Expression of Frankia nif genes in nodules of Alnus glutinosa

Expression of Frankia genes involved in nitrogen fixation was studied in Alnus glutinosa nodules using the in situ hybridization technique. The results show that high level expression of nif genes does not occur immediately upon infection of cortical cells by Frankia. Also, only in the infected cells near the tips of the nodule lobes, nif genes are expressed at high levels. In the majority of infected cells, nif gene expression is rather low.     

A comparison of symbiotic nitrogen fixation in Scotland inAlnus glutinosa andAlnus rubra

Summary Alnus glutinosa andAlnus rubra growing in the field in Scotland show specific nitrogenase activities of the same order of magnitude. The period of maximum potential nitrogenase activity coincides with that of maximum growth in late Spring and Summer. It is suggested that the retention of nitrogenase activity into the Autumn when growth has virtually ceased may be important as a contribution to the nitrogenous reserves of the tree.Bioassay of different Scottish soils, all collected from the locality of natural stands ofAlnus glutinosa, showed wide variation in the nodulation of seedlings, although generally a soil poor for nodulation ofAlnus glutinosa generally gave poor nodulation ofAlnus rubra. Soils of pH 4.5 to 6.5, best suited for growth and nitrogen fixation of the two species, often gave nodules showing highest specific nitrogen fixing activity. Young (2 to 3 year old) plants in glasshouse or controlled environment cabinet, inoculated withAlnus glutinosa endophyte, differed from mature field grown plants, however, sinceAlnus rubra required a much larger (up to 2.5 times) mass of root nodules to fix a unit quantity of N. Microscopic comparison of the nodules of glasshouse plants showed that the proportion of cells containing the vesicular (nitrogen fixing) form of the endophyte was only slightly lower inAlnus rubra than inAlnus glutinosa and it is suggested that the differences in specific nitrogen fixing activity between the two species may reflect some incompatibility of function of theAlnus glutinosa endophyte when in symbiosis withAlnus rubra.     

Variable compatibility of clonedAlnus glutinosa ecotypes against ineffectiveFrankia strains

Nodulation tests onin-vitro propagated clones ofAlnus glutinosa ecotypes (forest ecotype, pioneer ecotype) withFrankia strains originating from both ecotypes indicated differences in host-plant compatibility. Inoculated plants of the pioneer ecotype clone were not infected by strains, that were unable to fix nitrogen in pure culture. Nodulation could only be induced on the clone of the forest ecotype, but no nitrogen-fixing activity could be detected. Ultra-structural observations of the nodules by SEM and TEM indicated that ineffectivity of these strains was correlated with the lack of vesicles in the infected cells. Cells were only filled with hyphae: neither sporangia nor vesicles could be detected. In contrast, effective nodules could be obtained on both alder clones after inoculation with an effective strain, showing normal development of vesicle clusters in infected cells. In pure culture the ineffective strains produced no vesicles; sporangia were found only during early stage of growth. The results demonstrate the existence ofFrankia strains which were either non-infective or ineffective on different clones ofAlnus glutinosa.     

A block in the endocytosis of Rhizobium allows cellular differentiation in nodules but affects the expression of some peribacteroid membrane nodulins

A transposon-induced mutant (T8-1) of Bradyrhizobium japonicum (61A76) was unable to develop into the nitrogen-fixing endosymbiotic form, the bacteroid. Comparison between this mutant and T5-95, an ineffective (non-nitrogen fixing, Fix^-) mutant, confirmed that the process of bacteroid development is a distinct phase of differentiation of the endosymbiont and is independent of nitrogen fixation activity. The T8-1 mutant was able to induce normal-size nodules which differentiated two plant cell types and contained numerous infection threads. However, the infected cells were devoid of bacteroids. Electron microscopy revealed that the ends of the infection threads were broken down in a normal manner once the thread had penetrated the cells, but the mutant was not internalized by endocytosis. The lack of peribacteroid membrane (PBM) in nodules induced by this mutant was correlated with a reduced level of expression of plant genes coding for PBM nodulins. These genes were expressed in the T5-95 mutant, showing that the low expression in T8-1 was not due to the lack of nitrogen fixation. One of the PBM nodulins, nodulin-26, was found at normal levels in the nodules which lack PBM, suggesting that there are at least two developmental stages in PBM biosynthesis. These data suggest that a coordination of plant and Rhizobium gene expression is required for the release and internalization of bacteria into the PBM compartments of infected cells of nodules.author for correspondence     

Appearance of a novel form of plant glutamine synthetase during nodule development in Phaseolus vulgaris L.

The activities of glutamine synthetase (GS), nitrogenase and leghaemoglobin were measured during nodule development in Phaseolus vulgaris infected with wild-type or two non-fixing (Fix^-) mutants of Rhizobium phaseoli. The large increase in GS activity which was observed during nodulation with the wild-type rhizobial strain occurred concomitantly with the detection and increase in activity of nitrogenase and the amount of leghaemoglobin. Moreover, this increase in GS was found to be due entirely to the appearance of a novel form of the enzyme (GS_n1) in the nodule. The activity of the form (GS_n2) similar to the root enzyme (GS_r) remained constant throughout the experiment. In nodules produced by infection with the two mutant strains of Rhizobium phaseoli (JL15 and JL19) only trace amounts of GS_n1 and leghaemoglobin were detected.Abbreviations DEAE-Sephacel diethylaminoethyl-Sephacel - GS glutamine synthetase     

Study of the contribution of the shoot and/or root ofAlnus sp. in the compatibility between the host and a Sp+ Frankia strain using a grafting technique

Two alder species,Alnus glutinosa (L.) Gaertn. andAlnus incana (L) Moench, were inoculated with a Sp⁺ Frankia homogenate obtained fromA. incana root nodules. This inoculum formed effective nodules on the original host plant and ineffective nodules onA. glutinosa. Grafts between the two alder species were made to determine which part of the plant is involved in this phenomenon. The results obtained indicate that the compatibility between Alnus andFrankia is restricted to the root system.     

Infectivity and effectivity of Frankia strains from the Rhamnaceae family on different actinorhizal plants

Ten strains of Frankia isolated from root nodules of plant species from five genera of the host family Rhamnaceae were assayed in cross inoculation assays. They were tested on host plants belonging to four actinorhizal families: Trevoa trinervis (Rhamnaceae), Elaeagnus angustifolia (Elaeagnaceae), Alnus glutinosa (Betulaceae) and Casuarina cunninghamiana (Casuarinaceae). All Frankia strains from the Rhamnaceae were able to infect and nodulate both T. trinervis and E. angustifolia. Strain ChI4 isolated from Colletia hystrix was also infective on Alnus glutinosa. All nodules showed a positive acetylene reduction indicating that the microsymbionts used as inoculants were effective in nitrogen fixation. The results suggest that Frankia strains from Rhamnaceae belong to the Elaeagnus-infective subdivision of the genus Frankia.     

Oxidation of the enzymes involved in nitrogen assimilation plays an important role in the cadmium-induced toxicity in soybean plants

Cadmium causes oxidative damage and hence affects nitrogen assimilation. In the present work we tested the relationship between the inactivation of the enzymes involved in nitrogen assimilation pathway (glutamine synthetase (GS)/glutamate synthase (GOGAT)) and the protein oxidation in nodules of soybean (Glycine max L.) plants under Cd²⁺ stress. Therefore, the effect of Cd²⁺ and reduced gluthatione (GSH) on GS and GOGAT activities, and protein abundance and oxidation were analyzed. Under the metal treatment, amino acids oxidative modification occurred, evidenced by the accumulation of carbonylated proteins, especially those of high molecular weight. When Cd²⁺ was present in the nutrient solution, although a decrease in GS and GOGAT activities was observed (17 and 52%, respectively, compared to controls), the protein abundance of both enzymes remained similar to control nodules. When GSH was added together with Cd²⁺ in the nutrient medium, it protected the nodule against Cd²⁺ induced oxidative damage, maintaining GS and GOGAT activities close to control values. These results allow us to conclude that the inactivation of the nitrogen assimilation pathway by Cd²⁺ in soybean nodules is due to an increment in GS and GOGAT oxidation that can be prevented by the soluble antioxidant GSH. Section Editor: H. Schat     

Nitrogen metabolism in the facultative methylotroph Arthrobacter P1 grown with various amines or ammonia as nitrogen sources

The metabolism of trimethylamine (TMA) and dimethylamine (DMA) in Arthrobacter P1 involved the enzymes TMA monooxygenase and trimethylamine-N-oxide (TMA-NO) demethylase, and DMA monooxygenase, respectively. The methylamine and formaldehyde produced were further metabolized via a primary amine oxidase and the ribulose monophosphate (RuMP) cycle. The amine oxidase showed activity with various aliphatic primary amines and benzylamine. The organism was able to use methylamine, ethylamine and propylamine as carbon-and nitrogen sources for growth. Butylamine and benzylamine only functioned as nitrogen sources. Growth on glucose with ethylamine, propylamine, butylamine and benzylamine resulted in accumulation of the respective aldehydes. In case of ethylamine and propylamine this was due to repression by glucose of the synthesis of the aldehyde dehydrogenase(s) required for their further metabolism. Growth on glucose/methylamine did not result in repression of the RuMP cycle enzyme hexulose-6-phosphate synthase (HPS). High levels of this enzyme were present in the cells and as a result formaldehyde did not accumulate. Ammonia assimilation in Arthrobacter P1 involved NADP-dependent glutamate dehydrogenase (GDH), NAD-dependent alanine dehydrogenase (ADH) and glutamine synthetase (GS) as key enzymes. In batch cultures both GDH and GS displayed highest levels during growth on acetate with methylamine as the nitrogen source. A further increase in the levels of GS, but not GDH, was observed under ammonia-limited growth conditions in continuous cultures with acetate or glucose as carbon sources.Abbreviations HPS hexulose-6-phosphate synthase - RuMP ribulose monophosphate - DMA dimethylamine - TMA trimethylamine - TMA-NO trimethylamine-N-oxide - ICL isocitrate lyase - GS glutamine synthetase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase - GOGAT glutamate synthase     

Distribution of spore-positive and spore-negative nodules in stands ofAlnus glutinosa andAlnus incana in Finland

Summary The distribution of spore positive (Sp⁺) and spore negative (Sp⁻) nodules on the two native alder species (A. incana andA. glutinosa) in Finland was investigated. Nodules were collected throughout the country from different ecosystems (forests, swamps, lake- sea- and riversides, old pastures and fields as well as from alder plantations). OnA. incana Sp⁺ nodules predominated, whereas onA. glutinosa the vast majority of the nodules were of the Sp⁻ type. Sp⁺ nodules onA. glutinosa were found only at sites where the two alder species grew close together. This distribution pattern indicates an association of nodule type with alder species, the reasons for which are discussed. Indications of saprophytic growth in the Sp⁻ strain were also found.     

Immunocytochemical study of phosphoenolpyruvate carboxylase in nodulated Alnus glutinosa

The activities of phosphoenolpyruvate carboxylase (PEP carboxylase, EC 4.1.1.3.1) have been investigated in various organs of young nodulated Alnus glutinosa. The root nodules exhibited the highest specific enzyme activity when compared with the one in roots and leaves. Furthermore, in the root nodules the PEP carboxylase was predominantly localized in the cytosol of the large cortical cells containing the endophyte vesicles.Abbreviations PEP carboxylase phosphoenolpyruvate carboxylase - MDH malate dehydrogenase - PVP polyvinylpyrrolidone - PBS phosphate buffer saline     

Forcing expression of a soybean root glutamine synthetase gene in tobacco leaves induces a native gene encoding cytosolic enzyme

Glutamine synthetase (GS; EC 6.3.1.2) is present in different subcellular compartments in plants. It is located in the cytoplasm in root and root nodules while generally present in the chloroplasts in leaves. The expression of GS gene(s) is enhanced in root nodules and in soybean roots treated with ammonia. We have isolated four genes encoding subunits of cytosolic GS from soybean (Glycine max L. cv. Prize). Promoter analysis of one of these genes (GS15) showed that it is expressed in a root-specific manner in transgenic tobacco and Lotus corniculatus, but is induced by ammonia only in the legume background. Making the GS15 gene expression constitutive by fusion with the CaMV-35S promoter led to the expression of GS in the leaves of transgenic tobacco plants. The soybean GS was functional and was located in the cytoplasm in tobacco leaves where this enzyme is not normally present. Forcing this change in the location of GS caused concomitant induction of the mRNA for a native cytosolic GS in the leaves of transgenic tobacco. Shifting the subcellular location of GS in transgenic plants apparently altered the nitrogen metabolism and forced the induction in leaves of a native GS gene encoding a cytosolic enzyme. The latter is normally expressed only in the root tissue of tobacco. This phenomenon may suggest a hitherto uncharacterized metabolic control on the expression of certain genes in plants.     

地黄C3H基因的克隆及生物信息学分析

香豆酸-3-羟化酶属于植物中最大的蛋白酶细胞色素P450家族之一,在植物生命活动中发挥着重要作用。为了解地黄香豆酸-3-羟化酶基因RgC3H合成毛蕊花糖苷的功能,该研究基于地黄代谢组学分析获得KEGG途径中的C3H,采用多重比对在NCBI中获得同源基因的一个保守序列,并基于该保守序列和地黄SRA数据库,采用电子克隆和RT-PCR克隆技术获得地黄C3H基因全长CDS(RgC3H),对其进行生物信息学分析。结果表明:RgC3H基因全长为1 530 bp,且编码一个含509个氨基酸、分子量为57.91 kD、无信号肽的蛋白质; 基于氨基酸序列的结构分析显示,RgC3H有一个保守区域-P450结构域; 系统进化分析结果显示,RgC3H与芝麻和猴面花的C3H基因具有很高的同源性。上述结果为进一步研究RgC3H基因在地黄毛蕊花糖苷生物合成途径中的作用奠定了基础。     

Isolation of infective and effective Frankia strains from root nodules of Alnus acuminata (Betulaceae)

Two Frankia strains were isolated from root nodules of Alnus acuminata collected in the Tucumano-oranense forest, Argentina. Monosporal cultures were obtained by plating a spore suspension of each strain and isolating a single colony. The strains (named AacI and AacIII) showed branched mycelia with polymorphic sporangia and NIR-vesicles. They differed in their ability to use carbon sources: the AacI strain grew well on pyruvate, while the AacIII strain grew on mineral medium supplemented with glucose or, alternatively, with sucrose. The two strains were sensitive to oleandomycin, erythromycin, kanamycin, penicillin G, streptomycin and chloramphenicol at 5 μg/ml. The AcIII strain exhibited a moderate resistance to rifampicin, ampicillin and vancomycin. The nitrogenase activity in vitro of the strains was significantly higher in basal medium without nitrogen than that determined in the presence of ammonium chloride. Both strains were infective on seedlings of Alnus glutinosa, inducing an approximately similar percentage of nodulated plants (80%), although strain AacIII produced a higher number of nodules per plant (≤15) than strain AacI (≤6). They were also effective for nitrogen fixation in planta, determined by the acetylene reduction assay. This revised version was published online in July 2006 with corrections to the Cover Date.     

Soil properties and actinorhizal vegetation influence nodulation of Alnus glutinosa and Elaeagnus angustifolia by Frankia

Nodulation (mean number of nodules per seedling) was 5 times greater for Elaeagnus angustifolia than for Alnus glutinosa overall when seedlings were grown in pots containing either an upland or an alluvial soil from central Illinois, USA. However, the upland Alfisol had 1.3 times greater nodulation capacity for A. glutinosa than for E. angustifolia. The presence of A. glutinosa trees on either soil was associated with a two-fold increase in nodulation capacity for E. angustifolia. Nodulation increases for soils under A. glutinosa were obtained for A. glutinosa seedlings in the Alfisol, but decreased nodulation for A. glutinosa seedlings occurred in the Mollisol. Greatest nodulation of E. angustifolia seedlings occurred near pH 6.6 for soil pH values ranging from 4.9 to 7.1, while greatest nodulation of A. glutinosa occurred at pH 4.9 over the same pH range. Nodulation was not affected by total soil nitrogen concentrations ranging from 0.09 to 0.20%. Mollisol pH was significantly lower under A. glutinosa trees than under E. angustifolia trees. For 4- to 8-year-old field-grown trees, A. glutinosa nodule weights were negatively correlated with soil pH, while for similar aged E. angustifolia trees nodulation in the acidic Alfisol was not detected.     

A novel Δ-pyrroline-5-carboxylate synthetase gene of Medicago truncatula plays a predominant role in stress-induced proline accumulation during symbiotic nitrogen fixation

Proline accumulates in environmentally stressed plant cells including those of legume roots and nodules, but how its level is regulated is poorly understood. Δ¹-Pyrroline-5-carboxylate synthetase (P5CS), the committed-step enzyme of proline biosynthesis, is encoded by two duplicated genes in many plants. Here, we isolated MtP5CS3, a third gene, from Medicago truncatula, whose predicted polypeptide sequence is highly similar to those of previously isolated MtP5CS1 and MtP5CS2 except an extra amino-terminal segment. MtP5CS3 was strongly expressed under salinity and drought in shoots and nodulating roots, while MtP5CS1 was constitutive and MtP5CS2 induced by abscisic acid. Under salinity, MtP5CS3 promoter was more active than those of MtP5CS1 and MtP5CS2, as shown by GUS fusions. Translationally fused MtP5CS1-GFP was localized in the cytoplasm, whereas significant proportions of MtP5CS2-GFP and MtP5CS3-GFP were co-localized with rubisco small subunit protein-fused RFP in transformed hairy root cells. Under salinity, RNA silencing of MtP5CS1 or MtP5CS2 strongly induced MtP5CS3 expression, while that of MtP5CS3 decreased free proline content and nodule number. Consistently, Mtp5cs3, a loss-of-function mutant, accumulated much less proline, formed fewer nodules, and fixed nitrogen significantly less efficiently than the wild type under salinity. Thus, MtP5CS3 plays a critical role in regulating stress-induced proline accumulation during symbiotic nitrogen fixation.     

Spatial relationships between uninfected and infected cells in root nodules of soybean

In soybean (Glycine max (L.) Merr.) the uninfected cells of the root nodule are responsible for the final steps in ureide production from recently fixed nitrogen. Stereological methods and an original quantitative method were used to investigate the organization of these cells and their spatial relationships to infected cells in the central region of nodules of soybean inoculated with Rhizobium japonicum strain USDA 3I1B110 and grown with and without nitrogen (as nitrate) in the nutrient medium. The volume occupied by the uninfected tissue was 21% of the total volume of the central infected region for nodules of plants grown without nitrate, and 31% for nodules of plants grown with nitrate. Despite their low relative volume, the uninfected cells outnumbered the much larger infected cells in nodules of plants grown both without and with nitrate. The surface density of the interface between the ininfected and infected tissue in the infected region was similar for nodules in both cases also, the total range being from 24 to 26 mm²/mm³. In nodules of plants grown without nitrate, all sampled infected cells were found to be in contact with at least one uninfected cell. The study demonstrates that although the uninfected tissue in soybean nodules occupies a relatively small volume, it is organized so as to produce a large surface area for interaction with the infected tissue.     

氮素水平对茶树叶片氮代谢关键酶活性及非结构性碳水化合物的影响

以福鼎大白茶(FD)、保靖黄金茶1号(HJ1)、白毫早(BHZ)为材料,设置不施氮N_0(0 g)、低氮N1(11 g)、中氮N_2(22 g)和高氮N_3(33 g)4个氮素水平的盆栽实验,研究了铵态氮对3个品种茶树的根系活力、氮代谢关键酶及非结构性碳水化合物(NSC)的影响。结果表明:随着施氮水平的提高,N_2、N_3处理的茶树根系活力较对照N0显著增加(P0.05),但二者间无显著差异;叶片谷氨酰胺合成酶(GS)、谷氨酸合成酶(GOGAT)活性总体呈上升趋势;与对照比较,茶树叶片全氮和可溶性蛋白含量增加,其中HJ1在N_2和N_3处理后显著增加(P0.05);在3个茶树品种中,非结构性碳水化合物中可溶性总糖含量均呈上升趋势,淀粉含量具有品种特异性,施氮处理后3个茶树品种氮代谢关键酶活性及NSC含量变化存在差异,以HJ1的氮同化关键酶GS、GOGAT酶活性较高、根系活力较强,氮代谢产物显著增加,表明其具有较高的氮同化速率。施氮后HJ1的总NSC的含量及碳氮比的变化幅度较另外2个品种小,能够更好的保持碳氮平衡,游离氨基酸含量增幅较高,品质更优。因此,通过茶树氮代谢关键酶活性及非结构性化合物的研究能为茶树品种的品质评价以及提高茶树的品质和氮素利用效率提供依据。