Genotypic variation of N2-fixing common bean (Phaseolus vulgaris L.) in response to iron deficiency

In calcareous soils, the yield of grain legumes is often limited by the lower availability of iron (Fe), especially when they depend upon symbiosis with root nodule bacteria for their N nutrition. In order to explore the variability of responses of N(2)-fixing common bean to Fe deficiency the common bean white-seeded lines Striker and Coco blanc, and coloured-seeded lines SVM-29-21 and ARA14 were inoculated with Rhizobium tropici (CIAT 899) and cultivated hydroaeroponically with a N-free nutrient solution supplied or not with 45microM Fe. Differences among lines were observed: Fe-deficiency-induced-chlorosis on young leaves was earlier and more severe in some lines than others. Nodule development and N(2)-fixing capacity was less affected in line ARA14 which preferentially allocated Fe towards nodules. Results suggest that Fe use efficiency for symbiotic nitrogen fixation (FeUE SNF) could be used to screen tolerant bean lines to Fe deficiency in condition of symbiotic nitrogen fixation.     

攀枝花苏铁(Cycas panzhihuaensis)的根瘤和固氮作用

 分布在南亚热带金沙江干热河谷的攀枝花苏铁,普遍受蓝细菌侵染形成特殊的多级分枝珊瑚状根瘤簇。当年生树苗活瘤重可达8克／株,100年生370克／株。固氮活性在秋季一般为1.8—11.1μmol C2H4／g·f·w·h-1,它明显受光照和湿度影响,昼夜动态是白天活性明显比夜间高。苏铁固氮量从0.64—18.69毫克／株·小时,它在生态系统的氮循环中起良好作用。     

Effect of inoculation and leaf litter amendment on establishment of nodule-forming Frankia populations in soil

High-N(2)-fixing activities of Frankia populations in root nodules on Alnus glutinosa improve growth performance of the host plant. Therefore, the establishment of active, nodule-forming populations of Frankia in soil is desirable. In this study, we inoculated Frankia strains of Alnus host infection groups I, IIIa, and IV into soil already harboring indigenous populations of infection groups (IIIa, IIIb, and IV). Then we amended parts of the inoculated soil with leaf litter of A. glutinosa and kept these parts of soil without host plants for several weeks until they were spiked with [(15)N]NO(3) and planted with seedlings of A. glutinosa. After 4 months of growth, we analyzed plants for growth performance, nodule formation, specific Frankia populations in root nodules, and N(2) fixation rates. The results revealed that introduced Frankia strains incubated in soil for several weeks in the absence of plants remained infective and competitive for nodulation with the indigenous Frankia populations of the soil. Inoculation into and incubation in soil without host plants generally supported subsequent plant growth performance and increased the percentage of nitrogen acquired by the host plants through N(2) fixation from 33% on noninoculated, nonamended soils to 78% on inoculated, amended soils. Introduced Frankia strains representing Alnus host infection groups IIIa and IV competed with indigenous Frankia populations, whereas frankiae of group I were not found in any nodules. When grown in noninoculated, nonamended soil, A. glutinosa plants harbored Frankia populations of only group IIIa in root nodules. This group was reduced to 32% +/- 23% (standard deviation) of the Frankia nodule populations when plants were grown in inoculated, nonamended soil. Under these conditions, the introduced Frankia strain of group IV was established in 51% +/- 20% of the nodules. Leaf litter amendment during the initial incubation in soil without plants promoted nodulation by frankiae of group IV in both inoculated and noninoculated treatments. Grown in inoculated, amended soils, plants had significantly lower numbers of nodules infected by group IIIa (8% +/- 6%) than by group IV (81% +/- 11%). On plants grown in noninoculated, amended soil, the original Frankia root nodule population represented by group IIIa of the noninoculated, nonamended soil was entirely exchanged by a Frankia population belonging to group IV. The quantification of N(2) fixation rates by (15)N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N(2)-fixing capacity than populations belonging to group IIIa under the conditions applied. These results show that through inoculation or leaf litter amendment, Frankia populations with high specific N(2)-fixing capacities can be established in soils. These populations remain infective on their host plants, successfully compete for nodule formation with other indigenous or inoculated Frankia populations, and thereby increase plant growth performance.     

大叶相思的结瘤固氮和吸氢酶活性

本文报道大叶相思的结瘤固氮和氢酶活性的研究结果。大叶相思结瘤状况与其他含羞草亚科的树种相似,刚形成的幼瘤为单生球状或椭圆形,以后顶端伸长或分叉,呈分叉状、姜状和扇状。同一植株的不同成熟度根瘤的固氮活性也不同,成熟壮瘤固氮活性最高,幼瘤次之,衰老瘤最低。不同立地条件下种植的大叶相思根瘤固氮活性虽有差异,但生长在pH4.7的酸性红壤中的大叶相思,根瘤固氮活性仍具有较高水平。大叶相思根瘤固氮活性也有明显的季节变化,夏秋较高,春冬较低。根瘤离体后7小时内固氮活性变化不大,甚至在离体后21小时内仍维持一定水平的固氮活性。大叶相思根瘤具有吸氢酶,其吸H_2活性在最初3小时内随时间延长而升高,3—7小时内仍维持一定水平。在根瘤固氮系统中注入外源分子H_2,可提高固氮酶活性,外源H_2最适浓度为7.5％。由于其根瘤具有催化吸收分子H_2的氢酶系统,能吸收利用固氮反应所放出的大量H_2,因而能更有效地利用光合产物于固氮过程。大叶相思根瘤离体后能较长时间维持高的固氮活性水平,可能与其吸H_2酶系统有关。     

NADP+ -dependent malic enzyme of Rhizobium meliloti.

The bacterium Rhizobium meliloti, which forms N2-fixing root nodules on alfalfa, has two distinct malic enzymes; one is NADP+ dependent, while a second has maximal activity when NAD+ is the coenzyme. The diphosphopyridine nucleotide (NAD+)-dependent malic enzyme (DME) is required for symbiotic N2 fixation, likely as part of a pathway for the conversion of C4-dicarboxylic acids to acetyl coenzyme A in N2-fixing bacteroids. Here, we report the cloning and localization of the tme gene (encoding the triphosphopyridine nucleotide [NADP+]-dependent malic enzyme) to a 3.7-kb region. We constructed strains carrying insertions within the tme gene region and showed that the NADP+ -dependent malic enzyme activity peak was absent when extracts from these strains were eluted from a DEAE-cellulose chromatography column. We found that NADP+ -dependent malic enzyme activity was not required for N2 fixation, as tme mutants induced N2-fixing root nodules on alfalfa. Moreover, the apparent NADP+ -dependent malic enzyme activity detected in wild-type (N2-fixing) bacteroids was only 20% of the level detected in free-living cells. Much of that residual bacteroid activity appeared to be due to utilization of NADP+ by DME. The functions of DME and the NADP+ -dependent malic enzyme are discussed in light of the above results and the growth phenotypes of various tme and dme mutants.     

Facultative nitrogen fixation by canopy legumes in a lowland tropical forest

Symbiotic dinitrogen (N₂) fixation is often invoked to explain the N richness of tropical forests as ostensibly N₂-fixing trees can be a major component of the community. Such arguments assume N₂ fixers are fixing N when present. However, in laboratory experiments, legumes consistently reduce N₂ fixation in response to increased soil N availability. These contrasting views of N₂ fixation as either obligate or facultative have drastically different implications for the N cycle of tropical forests. We tested these models by directly measuring N₂-fixing root nodules and nitrogenase activity of individual canopy-dominant legume trees (Inga sp.) across several lowland forest types. Fixation was substantial in disturbed forests and some gaps but near zero in the high N soils of mature forest. Our findings suggest that canopy legumes closely regulate N₂ fixation, leading to large variations in N inputs across the landscape, and low symbiotic fixation in mature forests despite abundant legumes.     

Alder and lupine enhance nitrogen cycling in a degraded forest soil in Northern Sweden

Positive effects of legumes and actinorhizal plants on N-poor soils have been observed in many studies but few have been done at high latitudes, which was the location of our study. We measured N₂ fixation and several indices of soil N at a site near the Arctic Circle in northern Sweden. More than 20 years ago lupine (Lupinus nootkatensis Donn) and gray alder (Alnus incana L. Moench) were planted on this degraded forest site. We measured total soil N, net N mineralization and nitrification with a buried bag technique, and fluxes of NH⁺ ₄ and NO^– ₃ as collected on ion exchange membranes. We also estimated N₂ fixation activity of the N₂-fixing plants by the natural abundance of ¹⁵N of leaves with Betula pendula Roth. as reference species. Foliar nitrogen in the N₂-fixing plants was almost totally derived from N₂ fixation. Plots containing N₂-fixing species generally had significantly higher soil N and N availability than a control plot without N₂-fixing plants. Taken together, all measurements indicated that N₂-fixing plants can be used to effectively improve soil fertility at high latitudes in northern Sweden.     

Rhizobial factors required for stem nodule maturation and maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 symbiosis

The molecular and physiological mechanisms behind the maturation and maintenance of N(2)-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N(2)-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N(2) fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C(4) metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.     

The soybean NRAMP homologue,GmDMT1, is a symbiotic divalent metal transporter capable of ferrous iron transport

Iron is an important nutrient in N2-fixing legume root nodules. Iron supplied to the nodule is used by the plant for the synthesis of leghemoglobin, while in the bacteroid fraction, it is used as an essential cofactor for the bacterial N2-fixing enzyme, nitrogenase, and iron-containing proteins of the electron transport chain. The supply of iron to the bacteroids requires initial transport across the plant-derived peribacteroid membrane, which physically separates bacteroids from the infected plant cell cytosol. In this study, we have identified Glycine max divalent metal transporter 1 (GmDmt1), a soybean homologue of the NRAMP/Dmt1 family of divalent metal ion transporters. GmDmt1 shows enhanced expression in soybean root nodules and is most highly expressed at the onset of nitrogen fixation in developing nodules. Antibodies raised against a partial fragment of GmDmt1 confirmed its presence on the peribacteroid membrane (PBM) of soybean root nodules. GmDmt1 was able to both rescue growth and enhance 55Fe(II) uptake in the ferrous iron transport deficient yeast strain (fet3fet4). The results indicate that GmDmt1 is a nodule-enhanced transporter capable of ferrous iron transport across the PBM of soybean root nodules. Its role in nodule iron homeostasis to support bacterial nitrogen fixation is discussed.     

A study of nodulation and nitrogen fixation of alder on the purplish soils in China

Summary The alder has a perennial nodule cluster. The nodule amount on the roots increases with tree age. The N₂-fixing activity of nodules decreases with nodule age. Purple coloured soils with various soil pHs and CaCO₃ contents are, in the main, the ones which influence nodulation and N₂-fixing. Higher N₂-fixing capacity existed in the neutral and low calcium soils. High calcium soils and acid soils can restrain nodulation and the N₂-fixing rate significantly. On the slope, where calcarous light loams are found, the annual nitrogen fixation capacity of alder and cypress mixed plantations, less than 10 years old, is 16 or 17 kg/ha yr, but in the valley, a pure alder plantation can reach 40 kg/ha yr.