Effect of precursor feeding on alkaloid accumulation by a strictosidine synthase over-expressing transgenic cell line S1 of Catharanthus roseus

The transgenic S1 cell line of Catharanthus roseus (L.) G. Don has been used to study possible rate limiting steps in the terpenoid indole alkaloid (TIA) biosynthesis. Line S1 carries a recombinant, over-expressed version of the endogenous Str gene which encodes strictosidine synthase (STR; EC 4.3.3.2). STR catalyzes the stereospecific condensation of tryptamine and secologanin to strictosidine. Various concentrations and combinations of biosynthetic indole precursors L-tryptophan, tryptamine, and iridoid precursors loganin and secologanin were added to the cell suspension cultures of line S1. The largest TIA accumulation occurred when the precursor was supplied at the time of inoculation of the cells into the production medium. Line S1 could supply tryptamine endogenously up to 0.8 mM loganin feeding. The enhancement of the accumulation of TIAs by addition of loganin indicates a limitation in the terpenoid pathway. Supplying tryptamine or tryptophan along with the iridoid precursors resulted in even further increase of alkaloid accumulation. Under optimal conditions, cultures of line S1 accumulated about 600 mol l^–1 of TIAs. Also, the conversion of strictosidine into other TIAs further down the pathway seems to be a limiting step. Considering the mass balance of the intermediates fed and TIAs recovered, several yet unknown pathways must be involved in channeling away intermediates from the TIA pathway and in the breakdown of the TIAs. Our results suggest that high rates of tryptamine synthesis can still take place under conditions of low TDC activity and the flux towards tryptamine is induced by loganin feeding. However, accumulation of tryptamine seems to reduce the flux through feedback inhibition.     

Effect of precursor feeding on alkaloid accumulation by a tryptophan decarboxylase over-expressing transgenic cell line T22 of Catharanthus roseus

To obtain more insight into the regulation of terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus (L.) G. Don cell cultures and particularly to identify possible rate limiting steps, a transgenic cell line over-expressing tryptophan decarboxylase (Tdc), and thus having a high level of tryptamine, was fed with various amounts of precursors (tryptophan, tryptamine, loganin and secologanin) in different time schedules and analyzed for TIA production. When these precursors were added to this culture it was found that the optimal time for supplying the precursors was at inoculation of the cells into the production medium. Alkaloid accumulation by line T22 was enhanced by addition of loganin or secologanin; however, the secologanin feeding was less effective. Tryptamine or tryptophan alone had no effect on TIA accumulation. The over-expression of Tdc causes this cell line to produce quite large quantities of alkaloids after feeding loganin or secologanin. However, in combination with tryptophan or tryptamine, feeding of these precursors resulted in an even further increase of alkaloid accumulation and under optimal conditions line T22 accumulated around 1200 micromol l(-1) of TIAs whereas the control cultures accumulated less than 10 micromol l(-1) TIAs.     

Effects of alkaloid precursor feeding and elicitation on the accumulation of secologanin in a Catharanthus roseus cell suspension culture

In a Catharanthus roseus cell line accumulating secologanin, time-course studies on the uptake of loganin and the in vivo conversion to secologanin were performed. Four-day-old cells converted 100% of the fed loganin to secologanin within 24 hours, showing that this step is unlikely to be limiting for alkaloid accumulation. Thirteen-day-old cells also took up loganin, but only about 25% was recovered as secologanin. A saturation in the uptake of loganin and in the conversion of loganin into secologanin was observed after feeding increasing amounts of loganin. Elicitation by cellulase and pectinase decreased the cellular contents of secologanin and strictosidine whereas it increased the tryptamine content. In addition, the uptake of loganin in elicited cells was blocked. In vitro assays with protein extracts of elicited Catharanthus roseus cells indicated the activation of secologanin degrading enzyme(s). Feeding of tryptophan did not result in any increase in alkaloid contents, despite its complete uptake. Tryptamine feeding led to increased strictosidine contents, but ajmalicine levels remained unchanged. This revised version was published online in June 2006 with corrections to the Cover Date.     

通过组织培养筛选高含量喜树碱细胞系

测定不同生长期喜树苗各器官中喜树碱的含量。其中新生叶和根中的喜树碱的含量较高,然而,喜树碱的含量随叶和枝条生长期的延长而逐渐下降。以喜树碱高含量的幼叶为材料,进行了愈伤组织的诱导和培养,并通过优良细胞系的筛选,使愈伤组织中喜树碱的含量提高到0．02％。     

Effects of Chemical and Physical Conditions on Growth of Camptotheca acuminata Cell Cultures

Callus was induced from Camptotheca acuminata, which produces an antitumor alkaloid, carnptothecin. Using the Murashige and Skoogs’ medium as the basal, cultural conditions were examined for C. acuminata suspension cultures. As a result, a medium, containing 0.1 mg/liter 2,4-D, 3 mg/liter kinetin and 0.05 mg/liter GA₃, was established as a medium that gave the best cell growth in suspension cultures. In addition, conditioning of medium and addition of 0.115 mm l-Trp and l-Phe to medium promoted remarkably growth of cell suspensions.     

Characterization of callus formation and camptothecin production by cell lines of Camptotheca acuminata

Both incubation temperature and photosynthetic radiation affected morphogenesis, callus culture and plantlet culture of sweet orange (Citrus sinensis) cultured in vitro. Bud culture from nodal stem segments, regeneration of shoots and buds from internode stem segments and induction of primary callus were near optimal at incubation temperatures between 21–30°C. The optimal temperature for root formation was 27°C with temperatures above and below being clearly deleterious. Incubation in the dark or under low photosynthetic photon flux density (PPFD) was beneficial for callus induction and growth and also favored the production of rooted plantlets from bud cultures. Incubation in the dark improved considerably the regeneration of shoots and buds from internode segments and the recovery of whole plants. No off-types, as determined by protein and isoenzyme analysis, were observed among plantlets recovered from bud cultures or from regeneration of shoots from internode stem segments.     

Cu2+对喜树愈伤细胞中喜树碱合成的影响

喜树碱是一种从木本植物喜树(Camptothecaacuminata)中分离得到的抗癌活性物质,通过细胞培养合成喜树碱是喜树碱生产的一条重要途径。研究Cu~(2 )对喜树愈伤细胞培养中喜树碱积累的影响,结果表明,在B5培养基中添加0.008mg/mLCuCl2时,对喜树愈伤细胞的生长没有明显影响,但是对喜树愈伤细胞合成喜树碱的促进作用最强,喜树碱含量比未诱导前增加了约30倍。Cu~(2 )阻止培养细胞后期过氧化物酶活力的下降,并抑制花青素的生成。与黑暗培养相比,光照条件下添加Cu~(2 )更利于喜树愈伤细胞诱导合成喜树碱。     

Establishment of Camptotheca acuminata regeneration from leaf explants

Plantlet regeneration through shoot formation from young leaf explant-derived callus of Camptotheca acuminata is described. Calli were obtained by placing leaf explants on Woody plant medium (WPM) supplemented with various concentrations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Callus induction was observed in all media evaluated. On the shoot induction medium, the callus induced on the WPM medium containing 19.8 μM BA and 5.8 μM NAA was the most effective, providing high shoot regeneration frequency (70.3 %) as well as the highest number of shoots (11.2 shoots explant⁻¹). The good rooting percentage and root quality (98 %, 5.9 roots shoot⁻¹) were achieved on WPM medium supplemented with 9.6 μM indole-3-butyric acid (IBA). 96 % of the in vitro rooted plantlets with well developed shoots and roots survived transfer to soil.     

影响喜树组织培养苗离体生根的因素

为了建立有效的喜树(Camptotheca acuminata)组培苗生根系统,提高其移栽成活率及适应性,用不同生长素种类及浓度、不同蔗糖浓度及不同培养基对喜树组培苗不定根形成影响以及移栽初期根系发育状况进行了研究.结果发现:1)生长素种类和浓度明显影响喜树组培苗不定根形成,在含有IBA0.5 mg·L-1培养基中取得了最佳生根效果,生根率达到了98%,外植体平均生根数为5.9条/株;2)不同浓度蔗糖对喜树组培苗生根也有一定影响,在10～30 g·L-1范围内,随着蔗糖浓度增加,生根百分率和生根数量都有增加,蔗糖浓度达到30 g·L-1时,生根百分率为95%,外植体平均生根数为5.4条/株;蔗糖浓度在40 g·L-1时,表现出对生根抑制作用;3)在基本培养基对喜树组培苗生根影响研究中发现,MS培养基对根形成表现出一定抑制作用;1/2MS和WPM培养基均适合喜树组培苗生根;4)根系发育正常的喜树组培苗移栽后成活率可达96%,但组培苗根系根毛系统发育较差.组培苗单位叶面积根尖数量显著低于对照实生苗,而且此参数与叶片气孔导度呈显著正相关.这种较差根系发育导致叶片气孔导度过低可能是组培苗叶片光合能力较低的重要原因.     

The effect of growth regulators and sucrose on anthocyanin production in Camptotheca acuminata cell cultures.

The effect of different concentrations of growth regulators and sucrose on anthocyanin production in cell suspension cultures of Camptotheca acuminata Decaisne (Nyssaceae) was described for the first time and qualitatively and quantitatively evaluated. Anthocyanin production was significantly greater in the presence of kinetin, compared to benzyladenine, with the greatest concentration observed in the presence of 2 microM kinetin. No significant differences in anthocyanin production were observed when comparing 2,4-dichlorophenoxyacetic acid to alpha-naphthaleneacetic acid, except when using 2 microM, 2,4-dichlorophenoxyacetic acid, which resulted in greater anthocyanin production. High sucrose concentration enhanced the production of anthocyanins. Based on the absence of anthocyanin production in the dark, we concluded that light was essential for stimulating anthocyanin production. The optimised medium consisted of: 2 microM kinetin, 2 microM 2,4-dichlorophenoxyacetic acid and 292 mM sucrose. HPLC/DAD and HPLC/MS analyses revealed that the main anthocyanin was Cy 3-O-galactoside and that the minor derivative was Cy 3-O-glucoside.     

Effects of Benzyladenine and Naphthalene Acetic Acid on Growth and Camptothecin Accumulation in Camptotheca acuminata Seedlings

The effects of the cytokinin benzyladenine (BA) and the auxin naphthalene acetic acid (NAA) on Camptotheca acuminata Decaisne growth and camptothecin (CPT) accumulation (leaf CPT concentration and total leaf CPT yield) were studied in a hydroponic culture system for three weeks. Increasing BA concentrations from 0 to 3 mg l^–1 in growth medium decreased plant height, stem weight, and leaf weight but increased root weight. High BA levels (1 and 3 mg l^–1) increased leaf CPT concentration (% of dry weight), whereas BA applications had no effect on total leaf CPT yield, the product of leaf CPT concentration and total leaf dry weight per seedling. There was a positive correlation between root weight and leaf CPT concentration under BA treatments. NAA supplementations (from 0.5 to 4 mg l^–1) to growth medium reduced plant height, leaf number, leaf length, specific leaf weight, plant weight, stem weight, and leaf weight compared with the NAA control. Meanwhile, there were no differences in plant height, leaf length, and specific leaf weight among the NAA supplementations. NAA applications had no effect on leaf CPT concentration and NAA applications decreased total leaf CPT yield. There were negative correlations between leaf number and leaf CPT concentration, leaf length and leaf CPT concentration under NAA treatments. Our results suggest that BA applications from 0.3 to 3 mg l^–1 are not helpful for achieving high total leaf CPT yield and NAA applications from 0.5 to 4 mg l^–1 decrease total leaf CPT yield.     

不同种源喜树幼枝中喜树碱的含量

通过HPLC测定了喜树(Camptotheca acuminata)不同器官及不同种源幼枝中喜树碱的含量.结果表明,喜树碱含量在叶、种子和幼枝(幼叶和幼茎)中较高,木质部中较少,髓中最低.不同种源的幼枝中,来自成都种源喜树的喜树碱含量最高.通过比较,两年生喜树各器官中喜树碱含量普遍比一年生高,幼枝中顶枝的喜树碱含量比侧枝高.     

Molecular cloning and characterization of a novel stem-specific gene from Camptotheca acuminata

In higher plants, P450s participate in the biosynthesis of many important secondary metabolites. Here we reported for the first time the isolation of a new cytochrome P450 cDNA that expressed in a stem-specific manner from Camptotheca acuminata (designated as CaSS), a native medicinal plant species in China, using RACE-PCR. The full-length cDNA of CaSS was 1735 bp long containing a 1530 bp open reading frame (ORF) encoding a polypeptide of 509 amino acids. Bioinformatic analysis revealed that CASS contained a heme-binding domain PFGXGRRXCX and showed homology to other plant cytochrome P450 monooxygenases and hydroxylases. Southern blotting analysis revealed that there was only one copy of the CaSS present in the genome of Camptotheca acuminata. Northern blotting analysis revealed that CaSS expressed, in a tissuespecific manner, highly in stem and lowly in root, leaf and flower. Our study suggests that CaSS is likely to be involved in the phenylpropanoid pathway.     

Clonal propagation of Camptotheca acuminata through shoot bud culture

The chinese tree Camptotheca acuminata produces the anti-cancer and anti-retroviral drug camptothecin. Methods were developed for the clonal propagation of this important medicinal plant through shoot bud culture. Shoot buds were excised from 25 to 30 day old seedlings, presoaked for 48 h in three different liquid media containing either BA (2.22–17.4 M), kinetin (2.32–18.58 M), or thidiazuron (0.1–10 M) and were subsequently cultured on semi-solid medium of the same composition. Multiple shoots only developed from the 6-benzyladenine presoaked explants with the maximum number of shoots initiated from buds presoaked in and grown on B5 medium containing 17.4 M 6-benzyladenine. Individual shoots were removed from clusters and rooted on B5 supplemented with indole-3-butyric acid (4.9–19.6 M). The lowest concentration of indole-3-butyric acid (4.9 M) gave the highest percentage of rooting (82%) and the shortest root initiation period (18 d). Over 90% of the in vitro rooted plantlets survived transfer to soil.Abbreviations BA 6-benzyladenine - B5 Gamborg's B5 medium (Gamborg et al., 1968) - CPT camptothecin - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - LS Linsmaier & Skoog medium (Linsmaier & Skoog, 1965) - MS Murashige & Skoog (Murashige & Skoog, 1962) - NAA I-naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - WPM woody plant medium (Lloyd & McCown, 1981)     

Dependence of UV-B-induced camptothecin production on nitrate reductase-mediated nitric oxide signaling in Camptotheca acuminata suspension cell cultures

UV-B irradiation induced production of secondary metabolites in plant cells. However, the mechanisms of UV-B-induced secondary metabolite production remained largely unknown. Here we report that UV-B treatment stimulated nitric oxide (NO) generation and camptothecin (CPT) production in Camptotheca acuminata cells. To investigate the origin of the UV-B-triggered NO and the role of NO in UV-B-induced CPT production, we assayed the responses of nitrate reductase (NR) and NO synthase (NOS) activities of the cells to UV-B exposure and examined the effects of NR and NOS inhibitors on CPT production in UV-B-treated cells. The data showed that UV-B irradiation enhanced NR activities in the cells. Pretreatment with NR inhibitors tungstate and okadaic acid not only suppressed the UV-B-triggered NR activities but also inhibited the UV-B-induced NO generation and CPT production in the cells. In contrast, UV-B irradiation had no effects on NOS activity of the cells and treatment of NOS inhibitor did not suppress UV-B-induced CAT production. Together, the results demonstrated that NR activity was essential for UV-B-triggered NO generation and that NR-mediated NO signaling was involved in UV-B-induced CPT production in C. acuminata cells.     

中国特有植物喜树的雌性不育形态解剖学研究

为阐明雌性不育与喜树（Camptotheca acuminata Decne.）开花期花序大量掉落的关系,本文采用数码体视显微镜及石蜡切片技术,对各个发育时期的喜树的花进行解剖学观察。喜树花均为两性花,雄蕊发育正常,雌蕊柱头有三种不同形态,即上位、中位和下位;柱头上位的小花,花柱伸长正常,柱头三裂,外翻,子房膨大,胚珠饱满,胚囊发育正常;柱头中位的小花,雌蕊选择性败育,柱头和花柱伸长缓慢,柱头外翻异常,局部子房细胞液泡化,胚珠退化,胚囊发育异常;柱头下位的小花,花柱退化,柱头不露,珠被细胞发育异常,大孢子母细胞未能减数分裂,胚珠萎缩,子房外形瘦长,室内中空。喜树雌性器官的发育与其柱头在子房上的表现形态有着严格的对应关系,雌性不育发生于大孢子母细胞减数分裂前后,而且雌性不育是喜树花序掉落的内部原因之一。     

Influence of auxins on alkaloid accumulation by a transgenic cell line of Catharanthus roseus

The transgenic cell line of Catharanthus roseus (L.) G. Don S10 was used to study the effect of the presence of the synthetic auxins naphthalene acetic acid and 2,4-dichlorophenoxyeacetic acid in the culture medium on the accumulation of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively over-expressed version of the endogenous strictosidine synthase gene. The experiments were carried out using a two-stage culture system, consisting of a growth phase of 7 to 10 days and a production phase of 14 or 30 days. The hormonal composition was a crucial factor when formulating both the growth and the production media. It was determined that the presence of naphthalene acetic acid during the production phase led to lower levels of alkaloid accumulation. The presence of 2,4-dichlorophenoxyacetic acid in the growth medium reduced culture aggregation and repressed secondary metabolism. Cultures grown in medium containing 2,4-dichlorophenoxyacetic acid showed reduced capacity to supply biosynthetic precursors, which resulted in low levels of accumulation of terpenoid indole alkaloids. Cultures grown in 2,4-D-containing medium showed reduced capacity to supply biosynthetic precursors and higher rates of catabolic activity, which resulted in low levels of TIA accumulation. The expression of the gus and strictosidine synthase transgenes, measured at the enzyme level, was similarly high under all conditions tested. This revised version was published online in June 2006 with corrections to the Cover Date.     

喜树幼枝的喜树碱积累及其组织内定位

对喜树幼枝发育过程中各组织结构变化的观察和喜树碱含量的分析,以及荧光显微技术对喜树碱的定位研究,结果表明,幼茎和幼叶表面的单细胞腺毛和幼茎及幼叶内部由1～2层细胞包围而成的分泌道是喜树碱的主要积累部位.