Is cytoplasmic pH involved in the regulation of cell cycle in plants?

Modifications of cytoplasmic pH has biological significance in animal and plant cell development. Many observations suggest an important function of cytopiasmic pH in mitotic signalling in animal ceils. In Bidens pilosa cultivated under white light, acidification of cytoplasm, observed after mechanical trauma, is associated with an inhibition of DNA synthesis and a decrease in mitotic frequency. In contrast, in Bidens pilosa cultivated under blue light, mechanical stimulation induces an increase of cytoplasmic pH and stimulation of DNA duplication and mitotic activity. A correlation has been established between transient variations of cytoplasmic pH and rapid modification in cell development. The critical role of cytoplasmic pH in the regulation of the cell cycle in plants is discussed.     

Effects of external pH,fusicoccin and butyrate on the cytoplasmic pH in barley root tips measured by 31P-nuclear magnetic resonance spectroscopy

³¹P-Nuclear magnetic resonance spectroscopy was used to measure the cytoplasmic pH (pH_c) in barley (Hordeum vulgare L.) root tips. As the external pH was raised from 4–10, pH_c was found to increase from 7.44 to 7.75. The sensitivity of pH_c to changes in external pH decreased with increasing external pH. Metabolic inhibition by sodium azide caused pH_c to fall by 0.3 units. Addition of 10 mM butyrate resulted in a gradual decline in pH_c, by approx. 0.3 units over 90 min. At a concentration of 1 mM, butyrate had no effect on pH_c even after 2 h. Fusicoccin caused pH_c to rise by 0.1–0.2 units. In maize (Zea mays L.) root tips, pH_c was shown to have a similar sensitivity to fusicoccin. The results are discussed in relation to the regulation of pH_c and the possible role of pH_c in determining transmembrane electrical potential differences.Abbreviations and symbols FC Fusicoccin - NMR nuclear magnetic resonance - p.d. membrane electrical potential difference - pH_c cytoplasmic pH - P1 inorganic phosphate - chemical shift     

QQT proteins colocalize with microtubules and are essential for early embryo development in Arabidopsis

During Arabidopsis embryogenesis, the control of division between daughter cells is critical for pattern formation. Two embryo-defective (emb) mutant lines named quatre-quart (qqt) were characterized by forward and reverse genetics. The terminal arrest of qqt1 and qqt2 embryos was at the octant stage, just prior to the round of periclinal divisions that establishes the dermatogen stage . Homozygous embryos of a weaker allele of qqt1 were able to divide further, resulting in aberrant periclinal divisions. These phenotypic analyses support an essential role of the QQT proteins in the correct formation of the tangential divisions. That an important proportion of qqt1 embryos were arrested prior to the octant stage indicated a more general role in cell division. The analysis of QQT1 and QQT2 genes revealed that they belong to a small subgroup of the large family encoding ATP/GTP binding proteins, and are widely conserved among plants, vertebrates and Archaea. We showed that QQT1 and QQT2 proteins interact with each other in a yeast two-hybrid system, and that QQT1 and QQT2 tagged by distinct fluorescent probes colocalize with microtubules during mitosis, in agreement with their potential role in cell division and their mutant phenotype. We propose that QQT1 and QQT2 proteins participate in the organization of microtubules during cell division, and that this function is essential for the correct development of the early embryo.     

Physical Limits on the Precision of Mitotic Spindle Positioning by Microtubule Pushing forces

Tissues are shaped and patterned by mechanical and chemical processes. A key mechanical process is the positioning of the mitotic spindle, which determines the size and location of the daughter cells within the tissue. Recent force and position‐fluctuation measurements indicate that pushing forces, mediated by the polymerization of astral microtubules against­ the cell cortex, maintain the mitotic spindle at the cell center in Caenorhabditis elegans embryos. The magnitude of the centering forces suggests that the physical limit on the accuracy and precision of this centering mechanism is determined by the number of pushing microtubules rather than by thermally driven fluctuations. In cells that divide asymmetrically, anti‐centering, pulling forces generated by cortically located dyneins, in conjunction with microtubule depolymerization, oppose the pushing forces to drive spindle displacements away from the center. Thus, a balance of centering pushing forces and anti‐centering pulling forces localize the mitotic spindles within dividing C. elegans cells.     

TORC1 signaling regulates cytoplasmic pH through Sir2 in yeast

Glucose controls the phosphorylation of silent information regulator 2 (Sir2), a NAD⁺‐dependent protein deacetylase, which regulates the expression of the ATP‐dependent proton pump Pma1 and replicative lifespan (RLS) in yeast. TORC1 signaling, which is a central regulator of cell growth and lifespan, is regulated by glucose as well as nitrogen sources. In this study, we demonstrate that TORC1 signaling controls Sir2 phosphorylation through casein kinase 2 (CK2) to regulate PMA1 expression and cytoplasmic pH (pHc) in yeast. Inhibition of TORC1 signaling by either TOR1 deletion or rapamycin treatment decreased PMA1 expression, pHc, and vacuolar pH, whereas activation of TORC1 signaling by expressing constitutively active GTR1 (GTR1Q65L) resulted in the opposite phenotypes. Deletion of SIR2 or expression of a phospho‐mutant form of SIR2 increased PMA1 expression, pHc, and vacuolar pH in the tor1Δ mutant, suggesting a functional interaction between Sir2 and TORC1 signaling. Furthermore, deletion of TOR1 or KNS1 encoding a LAMMER kinase decreased the phosphorylation level of Sir2, suggesting that TORC1 signaling controls Sir2 phosphorylation. It was also found that Sit4, a protein phosphatase 2A (PP2A)‐like phosphatase, and Kns1 are required for TORC1 signaling to regulate PMA1 expression and that TORC1 signaling and the cyclic AMP (cAMP)/protein kinase A (PKA) pathway converge on CK2 to regulate PMA1 expression through Sir2. Taken together, these findings suggest that TORC1 signaling regulates PMA1 expression and pHc through the CK2–Sir2 axis, which is also controlled by cAMP/PKA signaling in yeast.     

Analysis of Different Methodological Approaches to Measuring Microtubule Length in the Cytoplasm of Cultured Cells

It is generally assumed that microtubules in tissue culture cells extend from the centrosome to cell periphery, and the length of individual microtubules averages several dozens of microns. However, direct electron-microscopic measurements have cast some doubt on this assumption. In this study, the average length of microtubules in cultured Vero cells was estimated using a combined approach. The length of free cytoplasmic and centrosomal microtubules was determined by means of electron microscopy in serial sections; concurrently, the length of free microtubules in the lamella was measured in preparations stained with tubulin antibodies (an indirect immunofluorescent method), by tracing saltatory particle movements along the microtubules in living cells. According to the data of immunofluorescent microscopy, microtubule length in the lamella averaged 4.57 ± 3.69 m. However, since two or more microtubules can overlap, their length may be slightly overestimated by this method. On the other hand, saltatory movements are easy to monitor and measure fairly accurately, but their range may be shorter than the actual microtubule length because of a limited processiveness of motors (kinesin and dynein). On average, the trajectories of saltatory movements in living cells were 3.85 ± 0.72 m long. At the electron-microscopic level, microtubule length was analyzed using pseudo-three-dimensional reconstructions of the microtubule systems around the centrosome and in the lamella. The length of free microtubules in the lamella reached 18 m, averaging 3.33 ± 2.43 m; the average length of centrosomal microtubules was 1.49 ± 0.82 m. Good correspondence between the data on microtubule length and arrangement obtained by different methods allows the conclusion that most of the free microtubules in Vero cells actually have a length of 2–5 m; i.e., they are much shorter than the cell radius (about 25 m). Microtubules extending from the centrosome are shorter still and do not reach the cell periphery. Thus, most microtubules in the lamella of Vero cells are free and their ordered arrangement is not associated with their attachment to the centrosome.     

Microtubule Associated Proteins in Plants and the Processes They Manage

Microtubule associated proteins （MAPs） are proteins that physically bind to microtubules in eukaryotes. MAPs play important roles in regulating the polymerization and organization of microtubules and in using the ensuing microtubule arrays to carry out a variety of cellular functions. In plants, MAPs manage the construction, repositioning, and dismantling of four distinct microtubule arrays throughout the cell cycle. Three of these arrays, the cortical array, the preprophase band, and the phragmoplast, are prominent to plants and are responsible for facilitating cell wall deposition and modification, transducing signals, demarcating the plane of cell division, and forming the new cell plate during cytokinesis. This review highlights important aspects of how MAPs in plants establish and maintain microtubule arrays as well as regulate cell growth, cell division, and cellular responses to the environment.     

Confocal ratio imaging of cytoplasmic pH during germ tube growth and appressorium induction by Magnaporthe grisea

The role of cytosolic pH (pH_c) in growing germ tubes of the filamentous fungus Magnaporthe grisea was analysed by confocal ratio imaging of the pH-sensitive fluorescent dye 5(6)-carboxyseminaphthorhodafluor-1 (SNARF-1). The cytosol of these cells was successfully loaded with the acetoxymethyl ester of the dye and the pH_c was visualized and quantified during conidium germination, germ tube growth and appressorium induction by simultaneous dual-emission confocal ratio imaging. Calibrations of the free acid in vitro and calibrations in vivo produced results indicating a similar dynamic response in the pH range 6.0–8.0 for both methods. The pH_c in growing germ tubes was consistently pH 7.2±0.1 during all developmental stages analysed. Only slight changes in pH_c (<0.1 pH unit) were found in response to alkaline external pH (pH 8.0). No changes in pH_c occurred in response to an acidic extracellular pH (pH 6.0) or to the presence of nutrients. There was no observation of either pronounced gradients or changes in pH_c in growing germ tubes accompanying conidium germination, germ tube growth or early appressorium formation.     

水稻雄性不育系珍汕97A小孢子发育过程中的微管骨架

水稻(Oryza sativa L.)雄性不育系珍汕97A、保持系珍汕97B和恢复系测64三系小孢子发生过程的研究表明:恢复系测64小孢子母细胞细胞质浓,有明显的微管荧光围绕着细胞核.小孢子母细胞经两次减数分裂形成四分体.四分体和小孢子的微管从细胞核表面向胞质周缘延伸,形成放射性排列格局,花粉发育正常.细胞质中有少量点状微管荧光.保持系珍汕97B小孢子发生过程的细胞形态和微管结构与恢复系测64相似,但细胞质中的点状微管荧光多一些.雄性不育系珍汕97A小孢子发生早期,小孢子母细胞内出现液泡,核中染色质凝集,微管荧光很弱,没有清晰的微管丝结构,细胞质中有许多点状微管荧光等不正常现象.小孢子母细胞经过减数分裂形成的四分体也没有清晰的丝状微管结构.随后,所有的小孢子迅速败育.雄性不育系珍汕97A在小孢子母细胞发生的很早时期,微管结构就明显不正常.     

The Aurora-A/TPX2 Axis Directs Spindle Orientation in Adherent Human Cells by Regulating NuMA and Microtubule Stability

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Short-term pH regulation in plants

Cellular pH regulation consists of two features: (i) Long-term pH homeostasis, which ensures that all H⁺ or OH⁻ produced in excess is ultimately removed from the cell and which requires metabolic energy; (ii) short-term reactions of the cell(s) to sudden shifts in intracellular pH, in order to prevent acute disturbances of metabolism. Recent progress in measuring and understanding of mainly short-term cellular regulation is summarized, including cellular responses to pH loads that arise from different sources such as external pH, weak acids/bases, protonophores, metabolic inhibitors, H⁺/cotransport, light and phytohormones. Whereas the plasma membrane H⁺ pump and metabolic adjustments may serve both long- and short-term pH control, physico-chemical buffering and the translocation of H⁺ from and to cellular compartments render only time-limited capacity for the neutralization of pH loads and seem exhausted within minutes. In spite of the widespread opinion that, because of tight regulation, intracellular pH does not vary with time, there is good evidence for long-lasting pH changes in plant cells, i.e. after hormonal stimulation, light/dark changes or carboxylation during crassulacean acid metabolism (CAM). This emphasizes that cytoplasmic pH, besides being well regulated, is essential not only for the regulation of membrane transport but also as a cellular messenger.     

水稻雄性不育系珍汕97A小孢子发育过程中的微管骨架

水稻（Oryza sativaL.)雄性不育系珍汕97A,保持系珍汕97B和恢复系测64三系小孢子发生过程的研究表明;恢复系测64小孢子母细胞细胞质浓,有明显的微管荧光围绕着细胞核。小孢子母细胞经两次减数分裂形成四分体。四分体和小孢子的微管从细胞核表面向胞质周缘延伸,形成放射性排列格局,花粉发育正常。细胞质中有少量点状微管荧光,保持系珍汕97B小孢子发生过程的细胞形态和微管结构与恢复系测64相似。但细胞质中的点状微管荧光多一些。雄性不育系珍汕97A小孢子发生早期,小孢子母细胞内出现液泡,核中染色质凝集,微管荧光很弱,没有清晰的微管丝结构。细胞质中有许多点状微管荧光等不正常现象。小孢子母细胞经过减数分裂形成的四分体也没有清晰的丝状微管结构。随后,所有的小孢子迅速败育,雄性不育系珍汕97A在小孢子母细胞发生的很早时期,微管结构就明显不正常。     

Auxin-binding-protein antibodies and peptides influence stomatal opening and alter cytoplasmic pH

Previous work has shown that stomatal opening induced by indole-3-acetic acid (IAA) in epidermal strips of the orchid Paphiopedilum tonsum L. is preceded by a reduction in cytoplasmic pH (pH_i) of the guard cells. We now report that Fab fragments of an auxin-agonist antibody (D16), directed against a putative auxin-binding domain of the auxin-binding protein ABP1, induce stomatal opening and decrease guard-cell pH_i, as monitored with the acetomethoxy ester of the ratiometric pH indicator Snarf-1. Similar activity was shown by a monoclonal antibody against the same domain. The C-terminal dodecapeptide, Pz152–163 of maize ABP1 (ABPzm1) induced guard-cell alkalinization and closed stomata, as did Fab fragments of a monoclonal antibody (MAC 256) recognising the C-terminal region of ABPzm1. By implicating, for the first time, an auxin-binding protein in mediation of an auxin-dependent physiological response, these findings strongly support an auxin-receptor role for ABP1. Received: 23 December 1997 / Accepted: 16 January 1998     

Cryoconcentration of flavonoid extract for enhanced biophotovoltaics and pH sensitive thin films

Flavonoids are important value added products for dye sensitized solar cells biosensors, functional foods, medicinal supplements, nanomaterial synthesis, and other applications. Brassica oleracea contains high levels of anthocyanins in leaf sap vacuoles, and there are many viable extraction techniques that vary in terms of simplicity, environmental impact, cost, and extract photochemical/electrochemical properties. The efficiency of value added biotechnologies from flavonoid is a function of anthocyanin activity/concentration and molecule stability (i.e., ability to retain molecular resonance under a wide range of conditions). In this paper, we show that block cryoconcentration and partial thawing of anthocyanin from B. oleracea is a green, facile, and highly efficient technique that does not require any special equipment or protocols for producing enhanced value added products. Cryoconcentration increased anthocyanin activity and total phenol content approximately 10 times compared with common extraction techniques. Cryoconcentrated extract had enhanced electrochemical properties (higher oxidation potential), improved chroma, and higher UV absorbance than extract produced with other methods for a pH range of 2–12, with minimal effect on the diffusion coefficient of the extract. As a proof of concept for energy harvesting and sensor applications, dye sensitized solar cells and pH‐sensitive thin films were prepared and tested. These devices were comparable with other recently published biotechnologies in terms of efficacy, but did not require expensive/environmentally detrimental extraction or concentration methods. This low cost, biorenewable, and simple method can be used for development of a variety of value added products. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:206–217, 2018     

Mechanisms of cytoplasmic pH regulation in alkaliphilic strains of Bacillus

The central challenge for extremely alkaliphilic Bacillus species is the need to establish and sustain a cytoplasmic pH that is over two units lower than the highly alkaline medium. Its centrality is suggested by the strong correlation between the growth rate in the upper range of pH for growth, i.e., at values above pH 10.5, and the cytoplasmic pH. The diminishing growth rate at extremely high pH values correlates better with the rise in cytoplasmic pH than with other energetic parameters. There are also general adaptations of alkaliphiles that are crucial prerequisites for pH homeostasis as well as other cell functions, i.e., the reduced basic amino acid content of proteins or segments thereof that are exposed to the medium, and there are other challenges of alkaliphily that emerge from solution of the cytoplasmic pH problem, i.e., reduction of the chemiosmotic driving force. For cells growing on glucose, strong evidence exists for the importance of acidic cell wall components, teichuronic acid and teichuronopeptides, in alkaliphily. These wall macromolecules may provide a passive barrier to ion flux. For cells growing on fermentable carbon sources, this and other passive mechanisms may have a particularly substantial role, but for cells growing on both fermentable and nonfermentable substrates, an active Na1-dependent cycle is apparently required for alkaliphily and the alkaliphile's remarkable capacity for pH homeostasis. The active cycle involves primary establishment of an electrochemical gradient via proton extrusion, a secondary electrogenic Na⁺/H⁺ antiport to achieve net acidification of the cytoplasm relative to the outside pH, and mechanisms for Na⁺ re-entry. Recent work in several laboratories on the critical antiporters involved in this cycle has begun to clarify the number and characteristics of the porters that support active mechanisms of pH homeostasis. Received: August 1, 1997 / Accepted: August 5, 1995     

Outward-rectifying K+ channel activities regulate cell elongation and cell division of tobacco BY-2 cells

Potassium ions (K⁺) are required for plant growth and development, including cell division and cell elongation/expansion, which are mediated by the K⁺ transport system. In this study, we investigated the role of K⁺ in cell division using tobacco BY-2 protoplast cultures. Gene expression analysis revealed induction of the Shaker -like outward K⁺ channel gene, NTORK1 , under cell-division conditions, whereas the inward K⁺ channel genes NKT1 and NtKC1 were induced under both cell-elongation and cell-division conditions. Repression of NTORK1 gene expression by expression of its antisense construct repressed cell division but accelerated cell elongation even under conditions promoting cell division. A decrease in the K⁺ content of cells and cellular osmotic pressure in dividing cells suggested that an increase in cell osmotic pressure by K⁺ uptake is not required for cell division. In contrast, K⁺ depletion, which reduced cell-division activity, decreased cytoplasmic pH as monitored using a fluorescent pH indicator, SNARF-1. Application of K⁺ or the cytoplasmic alkalizing reagent (NH₄)₂SO₄ increased cytoplasmic pH and suppressed the reduction in cell-division activity. These results suggest that the K⁺ taken up into cells is used to regulate cytoplasmic pH during cell division.