Amino-acid cycling drives nitrogen fixation in the legume-Rhizobium symbiosis

The biological reduction of atmospheric N2 to ammonium (nitrogen fixation) provides about 65% of the biosphere's available nitrogen. Most of this ammonium is contributed by legume-rhizobia symbioses, which are initiated by the infection of legume hosts by bacteria (rhizobia), resulting in formation of root nodules. Within the nodules, rhizobia are found as bacteroids, which perform the nitrogen fixation: to do this, they obtain sources of carbon and energy from the plant, in the form of dicarboxylic acids. It has been thought that, in return, bacteroids simply provide the plant with ammonium. But here we show that a more complex amino-acid cycle is essential for symbiotic nitrogen fixation by Rhizobium in pea nodules. The plant provides amino acids to the bacteroids, enabling them to shut down their ammonium assimilation. In return, bacteroids act like plant organelles to cycle amino acids back to the plant for asparagine synthesis. The mutual dependence of this exchange prevents the symbiosis being dominated by the plant, and provides a selective pressure for the evolution of mutualism.     

四棱豆根瘤和类菌体的光学及超微结构

通过光学和电子显微镜观察根瘤切片,作者发现四棱豆(Psophocarpus tetragonolobus(L)DC)的根瘤具有多个感染区,类菌体力椭圆形和不规则的长形菌.包膜(membraneenvelope)包围住一个或多个类菌体,感染细胞边缘的线粒体比未感染的细胞多.根瘤感染区细胞内的根瘤菌体分裂整个过程是在包膜内进行.首先是菌体的核区拉长,随后菌体细胞中央部分的细胞壁凹陷,并逐渐加深,最后一个根瘤菌分裂成两个菌体.     

Deregulation of a Ca2+/calmodulin-dependent kinase leads to spontaneous nodule development

Induced development of a new plant organ in response to rhizobia is the most prominent manifestation of legume root-nodule symbiosis with nitrogen-fixing bacteria. Here we show that the complex root-nodule organogenic programme can be genetically deregulated to trigger de novo nodule formation in the absence of rhizobia or exogenous rhizobial signals. In an ethylmethane sulphonate-induced snf1 (spontaneous nodule formation) mutant of Lotus japonicus, a single amino-acid replacement in a Ca2+/calmodulin-dependent protein kinase (CCaMK) is sufficient to turn fully differentiated root cortical cells into meristematic founder cells of root nodule primordia. These spontaneous nodules are genuine nodules with an ontogeny similar to that of rhizobial-induced root nodules, corroborating previous physiological studies. Using two receptor-deficient genetic backgrounds we provide evidence for a developmentally integrated spontaneous nodulation process that is independent of lipochitin-oligosaccharide signal perception and oscillations in Ca2+ second messenger levels. Our results reveal a key regulatory position of CCaMK upstream of all components required for cell-cycle activation, and a phenotypically divergent series of mutant alleles demonstrates positive and negative regulation of the process.     

A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals

Plants belonging to the legume family develop nitrogen-fixing root nodules in symbiosis with bacteria commonly known as rhizobia. The legume host encodes all of the functions necessary to build the specialized symbiotic organ, the nodule, but the process is elicited by the bacteria. Molecular communication initiates the interaction, and signals, usually flavones, secreted by the legume root induce the bacteria to produce a lipochitin-oligosaccharide signal molecule (Nod-factor), which in turn triggers the plant organogenic process. An important determinant of bacterial host specificity is the structure of the Nod-factor, suggesting that a plant receptor is involved in signal perception and signal transduction initiating the plant developmental response. Here we describe the cloning of a putative Nod-factor receptor kinase gene (NFR5) from Lotus japonicus. NFR5 is essential for Nod-factor perception and encodes an unusual transmembrane serine/threonine receptor-like kinase required for the earliest detectable plant responses to bacteria and Nod-factor. The extracellular domain of the putative receptor has three modules with similarity to LysM domains known from peptidoglycan-binding proteins and chitinases. Together with an atypical kinase domain structure this characterizes an unusual receptor-like kinase.     

刺槐根瘤发生的超微结构研究

通过扫描电镜和透射电镜观察刺槐（Ｒｏｂｉｎｉａｐｓｅｕｄｏａｃａｃｉａ）幼瘤的超微结构。刺槐根瘤中侵入线丰富、粗大，内具有高电子密度基质，基质中包埋大量根瘤菌。根瘤菌通过降解侵入线壁和从侵入线无壁泡中释放两种方式进入宿主细胞质     

Shoot control of root development and nodulation is mediated by a receptor-like kinase

In legumes, root nodule organogenesis is activated in response to morphogenic lipochitin oligosaccharides that are synthesized by bacteria, commonly known as rhizobia. Successful symbiotic interaction results in the formation of highly specialized organs called root nodules, which provide a unique environment for symbiotic nitrogen fixation. In wild-type plants the number of nodules is regulated by a signalling mechanism integrating environmental and developmental cues to arrest most rhizobial infections within the susceptible zone of the root. Furthermore, a feedback mechanism controls the temporal and spatial susceptibility to infection of the root system. This mechanism is referred to as autoregulation of nodulation, as earlier nodulation events inhibit nodulation of younger root tissues. Lotus japonicus plants homozygous for a mutation in the hypernodulation aberrant root (har1) locus escape this regulation and form an excessive number of nodules. Here we report the molecular cloning and expression analysis of the HAR1 gene and the pea orthologue, Pisum sativum, SYM29. HAR1 encodes a putative serine/threonine receptor kinase, which is required for shoot-controlled regulation of root growth, nodule number, and for nitrate sensitivity of symbiotic development.     

Sinorhizobium meliloti lsrB is involved in alfalfa root nodule development and nitrogen-fixing bacteroid differentiation

Rhizobia interact with host legumes to induce the formation of nitrogen-fixing nodules, which is very important in agriculture and ecology. The development of nitrogen-fixing nodules is stringently regulated by host plants and rhizobial symbionts. In our previous work, a new Sinorhizobium meliloti LysR regulator gene (lsrB) was identified to be essential for alfalfa nodulation. However, how this gene is involved in alfalfa nodulation was not yet understood. Here, we found that this gene was associated with prevention of premature nodule senescence and abortive bacteroid formation. Heterogeneous deficient alfalfa root nodules were induced by the in-frame deletion mutant of lsrB (lsrB1-2), which was similar to the plasmid-insertion mutant, lsrB1. Irregular senescence zones earlier appeared in these nodules where bacteroid differentiation was blocked at different stages from microscopy observations. Interestingly, oxidative bursts were observed in these nodules by DAB staining. The decreased expression of lipopolysaccharide core genes (lpsCDE) was correspondingly determined in these nodules. S. meliloti lipopolysaccharide is required for suppression of oxidative bursts or host cell defense. These findings demonstrate that the S. meliloti lsrB gene is involved in alfalfa root nodule development and bacteroid differentiation by suppressing oxidative bursts or defense responses in host cells.     

用^15N示踪标记法测定人工诱发黄瓜根瘤固氮酶活性

报道了用＾１５Ｎ示踪标记法对人工诱发黄大氮酶活性的测定结果，将长有人工诱发根瘤的黄瓜根系暴露在＾１５Ｎ２的气体中，部分根系浸在无氮培养液内，标记４８ｈ后，质谱分析测定结瘤黄瓜根＾１５Ｎ的丰度为０．４３１原子％＾１５Ｎ，样品为０３６９原子％＾１５Ｎ，对＾１５Ｎ示踪标记结果进行了统计学ｔ检验，ｔ＝３．１５〉６０．０１＝２．８１９，表明黄奶系根瘤内的细菌固氮作用所的氮与相比达９９．９％极显著性水平，同时     

A phosphate transporter expressed in arbuscule-containing cells in potato.

Arbuscular mycorrhizas are the most common non-pathogenic symbioses in the roots of plants. It is generally assumed that this symbiosis facilitated the colonization of land by plants. In arbuscular mycorrhizas, fungal hyphae often extend between the root cells and tuft-like branched structures (arbuscules) form within the cell lumina that act as the functional interface for nutrient exchange. In the mutualistic arbuscular-mycorrhizal symbiosis the host plant derives mainly phosphorus from the fungus, which in turn benefits from plant-based glucose. The molecular basis of the establishment and functioning of the arbuscular-mycorrhizal symbiosis is largely not understood. Here we identify the phosphate transporter gene StPT3 in potato (Solanum tuberosum). Functionality of the encoded protein was confirmed by yeast complementation. RNA localization and reporter gene expression indicated expression of StPT3 in root sectors where mycorrhizal structures are formed. A sequence motif in the StPT3 promoter is similar to transposon-like elements, suggesting that the mutualistic symbiosis evolved by genetic rearrangements in the StPT3 promoter.     

Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi

Arbuscular mycorrhizal (AM) fungi form mutualistic, symbiotic associations with the roots of more than 80% of land plants. The fungi are incapable of completing their life cycle in the absence of a host root. Their spores can germinate and grow in the absence of a host, but their hyphal growth is very limited. Little is known about the molecular mechanisms that govern signalling and recognition between AM fungi and their host plants. In one of the first stages of host recognition, the hyphae of AM fungi show extensive branching in the vicinity of host roots before formation of the appressorium, the structure used to penetrate the plant root. Host roots are known to release signalling molecules that trigger hyphal branching, but these branching factors have not been isolated. Here we have isolated a branching factor from the root exudates of Lotus japonicus and used spectroscopic analysis and chemical synthesis to identify it as a strigolactone, 5-deoxy-strigol. Strigolactones are a group of sesquiterpene lactones, previously isolated as seed-germination stimulants for the parasitic weeds Striga and Orobanche. The natural strigolactones 5-deoxy-strigol, sorgolactone and strigol, and a synthetic analogue, GR24, induced extensive hyphal branching in germinating spores of the AM fungus Gigaspora margarita at very low concentrations.     

Evolution of an obligate social cheater to a superior cooperator

Obligate relationships have evolved many times and can be parasitic or mutualistic. Obligate organisms rely on others to survive and thus coevolve with their host or partner. An important but little explored question is whether obligate status is an evolutionarily terminal condition or whether obligate lineages can evolve back to an autonomous lifestyle. The bacterium Myxococcus xanthus survives starvation by the social development of spore-bearing fruiting bodies. Some M. xanthus genotypes defective at fruiting body development in isolation can nonetheless exploit proficient genotypes in chimaeric groups. Here we report an evolutionary transition from obligate dependence on an altruistic host to an autonomous mode of social cooperation. This restoration of social independence was caused by a single mutation of large effect that confers fitness superiority over both ancestral genotypes, including immunity from exploitation by the ancestral cheater. Thus, a temporary state of obligate cheating served as an evolutionary stepping-stone to a novel state of autonomous social dominance.     

诱导不结瘤豆科树种结瘤固氮的研究

根据国内外有关诱导非豆科作物结瘤固氮的现状,结合作者几年来的工作,对诱导不结瘤豆科树种结瘤固氮的可行性进行了探讨,并报道了作者近年来人工诱导不结瘤的豆科树种—山皂荚(Gledtsia melanacantha)的试验结果。     

Disentangling nestedness from models of ecological complexity

Complex networks of interactions are ubiquitous and are particularly important in ecological communities, in which large numbers of species exhibit negative (for example, competition or predation) and positive (for example, mutualism) interactions with one another. Nestedness in mutualistic ecological networks is the tendency for ecological specialists to interact with a subset of species that also interact with more generalist species. Recent mathematical and computational analysis has suggested that such nestedness increases species richness. By examining previous results and applying computational approaches to 59 empirical data sets representing mutualistic plant–pollinator networks, we show that this statement is incorrect. A simpler metric—the number of mutualistic partners a species has—is a much better predictor of individual species survival and hence, community persistence. Nestedness is, at best, a secondary covariate rather than a causative factor for biodiversity in mutualistic communities. Analysis of complex networks should be accompanied by analysis of simpler, underpinning mechanisms that drive multiple higher-order network properties.     

河北省豆科植物根瘤菌资源的初步调查研究

通过对河北省全境豆科植物的结瘤和固氮状况进行调查 ,共采集到 2 4属 5 3种豆科植物的根瘤样品 2 3 5份 ,98.1的被调查豆科植物可自然结瘤 ,96.2的自然瘤可测到固氮酶活性 .其中海边香豌豆 (L athyrus maritimus)、野百合 (Crotalaria sessilif lora)、阴山胡枝子 (L athyrusinschanica)、山岩黄耆 (H edysarum alpinum)、辽西扁苜蓿 (Melilotus ruthenicus)的结瘤固氮情况为首次报道 .     

A receptor kinase gene regulating symbiotic nodule development

Leguminous plants are able to establish a nitrogen-fixing symbiosis with soil bacteria generally known as rhizobia. Metabolites exuded by the plant root activate the production of a rhizobial signal molecule, the Nod factor, which is essential for symbiotic nodule development. This lipo-chitooligosaccharide signal is active at femtomolar concentrations, and its structure is correlated with host specificity of symbiosis, suggesting the involvement of a cognate perception system in the plant host. Here we describe the cloning of a gene from Medicago sativa that is essential for Nod-factor perception in alfalfa, and by genetic analogy, in the related legumes Medicago truncatula and Pisum sativum. The identified 'nodulation receptor kinase', NORK, is predicted to function in the Nod-factor perception/transduction system (the NORK system) that initiates a signal cascade leading to nodulation. The family of 'NORK extracellular-sequence-like' (NSL) genes is broadly distributed in the plant kingdom, although their biological function has not been previously ascribed. We suggest that during the evolution of symbiosis an ancestral NSL system was co-opted for transduction of an external ligand, the rhizobial Nod factor, leading to development of the symbiotic root nodule.     

豆科树种根瘤菌共生体系的研究——Ⅱ.豆科树种根瘤菌分类地位

<正>据作者试验观察,豆科树种根瘤菌周生鞭毛、生长快的菌株,在YEM培养基上产生酸,在石蕊牛奶中也产生乳清层,如刺槐、紫穗槐、马蹄针、紫藤等的根瘤菌具有上述性状;极生或亚极生鞭毛、生长慢的菌株在YEM培养基中不产生酸,在石蕊牛奶中也不产生乳清层,如垂耳相思、花梨木、华楹、合欢等树种的根瘤菌具有这些性状。因此,豆科树种根瘤菌也应分列于二个属中(Rhizobium和Bradyrhizobium),这与Jordan提出的新分类系统基本一致。     

大豆根毛对大豆根瘤菌的生化吸附及其对结瘤影响的研究

本文研究证明大豆根毛与大豆根瘤菌具有专化性生化吸附作用。吸附主要发生在根毛端部。同一棵根上不是所有的根毛都能发生生化吸附,吸附与根毛的生长令期有关。用13种大豆根瘤菌株系与7个大豆品种根毛生化吸附实验表明,吸附率明显地存在有差异性,但这种差异与结瘤量无相关性。使用本研究发现的生化吸附率高而不能结瘤的1.174大豆根瘤菌株系与吸附率较低而结瘤力强的113—2或61A67大豆根瘤菌株系双接菌,证明能明显降低113—2或地61A76的结瘤量,表现出大豆根毛生化吸附具有竞争性抑制大豆根瘤菌的侵入、结瘤作用,对理论研究和实际应用具有一定重要意义。     

Isolation and application of effective nitrogen fixation rhizobial strains on low-phosphorus acid soils in South China

Soybean (Glycine max L.) is a very important food and oil crop in China. Legume-rhizobium symbiotic nitrogen (N) fixation is an important biological character and also the base of improving soil fertility of soybean. However, soybean production and development is severely limited in tropical and subtropical areas in China due to a lack of effective rhizobial inoculants adapting to low-phosphorus (P) acid soils. In the present study, 12 soybean rhizobial strains were isolated and purified from the nodules of two soybean genotypes contrasting in P efficiency, which were grown on different low-P acid soils with different soybean cultivation histories. Results from 16S rDNA sequence analysis showed that these 12 rhizobial strains belonged to the genus of Bradyrhizobium, which had higher nitrogenase activities compared to the control strain, Bradyrhizboium japonicum USDA110. A field experiment was carried out by applying rhizobial inoculants, a mixture of three rhizobial strains that showed the highest nitrogenase activity, on a typical low-P acid soil in South China. The results showed that, without inoculation, no nodules were formed in the three soybean genotypes tested; with inoculation, the nodulation rates in all were 100%. Inoculation with rhizobial inoculants not only made many nodules formed, but also increased soybean shoot biomass and yield, and improved nitrogen (N) and P nutrient status. Among which, shoot dry weight, N and P content of a soybean genotype, Huachun 3, inoculated with rhizobium were increased 154.3%, 152.4% and 163.2% compared to that without inoculation, respectively. We concluded that: (i) The effective indigenous rhizobial strains isolated in this study from soybeans on low-P acid soils in South China have the characters of broad host range, high nodulation efficiency, efficient N fixation, great low pH and low P tolerance. (ii) Soil environment and host types are the key factors to screen the effective rhizobial strains. Considering soil pH values and P efficiency of the host genotypes might increase the screening efficiency. (iii) Improving N status and facilitating root growth might be the mechanisms of increasing the P uptake in soybean plants inoculated with the effective rhizobial strains on low-P acid soils. (iv) Inoculation with the effective rhizobial inoculants could significantly improve growth, N and P content of soybean on low-P acid soils, which might be an effective approach to enhance soybean cultivation and development in these areas. Therefore, application and extension of inoculation techniques with effective rhizobial inoculants in legumes would result in great economical, environmental and ecological benefits. The authors contributed equally to this work Supported by National Key Basic Research and Development of China (Grant No. 2005CB120902), McKnight Foundation Collaborative Crop Research Program (USA) (Grant No. 05-780) and National Natural Science Foundation of China (Grant No. 30571111)     

樟子松人工林细根生产、周转和碳归还对施氮的响应

在河北塞罕坝樟子松林, 设置对照样方、低氮(20 kgN/(hm²·a))、中氮(50 kgN/(hm²·a))和高氮(100 kgN/(hm²·a))添加4种处理, 分0~10, 10~20和20~30 cm共3个土层, 系统地研究细根生产、周转、碳归还以及细根生产力(NPPfr)占生态系统净初级生产力(NPP)的比例对不同程度氮可获得性的响应, 结论如下: 1) 细根生产力在低氮下增大, 高氮下降低; 细根生产力占NPP的比例则相反, 在低氮下降低, 中氮下升高; 2) 随氮浓度增大, 细根生物量逐渐降低, 细根周转率增大, 细根碳归还先升(低、中氮)后降(高氮); 3) 施氮对细根生产力的影响随土壤深度的加深无显著变化, 施氮对细根周转率的影响在不同深度间则差异显著; 4) 结构方程模型表明, 氮添加通过对土壤碳氮含量、pH的影响而改变细根生产力, 通过对细根碳氮含量的影响改变细根周转率。