with native promoter also recovered the atmap65-3, but OsMAP65-3.2 with its own promoter had no effects. OsMAP65-3.1 but not OsMAP65-3.2 was actively expressed in tissues enriched with dividing cells. R1R2R3-Myb (MYB3R) transcription factors directly bound to the OsMAP65-3.1 promoter but not that of OsMAP65-3.2. Furthermore, osmap65-3.2 had no obvious phenotype, while either osmap65-3.1 or osmap65-3.1(+/-) was lethal. The eminent MTs around the daughter nuclei and cytokinesis defects were frequently observed in OsMAP65-3.1-defective plants. Taken together, our findings suggest that OsMAP65-3.1, rather than OsMAP65-3.2, plays essential roles in rice cytokinesis resulting from their differential expression which were passably directly regulated by OsMYB3Rs.

Defects in division plane positioning in the root meristematic zone affect cell organization in the differentiation zone.

Cell-division-plane orientation is critical for plant and animal development and growth. TANGLED1 (TAN1) and AUXIN-INDUCED IN ROOT CULTURES 9 (AIR9) are division-site-localized microtubule-binding proteins required for division-plane positioning. The single mutants tan1 and air9 of Arabidopsis thaliana have minor or no noticeable phenotypes, but the tan1 air9 double mutant has synthetic phenotypes including stunted growth, misoriented divisions and aberrant cell-file rotation in the root differentiation zone. These data suggest that TAN1 plays a role in non-dividing cells. To determine whether TAN1 is required in elongating and differentiating cells in the tan1 air9 double mutant, we limited its expression to actively dividing cells using the G2/M-specific promoter of the syntaxin KNOLLE (pKN:TAN1-YFP). Unexpectedly, in addition to rescuing division-plane defects, expression of pKN:TAN1-YFP rescued root growth and cell file rotation defects in the root-differentiation zone in tan1 air9 double mutants. This suggests that defects that occur in the meristematic zone later affect the organization of elongating and differentiating cells.

Plant cytokinesis and the construction of new cell wall.

Cytokinesis in plants is fundamentally different from that in animals and fungi. In plant cells, a cell plate forms through the fusion of cytokinetic vesicles and then develops into the new cell wall, partitioning the cytoplasm of the dividing cell. The formation of the cell plate entails multiple stages that involve highly orchestrated vesicle accumulation, fusion and membrane maturation, which occur concurrently with the timely deposition of polysaccharides such as callose, cellulose and cross-linking glycans. This review summarizes the major stages in cytokinesis, endomembrane components involved in cell plate assembly and its transition to a new cell wall. An animation that can be widely used for educational purposes further summarizes the process.

A Quantitative Method for Evaluating Phragmoplast Morphology.

Phragmoplasts are plant-specific microtubule structures that form cell plates at the cell division plane. During late anaphase, phragmoplasts emerge between daughter nuclei as the derivative of spindle microtubules, and centrifugally expand toward the cell cortex to build cell plates during telophase. Phragmoplasts are composed of short antiparallel microtubules decorated with various microtubule-associated proteins. Mutants of these microtubule-associated proteins exhibit defects in phragmoplast morphology. Quantification of phragmoplast morphology is indispensable for assessing the phenotypes of these mutants. Here, we describe a method to quantify the width of phragmoplasts.

Cytokinesis in fra2 Arabidopsis thaliana p60-Katanin Mutant: Defects in Cell Plate/Daughter Wall Formation.

Cytokinesis is accomplished in higher plants by the phragmoplast, creating and conducting the cell plate to separate daughter nuclei by a new cell wall. The microtubule-severing enzyme p60-katanin plays an important role in the centrifugal expansion and timely disappearance of phragmoplast microtubules. Consequently, aberrant structure and delayed expansion rate of the phragmoplast have been reported to occur in p60-katanin mutants. Here, the consequences of p60-katanin malfunction in cell plate/daughter wall formation were investigated by transmission electron microscopy (TEM), in root cells of the fra2 Arabidopsis thaliana loss-of-function mutant. In addition, deviations in the chemical composition of cell plate/new cell wall were identified by immunolabeling and confocal microscopy. It was found that, apart from defective phragmoplast microtubule organization, cell plates/new cell walls also appeared faulty in structure, being unevenly thick and perforated by large gaps. In addition, demethylesterified homogalacturonans were prematurely present in fra2 cell plates, while callose content was significantly lower than in the wild type. Furthermore, KNOLLE syntaxin disappeared from newly formed cell walls in fra2 earlier than in the wild type. Taken together, these observations indicate that delayed cytokinesis, due to faulty phragmoplast organization and expansion, results in a loss of synchronization between cell plate growth and its chemical maturation.

The Protein Phosphatase PP2A Plays Multiple Roles in Plant Development by Regulation of Vesicle Traffic-Facts and Questions.

The protein phosphatase PP2A is essential for the control of integrated eukaryotic cell functioning. Several cellular and developmental events, e.g., plant growth regulator (PGR) mediated signaling pathways are regulated by reversible phosphorylation of vesicle traffic proteins. Reviewing present knowledge on the relevant role of PP2A is timely. We discuss three aspects: (1) PP2A regulates microtubule-mediated vesicle delivery during cell plate assembly. PP2A dephosphorylates members of the microtubule associated protein family MAP65, promoting their binding to microtubules. Regulation of phosphatase activity leads to changes in microtubule organization, which affects vesicle traffic towards cell plate and vesicle fusion to build the new cell wall between dividing cells. (2) PP2A-mediated inhibition of target of rapamycin complex (TORC) dependent signaling pathways contributes to autophagy and this has possible connections to the brassinosteroid signaling pathway. (3) Transcytosis of vesicles transporting PIN auxin efflux carriers. PP2A regulates vesicle localization and recycling of PINs related to GNOM (a GTP-GDP exchange factor) mediated pathways. The proper intracellular traffic of PINs is essential for auxin distribution in the plant body, thus in whole plant development. Overall, PP2A has essential roles in membrane interactions of plant cell and it is crucial for plant development and stress responses.

A rice tubulin tyrosine ligase-like 12 protein affects the dynamic and orientation of microtubules.

The detyrosination/retyrosination cycle is the most common post-translational modification of α-tubulin. Removal of the conserved C-terminal tyrosine of α-tubulin by a still elusive tubulin tyrosine carboxypeptidase, and religation of this tyrosine by a tubulin tyrosine ligase (TTL), are probably common to all eukaryotes. Interestingly, for plants, the only candidates qualifying as potential TTL homologs are the tubulin tyrosine ligase-like 12 proteins. To get insight into the biological functions of these potential TTL homologs, we cloned the rice TTL-like 12 protein (OsTTLL12) and generated overexpression OsTTLL12-RFP lines in both rice and tobacco BY-2 cells. We found, unexpectedly, that overexpression of this OsTTLL12-RFP increased the relative abundance of detyrosinated α-tubulin in both coleoptile and seminal root, correlated with more stable microtubules. This was independent of the respective orientation of cortical microtubule, and followed by correspondingly changing growth of coleoptiles and seminal roots. A perturbed organization of phragmoplast microtubules and disoriented cell walls were further characteristics of this phenotype. Thus, the elevated tubulin detyrosination in consequence of OsTTLL12 overexpression affects structural and dynamic features of microtubules, followed by changes in the axiality of cell plate deposition and, consequently, plant growth.

Random chromosome distribution in the first meiosis of F1 disomic substitution line 2R(2D) x rye hybrids (ABDR, 4× = 28) occurs without bipolar spindle assembly.

The assembly of the microtubule-based spindle structure in plant meiosis remains poorly understood compared with our knowledge of mitotic spindle formation. One of the approaches in our understanding of microtubule dynamics is to study spindle assembly in meiosis of amphyhaploids. Using immunostaining with phH3Ser10, CENH3 and α-tubulin-specific antibodies, we studied the chromosome distribution and spindle organisation in meiosis of F1 2R(2D)xR wheat-rye hybrids (genome structure ABDR, 4× = 28), as well as in wheat and rye mitosis and meiosis. At the prometaphase of mitosis, spindle assembly was asymmetric; one half of the spindle assembled before the other, with simultaneous chromosome alignment in the spindle mid-zone. At diakinesis in wheat and rye, microtubules formed a pro-spindle which was subsequently disassembled followed by a bipolar spindle assembly. In the first meiosis of hybrids 2R(2D)xR, a bipolar spindle was not found and the kinetochore microtubules distributed the chromosomes. Univalent chromosomes are characterised by a monopolar orientation and maintenance of sister chromatid and centromere cohesion. Presence of bivalents did not affect the formation of a bipolar spindle. Since the central spindle was absent, phragmoplast originates from "interpolar" microtubules generated by kinetochores. Cell plate development occurred with a delay. However, meiocytes in meiosis II contained apparently normal bipolar spindles. Thus, we can conclude that: (1) cohesion maintenance in centromeres and between arms of sister chromatids may negatively affect bipolar spindle formation in the first meiosis; (2) 2R/2D rye/wheat chromosome substitution affects the regulation of the random chromosome distribution in the absence of a bipolar spindle.

Into another dimension: how streptophyte algae gained morphological complexity.

Land plants with elaborated three-dimensional (3D) body plans have evolved from streptophyte algae. The streptophyte algae are known to exhibit varying degrees of morphological complexity, ranging from single-celled flagellates to branched macrophytic forms exhibiting tissue-like organization. In this review, I discuss mechanisms by which, during evolution, filamentous algae may have gained 2D and eventually 3D body plans. There are, in principle, two mechanisms by which an additional dimension may be added to an existing algal filament or cell layer: first, by tip growth-mediated branching. An example of this mechanism is the emergence and polar expansion of root hairs from land plants. The second possibility is the rotation of the cell division plane. In this case, the plane of the forthcoming cell division is rotated within the parental cell wall. This type of mechanism corresponds to the formative cell division seen in meristems of land plants. This literature review shows that of the extant streptophyte algae, the Charophyceae and Coleochaetophyceae are capable of performing both mechanisms, while the Zygnematophyceae (the actual sister to land plants) show tip growth-based branching only. I finally discuss how apical cells with two or three cutting faces, as found in mosses, may have evolved from algal ancestors.