The physiological response of Artemisia annua L. to salt stress and salicylic acid treatment

Salinity has a great influence on plant growth and distribution. A few existing reports on Artemisia annua L. response to salinity are concentrated on plant growth and artemisinin content; the physiological response and salt damage mitigation are yet to be understood. In this study, the physiological response of varying salt stresses (50, 100, 200, 300, or 400 mM NaCl) on A. annua L. and the effect of exogenous salicylic acid (0.05 or 0.1 mM) at 300-mM salt stress were investigated. Plant growth, antioxidant enzyme activity, proline, and mineral element level were determined. In general, increasing salt concentration significantly reduced plant growth. Superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were stimulated by salt treatment to a higher enzyme activity in treated plants than those in untreated plants. Content of proline had a visible range of increment in the salt-treated plants. Distribution of mineral elements was in inconformity: Na⁺ and Ca²⁺ were mainly accumulated in the roots; K⁺ and Mg²⁺ were concentrated in leaves and stems, respectively. Alleviation of growth arrest was observed with exogenous applications of salicylic acid (SA) under salt stress conditions. The activity of SOD and POD was notably enhanced by SA, but the CAT action was suppressed. While exogenous SA had no discernible effect on proline content, it effectively inhibited excessive Na⁺ absorption and promoted Mg²⁺ absorption. Ca²⁺ and K⁺ contents showed a slight reduction when supplemented with SA. Overall, the positive effect of SA towards resistance to the salinity of A. annua will provide some practical basis for A. annua cultivation.     

Protective Effect of Artemisia annua L. Extract against Galactose-Induced Oxidative Stress in Mice

Artemisia annua L. (also called qinghao) has been well known as a source of antimalarial drug artemisinins. In addition, the herb was reported to have in vitro antioxidative activity. The present study investigated the protective effect of aqueous ethanol extract of Qinghao (AA extract) against D-galactose-induced oxidative stress in C57BL/6J mice. Feeding AA extract-containing diet lowered serum levels of malondialdehyde and 8-OH-dG that are biomarkers for lipid peroxidation and DNA damage, respectively. Furthermore, AA extract feeding enhanced the activity of NQO1, a typical antioxidant marker enzyme, in tissues such as kidney, stomach, small intestine, and large intestine. In conclusion, AA extract was found to have antioxidative activity in mouse model.     

Metabolic engineering of artemisinin biosynthesis in Artemisia annua L.

Artemisinin, a sesquiterpene lactone isolated from the Chinese medicinal plant Artemisia annua L., is an effective antimalarial agent, especially for multi-drug resistant and cerebral malaria. To date, A. annua is still the only commercial source of artemisinin. The low concentration of artemisinin in A. annua, ranging from 0.01 to 0.8% of the plant dry weight, makes artemisinin relatively expensive and difficult to meet the demand of over 100 million courses of artemisinin-based combinational therapies per year. Since the chemical synthesis of artemisinin is not commercially feasible at present, another promising approach to reduce the price of artemisinin-based antimalarial drugs is metabolic engineering of the plant to obtain a higher content of artemisinin in transgenic plants. In the past decade, we have established an Agrobacterium-mediated transformation system of A. annua, and have successfully transferred a number of genes related to artemisinin biosynthesis into the plant. The various aspects of these efforts are discussed in this review.     

青蒿组织培养及其快速繁殖研究

以青蒿幼叶、叶柄为外植体,研究其离体培养和试管苗再生途径。结果表明,以青蒿嫩叶为外植体,在MS+6-BA0.5mg/L+IBA0.5mg/L的培养基中可诱导出愈伤组织,诱导率达87%,并在此培养基中可以分化出芽,分化率为85%,将分化苗转移到MS+IBA0.5mg/L的培养基上,生根率高达93%。     

黄花蒿组培快繁与种质离体保存的研究

以带侧芽的黄花蒿(Artemisia annua L.)茎段为外植体,以MS为基本培养基,进行组织培养和种质保存研究.结果表明,培养基MS 6-BA 1.0 mg L-1 IBA 0.1 mg L-1、MS 6-BA 0.5 mg L-1 IBA 0.1 mg L-1和MS NAA0.1 nag L-1 IBA 0.5 rng L-1可分别用于黄花蒿的芽诱导、增殖和生根培养,培养20 d的增殖倍数为5.5倍,生根率98.3%.培养基MS CCC 1.0 mg L-1、MS CCC 2.0 nag L-1、MS PP3334.0 mg L-1可用作离体保存,连续保存200 d的存活率分别达72.3%、77.0%、69.2%.活力检测表明,黄花蒿种质经保存后的增殖、生根能力没有下降.因此,可通过诱导腋芽增殖建立黄花蒿快繁体系,及在培养基中添加CCC或PP333拼能使材料长期保存.     

反相高效液相色谱法测定青蒿中青蒿酸的含量

本文建立了反相高效液相色谱快速测定青蒿中青蒿酸含量的方法。色谱条件为:Diamonsil C18色谱柱(250 mm×4.6 mm,5μm),柱温为(30±1)℃,流动相采用乙腈与0.2%磷酸水溶液混合液(体积比65:35),流速1 mL/min,检测波长220 nm。标准曲线回归方程:Y=8.784573×10-8X-6.443559×10-5,r=0.9997,青蒿酸回收率为102.4%。实验证明该方法稳定可靠、精密度高、重现性好、简单可行,适用于青蒿酸的分析检测。     

Agrobacterium-mediated transformation of Artemisia absinthium L. (wormwood) and production of secondary metabolites

Hairy roots were obtained after infection of Artemisia absinthium shoots with Agrobacterium rhizogenes strains 1855 and LBA 9402. The susceptibility to hairy root transformation varied between plant genotypes and bacterial strains. Hairy roots showed macroscopic differences from control root cultures. Southern blot hybridization confirmed the integration of T-DNA from both p1855 and pBin19, while polymerase chain reaction analysis indicated the presence of the neomycin phosphotransferase gene in the hairy root genome. Subcultured transformed root lines grew well in selective B5 agar-solidified medium containing kanamycin or rifampicin and without hormones. Shake-flask experiments with fast-growing root lines showed that 40 g l^–1 was the best sucrose concentration for biomass production, yielding a 463-fold increase in dry weight after 28 days of culture. Great differences were found in the profiles of the essential oils isolated from normal and hairy roots. Gas chromatography/mass spectrometry analysis showed the oil produced by transformed cultures to be a mixture of 50 compounds with only one major component representing 37% of the oil content. Received: 19 March 1996 / Revision received: 15 July 1996 / Accepted: 13 December 1996     

不同小麦品种对UV-B辐射增强响应的生理特性差异

研究了大田条件下模拟增强UV-B辐射（500 KJ·m^-2,相当于昆明地区臭氧层减少20%）对10个小麦品种生理指标的影响以及小麦对UV B辐射响应的种内差异.结果表明,10个供试小麦品种中有6个品种的叶绿素含量显著下降,叶绿素a降低的程度大于叶绿素b,从而导致叶绿素a/b的比率下降.UV-B对小麦叶片内MDA和类黄酮的影响也具有种内差异,有5个品种的MDA含量显著上升, 2个品种的MDA含量显著下降;4个品种的类黄酮含量显著增加,2个品种的类黄酮含量显著减少.叶绿素和类黄酮含量变化与MDA含量均呈显著负相关关系,类黄酮与小麦UV-B抗性之间存在密切联系.     

Responses of Artemisia annua L. to lead and salt-induced oxidative stress

The Artemisia annua L. plants were subjected separately to NaCl (0–160 mM) and lead acetate (0–500 μM) at the age of 90 days (S1 treatment) and 120 days (S2 treatment). The treated plants were studied on 100, 130 and 160 days after sowing (DAS) in S1, and on 130 and 160 DAS in S2 treatments for lipid peroxidation rate, photosynthetic rate (Pn), chlorophyll content, artemisinin concentration and artemisinin yield in leaf samples and for total biomass accumulation. The treatments enhanced lipid peroxidation at all stages of plant growth and increased the concentration and yield of artemisinin at 100 and 130 DAS in S1 and S2, respectively, while other parameters declined at all growth stages. The magnitude of changes was greater in lead-treated than in salt-treated plants. Both treatments induced oxidative stress which might have damaged the photosynthetic apparatus resulting in a loss of chlorophyll content and a decline in photosynthetic rate, biomass accumulation and artemisinin production. The increase in artemisinin content, observed during the early phase of plant growth, might be due to a sudden conversion of its precursors (e.g. artemisinic acid/dihydroartemisinic acid) to artemisinin by activated oxygen species under oxidative stress.     

Agrobacterium tumefaciens-mediated transformation of Artemisia annua L. and regeneration of transgenic plants

Summary A transformation system was developed for Artemisia annua L. plants. Leaf explants from in vitro grown plants developed callus and shoots on medium with 0.05 mg/L naphthaleneacetic acid and 0.5 mg/L N⁶-benzyladenine after transformation with the C58C1 Rif^R (pGV2260) (pTJK136) Agrobacterium tumefaciens strain. A concentration of 20 mg/L kanamycin was added in order to select transformed tissue. Kanamycin resistant shoots were rooted on naphthaleneacetic acid 0.1 mg/L. Polymerase chain reactions and DNA sequencing of the amplification products revealed that 75% of the regenerants contained the foreign genes. 94% of the transgenic plants showed a -glucuronidase-positive response.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyladenine - GM germination medium - GMVIT germination medium with vitamins - GUS -glucuronidase - Kin kinetin (N⁶-furfurylaminopurine) - NAA -naphthaleneacetic acid - NPT II neomycin phosphotransferase II - PCR Polymerase Chain Reaction - T-DNA transfer-DNA - X-glucuronide 5-bromo-4-chloro-3-indolyl -D-glucuronide     

Metabolomic and physiological responses reveal multi-phasic acclimation of Arabidopsis thaliana to chronic UV radiation

Biochemical changes in vivo and pathway interactions were investigated using integrated physiological and metabolic responses of Arabidopsis thaliana L. to ultraviolet (UV) radiation (280–400 nm) at 9.96 kJ m⁻² d⁻¹ over the entire life cycle from seed to seed (8 weeks). Columbia-0 (Col-0) and a UV-B sensitive accession ( fah-1 ) showed significant ( P  < 0.001) reductions in leaf growth after 6 weeks. Col-0 recovered growth after 8 weeks, with recovery corresponding to a switch from production of phenylpropanoids to flavonoids. fah-1 failed to recover, indicating that sinapate production is an essential component of recovery. Epidermal features show that UV radiation caused significant ( P  < 0.001) increases in trichome density, which may act as a structural defence response. Stomatal indices showed a significant ( P  < 0.0001) reduction in Col-0 and a significant ( P  < 0.001) increase in fah-1 . Epidermal cell density was significantly increased under UV radiation on the abaxial leaf surface, suggesting that that a fully functioning phenylpropanoid pathway is a requirement for cell expansion and leaf development. Despite wild-type acclimation, the costs of adaptation lead to reduced plant fitness by decreasing flower numbers and total seed biomass. A multi-phasic acclimation to UV radiation and the induction of specific metabolites link stress-induced biochemical responses to enhanced acclimation.     

Isolation and characterization of culturable endophytic actinobacteria associated with Artemisia annua L.

Endophytic actinobacteria isolated from Artemisia annua were characterized and evaluated for their bioactivities. A total of 228 isolates representing at least 19 different genera of actinobacteria were obtained and several of them seemed to be novel taxa. An evaluation of antimicrobial activity showed that more isolates possessed activity towards plant pathogens than activity against other pathogenic bacteria or yeasts. High frequencies of PCR amplification were obtained for type I polyketide synthases (PKS-I, 21.1%), type II polyketide synthases (PKS-II, 45.2%) and nonribosomal peptide synthetases (NRPS, 32.5%). The results of herbicidal activity screening indicated that 19 out of 117 samples of fermentation broths completely inhibited the germination of Echinochloa crusgalli. This study indicated that endophytic actinobacteria associated with A. annua are abundant and have potentially beneficial and diverse bioactivities which should be pursued for their biotechnical promise.     

Growth, structure, stomatal responses and secondary metabolites of birch seedlings (Betula pendula) under elevated UV-B radiation in the field

Three-year-old birch (Betula pendula Roth.) seedlings were exposed, in the field, to supplemental levels of UV-B radiation. Control seedlings were exposed to ambient levels of UV radiation, using arrays of unenergized lamps. A control for UV-A radiation was also included in the experiment. Enhanced UV-B radiation had no significant effects on height growth, and shoot and root biomass of birch seedlings. Leaf expansion rate increased transiently in the middle of the growing period in enhanced UV-B- and UV-A-exposed plants; however, final leaf size and relative growth rate remained unaffected. Leaf thickness and spongy intercellular spaces were increased in UV-B-exposed seedlings along with increased density of glandular trichomes. At the ultrastructural level, enhanced UV-B increased the number of cytoplasmic lipid bodies, and abnormal membrane whorls were found. Both enhanced UV-B and UV-A radiation induced swelling of chloroplast thylakoids. Stomatal density and conductance were significantly increased by elevated UV-B radiation. UV-A radiation increased the length and width of stomata, whereas UV-B radiation had only a marginal effect on stomatal size. UV-A and enhanced UV-B radiation attenuated the appearance of necrotic spots in autumn, probably caused by the fungus Pyrenopeziza betulicola, suggesting a direct harmful effect of UV on pathogens or reduced susceptibility to pathogens in UV-exposed seedlings. Secondary metabolite analysis showed increases in (+)-catechin, quercetin, cinnamic acid derivative, apigenin and pentagalloylglucose in birch leaves under enhanced UV-B radiation. Negative correlations between apigenin, and particularly quercetin concentrations and lipid peroxidation levels indicated an antioxidant role of secondary metabolites in birch leaves exposed to UV-B radiation.     

黄花蒿培养细胞中青蒿素合成代谢的体外调节

黄花蒿培养细胞通过两步培养积累青蒿素．第１步在含有０．２～０．４ｍｇ／Ｌ６－苄基氨基嘌呤（６－ＢＡ）和３～４ｍｇ／Ｌ吲哚乙酸（ＩＡＡ）的Ｎ６培养基中进行细胞的增殖培养,第２步将培养好的细胞转入含０．２～０．４ｍｇ／Ｌ６－ＢＡ和０．２～０．４ｍｇ／ＬＩＡＡ的改良Ｎ６培养基中进行青蒿素的合成．青蒿素的合成量为１９０μｇ／ｇ干细胞左右．当在第２步培养中加入青蒿素合成前体青蒿酸,青蒿素合成量比仅靠激素诱导提高了３倍多．青蒿素的合成途径是植物固醇合成途径的分支途径,当在青蒿素合成过程即第２步培养中加入固醇生物合成抑制剂双氯苯咪唑和氯化氯胆碱处理,可使代谢向合成青蒿素的方向移动,青蒿素合成量明显提高．经２００ｍｇ／Ｌ氯化氯胆碱处理２ｄ,黄花蒿细胞合成青蒿素量为３７２μｇ／ｇ干细胞;经２０ｍｇ／Ｌ双氯苯咪唑处理４ｄ,黄花蒿细胞合成青蒿素量为１５４０μｇ／ｇ干细胞,比靠激素诱导提高了８倍多,与诱导脱分化细胞的黄花蒿叶中所含的青蒿素（３０００μｇ／ｇ干细胞）处于同一个数量级．以上结果表明：在通过植物激素调节可以合成青蒿素的黄花蒿培养细胞中,缺乏青蒿素合成前体是青蒿素合成量低的重要原因．因此,在青蒿素合成的过程中通过体外调节,     

Short-term UV-B radiation effects on morphology,physiological traits and accumulation of bioactive compounds in Prunella vulgaris L.

Prunella vulgaris plants (full-bloom stage) were subjected to short-term (15 days) UV-B radiation in a growth chamber. UV-B radiation was effective at enhancing morphological and biomass characteristics and decreasing chlorophyll contents of P. vulgaris. Treatment of P. vulgaris with artificial UV-B radiation significantly increased peroxidase (POD), superoxide dismutase (SOD) and glutathione (GSH) activities compared to the control treatment. UV-B radiation significantly increased the levels of hydrogen peroxide (H₂O₂), malondialdehyde (MDA) and proline in leaves of P. vulgaris compared to those of control plants. In addition, the contents of total flavonoids, rosmarinic acid, caffeic acid and hyperoside significantly increased under UV-B radiation. The total phenolic levels also increased under UV-B treatment. These results demonstrated that short-term UV-B radiation can enhance production of secondary metabolites in P. vulgaris, resulting in increased spica yield compared to that of control plants.     

Responses to UV-B radiation in Trifolium repens L. – physiological links to plant productivity and water availability

This study used comparisons across nine populations of Trifolium repens (white clover) in conjunction with drought to examine physiological responses to ultraviolet‐B radiation (UV‐B). Plants were exposed for 12 weeks to supplementation with 13.3 kJ m^?2 d^?1 UV‐B, accompanied by 4 weeks of drought under controlled environmental conditions. UV‐B increased the levels of UV‐B‐absorbing compounds and of flavonol glycosides and this effect was synergistically enhanced by water stress. These changes were more pronounced for the ortho‐dihydroxylated quercetin, rather than the monohydroxylated kaempferol glycosides. UV‐B increased leaf water potential (ψ_L) by 16% under drought and proline levels by 23% under well‐watered conditions. The intraspecific comparisons showed that higher UV‐B‐induced levels of UV‐B‐absorbing compounds, of quercetin glycosides and of ψ_L were linked to lower plant productivity and to higher UV‐B tolerance under well‐watered conditions. These findings suggest that: (1) slow‐growing T. repens ecotypes adapted to other stresses have higher capacity for physiological acclimation to UV‐B; and (2) that these attributes also contribute to decreased UV‐B sensitivity under drought.