Influence of host cultivars and Bradyrhizobium strains on the growth and symbiotic performance of soybean under salt stress

This paper examines the importance of salt tolerance of host cultivars, Bradyrhizobium strains, and host-Bradyrhizobium combinations on the symbiotic nitrogen fixation potential of soybean under NaCl and KCl salt stress. Plants were grown in a soil medium, and the experiments were conducted under controlled environment growth room conditions. Bradyrhizobium growth was examined in yeast-mannitol broth andB. japonicum strains tolerant of NaCl and KCl (80 mM) stress were identified. Soybean cultivar Williams, which was sensitive to salt stress, performed poorly both in growth and symbiotic nitrogen fixation, irrespective of whether it was matched with a tolerant or sensitive Bradyrhizobium strain. Tolerant cultivar Manchu sustained nodulation and nitrogen fixation, irrespective of whether it was matched with a tolerant or sensitive Bradyrhizobium strain. Evidence presented here suggests a need, first to select soybean cultivars that are tolerant to salt stress, and then to match them with tolerant and effective Bradyrhizobium strains.     

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.     

Insufficient nitrogen supply from symbiotic fixation reduces seasonal crop growth and nitrogen mobilization to seed in highly productive soybean crops

Nitrogen (N) supply can limit the yields of soybean [Glycine max (L.) Merr.] in highly productive environments. To explore the physiological mechanisms underlying this limitation, seasonal changes in N dynamics, aboveground dry matter (ADM) accumulation, leaf area index (LAI) and fraction of absorbed radiation (fAPAR) were compared in crops relying only on biological N₂ fixation and available soil N (zero-N treatment) versus crops receiving N fertilizer (full-N treatment). Experiments were conducted in seven high-yield environments without water limitation, where crops received optimal management. In the zero-N treatment, biological N₂ fixation was not sufficient to meet the N demand of the growing crop from early in the season up to beginning of seed filling. As a result, crop LAI, growth, N accumulation, radiation-use efficiency and fAPAR were consistently higher in the full-N than in the zero-N treatment, leading to improved seed set and yield. Similarly, plants in the full-N treatment had heavier seeds with higher N concentration because of greater N mobilization from vegetative organs to seeds. Future yield gains in high-yield soybean production systems will require an increase in biological N₂ fixation, greater supply of N from soil or fertilizer, or alleviation of the trade-off between these two sources of N in order to meet the plant demand.     

Metabolic flux maps comparing the effect of temperature on protein and oil biosynthesis in developing soybean cotyledons

Metabolic flux maps developed from ¹³C metabolic flux analysis (¹³C MFA) are effective tools for assessing the response of biological systems to genetic or environmental perturbations, and for identifying possible metabolic engineering targets. Experimental treatments were designed to distinguish between temperature effects prior to, and during incubation in vitro , on primary metabolism in developing soybeans. Biomass accumulation increased with temperature as did carbon partitioning into lipids. The flux through the plastidic oxidative pentose phosphate pathway (pgl^P) relative to sucrose intake remained fairly constant [∼56% (±24%)] when cotyledons were transferred from an optimum growth temperature to varying temperatures in in vitro culture, signifying a rigid node under these conditions. However, pgl^P flux ranged from 57 to 77% of sucrose intake when growth temperature in planta varied and were cultured in vitro at the same temperature (as the plant), indicating a flexible node for this case. The carbon flux through the anaplerotic reactions catalysed by plastidic malic enzyme (me^P), cytosolic phosphoenolpyruvate (PEP) carboxylase and the malate (Mal) transporter from the cytosol to mitochondrion varied dramatically with temperature and had a direct influence on the carbon partitioning into protein and oil from the plastidic pyruvate (Pyr) pool. These results of the in vitro culture indicate that temperature during early stages of development has a dominant effect on establishing capacity for flux through certain components of central carbon metabolism.     

Distribution of two isoforms of NADP-dependent isocitrate dehydrogenase in soybean (Glycine max)

Two different cDNAs that encode NADP-specific isocitrate dehydrogenase (NADP-IDH) isozymes of soybean (Glycine max) were characterized. The nucleotide sequences of the coding regions of these cDNAs have 74% identity to each other and give predicted amino acid sequences that have 83% identity to each other. Using PCR techniques, their coding regions were subcloned into a protein overexpression vector, pQE32, to yield pIDH4 and pIDH1, respectively. Both IDH4 and IDH1 enzymes were expressed in Escherichia coli as catalytically active His6 tagged proteins, purified to homogeneity by affinity chromatography on nickel chelate resin and rabbit polyclonal antibodies to each were generated. Surprisingly, antiserum to IDH4 did not react with IDH1 protein and IDH1 antiserum reacted only very weakly with IDH4 protein. IDH4 antibody reacts with a protein of expected molecular weight in cotyledon, young leaf, young root, mature root and nodules but the reaction with mature leaf tissue was low compared to other tissues. Western blot results show that IDH1 was not expressed in young roots but a protein that reacts with the IDH1 antibody was highly expressed in leaves, showing that there was tissue-specific accumulation of NADP-IDH isozymes in soybean.     

Dimensions and distribution of intercellular spaces in cryo-planed soybean nodules

The ability of legume nodules to regulate their permeability to gas diffusion has been attributed to physiological control over the size and distribution of gas-filed intercellular spaces within the nodule cortex. To examine the size and distribution of intercellular spaces and to determine whether they were filled with gas (high diffusion permeability) or liquid (low diffusion permeability), whole nodules were frozen in liquid nitrogen slush (-210°C), and then either cryo-fractured or cryo-planed before being examined by cold-stage scanning electron microscopy (SEM). The cryo-planed tissue was found to have many advantages over cryo-fractured nodules in providing images which were easier to interpret and quantify. Intercellular spaces throughout the nodule were examined in both tangential and medial planed faces. Since no differences were observed between views in either the size or shape of the open intercellular spaces, it was concluded that the intercellular spaces of nodules were not radially oriented as assumed in many mathematical models of gas diffusion. The inner cortex region in the nodules had the smallest intercellular spaces compared to other zones, and less than 10% of the intercellular spaces were occluded with any type of material in the central zone regions. Vacuum infiltration of nodules with salt solutions and subsequent cryo-planing for SEM examination showed that open and water-filled intercellular spaces could be differentiated. The potential is discussed for using this method to study the mechanism of diffusion barrier regulation in legume nodules.     

Pools of non-structural carbohydrates in soybean root nodules during water stress

The aim of this study was to examine how the pools of non-structural carbohydrates in soybean nodules are affected under water stress conditions depending on the nature of the symbiont strains with particular emphasis on the plant-borne carbohydrates sucrose and pinitol, and on trehalose, a compatible solute synthesized by the bacteroids. Soybean ( Glycine max [L.] Merr. cv. Maple Arrow) plants were inoculated with the nitrogen-fixing strains Bradyrhizobium japonicum 61-A-101 or USDA 110 spc4 and cultivated axenically under conditions in which nodules formed in an upper soil compartment while roots for water supply grew into a compartment filled with nutrient solution. When the nodules were well established (1 month post inoculation), 10% (w/v) PEG 6000 was added to the nutrient solution. This led to a slowly progressing, moderate water stress, as determined by measuring the decrease of transpiration, and to a decrease in nitrogen fixation. The pool sizes of the major non-structural nodule carbohydrates changed during progression of water stress. Sucrose, the major soluble carbohydrate in nodules of unstressed plants (2 and 4%, respectively of nodule dry weight depending on symbiont strain), strongly increased in nodules of stressed plants, reaching nearly 10% of dry weight. The activities of two major sucrose-consuming enzymes, sucrose synthase and alkaline invertase, decreased markedly in nodules of stressed plants. Starch decreased only transiently upon water stress. Pinitol, a cyclitol serving as compatible solute in many plants, increased more than 4 times, reaching about 1% of nodule dry weight during the stress. Trehalose, the major soluble carbohydrate synthesized by the bacteroids, increased in nodules colonized by USDA 110 spc4 from about 0.2 to 0.8% of nodule dry weight, while in nodules colonized by 61-A-101 it amounted to more than 1.5% of dry weight both with and without stress.     

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.     

Immunogold localization of glutamine synthetase in soybean leaves,roots, and nodules

Summary Immunogold labelling was used to detect the cellular and sub-cellular distribution of glutamine synthetase (GS) in nodulatedGlycine max var. maple arrow. The protein was detected in thin sections of tissue embedded in LR white acrylic resin by employing two polyclonal antibody preparations, one active chloroplastic GS, the other against the cytosolic form of the enzyme. In the mature leaf tissue, GS was visualized only in the chloroplasts, exclusively within the stroma matrix; in the root cortical tissue, the enzyme was distributed homogenously in the cytosol but with a slight preferential localization associated with certain endomembranes, whereas in the root nodules both cytosolic and plastidial compartments were labelled in infected and uninfected cells. Particular to the infected cells, the bacteroids' inner matrix reacted slightly to the GS antibody and a strong signal was preferentially localized on the bacteroids' outer envelope membranes. In general, GS was more concentrated in nodules as estimated by gold particle distribution, whether in the cytosol, plastids or on the bacteroid envelope membranes, than in either root tissue or leaf tissue. Although the cytoplasmic labelling density in nodules was similar in uninfected and infected cells, certain structural features in the latter (abundant cytosol, numerous GS-positive bacteroids and GS-reactive proplastids) contribute to a more enzyme-rich type than its uninfected counterpart.Abbrevation GS glutamine synthetase     

Structural changes in the innercortex cells of soybean root nodules are induced by short-term exposure to high salt or oxygen concentrations

After a 2 h exposure of intact soybean nodules to high concentrations of NaCl (100mol m_?3) or oxygen (8OkPa O₂), morphometric computations carried out using an image analysis technique on semi-thin sections showed that both treatments induced a decrease in the area of the inner-cortex cells, which were then characterized by a tangential elongation. In contrast, no significant change in area occurred in the middle-cortex cells although their elongation decreased. Electron microscopic observations showed that in the inner-cortex cells changes included the presence of wall infoldings, an enlarged periplasmic space and a lobate nucleus whose chromatin distribution differed from that of the control. Structural changes also occurred in the endoplasmic reticulum, microbodies, mitochondria and plastids. From several of these changes, which are similar to those noted in osmocontractil cells in response to external stimuli, it can be hypothesized that the inner cortex may provide a potential mechanism for the control of oxygen diffusion through the nodules.     

Nodule gas exchange and water potential response to rapid imposition of water deficit

The permeability (P) of the gaseous diffusion barrier in the nodules of soybean [Glycine max (L.) Merr.] decreases when water deficits are extended over a 7 to 10 d period. The mechanism controlling P changes is unclear, but may result from the release of water to intercellular pathways, and an associated change in the nodule water potential. The purpose of these experiments was to impose water deficit treatments rapidly in order to determine the early sequence of the responses of nodule water potential and nodule gas exchange without the complications that arise from long-term water deficit treatments. A vertical, split-root system was used to separate nodule drying effects from plant water deficits by replacing humidified air that was passed over upper root nodules in well-watered plants with dry air, or by replacing the nutrient solution that surrounded lower roots with -1.0 MPa polyethylene glycol (PEG) solution, or by a combination of the dry air and PEG treatments. The PEG treatment caused large decreases in both the components of nodule water potential and nodule relative water content, but there was no indication that these factors had immediate, direct effects on either nitrogenase activity or P. After 7 h of the PEG treatment a significant decrease in nitrogenase activity was found but no decrease in P was detected. These results indicate that changes in nitrogenase activity in response to water deficits precede decreases in P. Exposure of nodules to dry air in well-watered plants had no significant effect on either nitrogenase activity or P during the 7 h treatment.     

Long- and short-term responses of leaf carbohydrate levels and photosynthesis to decreased sink demand in soybean

Axillary buds and the apical portion of shoots of soybean [Glycine max (L.) Merr. cultivar Turchina] plants were trimmed to investigate long-term regulation of photosynthesis by sink demand at ambient CO₂ and 22 °C. Also, in intact and trimmed shoots, the CO₂ level was increased to 660 μmol mol^?1 and temperature was lowered to 5°C to examine the superimposed short-term responses of photosynthesis to low sink demand. Under growth conditions, trimming the shoots increased leaf photosynthesis and the levels of sucrose, glucose-6-phosphate (G6P) and 3-phosphoglycerate (PGA), as well as the G6P/fructose-6-phosphate (F6P) and sucrose/starch ratios, while it decreased the level of starch and the triose-phosphate (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, TP)/PGA ratio. Photosynthesis enhancement was accompanied by increased chlorophyll contents and ribulose-l,5-bisphosphate carboxylase oxygenase (Rubisco) activity. Sink removal consistently increased photosynthesis measured under a variety of conditions (growth CO₂ or a short-term change to 660 μmol mol_-1 CO₂; growth temperature or a short-term change to 5 °C), except when low temperature was combined with ambient CO₂; the increase in photosynthesis was higher under short-term elevated CO₂ than at ambient CO₂. In contrast with its effect at ambient CO₂, shoot trimming increased the levels of TP and ribulose-1,5-bisphosphate (RuBP) and the TP/PGA ratio under high-CO₂ conditions.     

Soil moisture and potassium affect the performance of symbiotic nitrogen fixation in faba bean and common bean

Potassium (K) is reported to improve plant's resistance against environmental stress. A frequently experienced stress for plants in the tropics is water shortage. It is not known if sufficient K supply would help plants to partially overcome the effects of water stress, especially that of symbiotic nitrogen fixation which is often rather low in the tropics when compared to that of temperate regions. Thus, the impact of three levels of fertilizer potassium (0.1, 0.8 and 3.0 mM K) on symbiotic nitrogen fixation was evaluated with two legumes under high (field capacity to 25% depletion) and low (less than 50% of field capacity) water regimes. Plants were grown in single pots in silica sand under controlled conditions with 1.5 mM N (¹⁵N enriched NH₄NO₃). The species were faba bean (Vicia faba L.), a temperate, amide producing legume and common bean (Phaseolus vulgaris L.), a tropical, ureide producing species. In both species, 0.1 mM K was insufficient for nodulation at both moisture regimes, although plant growth was observed. The supply of 0.8 or 3.0 mM K allowed nodulation and subsequent nitrogen fixation which appeared to be adequate for respective plant growth. High potassium supply had a positive effect on nitrogen fixation, on shoot and root growth and on water potential in both water regimes. Where nodulation occurred, variations caused by either K or water supply had no consequences on the percentage of nitrogen derived from the symbiosis. The present data indicate that K can apparently alleviate water shortage to a certain extent. Moreover it is shown that the symbiotic system in both faba bean and common bean is less tolerant to limiting K supply than plants themselves. However, as long as nodulation occurs, N assimilation from the symbiotic source is not selectively affected by K as opposed to N assimilation from fertilizer.     

大豆快生根瘤菌SMH12效应蛋白NopP在共生固氮过程中的功能

【目的】研究Sinorhizobium fredii SMH12中的nopP在共生固氮过程中的功能,为深入解析根瘤菌效应蛋白的菌植互作机理提供线索,进而为大豆高效根瘤菌的遗传改良提供一定的科学依据。【方法】利用生物信息学分析nopP的结构特征,构建nopP缺失、过表达和互补菌株,并对其进行共生表型分析;通过qRT-PCR分析nopP在共生过程中的时空表达特征,测定在接野生型和突变体的冀豆17中NIN、ENOD40、PR1、PR2和PR5的表达量;采用激光共聚焦显微镜观察NopP的亚细胞定位。【结果】根瘤菌的NopP不包含任何已知功能域,与病原体的任何Avr效应物没有同源性。nopP缺失之后对冀豆17和中黄13的根瘤固氮酶活均有显著影响,在瘤数上对冀豆17有显著增加,表明nopP突变后促进其与冀豆17和中黄13的共生固氮。qRT-PCR显示,nopP在自生条件下少量表达,在共生条件下表达量显著升高,尤其在接菌2 d后表达量达到最高,显示该基因可能与根瘤菌早期侵染相关。此外,发现NopP在烟草叶片和大豆根中均定位于细胞膜和细胞核。接种突变体的冀豆17根中NIN的表达量升高1.2倍,PR5的表达量降低3.6倍。【结论】效应蛋白NopP在与大豆共生过程中,参与根瘤菌的早期侵染以及在根瘤菌与豆科宿主植物之间的免疫防御反应中发挥重要功能。     

A small heat shock protein,GmHSP17.9, from nodule confers symbiotic nitrogen fixation and seed yield in soybean

Legume–rhizobia symbiosis enables biological nitrogen fixation to improve crop production for sustainable agriculture. Small heat shock proteins (sHSPs) are involved in multiple environmental stresses and plant development processes. However, the role of sHSPs in nodule development in soybean remains largely unknown. In the present study, we identified a nodule-localized sHSP, called GmHSP17.9, in soybean, which was markedly up-regulated during nodule development. GmHSP17.9 was specifically expressed in the infected regions of the nodules. GmHSP17.9 overexpression and RNAi in transgenic composite plants and loss of function in CRISPR-Cas9 gene-editing mutant plants in soybean resulted in remarkable alterations in nodule number, nodule fresh weight, nitrogenase activity, contents of poly β-hydroxybutyrate bodies (PHBs), ureide and total nitrogen content, which caused significant changes in plant growth and seed yield. GmHSP17.9 was also found to act as a chaperone for its interacting partner, GmNOD100, a sucrose synthase in soybean nodules which was also preferentially expressed in the infected zone of nodules, similar to GmHSP17.9. Functional analysis of GmNOD100 in composite transgenic plants revealed that GmNOD100 played an essential role in soybean nodulation. The hsp17.9 lines showed markedly more reduced sucrose synthase activity, lower contents of UDP-glucose and acetyl coenzyme A (acetyl-CoA), and decreased activity of succinic dehydrogenase (SDH) in the tricarboxylic acid (TCA) cycle in nodules due to the missing interaction with GmNOD100. Our findings reveal an important role and an unprecedented molecular mechanism of sHSPs in nodule development and nitrogen fixation in soybean.     

Antioxidative response to cadmium in roots and leaves of tomato plants

Treatment of tomato seedlings (Lycopersicon esculentum Mill. cv. 63/5 F1) with increasing CdCl₂ concentrations in the culture medium resulted in Cd accumulation more important in roots than in leaves. Biomass production was severely inhibited, even at low Cd concentration. Cd reduced chlorophyll content in leaves and enhanced lipid peroxidation. An increase in antioxidative enzyme (superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, glutathione reductase) activities was more pronounced in leaves than in roots, while catalase activity increased only in roots. In addition, changes in isoenzyme composition were observed using the non-denaturing polyacrylamid gel electrophoresis.