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Plant growth and development relies on the accurate positioning of the cell plate between dividing cells during cytokinesis. The cell plate is synthetized by a specialized structure called the phragmoplast, which contains bipolar microtubules that polymerize to form a framework with the plus ends at or near the division site. This allows the transport of Golgi-derived vesicles toward the plus ends to form and expand the cell plate. Actin filaments play important roles in cell plate expansion and guidance in plant cytokinesis at the late phase, but whether they are involved at the early phase is unknown. To investigate this further, we disrupted the actin filaments in cell cycle-synchronized tobacco BY-2 cells with latrunculin B (LatB), an actin polymerization inhibitor. We observed the cells under a transmission electron microscope or a spinning-disk confocal laser scanning microscope. We found that disruption of actin filaments by LatB caused the membrane vesicles at the equatorial plane of the cell plate to be dispersed rather than form clusters as they did in the untreated cells. The midzone constriction of phragmoplast microtubules also was perturbed in LatB-treated cells. The live cell imaging and kymograph analysis showed that disruption of actin filaments also changed the accumulation timing of NACK1 kinesin, which plays a crucial role in cell plate expansion. This suggests that there are two functionally different types of microtubules in the phragmoplast. Together, our results show that actin filaments regulate phragmoplast microtubules at the initial phase of plant cytokinesis., (� The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Arabidopsis ASYMMETRIC LEAVES2 (AS2) plays a critical role in leaf adaxial-abaxial partitioning by repressing expression of the abaxial-determining gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3). We previously reported that six CpG dinucleotides in its exon 6 are thoroughly methylated by METHYLTRASFERASE1, that CpG methylation levels are inversely correlated with ETT/ARF3 transcript levels and that methylation levels at three out of the six CpG dinucleotides are decreased in as2-1. All these imply that AS2 is involved in epigenetic repression of ETT/ARF3 by gene body DNA methylation. The mechanism of the epigenetic repression by AS2, however, is unknown. Here, we tested mutations of NUCLEOLIN1 (NUC1) and RNA HELICASE10 (RH10) encoding nucleolus-localized proteins for the methylation in exon 6 as these mutations enhance the level of ETT/ARF3 transcripts in as2-1. Methylation levels at three specific CpGs were decreased in rh10-1, and two of those three overlapped with those in as2-1. Methylation levels at two specific CpGs were decreased in nuc1-1, and one of those three overlapped with that in as2-1. No site was affected by both rh10-1 and nuc1-1. One specific CpG was unaffected by these mutations. These results imply that the way in which RH10, NUC1 and AS2 are involved in maintaining methylation at five CpGs in exon 6 might be through at least several independent pathways, which might interact with each other. Furthermore, we found that AS2 binds specifically the sequence containing CpGs in exon 1 of ETT/ARF3, and that the binding requires the zinc-finger-like motif in AS2 that is structurally similar to the zinc finger-CxxC domain in vertebrate DNA methyltransferase1.

The phosphorylation of proteins by protein kinases controls many cellular and physiological processes, which include intracellular signal transduction. However, the underlying molecular mechanisms of such controls and numerous substrates of protein kinases remain to be characterized. The mitogen-activated protein kinase (MAPK) cascade is of particular importance in a variety of extracellular and intracellular signaling processes. In plant cells, the progression of cytokinesis is an excellent example of an intracellular phenomenon that requires the MAPK cascade. However, the way in which MAPKs control downstream processes during cytokinesis in plant cells remains to be fully determined. We show here that comparisons, by two-dimensional difference gel electrophoresis, of phosphorylated proteins from wild-type Arabidopsis thaliana and mutant plants defective in a MAPK cascade allow identification of substrates of a specific MAPK. Using this method, we identified the PATELLIN2 (PATL2) protein, which has a SEC14 domain, as a substrate of MPK4 MAP kinase. PATL2 was concentrated at the cell division plane, as is MPK4, and had binding affinity for phosphoinositides. This binding affinity was altered after phosphorylation of PATL2 by MPK4, suggesting a role for the MAPK cascade in the formation of cell plates via regeneration of membranes during cytokinesis., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.)

Correct positioning of membrane proteins is an essential process in eukaryotic organisms. The plant hormone auxin is distributed through intercellular transport and triggers various cellular responses. Auxin transporters of the PIN-FORMED (PIN) family localize asymmetrically at the plasma membrane (PM) and mediate the directional transport of auxin between cells. A fungal toxin, brefeldin A (BFA), inhibits a subset of guanine nucleotide exchange factors for ADP-ribosylation factor small GTPases (ARF GEFs) including GNOM, which plays a major role in localization of PIN1 predominantly to the basal side of the PM. The Arabidopsis genome encodes 19 ARF-related putative GTPases. However, ARF components involved in PIN1 localization have been genetically poorly defined. Using a fluorescence imaging-based forward genetic approach, we identified an Arabidopsis mutant, bfa-visualized exocytic trafficking defective1 (bex1), in which PM localization of PIN1-green fluorescent protein (GFP) as well as development is hypersensitive to BFA. We found that in bex1 a member of the ARF1 gene family, ARF1A1C, was mutated. ARF1A1C localizes to the trans-Golgi network/early endosome and Golgi apparatus, acts synergistically to BEN1/MIN7 ARF GEF and is important for PIN recycling to the PM. Consistent with the developmental importance of PIN proteins, functional interference with ARF1 resulted in an impaired auxin response gradient and various developmental defects including embryonic patterning defects and growth arrest. Our results show that ARF1A1C is essential for recycling of PIN auxin transporters and for various auxin-dependent developmental processes.

Cytokinesis in plants is achieved by the formation of the cell plate. A pathway that includes mitogen-activated protein (MAP) kinase kinase kinase and MAP kinase kinase (MAPKK) plays a key role in the control of plant cytokinesis. We show here that a MAP kinase, MPK4, is required for the formation of the cell plate in Arabidopsis thaliana. Single mutations in MPK4 caused dwarfism and characteristic defects in cytokinesis, such as immature cell plates, which became much more prominent upon introduction of a mutation in MKK6/ANQ, the MAPKK for cytokinesis, into mpk4. MKK6/ANQ strongly activated MPK4 in protoplasts, and kinase activity of MPK4 was detected in wild-type tissues that contained dividing cells but not in mkk6/anq mutants. Fluorescent protein-fused MPK4 localized to the expanding cell plates in cells of root tips. Expansion of the cell plates in mpk4 root tips appeared to be retarded. The level of MPK11 transcripts was markedly elevated in mpk4 plants, and defects in the mpk4 mpk11 double mutant with respect to growth and cytokinesis were more severe than in the corresponding single mutants. These results indicate that MPK4 is the downstream target of MKK6/ANQ in the regulation of cytokinesis in Arabidopsis and that MPK11 is also involved in cytokinesis.

Cytokinesis is regulated to ensure the precise partitioning of cytoplasm and duplicated chromosomes to daughter cells. The NACK-PQR pathway, which includes NACK1 kinesin-like protein (KLP) and a mitogen-activated protein kinase (MAPK) cascade, plays a key role in cytokinesis in tobacco cells. Although HINKEL/AtNACK1 (HIK) KLP, ANP MAP kinase kinase kinases (MAPKKKs) and MKK6/ ANQ MAP kinase kinase (MAPKK) have been identified independently as regulators of cytokinesis in Arabidopsis thaliana, the involvement of HIK, ANPs and MKK6/ANQ in a regulatory cascade remains to be demonstrated. Here we provide details of the protein kinase pathway that controls cytokinesis in A. thaliana. Analysis of the subcellular distribution of six MAPKKs of A. thaliana that had been fused to green fluorescent protein revealed that only MKK6/ANQ protein was concentrated at the equatorial plane of the phragmoplast, at the site of localization of HIK. Expression of MKK6/ANQ in yeast cells replaced the growth-control function of the MAPKK encoded by yeast PBS2, provided that both ANP1 MAPKKK and HIK [or TETRASPORE/AtNACK2 (TES)] KLP were coexpressed, suggesting that ANP1 activates MKK6/ANQ in the presence of HIK (or TES). Coexpression of HIK and ANP3 (another member of the ANP MAPKKK family) weakly activated MKK6/ANQ but that of TES and ANP3 did not. MKK6/ANQ phosphorylated MPK4 MAPK in vitro to activate the latter kinase. Thus cytokinesis in A. thaliana is controlled by a pathway that consists of ANP MAPKKKs that can be activated by HIK and MKK6/ANQ MAPKK, with MPK4 MAPK being a probable target of MKK6/ANQ.

Nuclear DNA sequence data for diploid organisms are potentially a rich source of phylogenetic information for disentangling the evolutionary relationships of closely related organisms, but present special phylogenetic problems owing to difficulties arising from heterozygosity and recombination. We analyzed allelic relationships for two nuclear gene regions (phosphoenolpyruvate carboxykinase and elongation factor-1a), along with a mitochondrial gene region (NADH dehydrogenase subunit 5), for an assemblage of closely related species of carabid beetles (Carabus subgenus Ohomopterus). We used a network approach to examine whether the nuclear gene sequences provide substantial phylogenetic information on species relationships and evolutionary history. The mitochondrial gene genealogy strongly contradicted the morphological species boundary as a result of introgression of heterospecific mitochondria. Two nuclear gene regions showed high allelic diversity within species, and this diversity was partially attributable to recombination between various alleles and high variability in the intron region. Shared nuclear alleles among species were rare and were considered to represent shared ancestral polymorphism. Despite the presence of recombination, nuclear allelic networks recovered species monophyly more often and presented genetic differentiation patterns (low to high) among species more clearly. Overall, nuclear gene networks provide clear evidence for separate biological species and information on the phylogenetic relationships among closely related carabid beetles.

The mitogen-activated protein kinase (MAPK) cascade is involved in responses to biotic and abiotic stress in plants. In this study, we isolated a new MAPK, NtMPK4, which is a tobacco homolog of Arabidopsis MPK4 (AtMPK4). NtMPK4 was activated by wounding along with two other wound-responsive tobacco MAPKs, WIPK and SIPK. We found that NtMPK4 was activated by salicylic acid-induced protein kinase kinase (SIPKK), which has been isolated as an SIPK-interacting MAPK kinase. In NtMPK4 activity-suppressed tobacco, wound-induced expression of jasmonic acid (JA)-responsive genes was inhibited. NtMPK4-silenced plants showed enhanced sensitivity to ozone. Inversely, transgenic tobacco plants, in which SIPKK or the constitutively active type SIPKK(EE) was overexpressed, exhibited greater responsiveness to wounding with enhanced resistance to ozone. We further found that NtMPK4 was expressed preferentially in epidermis, and the enhanced sensitivity to ozone in NtMPK4-silenced plants was caused by an abnormal regulation of stomatal closure in an ABA-independent manner. These results suggest that NtMPK4 is involved in JA signaling and in stomatal movement.

Several components of mitogen-activated protein kinase (MAPK) cascades have been identified in higher plants and have been implicated in cellular responses to a wide variety of abiotic and biotic stimuli. Our recent work has demonstrated that a MAP kinase cascade is involved in the regulation of cytokinesis in plant cells. The MAP kinase cascade in tobacco includes NPK1 MAPK kinase kinase, NQK1 MAPK kinase, and NRK1 MAPK, and its activation is triggered by the binding of NACK1/2 kinesin-like protein to the NPK1 MAPK kinase kinase at the late M-phase of the cell cycle. We refer to this cascade as the NACK-PQR pathway. In this review, we introduce a mechanism for the regulation of plant cytokinesis, focusing on the role of the NACK-PQR pathway.

The human dad-1 cDNA homolog was isolated from rice plants. The amino acid sequence of the predicted protein product is well conserved in both animals and plants. This rice dad-1 homolog can rescue the temperature-sensitive dad-1 mutants of hamster cells from apoptotic death, suggesting that the rice dad-1 homolog also functions as a suppressor for programmed cell death.

Monoclonal antibodies were purified from the culture medium of hybridoma from systemic lupus erythematosus (SLE)-prone MRL/Mp-Ipr-Ipr mice with affinity chromatography. Binding activity of the antibodies to double-stranded DNA from salmon milt was actually shown in enzyme-linked immunosorbent assay (ELISA). Circular dichroism (CD) spectrum of DNA solution (150 mM NaCl, 20 mM Na2HPO4, pH 7.4) which showed B-type conformation, changed significantly by addition of the anti-DNA antibodies. These results indicate that conformational change of DNA occurred by binding of anti-DNA antibodies, forming DNA-anti-DNA antibody immune complex.

Certain nematode infections induce eosinophil infiltration and granulomatous responses in the lungs. To examine the role of mast cells in the development of lung lesions, normal +/+ and genetically mast cell-deficient Ws/Ws rats were infected with the nematode Nippostrongylus brasiliensis. In +/+ rats, numbers of eosinophils in bronchoalveolar lavage fluid (BALF) increased significantly 3-7 days after infection, and granulomatous responses composed of histiocytes/ macrophages and multinucleate giant cells were triggered in the lungs 3-14 days after infection. Challenge infection, which was carried out on day 28 after primary infection, induced much higher levels of granulomatous response than after primary infection, suggesting that the response is mediated at least in part by an immunological mechanism. In Ws/Ws rats, both the eosinophil percentage in BALF and the size of the granulomas in the lungs were significantly smaller than in +/+ rats after primary as well as after challenge infection. The amount of rat mast cell protease (RMCP) II in +/+ rat BALF was increased 1 day after primary infection and more significantly after challenge infection, suggesting that lung mucosal mast cells were activated more markedly after the challenge infection. In Ws/Ws rats, RMCP II was undetectable throughout the observation period. The time course of nematode migration in the lungs did not differ in +/+ and Ws/Ws rats. These results suggest that mast cell activation might be relevant to eosinophil infiltration and granulomatous response in the lungs, although the responses do not affect lung migration of the nematode.