Jordi Chan

EB1 reveals mobile microtubule nucleation sites in Arabidopsis

In plants, it is unclear how dispersed cortical microtubules are nucleated, polarized and organized in the absence of centrosomes. In Arabidopsis thaliana cells, expression of a fusion between the microtubule-end-binding protein AtEB1a and green fluorescent protein (GFP) results in labelling of spindle poles, where minus ends gather. During interphase, AtEB1a–GFP labels the microtubule plus end as a comet, but also marks the minus end as a site from which microtubules can grow and shrink. These minus-end nucleation sites are mobile, explaining how the cortical array can redistribute during the cell cycle and supporting the idea of a flexible centrosome in plants.

Cortical microtubule arrays undergo rotary movements in Arabidopsis hypocotyl epidermal cells.

Plant-cell expansion is controlled by cellulose microfibrils in the wall with microtubules providing tracks for cellulose synthesizing enzymes. Microtubules can be reoriented experimentally and are hypothesized to reorient cyclically in aerial organs, but the mechanism is unclear. Here, Arabidopsis hypocotyl microtubules wershee labelled with AtEB1a–GFP (Arabidopsis microtubule end-binding protein 1a) or GFP–TUA6 (Arabidopsis α-tubulin 6) to record long cycles of reorientation. This revealed microtubules undergoing previously unseen clockwise or counter-clockwise rotations. Existing models emphasize selective shrinkage and regrowth or the outcome of individual microtubule encounters to explain realignment. Our higher-order view emphasizes microtubule group behaviour over time. Successive microtubules move in the same direction along self-sustaining tracks. Significantly, the tracks themselves migrate, always in the direction of the individual fast-growing ends, but twentyfold slower. Spontaneous sorting of tracks into groups with common polarities generates a mosaic of domains. Domains slowly migrate around the cell in skewed paths, generating rotations whose progressive nature is interrupted when one domain is displaced by collision with another. Rotary movements could explain how the angle of cellulose microfibrils can change from layer to layer in the polylamellate cell wall.

Microtubules and the shape of plants to come

Plants control the direction of cell expansion as a way of shaping growth. Since their discovery in plants 40 years ago, microtubules have been suspected of forming a template that helps to regulate the direction of growth. The detailed mechanism, however, has been elusive, especially as plants lack a microtubule-organizing centre. Developmental mutants are now beginning to show how microtubules are organized and how this affects plant morphology.

Localization of the Microtubule End Binding Protein EB1 Reveals Alternative Pathways of Spindle Development in Arabidopsis Suspension Cells

In a previous study on Arabidopsis thaliana suspension cells transiently infected with the microtubule end binding protein AtEB1a–green fluorescent protein (GFP), we reported that interphase microtubules grow from multiple sites dispersed over the cortex, with plus ends forming the characteristic comet-like pattern. In this study, AtEB1a-GFP was used to study the transitions of microtubule arrays throughout the division cycle of cells lacking a defined centrosome. During division, the dispersed origin of microtubules was replaced by a more focused pattern with the plus end comets growing away from sites associated with the nuclear periphery. The mitotic spindle then evolved in two quite distinct ways depending on the presence or absence of the preprophase band (PPB): the cells displaying outside-in as well as inside-out mitotic pathways. In those cells possessing a PPB, the fusion protein labeled material at the nuclear periphery that segregated into two polar caps, perpendicular to the PPB, before nuclear envelope breakdown (NEBD). These polar caps then marked the spindle poles upon NEBD. However, in the population of cells without PPBs, there was no prepolarization of material at the nuclear envelope before NEBD, and the bipolar spindle only emerged clearly after NEBD. Such cells had variable spindle orientations and enhanced phragmoplast mobility, suggesting that the PPB is involved in a polarization event that promotes early spindle pole morphogenesis and subsequent positional stability during division. Astral-like microtubules are not usually prominent in plant cells, but they are clearly seen in these Arabidopsis cells, and we hypothesize that they may be involved in orienting the division plane, particularly where the plane is not determined before division.

Arabidopsis Cortical Microtubules Are Initiated along, as Well as Branching from, Existing Microtubules

The principles by which cortical microtubules self-organize into a global template hold important implications for cell wall patterning. Microtubules move along bundles of microtubules, and neighboring bundles tend to form mobile domains that flow in a common direction. The bundles themselves move slowly and for longer than the individual microtubules, with domains describing slow rotary patterns. Despite this tendency for colinearity, microtubules have been seen to branch off extant microtubules at ∼45°. To examine this paradoxical behavior, we investigated whether some microtubules may be born on and grow along extant microtubule(s). The plus-end markers Arabidopsis thaliana end binding protein 1a, AtEB1a-GFP, and Arabidopsis SPIRAL1, SPR1-GFP, allowed microtubules of known polarity to be distinguished from underlying microtubules. This showed that the majority of microtubules do branch but in a direction heavily biased toward the plus end of the mother microtubule: few grow backward, consistent with the common polarity of domains. However, we also found that a significant proportion of emergent comets do follow the axes of extant microtubules, both at sites of apparent microtubule nucleation and at cross-over points. These phenomena help explain the persistence of bundles and counterbalance the tendency to branch.

Generation of Spatial Patterns Through Cell Polarity Switching

A few simple rules are sufficient to keep neighboring stomata at a safe distance from one another. The mechanisms that generate dynamic spatial patterns within proliferating tissues are poorly understood, largely because of difficulties in unravelling interactions between cell specification, polarity, asymmetric division, rearrangements, and growth. We address this problem for stomatal spacing in plants, which offer the simplifying advantage that cells do not rearrange. By tracking lineages and gene activities over extended periods, we show that limited stem cell behavior of stomatal precursors depends on maintenance of the SPEECHLESS (SPCH) transcription factor in single daughter cells. Modeling shows how this property can lead to observed stereotypical stomata lineages through a postmitotic polarity-switching mechanism. The model predicts the location of a polarity determinant BASL over multiple divisions, which we validate experimentally. Our results highlight the dynamic two-way interactions between stem cells and their neighborhood during developmental patterning.

The parallel lives of microtubules and cellulose microfibrils.

A major breakthrough was the recent discovery that cellulose synthases really do move along the plasma membrane upon tracks provided by the underlying cortical microtubules. It emphasized the cytoplasmic contribution to cell wall organization. A growing number of microtubule-associated proteins has been identified and shown to affect the way that microtubules are ordered, with downstream effects on the pattern of growth. The dynamic properties of microtubules turn out to be key in understanding the behaviour of the global array and good progress has been made in deciphering the rules by which the array is self-organized.

Not so divided: the common basis of plant and animal cell division

Plant cells do not have centrioles and their mitosis is frequently likened to the chromosome-based mechanism seen in acentriolar animal cells. However, this is a false analogy. Although plants can use this mechanism, they generally divide by a method that uses bipolar mitotic caps, which is more similar to the canonical centrosome-based method of animals.

The rotation of cellulose synthase trajectories is microtubule dependent and influences the texture of epidermal cell walls in Arabidopsis hypocotyls.

Plant shoots have thick, polylamellate outer epidermal walls based on crossed layers of cellulose microfibrils, but the involvement of microtubules in such wall lamellation is unclear. Recently, using a long-term movie system in which Arabidopsis seedlings were grown in a biochamber, the tracks along which cortical microtubules move were shown to undergo slow rotary movements over the outer surface of hypocotyl epidermal cells. Because microtubules are known to guide cellulose synthases over the short term, we hypothesised that this previously unsuspected microtubule rotation could, over the longer term, help explain the cross-ply structure of the outer epidermal wall. Here, we test that hypothesis using Arabidopsis plants expressing the cellulose synthase GFP-CESA3 and show that cellulose synthase trajectories do rotate over several hours. Neither microtubule-stabilising taxol nor microtubule-depolymerising oryzalin affected the linear rate of GFP-CESA3 movement, but both stopped the rotation of cellulose synthase tracks. Transmission electron microscopy revealed that drug-induced suppression of rotation alters the lamellation pattern, resulting in a thick monotonous wall layer. We conclude that microtubule rotation, rather than any hypothetical mechanism for wall self-assembly, has an essential role in developing cross-ply wall texture.

Microtubules and CESA tracks at the inner epidermal wall align independently of those on the outer wall of light-grown Arabidopsis hypocotyls.

Microtubules are classically described as being transverse, which is perpendicular to the direction of cell elongation. However, fixation studies have indicated that microtubules can be variably aligned across the epidermis of elongating shoots. In addition, microtubules are reported to have different orientations on inner and outer epidermal surfaces, undermining the idea of hoop-reinforcement. Here, long-term movies of Arabidopsis seedlings expressing GFP–TUA6 allowed microtubule alignment to be directly correlated with the rate of elongation within individual growing cells. We also investigated whether microtubule alignment at the inner or the outer epidermal wall better reflected the growth rate. Movies confirmed that transverse microtubules form on the inner wall throughout elongation, but orientation of microtubules is variable at the outer wall, where they tend to become transverse only during episodes of accelerated growth. Because this appears to contradict the concept that circumferential arrays of transverse microtubules or microfibrils are essential for cell elongation, we checked the organisation of cellulose synthase tracks using GFP–CESA3 and found a similar mismatch between trajectories on inner and outer epidermal surfaces. We conclude that microtubule alignment on the inner wall appears to be a more stable predictor of growth anisotropy, whereas outer-wall alignment is more sensitive to the elongation rate.