Molecular and whole-plant responses to selection for enzyme activity in alfalfa root nodules: evidence for molecular compensation of aspartate aminotransferase expression

Summary The enzyme aspartate aminotransferase (AAT) plays a key role in the assimilation of fixed-N in alfalfa (Medicago sativa L.) root nodules. AAT activity in alfalfa nodules is due to the activity of two dimeric isozymes, AAT-1 and AAT-2, that are products of two distinct genes. Three forms of AAT-2 (AAT-2a, -2b, and-2c) have been identified. It was hypothesized that two alleles occur at the AAT-2 locus, giving rise to the three AAT-2 enzymes. In a prior study bidirectional selection for root nodule AAT and asparagine synthetase (AS) activities on a nodule fresh weight basis in two diverse alfalfa germ plasms resulted in high nodule enzyme activity subpopulations with about 20% more nodule AAT activity than low enzyme activity subpopulations. The objectives of the study presented here were to determine the inheritance of nodule AAT-2 production and to evaluate the effect of bidirectional selection for AAT and AS on AAT-2 allelic frequencies, the relative contributions of AAT-1 and AAT-2 to total nodule activity, nodule enzyme concentration, and correlated traits. Two alleles at the AAT-2 locus were verified by evaluating segregation of isozyme phenotypes among F₁ and S₁ progeny of crosses or selfs. Characterization of subpopulations for responses associated with selection was conducted using immunoprecipitation of in vitro nodule AAT activity, quantification of AAT enzyme protein by ELISA, and AAT activity staining of native isozymes on PAGE. Results indicate that selection for total AAT activity specifically altered the expression of the nodule AAT-2 isozyme. AAT-2 activity was significantly greater in high compared to low activity subpopulations, and high AAT subpopulations from both germ plasms had about 18% more AAT-2 enzyme (on a nodule fresh weight basis). No significant or consistent changes in AAT-2 genotypic frequencies in subpopulations were caused by selection for AAT activity. Since changes in AAT activity were not associated with changes in AAT-2 genotype, selection must have affected a change(s) at another locus (or loci), which indirectly effects the expression of nodule AAT.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products or vendors that might also be suitable     

Aspartate aminotransferase in effective and ineffective alfalfa nodules : cloning of a cDNA and determination of enzyme activity, protein, and mRNA levels

Aspartate aminotransferase (AAT) is a key plant enzyme affecting nitrogen and carbon metabolism, particularly in legume root nodules and leaves of C₄ species. To ascertain the molecular genetic characteristics and biochemical regulation of AAT, we have isolated a cDNA encoding the nodule-enhanced AAT (AAT-2) of alfalfa (Medicago sativa L.) by screening a root nodule cDNA expression library with antibodies. Complementation of an Escherichia coli AAT mutant with the alfalfa nodule AAT-2 cDNA verified the identity of the clone. The deduced amino acid sequence of alfalfa AAT-2 is 53 and 47% identical to animal mitochondrial and cytosolic AATs, respectively. The deduced molecular mass of AAT-2 is 50,959 daltons, whereas the mass of purified AAT-2 is about 40 kilodaltons as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein's N-terminal domain (amino acids 1-59) contains many of the characteristics of plastid-targeting peptides. We postulate that AAT-2 is localized to the plastid. Southern blot analysis suggests that AAT-2 is encoded by a small, multigene family. The expression of AAT-2 mRNA in nodules is severalfold greater than that in either leaves or roots. Northern and western blots showed that expression of AAT activity during effective nodule development is accompanied by a sevenfold increase in AAT-2 mRNA and a comparable increase in enzyme protein. By contrast, plant-controlled ineffective nodules express AAT-2 mRNA at much lower levels and have little to no AAT-2 enzyme protein. Expression of root nodule AAT-2 appears to be regulated by at least two events: the first is independent of nitrogenase activity; the second is associated with nodule effectiveness.     

Immunolocalization of ferritin in determinate and indeterminate legume root nodules

Summary In eukaryotic organisms ferritin is a protein involved in the storage of iron. The occurrence of ferritin and its relationship to the effectiveness of the nitrogen-fixing activity have been previously studied during the early stages of the nodule development by biochemical methods. We have used immunocytochemistry techniques to determine the precise location of ferritin and the behavior of this protein along the nodule development. The major localization was found in plastids and amyloplasts of infected and uninfected cells of the three legume nodules studied. A decrease of the immunolabelling was observed in infected cells of lupin and soybean senescing nodules and in the senescent zone of indeterminate alfalfa nodules. In the cortex of soybean and lupin nodules, ferritin increased during nodule ageing and the immunogold particles were mainly located in crystalline structures. The putative role of ferritin and plastids during nodule development is discussed.     

Aspartate aminotransferase in alfalfa root nodules : I. Purification and partial characterization

Aspartate aminotransferase (l-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1 [AAT]), a key enzyme in the assimilation of C and N compounds, was purified from the cytosol of alfalfa (Medicago sativa L.) root nodules. Isoforms that increased during nodule development, AAT-2a, AAT-2b, and AAT-2c, were purified greater than 447-fold to apparent homogeneity, and high titer polyclonal antibodies were produced. The native molecular weight of the AAT-2 isoforms was approximately 80 kilodatons with a subunit molecular weight of 40 kilodatons, indicating that the holoenzymes are dimers. The AAT-2 isoforms comprised approximately 0.4% of the total soluble nodule protein. The AAT specific activity was measured in leaf, stem, root, and nodule organs, and zymograms of each were compared. Enzyme activity was 4- to 37-fold greater in effective (nitrogen fixing) nodules than in leaves, stems, and roots. Effective nodule AAT-specific activity was 3- to 8-fold greater than that of plant-controlled ineffective nodules. No differences in K_m were observed between AAT-1 and AAT-2. Antibodies raised against AAT-2 were more selective against AAT-2 than AAT-1. Evidence obtained from zymograms suggests that the expression of alfalfa nodule AAT is controlled at two different gene loci, AAT-1 and AAT-2, resulting in different dimeric isoforms.     

Immunogold localization of nodule-enhanced phosphoenolpyruvate carboxylase in alfalfa

Phosphoenolpyruvate carboxylase (PEPC; EC 4-1-1-31) plays a paramount role in providing carbon for synthesis of malate and aspartate in alfalfa (Medicago sativa L.) root nodules. PEPC protein and activity levels are highly enhanced in N₂-fixing alfalfa nodules. To ascertain the relationship between the cellular location of PEPC and root nodule metabolism, enzyme localization was evaluated by immunogold cytochemistry using alfalfa nodule PEPC antibodies. Gold labelling patterns in effective nodules showed that PEPC is a cytosolic enzyme and is distributed relatively equally in infected and uninfected cells of the nodule symbiotic zone. A high amount of labelling was also observed in pericycle cells of the nodule vascular system. Labelling was also detected within inner cortical cells, but the density was reduced by 60%. When Lotus corniculatus was transformed with a chimeric gene consisting of the 5′-upstream region of the PEPC gene fused to β-glucuronidase (GUS), GUS staining in nodules was consistent with immunogold localization patterns. The occurrence of PEPC in both infected and uninfected cells of the symbiotic zone of effective nodules coupled to the reduced amounts in ineffective nodules suggests a direct role for this enzyme in supporting N₂-fixation. PEPC localization in the uninfected, interstitial cells of the symbiotic zone indicates that these cells may also have a role in nodule carbon metabolism. Moreover, the association of PEPC with the nodule vascular system implies a role for the enzyme in the transport of assimilates to and from the shoot.     

Aspartate Aminotransferase in Alfalfa Root Nodules : II. Immunological Distinction between Two Forms of the Enzyme

Aspartate aminotransferase (AAT), a key enzyme in the biosynthesis of aspartate and asparagine, occurs as two forms in alfalfa (Medicago sativa L.), AAT-1 and AAT-2. Both forms were purified to near homogeneity, and high titer polyclonal antibodies produced to the native proteins. Alfalfa AAT-1 was purified from root suspension culture cells, while AAT-2 was purified from effective root nodules. Antibodies prepared to AAT-1 and used as probes for western blots readily recognized native and SDS forms of AAT-1 but did not recognize either native or SDS forms of AAT-2. Conversely, antibodies to AAT-2 readily recognized native and SDS forms of AAT-2 but did not recognize AAT-1. Immunotitrations further confirmed the immunological distinction between AAT-1 and AAT-2. AAT-1 antibodies immunotitrated 100% of the in vitro activity of purified AAT-1 but had no effect on AAT-2 in vitro activity. Likewise, AAT-2 antibodies removed 100% of the in vitro activity of purified AAT-2 but did not affect AAT-1 in vitro activity. Sequential titration of total AAT activity from roots and nodules showed that AAT-1 comprised the major form (62%) of AAT in roots, while AAT-2 was the predominant form (90%) in nodules. Last, SDS-PAGE western blots showed that the molecular masses of AAT-1 and AAT-2 were 42 and 40 kilodaltons, respectively. These data indicate that AAT is under the control of at least two distinct genes in alfalfa.     

Location of two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L.

The two isoenzymes of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), previously identified in root nodules of Phaseolus vulgaris L., have both been shown to be located in root-nodule plastids. The nodule specific NADH-GOGAT II accounts for the majority of the activity in root nodules, and is present almost exclusively in the central tissue of the nodule. However about 20% of NADH-GOGAT I activity is present in the nodule cortex, at about the same specific activity as this isoenzyme is found in the central tissue. Glutamine synthetase (GS; EC 6.3.1.2) occurs predominantly as the polypeptide in the central tissue, whereas in the cortex, the enzyme is represented mainly by the polypeptide. Over 90% of both GS and NADH-GOGAT activities are located in the central tissue of the nodule and GS activity exceeds NADH-GOGAT activity by about twofold in this region. Using the above information, a model for the subcellular location and stoichiometry of nitrogen metabolism in the central tissue of P. vulgaris root nodules is presented.Abbreviations Fd-GOGAT ferredoxin-dependent glutamate synthase - GOGAT glutamate synthase - GS glutamine synthetase - NADH-GOGAT NADH-dependent glutamate synthase - IEX-HPLC ion-exchange high-performance liquid chromatography     

Spontaneous nodules induce feedback suppression of nodulation in alfalfa

A small subpopulation of alfalfa (Medicago saliva L.) plants grown without fixed nitrogen can develop root nodules in the absence of Rhizobium. Cytological studies showed that these nodules were organized structures with no inter- or intracellular bacteria but with the histological characteristics of a normal indeterminate nodule. Few if any viable bacteria were recovered from the nodules after surface sterilization, and when the nodular content was used to inoculate alfalfa roots no nodulation was observed. These spontaneous nodules were formed mainly on the primary roots in the region susceptible to Rhizobium infection between 4 and 6 d after seed imbibition. Spontaneous nodules appeared as early as 10 d after germination and emerged at a rate comparable to normal nodules. The formation of spontaneous nodules on the primary root suppressed nodulation in lateral roots after inoculation with R. meliloti RCR2011. Excision of spontaneous nodules at inoculation eliminated the suppressive response. Our results indicate that the presence of Rhizobium is not required for nodule organogenesis and the elicitation of feedback regulation of nodule formation in alfalfa.Abbreviation RT root tip This work was supported by an endowment to the Racheff Chair of Excellence of the University of Tennessee, and the Soybean Promotion Board, Haskinsville, Tenn., USA. We are indebted to Noel Gerahty for performing the acetylene-reduction assays, and Dr. E.T. Graham for allowing the use of microscope facilities.     

Quantitative,three-dimensional analysis of alfalfa egg cells in two genotypes: Implications for biparental plastid inheritance

In alfalfa (Medicago sativa L.), plastids are inherited biparentally. Patterns of plastid transmission vary according to the genotypes involved, but there is a strong bias in favor of male plastid transmission. Previous cytological studies on the male gametophyte of this species have not provided an adequate explanation for the differences in plastid transmission frequencies among genotypes. In the present study, we compared egg cells from genotypes classified as strong or weak plastid transmitters to determine whether plastid transmission strength is correlated with egg cell structure before fertilization. We found that plastids in the mature egg cells of the strong female (genotype 6–4) are significantly larger than in mature eggs of the weak female (genotype CUF-B), and that significantly more plastids are positioned in the apical portion of the mature egg cell of genotype 6–4 than in CUF-B. Immature eggs in the two genotypes show the same pattern as mature eggs with regard to plastid number and polarization. Since only the apical portion of the egg cell/zygote gives rise to the functional embryo, these results indicate that the potential input of female plastids, in terms of plastid size and number, may be an important factor in determining the inheritance patterns of these organelles in alfalfa.Support for this work by the United States Department of Agriculture under grant 88-37234-3876, the National Science Foundation under grant DCB-9103658, the Organized Research Fund of Northern Arizona University, and the Arizona Agricultural Experiment Station is gratefully acknowledged. We are indebted to Dr. Craig Caldwell, Northern Arizona University Computer Visualization Laboratory, for his expert help with the computer graphics.     

The distribution of aquaporin subtypes (PIP1, PIP2 and gamma-TIP) is tissue dependent in soybean (Glycine max) root nodules

BACKGROUND AND AIMS The inner cortical cells (IC-cells) of legume root nodules have been previously shown to regulate the resistance to nodule O2 diffusion by a rapid contraction/expansion mechanism, which controls the volume of intercellular spaces and their occlusion by a liquid phase. The expression of aquaporins in IC-cells was also found to be involved in this nodule O2 diffusion mechanism. The aim of this study was to compare the expression of plasma membrane intrinsic proteins (PIP) aquaporin isoforms with tonoplast intrinsic protein (gamma-TIP) in both IC-cells and adjacent cell types. METHODS: Using immunogold labelling in ultra-thin sections of Glycine max nodules, the expression of two PIP isoforms was observed and compared with the gamma-TIP pattern. KEY RESULTS: The plasma membrane aquaporins PIP1 and PIP2 were expressed more in IC-cells and endodermis than in pericycle and infected cells. The tonoplast aquaporin gamma-TIP has shown a distribution pattern similar to that of the PIPs. CONCLUSIONS: PIPs and gamma-TIP aquaporins are highly expressed in both plasmalemma and tonoplast of nodule IC-cells. This distribution is consistent with the putative role of water fluxes associated with the regulation of nodule conductance to O2 diffusion and the subsequent ATP-dependent nitrogenase activity. In the endodermis, these aquaporins might also be involved in nutrient transport between the infected zone and vascular traces.     

Feedback regulation of nodule formation in alfalfa

When high dosages of wild-type Rhizobium meliloti RCR2011 were inoculated at two different times, 24 h apart, onto either the primary roots of alfalfa (Medicago sativa L.) seedlings or onto lateral roots on opposite sides of a split-root system, the number of nodules generated by the second inoculum was much smaller than the number generated by the first inoculum. These results provide evidence that alfalfa has an active, systemic mechanism for feedback control of nodulation. Non-nodulating mutants and delayed, weakly nodulating mutants did not elicit a discernable suppression of nodulation by subsequently inoculated wild-type cells. An appreciable number of Rhizobium infections thus seem required to elicit the suppressive response. Mutants in nodulation regions II_b and II_a nodulated extensively in the initially susceptible region of the root, but nodule initiation by these mutants was 100–1000 times less efficient, respectively, than the parent. Nodules formed by these mutants emerged 1 d later than normal. The II_b mutants elicited a relatively strong suppression of nodulation in younger parts of the root, but region-II_a mutants elicited only a weak response. These results indicate that elicitation of the regulatory response need not be proportional to nodule formation and imply that genes in region II_a play an important role in elicitation. At high dosages, the region-II mutants induced the development of thick, short roots in a considerably higher percentage of plants than the wild-type bacteria. Nodules generated by wild-type isolates and region-II mutants did not emerge in strict acropetal sequence, probably because some infections developed more slowly than others. Prior exposure of the root to non-nodulating mutants resulted in nodulation by the parent in regions of the root otherwise too mature to be susceptible, indicating that exposure to these mutants may affect the sequence of root development.Abbreviations RT root tip - EH smallest emergent root hair - Tsr thick, short roots This is contribution No. 79-88 of the Ohio Agricultural Research and Development Center     

The occurrence of leghemoglobin protein in the uninfected interstitial cells of soybean root nodules

The distribution of leghemoglobin (Lb) in resin-embedded root nodules of soybean (Glycine max (L.) Merr.) was investigated using immunogold labeling. Using anti-Lb immunoglobulin G and protein A-gold, Lb or its apoprotein was detected both in cells infected by Bradyrhizobium japonicum and in uninfected interstitial cells. Leghemoglobin was present in the cytoplasm, exclusive of the organelles, and in the nuclei of both cell types. In a comparison of the density of labeling in adjacent pairs of infected and uninfected cells, Lb was found to be about four times more concentrated in infected cells. This is the first report of Lb in uninfected cells of any legume nodule; it raises the possibility that this important nodule-specific protein may participate in mediating oxygen flow to host plant organelles throughout the infected region of the nodule.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - kDA kilodalton - Lb leghemoglobin - TBST Tris-buffered saline plus Tween 20     

Differential accumulation of hydroxyproline-rich glycoproteins in bean root nodule cells infected with a wild-type strain or a C4-dicarboxylic acid mutant of Rhizobium leguminosarum bv. phaseoli

An antiserum raised against deglycosylated hydroxyproline-rich glycoproteins (HPGPs) from melon (Cucumis melo L.) was used to study the relationship between Rhizobium infection and induction of HRGPs in bean (Phaseolus vulgaris L.) root nodule cells infected with either the wild-type or a C₄-dicarboxylic acid mutant strain of Rhizobium leguminosarum bv. phaseoli. In effective nodules, where fixation of atmospheric dinitrogen is taking place, HRGPs were found to accumulate mainly in the walls of infected cells and in peribacteroid membranes surrounding groups of bacteroids. Internal ramifications of the peribacteroid membrane were also enriched in HRGPs whereas the peribacteroid space as well as the bacteroids themselves were free of these glycoproteins. In mutant-induced root nodules, HRGPs were specifically associated with the electron-dense, laminated structures formed in plastids as a reaction to infection by this mutant. The presence of HRGPs was also detected in the host cytoplasm. The aberrant distribution of HRGPs in infected cells of mutant-induced nodules likely reflects one aspect of the altered host metabolism in relation to peribacteroid-membrane breakdown. The possibility that the antiserum used for HRGP localization may have cross-reacted with ENOD 2 gene products is discussed in relation to amino-acid sequences and sites of accumulation.     

Development of N2-fixing nodules on the wetland legume Lotus uliginosus exposed to conditions of flooding

Seeds of the wetland legume, Lotus uliginosus , were germinated and grown in vermiculite which was either continuously flooded or well-drained. Plants from both treatments were infected by Mesorhizobium loti strain DUS341 via a 'classical' root hair pathway, although some flooded plants appeared to be infected via enlarged epidermal cells. Subsequent to infection by M. loti , nodule meristems, which had developed within the root outer cortex, were penetrated by infection threads that released bacteria into the meristematic cells. The infection threads and infection droplets were immunogold labelled with monoclonal antibodies (MAC265 and MAC236) that recognize epitopes (at approx. 155/170 and 170/210 kDa, respectively) on a glycoprotein component of the matrix that surrounded the bacteria within the threads or droplets. Although labelling of infection threads or infection droplets with MAC236 was stronger than that with MAC265, both antibodies strongly labelled material occluding intercellular spaces in the cortices of developing nodules that had not yet expressed nitrogenase (as determined by a lack of signal after immunogold labelling with an antibody raised against nitrogenase component II). After 60 d, nitrogenase activity, shoot and root dry weights, and nodule fresh weight per plant did not differ between the treatments. After a further 30 d submergence, the flooded stems developed extensive aerenchyma and there was profuse development of (nodulated) adventitious roots. Nodules also formed at the junction of adventitious roots and the subtending stem and these were connected vascularly to a small stalk of tissue which gave rise to both a nodule and an adventitious root. The flooded nodules had prominent lenticels, and possible air pathways from the atmosphere to the nitrogen-fixing bacteroids are discussed.     

Ultrastructural specialization for ureide production in uninfected cells of soybean root nodules

Summary Ultrastructural studies were conducted on root nodules of soybean (Glycine max) inoculated as seeds withRhizobium japonicum. The development of the large peroxisomes and abundant tubular endoplasmic reticulum (ER) characteristic of the uninfected interstitial cells was followed during nodule growth and maturation. Quantitative data on differences between the uninfected and infected cells in volumes and numbers of peroxisomes, plastids and mitochondria were analyzed statistically. The peroxisomes are 60 times greater in volume per unit cytoplasm in the uninfected cells than the small presumptive peroxisomes in the infected cells. Plastids are about equal in volume in the two types of cells. Mitochondria have 4 × the volume and 3 × the number of profiles per unit cytoplasm in the infected cells than in the uninfected. The observations are discussed in relation to published evidence that several enzymes involved in ureide production are localized in organelles of the uninfected cells. The uninfected cells are viewed as essential components in the symbiotic relationship between host and bacterium.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum     

Isolation and partial characterization of Fix− mutants from Rhizobium strain 32H1

Summary Eight ineffective mutant strains were isolated from N-methyl-N'-nitro-N-nitrosoguanidine mutagenized cultures of cowpea Rhizobium strain 32H1. Strains CR1, CR2, CR3, CR4, CR5 and CR6 induced more, but smaller, nodules than the wild type. With the exception of strain CR2, these mutant strains reduced less than 1% of the amount of acetylene reduced by the wild type, in both the free-living and symbiotic assays. Strain CR2 reduced acetylene in the free-living assay but not in the symbiotic assay. Strains CR7 and CR8 responded variably (5–20% of the wild type) in free-living and symbiotic acetylene reduction assays. Nodules also varied from small white to normal-sized pink nodules. The phenotypic characteristics of the mutant strains were consistant with all leguminous plants tested and were stable upon reisolation from nodules. Fully effective revertants were selected from 4 of the ineffective mutant strains by the use of the leguminous plant,Macroptilium lathyroides. Serology, patterns of resistance to anti-bacterial agents, phage-typing, and antibiotic resistance markers were used to confirm strain identification.Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service and the North Carolina Agricultural Research Service, Raleigh, North Carolina. Paper no. 8834 of the Journal Series of the North Carolina Agricultural Research Service at Raleigh.     

Rhizobium leguminosarum inoculation decreases damage to faba bean (Vicia faba) caused by broad bean mottle bromovirus and bean yellow mosaic potyvirus

Faba bean (Vicia faba) plants were inoculated with rhizobia and then their sap was infected with broad bean mottle bromovirus (BBMV) or bean yellow mosaic potyvirus (BYMV) in a field experiment. Both viral infections significantly decreased shoot and root dry weight, number of nodules, nodule dry weight, numbers of flowers and pods/plant, total plant N, grain yield and N₂ fixation. However, inoculation withRhizobium leguminosarum significantly increased all these parameters, both in healthy and virus-infected plants. Although BYMV was more destructive than BBMV, inoculation with rhizobia could be used, with other control measures, to limit damage by both viruses.The authors are with the Department of Biochemistry and Soil Science, Faculty of Agriculture, Shambat, Sudan.     

NADH-Glutamate synthase in alfalfa root nodules. Immunocytochemical localization

In root nodules of alfalfa (Medicago sativa L.), N₂ is reduced to NH₄⁺ in the bacteroid by the nitrogenase enzyme and then released into the plant cytosol. The NH₄⁺ is then assimilated by the combined action of glutamine synthetase (EC 6.3.1.2) and NADH-dependent Glu synthase (NADH-GOGAT; EC 1.4.1.14) into glutamine and Glu. The alfalfa nodule NADH-GOGAT protein has a 101-amino acid presequence, but the subcellular location of the protein is unknown. Using immunocytochemical localization, we determined first that the NADH-GOGAT protein is found throughout the infected cell region of both 19- and 33-d-old nodules. Second, in alfalfa root nodules NADH-GOGAT is localized predominantly to the amyloplast of infected cells. This finding, together with earlier localization and fractionation studies, indicates that in alfalfa the infected cells are the main location for the initial assimilation of fixed N₂.