Role of Chitinase and Chitin Oligosaccharides in Lignification Response of Cultured Carrot Cells Treated with Mycelial Walls

Chitinase activity was induced in cultured carrot cells by incubationwith mycelial walls of a fungus, Chaetomium globosum. Both intra-and extracellular chitinases were resolved into four componentsby gel filtration chromatography. The extracellular enzymesliberated soluble oligosaccharides of different sizes from insolublechitin, suggesting that these carrot chitinases are endo-hydrolases.The solubilized chitinase digests obtained from insoluble mycelialwalls of C. globosum and chitin were fractionated by gel filtrationchromatography, and the elicitor activity of each fraction forthe accumulation of phenolic acids in cultured carrot cellswas determined. In both solubilized fragments of fungal wallsand of chitin, elicitor-active oligosaccharides were distributedin many fractions, however, potent activity for inducing phenolicacid synthesis was observed in the high molecular weight fractions. (Received October 5, 1987; Accepted February 12, 1988)     

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     

Ethylene-induced Phenylalanine Ammonia-Lyase Activity in Carrot Roots

Ethylene enhanced the activity of phenylalanine ammonialyase in carrot (Daucus carota L., var. “Nauty”) root tissue. Slight increase in enzyme activity was exhibited by root discs incubated in ethylene-free air. It was probably due to the ethylene formed within the sliced tissue. Addition of ethylene to the air stream increased phenylalanine ammonia-lyase activity and the total protein content of the discs until maximum activity was reached after 36 to 48 hours of incubation. The continuous presence of ethylene was required to maintain high level of activity. Ethylene, at a concentration of 10 microliter per liter induced higher activity than at lower or higher concentrations. CO₂ partially inhibited the ethylene-induced activity. Cycloheximide or actinomycin D effectively inhibited the ethylene-induced activity in discs that had not previously been exposed to ethylene. The results appear to support the hypothesis that the mode of action of ethylene may involve both de novo synthesis of the enzyme protein and protection or regulation of activity of the induced enzyme.     

Protein Kinase Inhibitors Inhibit Stimulation of Inositol Phospholipid Turnover and Induction of Phenylalanine Ammonia-Lyase in Fungal Elicitor-Treated Tobacco Suspension Culture Cells

Elicitor prepared from Phytophthora nicotianae stimulated inositolphospholipid turnover and induced phenylalanine ammonia-lyaseactivity in tobacco suspension culture cells [Kamada and Muto(1994) Plant Cell Physiol. 35: 397]. Protein kinase inhibitors,K252a and staurosporine inhibited both responses. These resultssuggest that inositol phospholipid turnover plays an importantrole in PAL induction through protein kinases. In addition,their mode of inhibition were different, proposing that severaltypes of protein kinases are involved in these elicitor-inducedresponses. ¹Present address: The Johns Hopkins University School of Hygieneand Public Health, 615 N. Wolfe St., Baltimore, Maryland 21205,U.S.A. ²Present address: Nagoya University BioScience Center and GraduateSchool of Agricultural Sciences, Nagoya University, Chikusa-ku,Nagoya, 464-01 Japan.     

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.     

Fungal Elicitor-Mediated Responses in Pine Cell Cultures : III. Purification and Characterization of Phenylalanine Ammonia-Lyase

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is involved in the lignification of pine suspension cultures in response to an elicitor prepared from an ectomycorrhizal fungus. To elucidate the molecular basis of this response, PAL was purified to homogeneity from jack pine (Pinus banksiana) suspension cultures using anion-exchange and chromatofocussing fast protein liquid chromatography. Physical characterization of the enzyme revealed that pine PAL was similar to PAL from other plant sources. Pine PAL had a pH optimum of 8.8, an isoelectric point of 5.75, and a native molecular mass of 340 kilodaltons. The enzyme appears to be a tetramer composed of 77 kilodalton subunits. Chromatographic and western blot analyses were used to identify possible isoenzymic changes in pine PAL in response to elicitation and to determine the nature of the increase in PAL activity associated with inducible lignification in these cultures. Only one species of PAL was detected in P. banksiana cell cultures and increased quantities of this protein were correlated with the enhanced enzyme activity observed in elicited cultures. P. banksiana PAL was not feedback-inhibited by a wide range of phenolic compounds at micromolar concentrations, including the reaction product cinnamic acid. Our data suggest that a different set of metabolic and molecular controls must be in place for the regulation of PAL in pine.     

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)     

Partial Resistance of Carrot to Alternaria dauci Correlates with In Vitro Cultured Carrot Cell Resistance to Fungal Exudates

Although different mechanisms have been proposed in the recent years, plant pathogen partial resistance is still poorly understood. Components of the chemical warfare, including the production of plant defense compounds and plant resistance to pathogen-produced toxins, are likely to play a role. Toxins are indeed recognized as important determinants of pathogenicity in necrotrophic fungi. Partial resistance based on quantitative resistance loci and linked to a pathogen-produced toxin has never been fully described. We tested this hypothesis using the Alternaria dauci – carrot pathosystem. Alternaria dauci, causing carrot leaf blight, is a necrotrophic fungus known to produce zinniol, a compound described as a non-host selective toxin. Embryogenic cellular cultures from carrot genotypes varying in resistance against A. dauci were confronted with zinniol at different concentrations or to fungal exudates (raw, organic or aqueous extracts). The plant response was analyzed through the measurement of cytoplasmic esterase activity, as a marker of cell viability, and the differentiation of somatic embryos in cellular cultures. A differential response to toxicity was demonstrated between susceptible and partially resistant genotypes, with a good correlation noted between the resistance to the fungus at the whole plant level and resistance at the cellular level to fungal exudates from raw and organic extracts. No toxic reaction of embryogenic cultures was observed after treatment with the aqueous extract or zinniol used at physiological concentration. Moreover, we did not detect zinniol in toxic fungal extracts by UHPLC analysis. These results suggest that strong phytotoxic compounds are present in the organic extract and remain to be characterized. Our results clearly show that carrot tolerance to A. dauci toxins is one component of its partial resistance.     

Phenylalanine Ammonia-Lyase Activity and Anthocyanin Accumulation in Wounded Maize Mesocotyls

Activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5) and anthocyanin accumulation were determined in wounded maize (Zea mays L.) mesocotyls. Mesocotyls were wounded with aluminum oxide and were placed in a 15 h light: 9 h dark photoperiod or in the dark. Extractable enzyme activity increased in response to wounding in the photoperiod but not in the dark. Anthocyanin accumulation in mesocotyls placed in the photoperiod decreased in response to wounding. The results are discussed with reference to phenylalanine ammonia-lyase activity in mesocotyl tissue wounded or inoculated with fungal pathogens.     

Immunocytochemical Localization of Phenylalanine Ammonia-Lyase and Cinnamyl Alcohol Dehydrogenase in Differentiating Tracheary Elements Derived from Zinnia Mesophyll Cells

Secondary wall thickening is the most characteristic morphologicalfeature of the differentiation of tracheary elements. Isolatedmesophyll cells of Zinnia elegans L. cv. Canary Bird in differentiationmedium are converted to tracheary elements, which develop lignifiedsecondary wall thickenings. Using this system, we investigatedthe distribution of two enzymes, phenylalanine ammonia-Iyase(PAL) (EC 4.3.1.5 [EC] ) and cinnamyl alcohol dehydrogenase (CAD)(EC 1.1.1.195 [EC] ), by both biochemical and immunological methods.Both PAL and CAD appear to be key enzymes in the biosynthesisof lignin precursors, and they have been shown to be associatedwith the differentiation of tracheary elements. Cultured cellswere collected after various times in culture. The culture mediumwas separated from cells by centrifugation and designated fraction(1), the extracellular fraction. The collected cells were homogenizedand separated into four fractions: (2) cytosol; (3) microsomes;(4) cell walls (loosely bound material); and (5) cell walls(tightly bound material). PAL activity was detected in eachfraction. The extracellular fraction consistently had the greatestPAL activity. Moreover, PAL activity in the cytosolic fractionincreased rapidly prior to lignification, as it did in boththe microsomal and the cell wall (tightly bound) fractions duringlignification. Antisera against PAL and against CAD detectedthe proteins with molecular masses that corresponded to thoseof PAL and CAD in Zinnia. Immuno-electron microscopy revealedthat, in differentiating tracheary elements, PAL was dispersedin the cytoplasmic matrix and was located on Golgi-derived vesiclesand on the secondary wall thickenings. "Cell-free" immuno-lightmicroscopy supported the putative distribution of PAL on lignifyingsecondary walls. The pattern of distribution of CAD was similarto that of PAL. Thus, both PAL and CAD seemed to be localizedin secondary wall thickenings. From the results of both biochemicalassays and immunocytochemical staining, it appeared that atleast two types of PAL and CAD are present in differentiatingcells. One type of each enzyme is distributed in the cytosol,while the other is secreted from the Golgi apparatus and transportedby Golgi-derived vesicles to the secondary wall thickenings. (Received April 19, 1996; Accepted November 18, 1996)     

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     

Compartmentation of Phenolic Compounds and Phenylalanine Ammonia-Lyase in Leaves of Phyllanthus tenellus Roxb. and their Induction by Copper Sulphate

The compartmentation of phenolic compounds in mature leavesof Phyllanthus tenellus and their induction by copper sulphatewere analysed at histological and subcellular levels. Lightand electron microscopy studies demonstrated that the vacuolesof spongy cells were the main sites of phenolic accumulation.Spraying plants with copper sulphate induced punctated lesionsformed by groups of necrotic cells which accumulated brownishsubstances. Histochemical tests and fluorescence microscopyanalysis of the sprayed leaves indicated that the phenolic compoundsincreased in spongy cells within the lesions. Ultrastructuralanalyses showed that 3 h after elicitation, the organelles ofthe cells within the lesion started to collapse and the contentof phenolic substances increased in the vacuole of spongy cells.Antibody against phenylalanine ammonia-lyase (PAL) from parsleycross-reacted with the crude extract of P. tenellus leaves.Two isoforms, one of 65 kD and the other of 66 kD, were identified.Immunocytochemical studies showed that PAL was synthesized inthe palisade and spongy cells, mainly in the cytoplasm and chloroplasts.The phytotoxicity of Cu²⁺ions induced the accumulation of PALin sub-cellular compartments of palisade cells. PAL accumulationstarted to increase 3 h after elicitation and reached a maximumafter 6 h, decreasing 12 h post-induction. The increase of PALwas more evident in cells within the necrotic punctated regionsthan in surrounding cells. Since the vacuole of palisade cellsdid not accumulate phenolic compounds, the in situ studies suggestedthat the end products of PAL synthesis play a role in palisadecell wall reinforcement or might accumulate in other tissues.The symptoms induced by copper sulphate suggest that this abioticelicitor may be a useful tool in the understanding of the regulationof biosynthetic phenolic pathways inP. tenellus . Copyright2000 Annals of Botany Company Cell death, copper sulphate, heavy metal, immunolabelling, phenolic compounds, phenylalanine ammonia-lyase,Phyllanthus , transmission electron microscopy, ultrastructure     

Reduced Phenylalanine Ammonia-Lyase and Tyrosine Ammonia-Lyase Activities and Lignin Synthesis in Wheat Grown under Low Pressure Sodium Lamps

Wheat (Triticum aestivum L. cv Fremont) grown in hydroponic culture under 24-hour continuous irradiation at 560 to 580 micromoles per square meter per second from either metalhalide (MH), high pressure sodium (HPS), or low pressure sodium (LPS) lamps reached maturity in 70 days. Grain yields were similar under all three lamps, although LPS-grown plants lodged at maturity. Phenylalanine ammonia-lyase (PAL) and a tyrosine ammonia lyase (TAL) with lesser activity were detected in all extracts of leaf, inflorescence, and stem. Ammonia-lyase activities increased with age of the plant, and plants grown under the LPS lamp displayed PAL and TAL activities lower than wheat cultured under MH and HPS radiation. Greenhouse solar-grown wheat had the highest PAL and TAL activities. Lignin content of LPS-grown wheat was also significantly reduced from that of plants grown under MH or HPS lamps or in the greenhouse, showing a correlation with the reduced PAL and TAL activities. Ratios of far red-absorbing phytochrome to total phytochrome were similar for all three lamps, but the data do not yet warrant a conclusion about specific wavelengths missing from the LPS lamps that might have induced PAL and TAL activities in plants under the other lamps.     

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.     

胡桐悬浮培养细胞中苯丙氨酸解氨酶活性与红厚壳素产量的关系

胡桐CR2细胞悬浮培养过程中,苯丙氨酸解氨酶(PAL)活性上升后红厚壳素产量即增加.加入真菌诱导子后,PAL活性与红厚壳素产量呈一定的正相关.以PAL抑制物降低PAL活性后,红厚壳素产量也降低;诱导物与抑制物同时加入,PAL活性与红厚壳素产量均界于诱导物处理与未处理之间.     

Membrane Potential of Cultured Carrot Cells in Relation to the Synthesis of Anthocyanin and Embryogenesis

Membrane potentials of cultured carrot cells in culture mediumwere about –40 mV and did not change with addition ofsalts of addition (or depletion) of 2,4-dichlorophenoxyaceticacid (2,4-D). When the measurement was performed in the testmedium (containing low concentration of salts), the values werewidely distributed (from –60 to –110mV) and changedlargely with external concentration of K⁺ but not Mg²⁺ nor Ca²⁺.When the cells were fractionated by Ficoll density gradientcentrifugation, the membrane potential of the cells of higherdensity (> 14% Ficoll) was about –150 mV in the testmedium and did not change during embryogenesis with depletionof 2,4-D. The membrane potential of cells of lower density (bandingbetween 6– 10% Ficoll) was less negative (– 60 to– 110 mV) in the test medium. When these cells were transferredand cultured in medium containing zeatin but lacking 2,4-D,the membrane potential was shifted negatively by about 15 mVprior to anthocyanin synthesis. When 2,4-D was added to anthocyanin-synthesizingcells in the medium containing zeatin, a transient hyperpolarizationand subsequent depolarization of the membrane were observedprior to the inhibition of anthocyanin synthesis. (Received October 22, 1987; Accepted April 20, 1988)     

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