Uninoculated Roots Research Articles

Proteomic Analysis of Soybean Root Hairs After Infection by Bradyrhizobium japonicum

Infection of soybean root hairs by Bradyrhizobium japonicum is the first of several complex events leading to nodulation. In the current proteomic study, soybean root hairs after inoculation with B. japonicum were separated from roots. Total proteins were analyzed by two-dimensional (2-D) polyacrylamide gel electrophoresis. In one experiment, 96 protein spots were analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to compare protein profiles between uninoculated roots and root hairs. Another 37 spots, derived from inoculated root hairs over different timepoints, were also analyzed by tandem MS (MS/MS). As expected, some proteins were differentially expressed in root hairs compared with roots (e.g., a chitinase and phosphoenolpyruvate carboxylase). Out of 37 spots analyzed by MS/MS, 27 candidate proteins were identified by database comparisons. These included several proteins known to respond to rhizobial inoculation (e.g., peroxidase and phenylalanine-ammonia lyase). However, novel proteins were also identified (e.g., phospholipase D and phosphoglucomutase). This research establishes an excellent system for the study of root-hair infection by rhizobia and, in a more general sense, the functional genomics of a single, plant cell type. The results obtained also indicate that proteomic studies with soybean, lacking a complete genome sequence, are practical.

Identification of premycorrhiza-related plant genes in the association between Quercus robur and Piloderma croceum.

• An in vitro system with micropropagated oaks (Quercus robur) and the ectomycorrhizal fungus Piloderma croceum, which is characterized by a delayed mycorrhiza formation, was used to identify plant transcripts upregulated in the premycorrhizal phase. • Complementary DNA (cDNA) populations of uninoculated roots and fungal mycelium were subtracted from a cDNA population of inoculated roots. Differential expression was confirmed by reverse Northern and 50 clones for different polypeptides were found to be up-regulated. Twenty-nine clones were investigated in more detail. • For approximately half of the cDNA fragments no homologies could be identified in databases. The residual fragments code for polypeptides with homologies to known proteins involved in signal perception and transmission, stress responses, metabolism and growth. • Since many of the identified genes have not yet been described in the context of symbiotic events, their potential roles during early phases of the recognition process are discussed.

Analysis of expressed sequence tags from roots of resistant soybean infected by the soybean cyst nematode.

The soybean cyst nematode (SCN) Heterodera glycines is the most devastating pest of soybean in the U.S.A. The resistance response elicited by SCN in soybean is complex, and genes involved in the response to a large extent are unknown and not well characterized. We constructed cDNA libraries made from mRNA extracted from roots of the resistant soybean Glycine max L. Merr. 'Peking' at 12 h, 2 to 4 days, and 6 to 8 days post inoculation with the soybean cyst nematode, population NL1-RHp, similar to race 3. Expressed sequence tag analysis of the libraries provides rapid discovery of genes involved in the response of soybean to the nematode. A total of 3454 cDNA clones were examined from the three libraries, of which 25 cDNAs were derived from nematode RNA. The levels of certain stress-induced genes such as SAM22 and glutathione S-transferase (GST8) were elevated in the SCN-infected roots relative to uninoculated roots. Early defense response genes, particularly ascorbate peroxidase and lipoxygenase, were abundant in the 12-h library. By 6-8 days, the expression of most of those genes was not as abundant, whereas genes coding for unknown proteins and stress-induced proteins continued to be highly expressed. These ESTs and associated information will be useful to scientists examining gene and protein interactions between nematodes and plants.

Resistance locus pyramids alter transcript abundance in soybean roots inoculated with Fusarium solani f.sp. glycines.

Soybean Sudden Death Syndrome (SDS) is caused by Fusarium solani f.sp. glycines (Fsg). Six quantitative trait loci (QTLs), each conferring partial resistance to SDS, have been discovered in an Essex x Forrest recombinant inbred line (RIL) population, but their mode of action is not clear. This study aimed to identify genes (ESTs) whose mRNA transcripts were altered in abundance in soybean roots following inoculation of Fsg. Roots of the soybean variety Forrest (four resistance alleles) were inoculated with Fsg, and 14 days later RNA sequences that were differentially expressed relative to uninoculated roots were enriched using suppression subtraction and differential display. The abundance of these RNAs was quantified in inoculated and non-inoculated roots by macroarray hybridizations. A unigene set of 135 ESTs was identified and used in a further macroarray analysis. The abundance of 28 cDNA fragments was increased more than two-fold in inoculated compared to uninoculated roots of RIL 23 (six resistance alleles). In Forrest and Essex (two resistance alleles), the level of only one mRNA was increased two-fold in inoculated roots compared to the uninoculated roots. In Essex most of the mRNAs analyzed decreased in abundance (61/135 showed a two-fold decrease), while in Forrest most mRNA abundances did not change. Among the 28 cDNAs that revealed a two-fold or higher increase in mRNA abundance in RIL 23, 14% code for proteins known to be involved in plant defense, 21% in metabolism, 14% in cell structure and 4% in transport. Unannotated ESTs accounted for 43% of the genes, and 4% of the sequences were previously unknown. The plant defense-related genes that showed a differential response to Fsg inoculation suggested a role for the phenylproponoid pathway in soybean defense against Fsg. In Essex, genes involved in plant defense, cell wall synthesis, ethylene synthesis and metabolism were expressed at lower levels in inoculated roots. The difference in response between the 2-, 4- and 6-gene pyramids suggests that QTLs for SDS resistance serve to delay symptoms or confer resistance by maintaining or increasing the expression of specific genes after inoculation/infection.

Identification of a family of extensin-like glycoproteins in the lumen of rhizobium-induced infection threads in pea root nodules.

Rhizobium leguminosarum bv. viciae normally gains access to pea host cells through tubular cell wall ingrowths termed infection threads. Matrix glycoprotein (MGP), a major component of the infection thread lumen, is also secreted from the tips of uninoculated roots and can be released into solution under reducing conditions. Monoclonal antibody MAC265, which recognizes MGP through a carbohydrate epitope, was used for immunoaffinity purification of the glycoprotein from pea roots. Following treatment with chymotrypsin, a peptide fragment was obtained and subjected to N-terminal sequencing. Using PCR primers based on this sequence, cDNA clones were isolated with RNA from inoculated roots and nodules. DNA sequencing of 30 of these clones revealed a family of closely related and repetitive polypeptides with (hydroxy)proline-rich motifs. The cDNA sequences showed over 70% identity with the deduced amino acid sequences of plant extensins, particularly with VfNDS-E from Vicia faba and MtN12 from Medicago truncatula, both of which are strongly upregulated in legume root nodules. Root nodule extensins from pea were of variable length but showed strong sequence conservation of the N-terminus, of the C-terminus, and of a central domain comprising 33 amino acids that were sometimes reiterated. The distribution of tyrosine residues suggested the possible importance of intramolecular and intermolecular cross-linking. There was strong sequence conservation with MtN12 in the 3'-untranslated region, suggesting a possible involvement in posttranscriptional regulation of gene expression.

Cloning of nodule-specific cDNAs of Galega orientalis.

Differential display was applied in order to clone cDNAs expressed exclusively or predominantly in nodules, compared to uninoculated root tissue of Galega orientalis. Forty-five fragments were unique for nodule RNA. These fragments were reamplified and cloned. Six of them produced a nodule-specific signal on Northern hybridization. These six fragments were sequenced. Five of the sequenced fragments showed homology to nodulin-gene sequences in databases, among them Vicia faba mRNA for protein showing partial homology with Medicago sativa nodulin-25 (Nms25), Pisum sativum PsN466, V. faba CCP2 and CCP4, P. sativum ENOD3, and Maackia amurensis ENOD2. The remaining sequence had no significant homology with sequences in the databanks. Full-size cDNA for the homologue to V. faba mRNA for the protein showing partial homology with M. sativa nodulin-25 (Nms25) and P. sativum PsN466 were cloned and sequenced.

Effect of low root-zone temperature on nodule initiation in narrow-leafed lupin (Lupinus angustifolius L.)

The effect of low root-zone temperature on nodulation of Lupinus angustifolius [L.] cv. Yandee was studied using glasshouse experiments in which the effects of temperature on nodule initiation and subsequent nodule development could be assessed separately. Low temperature (7 and 12˚C compared with 25˚C) reduced the growth of both uninoculated plants supplied with adequate mineral N and inoculated plants reliant on fixation alone for their N. However, even at 25˚C, growth of inoculated plants compared with plants supplied with mineral N was limited, and at lower temperatures nodulation was severely inhibited. The most sensitive stage to low root-zone temperature was nodule initiation and there appeared to be a critical temperature between 7 and 12˚C at which initiation did not take place. Increasing the number of bacteria in inocula (from 5 × 103 to 5 × 107 viable cells/mL) did not overcome inhibition. A number of diverse cultivars of L. angustifolius showed the same response as cv. Yandee. Low temperature inhibition of nodule initiation could be overcome by addition of culture solution collected from around the roots of symbioses established at 25˚C. The culture solutions were only effective if the roots at 25˚C were inoculated or, if collected from around uninoculated roots of plants grown with mineral N, they were first exposed to a Bradyrhizobium suspension and then sterilised before addition to cultures at low temperature. The data indicate that both plant and bradyrhizobial factors are required for nodule initiation and that exudation of plant factors at low root-zone temperature is insufficient to stimulate production of the nodulation factors from Bradyrhizobium. At 25˚C, the nodulation zone of lupin roots bore many fractures in the epidermis and showed a high frequency of free root cap border cells, as well as a distinct matrix of extracellular material. These features were significantly reduced at 12˚C and essentially absent at 7˚C, indicating that at low temperature bacterial entry may be restricted.

Establishment of a root proteome reference map for the model legume Medicago truncatula using the expressed sequence tag database for peptide mass fingerprinting.

We have established a proteome reference map for Medicago truncatula root proteins using two-dimensional gel electrophoresis combined with peptide mass fingerprinting to aid the dissection of nodulation and root developmental pathways by proteome analysis. M. truncatula has been chosen as a model legume for the study of nodulation-related genes and proteins. Over 2,500 root proteins could be displayed reproducibly across an isoelectric focussing range of 4-7. We analysed 485 proteins by peptide mass fingerprinting, and 179 of those were identified by matching against the current M. truncatula expressed sequence tag (EST) database containing DNA sequences of approximately 105,000 ESTs. Matching the EST sequences to available plant DNA sequences by BLAST searches enabled us to predict protein function. The use of the EST database for peptide identification is discussed. The majority of identified proteins were metabolic enzymes and stress response proteins, and 44% of proteins occurred as isoforms, a result that could not have been predicted from sequencing data alone. We identified two nodulins in uninoculated root tissue, supporting evidence for a role of nodulins in normal plant development. This proteome map will be updated continuously (http://semele.anu.edu.au/2d/2d.html) and will be a powerful tool for investigating the molecular mechanisms of root symbioses in legumes.

Establishment of a root proteome reference map for the model legume Medicago truncatula using the expressed sequence tag database for peptide mass fingerprinting

We have established a proteome reference map for Medicago truncatula root proteins using two-dimensional gel electrophoresis combined with peptide mass fingerprinting to aid the dissection of nodulation and root developmental pathways by proteome analysis. M. truncatula has been chosen as a model legume for the study of nodulation-related genes and proteins. Over 2500 root proteins could be displayed reproducibly across an isoelectric focussing range of 4–7. We analysed 485 proteins by peptide mass fingerprinting, and 179 of those were identified by matching against the current M. truncatula expressed sequence tag (EST) database containing DNA sequences of approximately 105 000 ESTs. Matching the EST sequences to available plant DNA sequences by BLAST searches enabled us to predict protein function. The use of the EST database for peptide identification is discussed. The majority of identified proteins were metabolic enzymes and stress response proteins, and 44% of proteins occurred as isoforms, a result that could not have been predicted from sequencing data alone. We identified two nodulins in uninoculated root tissue, supporting evidence for a role of nodulins in normal plant development. This proteome map will be updated continuously (http://semele.anu.edu.au/2d/2d.html) and will be a powerful tool for investigating the molecular mechanisms of root symbioses in legumes.

Localization of H(+)-ATPases in soybean root nodules.

The localization of H(+)-ATPases in soybean (Glycine max L. cv. Stevens) nodules was investigated using antibodies against both P-type and V-type enzymes. Immunoblots of peribacteroid membrane (PBM) proteins using antibodies against tobacco and Arabidopsis H(+)-ATPases detected a single immunoreactive band at approximately 100 kDa. These antibodies recognized a protein of similar relative molecular mass in the crude microsomal fraction from soybean nodules and uninoculated roots. The amount of this protein was greater in PBM from mature nodules than in younger nodules. Immunolocalization of P-type ATPases using silver enhancement of colloidal-gold labelling at the light-microscopy level showed signal distributed around the periphery of non-infected cells in both the nodule cortex and nodule parenchyma. In the central nitrogen-fixing zone of the nodule, staining was present in both the infected and uninfected cells. Examination of nodule sections using confocal microscopy and fluorescence staining showed an immunofluorescent signal clearly visible around the periphery of individual symbiosomes which appeared as vesicles distributed throughout the infected cells of the central zone. Electron-microscopic examination of immunogold-labelled sections shows that P-type ATPase antigens were present on the PBM of both newly formed, single-bacteroid symbiosomes just released from infection threads, and on the PBM of mature symbiosomes containing two to four bacteroids. Immunogold labelling using antibody against the B-subunit of V-type ATPase from oat failed to detect this protein on symbiosome membranes. Only a very faint signal with this antibody was detected on Western blots of purified PBM. During nodule development, fusion of small symbiosomes to form larger ones containing multiple bacteroids was observed. Fusion was preceded by the formation of cone-like extensions of the PBM, allowing the membrane to make contact with the adjoining membrane of another symbiosome. We conclude that the major H(+)-ATPase on the PBM of soybean is a P-type enzyme with homology to other such enzymes in plants. In vivo, this enzyme is likely to play a critical role in the regulation of nutrient exchange between legume and bacteroids.

Symbiosis-stimulated chitinase isoenzymes of soybean (Glycine max (L.) Merr.)

and to be colonized by arbuscular mycorrhiza (AM ) fungi (Harrison, 1997). Often, nodule formation by a Isoforms of endochitinase in soybean were studied in given bacterial strain is restricted to only a few host plant relation to root symbiosis. Five selected cultivars dif- genera, and varieties of a given legume species may diVer fering in their nodulation potential were inoculated in nodule formation when inoculated with a given strain. with two strains of Bradyrhizobium japonicum, the Specifically in soybean, allelic genes have been described broad host-range Rhizobium sp. NGR234, and with the which restrict nodulation of certain genotypes by certain mycorrhizal fungus Glomus mosseae. Total chitinase Bradyrhizobium strains ( Vest, 1970; Qian et al., 1996). activity in nodules was up to 7-fold higher than in Other, ‘broad host-range’ rhizobia exist: Rhizobium sp. uninoculated roots and in mycorrhizal roots. The chi- NGR234 is such an example, and it is able to induce tinase activity in nodules varied depending on the nodules on more than 110 genera of legumes as well

Citrus root responses to localized drying soil: A new approach to studying mycorrhizal effects on the roots of mature trees

Because fine roots tend to be concentrated at the soil surface, exposure to dry surface soil can have a large influence on patterns of root growth, death and respiration. We studied the effects of arbuscular mycorrhizas (AM) formation on specific root length (SRL), respiration and mortality of fine roots of bearing red grapefruit (Citrus paradisi Macf.) trees on Volkamer lemon (C. volkameriana Tan. & Pasq.) rootstock exposed to drying soil. For each tree, the fine roots were removed from two woody lateral roots, the roots were surface sterilized and then each woody root was placed in a separate pair of vertically divided and independently irrigated soil compartments. The two split-pot systems were filled with sterilized soil and one was inoculated with arbuscular mycorrhizal fungi (Glomus etunicatum/G. intraradices). New fine lateral roots that emerged from the woody laterals were permitted to grow inside the pots over a 10-month period. Irrigation was then removed from the top compartment for a 15-week period. At the end of the study, roots inoculated with AM fungi exhibited about 20% incidence of AM formation, whereas the uninoculated roots were completely void of AM fungi. Arbuscular mycorrhizal roots exhibited lower SRL, lower root/soil respiration and about 10% lower fine root mortality than nonmycorrhizal roots after 15 weeks of exposure to dry surface soil. This study demonstrates the feasibility of examining mycorrhizal effects on the fine roots of adult trees in the field using simple inexpensive methods.

Studying early nodulin gene ENOD40 expression and induction by nodulation factor and cytokinin in transgenic alfalfa.

ENOD40, an early nodulin gene, is expressed following inoculation with Rhizobium meliloti or by adding R. meliloti-produced nodulation (Nod) factors or the plant hormone cytokinin to uninoculated roots. We isolated two MsENOD40 clones, designated MsENOD40-1 and MsENOD40-2, with distinct promoters from an alfalfa (Medicago sativa cv Chief) genomic library. The promoters were fused to the reporter gene uidA (gus), and the constructs were introduced into alfalfa. We observed that the MsENOD40-1 construct was expressed almost exclusively under symbiotic conditions. The MsENOD40-2 construct was transcribed under both symbiotic and nonsymbiotic conditions and in nonnodular and nodular tissues. Both MsENOD40 promoter-gus constructs were similarly expressed as nodules developed, and both were expressed in roots treated with 6-benzylaminopurine or purified Nod factor. However, no blue color was detected in nodule-like structures induced by the auxin transport inhibitor N-1-(naphthyl)phthalamic acid on roots of plants containing the MsENOD40-1 promoter construct, whereas pseudonodules from plants containing the MsENOD40-2 promoter construct stained blue. A 616-bp region at the distal 5' end of the promoter is important for proper spatial expression of MsENOD40 in nodules and also for Nod-factor and cytokinin-induced expression.

Expression of early nodulin genes in alfalfa mycorrhizae indicates that signal transduction pathways used in forming arbuscular mycorrhizae and Rhizobium-induced nodules may be conserved.

Transcripts for two genes expressed early in alfalfa nodule development (MsENOD40 and MsENOD2) are found in mycorrhizal roots, but not in noncolonized roots or in roots infected with the fungal pathogen Rhizoctonia solani. These same two early nodulin genes are expressed in uninoculated roots upon application of the cytokinin 6-benzylaminopurine. Correlated with the expression of the two early nodulin genes, we found that mycorrhizal roots contain higher levels of trans-zeatin riboside than nonmycorrhizal roots. These data suggest that there may be conservation of signal transduction pathways between the two symbioses-nitrogen-fixing nodules and phosphate-acquiring mycorrhizae.

The effect of plant age and nodulation on the isoflavonoid content of red clover ( Trifolium pratense)

The effect of age and inoculation with nodulating bacteria on the isoflavonoid content of red clover ( Trifolium pratense L.) has been determined. Both roots and foliage contained a complex range of conjugated isoflavonoids with formononetin-7-O-glucoside-6"-O-malonate (FGM), maackian 3-O-glucoside-6"-O-malonate (MKGM), four acidic (presumed malonyiated) 7-O-glycosides of biochanin A (BGMs), and an acidic (presumed malonyiated) glycoside of an unknown 5,7-dihydroxy-containing isoflavonoid metabolite termed UN 1GM being the major metabolites in all plant parts. In contrast, the concentrations of the corresponding isoflavonoid aglycones always remained low. In the foliage, the order of abundance of the conjugates was FGM > BGM > UNI GM > MKGM, while in the roots of plants up to 15 days old the order was FGM > BGM = MKGM > UN 1GM, changing to MKGM > FGM > BGM > UN 1 GM in the older roots. In all plant parts the concentrations of these various conjugates were affected differently by plant growth and nodulation. In the foliage, BGM, FGM and UN 1 GM increased with age and this accumulation was partially suppressed by nodulation. In uninoculated roots MKGM accumulated steadily with age, while overall the concentrations of FGM, BGM and UN1GM remained unchanged. Nodulation suppressed the accumulation of MKGM and resulted in a decline in FGM content. These results suggest that nodulation not only affects isoflavonoid accumulation in the roots but can also systemically regulate isoflavonoid metabolism in the foliage.

Properties of invertases in mycelium of Hymenoscyphus ericae and in endomycorrhizal association with cranberry seedlings

The properties of extracellular, soluble and insoluble invertases in mycelium of Hymenoscyphus ericae an ericoid mycorrhizal fungus, were investigated. Soluble and insoluble acid invertases were more active in sucrose- compared with glucose-grown mycelium, whereas extracellular invertase activities were similar in both glucose- and sucrose-grown cultures. Acid (pH 5·5) and alkaline (pH 9·0) invertases were detected in soluble and insoluble fractions but extracellular activity was predominantly acid (pH 5·5). Insoluble alkaline invertase was released completely by freeze-thawing. Freeze-thawing and Triton X-100 released similar proportions of acid invertase from the wall (42 and 31% respectively). The K m values varied from 9·1 m m (extracellular acid invertase) to 139·3 m m sucrose (soluble alkaline invertase). Fructose inhibited soluble alkaline invertase completely but it competitively inhibited soluble acid invertase. Increased soluble acid invertase activity was demonstrated in endomycorrhizas compared with uninoculated roots of cranberry.

Lectin genes are expressed throughout root nodule development and during nitrogen‐fixation in the Rhizobium—Medicago symbiosis

In order to study the spatio‐temporal patterns of lectin gene expression throughout the various developmental stages of the Rhizobium—legume interaction, promoter‐gusA fusions for each of three lectin genes from Medicago truncatula (Mtlec1, 2 and 3) have been introduced into transgenic Medicago varia plants. Histochemical staining for β‐glucuronidase activity has shown that Mtlec1 and Mtlec3 are expressed independently of symbiosis in inner cortical and vascular tissues of the root, respectively. Transcription of Mtlec3 in the root stele is markedly enhanced following inoculation with Rhizobium meliloti. Furthermore, both Mtlec1 and Mtlec3 are transcribed in dividing cells of the root nodule primordium, whether elicited by R. meliloti or by purified nodulation (Nod) factors. These findings suggest that these two lectins are involved in developmental processes early during nodule ontogeny. In contrast, Mtlec2 is not expressed in uninoculated roots or during early stages of the symbiotic interaction. At later stages, all three lectin promoters are functional in mature nodules. In this context, lectin gene expression is restricted to non‐infected cells of both central and certain peripheral tissues and appears to be correlated with the nitrogen‐fixing capacity of the nodule.

Total sterols in root-knot nematode Meloidogyne incognita infected cotton Gossypium hirsutum (L.) plant roots

Two near-isogenic breeding lines of Gossypium hirsutum (L.) (upland cotton) were inoculated with eggs of Meloidogyne incognita (Kofoid and White) Chitwood (RKN) in greenhouse pots. One line was susceptible (ST-213) and the other was resistant (81–249) to RKN. Eight days after inoculation, roots of plants and their controls were analyzed for total sterols by GLC and GLC-MS. The total content in roots of uninoculated roots of susceptible plants was slightly higher than in the roots of uninoculated resistant plants, but significantly lower after inoculation. By comparison, the sterols in roots of the resistant line were significantly higher at 8 days after inoculation. The major sterols in both susceptible and resistant lines were stigmasterol, sitosterol and campesterol, with a lesser amount of cholesterol and a trace of stigmastanol. Because the amounts and distribution of the sterols were similar in susceptible and resistant lines, they do not appear to be a factor associated with RKN growth and development. Sitosterol was the most abundant sterol in RKN eggs which also contained lesser amounts of cholesterol and stigmasterol. However, no stanols were found in RKN eggs.

Boric acid-phenolic relationships within the Pinus echinata-Pisolithus tinctorius ectomycorrhizal association.

At germination, container-grown shortleaf pine seedlings were inoculated with Pisolithus tinctorius (Pers.) Coker & Couch or left uninoculated, and both groups were fertilized semiweekly with a modified Hoagland's solution supplemented with 0 or 0.4 mM boric acid. After 12, 16 and 24 weeks, seedling root tissue was analyzed for ectomycorrhizal colonization, phenolic concentration and phenoloxidase activity. In addition, phenoloxidase activity was assayed in P. tinctorius that had been cultured in a liquid medium containing boric acid. Inoculation with P. tinctorius increased the root phenolic concentration of 16- and 24-week-old seedlings, and increased root phenoloxidase activity in 12-, 16- and 24-week-old seedlings. Fertilization with boric acid reduced the phenolic concentration of P. tinctorius ectomycorrhizae after 24 weeks. Although boric acid fertilization did not affect the phenoloxidase activity of 12-, 16- and 24-week-old inoculated roots, or that of 16- and 24-week-old uninoculated roots, it increased the phenoloxidase activity of P. tinctorius grown in vitro and 12-week-old uninoculated roots. We conclude that boric acid fertilization influences the phenolic relations of the shortleaf pine-P. tinctorius ectomycorrhizal association, possibly through a boric acid-induced increase in phenoloxidase activity.

Nitrogenase activity is required for the activation of iron‐stress response in iron‐inefficient T203 soybean

Abstract Iron (Fe)‐stress response and symbiotic nitrogen (N2) fixation are chemical reduction processes within the roots which interact to the extent that nodulated, Fe‐stressed, Fe‐inefficient soybean initiates Fe‐stress response, but similar nonnodulated soybean does not. The objective of this study was to examine the relationship between nitrogenase activity and Fe‐stress response in the Fe‐inefficient T203 soybean by inoculating plants with effective or ineffective strains of Bradyrhizobium japonicum on either side of split‐root systems. Half the roots of each plant were either inoculated with effective or ineffective strains or left uninoculated. Nodules formed on roots inoculated with the ineffective B. japonicum strain, SM5, were similar in number and shape to those on roots inoculated with the effective strain, 61A209, but roots inoculated with SM5 were free of nitrogenase activity. Hydrogen ion and reductant were not released from uninoculated roots or roots inoculated with SM5, but were release...