植物类胡萝卜素生物合成及功能

详述了植物类胡萝卜素生物合成途径,并从突破类胡萝卜素合成途径中上游瓶颈限制、类胡萝卜素代谢各分支途径的改造、提高植物细胞对类胡萝卜素物质积累能力三个方面探讨了类胡萝卜素生物合成酶基因在植物基因工程中的研究现状,最后对植物类胡萝卜素代谢的研究前景进行了展望。     

Photosynthetic light-harvesting function of carotenoids in higher-plant leaves exposed to high light irradiances

Exposure of barley (Hordeum vulgare L.) leaves to strong white light (1500 μmol photons · m⁻² · s⁻¹) decreased the quantum yield of photosynthetic oxygen evolution in green light preferentially absorbed by carotenoids (Φ-510) but not in red light exclusively absorbed by chlorophylls (Φ-650). This phenomenon was observed to be (i) rapidly induced (within a few minutes), (ii) slowly reversible in darkness (within about 1 h), (iii) insensitive to dithiothreitol and (iv) maximally induced by photon flux densities higher than about 1000 μmol · m⁻² · s⁻¹. Determination of the carotenoid composition of the major light-harvesting complex of PSII (LHCII) and analysis of the thylakoid membrane lipid fluidity before and after strong illumination of barley leaves in the presence or the absence of dithiothreitol showed that the light-induced decrease in the Φ-510/Φ-650 ratio did not require the physical detachment of carotenoids from the pigment antennae. Compared to barley plants grown under moderate light and temperature conditions, plants grown in sustained high irradiance at elevated temperature exhibited (i) a lower Φ-510/Φ-650 ratio, (ii) a reduced size of the functional PSII pigment antenna in green light (but not in red light) and (iii) a marked increase in the amount of free carotenoids found in non-denaturing Deriphat-containing electrophoretic gels of thylakoid membranes. Similarly, the Φ-510/Φ-650 ratio of the LHCII-deficient chlorina-f2 barley mutant was very low compared to the wild type. Separation and quantification of the cis/trans carotenoid isomers of barley leaves revealed that strong illumination did not induce pronounced cis-trans isomerization of xanthophylls. Taken together, the data suggest that the efficiency of energy transfer from carotenoids to chlorophylls varies with the light environment both in the short term and in the long term, with excess light energy noticeably inhibiting the photosynthetic light-harvesting function of carotenoids. The photoprotective significance of this carotenoid decoupling from the chlorophyll antennae is discussed. Received: 28 July 1997 / Accepted: 25 October 1997     

KEGG数据库在生物合成研究中的应用

KEGG(Kyoto Encyclopedia of Genes and Genomes)提供了一个操作平台,即以基因组信息(GENES)和化学物质信息(LIGAND)为构建模块,通过代谢网络(PATHWAY)将基因组和生物系统联系起来,然后根据功能等级进行归纳分类(BRITE)。KEGG还为各种组学研究提供相关软件,用于代谢途径重建、遗传分析和化合物比对。作为一个综合数据库,KEGG不仅指导生物燃料、药物和新材料等生物基化学品的合成,而且致力于研究日趋严重的环境问题。系统介绍了KEGG数据库的结构、功能及其相关工具的最新进展,并展望在生物合成中的应用前景。     

链球菌合成透明质酸的研究进展

透明质酸是一种具有极大商业价值的线性、高分子粘多糖,微生物的生物合成是透明质酸来源的首选。主要综述链球菌合成透明质酸的研究概况,分析了目前存在的主要问题及解决途径,并对其前景进行了展望。     

Evidence for simply inherited dominant resistance to Meloidogyne javanica in carrot

Root-knot nematodes (Meloidogyne spp.) are serious pests of carrot (Daucus carota L.) worldwide. While soil treatment with nematicides is the primary means for managing nematodes in carrot, there is a need to identify and introduce host plant resistance for crop improvement. This study was conducted to determine the inheritance of resistance to root-galling and reproduction by M. javanica (Treub) Chitwood in a selection (BR-1252) of carrot variety Brasilia. F₂, F₃, F₄, and BC₁ progenies from the cross BR-1252×B6274 (a susceptible inbred line) were screened in pot tests for reaction to M. javanica. The observed reactions based on galling and egg production on fibrous roots gave segregation patterns in all tests that were consistent with relatively simply inherited dominant resistance. Field testing in progress indicates that this resistance is very effective against both M. javanica and M. incognita. A single gene model fits the observed data acceptably well in F₃ generations. However, the range of 3% to 51% susceptible plants in segregating F₃ families and 1% to 47% in segregating F₄ families is much wider than the 25% expected with a single-gene model, and linked duplicate factors in the coupling phase could also explain the observed segregation patterns. The variation in percentage susceptibility among these families did not clearly cluster into three expected categories (25% S, 20.25% S, and 0.25% S for a 10-cM linkage distance, or 25% S, 16% S and 1% S for 20 cM), but it did tend to occur over the same range. Thus a 10-cM to 20-cM-linked duplicate factor model cannot be dismissed at this time. Egg production data in the F₂, F₃, and F₄ families provided evidence for slightly lower resistance expression in the heterozygous condition. Thus, while overall expressed in a dominant fashion, the resistance does exhibit some allelic dosage response. Received: 14 June 1999 / Accepted: 7 July 1999     

Action of light on accumulation of carotenoids and chlorophylls in the milo shoot (Sorghum vulgare Pers.)

We have performed a comprehensive study on the mechanism of regulation of carotenogenesis by light in the shoot of Sorghum vulgare. Our work shows that carotenoid accumulation is simultaneously controlled by phytochrome (P_fr) and by the availability of chlorophyll. Throughout plastidogenesis light dependent chlorophyll and carotenoid accumulation are interdependent processes: Accumulation of chlorophyll in natural light requires the presence of carotenoids; likewise, accumulation of considerable amount of carotenoids depends on the availability of chlorophyll. However, in both cases the efficiency of the biosynthetic pathway, the potential biosynthetic rates (capacities) are determined by phytochrome. A push and pull model of carotenogenesis advanced previously (Frosch and Mohr 1980, Planta 148, 279) to explain carotenogenesis in the mustard (Sinapis alba) seedling also applies to the monocotyledonous milo (Sorghum vulgare) seedling. Therefore, we suggest that the model applies to carotenogenesis in higher plants in general.Abbreviations Chl chlorophyll(s) - PChl protochlorophyll(ide) - HIR High irradiance response (of phytochrome) - P_fr far-red absorbing, physiologically active form of phytochrome - P red absorbing physiologically inactive form of phytochrome - P_tot total phytochrome - i.e. [P_r]+[P_fr] =[P_fr]+[P_tot], wavelength dependent photoequilibrium of the phytochrome system - RL red light - FR far-red light     

Metabolic engineering of novel ketocarotenoid production in carrot plants

Carotenoids constitute a vast group of pigments that are ubiquitous throughout nature. Carrot (Daucus carota L.) roots provide an important source of dietary beta-carotene (provitamin A), alpha-carotene and lutein. Ketocarotenoids, such as canthaxanthin and astaxanthin, are produced by some algae and cyanobacteria but are rare in plants. Ketocarotenoids are strong antioxidants that are chemically synthesized and used as dietary supplements and pigments in the aquaculture and neutraceutical industries. We engineered the ketocarotenoid biosynthetic pathway in carrot tissues by introducing a beta-carotene ketolase gene isolated from the alga Haematococcus pluvialis. Gene constructs were made with three promoters (double CaMV 35S, Arabidopsis-ubiquitin, and RolD from Agrobacterium rhizogenes). The pea Rubisco small sub-unit transit peptide was used to target the enzyme to plastids in leaf and root tissues. The phosphinothricin acetyl transferase (bar) gene was used as a selectable marker. Following Agrobacterium-mediated transformation, 150 plants were regenerated and grown in a glasshouse. All three promoters provided strong root expression, while the double CaMV 35S and Ubiquitin promoters also had strong leaf expression. The recombinant ketolase protein was successfully targeted to the chloroplasts and chromoplasts. Endogenous expression of carrot beta-carotene hydroxylases was up-regulated in transgenic leaves and roots, and up to 70% of total carotenoids was converted to novel ketocarotenoids, with accumulation up to 2,400 microg/g root dry weight. Astaxanthin, adonirubin, and canthaxanthin were most prevalent, followed by echinenone, adonixanthin and beta-cryptoxanthin. Our results show that carrots are suitable for biopharming ketocarotenoid production for applications to the functional food, neutraceutical and aquaculture industries.     

On photoprotective mechanisms of carotenoids in light harvesting complex

Carotenoids in light harvesting complex (LHC) play an important role in preventing plants photodamage caused by excess light. Non-photochemical quenching (NPQ) is an important mechanism adopted by plants to deal with high light intensity and the major component is referred to as energy dependent quenching (qE). Despite numerous studies have been devoted to investigating the site and mechanism of qE, there are still much debate on these topics. In this article, we discussed the possible site and underlying mechanism of qE based on the structural similarity of carotenoids. Moreover, being as good antioxidants, carotenoids’ potential protective effects against LHC photo-oxidation by quenching active oxygen species or triplet excited state chlorophyll are also discussed.     

Exploitation of the resistance to carrot fly in the wild carrot species Daucus capillifolius

Under field conditions a wild Daucus species from Libya, D. capillifolius, supported less than one tenth as many carrot flies (Psila rosae) as the susceptible carrot cultivar Danvers Half Long 126. Breeding lines developed from crosses between D. capillifolius and three different carrot types were grown in a series of field experiments at Wellesbourne between 1980 and 1989. Each year selections were made for agronomic quality and/or for increased resistance to carrot fly. The programme produced lines which for size, shape and colour represented most of the commercially-important carrot types. Some of these lines were also significantly more resistant to carrot fly than selections from the partially-resistant cv. Sytan. However, the best lines were not as resistant as the wild parent. The highest quality resistant lines were sold to seed companies for variety production.     

Evidence that the plastid signal and light operate via the samecis-acting elements in the promoters of nuclear genes for plastid proteins

Nuclear-encoded genes for proteins of the photosynthetic maschinery represent a particular subset of genes. Their expression is cooperatively stimulated by discrete factors including the developmental stage of plastids and light. We have analyzed in transgenic tobacco the plastid- and light-dependent expression of a series of 5 promoter deletions of various nuclear genes from spinach, of fusions of defined promoter segments with the 90-bp 35S RNA CaMV minimal promoter, as well as with mutations in sequences with homologies to characterizedcis-elements, to address the question of whether the plastid signal and light operate via the same or differentcis-acting elements. In none of the 160 different transgenic lines (representing 32 promoter constructs from seven genes) analyzed, could significant differences be identified in the responses to the two regulatory pathways. The data are compatible with the idea that both signals control the expression of nuclear genes for plastid proteins via the samecis-acting elements.     

Influence of light on ascorbate formation and metabolism in apple fruits

To further understand the regulatory mechanism of light on the formation of ascorbic acid (AsA) in the sink organs of plants, a systematical investigation on AsA levels, activities of two key biosynthsis enzymes and their mRNA expression as well as the recycling was performed in the fruits of apple (Malus domestica Borkh), under different levels of shade. After the whole trees were shaded with the sun-light about 50–55% for 20 days, AsA levels were significantly decreased in fruit peel, flesh and leaves, while mRNA expression levels and activities of l-galactose dehydrogenase (l-GalDH, EC 1.1.1.117) and l-galactono-1,4-lactone dehydrogenase (l-GalLDH, EC 1.3.2.3) as well as activities of recycling enzymes was clearly declined in the leaf and peel but not in the flesh. By shading fruits only for 20 days, AsA levels, relative mRNA levels and activities of l-GalDH and l-GalLDH as well as activities of recycling enzymes all showed obvious decrease in the peel, but not in the flesh. However, their levels in the peel were markedly increased after the full shade was removed and re-exposed these fruits on natural light for 5 days. It is concluded that light affects AsA biosynthesis and recycling in the peel and leaf, but did not in the fresh. Results also suggest that apple fruit is potential to biosynthesize AsA via the l-galactose pathway, and AsA content in the fruits may depend partly on levels of AsA or other photochemistry controlled by light in the leaves.     

Biosynthesis of phenolic compounds and its regulation in apple

This paper summarises the information on the occurrence of phenolic compounds in apple Malus domestica leaves and fruits with special reference to their developmental changes and regulation of biosynthesis. Besides the ontogenetic variation in biosynthesis and accumulation, the stress-induced and pathogenesis-related changes are emphasised. Aspects of commercial importance are addressed, ranging from fruit colouration, through disease resistance, to the direct use of apple tissues, as raw material for the extraction of bioactive phenolic compounds.     

Role of nitrogen in the photoinduction of protoperithecia and carotenoids in Neurospora crassa

Nitrogen, as KNO₃ or NH₄NO₃, can inhibit the photoinduction of protoperithecia in Neurospora crassa when present in the medium at a high concentration but does not inhibit the photoinduction of carotenoids. The point at which the presence of high nitrogen levels is no longer inhibitory is 5 h after illumination.Abbreviations al albino mutant - wc white-collar mutant - WM Westergaard and Mitchell (1947) medium Dedicated to Professor Wilhelm Nultsch in honour of his 60th birthday     

An Introduction to the Biosynthesis of Chemicals Used in Plant-Microbe Communication

Abstract Plants accumulate a diverse array of natural products, which can serve either to defend the plant against various microbes in its environment or to attract various microbes, both beneficial and pathogenic. Plants must also attract pollinators, repel or poison herbivores, compete with other plant species, and protect themselves from environmental dangers such as high light intensities. Some compounds have been implicated in playing a role in multiple interactions. Although the structures vary immensely in size and complexity, most are derived from a limited number of core biosynthetic pathways. This review briefly summarizes the biosynthetic origins of phenylpropanoid (including simple phenolics, flavonoids, anthocyanins and isoflavonoids), polyacetate, terpenoid, and alkaloid classes of metabolites. Compounds reported to be important in plant-microbe, plant-animal, and plant-plant interactions will be given as examples of each of these classes. Other aspects of biosynthesis also will be discussed, including the timing or location of biosynthesis, the potential for genetic manipulation of these pathways, and various questions regarding the biosynthesis of these compounds.     

Foraging for nutrients, responses to changes in light, and competition in tropical deciduous tree seedlings

We evaluated (1) the responses of two co-occurring tropical tree species, Heliocarpuspallidus and Caesalpiniaeriostachys, to changes in light, (2) the ability of these species to search for and exploit a fertilized soil patch, (3) the relationship between the capacity to forage for a fertilized patch and the capacity to respond to changes in light availability and (4) how the relationship between light and nutrient acquisition influenced the competitive interactions between these species. Plants of the two species were exposed to a factorial combination of high (H) and low (L) light intensity and fertilized (+Fp) and unfertilized (−Fp) nutrient patches for 50 days. Half of the plants from H were then transferred to L (HL treatment), and half of the plants from L were transferred to H (LH). The remaining plants were kept in their original light condition and grown for another 50 days. Plants were grown in these light and patch treatments alone (one plant per pot) and in interspecific competition (one plant per species resulting in two plants per pot). Both species exploited fertilized patches by increasing root biomass and length in the patch. This enhanced plant productivity and growth rate mainly under LH and HH conditions for Heliocarpus and the HH condition for Caesalpinia). When plants in the HH light environment were grown with an unfertilized patch, plant biomass and relative growth rates (RGRs) were even lower than␣under the LL light environment [(HH–Fp)<LL]. However, the combined activity of shoot and roots when above- and below-ground resources were temporally and spatially heterogeneous influenced plant productivity and growth rate. The benefit from light increase (LH) was reduced when grown with an unfertilized patch. Larger reductions in root biomass, length and density in the patch, and in plant biomass and RGR, were exhibited by Heliocarpus than by Caesalpinia. These results suggest a close relationship between root foraging and light capture, where the benefit of the exploitation of the patch will be reflected in whole-plant benefit, if enough light is captured above-ground. In addition, the results suggest a change in the expected plant responses to light due to heterogeneity in soil nutrients, even though the fertilized patch was only a small proportion of the total soil volume. Leaf characteristics such as specific leaf area responded only to light conditions and not to patchily distributed nutrients. Root characteristics responded more strongly to nutrient heterogeneity. Competition modified the pattern of foraging under both high- and low-light conditions in Heliocarpus by 50 days, and the ability to forage for a fertilized patch under LL after 100 days of growth for Caesalpinia. Even though plant growth and productivity are greatly reduced under low-light conditions (HL and LL), competition modifies the ability of species to forage for a rich patch (especially for the fast-growing species Heliocarpus). Received: 24 November 1997 / Accepted: 15 June 1998     

Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells

Summary CAR and C1, two carrot (Daucus carota L.) suspension cultures of different genotypes, were subjected to stepwise selection for tolerance to the herbicide glyphosate [(N-phosphonomethyl)glycine]. The specific activity of the target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), as well as the mRNA level and copy number of the structural gene increased with each glyphosate selection step. Therefore, the tolerance to glyphosate is due to stepwise amplification of the EPSPS genes. During the amplification process, DNA rearrangement did not occur within the EPSPS gene of the CAR cell line but did occur during the selection step from 28 to 35 mM glyphosate for the C1 cell line, as determined by Southern hybridization of selected cell DNA following EcoRI restriction endonuclease digestion. Two cell lines derived from a previously selected glyphosate-tolerant cell line (PR), which also had undergone EPSPS gene amplification but have been maintained in glyphosate-free medium for 2 and 5 years, have lost 36 and 100% of the increased EPSPS activity, respectively. Southern blot analysis of these lines confirms that the amplified DNA is relatively stable in the absence of selection. These studies demonstrate that stepwise selection for glyphosate resistance reproducibly produces stepwise amplification of the EPSPS genes. The relative stability of this amplification indicates that the amplified genes are not extrachromosomal.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiothreitol - EPSPS 5-enolpyruvylshikimate-3-phosphate synthase - I₅₀ 50% inhibitory concentration - Kb Kilobase (pairs) - PEP phosphoenolpyruvate - PMSF phenylmethylsulfonyl fluoride - PVPP polyvinylpolypyrrolidone - S-3-P shikimate-3-phosphate     

An evaluation of gene expression during somatic embryogenesis of two temperature-sensitive carrot variants unable to complete embryo development

The synthesis of putative stage-specific polypeptides during somatic embryogenesis of the carrot ( Daucus carota L. cv. Danvers) was investigated in the temperature-sensitive variants OB-2 and OB-3. These variants undergo normal embryo development to produce mature plantlets at the permissive temperature (24°C), but are arrested at the oblong stage to form elongated embryos without cotyledons at the restrictive temperature (33°C). Using two-dimensional polyacrylamide gel electrophoresis of in vivo labelled polypeptides, the patterns of stage-specific polypeptides in both lines were compared in: (1) oblong embryos grown at continuous 24°C vs oblong embryos exposed to 33°C during their temperature-sensitive period (i.e. embryos of identical morphology but different developmental fates); and (2) heartshaped embryos grown at constant 24°C vs enlarged oblong embryos exposed to 33°C during their temperature-sensitive period (i.e. embryos of the same age but different morphologies). The 22 putative stage-specific, polypeptides observed in this study fall into four classes: (1) line-specific, (2) age-specific, (3) unsynchronized, and (4) synchronized polypeptides. Only the last class, which consists of 4 polypeptides, exhibits synthesis patterns which are consistent with the polypeptides being causally involved in somatic embryo development. It is concluded that stage-specific behavior as assayed by PAGE analyses of simple 'present or absent' comparisons is insufficient to identify most of the polypeptides that may be relevant for somatic embryogenesis.     

体外组装类胡萝卜素对外周捕光复合体(LH2)能量传递活性的影响

【目的】探求不产氧光合细菌(APB)外周捕光复合体(LH2)中类胡萝卜素(Car)结构和能量传递效率的关系和规律。【方法】通过二苯胺(DPA)抑制Car合成的方法从固氮红细菌134K20中获得部分缺失Car的LH2 (LC-LH2);采用TLC和HPLC法从3种APB中制备球形烯(SE)、玫红品(RP)和奥氏酮(OK) 3种Car;在含0.1%十二烷基二甲基胺氧化物(LDAO)的10 mmol/L Tris-HCl (pH 8.0)缓冲液中采用超声孵育法分别将这3种Car与LC-LH2体外组装,采用吸收光谱法、拉曼光谱法和荧光光谱法对组装LH2进行结构与功能分析。【结果】制备的部分缺失Car的LH2中,SE缺失率约为64.7%。这3种共轭长度、取代基的极性不同的Car均能与这种部分缺失SE的LH2自组装,Car组装率约在24.0%?29.4%之间,其中SE和OK的组装率高于RP。与部分缺失Car LH2中原有SE构象一致,重组的Car在LH2中也呈现较为伸展的平面构象。LH2中重组Car到细菌叶绿素(BChl)的能量传递效率由高到低的顺序依次为SE-LH2>RP-LH2>OK-LH2,与Car共轭体系大小的关系一致,而与Car极性大小没有明显的关系。【结论】在组装的LH2中Car采用平面构象与脱辅基蛋白结合,Car共轭长度仍是决定和影响LH2中Car-BChl能量传递效率的主要因素,而Car的取代基和极性影响较小。