一氧化氮对豆科植物结瘤及固氮的影响机制

豆科植物-根瘤菌共生过程受双方基因复杂且精细的调控, 能够产生特异的根瘤结构并可将大气中的惰性氮气(N₂)转化为可被植物直接利用的氨态氮。结瘤与固氮受多种因素影响, 其中, 一氧化氮(NO)作为一种自由基反应性气体信号分子, 可参与调节植物的许多生长发育过程, 如植物的呼吸、光形态建成、种子萌发、组织和器官发育、衰老以及响应各种生物及非生物胁迫。在豆科植物中, NO不仅影响寄主与菌共生关系的建立, 还参与调控根瘤菌对氮气的固定并提高植株氮素营养利用效率。该文主要从豆科植物及共生菌内NO的产生、降解及其对结瘤、共生固氮的影响和对环境胁迫的响应, 阐述了NO调控豆科植物共生体系中根瘤形成和共生固氮过程的作用机制, 展望了NO信号分子在豆科植物共生固氮体系中的研究前景。     

豆科植物SHR-SCR模块——根瘤“奠基细胞”的命运推手

豆科植物-根瘤菌共生固氮是可持续性农业氮肥的最重要来源。根瘤作为豆科植物共生固氮的一种特化植物侧生器官, 提供了根瘤菌生物固氮必需的微环境, 是根瘤菌的安身之本, 因此, 根瘤的正常发育是实现豆科植物-根瘤菌共生固氮的结构基础。根瘤器官的从头发生主要起始于根瘤菌诱导的根皮层细胞分裂。通常认为豆科植物的根皮层具备有别于非豆科植物根皮层的某种特异属性, 从而响应根瘤菌并与之建立固氮共生, 但长期以来该属性决定的分子机制一直不明确。近日, 中国科学院分子植物科学卓越创新中心王二涛团队以蒺藜苜蓿(Medicago truncatula)等豆科植物和拟南芥(Arabidopsis thaliana)等非豆科植物为研究对象, 发现豆科植物中保守的SHR-SCR干细胞模块决定了其皮层细胞分裂潜能从而赋予根瘤器官发生的命运。该研究揭示了豆科植物根瘤发育的全新机制, 提供了研究和理解植物-根瘤菌固氮共生进化的重要线索, 对提高豆科作物固氮效率和非豆科作物固氮工程具有重要意义。     

共生固氮体系中的豆科植物基因

豆科根瘤菌发现的近百年历史以来,共生固氮作用一直受到人们的瞩目。近廿几年来对根痛瘤—豆科植物共生体的研究进展迅速,对共生体中根瘤菌本身的固氮基因(nif)和结瘤基因的编码、定位等有了较深入的了解。然而,共生体系中基因的调控是比较复杂的,环境因素和寄生植物基因对共生固氮的调控也起着重要作用。人们对豆科寄主结瘤和固氮遗传进行了一系列研究,并力图选育高固氮的豆科品种资源。本文仅就豆科植物—根瘤菌共生固氮体系中寄主植物基因及它在共生固氮体系研究中的作用和意义作简要的概述。     

豆科植物早期共生信号转导的研究进展北大核心CSCD

豆科植物与根瘤菌建立特异的共生关系,在寄主根部产生固氮根瘤。此过程包含了共生信号识别与传递、根瘤菌侵染、根瘤形成以及固氮功能实现等生物学事件。研究人员已经从2种豆科模式植物蒺藜苜蓿(Medicago truncatula)和百脉根(Lotus japonicus)的共生固氮体系中,筛选到许多与根瘤菌共生相关的突变体及其相对应的功能基因,建立起包含结瘤因子识别、共生信号传递和转录响应在内的早期共生信号途径。该文对豆科植物早期共生信号途径的新进展进行了综述。     

豆科植物早期共生信号转导的研究进展

豆科植物与根瘤菌建立特异的共生关系,在寄主根部产生固氮根瘤。此过程包含了共生信号识别与传递、根瘤菌侵染、根瘤形成以及固氮功能实现等生物学事件。研究人员已经从2种豆科模式植物蒺藜苜蓿（Medicago truncatula）和百脉根（Lotus japonicus）的共生固氮体系中,筛选到许多与根瘤菌共生相关的突变体及其相对应的功能基因,建立起包含结瘤因子识别、共生信号传递和转录响应在内的早期共生信号途径。该文对豆科植物早期共生信号途径的新进展进行了综述。     

植物非共生血红蛋白的研究进展

植物非共生血红蛋白（nsHb）基因在植物界广泛存在。许多生物和非生物胁迫可以诱导nsHb的表达。nsHb在植物的生长发育和逆境胁迫中具有重要功能,其作用机制与NO的代谢密切相关。文章综述了非共生血红蛋白的表达特性、生物学功能及其作用机制等方面的研究进展。     

南山麻黄属的血红蛋白:有关植物血红蛋白的起源

在非豆科植物南山麻黄属(Parasponia)根瘤菌形成的固氮根瘤中提纯血红蛋白(Appleby等,1983),使人们怀疑这样一个假说,即,可能通过同一级别基因传递的单一作用所产生的根瘤豆血红蛋白和豆血红蛋白是一种渗入的杂物而不是植物共生固氮作用的必需物。南山麻黄属血红蛋白和氧可逆结合(Appleby等,1983)意味着:在体内,它象豆科植物共生现象中的豆血红蛋白一样,起着携带氧气的作用。因此,在共生固氮遗传工程方面,存在着血红蛋白的可能必要条件而引出几个问题: (1)南山麻黄属血红蛋白是植物或细菌的产物?什么因子控制它的表达? (2)它和豆科植物根瘤豆血红蛋白的遗传起源是否相同?     

The Legume SHR-SCR Module Predetermines Nodule Founder Cell Identity

豆科植物-根瘤菌共生固氮是可持续性农业氮肥的最重要来源。根瘤作为豆科植物共生固氮的一种特化植物侧生器官, 提供了根瘤菌生物固氮必需的微环境, 是根瘤菌的安身之本, 因此, 根瘤的正常发育是实现豆科植物-根瘤菌共生固氮的结构基础。根瘤器官的从头发生主要起始于根瘤菌诱导的根皮层细胞分裂。通常认为豆科植物的根皮层具备有别于非豆科植物根皮层的某种特异属性, 从而响应根瘤菌并与之建立固氮共生, 但长期以来该属性决定的分子机制一直不明确。近日, 中国科学院分子植物科学卓越创新中心王二涛团队以蒺藜苜蓿(Medicago truncatula)等豆科植物和拟南芥(Arabidopsis thaliana)等非豆科植物为研究对象, 发现豆科植物中保守的SHR-SCR干细胞模块决定了其皮层细胞分裂潜能从而赋予根瘤器官发生的命运。该研究揭示了豆科植物根瘤发育的全新机制, 提供了研究和理解植物-根瘤菌固氮共生进化的重要线索, 对提高豆科作物固氮效率和非豆科作物固氮工程具有重要意义。     

放线菌共生固氮的研究

Frankia属是能与非豆科植物共生结瘤固氮的放线菌。目前全球报道有8科24属230多个种的木本双子叶植物与Frankia菌共生结瘤固氮。这类植物统称放线菌结瘤植物。它分布广,适应性强,固氮能力强,是陆地生态系统中的重要供氮系统。70年代,在Bond[10]的倡导下,国际生物学规划IBP从1967至1976年间开展了全球性的双子叶植物结瘤资源调查。当时发现在非豆科木本植物中有8科13个属157个种有结瘤固氮作用,但因未获得内生菌的离体培养,研究进展缓慢,工作停留在资源调查阶段。1978年Callaham[11]从香蕉木中分离出内生菌,离体培养的成功,使共…     

辽宁省豆科结瘤植物及其根瘤菌资源调查

豆科植物作为一种共生固氮植物 ,能与根瘤菌共生结瘤固定大气中的氮素 ,因此受到科学家的广泛关注 ,并对其开展了多方面的研究工作。在固氮机理 ,共生代谢及固氮菌遗传学方面都取得了重要的进展。但是 ,已经被研究的与根瘤菌具有共生关系的豆科植物还不足自然界中已知豆科植物种类的 0 5% [1 ] ,因此 ,豆科结瘤植物及其根瘤菌资源的调查研究工作更显得尤为重要。辽宁省是我国豆科植物种类较为丰富地区之一[2～6] ,而且均为我国主要的经济作物 ,如大豆、菜豆、豇豆、落花生等。多年来人们在不断地利用这些豆科植物资源造福于人类。但是 ,对…     

Truncated hemoglobins in actinorhizal nodules of Datisca glomerata

Three types of hemoglobins exist in higher plants, symbiotic, non-symbiotic, and truncated hemoglobins. Symbiotic (class II) hemoglobins play a role in oxygen supply to intracellular nitrogen-fixing symbionts in legume root nodules, and in one case ( Parasponia Sp.), a non-symbiotic (class I) hemoglobin has been recruited for this function. Here we report the induction of a host gene, dgtrHB1, encoding a truncated hemoglobin in Frankia-induced nodules of the actinorhizal plant Datisca glomerata. Induction takes place specifically in cells infected by the microsymbiont, prior to the onset of bacterial nitrogen fixation. A bacterial gene (Frankia trHBO) encoding a truncated hemoglobin with O (2)-binding kinetics suitable for the facilitation of O (2) diffusion ( ) is also expressed in symbiosis. Nodule oximetry confirms the presence of a molecule that binds oxygen reversibly in D. glomerata nodules, but indicates a low overall hemoglobin concentration suggesting a local function. Frankia trHbO is likely to be responsible for this activity. The function of the D. glomerata truncated hemoglobin is unknown; a possible role in nitric oxide detoxification is suggested.     

Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development

Hemoglobins are ubiquitous in nature and among the best-characterized proteins. Genetics has revealed crucial roles for human hemoglobins, but similar data are lacking for plants. Plants contain symbiotic and nonsymbiotic hemoglobins; the former are thought to be important for symbiotic nitrogen fixation (SNF). In legumes, SNF occurs in specialized organs, called nodules, which contain millions of nitrogen-fixing rhizobia, called bacteroids. The induction of nodule-specific plant genes, including those encoding symbiotic leghemoglobins (Lb), accompanies nodule development. Leghemoglobins accumulate to millimolar concentrations in the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. Although widely accepted, this hypothesis has never been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus. This caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of bacterial nitrogenase protein, and absence of SNF. However, LbRNAi plants grew normally when fertilized with mineral nitrogen. These data indicate roles for leghemoglobins in oxygen transport and buffering and prove for the first time that plant hemoglobins are crucial for symbiotic nitrogen fixation.     

固氮相关的两个植物基因转化烟草及其表达

豆科植物凝集和血红蛋白分别在植物识别其相应的根瘤菌和在根瘤内降低氧分压保护固氮酶的共生固氮作用中起重要作用。将豌豆（Ｐｉｓｕｍ ｓａｔｉｖａ Ｌ．）凝集素基因（ｐｌ）和Ｐａｒａｑｓｐｏｎｉａ ａｎｄｅｒｓｏｎｉｉ血红蛋白基因（ｐｈｂ）构建到同一植物表达载体上,通过根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ（Ｓｍｉｔｈ ｅｔ Ｔｏｗｎｓｅｎｄ）Ｃｏｎｎ）介导法转化烟草（Ｎｉｃｓ     

Transformation of Two Nitrogen-fixation-related Plant Genes into Tobacco and Their Expressions

Lectins and leghemoglobins in legumes play the important roles, respectively, in recognition of host plants to their own rhizobia, and lowering the oxygen partial pressure surround the bacteroids and protecting nitrogenase from oxygen in symbiotic nitrogen-fixing nodules.In order to investigate the non-leguminous recognition of rhizobial bacteria relating to nitrogen fixation, plant expression vectors containing pea lectin gene (pl) and Parasponia hemoglobin gene (phb) have been, respectively, constructed in a plasmid and the plasmid has been introduced into tobacco (Nicotiana tabacum L.) using Agrobacterium tumefaciens (Smith et Townsend) Conn as a vehicle for transformation. PCR and Southern blot demonstrated that the two genes were integrated into the genome of the tobacco plants. Histochemical staining for GUS activity, Western blotting,and in situ hybridization of pea lectin showed that they were expressed at translational level in the plants. These results may provide a clue for exploring whether Rhizobium leguminosarum bv. viciae could extend its host range and make the transgenic tobacco plants have the possibility of being symbiotic, or associative to nitrogen fixation.     

Insights into the history of the legume‐betaproteobacterial symbiosis

The interaction between legumes and rhizobia has been well studied in the context of a mutualistic, nitrogen‐fixing symbiosis. The fitness of legumes, including important agricultural crops, is enhanced by the plants’ ability to develop symbiotic associations with certain soil bacteria that fix atmospheric nitrogen into a utilizable form, namely, ammonia, via a chemical reaction that only bacteria and archaea can perform. Of the bacteria, members of the alpha subclass of the protebacteria are the best‐known nitrogen‐fixing symbionts of legumes. Recently, members of the beta subclass of the proteobacteria that induce nitrogen‐fixing nodules on legume roots in a species‐specific manner have been identified. In this issue, Bontemps et al. reveal that not only are these newly identified rhizobia novel in shifting the paradigm of our understanding of legume symbiosis, but also, based on symbiotic gene phylogenies, have a history that is both ancient and stable. Expanding our understanding of novel plant growth promoting rhizobia will be a valuable resource for incorporating alternative strategies of nitrogen fixation for enhancing plant growth.     

豌豆凝集素和血红蛋白基因对水稻的转化和表达

为了扩大根瘤菌的突破产范围和试探根瘤菌在非豆科植物上的固所为作用,将豌豆凝集素基因（pl）和Parasponia andersonii血红蛋白基因 （phb）构建在同一个植物表达载体上,用基因枪法将其导入水稻（Oryza sativa L.ssp.japonica）。经PCR扩增和Southern杂匀分析,证明外源目的基因已整合到水稻基因组中。GUS组织化学染色及豌豆凝集素基因的Western印迹实验和表达产物的原位杂交,证实外源基因在转基因水稻中表达。在40个转化植株中18株有pl和phb基因的PCR产物,得率为45％。再用18株植物做pl基因的Western blot检测,有3株有翻译表达,占40株的7.5％,18株的17％。为水稻与根瘤菌的相互作用和固氮作用的可能性研究奠定了一定的基础。     

Transformation of Pea Lectin Gene and Parasponia Haemoglobin Gene into Rice and Their Expressions

Lectin and leghemoglobin in legumes play the important roles, respectively, in recognition of host plants to their rhizobial bacteria, and lowering the oxygen partial pressure around bacteroids and protecting nitrogenase from oxygen in symbiotic nitrogen-fixing nodules. In order to extend the host range of the rhizobial bacteria and to make them fix nitrogen in non-legumes, pea lectin gene ( pl ) and Parasponia hemoglobin gene ( phb ) have been constructed into a plant expression vector (pCBHUL) and the vector pCBHUL was introduced into rice calli from immature young embryos by particle bombardment. After the calli were regenerated into plantlets on the resistant-selecting media containing hygromycin, they were identified by PCR and Southern blot hybridization. It was indicated that the pl and phb genes were integrated into nucleic genome of the transformed rice plants. GUS activity and the product of the pl gene were determined by GUS staining, Western blot and in situ hybridization at translational level. Eighteen out of 40 plants resistant to hygromycin were positively identified by PCR analysis with the rate of 45%. The pl gene was expressed in 3 out of 18 plants with 17% and 7.5%in 40 plants. The results may provide a clue for exploring whether Rhizobium leguminosarum bv. viceae could extend its host range and make the transgenic rice plants have the possibility of being symbiotic, or associative to nitrogen fixation.     

Symbiotic and nonsymbiotic hemoglobin genes of Casuarina glauca.

Casuarina glauca has a gene encoding hemoglobin (cashb-nonsym). This gene is expressed in a number of plant tissues. Casuarina also has a second family of hemoglobin genes (cashb-sym) expressed at a high level in the nodules that Casuarina forms in a nitrogen-fixing symbiosis with the actinomycete Frankia. Both the nonsymbiotic and symbiotic genes retained their specific patterns of expression when introduced into the legume Lotus corniculatus. We interpret this finding to mean that the controls of expression of the symbiotic gene in Casuarina must be similar to the controls of expression of the leghemoglobin genes that operate in nodules formed during the interaction between rhizobia and legumes. Deletion analyses of the promoters of the Casuarina symbiotic genes delineated a region that contains nodulin motifs identified in legumes; this region is critical for the controlled expression of the Casuarina gene. The finding that the nonsymbiotic Casuarina gene is also correctly expressed in L. corniculatus suggests to us that a comparable non-symbiotic hemoglobin gene will be found in legume species.     

Determination of nitrogen fixation effectiveness in selected <Emphasis Type="Italic">Medicago truncatula</Emphasis> isolates by measuring nitrogen isotope incorporation into pheophytin

Effectiveness is a term used to describe the input that a bacterial nitrogen-fixing symbiosis makes to plant nitrogen metabolism. In legumes, effectiveness is considered a polymorphic trait where specific interactions between the plant and symbiotic rhizobia contribute to the success of the interaction. Evaluation of effectiveness using model legumes like Medicago truncatula may open new avenues for genetic studies. In previous work, an isotope dilution mass spectrometry method, which uses the effect of nitrogen fixation on the nitrogen isotope composition of chlorophyll in plants grown on ¹⁵N fertilizer as a measure of effectiveness, was developed for estimating the contribution of symbiotic nitrogen fixation to plant nitrogen content. This ¹⁵N-dilution assay was used to evaluate the level of nitrogen fixation effectiveness in three Medicago truncatula lines that have been used as parents in generating recombinant inbred lines. Three Sinorhizobium meliloti strains, USDA 1600, 102F51 and MK506, differ in this measure of effectiveness on three lines of M. truncatula: Jemalong A17, DZA315.16 and F83005.5. Plant–rhizobia combinations grown in two different conditions showed comparable differences in effectiveness.