Radially arranged cortical microtubules are a prominent feature of guard cells. Guard cells expressing GFP-tubulin showed consistent changes in the appearance of microtubules when stomata opened or closed. Guard cells showed fewer microtubule structures as stomata closed, whether induced by transfer to darkness, ABA, hydrogen peroxide, or sodium hydrogen carbonate. Guard cells kept in the dark (closed stomata) showed increases in microtubule structures and stomatal aperture on light treatment. GFP-EB1, marking microtubule growing plus ends, showed no change in number of plus ends or velocity of assembly on stomatal closure. Since the number of growing plus ends and the rate of plus-end growth did not change when microtubule structure numbers declined, microtubule instability and/or rearrangement must be responsible for the apparent loss of microtubules. Guard cells with closed stomata showed more cytosolic GFP-fluorescence than those with open stomata as cortical microtubules became disassembled, although with a large net loss in total fluorescence. Microtubule-targeted drugs blocked guard-cell function in Vicia and Arabidopsis. Oryzalin disrupted guard-cell microtubules and prevented stomatal opening and taxol stabilized guard-cell microtubules and delayed stomata! closure. Gas exchange measurements indicated that the transgenes for fluorescent-labeled proteins did not disrupt normal stomata! function. These dynamic changes in guard-cell microtubules combined with our inhibitor studies provide evidence for an active role of microtubules in guard-cell function.

Microtubules in eukaryotic cells are nucleated from ring-shaped complexes that contain ?-tubulin and a family of homologous ?-tubulin complex proteins (GCPs), but the subunit composition of the complexes can vary among fungi, animals and plants. Arabidopsis GCP3-interacting protein 1 (GIP1), a small protein with no homology to the GCP family, interacts with GCP3 in vitro, and is a plant homolog of vertebrate mitotic-spindle organizing protein associated with a ring of ?-tubulin 1 (MOZART1), a recently identified component of the ?-tubulin complex in human cell lines. In this study, we characterized two closely related Arabidopsis GIP1s: GIP1a and GIP1b. Single mutants of gip1a and gip1b were indistinguishable from wild-type plants, but their double mutant was embryonic lethal, and showed impaired development of male gametophytes. Functional fusions of GIP1a with green fluorescent protein (GFP) were used to purify GIP1a-containing complexes from Arabidopsis plants, which contained all the subunits (except NEDD1) previously identified in the Arabidopsis ?-tubulin complexes. GIP1a and GIP1b interacted specifically with Arabidopsis GCP3 in yeast. GFP-GIP1a labeled mitotic microtubule arrays in a pattern largely consistent with, but partly distinct from, the localization of the ?-tubulin complex containing GCP2 or GCP3 in planta. In interphase cortical arrays, the labeled complexes were preferentially recruited to existing microtubules, from which new microtubules were efficiently nucleated. However, in contrast to complexes labeled with tagged GCP2 or GCP3, their recruitment to cortical areas with no microtubules was rarely observed. These results indicate that GIP1/MOZART1 is an integral component of a subset of the Arabidopsis ?-tubulin complexes.

The brassinosteroid (BR) phytohormones play crucial roles in regulating plant cell growth and morphogenesis, particularly in hypocotyl cell elongation. The microtubule cytoskeleton is also known to participate in the regulation of hypocotyl elongation. However, it is unclear if BR regulation of hypocotyl elongation involves the microtubule cytoskeleton. In this study, we demonstrate that BRs mediate hypocotyl cell elongation by influencing the orientation and stability of cortical microtubules. Further analysis identified the previously undiscovered Arabidopsis thaliana MICROTUBULE DESTABILIZING PROTEIN40 (MDP40) as a positive regulator of hypocotyl cell elongation. BRASSINAZOLE-RESISTANT1, a key transcription factor in the BR signaling pathway, directly targets and upregulates MDP40. Overexpression of MDP40 partially rescued the shorter hypocotyl phenotype in BR-deficient mutant de-etiolated-2 seedlings. Reorientation of the cortical microtubules in the cells of MDP40 RNA interference transgenic lines was less sensitive to BR. These findings demonstrate that MDP40 is a key regulator in BR regulation of cortical microtubule reorientation and mediates hypocotyl growth. This study reveals a mechanism involving BR regulation of microtubules through MDP40 to mediate hypocotyl cell elongation.

Cells from all kingdoms of life adopt a dizzying array of fascinating shapes that support cellular function. Amoeboid and spherical shapes represent perhaps the simplest of geometries that may minimize the level of growth control required for survival. Slightly more complex are rod-shaped cells, from microscopic bacteria to macroscopic plants, which require additional mechanisms to define a cell's longitudinal axis, width, and length. Recent evidence suggests that many rod-shaped, walled cells achieve elongated growth through chiral insertion of cell-wall material that may be coupled to a twisting of the cell body. Inspired by these observations, biophysical mechanisms for twisting growth have been proposed that link the mechanics of intracellular proteins to cell shape maintenance. In this review, we highlight experimental and theoretical work that connects molecular-scale organization and structure with the cellular-scale phenomena of rod-shaped growth.

The Medicago truncatula DMI2 gene encodes a leucine-rich repeat receptor-like kinase that is essential for symbiosis with nitrogen-fixing rhizobia. While phenotypic analyses have provided a description for the host's responses mediated by DMI2, a lack of tools for in vivo biochemical analysis has hampered efforts to elucidate the mechanisms by which DMI2 mediates symbiotic signal transduction. Here, we report stably transformed M. truncatula lines that express a genomic DMI2 construct that is fused to a dual-affinity tag containing three copies of the hemagglutinin epitope and a single StrepII tag (gDMI2:HAST). gDMI2: HAST complements the dmi2-1 mutation, and transgenic plants expressing this construct behave similarly to wildtype plants. We show that the expression patterns of gDMI2:HAST recapitulate those of endogenous DMI2 and that we can detect and purify DMI2:HAST from microsomal root and nodule extracts. Using this line, we show that DMI2 resides in a high molecular weight complex, which is consistent with our observation that DMI2:GFP localizes to plasma membrane associated puncta and cytoplasmic vesicles. We further demonstrate that Nod factor (NF) perception increases the abundance of DMI2 vesicles. These tools should be a valuable resource for the Medicago community to dissect the biochemical function of DMI2.

Background: Ordered cortical microtubule (MT) arrays play a critical role in the spatial control of cell division and expansion and are essential for plant growth, morphogenesis, and development. Various developmental, hormonal, and mechanical signals and a large number of MT-associated proteins are known to impact cortical MT organization, but the underlying mechanisms remain poorly understood. Our previous studies show that auxin signaling, which is mediated by the ROP6 Rho GTPase and its effector RIC1, promotes the ordering of cortical MTs in pavement cells, but it is unknown how RIC1 controls the organization of cortical MTs into well-ordered arrays.

The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-g-tubulin complex protein2-tagged g-nucleation complexes (g-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving g-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation.

The advent of fluorescent proteins and access to modern imaging technologies have dramatically accelerated the pace of discovery in plant cell biology. Remarkable new insights into such diverse areas as plant pathogenesis, cytoskeletal dynamics, sugar transport, cell wall synthesis, secretory control, and hormone signaling have come from careful examination of living cells using advanced optical probes. New technologies, both commercially available and on the horizon, promise a continued march toward more quantitative methods for imaging and for extending the optical exploration of biological structure and activity to molecular scales. In this review, we lay out fundamental issues in imaging plant specimens and look ahead to several technological innovations in molecular tools, instrumentation, imaging methods, and specimen handling that show promise for shaping the coming era of plant cell biology.