Bacteroids Isolated from Ineffective Nodules of Pisum sativum Mutant E135 (syml3) Lack Nitrogenase Activity but Contain the Two Protein Components of Nitrogenase

Pea mutant E135 (sym15) forms ineffective (Fix^–) nodulesthat lack nitrogen fixing activity. To determine the developmentalstep blocked in E135 nodules we studied the nitrogenase activitiesin isolated bacteroids and in cell-free extracts of bacteroids,and measured the two components of nitrogenase protein in bacteroids.Bacteroids prepared anaerobically from E135 nodules showed noacetylene reduction activity in the presence and absence ofmyoglobin. Furthermore, no acetylene reduction activity by cell-freeextracts of E135 bacteroids was detected in the presence ofATP-generating system and dithionite. However, immuno-blottinganalyses revealed the presence of nitrogenase components I andII in E135 nodule bacteroids. These results suggest that a hostplant gene is involved in the expression of nitrogenase activityin symbiotic bacteria. (Received May 11, 1998; Accepted August 7, 1998)     

Pleiotropic Effects of brz: A Mutation in Pisum sativum (L.) cv ;Sparkle' Conditioning Decreased Nodulation and Increased Iron Uptake and Leaf Necrosis

Treatment of Pisum sativum (L.) cv `Sparkle' with ethylmethane sulfonic acid produced a stable mutant, E107, which forms few nodules. The mutant allele exhibits other pleiotropic properties including bronze necrotic spots on the leaflets and high accumulation of iron in the shoot. The mutant phenotype is under monogenic recessive control. The gene, designated brz (bronze), is nonallelic with two other genes conditioning necrotic spots on leaves of other mutants of P. sativum. The brz allele was located on chromosome 4 by linkage with wax production controlled by alleles at the was locus.     

Comparison of Enzymes Involved in Carbon and Nitrogen Metabolism in Normal Nodules and Ineffective Nodules Induced by a Pea Mutant E135 (sym 13)

Activities of enzymes involved in carbon and nitrogen metabolismwere examined in nodules of normal pea (Pisum sativum L. cv.Sparkle) and an ineffective plant mutant E135 (sym 13). Specificactivities of some enzymes were lower in ineffective nodulesthan in effective nodules. However, there were no major differencesbetween respective bacteroid fractions. ¹Present address: Department of Life Science, Aichi Universityof Education, Kariya, Aichi, 448 Japan     

Gene mapping related to yellow green leaf in a mutant line in rice (Oryza sativa L.)

A mutant, which derived from the restorer line Jinhui10 treated with EMS, showed completely yellow green leaves, and it had low chlorophyll content and poor agronomic characteristics during the growing stage. The F1 plants from the cross between normal × the mutant showed normal green leaves, and the segregation ratio of normal to yellow green leaves was 3 : 1 in F₂ population. It indicated that the trait was controlled by a single recessive nuclear gene, temporarily designated asygl3. The geneygl3 was mapped between RM468 and RM3684 with genetic distances 8.4 cM and 1.8 cM on chromosome 3. This result would be used as genetic information for fine mapping and map-based cloning ofygl3 gene.     

Comparison of the Protein Composition and Enzymatic Activities during Development between Effective and Plant-Determined Ineffective Nodules in Pea

The protein composition and enzymatic activities during developmentof ineffective nodules, produced by mutant E135 (sym 13) ofpea (Pisum sativum L.), were compared with those of the nitrogen-fixingnodules of the normal parent, the Sparkle cultivar. The proteincomposition of 3-week-old E135 nodules, as determined by SDS-polyacrylamidegel electrophoresis, was quite similar to that of Sparkle nodules.After 4 weeks, however, the intensities of bands of 15-, 38-,and 87-kDa polypeptides were lower in the case of E135 nodules.Western blot analysis using a "nodule-specific" antiserum revealedthat most nodulins could be detected in 3-week-old E135 nodules,but a 35.5-kDa nodulin disappeared after 5 weeks and severalnovel peptides ranging in molecular weight from 26 to 31 kDaappeared after 6 weeks in E135 nodules. The activities of glutaminesynthetase, glutamate synthase, alanine-pyruvate aminotransferase,sucrose synthase, and phosphoenolpyruvate carboxylase increasedduring development of Sparkle nodules, but such increases werenot found in E135 nodules after 5 weeks. These results showthat the nodules of E135 begin to develop normally but differfrom those of Sparkle within 4 weeks, indicating that, duringearly stages of nodule development, the protein compositionand activities of enzymes involved in carbon and nitrogen metabolismare not regulated by the presence or absence of nitrogenaseactivity. (Received February 26, 1993; Accepted May 19, 1993)     

Induced symbiosis mutants of pea (Pisum sativum) and sweetclover (Melilotus alba annua)

Treatment of Pisum sativum (L.) cv. ‘Sparkle’ with ethylmethane sulfonic acid or γ or neutron radiation induced stable mutants which do not form nodules, or form few nodules. Six mutants were at the previously described sym 5 locus. Non-nodulating mutants of Melilotus alba annua (Desr.) cv. U389 were obtained by treatment with ethylmethane sulfonic acid (EMS) or neutron radiation, but not by γ radiation or azide.     

sym 18. A novel gene conditioning altered strain specificity in Pisum sativum cv. ‘Sparkle’

An induced mutant of pea Pisum sativum cv. Sparkle forms few nodules with R. leguminosarum bv. viciea from temperate regions, exemplified by strain PRE, but nodulates normally with some rhizobia from Middle East soils, exemplified by strain TOM. The mutant gene is not an allele of sym2, found in the primitive cultivar Afghanistan. Mutant line E54 has a specificity similar to Afghanistan but forms more nodules with temperate strains, especially PF₂ which nodulates Afghanistan only poorly. The new phenotype is conditioned by gene sym18, which can act as recessive or semi-dominant depending on the rhizobial strain. Also sym18 is distinguished from sym 2 by its location on a different linkage group. Sym18 was mapped 9cM from k on linkage group II.     

A Mutation in Vicia faba Results in Ineffective Nodules with Impaired Bacteroid Differentiation and Reduced Synthesis of Late Nodulins

Although numerous reports have documented the effect of bacterially-inducedineffectiveness on root nodule structure, function, and plantgene expression, few studies have detailed the effect of theplant genome on similar parameters. In this report effective(N₂-fixing) broadbean {Vicia faba L.) and plant-controlled ineffective(non-N₂-fixing) broadbean recessive for the sym-1 gene werecompared for nodule structure, developmental expression of noduleenzyme activities, enzyme proteins, and mRNAs involved in Nassimilation, leghemoglobin (Lb) synthesis, and acetylene reductionactivity (ARA). During development of effective wild-type nodules,glutamine synthetase (GS), aspartate aminotransferase (AAT),phosphoenolpyruvate carboxylase (PEPC) and NADH-glutamate synthase(GOGAT) activities and enzyme proteins increased coincidentwith nodule ARA. The increases in GS, AAT, and PEPC were associatedwith increased synthesis of mRNAs for these proteins. Synthesisof Lb polypeptides and mRNAs during development of effectivenodules was similar to that of GS, AAT, and PEPC. By contrast,ineffective sym-1 nodules displayed little or no ARA and hadneither the increases in enzyme activities nor enzyme proteinsand mRNAs as seen for effective nodules. The effect of the sym-1gene appeared to occur late in nodule development at eitherthe stage of bacterial release from infection threads or differentiationof bacteria into bacteroids. High in vitro enzyme activities,enzyme polypeptides, and mRNA levels in parental effective noduleswere dependent upon a signal associated with effective bacteroidsthat was lacking in sym-1 nodules. Nodule organogenesis didnot appear to be a signal for the induction of GS, PEPC, AAT,and Lb expression in sym-1 nodules. Key words: Vicia faba, mutation, sym-1 gene, nodules     

Symbiotic mutants deficient in nodule establishment identified after T-DNA transformation of Lotus japonicus

Nitrogen-fixing root nodules develop on legumes as a result of an interaction between host plants and soil bacteria collectively referred to as rhizobia. The organogenic process resulting in nodule development is triggered by the bacterial microsymbiont, but genetically controlled by the host plant genome. Using T-DNA insertion as a tool to identify novel plant genes that regulate nodule ontogeny, we have identified two putatively tagged symbiotic loci, Ljsym8 and Ljsym13, in the diploid legume Lotus japonicus. The sym8 mutants are arrested during infection by the bacteria early in the developmental process. The sym13 mutants are arrested in the final stages of infection, and ineffective nodules are formed. These two plant mutant lines were identified in progeny from 1112 primary transformants obtained after Agrobacterium tumefaciens T-DNA-mediated transformation of L. japonicus and subsequent screening for defects in the symbiosis with Mesorhizobium loti. Additional nontagged mutants arrested at different developmental stages were also identified and genetic complementation tests assigned all the mutations to 16 monogenic symbiotic loci segregating recessive mutant alleles. In the screen reported here independent symbiotic loci thus appeared with a frequency of ～1.5%, suggesting that a relatively large set of genes is required for the symbiotic interaction.     

Structural and functional organization of the plasmid regulons of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> symbiotic genes

The structure of the plasmid locus containing the sym-genes (nod-, nif-, and fix-operons) was investigated in eight Rhizobium leguminosarum strains differing in their origin and host specificity, including five strains of the viciae biovar—symbionts of pea (3), forage beans (1), and Vavilovia (1)—as well as three strains of the biovar trifolii (clover symbionts). Strains of R. leguminosarum bv. viciae, which possess the nodX gene (controlling acetylation of the Nod factor, which is responsible for the ability of rhizobia to form symbioses with a broad spectrum of hosts, including the “Afghan” pea lines, homozygous by the allele sym^2A), are characterized by a less compact location of the sym-genes than the strains lacking the nodX gene. The size of the symbiotic cluster in the strains possessing nodX was 94.5 ± 3.5 kb, with the share of the sym-genes of 36.5 ± 1.5%, while for the strains lacking nodX these values were 61.7 ± 3.7 kb and 56.3 ± 1.4%, respectively (significant difference at P ₀ < 0.01). Syntenic structures were revealed in the symbiotic regions of strains Vaf12, UPM1131, and TOM, as well as syntenic structures of non-symbiotic regions in strains Vaf12, TOM, and WSM1689. The correlation coefficients between the matrices of genetic distances in the analyzed strains for the nodABC, nifHDK, and fixABC operons were on average 0.993 ± 0.002, while their values for the plasmid sites located between the sym-genes were considerably less (0.706 ± 0.010). In these regions, 21 to 27% of the genes were involved in amino acid transport and metabolism, which was substantially higher than the average for the genome of R. leguminosarum bv. viciae (11–12%). These data suggest that the evolution of R. leguminosarum bv. viciae, defined by narrowing of the host specificity (associated with a loss of the nodX gene), was accompanied by reduction of the regions of plasmids located between the sym-genes, as well as by specialization of these areas to perform the functions related to symbiotic nitrogen fixation. The observed increase of density in the cluster of sym-genes may be associated with intensification of their horizontal transfer in the populations of rhizobia, which determines the speed of evolution of the symbiotic system.     

Sequential functioning of Sym-13 and Sym-31, two genes affecting symbiosome development in root nodules of pea (Pisum sativum L.)

Two Fix^? mutants of pea (Pisum sativum L.) which are unable to fix molecular nitrogen, E135f (sym-13) and Sprint-2Fix^? (sym-31), were crossed to create the doubly homozygous recessive line, named RBT (sym-13, sym-31). The ultrastructural organization of nodules of the RBT line was compared with that of each of the two parental mutant lines and with the original wild-type genotypes of the cultivars Sparkle and Sprint-2. It was shown that the RBT line is similar to the mutant line Sprint-2Fix^? in having abnormal symbiosome composition and bacteroids with relatively undifferentiated morphology. Because the phenotypic manifestation of the sym-31 mutant allele suppresses the phenotypic manifestation of the sym-13 mutant allele, it is concluded that the function of the gene Sym-31 (which is mutated in the Sprint-2Fix^? line) is necessary at an earlier stage of symbiosome development than the gene Sym-13 (which is mutant in the E135f line).     

Linkage mapping of the locus responsible for male pseudohermaphroditism (mp) on rat chromosome 7.

TF is a mutant rat strain showing male pseudohermaphroditism controlled by an autosomal single recessive gene (mp). The affected rats show lack of Leydig cells and androgen deficiency. In this study, we performed linkage analysis using F(2) progeny of crosses between TF and BN strains to determine the chromosomal localization of the mp locus. The mp locus was mapped in a 4 cM region of the distal region of rat chromosome 7 between D7Rat3 and D7Rat115 or D7Rat94. Comparison of the linkage map with corresponding regions of the published rat genome sequence revealed several candidate genes for the mp mutation, including the Dhh, Tegt, Gdp3, and Amhr2 genes.     

The pea (Pisum sativum L.) genes sym33 and sym40 control infection thread formation and root nodule function

Two novel non-allelic mutants that were unable to fix nitrogen (Fix^?) were obtained after EMS (ethyl methyl sulfonate) mutagenesis of pea (Pisum sativum L.). Both mutants, SGEFix^?–1 and SGEFix^?–2, form two types of nodules: SGEFix^?–1 forms numerous white and some pink nodules, while mutant SGEFix^?–2 forms white nodules with a dark pit at the distal end and also some pinkish nodules. Both mutations are monogenic and recessive. In both lines the manifestation of the mutant phenotype is associated with the root genotype. White nodules of SGEFix^?–1 are characterised by hypertrophied infection threads and infection droplets, mass endocytosis of bacteria, abnormal morphological differentiation of bacteroids, and premature degradation of nodule symbiotic structures. The structure of the pink nodules of SGEFix^?–1 does not differ from that of the parental line, SGE. White nodules of SGEFix^?–2 are characterised by “locked” infection threads surrounded with abnormally thick plant cell walls. In these nodules there is no endocytosis of bacteria into host-cell cytoplasm. The pinkish nodules of SGEFix^?–2 are characterised by virtually undifferentiated bacteroids and premature degradation of nodule tissues. Thus, the novel pea symbiotic genes, sym40 and sym33, identified after complementation analysis in SGEFix^?–1 and SGEFix^?–2 lines, respectively, control early nodule developmental stages connected with infection thread formation and function.     

Bacteriocin-Like Substances Produced by Rhizobium japonicum and Other Slow-Growing Rhizobia

Bacteriocin-like substances were commonly produced by slow-growing Rhizobium japonicum and cowpea rhizobia on an L-arabinose medium. Antagonism between strains of R. japonicum was not detected in vitro; however, such strains were often sensitive to some bacteriocins produced by cowpea rhizobia. Inhibitory zones (2 to 8 mm from colony margins), produced by 58 of 66 R. japonicum test strains, were reproducibly detected with Corynebacterium nebraskense as an indicator. Quantitative production was not related to symbiotic properties of effective strains, since nine noninfective strains and one ineffective strain produced bacteriocin. Eight R. japonicum strains that did not produce bacteriocin nevertheless formed effective nodules on soybeans. R. japonicum strains that produced bacteriocin in vitro had no antagonistic effect on nonproducer strains during soybean nodulation. Under controlled conditions, a nonproducer (3I1b135) predominated over a bacteriocin producer (3I1b6) when inoculated at 1:1 and 1:9 ratios. Depending on the particular ratio, up to 38% of the total nodules formed were infected with mixed combinations. The bacteriocin(s) had a restricted host range and antibiotic-like properties which included the ability to be dialyzed and resistance to heat (75 to 80°C, 30 min), Pronase, proteinase K, trypsin, ribonuclease, and deoxyribonuclease. R. japonicum strains representing genetic, serological, cultural, and geographic diversity were differentiated into three groups on the basis of bacteriocin production.     

拟南芥白化突变体心口的基因定位与分析

EMS30是拟南芥经甲基磺酸乙酯（EMS）诱变得到的白化突变体。该突变体的叶绿体结构存在严重缺陷,同时伴随叶绿素缺失。遗传分析显示EMS30突变体的突变表型受隐性单基因控制。采用图位克隆的方法对EMS30突变基因进行定位的结果显示,该基因位于拟南芥第一条染色体的分子标记F21M12和F14N23之间的96kb区间内,该区间包含25个基因。通过生物信息学分析发现,该区间内有3个基因定位在叶绿体或与叶绿体发育相关。这些结果有助于该基因的克隆,为阐释叶绿体发育提供线索。     

Construction of highly-effective symbiotic bacteria: Evolutionary models and genetic approaches

Using the example of N₂-fixing legume-rhizobial symbiosis, we demonstrated that the origin and evolution of bacteria symbiotic for plants involve: (i) the formation of novel sym gene systems based on reorganizations of the bacterial genomes and on the gene transfer from the distant organisms; (ii) the loss of genes encoding for functions that are required for autonomous performance but interfere with symbiotic functions (negative regulators of symbiosis). Therefore, the construction of effective rhizobia strains should involve improvement of sym genes activities (for instance, nif, fix, and dct genes encoding for nitrogenase synthesis or for the energy supply of N₂ fixation), as well as the inactivation of negative regulators of symbiosis identified in our lab (eff genes encoding for the transport of sugars and the production of polysaccharides and storage compounds, as well as for oxidative-reductive processes).     

Genetic Analysis Reveals the Essential Role of Nitrogen Phosphotransferase System Components in Sinorhizobium fredii CCBAU 45436 Symbioses with Soybean and Pigeonpea Plants

The nitrogen phosphotransferase system (PTS^Ntr) consists of EI^Ntr, NPr, and EIIA^Ntr. The active phosphate moiety derived from phosphoenolpyruvate is transferred through EI^Ntr and NPr to EIIA^Ntr. Sinorhizobium fredii can establish a nitrogen-fixing symbiosis with the legume crops soybean (as determinate nodules) and pigeonpea (as indeterminate nodules). In this study, S. fredii strains with mutations in ptsP and ptsO (encoding EI^Ntr and NPr, respectively) formed ineffective nodules on soybeans, while a strain with a ptsN mutation (encoding EIIA^Ntr) was not defective in symbiosis with soybeans. Notable reductions in the numbers of bacteroids within each symbiosome and of poly-β-hydroxybutyrate granules in bacteroids were observed in nodules infected by the ptsP or ptsO mutant strains but not in those infected with the ptsN mutant strain. However, these defects of the ptsP and ptsO mutant strains were recovered in ptsP ptsN and ptsO ptsN double-mutant strains, implying a negative role of unphosphorylated EIIA^Ntr in symbiosis. Moreover, the symbiotic defect of the ptsP mutant was also recovered by expressing EI^Ntr with or without the GAF domain, indicating that the putative glutamine-sensing domain GAF is dispensable in symbiotic interactions. The critical role of PTS^Ntr in symbiosis was also observed when related PTS^Ntr mutant strains of S. fredii were inoculated on pigeonpea plants. Furthermore, nodule occupancy and carbon utilization tests suggested that multiple outputs could be derived from components of PTS^Ntr in addition to the negative role of unphosphorylated EIIA^Ntr.