Somatic Embryogenesis in Cultured Carrot Cells

Somatic embryogenesis in cultured plant cells is an ideal system for investigating the whole process of differentiation and development from single cells to whole plants, and especially the molecular mechanism of expression of totipotency. This review reports recent progress the studies on somatic embryogenesis.     

Phenylalanine Ammonia-Lyase Genes Involved in Anthocyanin Synthesis and the Regulation of its Expression in Suspension Cultured Carrot Cells

D and is also induced rapidly and transiently by transfer of cells to fresh medium and lowering the cell density. From the carrot genomic library, four clones of PAL genes, gDcPAL1,2,3 and 4, were obtained. Analyses of nucleotide sequences revealed that only the gDcPAL3 gene is responsible for the induction of anthocyanin synthesis by 2,4-D. Several cis-elements, boxes M, P, A, L, and G, exist in the proximal promoter region of gDcPAL3. Transient expression experiments in carrot protoplasts using deletion mutants of the proximal promoter region of gDcPAL3 gene showed that boxes M and L, both of which contain core sequences of the Myb binding sites, might play an important role in gDcPAL3 promoter activity. Four myb cDNAs, Dcmyb8,10,12 and 14 were obtained from a carrot subtracted cDNA library and their structure and expression patterns were analyzed. In addition to the analysis of the proximal region of gDcPAL3 promoter, the possibility of the regulation of gene expression by genomic DNA structure and chromatin modification in metabolic differentiation is discussed. Received 10 June 2000/ Accepted in revised form 1 July 2000     

Sources of Nitrogen Supporting Growth and Embryogenesis in Cultured Wild Carrot Tissue

This paper seeks to calarify conflicting reports on the nitrogen requirements for in vitro embryogenesis in Daucus carota. Tissue derived from petiole explants of the wild strain of this species were tested with a variety of sources of cellular nitrogen under conditions otherwise favorable for in vitro embryogenesis. The use of very small, sieved and well-washed inocula reduced the carry-over of soluble materials with the inoculum. Embryo yield was quantified by direct counting of samples. Nitrate at concentrations ranging from 5 to 95 mM KNO₃ supportes only weak growth and very low embryogenesis under the exacting conditions of these experiments. As little as 0.1 mM NH₄Cl added to a nitrate medium allows some embryogenesis and 10 mM NH₄Cl is near optimal when KNO₃ is in the range of 12 to 40 mM concentration. Glutamine, glutamic acid, urea and alanine can individually partially replace NH₄Cl as a supplement to KNO₃. Glutamine, alanine, and possibly glutamic acid can serve as sole sources of nitrogen supporting both good growth and embryogenesis. It was concluded that a reduced nitrogen source is required, at least as a supplement to nitrate, for rapid growth and for in vitro embryogenesis of cultured wild carrot tissue. The relationship of pH of the culture medium to growth and embryogenesis was explored and optima observed at approximately pH 5.4 for both processes.     

Ion Levels and Membrane Potential in Chick Heart Tissue and Cultured Cells

Intracellular concentrations of sodium and potassium as well as resting potentials and overshoots have been determined in heart tissue from chick embryos aged 2–18 days. Intracellular potassium declined from 167 mM at day 2 to 117–119 mM at days 14–18. Intracellular sodium remained nearly constant at 30–35 mM during the same period. The mean resting potential increased from -61.8 mV at day 3 to about -80 mV at days 14–18. The mean overshoot during the same period increased from 12 to 30 mV. P_Na/P_K calculated from the ion data and resting potentials declined from 0.08 at day 3 to 0.01 at days 14–18. Thus, the development of embryonic chick heart during days 2–14 is characterized by a declining intracellular potassium concentration and an increasing resting potential and overshoot. Heart cells from 7- to 8-day embryos, cultured either in monolayer or reassociated into aggregates, were compared with intact tissue of the same age. The intracellular concentrations of sodium and potassium were similar in the three preparations and cultured cells responded to incubation in low potassium medium or treatment with ouabain in a manner similar to that of intact tissue. Resting potentials and overshoots were also similar in the three preparations.     

Factors Affecting Phytoalexin Production in Cultured Carrot Cells

The production of 6-methoxymellein, a phytoalexin, in culturedcarrot cells under various growth conditions was studied usingtwo induction methods; by adding partial hydrolysates obtainedby treating the cells with pectinase or trypsin, or by directlyadding these enzymes to growing cells to cause the release ofcellular components as endogenous elicitor. 6-Methoxymellein production depended greatly on the cell cultureage. Maximal production was found in cells at the early stationaryphase, while actively dividing cells had only negligible amounts.Release of elicitor from the cells by pectinase or trypsin wasalso influenced by the culture stage. Effective elicitor wasobtained only from cells in the late logarithmic and early stationaryphases. 6-Methoxymellein production required the presence of 2,4-D.IAA could not substitute for 2,4-D, though partial hydrolysatesprepared from these cells grown with IAA or without auxin showedsignificant elicitor activity. On the other hand, the productionwas inhibited by actinomycin D or cycloheximide, suggestingthat de novo syntheses of RNA and protein are required for thephytoalexin production. (Received November 17, 1984; Accepted March 2, 1985)     

Light-Induced Synthesis of Anthocyanin in Carrot Cells in Suspension: I. THE FACTORS AFFECTING ANTHOCYANIN PRODUCTION

Takeda, J. 1988. Light-induced synthesis of anthocyanin in carrotcells in suspension. I. The factors affecting anthocyanin production.—J.exp. Bot. 39: 1065–1077. A light-triggered anthocyanin-synthesizing system was establishedfor carrot cells in suspension. A few days after transfer ofthe cells to a 2,4-dichlorophenoxyacetic acid (2,4-D)-free mediumin the dark, light irradiation triggered anthocyanin synthesisand concomitantly stopped expansion growth. Over 90% of thecells synthesized anthocyanin without cell division. By loweringthe concentration of phosphate or both nitrogen and phosphateand delaying the time of onset of irradiation, the productionof anthocyanin per cell increased to a maximum level of 0–8µmol anthocyanin per 10⁶ cells. A change in the physiologicalstate of cells (light-insensitive to light-sensitive state)induced by the transfer to 2,4-D-free medium is suggested tobe a prerequisite for the light-triggered synthesis of anthocyanin. Key words: Anthocyanin production, cultured cells, Daucus carota, light-triggered, 2,4-D     

Callose Synthesis in Spirostanol Treated Carrot Cells is Not Triggered by Cytosolic Calcium, Cytosolic pH or Membrane Potential Changes

Carrot (Daucus carota L.) cell suspensions were treated witha spirostanol saponin from Yucca. This saponin is an elicitorof callose synthesis. Irrespectively of the mode of action ofspirostanol on the callose synthase activity itself, the spirostanol-inducedcallose synthesis in carrot is not preceded by changes in membranepotential, cytosolic free calcium or cytosolic pH. The inabilityof modulators of cytosolic free calcium content (verapamil,nifedipine and Br-A23187), EGTA and a proton pump inhibitor(vanadate) to inhibit or induce callose formation is consistentwith a calcium- and pH-independent mechanism for callose deposition. (Received March 20, 1995; Accepted July 18, 1995)     

Membrane Potential Measurements in Cultured Intestinal Villi

Fragmented epithelia of newborn rat small intestine were successfully cultured for periods of up to 4 weeks. Stable intracellular recordings of membrane potential were obtained from these cultured cells. Membrane resting potential varied according to cell location along a villus. The potentials ranged from -70 to -15 mV, being highest at the tip of the villus. The mean resting potential and membrane resistance were -72.4 mV and 8.6 M Ω, respectively. The membrane potential was markedly dependent on the extracellular K⁺ concentration ([K]₀], but not significantly on [Na]₀ and [Cl]₀-Deprivation of Ca²⁺ from the surrounding medium depolarized the membrane by 20 mV. When the cells were cooled down to 6°C, membrane potential was reduced by 40 mV. Based on these data, basic mechanisms underlying the resting potential are discussed in connection with cell differentiation or maturation.     

桑葚花色苷提取物对乳腺癌细胞凋亡及线粒体膜电位的影响

目的:观察桑葚花色苷提取物对人乳腺癌细胞株MDA-MB-453、MDA-MB-231和MCF-7细胞凋亡及线粒体膜电位的影响.方法:利用超声辅助乙醇萃取法提取桑葚花色苷,pH示差法测定提取物花色苷总含量,以50、100和150 mg/mL桑葚花色苷提取物作用三种乳腺癌细胞MDA-MB-231、MDA-MB-453和MCF-7 24h,采用Annexin V/PI双染流式细胞分析法检测细胞凋亡水平变化,JC-1探针染色激光共聚焦扫描显微镜观察MDA-MB-453细胞线粒体膜电位水平变化.结果:凋亡分析结果表明,桑葚花色苷提取物作用后三种乳腺癌细胞凋亡率均升高,显示出促凋亡效应,且具有剂量-效应关系,100和150 mg/mL组凋亡率显著升高(P＜0.05).激光共聚焦扫描显微镜检测结果显示,桑葚花色苷提取物作用24h,可使MDA-MB-453细胞线粒体膜电位显著下降,表现为红色/绿色荧光的比值显著降低(P＜0.05).结论:桑葚花色苷提取物可显著降低乳腺癌细胞线粒体膜电位,并促发细胞凋亡.     

Breakdown of Phosphatidylinositol during the Elicitation of Phytoalexin Production in Cultured Carrot Cells

Elicitor-induced production of the phytoalexin, 6-methoxymellein, in cultured carrot cells was appreciably depressed by the calmodulin inhibitors N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and trifluoperazine. An inhibitor of Ca²⁺-phospholipid dependent protein kinase (protein kinase C), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, also inhibited the phytoalexin production in carrot. Both phorbol ester and synthetic diacylglycerol, activators of protein kinase C, showed an ability to induce 6-methoxymellein production even in the absence of elicitor. Phosphatidylinositol-degrading phospholipase activity increased in elicitor-treated carrot cells without a notable lag, and a product of this reaction, inositol trisphosphate, appeared to increase in parallel with the phospholipase activity. These results suggest that breakdown of phosphatidylinositol takes place in the elicitor-treated carrot cells. The messengers liberated from the phospholipid in the plasma membrane may participate in the elicitation process by controlling the activity of protein kinase C-like enzyme(s) and Ca²⁺-mediated processes including calmodulin.     

Effects of Growth Regulators on the Induction of Anthocyanin Synthesis in Carrot Suspension Cultures

The effects of plant growth regulators were investigated onanthocyanin synthesis induced by removing auxin from carrotsuspension cultures. Of the auxins tested, 2,4-D showed thestrongest inhibiting effect on anthocyanin synthesis and hadthe strongest promoting effect on undifferentiated growth. When2,4-D was added to anthocyanin synthesizing cells, in whichcell division had ceased, anthocyanin synthesis was repressedimmediately, accumulated anthocyanin disappeared and cell divisionresumed. All cytokinins examined promoted anthocyanin synthesisin the absence of auxin. Both gibberellic acid (GA₃) and abscisicacid inhibited anthocyanin synthesis in media lacking 2,4-D,though GA³ showed no effect on cell division. These effectsof growth regulators on anthocyanin synthesis are similar tothose reported for their effects on embryogenesis [Fujimuraand Komamine (1975) Plant Sci. Lett. 5: 359, (1979) Z. Pflanzenphysiol.95: 13, (1980) Z. PJlanzenphysiol. 99: 1]. The relationshipbetween the induction of anthocyanin synthesis, metabolic differentiation,and embryogenesis are discussed. ¹ Present address: Department of Biology, College of Arts andSciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo153, Japan. ² Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Miyagi 980, Japan. (Received November 28, 1985; Accepted July 23, 1986)     

Anthocyanin Production in Cultured Euphorbia millii Cells Using Film Culture Vessels

The anthocyanin production of cultured Euphorbia millii cells using envelope-shaped film culture vessels increased with the decrease of film thickness. The optimum temperature for anthocyanin production under the illumination was at 22°C. The anthocyanin production was increased by 2 times with mild agitation.     

Light-Triggered Changes of Membrane Potential in Cells of Anthoceros punctatus and their Relation to Activation of Chloroplast ATPase

Post-illumination transients of the membrane potential wererevealed in cells of Anthoceros punctatus upon short (1–2s) irradiation with photosynthetically active light. An initialfast depolarization of the cell by 20–30 mV after a lagperiod of 2 s and a subsequent slow repolarization were recordedwith micro-electrodes positioned both in the chloroplast andin transparent parts of the cell. The potential changes of similarkinetics were also observed upon continuous illumination. Post-illuminationpotential changes were abolished by 3-(3, 4-dichlorophenyl)-l,l-dimethylurea and dicyclohexylcarbodiimide and were stimulatedby the addition of NH₄C1. It is assumed that the light-triggeredpotential change across the plasmalemma of A. punctatus aredetermined by the light activation of chloroplast ATPase. Thisassumption is further supported by observations of post-illuminationtransient increase of the chlorophyll fluorescence in preparationsof A. punctatus. Key words: Cell membrane potential, Chloroplast, Photosynthesis     

The Localization in Cultured Carrot Cells of Factors that Induce their Growth

Various previously recognized parts of the complex of growthfactors present in the liquid endosperm of the coconut or inimmature fruits of Aesculus woerlitzensis were generally tritiated.The labeled growth factors were applied singly to culture mediawhich contained balanced requirements that had caused carrotexplants to proliferate and grow in accordance with combinationsof growth factors supplied. By the usc of electron microscopyand autoradiography, the radioactivity from each source wasdetected in the cells and its density and distribution, in theform of developed grains over different cellular compartmentsand organelles, was determined. The tabulated data relate tofour labeled sources as observed over seven cellular compartmentsunder six experimental treatments. Electron micrographs alsoshow how the radioactivity from the various sources relatedto organization of the cells. The distribution of radioactivity within the cells varied withthe source. Both ³H-myo-inositol and the tritiated growth factorsfrom Aesculus (³H-AF_1Aesc) with which it interacts (as in so-calledGrowth Promoting System I) contributed radioactivity, preferentially,to cell walls and sites of their formation in culturcd carrotcells. Both ³H-IAA and ³H-zatin (as in so-called Growth PromotingSystem II) contributed their radioactivity preferentially tothe nucleoli of the cultured cells. Some other conspicuous distributionsof radioactivity (e.g. from ³H-AF_1Aesc to plastids and from³H-IAA to the interstitial substance, i.e. middle lamella, whereenlarging cells separate) involved these tritiated moietieswithout regard to their counterparts in Growth Promoting SystemsI and II, respectively. The problems raised by such multiple effects due to differentgrowth factors acting singly and in combinations at differentcell sites are both recognized and discussed. growth factors, Aesculus woerlitzeensis, autoradiography, tritiation, cell sites, carrot, Daucus carota, coconut, electron microscopy     

Functions of the Cell Wall in the Interactions of Plant Cells: Analysis Using Carrot Cultured Cells

Interactions between cells and between tissues are importantin the development and morphogenesis of higher plants. Attemptsto characterize the role of the cell wall in such interactionshave benefited from the use of carrot (Daucus carota L.) culturedcells in vitro as a model system. The development of carrotcells in culture can be divided into three processes: the acquisitionof embryogenic competence; the development of the embryo; andthe maturation and dormancy of the embryo. Induction of non-embryogeniccallus is accompanied by weakened intercellular attachment,decreased levels of endogenous ABA and a decrease in responsivenessto exogenous ABA. Cell wall polysaccharides are known to beinvolved in various developmental and morphogenetic events.In carrot cultured cells, possible roles in intercellular attachmenthave been proposed for arabinan and xylose in the neutral sugarregions of pectins, and various extracellular proteins havebeen shown to be involved in somatic embryogenesis in vitro.Some of these proteins are also present around and/or in zygoticembryos, possibly being involved in the formation and functionsof zygotic embryos and seeds. A 57-kDa extracellular solubleglycoprotein that binds to insulin-like peptides and an 18-kDaextracellular insoluble cystatin that inhibits the proteinasesof germinating seeds of carrot might be involved in cellularsignal transduction and inter-tissue interaction, respectively,in carrot seeds. ¹ Recipient of the JSPP Young Investigator Award, 1997     

Membrane Potentials of Heterotrophically Cultured Tobacco Cells

The electrophysiological properties of the membrane of Nicotianatabacum var. Sarnsun cultured cells were determined using amicroelectrode technique in standard medium containing 1 mMKC1, 1 mM NaCl and 1 mu CaCl₂ at pH 7. Tobacco callus was derivedfrom the pith (E_m=–104.4%16.2 mV). The membrane potentialsof the callus cells did not show a symmetrical Gaussian distributionbut were scattered over a wide range. The percentage of highmembrane potential cells increased as the subculture was continueduntil about 11 months and then decreased. The response of themembrane potential to electric stimulus, ionic composition,metabolic inhibitors, sugars and amino acids was characteristicof high (E_m=–{small tilde}–160 220 mV; H-cells)and low (E_m=–80{small tilde}–90 mV; L-cells) membranepotential cells. The membrane potential of H-cells was largelydepolarized by addition of CN^–, carbonium cyanide m-chlorophenylhydrazone,decyclohexylcarbodiimide, and triphenyltin chloride and transientlydepolarized by addition of glucose, galactose, mannose or sucrose,and D-alanine, L-alanine or Llysine, but the membrane potentialof L-cells was not. (Received December 3, 1982; Accepted March 16, 1983)     

Membrane Potential and Resistance in Relation to Cytoplasmic pH in Nitellopsis

The responses of membrane potential and membrane resistanceof tonoplast-free Nitellopsis cells to step changes of internalpH (pH_i) from 7 were studied during continuous perfusion withmedia containing either 1 mu ATP or no ATP. Whether ATP waspresent or not, the time course of E_m responses was composedof an initial rapid change (initial phase) and a subsequentslow change (second phase). At the end of the second phase,E_m attained nearly stable values. When E_m values of ATP-containingcells obtained at the peak of the initial phase were plottedagainst pH_i, E_m was found to hyperpolarize most at pH_i 6.5.This was also found for steady E_m values measured at the endof the second phase. The E_m values of ATP-lacking cells werealmost insensitive to pH_i changes between 4 and 8, but morepositive than those of ATP-containing cells at pH_i 4–7.5.Above pH_i 8, no difference in E_m was observed between the twotypes of cells. In this range of pH_i, the E_m change in the initialphase amounted to about 60 mV per unit of pH_i change. The light-induced hyperpolarization still occurred at pH_i 6.5where the electrogenic potential was maximal, and over the widepH_i range, from 6.0 to 7.5 even when pH_i was strongly buffered.Thus, we concluded that the pH_i change may not be the causeof light-induced hyperpolarization. (Received December 5, 1983; Accepted June 18, 1984)     

Proteins binding to DNA and their Relation to Growth in Cultured Mammalian Cells

Analysis of the proteins of mouse fibroblasts which can bind to DNA suggests that one of them may control DNA synthesis.     

Oscillations of the Membrane Potential of Pulvinar Motor Cells In Situ in Relation to Leaflet Movements of Desmodium motorium

The ultradian rhythmic movement of the lateral leaflets of Desmodiummotorium is accompanied by rhythmic changes of the extra- andintracellular electrical potentials in the pulvinus, which aremeasured in situ in the pulvinus against the bathing solutionof the petiole. Extra- and intracellular potentials oscillatewith 180'b0 phase difference to each other, as shown by simultaneousmeasurements of both types of potentials in the abaxial partof the pulvinus. Light-induced changes of these potentials movein opposite directions. The in situ membrane potential of themotor cells of the pulvinus was calculated from the differencebetween the extra- and intracellular potentials. It was foundto oscillate between –136 and –36 mV, in phase withthe intracellular and inverse to the extracellular potential.The phase relationship between the leaflet movement rhythm andthe in situ membrane potential rhythm was as follows: downwardmovement is preceded and accompanied by a strong depolarization,upward movement by hyperpolarization. Our results suggest that membrane depolarization in pulvinarmotor cells of Desmodium motorium drives and controls potassiumefflux and hyperpolarization potassium influx via potassiumchannels. Key words: Desmodium pulvinus, leaf movement, pulvinar motor cells, electrical potential     

Evaluating Mitochondrial Membrane Potential in Cells

Permeant cationic fluorescent probes are widely employed to monitor mitochondrial transmembrane potential and its changes. The application of such potential-dependent probes in conjunction with both fluorescence microscopy and fluorescence spectroscopy allows the monitoring of mitochondrial membrane potential in individual living cells as well as in large population of cells. These approaches to the analysis of membrane potential is of extremely high value to obtain insights into both the basic energy metabolism and its dysfunction in pathologic cells. However, the use of fluorescent molecules to probe biological phenomena must follow the awareness of some principles of fluorescence emission, quenching, and quantum yield since it is a very sensitive tool, but because of this extremely high sensitivity it is also strongly affected by the environment. In addition, the instruments used to monitor fluorescence and its changes in biological systems have also to be employed with cautions due to technical limits that may affect the signals. We have therefore undertaken to review the most currently used analytical methods, providing a summary of practical tips that should precede data acquisition and subsequent analysis. Furthermore, we discuss the application and feasibility of various techniques and discuss their respective strength and weakness.