A Novel ABA Insensitive Mutant of Lotus japonicus with a Wilty Phenotype Displays Unaltered Nodulation Regulation

An ABA insensitive mutant, Beyma, was isolated in Lotus japonicus MG-20 from an EMS mutagenesis population using root growth inhibition to applied ABA as the screening criterion. （The name ＂Beyma＂ was taken from the Australian Aboriginal language, Wagiman, beyma, meaning ‘drying up＇.） The stable mutant that segregates as a dominant Mendelian mutation is insensitive to ABA induced inhibition of germination, vegetative growth, stomatal opening, as well as nodulation. Tissue ABA levels were normal, suggesting a sensitivity rather than biosynthesis mutation. It is slow-growing （50-70% of wild-type MG-20） and has a near-constitutive wilty phenotype associated with its inability to regulate stomatal opening. Whilst showing a wide range of ABA insensitive phenotypes, Beyma did not show alteration of nodule number control, as, in the absence of added ABA, the number and patterning （but not size） of nodules formed in the mutant were similar to that of MG-20. Split root experiments on MG-20 showed that application of ABA on one side of the root inhibited nodulation locally but not systemically. We propose that ABA is not involved directly in systemic autoregulation of nodulation （AON）.     

The integral membrane protein SEN1 is required for symbiotic nitrogen fixation in Lotus japonicus nodules

Legume plants establish a symbiotic association with bacteria called rhizobia, resulting in the formation of nitrogen-fixing root nodules. A Lotus japonicus symbiotic mutant, sen1, forms nodules that are infected by rhizobia but that do not fix nitrogen. Here, we report molecular identification of the causal gene, SEN1, by map-based cloning. The SEN1 gene encodes an integral membrane protein homologous to Glycine max nodulin-21, and also to CCC1, a vacuolar iron/manganese transporter of Saccharomyces cerevisiae, and VIT1, a vacuolar iron transporter of Arabidopsis thaliana. Expression of the SEN1 gene was detected exclusively in nodule-infected cells and increased during nodule development. Nif gene expression as well as the presence of nitrogenase proteins was detected in rhizobia from sen1 nodules, although the levels of expression were low compared with those from wild-type nodules. Microscopic observations revealed that symbiosome and/or bacteroid differentiation are impaired in the sen1 nodules even at a very early stage of nodule development. Phylogenetic analysis indicated that SEN1 belongs to a protein clade specific to legumes. These results indicate that SEN1 is essential for nitrogen fixation activity and symbiosome/bacteroid differentiation in legume nodules.     

Nodulation and nitrogen fixation in extreme environments

Biological nitrogen fixation is a phenomenon occurring in all known ecosystems. Symbiotic nitrogen fixation is dependent on the host plant genotype, theRhizobium strain, and the interaction of these symbionts with the pedoclimatic factors and the environmental conditions. Extremes of pH affect nodulation by reducing the colonization of soil and the legume rhizosphere by rhizobia. Highly acidic soils (pH<4.0) frequently have low levels of phosphorus, calcium, and molybdenum and high concentrations of aluminium and manganese which are often toxic for both partners; nodulation is more affected than host-plant growth and nitrogen fixation. Highly alkaline soils (pH>8.0) tend to be high in sodium chloride, bicarbonate, and borate, and are often associated with high salinity which reduce nitrogen fixation. Nodulation and N-fixation are observed under a wide range of temperatures with optima between 20–30°C. Elevated temperatures may delay nodule initiation and development, and interfere with nodule structure and functioning in temperate Iegumes, whereas in tropical legumes nitrogen fixation efficiency is mainly affected. Furthermore, temperature changes affect the competitive ability ofRhizobium strains. Low temperatures reduce nodule formation and nitrogen fixation in temperate legumes; however, in the extreme environment of the high arctic, native legumes can nodulate and fix nitrogen at rates comparable to those observed with legumes in temperate climates, indicating that both the plants and their rhizobia have successfully adapted to arctic conditions. In addition to low temperatures, arctic legumes are exposed to a short growing season, a long photoperiod, low precipitation and low soil nitrogen levels. In this review, we present results on a number of structural and physiological characteristics which allow arctic legumes to function in extreme environments.     

The sulfate transporter SST1 is crucial for symbiotic nitrogen fixation in Lotus japonicus root nodules

Symbiotic nitrogen fixation (SNF) by intracellular rhizobia within legume root nodules requires the exchange of nutrients between host plant cells and their resident bacteria. Little is known at the molecular level about plant transporters that mediate such exchanges. Several mutants of the model legume Lotus japonicus have been identified that develop nodules with metabolic defects that cannot fix nitrogen efficiently and exhibit retarded growth under symbiotic conditions. Map-based cloning of defective genes in two such mutants, sst1-1 and sst1-2 (for symbiotic sulfate transporter), revealed two alleles of the same gene. The gene is expressed in a nodule-specific manner and encodes a protein homologous with eukaryotic sulfate transporters. Full-length cDNA of the gene complemented a yeast mutant defective in sulfate transport. Hence, the gene was named Sst1. The sst1-1 and sst1-2 mutants exhibited normal growth and development under nonsymbiotic growth conditions, a result consistent with the nodule-specific expression of Sst1. Data from a previous proteomic study indicate that SST1 is located on the symbiosome membrane in Lotus nodules. Together, these results suggest that SST1 transports sulfate from the plant cell cytoplasm to the intracellular rhizobia, where the nutrient is essential for protein and cofactor synthesis, including nitrogenase biosynthesis. This work shows the importance of plant sulfate transport in SNF and the specialization of a eukaryotic transporter gene for this purpose.     

Symbiotic Nitrogen Fixation at the Late Stage of Nodule Formation in Lotus japonicus and Other Legume Plants

Lotus japonicus nodules are reviewed, and current research data on Nod-35 (uricase) gene, including that of Lotus japonicus as an example of a late nodulin gene are presented. Received 5 September 2000/ Accepted in revised form 2 October 2000     

Enhanced Nodulation and Nitrogen Fixation by a Revertant of a Nodulation-Defective Bradyrhizobium japonicum Tryptophan Auxotroph

In greenhouse studies, the symbiotic properties of a prototrophic revertant (TA11 NOD⁺) of a nodulation defective tryptophan auxotroph of Bradyrhizobium japonicum were compared with those of the normally nodulating wild-type strain, B. japonicum I-110 ARS. Strain I-110 ARS was the parent of auxotrophic mutant TA11. Plants inoculated with TA11 NOD⁺ contained significantly more nitrogen per plant than did plants inoculated with wild-type bacteria (275.9 ± 35 versus 184 ± 18 mg). Also, plants that received the revertant were larger, averaging 8.4 ± 0.9 g (dry weight) versus 6.4 ± 0.6 g for those that received the wild-type bacterial strain. Additionally, plants that received the NOD⁺ strain had 56% more nodules and 41% more nodule mass than did control plants. With both inocula, average nodule size and amount of nitrogen fixed per gram of nodule were about the same. These data indicated that the improvement in nitrogen fixation observed with the TA11 NOD⁺ resulted from an increase in the overall nodule number. The physiological basis for this increase in nodulation is not known, but enhanced tryptophan catabolism does not appear to be involved.     

Overexpression of class 1 plant hemoglobin genes enhances symbiotic nitrogen fixation activity between Mesorhizobium loti and Lotus japonicus

Plant hemoglobins (Hbs) have been divided into three groups: class 1, class 2, and truncated Hbs. The various physiological functions of class 1 Hb include its role as a modulator of nitric oxide (NO) levels in plants. To gain more insight into the functions of class 1 Hbs, we investigated the physical properties of LjHb1 and AfHb1, class 1 Hbs of a model legume Lotus japonicus and an actinorhizal plant Alnus firma , respectively. Spectrophotometric analysis showed that the recombinant form of the LjHb1 and AfHb1 proteins reacted with NO. The localization of LjHb1 expression was correlated with the site of NO production. Overexpression of LjHb1 and AfHb1 by transformed hairy roots caused changes in symbiosis with rhizobia. The number of nodules formed on hairy roots overexpressing LjHb1 or AfHb1 increased compared with that on untransformed hairy roots. Furthermore, nitrogenase activity as acetylene-reduction activity (ARA) of LjHb1- or AfHb1 -overexpressing nodules was higher than that of the vector control nodules. Microscopic observation with a NO-specific fluorescent dye suggested that the NO level in LjHb1 - and AfHb1 -overexpressing nodules was lower than that of control nodules. Exogenous application of a NO scavenger enhanced ARA in L. japonicus nodule s , whereas a NO donor inhibited ARA. These results suggest that the basal level of NO in nodules inhibits nitrogen fixation, and overexpression of class 1 Hbs enhances symbiotic nitrogen fixation activity by removing NO as an inhibitor of nitrogenase.     

Effect of abscisic acid on symbiotic nitrogen fixation activity in the root nodules of Lotus japonicus

The phytohormone abscisic acid (ABA) is known to be a negative regulator of legume root nodule formation. By screening Lotus japonicus seedlings for survival on an agar medium containing 70 µM ABA, we obtained mutants that not only showed increased root nodule number, but also enhanced nitrogen fixation. The mutant was designated enf1 (enhanced nitrogen fixation 1) and was confirmed to be monogenic and incompletely dominant.In long-term growth experiments with M. loti, although some yield parameters were the same for both enf1 and wild-type plants, both the dry weight and N content of 100 seeds and entire enf1 plants were significantly larger compared than those traits in wild-type seeds and plants. The augmentation of the weight and N content of the enf1 plants most likely reflects the increased N supplied by the additional enf1 nodules and the concomitant increase in N fixation activity.We determined that the endogenous ABA concentration and the sensitivity to ABA of enf1 were lower than that of wild-type seedlings. When wild-type plants were treated with abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase (NCED), which results in reduced ABA content, the N fixation activity of abamine-treated plants was elevated to the same levels as enf1. We also determined that production of nitric oxide (NO) in enf1 nodules was decreased. We conclude that endogenous ABA concentration not only regulates nodulation, but also nitrogen fixation activity by decreasing NO production in nodules.Key words: Lotus japonicus, symbiotic nitrogen fixation, nitric oxide, ABA, root nodulePhytohormones are known to be important for regulating the number of nodules established on the root of legumes.¹ For example, ethylene is a well-known negative regulator of nodulation, influencing the earliest stages from the perception of Nod factor to the growth of infection threads.^2–4 In contrast, cytokinin is a positive regulator of nodulation. The cytokinin insensitive mutant hit1 (loss-of-function) of Lotus japonicus and the snf2 (gain-of-function) mutants of Medicago truncatula provide genetic evidence demonstrating that cytokinin plays a critical role in the activation of nodule primordia.^5–7Abscisic acid (ABA), added at concentrations that do not affect plant growth, also negatively regulates nodulation in some legumes.^8–11 Recently, Medicago truncatula overexpressing abi1-1, a gene that encodes a mutated protein phosphatase of the type IIC class derived from Arabidopsis and that suppresses the ABA signaling pathway,^12,13 was shown to exhibit ABA insensitivity as well as a hypernodulating phenotype.¹⁴In this study, we isolated a Lotus japonicus (Miyakojima MG20) mutant that showed an increased root nodule phenotype and a lowered sensitivity to ABA, and proceeded to carry out its characterization. This mutant, named enf1 (enhanced nitrogen fixation 1) exhibit enhanced symbiotic N fixation activity. Most legume N fixation activity mutants, such as ign1, sen1 and sst1, are Fix-.^15–17At first, to obtain ABA-insensitive or low-sensitive mutants of Lotus japonicus, we treated Miyakojima MG20 with EMS to induce base substitutions randomly in the genome. M3 seeds were sown on an agar-solidified medium containing 70 µM ABA, a concentration that inhibits the germination of wild-type MG20 seeds. M4 plants obtained by the screening were inoculated with rhizobia (Mesorhizobium loti MAFF303099), and the number of nodules per plant was counted 35 days after inoculation (DAI). Plant No. 12 not only formed more root nodules than did the wild-type MG20 plants, but surprisingly it also exhibited increased nitrogen fixation activity per plant. Both mutant phenotypes were stably inherited in the M4 and M5 generation. Back-crossing mutant No. 12 to wild-type MG20 yielded 153 F2 progeny from which a line that showed the highest N fixation activity and more nodules per plant was derived. This line was designated enf1 (enhanced nitrogen fixation 1).At 28 DAI, the number of nodules formed on enf1 roots was approximately 1.7 times greater than that of MG20, and the N fixation activity per enf1 plant was elevated 1.8 times over that of the wildtype plants. Because the N fixation activity per unit of enf1 nodule weight was also increased 1.7 times, we concluded that the increased N fixation activity was not solely due to the enhanced number of root nodules.The endogenous ABA concentration and the sensitivity to ABA of enf1 were lower than those of wild-type seedlings. ABA is believed to regulate early nodulation stages negatively by inhibiting Nod factor signaling, bacterial infection, and nodule initiation.^14,18 Elongated ITs were more common in enf1 root hairs at later stages of development (8–12 DAI). Furthermore, ITs were detected in nodule primordia more frequently in enf1 compared to MG20. These results suggest that the earliest stages of nodule development are not as strongly inhibited in enf1 as they are in wild-type MG20.Because enf1 had a low endogenous ABA concentration, we hypothesized that the decrease in ABA concentration caused the elevation of N fixation activity. To test this hypothesis, we treated wild-type plants at 28 DAI with 20 µM abamine, a specific inhibitor of ABA synthesis.¹⁹ After a three day-treatment period, acetylene reduction activity was measured. Such short treatment periods of abamine are not expected to induce new nodule development. Wild-type plants treated with abamine had a reduced endogenous ABA concentration in roots, to about one-fourth of the level of control plants. However, N fixation activity was elevated to about 170% over the non-treated controls (Fig. 1A and B). This result phenocopies enf1, which shows decreased endogenous ABA concentration as well as elevated N fixation activity. These results strongly suggest that the decrease in endogenous ABA concentration in enf1 was responsible for the increased levels of N fixation activity. Applying 0.5 µM ABA did not result in a further increase in N fixation activity even though the endogenous ABA concentrations are presumed to increase (Fig. 1A and B).Open in a separate windowFigure 1Effects of ABAconcentration on nitrogen fixation activity. M. loti-inoculated plants were grown for 28 days on vermiculite-filled pots supplied with B & D medium. Plant roots 28 DAI were treated with 0.5 µM ABA, 20 µM abamine, with both ABA and abamine, or were untreated (B & D medium control), respectively, for 3 days. (A) ARA per nodule weight. (B) ABA concentration in root. At least 15 plants were used in acetylene reduction assay. Four different plants were used for the measurement of ABA concentration and 3 repeats were performed. Error bars indicate the standard error, and the significance of differences between untreated control and treated values was determined by the two-tailed multiple t-test with Bonferroni correction following ANOVA (three comparisons in four groups), *p < 0.05, **p < 0.01.Nitric oxide (NO) is known as a strong inhibitor of N fixation activity,²⁰ as well as a signal component in ABA signaling pathway.^21,22 NO production in root nodules formed by enf1 21 DAI and 28 DAI was examined by using the fluorescent dye diaminofluorescein-FM (DAF-FM), a NO specific detector, and relative fluorescence unit (RFU) values were estimated. The RFU values of enf1 nodules 21 DAI were clearly decreased compared with that of MG20; this trend was more obvious at 28 DAI. Moreover, the effect of reduced ABA concentration caused by treatment with abamine on NO production was analyzed (Fig. 2). When nodules formed on the roots of 28-d-old plants were treated, the RFU value of the enf1 mutant was almost the same for (−) abamine and (+) abamine-treated, whereas, the RFU value of abamine-treated MG20 plants was significantly reduced compared to untreated MG20 (Fig. 2). These results strongly suggest that decreased production of NO caused by the low concentration of ABA in enf1 nodules was responsible for the increase in N fixation activity.Open in a separate windowFigure 2NO production in nodules. Quantification of nitric oxide in nodules that were treated with abamine. Nodules on the root of 28-day-old plants were treated with 20 µM abamine for 3 days. Relative fluorescent units (RFU) per nodule fresh weight at 515 nm, normalized against MG20 plants, are shown. The data represent the average ± standard error of 3 independent experiments derived from nodules of 6 to 8 plants. The significance of differences among the four groups was determined by the two-tailed multiple t-test with Bonferroni correction following ANOVA (six comparisons in four groups) and the different letters refer to significant differences at p < 0.01.Until now, the majority of symbiotic mutants that have been described represents loss of or defects in root nodule formation.^6,23,24 Many of these mutants induce nodules that are Fix-.^15–17 Although reports of mutants that show increased root nodule number^25–28 or spontaneous root nodule formation exist,^7,29 reports concerning mutations where N fixation activity is elevated without deleterious effects on plant growth and development are limited. One exception is the L. japonicus rdh1 mutant, which also exhibits a hypernodulation and enhanced nitrogen fixation phenotype.³⁰In this report, we have shown that mutating the ENF1 gene leads to an elevation of N fixation activity without accompanying adverse growth effects. In long-term growth experiments, some yield parameters were the same for both enf1 and wild-type plants, but both the dry weight and N content of 100 seeds and entire enf1 plants were significantly larger compared to those parameters in wild-type seeds and plants. These results strongly suggest that more nitrogen is fixed in the enf1 mutant than in wild-type plants. Therefore, this gene should be an important target for molecular breeding. We have determined that ENF1 gene is inherited in a monogenic and incompletely dominant manner. Our future work will identify the gene responsible for these positive growth effects.     

Growth and nitrogen fixation in Lotus japonicus and Medicago truncatula under NaCl stress: nodule carbon metabolism

Lotus japonicus and Medicago truncatula model legumes, which form determined and indeterminate nodules, respectively, provide a convenient system to study plant-Rhizobium interaction and to establish differences between the two types of nodules under salt stress conditions. We examined the effects of 25 and 50mM NaCl doses on growth and nitrogen fixation parameters, as well as carbohydrate content and carbon metabolism of M. truncatula and L. japonicus nodules. The leghemoglobin (Lb) content and nitrogen fixation rate (NFR) were approximately 10.0 and 2.0 times higher, respectively, in nodules of L. japonicus when compared with M. truncatula. Plant growth parameters and nitrogenase activity decreased with NaCl treatments in both legumes. Sucrose was the predominant sugar quantified in nodules of both legumes, showing a decrease in concentration in response to salt stress. The content of trehalose was low (less than 2.5% of total soluble sugars (TSS)) to act as an osmolyte in nodules, despite its concentration being increased under saline conditions. Nodule enzyme activities of trehalose-6-phosphate synthase (TPS) and trehalase (TRE) decreased with salinity. L. japonicus nodule carbon metabolism proved to be less sensitive to salinity than in M. truncatula, as enzymatic activities responsible for the carbon supply to the bacteroids to fuel nitrogen fixation, such as sucrose synthase (SS), alkaline invertase (AI), malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC), were less affected by salt than the corresponding activities in barrel medics. However, nitrogenase activity was only inhibited by salinity in L. japonicus nodules.     

Nitrogen fixation is synchronized with carbon metabolism in Lotus japonicus and Medicago truncatula nodules under salt stress

Abstract

In the present work, the response to NaCl applied at the vegetative stage to Medicago truncatula and Lotus japonicus has been evaluated in order to ascertain whether the effect of salt stress on nitrogen fixation is due to a limitation on nodular carbon metabolism. Results show maximum sucrose synthase (SS) and alkaline invertase (AI) activities were obtained at the vegetative stage, when maximum nitrogenase activity was detected in both species. SS activity decreased with the salt treatment, providing evidence of the regulatory role of this enzyme for the carbon supply to the bacteroids. Phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) activities could account for higher nitrogen fixation efficiency detected in L. japonicus nodules and isocitrate dehydrogenase (ICDH) activity compensated for the carbon limitations that occur under salt stress. These results support that nitrogenase inhibition in nodules experiencing salt stress is doubt to a carbon flux shortage, as result of carbon metabolism enzymes activities down-regulation.     

Abscisic Acid Suppresses the Highly Occurred Somatic Homologous Recombination in Arabidopsis rfc1 Mutant

The phytohormone abscisic acid (ABA) regulates many aspects of plant growth, including seed germination, root growth and cell division. Previous study indicates that ABA treatment increases DNA damage and somatic homologous recombination (HR) in Arabidopsis abo4/pol ? (aba overly-sensitive 4 /DNA polymerase ?) mutants. DNA replication factor C (RFC) complex is required for loading PCNA (Proliferating Cell Nuclear Antigen) during DNA replication. The defect in RFC1, the largest subunit of RFC, causes the high HR and DNA damage sensitivity in Arabidopsis. Here we found that like pol ε/abo4, rfc1 is sensitive to ABA in both ABA-inhibiting seed germination and root growth. However, ABA treatment greatly reduces HR and also reduces the expression of the DNA-damaged marker genes in rfc1. These results suggest that RFC1 plays critical roles in ABA-mediated HR in Arabidopsis.     

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。     

Nodulation and nitrogen fixation mutants of pea,Pisum sativum

Summary The 36 mutants which did not nodulate and 24 mutants which formed inefficient nodules with no or very low acetylene reduction activity were isolated among 86,000 M₂-seedlings of Finale pea, Pisum sativum L., after treatment with chemical mutagens. One mutant was found for approximately every 50 chlorophyll mutants. Most mutations were induced by ethyl methanesulfonate; some by diethyl sulfate, ethyl nitrosourea and acidified sodium azide. Putative mutants were selected as nitrogen deficient plants, yellowing from the bottom and up, when M₂ seedlings were grown in sand with a Rhizobium mixture and PK fertilizer. The mutants were verified in the M₃ generation by acetylene reduction assay on intact plants.     

类黄酮激活根瘤菌在油菜上结瘤和固氮的研究初报

１引言自然界大多数植物不具备共生固氮系统．在非豆科中除Ｐａｒａｓｐｏｎｉａ［８］外,至今还未发现其它非豆科植物能像豆科植物一样与根瘤菌形成共生．聂延富报道了２．４Ｄ能使小麦根系诱导结瘤的作用［２］．ＡｌＭａｌｅｎ等研究了根瘤菌在水稻和油菜幼苗根系...     

3-Phosphoglycerate dehydrogenase in Mesorhizobioum loti is essential for maintaining symbiotic nitrogen fixation of Lotus japonicus root nodules

Mesorhizobium loti is a Gram negative bacterium that induces N₂-fixing root nodules on the model legume Lotus japonicus. Proteomic analysis in M. loti indicated that 3-phosphoglycerate dehydrogenase (EC. 1.1.1.95, PHGDH) protein content was 2.2 times higher in bacteroids than in cultured bacteria. A M. loti mutant (STM5) with a transposon insertion in the PHGDH gene, mll3875, showed an absolute dependence on serine or glycine in minimal medium for growth. When L. japonicus plants were infected with STM5, the roots formed nodules in numbers comparable to those formed by wild type M. loti; however, the nodules showed very low acetylene reduction activity, and significant starch granule accumulation was observed in the uninfected cells. In such nodules, vast necrosis occurred in the central tissue of the nodules, although bacteroids were detected in the infected cell of the nodules. These data indicate that serine or glycine biosynthesis by PHGDH is important for maintaining symbiosis and nitrogen fixation in L. japonicus nodules.