Two microtubule-associated proteins of the Arabidopsis MAP65 family function differently on microtubules

The organization and dynamics of microtubules are regulated by microtubule-associated proteins, or MAPs. In Arabidopsis (Arabidopsis thaliana), nine genes encode proteins of the evolutionarily conserved MAP65 family. We proposed that different MAP65s might have distinct roles in the interaction with microtubules. In this study, two AtMAP65 proteins, AtMAP65-1 and AtMAP65-6, were chosen to test this hypothesis in vitro. Although both fusion proteins were able to cosediment with microtubules in vitro, different properties on tubulin polymerization and microtubule bundling were observed. AtMAP65-1 was able to promote tubulin polymerization, enhance microtubule nucleation, and decrease the critical concentration for tubulin polymerization. It also induced the formation of large microtubule bundles by forming cross-bridges between microtubules evenly along the whole length of microtubules. In the presence of AtMAP65-1, microtubule bundles were more resistant to cold and dilution treatments. AtMAP65-6, however, demonstrated no activity in promoting tubulin polymerization and stabilizing preformed microtubules. AtMAP65-6 induced microtubules to form a mesh-like network with individual microtubules. Cross-bridge-like interactions were only found at regional sites between microtubules. The microtubule network induced by AtMAP65-6 was more resistant to high concentration of NaCl than the bundles induced by AtMAP65-1. Purified monospecific anti-AtMAP65-6 antibodies revealed that AtMAP65-6 was associated with mitochondria in Arabidopsis cells. It was concluded that these two MAP65 proteins were targeted to distinct sites, thus performing distinct functions in Arabidopsis cells.     

The Arabidopsis microtubule-associated protein AtMAP65-1: molecular analysis of its microtubule bundling activity

The 65-kD microtubule-associated protein (MAP65) family is a family of plant microtubule-bundling proteins. Functional analysis is complicated by the heterogeneity within this family: there are nine MAP65 genes in Arabidopsis thaliana, AtMAP65-1 to AtMAP65-9. To begin the functional dissection of the Arabidopsis MAP65 proteins, we have concentrated on a single isoform, AtMAP65-1, and examined its effect on the dynamics of mammalian microtubules. We show that recombinant AtMAP65-1 does not promote polymerization and does not stabilize microtubules against cold-induced microtubule depolymerization. However, we show that it does induce microtubule bundling in vitro and that this protein forms 25-nm cross-bridges between microtubules. We further demonstrate that the microtubule binding region resides in the C-terminal half of the protein and that Ala409 and Ala420 are essential for the interaction with microtubules. Ala420 is a conserved amino acid in the AtMAP65 family and is mutated to Val in the cytokinesis-defective mutant pleiade-4 of the AtMAP65-3/PLEIADE gene. We show that AtMAP65-1 can form dimers and that a region in the N terminus is responsible for this activity. Neither the microtubule binding region nor the dimerization region alone could induce microtubule bundling, strongly suggesting that dimerization is necessary to produce the microtubule cross-bridges. In vivo, AtMAP65-1 is ubiquitously expressed both during the cell cycle and in all plant organs and tissues with the exception of anthers and petals. Moreover, using an antiserum raised to AtMAP65-1, we show that AtMAP65-1 binds microtubules at specific stages of the cell cycle.     

Arabidopsis microtubule-associated protein AtMAP65-2 acts as a microtubule stabilizer

Nine genes that encode proteins of the MAP65 family have been identified in the Arabidopsis thaliana genome. In this study, we reported that AtMAP65-2, a member of the AtMAP65 family, could strongly stabilize microtubules (MTs). Bacterially-expressed AtMAP65-2 fusion proteins induced the formation of large MT bundles in vitro. Although AtMAP65-2 showed little effect on MT assembly or nucleation, AtMAP65-2 greatly stabilized MTs that were subjected to low-temperature treatment in vitro. Analyses of truncated versions of AtMAP65-2 indicated that the region that encompassed amino acids 495–578, which formed a flexible extended loop, played a crucial role in the stabilization of MTs. Analysis of suspension-cultured Arabidopsis cells that expressed the AtMAP65-2-GFP fusion protein showed that AtMAP65-2 co-localized with MTs throughout the cell cycle. Cortical MTs that were decorated with AtMAP65-2-GFP were more resistant to the MT-disrupting drug propyzamide and to ice treatment in vivo. The results of this study demonstrate that AtMAP65-2 strongly stabilizes MTs and is involved in the regulation of MT organization and dynamics. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. H. Li and X. Zeng have contributed equally to this paper and are considered as joint first authors.     

The AtMAP65-1 cross-bridge between microtubules is formed by one dimer

The microtubule-associated protein AtMAP65-1 from Arabidopsis thaliana dimerizes and forms 25 nm cross-bridges between microtubules, but the exact mechanism is unknown. Here, we used the predicted three-dimensional structure of AtMAP65-1 as a basis for analyzing the actual cross-bridging in detail. Fold-recognition predicts that AtMAP65-1 contains four coiled-coil domains and a flexible extended loop. The length of these coiled-coil domains is about 25 nm, suggesting that one molecule could span the gap, hence forming an antiparallel overlapping dimer instead of an end-to-end dimer. We then tested this model by using truncations of AtMAP65-1. EDC {[3-(dimethylamino) propyl] carbodiimide} cross-linking analysis indicated that the N-terminus of the rod domain of AtMAP65-1 (amino acids 1-339) binds to the C-terminus of the rod domain (amino acids 340-494) and also participates in connecting the two antiparallel proteins in the cross-bridge. Nevertheless, microtubules can still form bundles in the presence of AtMAP65-1 340-587 (amino acids 340-587) or AtMAP65-1 1-494 (amino acids 1-494). Comparing the cold stability of microtubule bundles induced by full-length AtMAP65-1 with that of AtMAP65-1 340-587 or AtMAP65-1 1-494, we conclude that AtMAP65-1 495-587 acts as a flexible extended loop, playing a crucial role in binding to and stabilizing microtubules in the AtMAP65-1 cross-bridge.     

In vivo dynamics and differential microtubule-binding activities of MAP65 proteins

Plant cells produce different microtubule arrays that are essential for cell division and morphogenesis without equivalent in other eukaryotes. Microtubule-associated proteins influence the behavior of microtubules that is presumed to culminate into transitions from one array to another. We analyzed the microtubule-binding properties of three Arabidopsis (Arabidopsis thaliana) members, AtMAP65-1, AtMAP65-4, and AtMAP65-5, in live cells using laser scanning confocal microscopy. Depending on the overall organization of the cortical array, AtMAP65-1-GFP (green fluorescent protein) and AtMAP65-5-GFP associated with a subset of microtubules. In cells containing both coaligned and oblique microtubules, AtMAP65-1-GFP and AtMAP65-5-GFP tended to be associated with the coaligned microtubules. Cortical microtubules labeled with AtMAP65-1-GFP and AtMAP65-5-GFP appeared as thick bundles and showed more resistance to microtubule-destabilizing drugs. The polymerization rates of AtMAP65-1-GFP and AtMAP65-5-GFP microtubules were similar to those of tubulin-GFP marked microtubules but were different from AtEB1a-GFP, a microtubule plus-end-binding EB1-like protein that stimulated polymerization. By contrast, depolymerization rates of AtMAP65-1-GFP- and AtMAP65-5-GFP-labeled microtubules were reduced. AtMAP65-1-GFP associated with polymerizing microtubules within a bundle, and with fixed microtubule termini, suggesting that AtMAP65-1's function is to bundle and stabilize adjacent microtubules of the cortex. Polymerization within a bundle took place in either direction so that bundling occurred between parallel or antiparallel aligned microtubules. AtMAP65-4-GFP did not label cortical microtubules or the preprophase band, despite continuous expression driven by the 35S promoter, and its subcellular localization was restricted to microtubules that rearranged to form a spindle and the polar sides of the spindle proper. The expression of AtMAP65-4 peaked at mitosis, in agreement with a function related to spindle formation, whereas AtMAP65-1 and AtMAP65-5 were expressed throughout the cell cycle.     

植物微管结合蛋白AtMAP65-1在拟南芥气孔保卫细胞中的定位(简报)

植物细胞微管骨架的不同排列方式对细胞的生长分化及形态建成具有重要意义,微管的这种动态组织行为不仅需要自身的组成蛋白-微管蛋白(tubulin),还要有微管辅助蛋白MAPs(Microtubule-associated proteins)的参与[1,2]。即MAPs是一类能够与微管骨架特异结合并调节其动态装配过程及其结构、进而影响微管功能的蛋白大分子。其中,MAP65是最先在烟草悬浮细胞BY-2中纯化出来的、分子量约为65KDa的一个微管结合蛋白家族。     

Putative Arabidopsis homologues of metazoan coiled-coil cytoskeletal proteins

The Arabidopsis thaliana genome encodes about 386 proteins with coiled-coil domains of at least 50 amino acids in length. In mammalian systems, many coiled-coil proteins are part of various cytoskeletal networks including intermediate filament protein, actin-binding proteins and MAP (microtubule-associated proteins). Immunological evidence suggests that some of these cytoskeletal proteins, such as lamins, keratins and tropomyosins, may be conserved in Arabidopsis. However, coiled-coil proteins are of low complexity, and thus, traditional sequence comparison algorithms, such as BLAST may not detect homologies. Here, we use the PROPSEARCH algorithm to detect putative coiled-coil cytoskeletal protein homologues in Arabidopsis. This approach reveals putative intermediate filament protein homologues of filensin, lamin and keratin; putative actin-binding homologues of ERM (ezrin/radixin/moesin), periplakin, utrophin, tropomyosin and paramyosin, and putative MAP homologues of restin/CLIP-170 (cytoplasmic linker protein-170). We suggest that the AtFPP (Arabiopsis thaliana filament-like plant protein) and AtMAP70 (Arabidopsis microtubule-associated protein 70) families of coiled-coil proteins may, in fact, be related to lamins and function as intermediate filament proteins.     

Mini spindles, the XMAP215 homologue, suppresses pausing of interphase microtubules in Drosophila

Drosophila Mini spindles (Msps) protein belongs to a conserved family of microtubule-associated proteins (MAPs). Intriguingly, this family of MAPs, including Xenopus XMAP215, was reported to have both microtubule stabilising and destabilising activities. While they are shown to regulate various aspects of microtubules, the role in regulating interphase microtubules in animal cells has yet to be established. Here, we show that the depletion or mutation of Msps prevents interphase microtubules from extending to the cell periphery and leads to the formation of stable microtubule bundles. The effect is independent of known Msps regulator or effector proteins, kinesin-13/KinI homologues or D-TACC. Real-time analysis revealed that the depletion of Msps results in a dramatic increase of microtubule pausing with little or no growth. Our study provides the first direct evidence to support a hypothesis that this family of MAPs acts as an antipausing factor to exhibit both microtubule stabilising and destabilising activities.     

AtMAP65-1 binds to tubulin dimers to promote tubulin assembly

In Arabidopsis thaliana, the microtubule-associated protein AtMAP65-1 shows various functions on microtubule dynamics and organizations. However, it is still an open question about whether AtMAP65-1 binds to tubulin dimers and how it regulates microtubule dynamics. In present study, the tubulin-binding activity of AtMAP65-1 was investigated. Pull-down and co-sedimentation experiments demonstrated that AtMAP65-1 bound to tubulin dimers, at a molar ratio of 1 : 1. Cross-linking experiments showed that AtMAP65-1 bound to tubulin dimers by interacting with alpha-tubulin of the tubulin heterodimer. Interfering the bundling effect of AtMAP65-1 by addition of salt and monitoring the tubulin assembly, the experiment results indicated that AtMAP65-1 promoted tubulin assembly by interacting with tubulin dimers. In addition, five truncated versions of AtMAP65-1, namely AtMAP65-1 deltaN339 (amino acids 340-587); AtMAP65-1 deltaN494 (amino acids 495-587); AtMAP65-1 340-494 (amino acids 340-494); AtMAP65-1 deltaC495 (amino acids 1-494) and AtMAP65-1 deltaC340 (amino acids 1-339), were tested for their binding activities and roles in tubulin polymerization in vitro. Four (AtMAP65-1 deltaN339, deltaN494, AtMAP65-1 340-494 and deltaC495) from the five truncated proteins were able to co-sediment with microtubules, and three (AtMAP65-1 deltaN339, deltaN494 and AtMAP65-1 340-494) of them could bind to tubulin dimers in vitro. Among the three truncated proteins, AtMAP65-1 deltaN339 showed the greatest activity to promote tubulin polymerization, AtMAP65-1 deltaN494 exhibited almost the same activity as the full length protein in promoting tubulin assembly, and AtMAP65-1 340-494 had minor activity to promote tubulin assembly. On the contrast, AtMAP65-1 deltaC495, which bound to microtubules but not to tubulin dimers, did not affect tubulin assembly. Our study suggested that AtMAP65-1 might promote tubulin assembly by binding to tubulin dimers in vivo.     

The plant microtubule-associated protein AtMAP65-3/PLE is essential for cytokinetic phragmoplast function

Directional cell expansion in interphase and nuclear and cell division in M-phase are mediated by four microtubule arrays, three of which are unique to plants: the interphase array, the preprophase band, and the phragmoplast. The plant microtubule-associated protein MAP65 has been identified as a key structural component in these arrays. The Arabidopsis genome has nine MAP65 genes, and here we show that one, AtMAP65-3/PLE, locates only to the mitotic arrays and is essential for cytokinesis. The Arabidopsis pleiade (ple) alleles are single recessive mutations, and we show that these mutations are in the AtMAP65-3 gene. Moreover, these mutations cause C-terminal truncations that abolish microtubule binding. In the ple mutants the anaphase spindle is normal, and the cytokinetic phragmoplast can form but is distorted; not only is it wider, but the midline, the region where oppositely oriented microtubules overlap, is unusually expanded. Here we present data that demonstrate an essential role for AtMAP65-3/PLE in cytokinesis in plant cells.     

Modulated targeting of GFP-AtMAP65-1 to central spindle microtubules during division

AtMAP65-1 bundles cortical microtubules and we examined how this property is regulated during division in time-lapse studies of Arabidopsis suspension cells expressing GFP-AtMAP65-1. Spindle fluorescence is diffuse during metaphase, restored to the central spindle at anaphase and then compacted at the midline during late anaphase/early telophase. However, mutagenesis of the microtubule-associated protein (MAP) consensus Cdk site to a non-phosphorylatable form allows premature decoration of microtubules traversing the central region of the metaphase spindle without affecting the timing of the subsequent compaction. This suggests that mutagenesis does not affect compaction but does affect a phosphorylation/dephosphorylation switch that normally targets AtMAP65-1 to the central spindle at the metaphase/anaphase transition. GFP-AtMAP65-1 continues to label the midline of the early phragmoplast, suggesting a structural continuity with the central spindle - both structures being composed of anti-parallel microtubules. However, once the cytokinetic apparatus expands into a ring the MAP becomes depleted at the midline. Despite this, cytokinesis is not arrested and membrane and callose are deposited at the cell plate. It is concluded that AtMAP65-1 plays a role in the central spindle at anaphase to early cytokinesis but is not essential at the midline of the phragmoplast at later stages.     

Microtubule-associated proteins bind specifically to the 70-kDa neurofilament protein

Morphological and biochemical evidence have suggested that the components of the neuronal cytoskeleton, microtubules and neurofilaments (NF), interact with each other. Microtubule-associated proteins (MAPs) are plausible candidates for mediating some of these interactions and have been shown to bind to neurofilaments, as well as induce the formation of a viscous complex between neurofilaments and microtubules. By binding 32P-labeled MAPs to neurofilament proteins, which were transferred electrophoretically to nitrocellulose, we determined that, of the three neurofilament subunits, only the core NF70 subunit bound MAPs. The binding to electrophoretically transferred NF70 was specific, saturable, and reversible. Binding parameters were estimated by binding 32P-labeled MAPs to purified NF70 immobilized on nitrocellulose. Approximately 1 mol of MAPs bound per 45 +/- 15 mol of NF70 with an approximate Kd approximately 2.0 +/- 0.9 X 10(-7) M (n = 8). Reassembled filaments in suspension were used to confirm the specific binding. Tubulin and NF70 apparently bind to different sites on MAPs.     

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.     

Putative microtubule-associated proteins from the Arabidopsis genome

Summary. Plant microtubule-associated proteins (MAPs) are important in modulating the function of the microtubule cytoskeleton. Various plant MAPs have already been described. However, because of the complexity of the plant microtubule cytoskeleton and its responses to developmental and environmental stimuli, there are undoubtedly many more MAPs to be discovered. We have used a literature search and the BLAST protein comparison program to identify which model MAPs from other taxa have close homologues in Arabidopsis thaliana. The search revealed Arabidopsis homologues of 14 model MAPs, with E values (numbers of proteins that will match the model protein merely by chance) of <1×10^–10 and homologous domains spanning 98–599 amino acid residues, representing 57.1–97.0% of the model MAP sequence, as well as 22.5–72.8% amino acid identities and 76.3–96.2% conservation of secondary structure in the homologous domain. All of the Arabidopsis homologues have either a full cDNA clone or an expressed sequence tag in the GenBank database and therefore are expressed. The proteins are likely to regulate a variety of functions, including tubulin folding, microtubule nucleation and polymerisation dynamics, microtubule-dependent cell cycle control, organisation of microtubule arrays, interaction of microtubules with plasma-membrane-associated protein complexes, and interactions with various other proteins. The exact functions of these putative MAPs in the plant cell remain to be elucidated empirically. The identification of these putative MAPs opens new avenues for the investigation of the complexities of the plant microtubule cytoskeleton.Present address: School of Biological Sciences, University of Manchester, Manchester, United Kingdom.Correspondence and reprints: School of Biological Sciences A12, University of Sydney, NSW 2006, Australia.Received October 21, 2002; accepted December 30, 2002; published online September 23, 2003     

Higher plants possess two different types of ATX1-like copper chaperones

Copper (Cu) chaperones constitute a family of small Cu+-binding proteins required for Cu homeostasis in eukaryotes. The ATX1 family of Cu chaperones specifically delivers Cu to heavy metal P-type ATPases. The plant Arabidopsis thaliana expresses the ATX1-like Cu chaperone CCH, which exhibits a plant-specific carboxy-terminal domain (CTD) with unique structural properties. We show that CCH homologues from other higher plants contain CTDs with structural properties similar to Arabidopsis CCH. Furthermore, we identify a new ATX1-like Cu chaperone in Arabidopsis, AtATX1, which functionally complements yeast atx1Delta and sod1Delta associated phenotypes, and localizes to the cytosol of Arabidopsis cells. Interestingly, AtATX1, but not full-length CCH, interacts in vivo with the Arabidopsis RAN1 Cu-transporting P-type ATPase by yeast two-hybrid. We propose that higher plants express two types of ATX1-like Cu chaperones: the ATX1-type with a predominant function in Cu delivery to P-type ATPases, and the CCH-type with additional CTD-mediated plant-specific functions.     

A member of a novel Arabidopsis thaliana gene family of candidate Mg2+ ion transporters complements a yeast mitochondrial group II intron-splicing mutant

Autocatalytic activity of some group II introns has been demonstrated in vitro, but helper functions such as the yeast MRS2 protein are essential for splicing in vivo. In our search for such helper factors in plants, we pursued the cloning of two Arabidopsis thaliana homologues, atmrs2-1 and atmrs2-2. Atmrs2-1, but not atmrs2-2, complements the yeast deletion mutant of mrs2, and this is congruent with the prediction of two adjacent transmembrane stretches in AtMRS2-1 and yeast MRS2 but not in AtMRS2-2. This complementation depends on fusion of the native yeast mitochondrial import sequence to atmrs2-1. A differing, non-mitochondrial, cellular targeting in Arabidopsis is supported by the analysis of green fluorescent protein fusion constructs after transient transformation into plant protoplasts. Further members of what now appears to be a family of 10 mrs2 homologues are identified in the Arabidopsis genome. Similarity searches with the PSI-BLAST algorithm in the protein database fail to identify homologues of this novel gene family in any eukaryotes other than yeasts, but do identify its distant relatedness to the corA group of bacterial magnesium transporters. In line with this observation, intramitochondrial magnesium concentrations are indeed restored to wild-type levels in the yeast mutant on complementation with atmrs2-1.     

An SC35-like protein and a novel serine/arginine-rich protein interact with Arabidopsis U1-70K protein

The U1 small nuclear ribonucleoprotein 70-kDa protein, a U1 small nuclear ribonucleoprotein-specific protein, has been shown to have multiple roles in nuclear precursor mRNA processing in animals. By using the C-terminal arginine-rich region of Arabidopsis U1-70K protein in the yeast two-hybrid system, we have identified an SC35-like (SR33) and a novel plant serine/arginine-rich (SR) protein (SR45) that interact with the plant U1-70K. The SR33 and SR45 proteins share several features with SR proteins including modular domains typical of splicing factors in the SR family of proteins. However, both plant SR proteins are rich in proline, and SR45, unlike most animal SR proteins, has two distinct arginine/serine-rich domains separated by an RNA recognition motif. By using coprecipitation assays we confirmed the interaction of plant U1-70K with SR33 and SR45 proteins. Furthermore, in vivo and in vitro protein-protein interaction experiments have shown that SR33 protein interacts with itself and with SR45 protein but not with two other members (SRZ21 and SRZ22) of the SR family that are known to interact with the Arabidopsis full-length U-70K only. A Clk/Sty protein kinase (AFC-2) from Arabidopsis phosphorylated four SR proteins (SR33, SR45, SRZ21, and SRZ22). Coprecipitation studies have confirmed the interaction of SR proteins with AFC2 kinase, and the interaction between AFC2 and SR33 is modulated by the phosphorylation status of these proteins. These and our previous results suggest that the plant U1-70K interacts with at least four distinct members of the SR family including SR45 with its two arginine/serine-rich domains, and the interaction between the SR proteins and AFC2 is modulated by phosphorylation. The interaction of plant U1-70K with a novel set of proteins suggests the early stages of spliceosome assembly, and intron recognition in plants is likely to be different from animals.     

Arabidopsis thaliana MAP65-1 and MAP65-2 function redundantly with MAP65-3/PLEIADE in cytokinesis downstream of MPK4

Plant cytokinesis occurs by the growth of cell plates from the interior to the periphery of the cell. These dynamic events in cytokinesis are mediated by a plant-specific microtubule (MT) array called the phragmoplast, which consists of bundled antiparallel MTs between the two daughter nuclei. The NACK-PQR pathway, a NACK1 kinesin-like protein and mitogen activated protein kinase (MAPK) cascade, is a key regulator of plant cytokinesis through the regulation of phragmoplast MTs. The MT-associated protein MAP65 has been identified as one of the structural components of MT assays involved in cell division, and we recently showed that Arabidopsis AtMAP65-3/PLEIADE (PLE) is a substrate of MPK4 that is a component of the NACK-PQR pathway in Arabidopsis. Here we show that AtMAP65-1 and AtMAP65-2 are also phosphorylated by MPK4. AtMAP65-1 and AtMAP65-2 that localize to the phragmoplast were phosphorylated by MPK4 in vitro. Although mutants of the Arabidopsis AtMAP65-1 and AtMAP65-2 genes exhibited a wild-type phenotype, double mutations of AtMAP65-3 and AtMAP65-1 or AtMAP65-2 caused more severe growth and cytokinetic defects than the single atmap65-3/ple mutation. These results suggest that AtMAP65-1 and AtMAP65-2 also function in cytokinesis downstream of MPK4.Key words: MAP65, microtubule-associated protein, MAPK, cytokinesis, phragmoplast, microtubule, arabidopsisMitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules in eukaryotes, and are involved in various signaling processes in plant development, cell division and responses to endogenous or exogenous stimuli.¹ The NACK-PQR pathway, one of the best-characterized MAPK cascades in plants, has been identified as a key regulator of plant cytokinesis in tobacco. This pathway is composed of NPK1 MAPK kinase kinase (MAPKKK), NQK1/NtMEK1 MAPK kinase (MAPKK), NRK1/NTF6 MAPK and NACK1 kinesin-like protein, an activator of NPK1 MAPKKK.^2–5 During cytokinesis, all these components are localized on the equator of the phragmoplast, which is the plant-specific cytokinetic apparatus organized by microtubules (MTs). Downstream of this pathway, tobacco MAP65-1, an MT-associated protein, is phosphorylated by NRK1/NTF6 MAPK and phosphorylated MAP65-1 is localized to the equator of the phragmoplast.⁶ Phosphorylation of MAP65-1 by NRK1/NTF6 decreases the ability of MAP65-1 to bundle MTs, suggesting that the NACK-PQR pathway regulates expansion of the phragmoplast through the phosphorylation of MAP65.⁶The NACK-PQR pathway also seems to be conserved in Arabidopsis and rice. Several orthologs of components of the NACK-PQR pathway except for MAPK have been identified independently as regulators of cytokinesis in these plants.^3,5,7–14 Recently we reported that ANP MAPKKKs, MPK6/ANQ MAPKK and MPK4 MAPK biochemically constitute the MAPK pathway and HINKEL/AtNACK1 functions as an activator of ANP MAPKKKs.¹⁵ In addition, we revealed that MPK4 MAPK is localized to cell plates during cytokinesis, is required for cytokinesis in Arabidopsis and phosphorylates AtMAP65-3.¹⁶ Although AtMAP65-3 is proposed to be involved in cytokinesis,^17,18 and AtMAP65-1 is supposed to be a substrate of MPK4 based on a series of experiments,^6,19,20 the involvement in cytokinesis of other closely related members of the Arabidopsis MAP65 family, AtMAP65-1 and AtNAP65-2, has yet to be tested. In this report, we suggest redundant functions of these MAP65 molecules in cytokinesis of Arabidopsis.     

The Arabidopsis HINKEL gene encodes a kinesin-related protein involved in cytokinesis and is expressed in a cell cycle-dependent manner

Plant cytokinesis starts in the center of the division plane, with vesicle fusion generating a new membrane compartment, the cell plate, that subsequently expands laterally by continuous fusion of newly arriving vesicles to its margin. Targeted delivery of vesicles is assisted by the dynamic reorganization of a plant-specific cytoskeletal array, the phragmoplast, from a solid cylinder into an expanding ring-shaped structure. This lateral translocation is brought about by depolymerization of microtubules in the center, giving way to the expanding cell plate, and polymerization of microtubules along the edge. Whereas several components are known to mediate cytokinetic vesicle fusion [8-10], no gene function involved in phragmoplast dynamics has been identified by mutation. Mutations in the Arabidopsis HINKEL gene cause cytokinesis defects, such as enlarged cells with incomplete cell walls and multiple nuclei. Proper targeting of the cytokinesis-specific syntaxin KNOLLE [8] and lateral expansion of the phragmoplast are not affected. However, the phragmoplast microtubules appear to persist in the center, where vesicle fusion should result in cell plate formation. Molecular analysis reveals that the HINKEL gene encodes a plant-specific kinesin-related protein with a putative N-terminal motor domain and is expressed in a cell cycle-dependent manner similar to the KNOLLE gene. Our results suggest that HINKEL plays a role in the reorganization of phragmoplast microtubules during cell plate formation.     

Identification of nine sucrose nonfermenting 1-related protein kinases 2 activated by hyperosmotic and saline stresses in Arabidopsis thaliana

Several calcium-independent protein kinases were activated by hyperosmotic and saline stresses in Arabidopsis cell suspension. Similar activation profiles were also observed in seedlings exposed to hyperosmotic stress. One of them was identified to AtMPK6 but the others remained to be identified. They were assumed to belong to the SNF1 (sucrose nonfermenting 1)-related protein kinase 2 (SnRK2) family, which constitutes a plant-specific kinase group. The 10 Arabidopsis SnRK2 were expressed both in cells and seedlings, making the whole SnRK2 family a suitable candidate. Using a family-specific antibody raised against the 10 SnRK2, we demonstrated that these non-MAPK protein kinases activated by hyperosmolarity in cell suspension were SnRK2 proteins. Then, the molecular identification of the involved SnRK2 was investigated by transient expression assays. Nine of the 10 SnRK2 were activated by hyperosmolarity induced by mannitol, as well as NaCl, indicating an important role of the SnRK2 family in osmotic signaling. In contrast, none of the SnRK2 were activated by cold treatment, whereas abscisic acid only activated five of the nine SnRK2. The probable involvement of the different Arabidopsis SnRK2 in several abiotic transduction pathways is discussed.