Role of nitric oxide in UV-B-induced activation of PAL and stimulation of flavonoid biosynthesis in Ginkgo biloba callus

The role of nitric oxide (NO) in UV-B-induced secondary metabolite accumulation in Ginkgo biloba callus was investigated. Overall, UV-B irradiation induced multiple biological responses in callus of G. biloba, including increased both NO production and nitric oxide synthase (NOS) activity, and subsequent activation of phenylalanine ammonium lyase (PAL) and synthesis of flavonoids. Application of NO via the donor sodium nitroprusside (SNP) enhanced UV-B-induced PAL activity and increased accumulation of flavonoids in G. biloba callus. Both, the NOS inhibitor l-NAME (N (G)-nitro-l-arginine methyl ester) and the NO scavenger c-PTIO (2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide) reduced the production of NO. Moreover, UV-B-induced increase of PAL activity and flavonoid accumulation were suppressed by l-NAME and c-PTIO. These findings suggested a causal relationship between NO release and both PAL activity and flavonoid accumulation under UV-B irradiation. In addition, it also indicated that NO, produced via NOS-like activity in ginkgo callus subjected to UV-B irradiation, might act as an essential signaling molecule for triggering the activation of PAL and synthesis of flavonoids. Additionally, a guanylyl cyclase inhibitor 6-anilino-5,8-quinolinequinone (LY-83583) prevented both UV-B- and SNP-induced enhancement of PAL activation and flavonoid biosynthesis thus suggesting that the NO function was mediated by cyclic guanosine 5’-monophosphate. However, these effects of c-PTIO, l-NAME, and LY-83583 were partial, thus suggesting that there were NO-independent pathways in UV-B signaling networks. Gangping Hao and Xihua Du are contributed equally to this article.     

不同光质对红花檵木愈伤组织生长及黄酮类物质含量的影响

该试验以红花檵木叶片诱导的愈伤组织为试验材料,以黑暗(CK)为对照,分别进行白光(W)、红光(R)、蓝光(B)、蓝紫光(BP)、蓝光+UV-A(B+UV A)和UV-A处理15d和30 d,通过对愈伤组织的生长、生理指标、花色素苷和总黄酮含量的比较分析,探讨光质对红花檵木愈伤组织生长和黄酮类物质含量的影响.结果 显示:...     

Effects of light and plant growth regulators on the biosynthesis of vindoline and other indole alkaloids in Catharanthus roseus callus cultures

A callus strain with stable ability for vindoline synthesis was selected from many prepared Catharanthus roseus leaf calli to study the regulation of vindoline biosynthesis as well as other indole alkaloids. It was shown that light and plant growth regulators significantly influenced the biosynthesis of vindoline and other alkaloids as well as acidic and basic peroxidase activities. Light promoted vindoline and serpentine biosynthesis, and stimulated plastid development and peroxidase activity. However, 2,4-D suppressed the biosynthesis of all indole alkaloids and peroxidase activity. Our results suggest that light or plant hormones regulate vindoline, serpentine and other alkaloid biosynthesis and accumulation by influencing peroxidase activity and the differentiation status of callus cultures, especially chloroplast development. Some possible relationships between serpentine or vindoline biosynthesis and peroxidase activity are proposed.     

Callus and cell suspension culture of <Emphasis Type="Italic">Viola odorata</Emphasis> as in vitro production platforms of known and novel cyclotides

Callus from Opuntia streptacantha (cv. Tuna loca), Opuntia megacantha (cv. Rubí reina), and Opuntia ficus-indica (cv. Rojo vigor) were exposed to jasmonic acid (JA) and abiotic stress (drought and UV light) to improve the metabolite production. The callus growth curves, phenolic acids and flavonoids content, antioxidant activity and phenylalanine ammonia lyase (PAL) activity were analyzed under normal and stress conditions. In O. streptacantha callus, the phenolics concentration increased 1.6 to 3 times times in presence of 5% PEG or after irradiation with UV light for 240 min, respectively, while flavonoids triplicate with UV light. A significant increase in antioxidant activity was observed in calli from the three Opuntia species in media with 50 µM JA. The relationships between metabolites/PAL activity, and metabolites/antioxidant activity were analyzed using a surface response methodology. Results showed that PAL activity, induced with PEG and UV, correlated with flavonoids content in O. megacantha and O. ficus-indica calli; PAL activity was related to both flavonoids and phenolics compounds in O. ficus-indica and O. megacantha calli exposed to JA, but only to flavonoids in O. streptacantha callus. In general, the JA stimulated simultaneously the metabolic pathways for phenolics and flavonoids synthesis, while abiotic stress induced mainly flavonoids route. As the stressed Opuntia calli exhibited as high antioxidant activity as cladodes, they are a promising system for research on antioxidant biosynthesis and/or to identify new compounds with antioxidant properties.     

Light-enhanced caffeic acid derivatives biosynthesis in hairy root cultures of <Emphasis Type="Italic">Echinacea purpurea</Emphasis>

Light plays an important role in almost all plant developmental processes and provides the fundamental building blocks for growth, development, primary and secondary metabolism. The effects of light on growth rate and caffeic acid derivative (CADs) biosynthesis in hairy root cultures of Echinacea purpurea (Moench) were assessed. Light-grown hairy roots accumulated increased levels of anthocyanins, which became visible in outer cell layer of the cortex as a ring of purple color. The light-grown root cultures also had radially thickened morphology compared with the dark-grown controls. The growth rate and cell viability of the hairy root cultures in light did not show obvious difference in comparison with those in dark. However, biosynthesis of CADs including cichoric acid, caftaric acid, chlorogenic acid and caffeic acid was significantly increased in hairy root cultures grown in the light. The enhanced accumulation of CADs and anthocyanins in E. purpurea hairy root cultures was correlated to an observed light-stimulated activity of phenylalanine ammonium lyase (PAL).     

Fungal endophytes-induced abscisic acid is required for flavonoid accumulation in suspension cells of <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Treatment of suspension cells of Ginkgo biloba with fungal endophytes resulted in accumulation of flavonoids, increased abscisic acid (ABA) production and activation of phenylalanine ammonia-lyase (PAL). Fluridone, an inhibitor of ABA biosynthesis, was effective in inhibiting fungal endophytes-induced ABA biosynthesis, increase of PAL activity and flavonoids accumulation. Moreover, exogenous application of ABA enhanced PAL activity and increased accumulation of flavonoids in G. biloba cells with or without fungal endophytes elicitor. These finding suggest a causal relationship between ABA release and both PAL activity and flavonoid accumulation under fungal endophytes treatment and that ABA is involved in fungal endophytes-induced flavonoids accumulation in this plant.     

l-Phenylalanine ammonia-lyase in suspension culture cells of spruce (Picea abies)

The activity of l-phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) was determined in seedlings, callus cells, cell suspension cultures and in young needles of spruce (Picea abies) (L.) (Karst). PAL activity increased up to 10 fold in response to transferring suspension cultured cells into new cultivation medium. PAL was also induced about 10 fold when callus cells were transferrd into liquid medium. The increase was transient and it required the presence of a carbohydrate.In cell suspension cultures, grown in the dark (white cells), but not in light-grown cultures (green cells), PAL activity was induced up to 30 fold by UV-light.With a cell wall preparation of Rhizosphaera kalkhoffii, a forest pathogenic fungus, used as elicitor, the activity of PAL could be induced more than 10 fold. The degree of induction depended on the elicitor concentration. Induction was prevented by cycloheximide but not by actinomycin D.     

Anthocyanin accumulation and changes in activities of phenylalanine ammonia-lyase and chalcone synthase in roselle (Hibiscus sabdariffa L.) callus cultures

Time-course changes in anthocyanin accumulation, phenylalanine ammonia-lyase activity and chalcone synthase activity were examined in roselle callus tissues incubated under different culture conditions. Phenylalanine ammonia-lyase activity was not affected by either the kind of auxin supplemented to the medium or light regime. In contrast, chalcone synthase activity was markedly suppressed when the callus was cultured with a medium containing indole-3-acetic acid instead of 2,4-dichlorophenoxyacetic acid (2,4-D) or in the dark. The results imply that in roselle callus cultures chalcone synthase plays a more important role in anthocyanin biosynthesis regulated by 2,4-D and light irradiation than phenylalanine ammonialyase.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - PAL phenylalanine ammonia-lyase - CHS chalcone synthase     

Formation of heartwood substances in the stem of Robinia pseudoacacia L.

Summary The activities of two key enzymes in flavonoid biosynthesis, phenylalanine ammonia-lyase (PAL, E.C. 4.3.1.5) and chalcone synthase (CHS, E.C. 2.3.1.74) were determined in the trunkwood of Robinia pseudoacacia L. The trees under investigation were cut at different times of the year (September, November, January and April). At all times PAL is active, both in the youngest wood layer (the outermost growth ring) and at the sapwood heartwood boundary. On the other hand, CHS is active exclusively in the vicinity of the heartwood boundary. The results indicate that PAL is involved both in the formation of lignin (outermost annual ring), and in flavonoid biosynthesis (heartwood boundary). Highest activity of both PAL and CHS could be measured at the sapwood heartwood boundary in the tree felled in November, indicating that heartwood formation was occurring mainly at that time. The flavonoids accumulated in the heartwood are obviously formed in situ and seem to be transported only to a minor extent — if at all — via the phloem and the ray cells to the heartwood.     

Expression profile of a PAL gene from Astragalus membranaceus var. Mongholicus and its crucial role in flux into flavonoid biosynthesis

An important traditional Chinese medicine herb, Astragalus membranaceus var. Mongholicus, whose dried root is known as Radix astragali (“Huangqi” in Chinese), has high flavonoid content as an essential active constituent. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) catalyzes the first and also a rate-limiting step in phenylpropanoid pathway, which supplies precursors for a variety of secondary metabolites including flavonoids. A PAL gene, designated AmPAL1 (GenBank accession no. AY986506), was isolated from A. membranaceus var. Mongholicus with a full-length cDNA of 2562 nucleotides and an open reading frame of 2154 bp. Northern blot analysis revealed that AmPAL1 expressed universally in different organs, and its expression was markedly induced by UV irradiation, mechanical wounding, and white light irradiation on etiolated seedlings, with some distinctive responsive properties. Content of a typical flavonoid, quercetin, in A. membranaceus var. Mongholicus of different ages correlated with PAL enzymatic activity. Transgenic tobacco plants harboring AmPAL1 under the control of the CaMV35S promoter showed significantly increased PAL activity and correlatively increased quercetin content than those in non-transformed plants. These results indicate that PAL is maybe a key point for flux into flavonoid biosynthesis in the genetic control of secondary metabolism in A. membranaceus var. Mongholicus.     

Enhancement of phenylethanoid glycosides biosynthesis in cell cultures of <Emphasis Type="Italic">Cistanche deserticola</Emphasis> by osmotic stress

The effect of osmotic stress on cell growth and phenylethanoid glycosides (PeGs) biosynthesis was investigated in cell suspension cultures of Cistanche deserticola Y. C. Ma, a desert medicinal plant grown in west region of China. Various initial sucrose concentrations significantly affected cell growth and PeGs biosynthesis in the suspension cultures, and the highest dry weight and PeGs accumulation reached 15.9 g l⁻¹-DW and 20.7 mg g⁻¹-DW respectively at the initial osmotic stress of 300 mOsm kg⁻¹ where the sucrose concentration was 175.3 mM. Stoichiometric analysis with different combinations of sucrose and non-metabolic sugar (mannitol) or non-sugar osmotic agents (PEG and NaCl) revealed that osmotic stress itself was an important factor for enhancing PeGs biosynthesis in cell suspension cultures of C. deserticola. The maximum PeGs contents of 26.9 and 23.8 mg g⁻¹-DW were obtained after 21 days at the combinations of 87.6 mM sucrose with 164.7 mM mannitol (303 mOsm kg⁻¹) or 20 mM PEG respectively, which was higher than that of C. deserticola cell cultures grown under an initial sucrose concentration of 175.3 mM after 30 days. The stimulated PeGs accumulation in the cell suspension cultures was correlated to the increase of phenylalanine ammonium lyase (PAL) activity induced by osmotic stress.     

Canavanine metabolism in tissue cultures of Canavalia lineata

The greening of callus was achieved by modulating the medium's growth regulator concentrations under continuous light. Canavalia lineata (L.) DC. calluses formed chlorophyll when they were exposed to continuous light in the presence of benzylaminopurine and indole-3-acetic acid. Canavanine and canaline were detected in the green callus. But only canaline was detected in the white callus grown in the dark. Feedings of canaline to suspension cultures showed that the green suspended cells were capable of de novo biosynthesis of canavanine, but the white suspended cells were not. Exogeneously supplied canavanine was used to produce canaline and homoserine by the white suspended cells. Arginase activity was induced by the addition of arginine or canavanine to the medium, and canaline reductase activity was induced by the addition of canaline but not with ornithine in the white suspended cells.Abbreviations BA benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - OPA o-phthaldialdehyde - PC Phillips & Collins (1979) medium     

pH对槐角愈伤组织黄酮类化合物产量的影响

培养基酸碱度是影响植物生长和次生代谢的重要因素之一。将生长稳定的国槐槐角愈伤组织在不同pH值的B5悬浮培养基中培养,比较其生长状况、生物量及苯丙氨酸转氨酶活性和黄酮类化合物的产量。结果表明:pH为6.6时,国槐细胞生长状况好,且不同pH下的苯丙氨酸转氨酶活性差异显著;培养基呈弱酸性(即6.6±0.05)时,细胞黄酮类化合物含量较高。综合生长天数和产量,最佳的收获时期为继代后的第25～30天。     

NO对银杏悬浮细胞生长及黄酮类物质合成的影响

以硝普钠(sodium nitroprusside,SNP)为一氧化氮(NO)的供体,向银杏悬浮细胞培养液中加入不同浓度的SNP,研究外源NO对银杏悬浮细胞生长状况、过氧化氢酶(CAT)活性、苯丙氨酸解氨酶(PAL)活性和黄酮类物质生物合成的影响.结果表明,低浓度SNP有利于银杏悬浮细胞生长,而高浓度SNP可以促进黄酮类物质的合成.银杏悬浮细胞在添加0.5和10 mmol/L SNP的培养基中培养16 d时,细胞干重分别为对照组的134%和73%;在添加10 mmol/L SNP的培养基中培养20 d时,细胞中黄酮类物质的含量为对照组的136%.同时,10 mmol/L SNP促进银杏悬浮细胞PAL和CAT活性显著升高.NO专一性淬灭剂c-PITO(carboxyl phenyltetramethylimidazoleoxide)抑制SNP对银杏悬浮细胞生长、CAT活性、PAL活性和黄酮类物质含量的促进作用,说明SNP是通过其分解产物NO影响细胞生长和黄酮类物质的合成.根据这些结果推测,NO可能通过触发银杏悬浮细胞的防卫反应,激活了细胞中黄酮类物质的生物合成途径.     

Programmed cell death in Datura innoxia Mill. callus cultures cultivated in light and in dark

It has been established that dimedone in solid culture medium influencedthe growth of Datura innoxia Mill. callus tissues and theapoptotic processes of cells. This formaldehyde (HCHO) capture reagent appearsto modify the metabolism of plant cells, resulting in quantitative changes inthe apoptotic index (A_i). Apoptotic cells were detected insix-week-old callus tissues by the TUNEL reaction. The amount of TUNEL positivenuclei showed a characteristic spatial distribution. Enhanced DNA fragmentationwas observed in the cell layers close to the surface of the cultures. ElevatedA_i was determined in cultures grown in the dark compared to thetissues grown in the light. High doses of dimedone considerably decreasedapoptosis in tissue cultures under both light and dark conditions.     

Effects of sodium alginate elicitation on secondary metabolites and antioxidant activity of safflower genotypes under in vitro salinity stress

The present study was aimed at investigating the effects of different concentrations of sodium alginate (NaAlg) (0.075 and 0.15% (w/v)) on the production of secondary metabolites (SMs) and antioxidant activity of seven safflower genotypes under in vitro salinity stress. The results showed that total phenolic content (TPC), total flavonoids (TFD), total flavonols (TFL), anthocyanin (Ant), total antioxidant capacity (TAC), phenylalanine ammonia-lyase (PAL), catalase (CAT) activity, and lipid peroxidation significantly increased under salinity stress consisting of the concentration of 1.5% (w/v) of NaCl, but callus growth traits decreased. The highest amount of TPC, Ant, and callus growth traits was observed under the elicitation of the sample with the concentration of 0.075(%) NaAlg under salinity stress, but the highest amount for TFD, TFL, CAT, PAL, and TAC was observed under elicitation of the sample with the concentration of 0.15% of NaAlg under salinity stress. This indicated the superiority of NaAlg for elicitation to increase SMs in safflower under salinity stress. Overall, the results showed that genotypes of Mex.22-191 and GE₆₂₉₁₈ could be processed to produce SMs by eliciting NaCl in safflower as an important medicinal plant at cellular level. A positive and significant correlation between CAT and TPC was observed and indicates that phenolic compounds are the major contributors to the antioxidant potential in safflower. This new elicitor introduced new ways to select and exploit the best NaAlg concentration to develop SMs that are of tremendous importance in terms of commercial purposes along with medical features in safflower at cellular level.

     

Intracellular location of NADP+-linked malic enzyme in C3 plants

Ultraviolet light induces anthocyanin biosynthesis in cell cultures of an Afghan cultivar of Daucus carota (Daucus carota L. ssp. sativus). Simultaneous treatment with a fungal elicitor from Pythium aphanidermatum results in an inhibition of the catalytic activity of chalcone synthase (CHS), which in turn correlates with an inhibition of anthocyanin biosynthesis. On immunoblots, one isoenzyme (40 kDa) of CHS disappears upon elicitor treatment. On an mRNA level, only the mRNA for the 40-kDa-CHS is active after treatment with ultraviolet light. After inhibition of anthocyanin biosynthesis by the elicitor the enzyme protein disappears and the CHS mRNA is strongly diminished. This inhibition depends on the concentration of the elicitor. In addition, elicitor treatment leads to an induction of the general phenylpropanoid pathway as well as to the accumulation of 4-hydroxybenzoic acid which is covalently bound to wall polysaccharides of the carrot cells. The possible function of phenylalanine ammonia-lyase in providing precursors for 4-hydroxybenzoic acid is discussed.Abbreviations CHI chalcone isomerase - CHS chalcone synthase - PAL phenylalanine ammonia-lyase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We are grateful to Professor K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for providing us with antisera to CHS and PAL, respectively. This work was supported by a grant from the Deutsche Forschungsgemeinschaft and scholarships from the Friedrich-Ebert-Stiftung (J. G.), the Landesgraduierten-förderungsgesetz Baden-Württemberg (J.-P. S) and the Gerhard-Rösch-Stiftung (D. S.). We thank R. Hofmann for her excellent technical assistance.     

Variation in the growth and biosynthetic activity of cloned cell cultures of Capsicum frutescens and their response to an exogenously supplied elicitor

Twenty clones established from single cells of a suspension culture of Capsicum frutescens were maintained as callus and in suspension over a sixteen week culture period. These clones exhibited marked differences in growth, chlorophyll and chloroform-soluble phenolic content which became more apparent with increasing time in culture. Clones in suspension exhibited a more rapid change in morphology and biosynthetic activity than those cultured as callus. Elicitation increased PAL activity, reduced the incorporation of L-[U-¹⁴C] phenylalanine into the chloroform-soluble fraction of the culture medium and increased incorporation into the methanol-soluble fraction of the cells in ten suspension clones. Differences to elicitation were observed among clones; in particular the faster growing isolates incorporated more radioactive label into soluble phenolics that remain in the cells than those that are released into the medium. The implications of these results are discussed.Abbreviations SH Schenk & Hildebrandt - PAL phenylalanine ammonia-lyase - RGR relative growth rate - TCC total chlorophyll content - HPLC high performance liquid chromatography     

Polyamine levels as related to growth,differentiation and senescence in protoplast-derived cultures of Vigna aconitifolia and Avena sativa

We have previously reported that aseptically cultured mesophyll protoplasts of Vigna divide rapidly and regenerate into complete plants, while mesophyll protoplasts of Avena divide only sporadically and senesce rapidly after isolation. We measured polyamine titers in such cultures of Vigna and Avena, to study possible correlations between polyamines and cellular behavior. We also deliberately altered polyamine titer by the use of selective inhibitors of polyamine biosynthesis, noting the effects on internal polyamine titer, cell division activity and regenerative events.In Vigna cultures, levels of free and bound putrescine and spermidine increased dramatically as cell division and differentiation progressed. The increase in bound polyamines was largest in embryoid-forming callus tissue while free polyamine titer was highest in root-forming callus. In Avena cultures, the levels of total polyamines decreased as the protoplast senesced. The presence of the inhibitors -difluoromethyl-arginine (specific inhibitor of arginine decarboxylase) and dicyclohexylamine (inhibitor of spermidine synthase) reduced cell division and organogenesis in Vigna cultures. Addition of low concentration of polyamines to such cultures containing inhibitors or removal of inhibitors from the culture medium restored the progress of growth and differentiation with concomitant increase in polyamine levels.     

5‐Aminolevulinic acid promotes callus growth and paclitaxel production in light‐grown Taxus cuspidata suspension cultures

Cultured plant cells generally produce low levels of secondary metabolites, and elicitors of secondary metabolites usually inhibit callus growth. The aim of this study was to determine the effect of 5‐aminolevulinic acid (ALA), a chlorophyll precursor that promotes plant growth, on callus induction from leaves of Taxus cuspidata, and on callus growth on solid medium. ALA at 0.76, 7.6, and 76 μM had similar effects on callus induction and growth, while ALA at 760 μM had negative effects. Next, the effects of ALA concentrations on callus growth and paclitaxel production in suspension cultures in the dark were evaluated. The results showed that 0.76 and 7.6 μM ALA stimulated growth and paclitaxel production, while 76 μM ALA had negative effects. ALA is thought to promote cellular activity under light conditions. Therefore, the effects of light intensity on callus growth and paclitaxel production in the presence of ALA were evaluated. Our results showed that the best conditions for callus growth and paclitaxel production were 7.6 μM ALA under photosynthetically active radiation of 12 μmol photons m^?2 s^?1. Callus growth and paclitaxel production were inhibited under stronger light (24 μmol photons m^?2 s^?1). Together, these results show that ALA promoted callus growth and the production of paclitaxel by light‐grown cultured T. cuspidata cells.