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.

The phosphoinositide signature guides the final step of plant cytokinesis.

Plant cytokinesis, which fundamentally differs from that in animals, requires the outward expansion of a plasma membrane precursor named the cell plate. How the transition from a cell plate to a plasma membrane occurs remains poorly understood. Here, we report that the acquisition of plasma membrane identity occurs through lateral patterning of the phosphatidylinositol 4,5-bisphosphate PI(4,5)P2 at the newly formed cell plate membrane. There, the phosphoinositide phosphatase SAC9 emerges as a key regulator, colocalizing with and regulating the function of the microtubule-associated protein MAP65-3 at the cell plate leading zone. In sac9-3 mutant, the polar distribution of PI(4,5)P2 at the cell plate is altered, leading to ectopic recruitment of the cytokinesis apparatus and formation of an additional cell plate insertion site. We propose that at the cell plate, SAC9 drives the depletion of PI(4,5)P2, which acts as a polar cue to spatially separate cell plate expansion from the acquisition of plasma membrane identity during final step of cytokinesis.

Studying Cell Division Plane Positioning in Early-Stage Embryos.

Unraveling the mechanisms that govern division plane orientation is a major challenge to understand plant development. In this respect, the Arabidopsis early embryo is a model system of choice since embryogenesis is relatively simple and cell division planes orientation is highly predictable. Here we present an integrated set of protocols to study 3D cell division patterns in early-stage Arabidopsis embryos that combine both cellular and sub-cellular localization of selected protein markers with spatial organization of cells, cytoskeleton, and nuclei.

Quantification of Cell Division Angles in the Arabidopsis Root.

In many plant tissues, division plane orientation within cell files is highly predictable since all cells divide almost perpendicular to the cell file axis. Many mutations can affect division plane orientation, and the quantification of the deviation from the expected transverse orientation in various genetic backgrounds is thus an important issue.While several software tools have been proposed for the quantification of cellular morphology in plant tissues, none of them allowed investigating division plane orientation. We propose here a complete method for measuring orientation of division planes in 2D, using an open-source ImageJ plugin named "Cell File Angles." The method comprises the staining of cell wall within whole mount roots with the calcofluor dye, the acquisition of 3D Z-stacks of the stained roots, and the measurement of cell wall orientation using image processing algorithms and semi-automated analysis.

B1-type cyclins control microtubule organization during cell division in Arabidopsis.

Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development-specific requirements of B1-type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1-type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1-type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA-TUBULIN COMPLEX PROTEIN 3-INTERACTING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1-type cyclin complexes and further genetic analyses support a potential role in the regulation of GIP1 by CYCB1s.

The preprophase band of microtubules controls the robustness of division orientation in plants.

Controlling cell division plane orientation is essential for morphogenesis in multicellular organisms. In plant cells, the future cortical division plane is marked before mitotic entry by the preprophase band (PPB). Here, we characterized an Arabidopsis trm (TON1 Recruiting Motif) mutant that impairs PPB formation but does not affect interphase microtubules. Unexpectedly, PPB disruption neither abolished the capacity of root cells to define a cortical division zone nor induced aberrant cell division patterns but rather caused a loss of precision in cell division orientation. Our results advocate for a reassessment of PPB function and division plane determination in plants and show that a main output of this microtubule array is to limit spindle rotations in order to increase the robustness of cell division.

Studying Cell Division Plane Positioning in Early-Stage Embryos.

Unraveling the mechanisms that govern division plane orientation is a major challenge to understand plant development. In this respect, the Arabidopsis early embryo is a model system of choice since embryogenesis is relatively simple and cell division planes orientation is highly predictable. Here, we present an integrated set of protocols to study 3D cell division patterns in early-stage Arabidopsis embryos that combine both cellular and sub-cellular localization of selected protein markers with spatial organization of cells, cytoskeleton, and nuclei.

A protein phosphatase 2A complex spatially controls plant cell division.

In the absence of cell migration, the orientation of cell divisions is crucial for body plan determination in plants. The position of the division plane in plant cells is set up premitotically via a transient cytoskeletal array, the preprophase band, which precisely delineates the cortical plane of division. Here we describe a protein complex that targets protein phosphatase 2A activity to microtubules, regulating the transition from the interphase to the premitotic microtubule array. This complex, which comprises TONNEAU1 and a PP2A heterotrimeric holoenzyme with FASS as regulatory subunit, is recruited to the cytoskeleton via the TONNEAU1-recruiting motif family of proteins. Despite the acentrosomal nature of plant cells, all members of this complex share similarity with animal centrosomal proteins involved in ciliary and centriolar/centrosomal functions, revealing an evolutionary link between the cortical cytoskeleton of plant cells and microtubule organizers in other eukaryotes.

The Arabidopsis TRM1-TON1 interaction reveals a recruitment network common to plant cortical microtubule arrays and eukaryotic centrosomes.

Land plant cells assemble microtubule arrays without a conspicuous microtubule organizing center like a centrosome. In Arabidopsis thaliana, the TONNEAU1 (TON1) proteins, which share similarity with FOP, a human centrosomal protein, are essential for microtubule organization at the cortex. We have identified a novel superfamily of 34 proteins conserved in land plants, the TON1 Recruiting Motif (TRM) proteins, which share six short conserved motifs, including a TON1-interacting motif present in all TRMs. An archetypal member of this family, TRM1, is a microtubule-associated protein that localizes to cortical microtubules and binds microtubules in vitro. Not all TRM proteins can bind microtubules, suggesting a diversity of functions for this family. In addition, we show that TRM1 interacts in vivo with TON1 and is able to target TON1 to cortical microtubules via its C-terminal TON1 interaction motif. Interestingly, three motifs of TRMs are found in CAP350, a human centrosomal protein interacting with FOP, and the C-terminal M2 motif of CAP350 is responsible for FOP recruitment at the centrosome. Moreover, we found that TON1 can interact with the human CAP350 M2 motif in yeast. Taken together, our results suggest conservation of eukaryotic centrosomal components in plant cells.

The function of TONNEAU1 in moss reveals ancient mechanisms of division plane specification and cell elongation in land plants.

The preprophase band (PPB) is a transient ring of microtubules that forms before mitosis in land plants, and delineates the cytokinetic division plane established at telophase. It is one of the few derived traits specific to embryophytes, in which it is involved in the spatial control of cell division. Here we show that loss of function of Physcomitrella patens PpTON1 strongly affects development of the moss gametophore, phenocopying the developmental syndrome observed in Arabidopsis ton1 mutants: mutant leafy shoots display random orientation of cell division and severe defects in cell elongation, which are correlated with absence of PPB formation and disorganization of the cortical microtubule array in interphase cells. In hypomorphic Ppton1 alleles, PPB are still formed, whereas elongation defects are observed, showing the dual function of TON1 in organizing cortical arrays of microtubules during both interphase and premitosis. Ppton1 mutation has no impact on development of the protonema, which is consistent with the documented absence of PPB formation at this stage, apart from alteration of the gravitropic response, uncovering a new function of TON1 proteins in plants. Successful reciprocal cross-complementation between Physcomitrella and Arabidopsis shows conservation of TON1 function during land plant evolution. These results establish the essential role of the PPB in division plane specification in a basal land plant lineage, and provide new information on the function of TON1. They point to an ancient mechanism of cytoskeletal control of division plane positioning and cell elongation in land plants.

Arabidopsis TONNEAU1 proteins are essential for preprophase band formation and interact with centrin.

Plant cells have specific microtubule structures involved in cell division and elongation. The tonneau1 (ton1) mutant of Arabidopsis thaliana displays drastic defects in morphogenesis, positioning of division planes, and cellular organization. These are primarily caused by dysfunction of the cortical cytoskeleton and absence of the preprophase band of microtubules. Characterization of the ton1 insertional mutant reveals complex chromosomal rearrangements leading to simultaneous disruption of two highly similar genes in tandem, TON1a and TON1b. TON1 proteins are conserved in land plants and share sequence motifs with human centrosomal proteins. The TON1 protein associates with soluble and microsomal fractions of Arabidopsis cells, and a green fluorescent protein-TON1 fusion labels cortical cytoskeletal structures, including the preprophase band and the interphase cortical array. A yeast two-hybrid screen identified Arabidopsis centrin as a potential TON1 partner. This interaction was confirmed both in vitro and in plant cells. The similarity of TON1 with centrosomal proteins and its interaction with centrin, another key component of microtubule organizing centers, suggests that functions involved in the organization of microtubule arrays by the centrosome were conserved across the evolutionary divergence between plants and animals.

Molecular encounters at microtubule ends in the plant cell cortex.

The cortical arrays that accompany plant cell division and elongation are organized by a subtle interplay between intrinsic properties of microtubules, their self-organization capacity and a variety of cellular proteins that interact with them, modify their behaviour and drive organization of diverse, higher order arrays during the cell cycle, cell growth and differentiation. As a polar polymer, the microtubule has a minus and a plus end, which differ in structure and dynamic characteristics, and to which different sets of partners and activities associate. Recent advances in characterization of minus and plus end directed proteins provide insights into both plant microtubule properties and the way highly organized cortical arrays emerge from the orchestrated activity of individual microtubules.

Gamma-tubulin is essential for microtubule organization and development in Arabidopsis.

The process of microtubule nucleation in plant cells is still a major question in plant cell biology. gamma-Tubulin is known as one of the key molecular players for microtubule nucleation in animal and fungal cells. Here, we provide genetic evidence that in Arabidopsis thaliana, gamma-tubulin is required for the formation of spindle, phragmoplast, and cortical microtubule arrays. We used a reverse genetics approach to investigate the role of the two Arabidopsis gamma-tubulin genes in plant development and in the formation of microtubule arrays. Isolation of mutants in each gene and analysis of two combinations of gamma-tubulin double mutants showed that the two genes have redundant functions. The first combination is lethal at the gametophytic stage. Disruption of both gamma-tubulin genes causes aberrant spindle and phragmoplast structures and alters nuclear division in gametophytes. The second combination of gamma-tubulin alleles affects late seedling development, ultimately leading to lethality 3 weeks after germination. This partially viable mutant combination enabled us to follow dynamically the effects of gamma-tubulin depletion on microtubule arrays in dividing cells using a green fluorescent protein marker. These results establish the central role of gamma-tubulin in the formation and organization of microtubule arrays in Arabidopsis.

The Arabidopsis TONNEAU2 gene encodes a putative novel protein phosphatase 2A regulatory subunit essential for the control of the cortical cytoskeleton.

In Arabidopsis ton2 mutants, abnormalities of the cortical microtubular cytoskeleton, such as disorganization of the interphase microtubule array and lack of the preprophase band before mitosis, markedly affect cell shape and arrangement as well as overall plant morphology. We present the molecular isolation of the TON2 gene, which is highly conserved in higher plants and has a vertebrate homolog of unknown function. It encodes a protein similar in its C-terminal part to B" regulatory subunits of type 2A protein phosphatases (PP2As). We show that the TON2 protein interacts with an Arabidopsis type A subunit of PP2A in the yeast two-hybrid system and thus likely defines a novel subclass of PP2A subunits that are possibly involved in the control of cytoskeletal structures in plants.

Comparison of the expression patterns of two small gene families of S gene family receptor kinase genes during the defence response in Brassica oleracea and Arabidopsis thaliana.

SFR2, a member of the S gene family of receptor kinases, has been shown to be rapidly induced by wounding and bacterial infection suggesting that this gene may play a role in the defence response in Brassica. In this study we have compared the response of SFR2 to that of two other members of the SFR gene family in Brassica (SFR1 and SFR3) and to the closely-related ARK genes of Arabidopsis. Different patterns of mRNA accumulation were observed for different members of these families. SFR1 transcripts only accumulated in response to bacterial infection and their abundance was not significantly affected by wounding. Neither treatment induced accumulation of SFR3 transcripts. ARK1 and ARK3 resembled SFR2 in that their mRNAs accumulated in response to both wounding and bacterial infection. Both SFR1 and SFR2 mRNAs accumulated in response to exogenously applied salicylic acid (SA) and SA was shown to be required for induction of expression from the SFR2 promoter in Arabidopsis. However, the timing of the increase in endogenous SA levels following bacterial infiltration in Brassica indicates that the accumulation of SFR mRNA in the first few hours after infiltration does not occur in response to an increase in SA levels. We discuss the possibility that induction of SFR gene expression by SA may contribute to potentialization of the defence response. Taken together with previous studies that indicate a possible role during development, the data presented here suggest that the SFR and ARK gene families may have overlapping roles in both defence and during development.