Sinorhizobium meliloti nifA gene exerts a pleiotropic effect on nodulation through the enhanced plant defense response

Sinorhizobium meliloti nifA gene is required for the expression of a bunch of nif and fix genes. Here, we report its pleiotropic effects on the nodule formation. Compared with wild type strain, nifA mutant sig- nificantly reduced nodule suppression rate in split-root system. The plants inoculated with mutant strain produced lower amount of daidzein and less necrotic cells on their roots. In addition, the defense genes failed to be evoked by nifA mutant at the early nodulation stage. These findings indicated that host defense response was one of the mechanisms mediated by nifA gene to regulate nodule formation during symbiosis. Even though nifA mutant could increase the number of nodules in host plant, it synthesized lower Nod factors than wild type. This suggested that nifA gene mediated multiple and diverse instances in nodulation formation.     

贵州省豆科结瘤固氮植物资源调查

通过对贵州省豆科植物结瘤固氮资源的调查,共采集根瘤标本242份,分属豆科植物42属103种及变种,其中蝶形花亚科、含羞草亚科、云实亚科植物的结瘤率分别为95%,75%和5%;采集的根瘤中,96%生于寄主的侧根,形状以球形、长柱形为主,颜色多为白色、褐色.对分离纯化的55株根瘤菌进行了回接试验,回接结瘤率为33%.生态条件是影响根瘤的大小、数量以及有效性的原因之一.     

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.     

Identification of genes induced during Medicago sativa nodule development by using the cDNA-AFLP technique

Under limited nitrogen conditions, rhizobia are ableto induce the formation of nitrogen-fixing root nodules on their leguminous plant host. This organogenetic process is triggered by a complex exchange of molecu- lar signals between the host plant and bac…     

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.     

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.     

HAR1 mediates systemic regulation of symbiotic organ development

Symbiotic root nodules are beneficial to leguminous host plants; however, excessive nodulation damages the host because it interferes with the distribution of nutrients in the plant. To keep a steady balance, the nodulation programme is regulated systemically in leguminous hosts. Leguminous mutants that have lost this ability display a hypernodulating phenotype. Through the use of reciprocal and self-grafting studies using Lotus japonicus hypernodulating mutants, har1 (also known as sym78), we show that the shoot genotype is responsible for the negative regulation of nodule development. A map-based cloning strategy revealed that HAR1 encodes a protein with a relative molecular mass of 108,000, which contains 21 leucine-rich repeats, a single transmembrane domain and serine/threonine kinase domains. The har1 mutant phenotype was rescued by transfection of the HAR1 gene. In a comparison of Arabidopsis receptor-like kinases, HAR1 showed the highest level of similarity with CLAVATA1 (CLV1). CLV1 negatively regulates formation of the shoot and floral meristems through cell-cell communication involving the CLV3 peptide. Identification of hypernodulation genes thus indicates that genes in leguminous plants bearing a close resemblance to CLV1 regulate nodule development systemically, by means of organ-organ communication.     

Present status and development on biological nitrogen fixation research in China

This presentation introduces the advances in biological nitrogen fixation research abroad, in particular,describes the great progress and achievements on its research in China as follows: collection of rhizobial resources and establishment of the largest database of Rhizobium in China, correction and development of Rhizobium taxonomy in international; discovery of a couple of nif genes, identification and unification of linkage among the nif gene operons of Klebsiella pneumoniae, finding of regulative mechanism of positive regulation nif gene and its sensitivity to oxygen,temperature; finding of the activity of nodulation gene nodD3 product in Sinorhizobium meliloti which is not controlled by flavonoid produced from its host alfalfa; finding of the association between expression of genes coding the products for carbon utilization and nitrogen metabolism and their regulations; chemical synthesis of nodulation factor of Sinorhizobium meliloti; constructions of engineered nitrogen fixers and utilization in practice based on the research of gene expression and regulation; chemical simulation of the structure and function of nitrogenase and bringing forward the model of nitrogenase active center for the first time in international and synthesis of model compounds which were paid attention by colleagues abroad. Finally, the development of nitrogen fixation research in China in future has been put forward, suggesting that the nif gene regulation and its role in providing crops with nitrogen element, signal transduction and molecular interactions between Rhizobium and legume, coupling between carbon and nitrogen metabolisms, nitrogen fixation and photosynthesis, and functional genomics of nitrogen-fixing nodule symbiosis, etc., would be actively worked on.     

A wound-induced small polypeptide gene family is upregulated in soybean nodules

Small peptides function as key signals in processes, such as plant cell differentiation, organ development and defenses to biotic stresses. A large number of small peptide precursor genes have been predicted from the analysis of the soybean (Glycine max) whole genome DNA sequence. However, most of these genes have unknown characteristics and functions. In this report, we systemically searched for the gene families of small peptide precursors that are up-regulated in soybean nitrogen-fixing root nodules. We found 212 genes (encoding peptides shorter than 150 amino acids) that were up-regulated, and among them, 79 genes belong to 38 multiple-gene families, but the other 133 genes are unique. Twenty-eight of 38 families are conserved in Arabidopsis, but the other 10 only exist in legumes. We also identified 16 out of the 38 members of the wound-induced polypeptide (WIP) gene family to be upregulated in nitrogen-fixing nodules. We further analyzed homologs of WIP genes in Medicago, Lotus, Arabidopsis and Oryza species and found that a few homologous genes from Medicago truncatula and Lotus japonicus were also upregulated in their nodules and some WIP genes were induced by specific fungal pathogens on soybean and rice. Structure prediction indicated that all WIP prepropeptides contain a conserved DUF3774 domain (including two hydrophobic regions) and most of them have an N-terminal signal sequence. Fluorescence microscopy analysis of two WIP prepropeptides fused to GFP revealed that these proteins are located on the plasma membrane of tobacco leaf cells. Interestingly, 34 soybean WIP genes are clustered onto three soybean chromosomes, different from known peptide gene families (such as CLE). Among them, 11 highly identical genes are aligned on the 6th chromosome, 12 on the 12th, and 11 on the 13th chromosomes. Most of WIP genes from the 12th chromosome share the highest identities with their homologs on the 13th chromosome, suggesting that ancestral WIP genes could have originated from the 13th chromosome, then spread onto the 12th chromosome by chromosome homologous recombination; the new WIP genes could have existed in multiple copies by gene duplication which then spread onto the 6th chromosome. In Arabidopsis and Oryza species, half of the WIP genes are also aligned on one chromosome and showed higher identity with those from the soybean 12th and 13th chromosomes, suggesting that WIP genes originated from one common ancestor.     

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.     

百脉根Rac1基因启动子的克隆与表达分析

百脉根小G蛋白Rac1基因促进共生结瘤过程,但其转录调控机制尚不明确.采用生物信息学方法对百脉根Rac1基因的启动子序列进行了预测,并对该启动子中含有的顺式调控元件进行了统计分析.克隆了约1.8kb的启动子片段,并构建了GUS融合的重组质粒p1391Z-Rac1Pro.通过百脉根毛根转化法获得转基因毛根,进一步利用组织化学染色法对Rac1基因在阳性毛根中的表达部位进行了研究.结果显示:该启动子除含有常见的转录调控保守元件TATA-box和CAAT-box外,还含有调控防御和胁迫、激素、光照等信号的应答元件.组织化学染色发现,Rac1基因在接种根瘤菌的根毛、根尖、根瘤原基皮层中表达量较高.     

黄腐酸对大豆内源激素的调控及结瘤能力的影响

为探讨黄腐酸提高大豆结瘤能力的作用机理,以辽宁慢生根瘤菌(Bradyrhizobium liaoningense)CCBAU 05525和冀豆17为材料,采用高效液相色谱法(HPLC)探究黄腐酸对大豆结瘤初期内源激素含量、分布及结瘤的影响.结果表明:黄腐酸质量浓度为500 mg/L时大豆结瘤数量最多,比对照组显著提高73.1%,同时该条件下激素含量和分布变化最为明显,具体表现为大豆根、茎、叶中玉米素(ZT)、生长素(IAA)、赤霉素(GA3)含量增加,根中脱落酸(ABA)含量减少,叶中ABA含量增加.而黄腐酸质量浓度为1 000 mg/L时则表现为抑制大豆结瘤. 上述结果为解析黄腐酸提高大豆结瘤固氮的机理、增加大豆产量提供了理论依据.     

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。     

八种豆科树种根瘤的形态与结构研究

对8种豆科树种根瘤的形态和结构进行观察,结果显示:根瘤形态多样,分有限根瘤和无限根瘤两类,根瘤形状、大小与根瘤类型之间有一定的相关性。豆科树种根瘤具有豆科植物根瘤的基本结构,即从外向内依次为皮层、维管束和含菌细胞区,但在皮层中发现有特殊的根瘤形成层细胞,可能与根瘤增粗及根瘤维管束的分化形成有关。豆科树种根瘤中维管束粗而发达,并具有木本植物根的中柱结构,包括有典型的环纹导管、筛管、纤维细胞等。     

Transcriptome analysis of Sinorhizobium meliloti nodule bacteria in nifA mutant background

Sinorhizobium meliloti is one genus of gram-nega- tive soil bacteria that can fix atmospheric nitrogen in root nodules of its symbiotic leguminous host plants[1]. Specific recognition and progressive differentiation ofboth bacteria and host cells are requ…