植物乙烯生物合成过程中活性氧的作用

大量的研究结果表明,活性氧参与植物乙烯生物合成过程具有明显的普遍性,超氧阴离子自由基是参与乙烯生物合成过程的主要活性氧。近年来研究的焦点主要从乙烯生物合成的关键调控酶ACC合酶及ACC氧化酶的酶活性、酶动力学特性、酶蛋白空间结构、酶基因表达水平等方面来阐明活性氧调控植物乙烯生物合成的机制。最新的研究表明：植物在各种正常或应激的生长条件下首先诱导了活性氧产生水平的变化,活性氧在基因或蛋白质水平上影响ACC合酶和ACC氧化酶的活性水平,从而调节乙烯的生物合成。本文首次综述了活性氧影响植物乙烯生物合成过程的最新研究进展,并对活性氧在植物乙烯生物合成中具有诱导与抑制并存的“双重性”作用进行了探讨。     

甘蔗ACC氧化酶基因片段的克隆与序列分析

1- 氨基环丙烷-1-羧酸（ACC）氧化酶是植物乙烯合成的一个关键酶,乙烯作为一种内源激素,对植物生长、老熟过程有多方面的调节作用。根据报道的各种植物ACC氧化酶氨基酸序列上前后两个保守区设计两个简并引物,以甘蔗总DNA为模板,通过PCR扩增到一个940bp的基因片段。将片段序列在MCBI的BLAST软件上进行同源性搜寻,显示的63个序列全部是ACC氧化酶基因,因而认为克隆到的片段就是甘蔗ACC氧化酶基因的一个成员。经对不同植物来源的ACC氧化酶基因家族进行比较分析,去除一个103bp的“内含子“后,推导的氨基酸序列为279个残基,占推测全长氨基酸残基总数的86％左右。经同源性分析,序列与毛竹和水稻ACC氧化酶的同源率达到86％。系统进化分析表明,该序列最先与水稻、其次和香蕉的ACC氧化酶聚类,然后再与双子叶植物的ACC氧化酶聚类,符合按形态特征分类的血缘关系。基因的获得对下一步了解乙烯的合成表达与甘蔗生长、成熟过程之间的关系奠定了基础。     

植物体中的过氧化物酶体

过氧化物酶体参与了包括氧化氢反应、长链脂肪酸的β-氧化等几乎所有的必需代谢途径。植物过氧化物酶体在植物体抗病和抗衰老过程中发挥作用。介绍了植物过氧化物酶体与亚硫酸盐氧化酶以及植物过氧化物酶体抗衰老、生物发生和动力学等方面的研究进展。     

原花青素聚合作用机理研究进展

原花青素是一类通过植物类黄酮次生代谢途径合成的聚多酚类化合物,它具有抗紫外线、抗病、抗虫,清除自由基,调节种子休眠和萌发等生理功能。该文对近年来国内外有关原花青素的结构类型和生物合成机制、原花青素聚合作用机理(包括:原花青素的聚合作用发生在植物细胞中央大液泡、缩合过程中延伸单元前体可能为无色花青素、黄烷-3-醇和花青素等假说)、以及推测参与催化聚合反应的缩合酶(包括:植物多酚氧化酶、植物漆酶和植物过氧化酶)等方面的研究进展进行综述,为深入研究原花青素生物合成机制提供资料。     

铜在植物生长发育中的作用

铜是植物正常生命活动所必需的 7种微量元素之一 ,参与植物生长发育过程中的多种代谢反应。铜是多酚氧化酶、抗坏血酸氧化酶、细胞色素氧化酶等的组成成分 ,参与植物体内的氧化还原过程。它也存在于叶绿体的质体蓝素中 ,参与光合作用的电子传递。1　植物对铜的吸收及其代谢植物通过根部从土壤中以离子形式吸收铜 ,也可通过叶面吸收。根部除了吸收溶解在土壤溶液中的铜以外 ,还能通过分泌出柠檬酸、苹果酸等有机酸以及呼吸作用形成的碳酸溶解难溶性物质以获取铜。影响根部吸收铜的因素除温度、通气状况、溶液浓度和离子间相互作用外 ,很重要…     

植物营养贮存蛋白及其生物学功能研究进展

植物营养贮存蛋白（vegetative storage proteins）是广泛存在于植物营养组织且含量丰富的蛋白,最初是作为植物氮源的临时贮存形式而被人们认识。然而,不同植物中的营养贮存蛋白的生化来源和生物学特性并不相同,并且除了营养贮存功能外,更重要的是这类蛋白在植物防御中也承担着多种多样的重要角色,或具有抗虫活性,或能够抑制病原细菌和病原真菌的生长,或参与植物防御过程中的信号转导等。对植物营养贮存蛋白在植物防御中作用机制的深入研究将使这类蛋白在新型生物农药的开发和植物抗病基因工程中具有广阔的应用前景。     

植物营养贮存蛋白及其生物学功能研究进展

植物营养贮存蛋白(vegetative storage proteins )是广泛存在于植物营养组织且含量丰富的蛋白, 最初是作为植物氮源的临时贮存形式而被人们认识。然而, 不同植物中的营养贮存蛋白的生化来源和生物学特性并不相同, 并且除了营养贮存功能外, 更重要的是这类蛋白在植物防御中也承担着多种多样的重要角色, 或具有抗虫活性, 或能够抑制病原细菌和病原真菌的生长, 或参与植物防御过程中的信号转导等。对植物营养贮存蛋白在植物防御中作用机制的深入研究将使这类蛋白在新型生物农药的开发和植物抗病基因工程中具有广阔的应用前景。     

甘油-3-磷酸酰基转移酶在植物脂质代谢、生长及逆境反应中的作用

甘油脂质是高等植物中含量最丰富的脂质,其种类包括磷脂、糖脂、油脂及胞外脂质等,广泛参与不同的生物过程。甘油-3-磷酸酰基转移酶(GPAT)利用各种脂肪族酰基或其衍生物作为底物催化甘油-3-磷酸(G3P)脂酰基化反应形成溶血磷脂酸(LPA),是脂质合成代谢途径中的限速酶。植物GPAT家族含有多个成员,根据其亚细胞定位、酶活性及底物选择性,拟南芥GPAT家族10个成员可分为3类。不同的GPAT具有独特的分子结构、活性调控及时空分布,并参与膜磷脂、甘油三脂、角质及软木脂合成代谢过程。研究表明极具分子异质性的GPAT在植物生长、发育和逆境胁迫反应过程中发挥着重要作用。     

豌豆叶绿体脂氧合酶活性与叶片衰老及膜脂过氧化作用的关系

豌豆叶绿体脂氧合酶(LOX)活性在连体叶片衰老过程中变化不大。ABA处理离体叶片2d叶绿体LOX活性升高,处理时间延长活性下降。抗氧化剂α-生育酚、谷胱甘肽、没食子酸丙酯抑制豌豆叶绿体LOX活性。脂质过氧化产物丙二醛对豌豆叶绿体LOX和大豆纯LOX-1的活性均有抑制作用,大豆LOX-1能促进离体豌豆叶绿体膜脂过氧化作用。因此,豌豆叶绿体LOX可能参与叶片衰老过程中叶绿体膜结构和功能的改变,又受膜脂过氧化产物的制约。     

改进的大豆脂肪氧化酶电泳显色方法

大豆种子富含脂肪氧化酶(lipoxygenase,简称LOX),约占其蛋白含量的1％。大豆LOX至少以三种同工酶存在,定名为L1、L2、L3a和L3b,后两种性质类似统归一种。大豆LOX能催化破碎种子中的不饱和脂肪酸(如亚油酸)氧化生成具有豆腥味的醛、醇类物质,从而大大降低大豆产品的品质与风味,     

Analysis of the subcellular localisation of lipoxygenase in legume and actinorhizal nodules

Plant lipoxygenases (LOXs; EC 1.13.11.12) catalyse the oxygenation of polyunsaturated fatty acids, linoleic (18:2) and α-linolenic acid (18:3(n-3)) and are involved in processes such as stress responses and development. Depending on the regio-specificity of a LOX, the incorporation of molecular oxygen leads to formation of 9- or 13-fatty acid hydroperoxides, which are used by LOX itself as well as by members of at least six different enzyme families to form a series of biologically active molecules, collectively called oxylipins. The best characterised oxylipins are the jasmonates: jasmonic acid (JA) and its isoleucine conjugate that are signalling compounds in vegetative and propagative plant development. In several types of nitrogen-fixing root nodules, LOX expression and/or activity is induced during nodule development. Allene oxide cyclase (AOC), a committed enzyme of the JA biosynthetic pathway, has been shown to localise to plastids of nodules of one legume and two actinorhizal plants, Medicago truncatula, Datisca glomerata and Casuarina glauca, respectively. Using an antibody that recognises several types of LOX interspecifically, LOX protein levels were compared in roots and nodules of these plants, showing no significant differences and no obvious nodule-specific isoforms. A comparison of the cell-specific localisation of LOXs and AOC led to the conclusion that (i) only cytosolic LOXs were detected although it is generally assumed that the (13S)-hydroperoxy α-linolenic acid for JA biosynthesis is produced in the plastids, and (ii) in cells of the nodule vascular tissue that contain AOC, no LOX protein could be detected.     

Creating lipoxygenases with new positional specificities by site-directed mutagenesis

In order to analyse the amino acid determinants which alter the positional specificity of plant lipoxygenases (LOXs), multiple LOX sequence alignments and structural modelling of the enzyme-substrate interactions were carried out. These alignments suggested three amino acid residues as the primary determinants of positional specificity. Here we show the generation of two plant LOXs with new positional specificities, a gamma-linoleneate 6-LOX and an arachidonate 11-LOX, by altering only one of these determinants within the active site of two plant LOXs. In the past, site-directed-mutagenesis studies have mainly been carried out with mammalian lipoxygenases (LOXs) [1]. In these experiments two regions have been identified in the primary structure containing sequence determinants for positional specificity. Amino acids aligning with the Sloane determinants [2] are highly conserved among plant LOXs. In contrast, there is amino acid heterogeneity among plant LOXs at the position that aligns with P353 of the rabbit reticulocyte 15-LOX (Borngr?ber determinants) [3].     

A lipoxygenase with dual positional specificity is expressed in olives (Olea europaea L.) during ripening

Plant lipoxygenases (LOXs) are a class of widespread dioxygenases catalysing the hydroperoxidation of polyunsaturated fatty acids. Although multiple isoforms of LOX have been detected in a wide range of plants, their physiological roles remain to be clarified. With the aim to clarify the occurrence of LOXs in olives and their contribution to the elaboration of the olive oil aroma, we cloned and characterized the first cDNA of the LOX isoform which is expressed during olive development. The open reading frame encodes a polypeptide of 864 amino acids. This olive LOX is a type-1 LOX which shows a high degree of identity at the peptide level towards hazelnut (77.3%), tobacco (76.3%) and almond (75.5%) LOXs. The recombinant enzyme shows a dual positional specificity, as it forms both 9- and 13-hydroperoxide of linoleic acid in a 2:1 ratio, and would be defined as 9/13-LOX. Although a LOX activity was detected throughout the olive development, the 9/13-LOX is mainly expressed at late developmental stages. Our data suggest that there are at least two Lox genes expressed in black olives, and that the 9/13-LOX is associated with the ripening and senescence processes. However, due to its dual positional specificity and its expression pattern, its contribution to the elaboration of the olive oil aroma might be considered.     

Biochemical and molecular characterization of hazelnut (Corylus avellana) seed lipoxygenases.

Plant lipoxygenases (LOXs) are a class of dioxygenases which display diverse functions in several physiological processes such as growth, development and response to biotic and abiotic stresses. Even though LOXs have been characterized from several plant species, the physiological role of seed LOXs is still unclear. With the aim to better clarify the occurrence of LOXs and their influence on hazelnut seed quality, we carried out the biochemical and molecular characterization of the main LOX isoforms expressed during seed development. A genomic clone containing a complete LOX gene was isolated and fully characterized. The 9887 bp sequence reported contains an open reading frame of 5334 bp encoding a putative polypeptide of 99 kDa. Semiquantitative RT-PCR carried out from RNAs extracted from seeds at different maturation stages showed that LOXs are mainly expressed at early developmental stages. These results were confirmed by LOX activity assays. Biochemical characterization of the reaction products of the hazelnut LOX indicated that it is a 9-LOX. Two cDNAs were isolated by RT-PCR carried out on total RNA from immature hazelnut seeds. Sequence analysis indicated that the two cDNAs are highly homologous (91.9% degree of identity) and one of these corresponded exactly to the genomic clone. The deduced amino acid sequences of the hazelnut LOXs showed that they are closely related to a previously reported almond LOX (79.5% identity) and, to a lesser extent, to some LOXs involved in plant responses to pathogens (cotton and tobacco LOXs, 75.5 and 74.6% identity, respectively). The physiological role of hazelnut LOXs and their role in influencing seed quality are also discussed.     

Cunxi Wang · Kevan P.C. Croft · David F. Hildebrand

 Auxin [α-naphthaleneacetic acid (NAA) or indole-3-acetic acid] can induce the expression of lipoxygenases (LOXs) in cultured immature zygotic embryo cotyledons of soybean [Glycine max. (L.) Merr]. These auxin-induced LOXs are different from those normally expressed in seeds but have the same isoelectric points (pI) as those found in seedlings. The pIs of the two seedling LOXs were determined to be 5.09 and 5.23. One of the auxin-induced LOXs has the same pI (5.09) and molecular mass (94 kDa) as seedling LOX4. The partial amino acid sequences from the purified NAA-induced pI-5.09 LOX are identical to those of LOX4. RNA protection assays showed that NAA induces the expression of LOX4 and LOX5 mRNAs in cultured embryo cotyledons where they are not normally expressed. Soybean genotypes with a polymorphic variant of LOX4 in hypocotyls showed the same variation as NAA-induced LOXs in the embryo cotyledons. These results demonstrate that the NAA-induced pI-5.09 LOX is seedling LOX4 and also suggest that auxin might be directly or indirectly involved in seedling LOX expression during seed germination. Received: 10 January 2000 / Revision received: 16 June 2000 / Accepted: 29 June 2000     

Effects of exogenous auxins on expression of lipoxygenases in cultured soybean embryos

The expression of lipoxygenases (LOXs) is known to be developmentally regulated in soybeans (Glycine max. [L.] Merr.). Hormones have been firmly established as being involved in the growth and developmental processes of a number of plant species. Correlation between the expression of LOXs and the development and germination of soybean embryos suggests that plant hormones may affect the expression of LOXs. The present studies were conducted to investigate the effects of exogenous auxins on the expression of LOX isozymes and LOX activities in cultured cotyledon tissues of immature soybean seeds. The results revealed that at least one of the more acidic nonembryo LOX isozymes was induced by either α-naphthaleneacetic acid or indoleacetic acid but not by 2,4-dichlorophenoxyacetic acid after 4 days' exposure. Levels of LOX-1, -2, and -3 proteins and activities were significantly decreased by 2,4-dichlorophenoxyacetic acid 10 days after explanting. S1 analysis showed that embryo LOX messenger RNAs were detectable in the tissues treated with each of the auxins. The reduced levels of the embryo LOX proteins may, therefore, be regulated at the levels of translation, posttranslational modification, or degradation. The more acidic isozymes induced by α-naphthaleneacetic acid showed enzymatic activity and shared the same molecular mass and isoelectric point values as the germination-associated LOX isozymes found in hypocotyls and radicles, suggesting that those LOXs are involved in germination competency of soybean embryos.     

Cloning of Lipoxygenase Genes from a Cyanobacterium, Nostoc punctiforme, and Its Expression in Eschelichia coli

Oxylipin metabolism represents one of the important hormonal and defensive mechanisms employed by plants, algae, or animals. It begins mostly with the reaction of lipoxygenases (LOXs), which catalyze the oxygenation of polyunsaturated fatty acids to form the corresponding hydroperoxides. At present, little information about LOXs in cyanobacteria has been reported. Herein, we report the first isolation of two LOX genes (NpLOX1 and NpLOX2) from a cyanobacterium, Nostoc punctiforme ATCC29133. Incubations of recombinant NpLOX1 and NpLOX2 proteins expressed in Eschelichia coli with linoleic acid resulted in the predominant formation of linoleic acid 13-S-hydroperoxide. Other C18 and C20 fatty acids could also be substrates for NpLOX enzymes. Phylogenetic analysis of NpLOX sequences showed that the NpLOX enzymes shared a high homology with LOX sequence of a bacterial pathogen, Pseudomonas aeruginosa, and these bacterial LOXs formed a subfamily distinct from those of plants, algae, and mammals.     

Heterologous Expression and Biochemical Characterization of Two Lipoxygenases in Oriental Melon,Cucumis melo var. makuwa Makino

Lipoxygenases (LOXs) are a class of non-heme iron-containing dioxygenases that catalyse oxidation of polyunsaturated fatty acids to produce hydroperoxidation that are in turn converted to oxylipins. Although multiple isoforms of LOXs have been detected in several plants, LOXs in oriental melon have not attracted much attention. Two full-length LOX cDNA clones, CmLOX10 and CmLOX13 which have been isolated from oriental melon (Cucumis melo var. makuwa Makino) cultivar “Yumeiren”, encode 902 and 906 amino acids, respectively. Bioinformatics analysis showed that CmLOX10 and CmLOX13 included all of the typical LOX domains and shared 58.11% identity at the amino acid level with each other. The phylogenetic analysis revealed that CmLOX10 and CmLOX13 were members of the type 2 13-LOX subgroup which are known to be involved in biotic and abiotic stress. Heterologous expression of the full-length CmLOX10 and truncated CmLOX13 in Escherichia coli revealed that the encoded exogenous proteins were identical to the predicted molecular weights and possessed the lipoxygenase activities. The purified CmLOX10 and CmLOX13 recombinant enzymes exhibited maximum activity at different temperature and pH and both had higher affinity for linoleic acid than linolenic acid. Chromatogram analysis of reaction products from the CmLOX10 and CmLOX13 enzyme reaction revealed that both enzymes produced 13S-hydroperoxides when linoleic acid was used as substrate. Furthermore, the subcellular localization analysis by transient expression of the two LOX fusion proteins in tobacco leaves showed that CmLOX10 and CmLOX13 proteins were located in plasma membrane and chloroplasts respectively. We propose that the two lipoxygenases may play different functions in oriental melon during plant growth and development.     

Ligand‐induced formation of transient dimers of mammalian 12/15‐lipoxygenase: A key to allosteric behavior of this class of enzymes?

Mammalian lipoxygenases (LOXs) have been implicated in cellular defense response and are important for physiological homeostasis. Since their discovery, LOXs have been believed to function as monomeric enzymes that exhibit allosteric properties. In aqueous solutions, the rabbit 12/15-LOX is mainly present as hydrated monomer but changes in the local physiochemical environment suggested a monomer-dimer equilibrium. Because the allosteric character of the enzyme can hardly be explained using a single ligand binding-site model, we proposed that the binding of allosteric effectors may shift the monomer-dimer equilibrium toward dimer formation. To test this hypothesis, we explored the impact of an allosteric effector [13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid] on the structural properties of rabbit 12/15-LOX by small-angle X-ray scattering. Our data indicate that the enzyme undergoes ligand-induced dimerization in aqueous solution, and molecular dynamics simulations suggested that LOX dimers may be stable in the presence of substrate fatty acids. These data provide direct structural evidence for the existence of LOX dimers, where two noncovalently linked enzyme molecules might work in unison and, therefore, such mode of association might be related to the allosteric character of 12/15-LOX. Introduction of negatively charged residues (W181E + H585E and L183E + L192E) at the intermonomer interface disturbs the hydrophobic dimer interaction of the wild-type LOX, and this structural alteration may lead to functional distortion of mutant enzymes.