Increases of secondary metabolite production in various plant cell cultures by co-cultivation with cork

Cork tissues increased secondary metabolite production of various plant cell cultures in a different manner from those of conventional elicitors. In Sophora flavescens and Glycyrrhiza glabra cultured cells, cork tissues increased the amounts of both lipophilic and hydrophilic flavonoids without affecting the cell growth, although elicitors such as copper ion and yeast extracts showed a clear inhibition of cell growth with the increasing amount of these lipophilic ones. The validity of this effect of cork tissues covered a wide range of aromatic compounds produced by suspension cell cultures derived from diverse plant species. Woody tissues of Japanese cypress had a very similar effect to that of cork. Partial purification of cork tissues suggested that the production-stimulating factor was present in the hemicellulose B fraction that was not included in the dedifferentiated cultured tissues.     

Efficient production and capture of 8-prenylnaringenin and leachianone G-biosynthetic intermediates of sophoraflavanone G--by the addition of cork tissue to cell suspension cultures of Sophora flavescens

It has previously been demonstrated that the addition of cork tissue to cell suspension cultures of Sophora flavescens stimulates the production of sophoraflavanone G, most of which has been recovered from the added cork tissue. In the present study, it was found that two precursors of sophoraflavanone G, 8-prenylnaringenin (sophoraflavanone B) and leachianone G, both of which have never been detected either in cultured cells or in the original plants, also accumulated in the added cork tissue. Thirteen minor flavonoids including three prenylated flavonoids, in addition to 8-prenylnaringenin and leachianone G, were isolated from the cork tissue co-incubated with S. flavescens cells. The new compounds flavescenones A, B and C, were determined to be (3R)-5, 7, 2'-trihydroxy-6-gamma, gamma-dimethylallyl-4', 5'-methylenedioxyisoflavanone; 5, 7, 2'-trihydroxy-6-gamma, gamma-dimethylallyl-4', 5'-methylenedioxyisoflavone and 2-[2',4'-dihydroxy-3'-(gamma-hydroxymethyl-gamma-methylallyl)phenyl]-5,6-methylenedioxybenzofuran, respectively, by means of spectroscopic analyses that included 2D-NMR techniques.     

Zn/Pb-tolerant lichens with higher content of secondary metabolites produce less phytochelatins than specimens living in unpolluted habitats

Many lichens can cope with heavy-metal stress, however, the mechanisms of lichen tolerance are still not fully understood. Some lichen secondary metabolites (depsides and depsidones), produced in lichens by the fungal symbiont and accumulated on the outer surface of its hyphae, are supposed to play an important role in the extracellular immoblilization of heavy metals. Lichen photobionts (algal partners in the symbiosis), although surrounded by the mycobiont hyphae, may also accumulate high amounts of trace metals. This can lead to physiological disruptions and morphological damage in algal cells and hence affect the lichen physiological status. We hypothesized that lichen species/specimens living in heavily polluted sites and showing HM tolerance possess a higher content of secondary metabolites than those living in unpolluted sites. Hence, their photobionts can be better protected from the excess of metal ions and need to produce less metal-complexing phytochelatins (PCn) to combat metal toxicity. Specimens of Hypocenomyce scalaris, Cladonia furcata and Lepraria spp. sampled from Zn/Pb-polluted and control sites were compared for the accumulation of Zn/Pb and secondary metabolites, as well as for their production of phytochelatins and glutathione in response to experimental Zn or Pb exposure. Generally, the lichen specimens sampled from the HM-polluted site contained higher amounts of Zn and Pb as well as lichen substances (different depsides and depsidones) than those from the control site. A strong positive correlation was found between the accumulation of secondary metabolites and Zn/Pb accumulation (R² = 0.98 and 0.63, respectively). For the first time, production of phytochelatins (PC_2-3) in response to Zn and Pb (50-200 μM) exposure was found in H. scalaris, L. elobata, L. incana and C. furcata. In both species of Lepraria also cysteine, a substrate for GSH and PCs synthesis was detected. The lichens from the polluted site produced under the same exposure conditions, or in response to higher metal concentrations, lower amounts of PCn than those sampled from the control site. It strongly suggests that less Zn and Pb ions reached the photobiont cells of the lichens containing higher amounts of secondary metabolites (lecanoric, fumarprotocetraric, stictic, constictic acids, antranorin). The results obtained support the putative role of some metabolites in heavy-metal tolerance of the lichens inhabiting metal-polluted habitats.     

Diterpenes from the brown algae Dictyota dichotoma and Dictyota linearis

Nineteen secondary metabolites of the brown alga Dictyota dichotoma (Huds.) Lam. and fifteen metabolites of the brown alga D. linearis (Ag.) Grev. were isolated and their chemical structures were elucidated on the basis of their NMR and mass spectral data. The diterpenes isopachydictyolal (1) from D. dichotoma and 4alpha-acetyldictyodial (2) from D. linearis are new natural products. The antiviral activity of metabolites isolated in adequate amounts was evaluated in laboratory assays against Herpes simplex virus I (HSV I) and Poliomyelitis Virus I, using Vero cells as hosts.     

玉米叶表皮短细胞发育过程的形态特征及栓质细胞作用研究

采用大田试验,直接撕表皮或对叶片进行固定处理,结合单染、复染、荧光染色等多种细胞学显色方法,利用光学显微镜、荧光显微镜和扫描电子显微镜系统观察玉米叶表皮短细胞的发生时期、发育过程、分布规律以及形态结构特征,研究K⁺和H₂O₂在栓质细胞中的分布变化与表皮其它细胞中K⁺和H₂O₂的分布及气孔器开关的关系,为进一步挖掘短细胞的新功能提供细胞学依据。结果表明：（1）短细胞是同步发生在玉米多叶位新表皮组织形成过程中,所有植株从第7新生叶,大部分第6叶,极少数第5叶的基部同时开始发生短细胞,之后新生的高位叶也均发生短细胞,并随着叶位的升高叶片各部位短细胞密度均增大,所有植株的1~4叶（因不再生长）均无短细胞出现。（2）初期发育的叶表皮细胞进行不对称分裂,生成相互交替的长、短细胞,有的短表皮细胞横（垂直叶脉）分裂,形成栓质细胞和硅质细胞对;栓质细胞基部与叶肉细胞相邻,硅质细胞嵌在栓质细胞和表皮细胞间偏上。（3）有短细胞发生的叶片,宏观背面发亮且覆有蜡质层,微观表皮细胞的着色特性发生了变化;栓质细胞为面包形柱状细胞,硅质细胞为哑铃形扁细胞。（4）气孔器张开时,栓质细胞中没有K⁺和H₂O₂的积累;气孔器关闭时,栓质细胞中积累了大量的K⁺和H₂O₂,且栓质细胞中K⁺和H₂O₂的积累始终与副卫细胞中K⁺和H₂O₂的积累变化一致,而硅质细胞和长细胞没有K⁺和H₂O₂的积累。该研究确定了玉米叶表皮短细胞发生的时期;展示了其发育过程的形态学变化特征;发现栓质细胞中K⁺和H₂O₂的积累随气孔器开关呈周期性变化,且与副卫细胞中K⁺和H₂O₂的积累变化保持一致。     

Sub-lethal levels of electric current elicit the biosynthesis of plant secondary metabolites

Many secondary metabolites that are normally undetectable or in low amounts in healthy plant tissue are synthesized in high amounts in response to microbial infection. Various abiotic and biotic agents have been shown to mimic microorganisms and act as elicitors of the synthesis of these plant compounds. In the present study, sub-lethal levels of electric current are shown to elicit the biosynthesis of secondary metabolites in transgenic and non-transgenic plant tissue. The production of the phytoalexin (+)-pisatin by pea was used as the main model system. Non-transgenic pea hairy roots treated with 30-100 mA of electric current produced 13 times higher amounts of (+)-pisatin than did the non-elicited controls. Electrically elicited transgenic pea hairy root cultures blocked at various enzymatic steps in the (+)-pisatin biosynthetic pathway also accumulated intermediates preceding the blocked enzymatic step. Secondary metabolites not usually produced by pea accumulated in some of the transgenic root cultures after electric elicitation due to the diversion of the intermediates into new pathways. The amount of pisatin in the medium bathing the roots of electro-elicited roots of hydroponically cultivated pea plants was 10 times higher 24 h after elicitation than in the medium surrounding the roots of non-elicited control plants, showing not only that the electric current elicited (+)-pisatin biosynthesis but also that the (+)-pisatin was released from the roots. Seedlings, intact roots or cell suspension cultures of fenugreek (Trigonella foenum-graecum), barrel medic, (Medicago truncatula), Arabidopsis thaliana, red clover (Trifolium pratense) and chickpea (Cicer arietinum) also produced increased levels of secondary metabolites in response to electro-elicitation. On the basis of our results, electric current would appear to be a general elicitor of plant secondary metabolites and to have potential for application in both basic and commercial research.     

Chlorophyll distribution in the stems and trunk of beech trees

The distribution of chlorophyll was examined in cross-sections of 2- and 6-year-old stems as well as in the bark of the stump trunk of beech trees, utilising chlorophyll autofluorescence. The investigations were conducted using a confocal microscope. The tests carried out on 2 – 6-year old stems showed a large presence of chlorophyll in the bark, in primary and secondary rays as well as in the pith, but smaller amounts in wood parenchyma cells. There was no chlorophyll in the cork, sclerenchyma: in wood in vessels, tracheids and fibers. A reduction in the chlorophyll content in 6-year-old stems was not observed. In the bark of the trunk, chlorophyll was found in large amounts directly under the cork and in vestigial amounts in the primary phloem.     

Cytokine regulation of adult human osteoblast-like cell prostaglandin biosynthesis

Prostaglandin (PG) biosynthesis by cytokine stimulated normal adult human osteoblast-like (hOB) cells was evaluated by thin layer chromatography, high performance liquid chromatography, and specific immunoassays. PGE₂ was the predominant PG formed under all incubation conditions tested. Control samples produced measurable amounts of PGE₂, and the measured level of this metabolite increased by 22-fold (from 7 to 152 ng/ml) following a 20 h treatment with the combination of TGFβ and tumor necrosis factor-α(TNF). The production of 6-keto-PGF_1α (the stable metabolite of prostacyclin) and of PGF_2α were each increased by about five-fold (from about 0.5 to 2.5 ng/ml) in samples treated with the cytokines. Thus, TGFβ and TNF exerted a regulation of hOB cell PG biosynthesis that was principally directed towards an increased PGE₂ biosynthesis, with lesser effects on the production of other PG metabolites. COX-2 mRNA levels were increased within 2 h of cytokine stimulation, reached a maximum at 6–12 h, and levels had appreciably diminished by 24 h after treatment. Both TGFβ and TNF could independently increase COX-2 mRNA levels and PG biosynthesis. However, the increased production of PGE₂ resulting from TNF stimulation was blocked by the addition of an interleukin-1β (IL-1β) neutralizing antibody, suggesting that TNF regulation of hOB cell PG synthesis was secondary to its capacity to increase hOB cell IL-1β production. TGFβ regulation of PG production was not affected by the addition of the neutralizing antibody. These studies support the proposition that PGs can be important autocrine/paracrine mediators of bone biology, whose production by hOB cells is responsively regulated by osteotropic cytokines. J. Cell. Biochem. 64:618–631. © 1997 Wiley-Liss, Inc.     

美登木根的显微结构及其内生真菌的分布

本文采用石蜡永久制片和光学显微摄像的方法对美登木(Maytenus confertiflorus)根的显微结构及其内生真菌的分布进行了研究。结果表明, 美登木根的次生结构由周皮和维管组织构成; 周皮由木栓层、木栓形成层和栓内层组成, 其中木栓层由5～6 列长形细胞组成; 维管组织中次生韧皮部所占根径的比例达46%, 其薄壁细胞中内含物较丰富, 次生木质部中分布有导管和木射线及少量木薄壁组织; 在美登木木栓层和次生韧皮部中分布有菌丝片段、膨大的菌丝、菌丝团及分生孢子; 内生真菌只在一定区域的皮层和次生韧皮部细胞中分布。     

远志根的发育解剖学研究

运用石蜡切片法对远志根的发育过程及1~3年生根的结构进行解剖学研究。结果显示:远志根的原分生组织由3群原始细胞组成,具有典型分生组织的细胞学特征。初生分生组织分化为根冠原、表皮原、皮层原和中柱原;初生结构由表皮、皮层和中柱组成,初生木质部为二原型。次生生长是依靠维管形成层和木栓形成层的活动完成,次生结构从外到内由周皮和次生维管组织组成;远志根次生结构特点为:次生韧皮部在次生维管组织中占主要部分,次生韧皮部中以韧皮薄壁细胞为主且其中储存有丰富的内含物,随着根龄的增加,韧皮薄壁细胞中的内含物也随之增加。3年生的主根中次生韧皮部薄壁细胞中的内含物最丰富;不同年份远志的主根随根龄的增加,周皮、次生韧皮部和次生木质部的面积都呈增加趋势,其中韧皮部和木质部的面积比值随根龄增长呈由小到大的变化,这是远志根的显著特点;根中的周皮发达,具有较厚的木栓层,次生木质部中导管和纤维发达,导管分布频率较高,并具有较大的口径。周皮和次生木质部的结构特征与远志的抗旱特性相适应。     

不同番茄种质叶表次生代谢物质

多毛番茄(Solanum habrochaites)为重要的番茄种质资源, 其叶表存在大量次生代谢物质, 对多种虫害具有趋避或/和毒害作用。利用气相色谱-质谱联用(GC-MS)技术分析栽培番茄(S. lycopersicum ) 9706与3份多毛番茄(LA2329、LA1777和PI134417)材料叶表次生代谢物质。结果表明, 3份多毛番茄叶表可检测到的次生代谢物质种类和总含量均高于普通番茄, 同时多毛番茄亚种间次生代谢物质的种类和含量也存在差异。普通番茄叶表次生代谢物质为3种单萜和3种倍半萜类物质, 其中单萜和倍半萜类物质分别占次生代谢物质总量的60.3%和39.7%。多毛番茄LA2329和LA1777叶表倍半萜类物质的种类和含量较高, 有些萜类物质具有物种特异性。如LA2329中含量最高的α-姜烯, 其含量为2 409.1 μg·g^–1; LA1777中含量较高的γ-榄香烯和E-β-法尼烯, 含量分别为573.3 μg·g^–1和289.9 μg·g^–1, 在其它番茄材料中未检测到这3种倍半萜类物质。PI134417中含量最高的是月桂酸乙酯, 其含量为5 312.8 μg·g^–1, 在普通番茄中这一物质未见报道。PI134417中甲基酮类物质含量也较高, 其中2-十一烷酮和2-十三烷酮的含量分别为689.8 μg·g^–1和1 459.7 μg·g^–1。研究结果可为番茄种质资源利用和次生代谢物质开发提供理论依据。     

濒危植物海南风吹楠营养器官解剖结构特征

该研究采用石蜡切片和光学显微技术,对海南风吹楠营养器官的解剖结构及其对环境的适应性进行了探讨。结果表明:海南风吹楠为典型异面叶,叶片中脉发达,中部分化出髓,上表皮外侧具角质层,内侧具1层内皮层,下表皮外侧无角质层,有气孔器分布,气孔器为双环型,略下陷;栅栏组织3~4层细胞,海绵组织4~6层细胞。茎的初生结构中表皮轻微角质化,维管束为外韧型,8~10个初生维管束围绕髓排列为1轮;茎的次生结构中,表皮外部角质层加厚,维管柱紧密排列连成环状,次生韧皮部和次生木质部发达,形成层细胞3~5层。根的初生结构中表皮细胞外壁加厚,外皮层细胞体积大,形状不规则,内侧具1层形成层,内皮层具凯氏带,初生木质部为多原型,呈辐射状排列。根的次生结构中木栓层细胞5~6层,木栓层内侧具1层木栓形成层,栓内层细胞3层。海南风吹楠营养器官具有一定耐阴和耐旱结构特征,同时与其生活的热带雨林沟谷中高温荫湿的环境相适应。     

美登木根的显微结构及其内生真菌的分布

本文采用石蜡永久制片和光学显微摄像的方法对美登木（Maytenus confertiflorus）根的显微结构及其内生真菌的分布进行了研究.结果表明,美登木根的次生结构由周皮和维管组织构成;周皮由木栓层、木栓形成层和栓内层组成,其中木栓层由5～6列长形细胞组成;维管组织中次生韧皮部所占根径的比例达46%,其薄壁细胞中内含物较丰富,次生木质部中分布有导管和木射线及少量木薄壁组织;在美登木木栓层和次生韧皮部中分布有菌丝片段、膨大的菌丝、菌丝团及分生孢子;内生真菌只在一定区域的皮层和次生韧皮部细胞中分布.     

闽楠营养器官的解剖结构及其生态适应性

采用石蜡切片和光学显微技术对闽楠(Phoebe bournei(Hemsl.)Yang)营养器官的解剖结构及其生态适应性进行了研究。结果显示,闽楠为典型异面叶,叶片中脉发达,维管束呈扇形,导管径向排列,韧皮部外侧有大量韧皮纤维分布。上表皮外侧具角质层,下表皮外侧无角质层,下表皮细胞呈犬牙状向外凸起,有表皮毛和气孔分布,气孔为双环型、外凸;栅栏组织由1层细胞组成,海绵组织由3~4层细胞组成。茎的初生结构中,表皮轻微角质化,厚角细胞5~6层,薄壁细胞5~7层,维管束为外韧型;茎的次生结构中,表皮外部角质层加厚,木栓层细胞3~4层,木栓形成层细胞1层,栓内层细胞2~3层,维管束紧密排列连成环状,次生韧皮部和次生木质部发达,形成层细胞2~3层。根的次生结构中木栓层细胞5~6层,木栓层内侧具1层木栓形成层,栓内层细胞2层。闽楠营养器官的解剖结构特征一方面呈现出阴生植物的特点,另一方面也对阳生和旱生环境具有一定的适应性。     

Plant in vitro culture for the production of antioxidants--a review

Plant in vitro cultures are able to produce and accumulate many medicinally valuable secondary metabolites. Antioxidants are an important group of medicinal preventive compounds as well as being food additives inhibiting detrimental changes of easily oxidizable nutrients. Many different in vitro approaches have been used for increased biosynthesis and the accumulation of antioxidant compounds in plant cells. In the present review some of the most active antioxidants derived from plant tissue cultures are described; they have been divided into the main chemical groups of polyphenols and isoprenoids, and several examples also from other chemical classes are presented. The strategies used for improving the antioxidants in vitro production efficiency are also highlighted, including media optimization, biotransformation, elicitation, Agrobacterium transformation and scale-up.     

The Effect of Cork Pieces on Pseudohypericin Production in Cells ofHypericum perforatumShoots

The stimulating effect of cork pieces on hypericin and pseudohypericin biosyntheses was studied in cells of shoots regenerated from the callus cultures of St. John's wort (Hypericum perforatumL.). The addition of the cork matrix slightly stimulated shoot growth and enhanced pseudohypericin biosynthesis about threefold (to 0.4 mg/g dry wt). Pseudohypericin production increased proportionally with the amount of cork material added (from 1 to 4 mg/ml of growth medium). Further increase in the amount of cork pieces inhibited both pseudohypericin production and shoot growth. Organic and aqueous extracts of cork pieces did not affect the production of these substances.     

Allometric analysis of the induced flavonols on the leaf surface of wild tobacco (Nicotiana attenuata)

Trichomes excrete secondary metabolites that may alter the chemical composition of the leaf surface, reducing damage caused by herbivores, pathogens and abiotic stresses. We examined the surface exudates produced by Nicotiana attenuata Torr. Ex Wats., a plant known to contain and secrete a number of secondary metabolites that are toxic or a deterrent to herbivorous insects. Extractions specific to the leaf surface, the trichomes, and the laminar components demonstrated the localization of particular compounds. Diterpene glycosides occurred exclusively in leaf mesophyll, whereas nicotine was found in both the trichomes and mesophyll. Neither rutin nor nicotine was found on the leaf surface. Quercetin and 7 methylated derivatives were found in the glandular trichomes and appeared to be excreted onto the leaf surface. We examined the elicitation of these flavonols on the leaf surface with a surface-area allometric analysis, which measures changes in metabolites independent of the effects of leaf expansion. The flavonols responded differently to wounding, methyl jasmonate (MeJA), herbivore attack and UV-C radiation, and the response patterns corresponded to their compound-specific allometries. Finding greater amounts of quercetin on younger leaves and reduced amounts after herbivore feeding and MeJA treatment, we hypothesized that quercetin may function as an attractant, helping the insects locate a preferred feeding site. Consistent with this hypothesis, mirids (Tupiocoris notatus) were found more often on mature leaves sprayed with quercetin at a concentration typical of young leaves than on unsupplemented mature leaves. The composition of metabolites on the leaf surface of N. attenuata changes throughout leaf development and in response to herbivore attack or environmental stress, and these changes are mediated in part by responses of the glandular trichomes.     

嫁接茄子根系分泌物变化及其对黄萎菌的影响

采用番茄为砧木嫁接茄子,经过GC-MS检测,研究了黄萎菌胁迫前后嫁接对茄子植株根系次生代谢的影响。通过比较黄萎菌胁迫前后茄子根系次生代谢物质的变化,探讨了嫁接在胁迫前后对植株根系次生代谢的调节作用,并对嫁接茄根系分泌物中丁二酸二甲酯对茄子黄萎菌及茄子种子萌发、幼苗生长的化感效应进行了研究。结果表明,黄萎菌胁迫前,嫁接影响了根系的次生代谢物质的分泌,表现为物质种类和数量的增加,各类物质相对含量改变。嫁接茄子根系分泌物中检测出9大类、66种物质,比自根茄处理多出4大类、33种物质。黄萎菌胁迫时,嫁接茄子田间表现出明显的抗病性;进一步对根系分泌物进行检测发现,嫁接茄根系分泌物中物质种类和相对含量与自根茄处理相比均有显著差别;嫁接茄根系分泌物中烃和酚醇类物质相对含量分别增加了3.25%和0.07%,苯类、茚类和脂肪酸酯类物质相对含量降低,降幅分别为2.62%、0.26%和0.07%。新出现了胺类物质,芴类物质。黄萎菌胁迫前后茄子根系次生代谢物质成分同样发生了变化。与接菌前嫁接茄子植株根系分泌物相比,接菌后嫁接处理的苯类、茚类、酚醇类和胺类物质相对含量增加,增幅分别为22.07%、1.72%、1.21%和0.34%;烃类和脂肪酸酯类物质相对含量降低了1.28%和21.75%;酮类、咔唑类和芴类物质消失。新增物质中以丁二酸二甲酯的相对含量最高,达14.38%。随后的生物检测结果显示,丁二酸二甲酯能够提高茄子田间抗病性,对黄萎菌菌丝生长起化感抑制作用,并促进了茄子种子的萌发和幼苗的生长,随着处理浓度升高作用效果增强,并在1 mmol/L处理时达到最佳作用效果。     

Overexpression of Shinorhizobium meliloti hemoprotein in Streptomyces lividans to enhance secondary metabolite production

It was found that Shinorhizobium meliloti hemoprotein (SM) was more effective than Vitreoscilla hemoglobin (Vhb) in promoting secondary metabolites production when overexpressed in Streptomyces lividans TK24. The transformant with sm (sm-transformant) produced 2.7-times and 3-times larger amounts of actinorhodin than the vhbtransformant in solid culture and flask culture, respectively. In both solid and flask cultures, a larger amount of undecylprodigiocin was produced by the sm-transformant. It is considered that the overexpression of SM especially has activated the pentose phosphate pathway through oxidative stress, as evidenced by an increased NADPH production observed, and that it has promoted secondary metabolites biosynthesis.     

Production of 8-epi-tomentosin by plant cell culture ofXanthium strumarium

This study was conducted to establish a plant cell culture system for the production of medically important secondary metabolites fromXanthium strumarium. The effects of plant growth regulators including NAA, 2,4-D, kinetin, and ABA were examined in terms of callus induction, maintenance of callus and suspension cultures. It was shown that callus was induced upon treatment with NAA while embryo was induced after treatment with 2,4-D. Callus formation was further improved by treatment with ABA and NAA. The level of callusing increased by 17–29% for the seed case, cotyledon, leaf, and hypocotyl and by 96% in the case of the root. Suspension cell lines were established using calli produced from cotyledon, hypocotyl and root and cultured at 25°C under light conditions. The cells grew up to 15 g/L with NAA 2 ppm, BA 2 ppm, and ABA 1 ppm treatment. Supernatants of suspension cultures of cell lines derived from coyledon and hypocotyl produced some distinctive secondary metabolites, one of which was identified as 8-epi-tomentosin, which belongs to the xanthanolides. The amounts of 8-epi-tomentosin produced by the cotyledon-and hypocotylderived cell lines were 13.4 mg/L and 11.0 mg/L, respectively.