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.

The actin and microtubule cytoskeletons regulate cell shape across phyla, from bacteria to metazoans. In organisms with cell walls, the wall acts as a primary constraint of shape, and generation of specific cell shape depends on cytoskeletal organization for wall deposition and/or cell expansion. In higher plants, cortical microtubules help to organize cell wall construction by positioning the delivery of cellulose synthase (CesA) complexes and guiding their trajectories to orient newly synthesized cellulose microfibrils. The actin cytoskeleton is required for normal distribution of CesAs to the plasma membrane, but more specific roles for actin in cell wall assembly and organization remain largely elusive. We show that the actin cytoskeleton functions to regulate the CesA delivery rate to, and lifetime of CesAs at, the plasma membrane, which affects cellulose production. Furthermore, quantitative image analyses revealed that actin organization affects CesA tracking behavior at the plasma membrane and that small CesA compartments were associated with the actin cytoskeleton. By contrast, localized insertion of CesAs adjacent to cortical microtubules was not affected by the actin organization. Hence, both actin and microtubule cytoskeletons play important roles in regulating CesA trafficking, cellulose deposition, and organization of cell wall biogenesis.

Environmental and hormonal signals cause reorganization of microtubule arrays in higher plants, but the mechanisms driving these transitions have remained elusive. The organization of these arrays is required to direct morphogenesis. We discovered that microtubule severing by the protein katanin plays a crucial and unexpected role in the reorientation of cortical arrays, as triggered by blue light. Imaging and genetic experiments revealed that phototropin photoreceptors stimulate katanin-mediated severing specifically at microtubule intersections, leading to the generation of new microtubules at these locations. We show how this activity serves as the basis for a mechanism that amplifies microtubules orthogonal to the initial array, thereby driving array reorientation. Our observations show how severing is used constructively to build a new microtubule array.

Based on comparative genomics, a list of proteins present in the green algal, flowering and nonflowering plant lineages, but not detected in nonphotosynthetic organisms, was assembled (Merchant et al., Science 318:245-250, 2007; Karpowicz et al., J Biol Chem 286:21427-21439, 2011). This protein grouping, previously designated the GreenCut, was established using stringent comparative genomic criteria; they are those Chlamydomonas reinhardtii proteins with orthologs in Arabidopsis thaliana, Physcomitrella patens, Oryza sativa, Populus tricocarpa and at least one of the three Ostreococcus species with fully sequenced genomes, but not in bacteria, yeast, fungi or mammals. Many GreenCut proteins are also present in red algae and diatoms and a subset of 189 have been identified as encoded on nearly all cyanobacterial genomes. Of the current GreenCut proteins (597 in total), approximately half have been studied previously. The functions or activities of a number of these proteins have been deduced from phenotypic analyses of mutants (defective for genes encoding specific GreenCut proteins) of A. thaliana, and in many cases the assigned functions do not exist in C. reinhardtii. Therefore, precise physiological functions of several previously studied GreenCut proteins are still not clear. The GreenCut also contains a number of proteins with certain conserved domains. Three of the most highly conserved domains are the FK506 binding, cyclophilin and PAP fibrillin domains; most members of these gene families are not well characterized. In general, our analysis of the GreenCut indicates that many processes critical to green lineage organisms remain unstudied or poorly characterized. We have begun to examine the functions of some GreenCut proteins in detail. For example, our work on the CPLD38 protein has demonstrated that it has an essential role in photosynthetic function and the stability of the cytochrome b (6) f complex.

Cell-cell communication and interaction is critical during fertilization and triggers free cytosolic calcium ([Ca2+](cyto)) as a key signal for egg activation and a polyspermy block in animal oocytes. Fertilization in flowering plants is more complex, involving interaction of a pollen tube with egg adjoining synergid cells, culminating in release of two sperm cells and their fusion with the egg and central cell, respectively. Here, we report the occurrence and role of [Ca2+](cyto) signals during the entire double fertilization process in Arabidopsis. [Ca2+](cyto) oscillations are initiated in synergid cells after physical contact with the pollen tube apex. In egg and central cells, a short [Ca2+](cyto) transient is associated with pollen tube burst and sperm cell arrival. A second extended [Ca2+](cyto) transient solely in the egg cell is correlated with successful fertilization. Thus, each female cell type involved in double fertilization displays a characteristic [Ca2+](cyto) signature differing by timing and behaviour from [Ca2+](cyto) waves reported in mammals.

The Chlamydomonas reinhardtii proton gradient regulation5 (Crpgr5) mutant shows phenotypic and functional traits similar to mutants in the Arabidopsis (Arabidopsis thaliana) ortholog, Atpgr5, providing strong evidence for conservation of PGR5-mediated cyclic electron flow (CEF). Comparing the Crpgr5 mutant with the wild type, we discriminate two pathways for CEF and determine their maximum electron flow rates. The PGR5/proton gradient regulation-like1 (PGRL1) ferredoxin (Fd) pathway, involved in recycling excess reductant to increase ATP synthesis, may be controlled by extreme photosystem I acceptor side limitation or ATP depletion. Here, we show that PGR5/PGRL1-Fd CEF functions in accordance with an ATP/redox control model. In the absence of Rubisco and PGR5, a sustained electron flow is maintained with molecular oxygen instead of carbon dioxide serving as the terminal electron acceptor. When photosynthetic control is decreased, compensatory alternative pathways can take the full load of linear electron flow. In the case of the ATP synthase pgr5 double mutant, a decrease in photosensitivity is observed compared with the single ATPase-less mutant that we assign to a decreased proton motive force. Altogether, our results suggest that PGR5/PGRL1-Fd CEF is most required under conditions when Fd becomes overreduced and photosystem I is subjected to photoinhibition. CEF is not a valve; it only recycles electrons, but in doing so, it generates a proton motive force that controls the rate of photosynthesis. The conditions where the PGR5 pathway is most required may vary in photosynthetic organisms like C. reinhardtii from anoxia to high light to limitations imposed at the level of carbon dioxide fixation.

The preprophase band (PPB) is a faithful but transient predictor of the division plane in somatic cell divisions. Throughout mitosis the PPBs positional information is preserved by factors that continuously mark the division plane at the cell cortex, the cortical division zone, by their distinct spatio-temporal localization patterns. However, the mechanism maintaining these identity factors at the plasma membrane after PPB disassembly remains obscure. The pair of kinesin-12 class proteins PHRAGMOPLAST ORIENTING KINESIN1 (POK1) and POK2 are key players in division plane maintenance. Here, we show that POK1 is continuously present at the cell cortex, providing a spatial reference for the site formerly occupied by the PPB. Fluorescence recovery after photobleaching analysis combined with microtubule destabilization revealed dynamic microtubule-dependent recruitment of POK1 to the PPB during prophase, while POK1 retention at the cortical division zone in the absence of cortical microtubules appeared static. POK function is strictly required to maintain the division plane identity factor TANGLED (TAN) after PPB disassembly, although POK1 and TAN recruitment to the PPB occur independently during prophase. Together, our data suggest that POKs represent fundamental early anchoring components of the cortical division zone, translating and preserving the positional information of the PPB by maintaining downstream identity markers.