刺激剂对植物细胞悬浮培养的影响

对刺激剂对植物细胞悬浮培养的影响及如何提高刺激剂的诱导率进行了综述。刺激剂对细胞悬浮培养的影响主要表现为酶活化、蛋白质含量改变、氧迸发、细胞程序性死亡、pH值改变、次生代谢产物积累等防御反应。提高刺激剂对悬浮培养细胞的诱导效率必须优化刺激剂的种类,刺激剂的浓度和加入时间,创建更好的诱导模式。     

Reduced protease activity in transformed rice cell suspension cultures expressing a proteinase inhibitor

In this study, we synthesized a synthetic serine proteinase inhibitor II gene (sPI-II) that harbored the chymotrypsin and trypsin inhibitor domains of the PI-II gene from Nicotiana alata. In an effort to reduce protease activity in a rice cell suspension culture, we first synthesized sPI-II using overlap PCR and then introduced the gene into a rice calli (Oryza sativa L. cv. Dongin) by particle bombardment-mediated transformation. The sPI-II gene was under the control of a rice alpha-amylase 3D promoter induced by sugar starvation. To verify the integration and expression of the sPI-II gene in the transformed rice cells, we employed genomic DNA PCR amplification and Northern blot analysis, respectively. The relative protease activity of the transformed cell suspension culture was reduced to approximately 23% when compared to the non-transformed culture. This indicates that a transformed suspension culture system expressing a proteinase inhibitor, may be a useful tool to protect against recombinant protein losses resulting from extracellular proteases.     

Effect of age of cell suspension cultures on susceptibility to a fungal elicitor

Fungal elicitor induced phytoalexin formation and the corresponding fluorescence transitions of the molecular probes pyranine and oxonol VI, in soybean (Glycine max Merr var Kent) and cotton (Gossypium arboreum L. Nanking) cell suspensions were both significantly affected by the age of the cells. During the lag phase and the beginning of the exponential growth phase both cultures exhibited stress responses (i.e. phytoalexin formation and molecular probe fluorescence transitions) in the absence of added elicitors. This behavior was termed autoelicitation because elicitation occurred without added external stimuli. In contrast, cells in the late exponential-early stationary phase were relatively unresponsive to elicitor. During intermediate growth periods the cell suspensions behaved optimally, producing no phytoalexins until stimulated with an elicitor. It would appear, therefore, that the culture period can be divided into 3 phases, with respect to susceptibility to fungal elicitors: a distinct autoelicitation period (immediately after transfer of the cells into fresh medium), followed by a period in which negligible amounts of phytoalexins are synthesized without elicitor, and culminating in a late period in which the cells respond poorly to elicitor. The onset and duration of these periods are somewhat different for soybean and cotton cells.     

Induction of sesquiterpenoid biosynthesis in tobacco cell suspension cultures by fungal elicitor

Large amounts of the sesquiterpenoid capsidiol accumulated in the media of tobacco (Nicotiana tabacum L. cv KY14) cell suspension cultures upon addition of fungal elicitor. Capsidiol accumulation was proportional to the amount of elicitor added. The accumulation of capsidiol was preceded by a transient increase in the capsidiol de novo synthesis rate as measured by the incorporation of exogenous [¹⁴C]acetate. Changes in 3-hydroxy-3-methylglutaryl-CoA reductase activity (HMGR; EC 1.1.1.34), an enzyme of general isoprenoid metabolism, paralleled the changes in [¹⁴C]acetate incorporation into capsidiol. Incubation of the cell cultures with mevinolin, a potent in vitro inhibitor of the tobacco HMGR enzyme activity, inhibited the elicitor-induced capsidiol accumulation in a concentration dependent manner. [¹⁴C]Acetate incorporation into capsidiol was likewise inhibited by mevinolin treatment. Unexpectedly, [³H] mevalonate incorporation into capsidiol was also partially inhibited by mevinolin, suggesting that mevinolin may effect secondary sites of sesquiterpenoid biosynthesis in vivo beyond HMGR. The data indicated the importance of the induced HMGR activity for capsidiol production in elicitor-treated tobacco cell suspension cultures.     

Production of a hepatoprotective cerebroside from suspension cultures of Lycium chinense

Suspension cultures derived from Lycium chinense Miller seedlings produced significant amounts of a hepatoprotective cerebroside. Callus was induced from the stem of aseptic seedlings of L. chinense and maintained on MS solid media supplemented with 1.0 ppm 2,4-D and 0.1 ppm kinetin. Suspension cultures were established, and the cells were grown in the same liquid media in the dark. Lyophilized cells were extracted with a combined reagent of chloroform and methanol (2:1, v/v). An aqueous suspension of the evaporated cell extract was partitioned with chloroform, and the chloroform layer was subjected to silicic acid column chromatography followed by semi-preparative reverse phase C8 high pressure liquid chromatography. The purified compound showed hepatoprotective activity comparable to that shown by silymarin, and the structure was identified as 1-O-(β-d-glucopyranosyl)-(2S,3R,4E,8Z)-2-N-2′-hydroxy-(palmitoyl)-4,8-sphingadiene on the basis of spectral data. The content of the compound in cultured cell was tenfold higher than that of the fruit of L. chinense. The biosynthesis of the compound in cultured cell systems appears to parallel cell growth. Received: 12 June 1998 / Revision received: 30 July 1998 / Accepted: 14 August 1998     

Fungal elicitor triggers rapid, transient, and specific protein phosphorylation in parsley cell suspension cultures

Treatment of suspension-cultured parsley (Petroselinum crispum) cells with fungal elicitor triggers rapid, transient and sequential phosphorylation of a number of proteins, as shown by electrophoretic analysis on two-dimensional gels. This response is rapidly reversed by removal of the elicitor from the medium and appears to be specific. It is not observed in cells exposed to other environmental stress factors, such as heat shock, UV irradiation or treatment with mercuric chloride. Pronase digestion of the elicitor has the same negative effect on protein phosphorylation as its previously demonstrated effect on the activation of some pathogen defense-related genes, suggesting a link between these two phenomena. Some of the changes in protein phosphorylation are among the earliest known events following elicitation. The phosphorylation of a neutral 45-kDa protein, which is found in both the microsomal and cytoplasmic fractions, can be observed as early as 1 min after the onset of elicitor treatment. The phosphorylation of a 26-kDa nuclear protein also starts increasing very early. The changes in protein phosphorylation in response to the elicitor are dependent on the presence of Ca2+ in the medium. Our data are compatible with the hypothesis that protein phosphorylation is involved in the signal transduction processes following elicitor recognition by parsley cells.     

Effects of fungal elicitor on lignin biosynthesis in cell suspension cultures of soybean

Soybean (Glycine max L.) cells cultured in B5 medium produce extremely low amounts of lignin. However, modification in the growth medium, by lowering the concentration of NO⁻₃ and PO²⁻₄, results in the lignification of these cells without affecting levels of cell wall-esterified 4-coumaric and ferulic acid. The production of an extracellular, macromolecular complex by the cultured soybean cells (Moore TS Jr 1973 Plant Physiol 51: 529-536) allows a rapid, nondestructive solubilization of the lignin which can be estimated by reaction with phloroglucinol in free solution. This system has been used to study the effects of fungal elicitor on the synthesis of lignin in soybean cells. The inclusion of very low levels of an elicitor fraction from the cell walls of Phytophthora megasperma in the medium in which lignification of the soybean cells occurs suppressed both the accumulation of extracellular lignin and phloroglucinol staining of the cell walls without affecting the levels of bound hydroxycinnamic acids. The activity profiles of phenylalanine ammonia-lyase (EC 4.3.1.5) and isoenzymes of 4-coumarate:CoA ligase (EC 6.2.1.12) were compared in lignifying and elicitor-treated cell cultures as was the activity of chalcone synthase, an enzyme of flavonoid biosynthesis. The measured activities of these enzymes in cell cultures treated with elicitor were considerably lower than in untreated cells.     

Improvement of phenylethanoid glycosides biosynthesis in Cistanche deserticola cell suspension cultures by chitosan elicitor

Effect of chitosan elicitor on growth and phenylethanoid glycosides (PeGs) accumulation in Cistanche deserticola cell suspension cultures was investigated. PeGs accumulation was dramatically improved by addition of selected chitosan at optimal elicitation conditions. Furthermore, a strategy of repeated addition of the chitosan elicitor for enhancing PeGs accumulation was developed. The chitosan elicitor of 10 mg l(-1)-medium repeatedly added on days 15 and 17 improved PeGs accumulation further, and the final PeGs production in the treated cell cultures of C. deserticola reached 364.6 mg l(-1), which was 3.4-fold higher than that of the control without elicitation. The increase of PeGs accumulation in C. deserticola cell suspension cultures was related to the increase of phenylalanine ammonium lyase activity stimulated by the chitosan elicitor.     

Methyltransferase activity in Allanthm altissima cell suspension cultures

Enzymes for the methylation of 1hydroxycanthin-6-one and a series of coumarins have been isolated from Ailanthus altissima cell suspension cultures. The coumarin methyltransferases methylate aesculetin to scopoletin and isoscopoletin, but not scopoletin, to scoparone. Fraxetin was methylated to isofraxidine but not to fraxidine and only fraxidine was methylated to 6,7,8-trimethoxycoumarin. These enzymes were studied throughout the culture growth cycle with two cell lines: 1, which produced 1-methoxycanthin-6-one as the major alkaloid and 2, in which canthin-6-one was the major alkaloid.Abbreviations UV ultraviolet - DEAE diethylaminoethyl - dw dry weight - MS mass spectrometry - NMR nuclear magnetic resonance - TLC thin layer chromatography     

Induced accumulation of triterpenoids in Scutellaria baicalensis suspension cultures using a yeast elicitor

When growth-phase cell suspension cultures of Scutellaria baicalensis were treated with 50 g of yeast elicitor preparation ml^–1, both oleanolic acid and ursolic acid transiently increased in the culture medium rather than in the cells. The maximal triterpenoid concentration was 13.7 mg l^–1 media approx. 35 h after treatment, whereas the maximum concentration was 2.1 mg l^–1 media after about 20 h following treatment with methyl jasmonate. Elicitor treatment also doubled phospholipase A₂ activity (25 pmol mg^–1 min) and the simultaneous treatment of aristolochic acid, a phospholipase A₂ inhibitor, inhibited triterpenoids accumulation as well as phospholipase A₂ activity.     

Expression of bioactive human interferon-gamma in transgenic rice cell suspension cultures

We investigated the possibility of producing the therapeutic recombinant cytokine, Interferon-gamma (IFN-), in transgenic rice cell (Oryza sativa, cultivar TNG67) suspension cultures. We tested expression of two vector constructs, each harboring an Amy3 leader peptide and a C-terminus His 6 tag fused to a human IFN- cDNA, one driven by a sucrose-starvation inducible promoter (rice Amy3 promoter) and the other by a constitutive maize ubiquitin promoter, in rice cell suspensions, introduced via Agrobacterium tumefaciens. There was a significant difference in the amounts of recombinant IFN- protein produced by the Ups and Amy cell lines, as cytosolic and secretory proteins respectively. Immunological analysis of IFN- recombinant protein conferred a dose-dependent anti-dengue virus activity in human A549 cells, similar to the commercial product. We discuss the attractive attributes of using rice cell suspension system for the expression of therapeutic recombinant IFN-.     

Effect of yeast elicitor on the secondary metabolism of Ti-transformed Salvia miltiorrhiza cell suspension cultures

 Salvia miltiorrhiza contains two groups of biologically active secondary metabolites termed phenolic compounds (e.g. rosmarinic acid) and tanshinones (e.g. cryptotanshinone). Their roles in plant defense responses were examined using a simplified system consisting of a yeast elicitor and a Ti C58 transformed S. miltiorrhiza cell line. Both dosage and time course studies were carried out on the effects of yeast elicitor on the formation of rosmarinic acid and cryptotanshinone. It was found that the yeast elicitor reduced the constituent level of rosmarinic acid (from ca. 5% to ca. 3.0% of dry cell weight) whereas the level of cryptotanshinone was enhanced greatly (from a negligible amount to ca. 20 mg/l). These results suggest that in S. miltiorrhiza, rosmarinic acid and cryptotanshinone may take part in plant passive and active defense responses, respectively, against pathogen attack. Cryptotanshinone was identified as a phytoalexin in S. miltiorrhiza for the first time. Results of the treatment of cell cultures with 2-aminoindan-2-phosphonic acid, a highly specific and potent inhibitor of phenylalanine ammonia-lyase (EC 4.3.1.5), indicated that this compound did not inhibit yeast elicitor induced tanshinone formation, but did inhibit rosmarinic acid biosynthesis. Received: 22 May 1999 / Revision received: 27 August / Accepted: 1 September 1999     

Elicitor induction of a microsomal 5-O-(4-coumaroyl)shikimate 3'-hydroxylase in parsley cell suspension cultures

Microsomal preparations from parsley cell suspension cultures challenged with an elicitor from Phytophthora megasperma f.sp. glycinea (Pmg) catalyze the formation of trans-5-O-caffeoylshikimate from trans-5-O-(4-coumaroyl)shikimate. Neither the cis isomer nor free 4-coumarate, 4-coumaroyl-CoA, or 5-O-(4-coumaroyl)quinate are substrates for this enzyme. The reaction is strictly dependent on NADPH as a reducing cofactor and on molecular oxygen. NADH, ascorbic acid, and 6,7-dimethyl-5,6,7,8-tetrahydropterine cannot substitute for NADPH. However, NADH enhances enzyme activity observed in the presence of NADPH. Cytochrome c and carbon monoxide inhibit the hydroxylation reaction, suggesting a cytochrome P-450-dependent mixed-function monooxygenase.     

Accumulation of 2,5-dimethoxy-1,4-benzoquinone in suspension cultures of Panax ginseng by a fungal elicitor preparation and a yeast elicitor preparation

Suspension cultures of Panax ginseng C.A. Meyer (Araliaceae) were treated with either an elicitor preparation from the culture broth of the phytopathogenic hyphomycete Botrytis cinerea or a yeast elicitor preparation, and the accumulation of a new compound, which was not detected in non-elicited cultures, was observed. The accumulated compound was isolated and shown to be 2,5-dimethoxy-1,4-benzoquinone by 1H-NMR, 13C-NMR and electron ionization (EI) mass spectra. While it is well known that this compound shows antibacterial activity against Staphylococcus aureus, its presence in ginseng root has not been reported to date. Levels of the compound in the media increased rapidly, reaching a maximum level of 65.10 +/- 4.96 microg/g fresh weight at approximately 12 h after treatment with the yeast elicitor preparation. The maximal level of the compound in medium from the culture treated with an elicitor preparation from the culture broth of B. cinerea was 46.13 +/- 10.42 microg/g fresh weight after 24 h of incubation.     

Increased secondary product formation in plant cell suspension cultures after treatment with a yeast carbohydrate preparation (elicitor)

A carbohydrate fraction isolated from yeast extract by ethanolic precipitation was used as an elicitor to induce secondary product formation in plant cell suspension cultures. The elicitor preparation is effective in inducing glyceollin isomer synthesis (up to 200 μg glyceollin per g dry wt) in cells of Glycine max and enhancing berberine biosynthesis (up to four-fold) in cells of Thalictrum rugosum. The response of the cell cultures to the elicitor treatment is dependent on the amount of carbohydrate per unit of biomass and on the physiological state of the cells. Cells are optimally induced in late exponential or early stationary growth phases.     

Production of functional recombinant bovine trypsin in transgenic rice cell suspension cultures

A synthetic bovine trypsinogen (sbTrypsinogen) was synthesized on the basis of rice-optimized codon usage via an overlap PCR strategy, prior to being expressed under the control of the sucrose starvation-inducible rice α-amylase 3D (RAmy3D) promoter. Secretion of trypsin into the culture medium was achieved by using the existing signal peptide. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin), mediated by Agrobacterium tumefaciens. The integration of the sbTrypsinogen gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification, and sbTrypsin expression in transgenic rice suspension cells was confirmed via Northern blot analysis. Western blot analysis detected glycosylated proteins in the culture medium, having masses from 24 to 26 kDa, following induction by sugar starvation. Proteolytic activity of the rice-derived trypsin was confirmed by gelatin zymogram, and was similar to that of the commercial bovine-produced trypsin. The yields of sbTrypsin that accumulated in the transgenic rice cell suspension medium were 15 mg/L at 5 days after sugar starvation.