Henrik Buschmann

Helical Growth of the Arabidopsis Mutant tortifolia1 Reveals a Plant-Specific Microtubule-Associated Protein

Plants can grow straight or in the twisted fashion exhibited by the helical growth of some climbing plants. Analysis of helical-growth mutants from Arabidopsis has indicated that microtubules are involved in the expression of the helical phenotype. Arabidopsis mutants growing with a right-handed twist have been reported to have cortical microtubules that wind around the cell in left-handed helices and vice versa. Microtubular involvement is further suspected from the finding that some helical mutants are caused by single amino acid substitutions in alpha-tubulin and because of the sensitivity of the growth pattern to anti-microtubule drugs. Insight into the roles of microtubules in organ elongation is anticipated from analyses of genes defined by helical mutations. We investigated the helical growth of the Arabidopsis mutant tortifolia1/spiral2 (tor1/spr2), which twists in a right-handed manner, and found that this correlates with a complex reorientation of cortical microtubules. TOR1 was identified by a map-based approach; analysis of the TOR1 protein showed that it is a member of a novel family of plant-specific proteins containing N-terminal HEAT repeats. Recombinant TOR1 colocalizes with cortical microtubules in planta and binds directly to microtubules in vitro. This shows that TOR1 is a novel, plant-specific microtubule-associated protein (MAP) that regulates the orientation of cortical microtubules and the direction of organ growth.

AtMAP70-5, a divergent member of the MAP70 family of microtubule-associated proteins, is required for anisotropic cell growth in Arabidopsis.

AtMAP70-5 is the most divergent of a recently described multigene family of plant-specific microtubule-associated proteins (MAPs). It is significantly smaller than other members and has several isoform-specific sequence features. To confirm that this protein still functions as a MAP we show that it directly binds microtubules in vitro and decorates microtubules in vivo. When added to tubulin polymerization assays, AtMAP70-5 increases the length distribution profile of microtubules indicating that it stabilizes microtubule dynamics. The overexpressed fusion protein perturbs cell polarity in cell suspensions by inducing extra poles for growth. Similarly, in Arabidopsis plants the overexpression of AtMAP70-5 causes epidermal cells to swell; it also stunts growth and induces right-handed organ twisting. RNAi-mediated downregulation of AtMAP70-5 results in reduced inflorescence stem length and diameter and individual cells are inhibited in their capacity for expansion. These observations suggest that the control over AtMAP70-5 expression levels is important in order to maintain axial polarity and to ensure regular extension of plant organs.

Helical Growth of the Arabidopsis Mutant tortifolia2 Does Not Depend on Cell Division Patterns but Involves Handed Twisting of Isolated Cells

Several factors regulate plant organ growth polarity. tortifolia2 (tor2), a right-handed helical growth mutant, has a conservative replacement of Arg-2 with Lys in the α-tubulin 4 protein. Based on a published high-resolution (2.89 Å) tubulin structure, we predict that Arg-2 of α-tubulin forms hydrogen bonds with the GTPase domain of β-tubulin, and structural modeling suggests that these contacts are interrupted in tor2. Consistent with this, we found that microtubule dynamicity is reduced in the tor2 background. We investigated the developmental origin of the helical growth phenotype using tor2. One hypothesis predicts that cell division patterns cause helical organ growth in Arabidopsis thaliana mutants. However, cell division patterns of tor2 root tips appear normal. Experimental uncoupling of cell division and expansion suggests that helical organ growth is based on cell elongation defects only. Another hypothesis is that twisting is due to inequalities in expansion of epidermal and cortical tissues. However, freely growing leaf trichomes of tor2 mutants show right-handed twisting and cortical microtubules form left-handed helices as early as the unbranched stage of trichome development. Trichome twisting is inverted in double mutants with tor3, a left-handed mutant. Single tor2 suspension cells also exhibit handed twisting. Thus, twisting of tor2 mutant organs appears to be a higher-order expression of the helical expansion of individual cells.

The role of MAP65-1 in microtubule bundling during Zinnia tracheary element formation

The MAP65 family of microtubule-associated proteins performs various functions at different stages of the cell cycle and differentiation. In this study, we have investigated the synchronous transdifferentiation of Zinnia mesophyll cells into tracheary elements in vitro. This allowed us to examine the role of the microtubule-associated protein MAP65 during the characteristic bunching of cortical microtubules that underlie the developing ribs of secondarily thickened cell wall. Immunofluorescence confirmed the microtubule bundles to be decorated with anti-MAP65 antibodies. Three Zinnia MAP65 genes were examined; the expression of ZeMAP65-1 was found to match that of the differentiation marker TED2 and both were found to be upregulated upon addition of inductive hormones. We cloned the full-length sequence of ZeMAP65-1 and found it to be most similar to other MAP65 isoforms known to bundle microtubules in other plant species. However, not all MAP65 proteins crosslink cortical microtubules and so, to confirm its potential bundling capacity, ZeMAP65-1 was transiently overexpressed in Arabidopsis suspension cells. This resulted in the super-bundling of microtubules in patterns resembling those in differentiating xylem cells. These findings establish that the MAP65-1 group of proteins is responsible for the bundling of cortical microtubules during secondary cell wall formation of xylogenesis as well as during the expansion of primary cell walls.

Arabidopsis KCBP interacts with AIR9 but stays in the cortical division zone throughout mitosis via its MyTH4-FERM domain

ABSTRACT The preprophase band of microtubules performs the crucial function of marking the plane of cell division. Although the preprophase band depolymerises at the onset of mitosis, the division plane is ‘memorized’ by a cortical division zone to which the phragmoplast is attracted during cytokinesis. Proteins have been discovered that are part of the molecular memory but little is known about how they contribute to phragmoplast guidance. Previously, we found that the microtubule-associated protein AIR9 is found in the cortical division zone at preprophase and returns during cell plate insertion but is absent from the cortex during the intervening mitosis. To identify new components of the preprophase memory, we searched for proteins that interact with AIR9. We detected the kinesin-like calmodulin-binding protein, KCBP, which can be visualized at the predicted cortical site throughout division. A truncation study of KCBP indicates that its MyTH4-FERM domain is required for linking the motor domain to the cortex. These results suggest a mechanism by which minus-end-directed KCBP helps guide the centrifugally expanding phragmoplast to the cortical division site.

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.

Microtubule dynamics in plant cells.

This chapter describes some of the choices and unavoidable compromises to be made when studying microtubule dynamics in plant cells. The choice of species still depends very much on the ability to produce transgenic plants and most work has been done in the relatively small cells of Arabidopsis plants or in tobacco BY-2 suspension cells. Fluorescence-tagged microtubule proteins have been used to label entire microtubules, or their plus ends, but there are still few minus-end markers for these acentrosomal cells. Pragmatic decisions have to be made about probes, balancing the efficacy of microtubule labeling against a tendency to overstabilize and bundle the microtubules and even induce helical plant growth. A key limitation in visualizing plant microtubules is the ability to keep plants alive for long periods under the microscope and we describe a biochamber that allows for plant cell growth and development while allowing gas exchange and reducing evaporation. Another major difficulty is the limited fluorescence lifetime and we describe imaging strategies to reduce photobleaching in long-term imaging. We also discuss methods of measuring microtubule dynamics, with emphasis on the behavior of plant-specific microtubule arrays.

Plant division: remembering where to build the wall.

Before mitosis, a band of microtubules accurately forecasts where the next cross-wall will be inserted but then depolymerizes. How is this division plane memorized until cytokinesis? The molecular memory is being uncovered.

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.

BiasViz: visualization of amino acid biased regions in protein alignments

About a third of all protein sequences have at least one composition biased region (CBR). Such regions might act as linkers between protein domains but often confer specific binding to various molecules; therefore, their characterization in terms of their boundaries and over-represented residues is important. Analysis of CBRs in a particular sequence can be time consuming if several types of biases have to be explored and their position visualized. Assessment of the significance of the detected CBRs can be approached by comparison to homologous protein sequences. To assist this procedure, we have developed BiasViz, a tool that allows to graphically studying local amino acid composition in protein sequences of a multiple sequence alignment.