Genetic engineering of shikonin biosynthesis hairy root cultures of Lithospermum erythrorhizon transformed with the bacterial ubiC gene

The biosynthetic pathway to 4-hydroxybenzoate (4HB), a precursor of the naphthoquinone pigment shikonin, was modified in Lithospermum erythrorhizon hairy root cultures by introduction of the bacterial gene ubiC. This gene of Escherichia coli encodes chorismate pyruvate-lyase (CPL), an enzyme that converts chorismate into 4HB and is not normally present in plants. The ubiC gene was fused to the sequence for a chloroplast transit peptide and placed under control of a constitutive plant promoter. This construct was introduced into L. erythrorhizon by Agrobacterium rhizogenes-mediated transformation.The resulting hairy root cultures showed high CPL activity. 4HB produced by the CPL reaction was utilized for shikonin biosynthesis, as shown by in vivo inhibition of the native pathway to 4HB with 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase. A feeding experiment with [1,7-13C2]shikimate showed that in the absence of AIP the artificially introduced CPL reaction contributed ca. 20% of the overall 4HB biosynthesis in the transgenic cultures. ubiC transformation did not lead to a statistically significant increase of shikonin formation, but to a 5-fold increase of the accumulation of menisdaurin, a nitrile glucoside which is presumably related to aromatic amino acid metabolism.     

Stable transformation of Lithospermum erythrorhizon by Agrobacterium rhizogenes and shikonin production of the transformants

Seedling hypocotyls of Lithospermum erythrorhizon were infected with Agrobacterium rhizogenes (strain 15834) harboring a binary vector with an intron-bearing the β-glucuronidase (GUS) gene driven by cauliflower mosaic virus (CaMV) 35S promoter as well as the hygromycin phosphotransferase (HPT) gene as the selection marker. About 20% of the hairy roots isolated were hygromycin resistant and had co-integrated GUS and HPT genes in their Lithospermum genomic DNA. Because GUS activity was detected in almost all the hygromycin-resistant root tissues, the CaMV 35S promoter seems to be ubiquitously active in L. erythrorhizon hairy roots. In pigment production medium M9, the hairy root cultures had shikonin productivity similar to that of cell suspension cultures of Lithospermum. They also showed light-dependent inhibition of shikonin biosynthesis similar to that of Lithospermum cell cultures. These findings suggest that this hairy root system transformable with A. rhizogenes is a suitable model system for molecular characterization of shikonin biosynthesis via reverse genetics. Received: 2 March 1998 / Revision received: 25 May 1998 / Accepted: 8 July 1998     

Regulation of shikonin production by glutamine in Lithospermum erythrorhizon cell cultures

Amino acid analysis has shown that Lithosperum erythrorhizon cell suspension cultures which are unable to produce shikonin derivatives in LS medium containing ammonium accumulate a large quantity of glutamine, as compared with shikonin-producing cells cultured in the production medium M9 containing nitrate as the sole nitrogen source. The addition of glutamine to M9 medium proved to be strongly inhibitory to shikonin production. Furthermore, culture experiments using an inhibitor of glutaminase suggested that shikonin synthesis is not inhibited by ammonium released from glutamine but by glutamine itself. These findings indicate that the repression of shikonin synthesis occurs in close association with an accumulation of glutamine in cultured cells grown in a medium containing ammonium.     

根癌农杆菌转化紫草的研究

紫草 (ＬｉｔｈｏｓｐｅｒｍｕｍｅｒｙｔｈｒｏｒｈｉｚｏｎＳｉｅｂ .ｅｔＺｕｃｃ)是传统中药。其根部含有萘醌类化合物—紫草素及其衍生物 ,具有显著的抗菌、抗炎、抗癌以及促进伤口愈合等生理活性。紫草素同时也是一种名贵化妆品染料。科学家对紫草的研究兴趣是基于其资源的缺乏及紫草植物本身所具有的一些特点 ;如 :紫草素及其衍生物的颜色特性可凭借肉眼观察 ,紫草素及其衍生物只在紫草的根部积累 ,紫草素合成的次生代谢途径受多种酶和外界条件 (光照 ,营养等 )的调节等。紫草细胞培养 (Ｆｕｊｉｔａ等 ,1983;叶和春等 ,1991)可以产…     

藻类活性物质对紫草细胞生长和色素形成的影响

本文研究了螺旋藻、栅列藻和织线藻的水提物对新疆紫草和硬紫草细胞生长和色素形成的作用。结果表明不同种类的藻的提取物对不同紫草细胞作用呈现差异。在生长阶段,对新疆紫草,上述3种藻的低浓度提取物促进生长但作用不大,高浓度的螺旋藻和栅列藻提取物强烈抑制生长;对硬紫草,各种藻提取液的所有浓度处理均有促生长作用。在色素形成阶段,连续用高浓度织线藻提取物处理可以加速新疆紫草色素形成,同时提高色素含量。低浓度的织线藻提取液处理能提高硬紫草色素含量。栅列藻的水提物对两种紫草的色素形成均起抑制作用。螺旋藻水提物的适当浓度可加速两种紫草的色素形成。B_5培养基中加高浓度织线藻水提物,可抑制新疆紫草生长阶段的色素形成。     

碳氮源对转基因鱼腥藻Anabaena sp.PCC7120培养的影响

对碳源、氮源种类和用量对转rhTNF-α基因鱼腥藻7120（Anabaena sp.PCC7120）培养的影响进行了研究,发现最适碳源为蔗糖,最适氮源为NaNO3,最佳用量分别为9g/L和2．25g/L,此时生物量远高于自养方式,达2．52g/L,比相同条件下在BG－11培养基培养高71．66％,ATNF－α表达量为16％－22％,生物活性为10^5U/mg。     

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     

Altered ecosystem carbon and nitrogen cycles by plant invasion: a meta-analysis

Plant invasion potentially alters ecosystem carbon (C) and nitrogen (N) cycles. However, the overall direction and magnitude of such alterations are poorly quantified. Here, 94 experimental studies were synthesized, using a meta-analysis approach, to quantify the changes of 20 variables associated with C and N cycles, including their pools, fluxes, and other related parameters in response to plant invasion. Pool variables showed significant changes in invaded ecosystems relative to native ecosystems, ranging from a 5% increase in root carbon stock to a 133% increase in shoot C stock. Flux variables, such as above-ground net primary production and litter decomposition, increased by 50-120% in invaded ecosystems, compared with native ones. Plant N concentration, soil NH+4 and NO-3 concentrations were 40, 30 and 17% higher in invaded than in native ecosystems, respectively. Increases in plant production and soil N availability indicate that there was positive feedback between plant invasion and C and N cycles in invaded ecosystems. Invasions by woody and N-fixing plants tended to have greater impacts on C and N cycles than those by herbaceous and nonN-fixing plants, respectively. The responses to plant invasion are not different among forests, grasslands, and wetlands. All of these changes suggest that plant invasion profoundly influences ecosystem processes.     

柞树林下菌根真菌对碳、氮营养的利用

研究了柞树林下菌根真菌对Ｃ、Ｎ营养的利用状况．结果表明,供试菌种对Ｃ源的利用较为广泛,葡萄糖、果糖为其最适碳源,平均生长量比对照高出４．４倍;供试菌种对有机氮的利用优于无机氮,平均生长量比无机氮源高出１．６倍,硝态无机氮源中平均生长量为对照的２．５倍,对铵态氮的利用较差,平均生长量仅为对照的２．２倍     

植物的光合作用与光合氮、碳代谢的耦联及调节

概述了光合作用反应与CO2同化和NO^-3/NO^-2还原的耦联关系,提出了应该从氮,碳代谢整合角度讨论作动和光合作用,以便根据生产目的,调节作物的氮,碳代谢,实现农业生产的高产,优质。     

Utilization of carbon and nitrogen reserves of alfalfa roots in supporting N2-fixation and shoot regrowth

Perennial legume such as alfalfa have the capacity to sustain shoot regrowth and some nodule N₂-fixation after removal (cutting) of shoots which contain practically all of the plant's photosynthetic capacity. The role of the roots in supporting these processes has not been fully described. Measurements were made of the nodules' responses to removal of shoots from 8-week-old seedlings in terms of N₂-fixation, as nitrogenase activity (NA) measured as acetylene reduction, dark CO₂ fixation, measured as in vitro phosphoenolpyruvate carboxylase (PEPC) activity, and total non-structural carbohydrate (NSC) content. These properties decreased and recovered in that sequence, which suggests that nodule NSC supported the substrate requirements of NA and PEPC immediately after cutting. The utilization and redistribution or root carbon and nitrogen, prelabeled with ¹⁴C and ¹⁵N, were also followed after cutting 8-week-old alfalfa seedlings. In the first 2 weeks of regrowth 12% of root C and 25% of root N were transferred for incorporation into new shoots. Up to 40% of the root C was used for plant respiration to support 28 days of shoot regrowth and N₂-fixation. The decline of N₂-fixation was slower after cutting and its minimum activity rose up 45% of pre-cut activity as root reserves were built up with plant age. Therefore, the stored reserves of nodulated roots play an important role in support of N₂-fixation after cutting.Contribution No. 1265 from Plant Research Center.Contribution No. 1265 from Plant Research Center.     

Feedback of carbon and nitrogen cycles enhances carbon sequestration in the terrestrial biosphere

The efforts to explain the ‘missing sink’ for anthropogenic carbon dioxide (CO₂) have included in recent years the role of nitrogen as an important constraint for biospheric carbon fluxes. We used the Nitrogen Carbon Interaction Model (NCIM) to investigate patterns of carbon and nitrogen storage in different compartments of the terrestrial biosphere as a consequence of a rising atmospheric CO₂ concentration, in combination with varying levels of nitrogen availability. This model has separate but closely coupled carbon and nitrogen cycles with a focus on soil processes and soil–plant interactions, including an active compartment of soil microorganisms decomposing litter residues and competing with plants for available nitrogen. Biological nitrogen fixation is represented as a function of vegetation nitrogen demand. The model was validated against several global datasets of soil and vegetation carbon and nitrogen pools. Five model experiments were carried out for the modeling periods 1860–2002 and 2002–2100. In these experiments we varied the nitrogen availability using different combinations of biological nitrogen fixation, denitrification, leaching of soluble nitrogen compounds with constant or rising atmospheric CO₂ concentrations. Oversupply with nitrogen, in an experiment with nitrogen fixation, but no nitrogen losses, together with constant atmospheric CO₂, led to some carbon sequestration in organismic pools, which was nearly compensated by losses of C from soil organic carbon pools. Rising atmospheric CO₂ always led to carbon sequestration in the biosphere. Considering an open nitrogen cycle including dynamic nitrogen fixation, and nitrogen losses from denitrification and leaching, the carbon sequestration in the biosphere is of a magnitude comparable to current observation based estimates of the ‘missing sink.’ A fertilization feedback between the carbon and nitrogen cycles occurred in this experiment, which was much stronger than the sum of separate influences of high nitrogen supply and rising atmospheric CO₂. The demand‐driven biological nitrogen fixation was mainly responsible for this result. For the modeling period 2002–2100, NCIM predicts continued carbon sequestration in the low range of previously published estimates, combined with a plausible rate of CO₂‐driven biological nitrogen fixation and substantial redistribution of nitrogen from soil to plant pools.     

Induction of benzoquinone formation by activated carbon in Lithospermum erythrorhizon cell suspension cultures

Cultured cells of Lithospermum erythrorhizon which were capable of producing red naphthoquinone (shikonin) derivatives on Linsmaier-Skoog's agar medium stopped synthesizing these compounds when grown in liquid medium without agar. However, when the liquid medium was supplemented with a small amount of activated carbon, the cells produced a new orange benzoquinone derivative, echinofuran B, which may be considered an abnormal metabolite of geranylquinol, the key intermediate in the biosynthesis of shikonin. A similar effect of activated carbon was also observed with a variant cell line incapable of producing shikonin derivatives even on the agar medium. By contrast, the callus cultures grown on the agar medium as well as the dried roots of the intact plant were found to contain a small amount of echinofuran C, another new benzoquinone related to echinofuran B, in addition to shikonin derivatives.     

Nitrogen fixation and the mass balances of carbon and nitrogen in ecosystems

Ecosystems with high rates of nitrogen fixation often have high loss rates through leaching or possibly denitrification. However, there is no formal theoretical context to examine why this should be the case nor of how nitrogen accumulates in such open systems. Here, we propose a simple model coupling nitrogen inputs and losses to carbon inputs and losses. The nitrogen balance of this model system depends on plant (nitrogen fixer) growth rate, its carrying capacity, N fixed/C fixed, residence time of nitrogen and carbon in biomass, litter decay rate, litter N/C, and fractional loss rate of mineralized nitrogen. The model predicts the requirements for equilibrium in a nitrogen-fixing system, and the conditions on nitrogen fixation and losses in order for the system to accumulate nitrogen and carbon. In particular, the accumulation of nitrogen and carbon in a nitrogen-fixing system depend on an interaction between residence time in vegetation and litter decay rate in soil. To reflect a possible increased uptake of soil nitrogen and decreased respiratory cost of symbiotic nitrogen fixers, the model was then modified so that fixation rate decreased and growth rate increased as nitrogen capital accumulated. These modifications had only small effects on carbon and nitrogen accumulation. This suggests that switching from uptake of atmospheric nitrogen to mineral soil nitrogen as nitrogen capital accumulates simply results in a trade-off between energetic limitations and soil nitrogen limitations to carbon and nitrogen accumulation. Experimental tests of the model are suggested.     

Rhizodeposition and C-partitioning of Lolium perenne in axenic culture affected by nitrogen supply and defoliation

This study investigated the effects of N-supply and partial defoliation on C-partitioning, root morphology and soluble rhizodeposition, for Lolium perenne grown in axenic sand culture systems percolated with nutrient solution. Plants were grown for 36 d in nutrient solutions with differing N concentrations (4 mM or 0.02 mM NH₄ ⁺NO₃ ^-), and effects of repeated defoliation to 4 cm were determined. The ‘low N’ supply reduced (P < 0.05) dry matter accumulation, with proportionately increased partitioning to the root systems. Root morphology was also altered at ‘low N’, with development of a finer root system, manifest as increased (P < 0.05) specific root length. Concurrent with these effects on growth of L. perenne, ‘low N’ increased (P < 0.05) exudation of C-compounds from roots on a per g root basis. Defoliation was found to increase exudation (P < 0.05) of soluble compounds for periods of 3-5 d following each cut, at both N-supply rates. The effects of N-supply and defoliation are of importance in understanding the coupling of plant productivity to nutrient cycling in soils with differing N availabilities and for grassland systems which are subject to grazing. This revised version was published online in June 2006 with corrections to the Cover Date.     

A model of the long-term response of carbon allocation and productivity of forests to increased CO2concentration and nitrogen deposition

We present a simple theoretical analysis of the long term response of forest growth and carbon allocation to increased atmospheric [CO₂] and N deposition. Our analysis is based on a recent model which predicts that plant light-use efficiency increases with [CO₂] but is independent of plant N supply. We combine that model with simple assumptions for nitrogen fluxes in the soil. A quasi-equilibrium analysis of the short term tree and soil pools is then used to develop a simple graphical depiction of the long term carbon and nitrogen supply constraints on total growth, stem growth and foliar allocation. Our results suggest that long-term growth responses to [CO₂] and N deposition depend strongly on the extent to which stem allocation and foliage allocation are coupled. At one extreme (‘no coupling’), when stem allocation is fixed and independent of foliage allocation, there is no response of total growth or stem growth to increased [CO₂] unless N deposition increases. At the other extreme (‘linear coupling’), when stem allocation is proportional to foliage allocation, there is a significant long-term increase in total growth following a doubling of [CO₂], even when N deposition is unchanged, but stem growth decreases because of a long-term decrease in foliage allocation. For both types of coupling, total growth and stem growth increase with increasing N deposition. In the case of linear coupling, however, the N deposition response of stem growth is significantly larger than that of total growth, because of a long-term increase in foliage allocation. We compare our results with those obtained previously from an alternative model of canopy light-use efficiency involving a dependence on the foliar N:C ratio in addition to [CO₂]. Our results highlight the need for more experimental information on (i) the extent to which canopy light-use efficiency is independent of N supply, and (ii) the relationship between foliage allocation and stem allocation.     

刺参对筏式贝藻养殖系统不同碳、氮负荷自污染物的生物清除

针对我国北方浅海筏式贝藻养殖系统自身污染输出导致的环境问题,以滤食性贝类生物沉积物和海藻粉的不同配比来模拟贝藻筏式养殖系统不同碳、氮负荷的颗粒自污染物,研究了刺参摄取这些颗粒物后的碳、氮收支,评估了其对碳和氮生源要素的生物清除潜力。结果表明,刺参对筏式贝藻养殖系统不同配比颗粒自污染物中的碳和氮具有较强的摄食能力,摄食率分别为35.77～181.18mgC·g-1·d-1和6.08～14.28mgN·g-1·d-1;颗粒自污染物中碳和氮的含量越高,其摄食碳、氮的能力越强。刺参摄取的碳以粪便碳形式排出居多(59.3%～97.1%),其次是呼吸消耗的碳(9.9%～37.3%),而用于生长的碳最少(-7.0%～6.1%);刺参摄取的氮主要用于排泄消耗为主(53.1%～63.1%),粪便氮次之(27.7%～39.2%),用于生长的氮最少(-2.3%～16.7%)。通过建立的碳、氮收支方程,估算出刺参对筏式贝藻养殖系统不同碳、氮负荷自污染物的生物清除效率(SE)分别为0.83～4.57mgC·g-1·d-1和0.28～0.76mgN·g-1·d-1;而且其清除效率随着颗粒自污染物中碳、氮含量的升高而增大,呈明显的正相关关系;清除效率和碳(C)、氮(N)含量之间回归关系可用SEC=0.7368+14.9488C和SEN=0.2281+0.2202N来描述。     

城市土壤活性碳、氮分布特征及影响因素

为揭示城市绿地土壤活性碳氮分布特征及影响因素,选取合肥市不同类型绿地(蜀山森林公园、公园绿地、道路绿地、学校绿地、居住区绿地、工厂绿地)土壤为研究对象,对其0 ～ 30 cm土壤微生物量碳(MBC)、微生物量氮(MBN)、溶解性有机碳(DOC)、溶解性有机氮(DON)等活性组分进行研究.结果表明,绿地类型对土壤活性碳氮含量影响显著(P＜0.05),各活性碳氮含量随土层深度的增加而降低.城区内各人工绿地土壤活性碳氮含量均低于郊区蜀山森林公园绿地:MBC下降了46.81％ ～ 64.39％,MBN下降了49.90％ ～80.13％,DOC下降了28.95％ ～45.52％,DON下降了5.67％ ～48.90％,表明土地利用变化是导致绿地土壤活性碳氮变化的主要因素.相关分析表明,研究区域内MBC与MBN、DON正相关(P＜0.01),MBN与DOC正相关(P＜0.01),DOC与DON正相关(P＜0.01).研究还发现,土壤pH与活性碳、氮间均呈负相关关系(P＜0.01),表明适当降低城市土壤碱性污染物的侵入有利于土壤活性碳氮的积累.     

氯嘧磺隆与尿素对土壤微生物生物量碳、氮及无机氮的影响

通过室内培养试验,研究了不同浓度氯嘧磺隆(20、200、2000 μg·kg-1土)单一施用及与尿素(120 mg· kg-1土)配合施用情况下,土壤微生物生物量碳、氮和土壤铵态氮、硝态氮随时间的动态变化规律.结果表明:各浓度氯嘧磺隆单独处理在整个培养期(60 d)中对微生物生物量碳、氮均有抑制作用,且浓度越高,后期抑制作用越强;各浓度氯嘧磺隆处理在培养前期对硝态氮、铵态氮没有明显影响,中期(15 d)能显著提高土壤中铵态氮的含量,后期(30 d后)显著提高了土壤中硝态氮的含量.尿素单独施用及与氯嘧磺隆配施均能在短时间内增加微生物生物量碳、氮,但随后配施处理的促进作用减弱;尿素单独和配施均能持久增加土壤中铵态氮、硝态氮含量.