Grant Calder

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.

Generation of Leaf Shape Through Early Patterns of Growth and Tissue Polarity

Shape-Shifting Signals Although orthogonal signaling systems seem to direct various developmental processes, few tissues remain in the same shape as they are at initiation to that of the final form. Arabidopsis leaves are free of the cell migrations that complicate animal development, and thus allowed Kuchen et al. (p. 1092) to track and model the trajectory of leaf growth under a variety of perturbations. Varying the values of parameters in their model produced outputs of different leaf shapes ranging from obcordate, ovate, and oval to elliptic, and offered predictions for genes that regulate the developmental process. The meristem at the growing tip of plants is home to stem cells and is the source of newly differentiating shoots and leaves. New leaves make their first appearance as bulges at the side of the dome-shaped meristem. Although these developmental events are under hormonal control, they also seem to be constrained by the physical properties of the meristem. Kierzkowski et al. (p. 1096) tested physical effects acting on the shoot apical meristem of growing tomato shoots that alter turgor pressure. Again, mathematical modeling combined with observations of plant tissue helped to define the different zones in the meristem that respond to diverse mechanical stimuli. A model for the development of leaf shape describes how it arises through oriented growth and tissue deformation. A major challenge in biology is to understand how buds comprising a few cells can give rise to complex plant and animal appendages like leaves or limbs. We address this problem through a combination of time-lapse imaging, clonal analysis, and computational modeling. We arrive at a model that shows how leaf shape can arise through feedback between early patterns of oriented growth and tissue deformation. Experimental tests through partial leaf ablation support this model and allow reevaluation of previous experimental studies. Our model allows a range of observed leaf shapes to be generated and predicts observed clone patterns in different species. Thus, our experimentally validated model may underlie the development and evolution of diverse organ shapes.

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.

Dynamic Behavior of Arabidopsis eIF4A-III, Putative Core Protein of Exon Junction Complex: Fast Relocation to Nucleolus and Splicing Speckles under Hypoxia

Here, we identify the Arabidopsis thaliana ortholog of the mammalian DEAD box helicase, eIF4A-III, the putative anchor protein of exon junction complex (EJC) on mRNA. Arabidopsis eIF4A-III interacts with an ortholog of the core EJC component, ALY/Ref, and colocalizes with other EJC components, such as Mago, Y14, and RNPS1, suggesting a similar function in EJC assembly to animal eIF4A-III. A green fluorescent protein (GFP)-eIF4A-III fusion protein showed localization to several subnuclear domains: to the nucleoplasm during normal growth and to the nucleolus and splicing speckles in response to hypoxia. Treatment with the respiratory inhibitor sodium azide produced an identical response to the hypoxia stress. Treatment with the proteasome inhibitor MG132 led to accumulation of GFP-eIF4A-III mainly in the nucleolus, suggesting that transition of eIF4A-III between subnuclear domains and/or accumulation in nuclear speckles is controlled by proteolysis-labile factors. As revealed by fluorescence recovery after photobleaching analysis, the nucleoplasmic fraction was highly mobile, while the speckles were the least mobile fractions, and the nucleolar fraction had an intermediate mobility. Sequestration of eIF4A-III into nuclear pools with different mobility is likely to reflect the transcriptional and mRNA processing state of the cell.

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.

Fungal Virulence and Development Is Regulated by Alternative Pre-mRNA 3′End Processing in Magnaporthe oryzae

RNA-binding proteins play a central role in post-transcriptional mechanisms that control gene expression. Identification of novel RNA-binding proteins in fungi is essential to unravel post-transcriptional networks and cellular processes that confer identity to the fungal kingdom. Here, we carried out the functional characterisation of the filamentous fungus-specific RNA-binding protein RBP35 required for full virulence and development in the rice blast fungus. RBP35 contains an N-terminal RNA recognition motif (RRM) and six Arg-Gly-Gly tripeptide repeats. Immunoblots identified two RBP35 protein isoforms that show a steady-state nuclear localisation and bind RNA in vitro. RBP35 coimmunoprecipitates in vivo with Cleavage Factor I (CFI) 25 kDa, a highly conserved protein involved in polyA site recognition and cleavage of pre-mRNAs. Several targets of RBP35 have been identified using transcriptomics including 14-3-3 pre-mRNA, an important integrator of environmental signals. In Magnaporthe oryzae, RBP35 is not essential for viability but regulates the length of 3′UTRs of transcripts with developmental and virulence-associated functions. The Δrbp35 mutant is affected in the TOR (target of rapamycin) signaling pathway showing significant changes in nitrogen metabolism and protein secretion. The lack of clear RBP35 orthologues in yeast, plants and animals indicates that RBP35 is a novel auxiliary protein of the polyadenylation machinery of filamentous fungi. Our data demonstrate that RBP35 is the fungal equivalent of metazoan CFI 68 kDa and suggest the existence of 3′end processing mechanisms exclusive to the fungal kingdom.

ATP‐dependent regulation of nuclear Ca2+ levels in plant cells

Localised alterations in cytoplasmic Ca2+ levels are an integral part of the response of eukaryotic cells to a plethora of external stimuli. Due to the large size of nuclear pores, it has generally been assumed that intranuclear Ca2+ levels reflect the prevailing cytoplasmic Ca2+ levels. Using nuclei prepared from carrot (Daucus carota L.) cells, we now show that Ca2+ can be transported across nuclear membranes in an ATP‐dependent manner and that over 95% of Ca2+ is accumulated into a pool releasable by the Ca2+ ionophore A.23187. ATP‐dependent nuclear Ca2+ uptake did not occur in the presence of ADP or ADPγS and was abolished by orthovanadate. Confocal microscopy of nuclei loaded with dextran‐linked Indo‐1 showed that the initial ATP‐induced rise in [Ca2+] occurs in the nuclear periphery. The occurrence of ATP‐dependent Ca2+ uptake in plant nuclei suggests that alterations of intranuclear Ca2+ levels may occur independently of cytoplasmic [Ca2+] changes.