Induction of rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures by yeast extract

Summary A transient increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after addition of yeast extract (YE) to the suspension cultures, reaching a maximum at 24 hr. The highest increase of the RA content (2.5-fold) was obtained when 6-day-old cells in the exponential growth phase were treated with YE. Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) activity increased rapidly, whereas tyrosine aminotransferase (TAT) activity was largely unaffected by the treatment. The incorporation of both ¹⁴C-phenylalanine and ¹⁴C-tyrosine into RA was enhanced in the YE-treated cells, consistent with increased synthesis of the ester.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - RA rosmarinic acid - YE yeast extract     

Elicitor-induced rosmarinic acid accumulation and secondary metabolism enzyme activities in Solenostemon scutellarioides

This study aimed to improve rosmarinic acid (RA) production in the whole plant culture of Solenostemon scutellarioides through elicitation. Amongst selected elicitors methyl jasmonate (MJ), salicylic acid (SA), and yeast extract (YE) caused significant elevation in RA accumulation. Elicitation with MJ (50 μM) and SA (50 μM) caused almost 1.7 and 1.4-fold increase in RA accumulation, respectively, within day 1. While YE (100 μg ml^?1) elicitation showed highest RA content (~1.5-fold) in day 3. Preceding the elicitor-induced RA accumulation, there was a notable alteration in the specific activities of RA biosynthetic enzymes viz. phenylalanine ammonia lyase, tyrosine aminotransferase, hydroxyl-phenylpyruvate reductase and rosmarinic acid synthase up on MJ (50 μM), SA (50 μM) and YE (100 mg ml^?1) elicitation. Based on differential responses of aforementioned enzymes, RA synthesis was further scaled up through combination of elicitors in pre-optimized doses. In synergy study, at a time exposure with MJ + SA + YE and MJ + SA followed by YE after 24 h has been found to produce significant elevation of RA (2.0 and 1.9-fold, respectively) within 24 h while later maintained a steady state increased level (~1.7 ± 0.2-fold) over control up to day 7.     

Rosmarinic acid formation and differential expression of tyrosine aminotransferase isoforms in Anchusa officinalis cell suspension cultures

Time-course changes in rosmarinic acid (RA) formation and activities of tyrosine aminotransferase (TAT) isoforms were examined in Anchusa officinalis suspension cultures. Three TAT isoforms (TAT-1, TAT-3, TAT-4) were resolved by Mono-Q anion-exchange column chromatography. The proportion of the TAT-3 activity within the total TAT activity remained high regardless of the growth stage of the cultured cells. TAT-1 activity was positively correlated with the rate of RA biosynthesis during linear growth stage of the culture cycle, while TAT-4 activity was rapidly induced in conjunction with transfer to fresh medium coincident with a transient increase in RA synthesis. Based on these results, as well as the substrate specificity of each TAT isoform, it was concluded that both TAT-1 and TAT-4 are closely involved in RA biosynthesis. TAT-1 controls conversion of tyrosine to 4-hydroxyphenyl pyruvate, and TAT-4 acts by participating in the formation of tyrosine and phenylalanine via prephenate.Abbreviations PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - RA rosmarinic acid     

Differential accumulation of xanthones in methyl-jasmonate- and yeast-extract-treated cell cultures of Centaurium erythraea and Centaurium littorale

Cell-suspension cultures of Centaurium erythraea and Centaurium littorale (Gentianaceae) respond to methyl jasmonate and yeast extract with a differential accumulation of xanthones. Methyl jasmonate induced the formation of 1-hydroxy-3,5,6,7-tetramethoxyxanthone, the amount of which increased in both cell cultures around 10 h after addition. A substantial increase in the activity of phenylalanine ammonia-lyase (PAL) was not observed. When challenged with yeast extract the cell cultures accumulated l,5-dihydroxy-3-methoxyxanthone. This appeared rapidly after addition of yeast extract in C. erythraea but its amount in C. littorale increased only after a lag phase of 25 h. While PAL activity in C. erythraea was strongly suppressed a fourfold increase in its activity was found in C. littorale. Both elicited xanthones accumulated intracellularly. A scheme for xanthone biosynthesis in the two cell cultures is proposed.     

Biosynthesis and accumulation of rosmarinic acid in suspension cultures ofColeus blumei

This communication reviews data on the accumulation and biosynthesis of rosmarinic acid in cell suspension cultures ofColeus blumei. The influence of the medium, mainly the carbohydrate source on growth and rosmarinic acid production in these cell cultures is described. The biosynthetic pathway of rosmarinic acid was elucidated inColeus blumei cell cultures: eight enzymatic activities are involved in the transformation of the precursors phenylalanine and tyrosine to the end product rosmarinic acid.Abbreviations CAH cinnamic acid 4-hydroxylase - 4CL 4-coumarate:CoA ligase - HPPR hydroxyphenylpyruvate reductase - 3-H hydroxycinnamoyl-hydroxyphenyllactate 3-hydroxylase - 3-H hydroxycinnamoyl-hydroxyphenyllactate 3-hydroxylase - PAL phenylalanine ammonia-lyase - RAS rosmarinic acid synthase (hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyl transferase) - TAT tyrosine aminotransferase     

The biosynthesis of rosmarinic acid in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei accumulate very high amounts of rosmarinic acid, an ester of caffeic acid and 3,4-dihydroxyphenyllactate, in medium with elevated sucrose concentrations. Since the synthesis of this high level of rosmarinic acid occurs in only five days of the culture period, the activities of the enzymes involved in the biosynthesis are very high. Therefore all the enzymes necessary for the formation of rosmarinic acid from the precursors phenylalanine and tyrosine could be isolated from cell cultures of Coleus blumei: phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, hydroxycinnamoyl:CoA ligase, tyrosine aminotransferase, hydroxyphenylpyruvate reductase, rosmarinic acid synthase and two microsomal 3- and 3-hydroxylases. The main characteristics of these enzymes of the proposed biosynthetic pathway of rosmarinic acid will be described.Abbreviations DHPL 3,4-dihydroxyphenyllactate - DHPP 3,4-dihydroxyphenylpyruvate - pHPL 4-hydroxyphenyllactate - pHPP 4-hydroxyphenylpyruvate - RA rosmarinic acid     

水杨酸对丹参培养细胞中迷迭香酸生物合成及其相关酶的影响

迷迭香酸(RA)是丹参中一种重要的酚酸类次生代谢物。为探讨水杨酸(SA)诱导子对丹参悬浮培养细胞中RA的生物合成及其相关酶的影响,考察了SA诱导子和酪氨酸氨基转移酶(TAT)的竞争性抑制剂(AOPP)对RA合成积累量、苯丙氨酸解氨酶(PAL)和TAT活性的影响。发现在培养的第6天用浓度为6.25 mg/L的SA处理后,PAL活性在诱导后4 h出现高峰,为对照组水平的124%;RA的积累量在诱导后8 h出现峰值(5.914±0.296)mg/g。用浓度为0.1μmol/L的AOPP处理,6 h后AOPP对TAT活性影响较小(与对照组无显著差异),但明显抑制了PAL活性(为对照组水平的44%),且在PAL活性明显降低的同时RA的积累量显著减少(4.709±0.204)mg/g。进一步用0.1μmol/L AOPP和6.25 mg/L SA共处理,AOPP对PAL的抑制作用可得到一定程度的缓解,且RA的积累量较AOPP单独处理的高。表明SA可以诱导丹参悬浮培养细胞中RA积累量的增加,且在RA合成过程中PAL的限速作用比TAT明显。     

咖啡酸和氯化钴对茉莉酸甲酯诱导抗病相关酶活性的影响

烟草愈伤组织在含咖啡酸（5 mmol/L）和/或CoCl2(10 mmol/L,乙烯合成抑制剂)的MS培养基上暗培养,同时用茉莉酸甲酯（1 mg/ml,简称MJ）处理愈伤组织。处理后测定乙烯、水杨酸和病程相关蛋白（PR）含量及一些抗病相关酶活性。MJ明显促进乙烯产生、增加水杨酸和PR蛋白含量,提高苯丙氨酸解氨酶（PAL）、β1,3-葡聚糖苷酶和几丁酶的活性。咖啡酸降低MJ对乙烯和水杨酸诱导,CoCl2明显降低MJ对乙烯的诱导,但没有明显影响MJ对水杨酸的诱导,两者都促进MJ诱导PAL活性而抑制MJ诱导β1,3-葡聚糖苷酶活性。咖啡酸明显影响MJ诱导内切几丁酶,几乎完全抑制对外切几丁酶的诱导;CoCl2对MJ诱导内切几丁酶没有影响,促进对外切几丁酶的诱导。实验结果表明,不同的抗病相关酶活性诱导有不同的信号传递途径,在所测几种酶的诱导中,水杨酸起主要作用,乙烯作用较小,MJ的诱导作用主要是由水杨酸所转导。     

Regulation of phenylalanine ammonia-lyase genes in carrot suspension cultured cells

Induction of anthocyanin synthesis occurs during metabolic differentiation in carrot suspension cultured cells grown in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), and is closely correlated with embryogenesis. Anthocyanin synthesis may also be induced by light-irradiation under different culture conditions. The phenylalanine ammonia-lyase (PAL) gene (TRN-PAL), which was transiently induced by the transfer effect, was also rapidly induced after light-irradiation. However, TRN-PAL was not involved in anthocyanin synthesis. A second PAL gene, ANT-PAL, was involved in anthocyanin synthesis. ANT-PAL was induced during metabolic differentiation in medium lacking 2,4-D parallel with the induction of chalcone synthase (CHS). PAL genes in the carrot genome are expressed differentially depending on the nature of the environmental stimulus, e.g. transfer effect and light, and other parameters which also affect anthocyanin synthesis.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - Luc firefly luciferase - PAL phenylalanine ammonia-lyase - UV ultraviolet     

Pisatin metabolism in pea (Pisum sativum L.) cell suspension cultures

Cell suspension cultures were established from germinating pea (Pisum sativum L.) seeds. This cell culture, which accumulated pisatin, consisted mostly of single cells containing a few cell aggregates. The cells responded to treatment with a yeast glucan preparation with transient accumulation of pisatin in both cells and culture media. Addition of pisatin to cell cultures resulted in increased synthesis of pisatin. Phenylalanine ammonia-lyase, chalcone synthase and isoflavone reductase activities were present in untreated cells. Upon treatment with an elicitor preparation the activities of the first two enzymes showed a rapid, transient increase up to 20 hours after treatment. Isoflavone reductase showed a major and minor peak at 16 and 36 h, respectively, after elicitor treatment. The time course of the enzyme activity and pisatin accumulation is consistent with an elicitor-mediated response.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - IFR isoflavone reductase - 2iP 6-(dimethylallylamino)-purine - MS Murashige & Skoog basal salt medium - PAL phenylalanine ammonia-lyase - PMSF phenylmethylsulfonyl fluoride - POPOP 1,4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene - PPO 2,5-diphenyloxazole     

Regulation of anthocyanin and lignin synthesis in Petunia hybrida cell suspensions

In Petunia hybrida cv. Violet 30 cell suspensions the phenylpropanoid pathway can be induced to produce lignin and anthocyanins. Orthovanadate addition leads to lignin accumulation, subculturing the cells using small inoculum sizes (<2 g fresh weight l^-1) gives rise to both anthocyanin and lignin production. Orthovanadate has a negative effect on cell growth. By replacing the medium, one day after orthovanadate addition, by medium without elicitor, we were able to restore growth without disturbing the lignin accumulation. The activity of phenylalanine ammonia-lyase (PAL) increased immediately after orthovanadate addition; this increase stopped upon medium replacement without affecting the lignin production. Reduction of the NAA concentration from 2 mg l^-1 to 0.1 mg l^-1, subsequent to the elicitation by orthovanadate or dilution stress, gave rise to a further increase in the production of lignin and anthocyanins respectively. Decreasing the NAA concentration without a prior elicitation, didn't have any effect on either PAL activity or product formation.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - BSA bovine serum albumine - FW fresh weight - NAA naphthaleneacetic acid - PAL phenylalanine ammonia-lyase - PPP phenyl propanoid pathway     

The estimation of phenylalanine ammonia-lyase (PAL) activity in intact cells of higher plant tissue

A previously described procedure for the estimation of relative activities of phenylalanine ammonia-lyase (EC 4.3.1.5) in intact plant cells (Amrhein et al. (1976) Planta 131, 33–40) was reexamined for its specificity and its applicability to various tissues. In buckwheat hypocotyl segments ³H is stereospecifically released from the pro-3S-position of L-[2,3-³H]phenylalanine and is thus due to phenylalanine ammonia-lyase activity. In buck wheat and sunflower leaf disks, however, ³H release occurs from both the 2- and 3-positions of the labeled substrate and can only partially be attributed to phenylalanine ammonia-lyase activity.Abbreviations AOA -aminooxyacetic acid - L-AOD L-aminoacid oxidase (EC 1.4.3.2) - D-AOD D-amino-acid oxidase (EC 1.4.3.3) - L-AOPP L--aminooxy--phenylpropionic acid - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - TAL tyrosine ammonia-lyase     

Optimization of elicitors and precursors to enhance valtrate production in adventitious roots of <Emphasis Type="Italic">Valeriana amurensis</Emphasis> Smir. ex Kom

Valeriana amurensis Smir. ex Kom. is a traditional Chinese medicinal plant as its roots serve as a rich source of valtrate, a compound with antifungal cytotoxic sedation and tranquilizing activities. In this study, an adventitious root culture of V. amurensis was established, and the effects of different elicitors, including salicylic acid (SA), chitosan (CH), jasmonic acid (JA), methyl jasmonate (MJ), yeast extract (YE), and yeast extract carbohydrate fraction (YECF) along with the precursor citronellal (CI) on production of valepotriates were assayed. MJ and JA highly promoted valtrate production; while, CH moderately promoted valtrate production. In contrast, CI, SA, YE, and YECF were ineffective in promoting valtrate production. Using a response surface methodology, optimal combinations of MJ, JA, and CH were shown to have strong synergistic effects on valtrate production, and this was subsequently confirmed in experimental studies.     

Evidence for p-hydroxybenzoate formation involving enzymatic phenylpropanoid side-chain cleavage in hairy roots of Daucus carota

In this study, methyl jasmonate (MJ)-elicited hairy root cultures of Daucus carota were explored to study the enzymatic route to p-hydroxybenzoic acid (p-HBA) biosynthesis. Treatment with 100muM MJ caused an enhanced accumulation of p-HBA as well as total phenolic content in elicited root lines as compared to untreated (controls) lines. Using cell-free extract as the source of crude enzymes, attempt was made to reveal the enzymatic route to p-HBA formation. The accumulation of p-HBA was preceded by a substantial upliftment of p-hydroxybenzaldehyde dehydrogenase (HBD) activity in elicited lines as compared to controls. A rapid 6-fold enhancement of phenylalanine ammonia-lyase (PAL) activity, the first enzyme of the phenylpropanoid pathway was also observed. Finally, we demonstrated here for the first time, in D. carota, the evidence of a quite unusual p-hydroxybenzaldehyde synthase (HBS)-type enzyme, which catalyzes the penultimate step of p-HBA biosynthesis by making phenylpropanoid side-chain cleavage of p-coumaric acid without involvement of any cofactor(s), but uplifted by supplementation of a thiol reagent such as DTT in the reaction buffer. This enzyme showed activity in a relatively broad pH range (7-8.4) and the temperature optimum was found to be at 34 degrees C. The MJ-treated roots showed highest HBS activity at 24h (52nkat/mg protein), which was nearly 5-fold higher than that in the control lines.     

The interaction of auxin and cytokinin in the induction of phenylalanine ammonia-lyase in suspension cultures of Phaseolus vulgaris

The dependence of phenylalanine ammonialyase (PAL) induction in bean suspension cultures on the concentration of naphthylacetic acid (NAA) and kinetin has been investigated and the timing of the effect of each hormone has been determined. NAA was required at an optimal concentration of 1 mg l^-1 2 days prior to the increase in PAL activity. Kinetin caused a prapid stimulation of the rate of PAL induction and the total amount of PAL induced in a concentration range of 0.1–0.5 mg l^-1 when it was supplied to the cells immediately prior to the expected rise in PAL activity. The inhibitory effect of 2 mg l^-1 NAA on PAL induction was overcome by an increased concentration of kinetin.Abbreviations NAA naphthylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3yl acetic acid - PAL phenylalanine ammonia-lyase (EC 4.3.1.5.)     

Improved cardenolide production in Calotropis gigantea hairy roots using mechanical wounding and elicitation

A hairy root culture system of Calotropis gigantea was established and effects of mechanical wounding (MW) and elicitors [methyl jasmonate (MJ), yeast extract (YE) and chitosan (CS)] on cardenolide production were investigated. All treatments stimulated the production of cardenolide in hairy root cultures of C. gigantea. CS was the most effective elicitor, followed by MJ. YE and MW also improved cardenolide yield in individual treatments. The highest cardenolide yield (1,050 ± 55 mg/l) was obtained after adding 50 mg CS/l for 20 days, which was 2.7-fold higher than the control.     

Differential induction of protein expression and benzophenanthridine alkaloid accumulation in Eschscholtzia californica suspension cultures by methyl jasmonate and yeast extract

Methyl jasmonate (MJ) and yeast extract (YE) induce protein expression and benzophenanthridine alkaloid accumulation in Eschscholtzia californica suspension cell cultures. One hundred microM MJ primarily induced dihydrosanguinarine 509.0+/-7.4 mg/l); 0.2 g/l YE induced sanguinarine (146.8+/- 3.8 mg/l) and an unknown compound. These results occur because dihydrobenzophenanthridine oxidase (DHBO) is induced by YE and not by MJ. YE and chitin (CHI) had similar effects on sanguinarine production and DHBO expression. Differential induction of secondary metabolites was shown in E. californica suspension cultures and the expression of proteins confirmed the metabolite results. Furthermore, treatment by various oligosaccharides helped us to understand the elicitation effect of YE in signal transduction pathways.