Chromosomal location of lectin genes indicates they are not the basis of Rhizobium strain specificity mutations identified in pea (Pisum sativum L.).

A lectin gene family is located on linkage group 7 in pea. The lectin genes are arranged as a cluster, with no recombination observed within the multigene family. A lectin-like cDNA clone, pEA207, and eight DNA fragments generated by random priming also were mapped in the region of the lectin genes. None of the known pea mutants altering Rhizobium leguminosarum strain specificity map to this region of the genome, and therefore their altered specificities appear not to be directly produced by mutations in the lectin genes.

Exogenous Ethylene Inhibits Nodulation of Pisum sativum L. cv Sparkle.

Exogenous ethylene inhibited nodulation on the primary and lateral roots of pea, Pisum sativum L. cv Sparkle. Ethylene was more inhibitory to nodule formation than to root growth; nodule number was reduced by half with only 0.07 muL/L ethylene applied continually to the roots for 3 weeks. The inhibition was overcome by treating roots with 1 mum Ag(+), an inhibitor of ethylene action. Exogenous ethylene also inhibited nodulation on sweet clover (Melilotus alba) and on pea mutants that are hypernodulating or have ineffective nodules. Exogenous ethylene did not decrease the number of infections per centimeter of lateral pea root, but nearly all of the infections were blocked when the infection thread was in the basal epidermal cell or in the outer cortical cells.

Pleiotropic Effects of sym-17 : A Mutation in Pisum sativum L. cv Sparkle Causes Decreased Nodulation, Altered Root and Shoot Growth, and Increased Ethylene Production.

R82 (sym-17), a stable mutant of Pisum sativum L. cv Sparkle, is described. The shoot growth of the mutant was less than that of its parent under light or dark growth conditions. Gibberellic acid treatment did not normalize the shoot growth of R82. The mutant had thick and short roots. It formed few nodules, but the specific nitrogenase activity was not affected. R82 produced and contained more ethylene than Sparkle. It also contained more free 1-amino-cyclopropane-1-carboxylic acid than did its parent in both the shoot and the root. The root tip of R82 had a lower activity of ethylene-forming enzyme than that of Sparkle, whereas the whole shoot of R82 had a similar activity. The sensitivity of R82 to exogenous ethylene was not more than that of Sparkle. Exogenous ethylene treatments did not make Sparkle mimic R82, and inhibitors of ethylene biosynthesis or action did not normalize the phenotype of R82. The data suggest that the primary effect of sym-17 is not the enhanced ethylene production.

Ethylene as a Possible Mediator of Light- and Nitrate-Induced Inhibition of Nodulation of Pisum sativum L. cv Sparkle.

Exposure of pea (Pisum sativum L. cv Sparkle) roots to light suppressed nodulation and induced an increase in ethylene production by roots. Dim light did not affect the number of infections per centimeter of root on the primary root, but most infections were blocked when the infection thread was in the epidermis or in the outer cortex. This is the same stage of infection on lateral roots that is blocked by exogenous ethylene. Silver, an inhibitor of ethylene action, increased nodule number on roots exposed to dim light. Exposure of pea roots to nitrate also suppressed nodulation and induced increased ethylene production by roots. However, induction of ethylene production from roots by nitrate was less than that induced by dim light. Nitrate decreased the number of infections per centimeter of lateral root. With 25 mm nitrate, about half of the infections that occurred were blocked in the epidermis or in the outer cortex. Silver did not reverse the inhibitory effects of nitrate on nodulation. Our data indicate that the inhibitory effect of light may occur via increased ethylene production, but they do not support the hypothesis that ethylene mediates nitrate control of nodule number. The possible role of ethylene in regulating nodule number should be studied in experiments in which light is excluded from the roots.

Ethylene Inhibitors Partly Restore Nodulation to Pea Mutant E 107 (brz).

E107 (brz) is a pleiotropic mutant of pea (Pisum sativum L. cv Sparkle) characterized by low nodulation, leaf necrosis, excessive ion accumulation, and decreased plant size. The defective nodulation of E107 was studied by light microscopy of lateral roots. The number of infections per centimeter of lateral root was only a third that of Sparkle. Moreover, most of the infections were aborted early; i.e. in only 14% of the infections did the infection thread penetrate beyond the epidermis. Nodulation of E107 was partly restored by treating the plant with the ethylene inhibitors aminoethoxyvinylglycine (AVG) or Ag(+). Treatment with Ag(+) did not increase the number of infections, but half of the infections went to completion. Ag(+) and AVG did not alter the size of the mutant, the accumulation of cations in its shoots, nor the leaf necrosis. Thus, in E107, nodule development can be uncoupled from other pleiotropic characteristics.

Light Microscopy Study of Nodule Initiation in Pisum sativum L. cv Sparkle and in Its Low-Nodulating Mutant E2 (sym 5).

We compared nodule initiation in lateral roots of Pisum sativum (L.) cv Sparkle and in a low-nodulating mutant E2 (sym 5). In Sparkle, about 25% of the infections terminated in the epidermis, a similar number stopped in the cortex, and 50% resulted in the formation of a nodule meristem or an emerged nodule. The mutant E2 (sym 5) was infected as often as was the parent, and it formed a normal infection thread. In the mutant, cell divisions rarely occurred in advance of the infection thread, and few nodule primordia were produced. Growing the mutant at a low root temperature or adding Ag(+) to the substrate increased the number of cell divisions and nodule primordia. We conclude that, in the E2 line, the infection process is arrested in the cortex, at the stage of initial cell divisions before the establishment of a nodule primordium.

Ethylene Inhibitors Restore Nodulation to sym 5 Mutants of Pisum sativum L. cv Sparkle.

The sym 5 mutants of pea, Pisum sativum L. cv Sparkle, do not differ in growth habit from their normal parent and nodulate poorly at a root temperature of 20 degrees C. If inhibitors of ethylene formation or action (Co(2+), aminoethoxyvinylglycine, or Ag(+)) are added to the substrate, nodulation of the sym 5 mutants is increased. Similar treatments of four other mutant sym lines do not restore nodulation. When Ag(+) is added to the substrate from 4 days before to 4 days after inoculation with rhizobia, nodulation of sym 5 mutants is increased. The roots of the mutant need only be exposed to Ag(+) for 4 hours to significantly increase nodule numbers. The content of free 1-aminocyclopropane-1-carboxylic acid and the production of ethylene in the lateral roots of sym 5 mutants do not differ from Sparkle.

sym 13-A Gene Conditioning Ineffective Nodulation in Pisum sativum.

Treatment of Pisum sativum (L.) cv. ;Sparkle' with ethyl methanesulfonic acid (EMS) produced a stable mutant, E135F, which forms small, white, ineffective nodules. These nodules exhibit histological zonation typical of an indeterminant nodule, e.g. meristematic, early symbiotic, late symbiotic, and senescent zones. Compared with the nitrogen fixing nodules of the parent, the zones are smaller and the nodules senesce prematurely. Bacteroids in E135F are less elongated and less differentiated than those in ;Sparkle.' The E135F mutant forms ineffective nodules when inoculated with nine different effective strains of Rhizobium leguminosarum and also when grown in a soil containing effective strains. The ineffective phenotype of E135F is under monogenic recessive control; the gene is designated sym 13. sym 13 was located on chromosome 2 by linkage with genes for shikimic dehydrogenase and esterase-2. The original selection E135F carried another mutation in heterozygous form at a separate locus, yielding some homozygous recessive nonnodulating progeny, E135N, in later generations. This indicates that EMS treatments may cause mutations at more than one sym gene. The gene conditioning non-nodulation in E135N was designated sym 14. It mapped to a locus on a different part of chromosome 2 by linkage to the gene for fumarase. The data demonstrate that sym genes are not necessarily closely linked.

Pleiotropic Effects of brz: A Mutation in Pisum sativum (L.) cv ;Sparkle' Conditioning Decreased Nodulation and Increased Iron Uptake and Leaf Necrosis.

Treatment of Pisum sativum (L.) cv ;Sparkle' with ethylmethane sulfonic acid produced a stable mutant, E107, which forms few nodules. The mutant allele exhibits other pleiotropic properties including bronze necrotic spots on the leaflets and high accumulation of iron in the shoot. The mutant phenotype is under monogenic recessive control. The gene, designated brz (bronze), is nonallelic with two other genes conditioning necrotic spots on leaves of other mutants of P. sativum. The brz allele was located on chromosome 4 by linkage with wax production controlled by alleles at the was locus.

Physiological Characteristics of Fe Accumulation in the ;Bronze' Mutant of Pisum sativum L., cv ;Sparkle' E107 (brz brz).

The pea (Pisum sativum L.) mutant, E107 (brz, brz) accumulated extremely high concentrations of Fe in its older leaves when grown in light rooms in either defined nutrient media or potting mix, or outdoors in soil. Leaf symptoms (bronze color and necrosis) were correlated with very high Fe concentrations. When E107 plants were grown in nutrient solutions supplied 10 mum Fe, as the Fe(III)-N,N'-ethylenebis[2-(2-hydroxyphenyl)glycine] chelate, their roots released higher concentrations of Fe(III) reducing substances to the nutrient media than did roots of the normal parent cv, ;Sparkle.' Reciprocal grafting experiments demonstrated that the high concentrations of Fe in the shoot was controlled by the genotype of the root. In short-term (59)Fe uptake studies, 15-day-old E107 seedlings exhibited higher rates of Fe absorption than did ;Sparkle' seedlings under Fe-adequate growth conditions. Iron deficiency induced accelerated short-term Fe absorption rates in both mutant and normal genotypes. Iron-treated E107 roots also released larger amounts of both protons and Fe(III) reductants into their nutrient media than did iron-treated ;Sparkle' roots. Furthermore, the mutant translocated proportionately more Fe to its shoot than did the parent regardless of Fe status.

An altered constitutive peptide in sym 5 mutants of Pisum sativum L.

Mutational analysis of Pisum sativum L. was used to search for constitutive proteins that might function in nodule formation. The sym 5 locus is a mutational hot spot, represented by seven independently derived mutant lines with decreased nodulation. Comparison of two-dimensional polyacrylamide gels of in vitro-translated root RNA showed a consistent difference in the migrational pattern of one peptide. In the nodulating parental cultivar 'Sparkle', a 66 kDa peptide had a pI of 5.9. In four of the five tested sym 5 mutants, the 66 kDa peptide had a more acidic pI of 5.8. This 66 kDa peptide is found in lateral root, tap root, and shoot. Its expression was independent of rhizobial inoculation, root temperature, or light.

Effects of carbohydrate on the internal oxygen concentration, oxygen uptake, and nitrogenase activity in detached pea nodules.

The interaction between carbon substrates and O(2) and their effects on nitrogenase activity (C(2)H(2)) were examined in detached nodules of pea (Pisum sativum L. cv "Sparkle"). The internal O(2) concentration was estimated from the fractional oxygenation of leghemoglobin measured by reflectance spectroscopy. Lowering the endogenous carbohydrate content of nodules by excising the shoots 16 hours before nodule harvest or by incubating detached nodules at 100 kPa O(2) for 2 hours resulted in a 2- to 10-fold increase in internal O(2), and a decline in nitrogenase activity. Conversely, when detached nodules were supplied with 100 millimolar succinate, the internal O(2) was lowered. Nitrogenase activity was stimulated by succinate but only at high external O(2). Oxygen uptake increased linearly with external O(2) but was affected only slightly by the carbon treatments. The apparent diffusion resistance in the nodule cortex was similar in all of the treatments. Carbon substrates can thus affect nitrogenase activity indirectly by affecting the O(2) concentration within detached nodules.

Measurement of the fractional oxygenation of leghemoglobin in intact detached pea nodules by reflectance spectroscopy.

A method is presented for the rapid measurement of the spectral properties of detached nodules of pea (Pisum sativum L. cv "Sparkle") by diffuse reflectance spectroscopy. After correcting the spectra for surface light scattering, the spectrum of leghemoglobin is obtained. From this, the fractional oxygenation of leghemoglobin and the internal O(2) concentration can be calculated. With this method, we determined internal O(2) while measuring nitrogenase activity (C(2)H(2)) in detached pea nodules over a range of external O(2) concentrations. Nitrogenase activity was maximum when leghemoglobin was 25% oxygenated, corresponding to a calculated free O(2) concentration of 45 nanomolar in infected cells. Advantages of this method over previous methods which employed transmitted light are: (a) many nodules can be assayed simultaneously, (b) nitrogenase activity (C(2)H(2)) can be determined at the same time as spectra are recorded, and (c) spectra can be obtained from nodules submerged in buffer containing metabolic effectors.

Metabolism under Microaerobic Conditions of Mitochondria from Cowpea Nodules.

A method is described for isolating mitochondria from nodules of cowpea (Vigna unguiculata [L.] Walp.) under completely anaerobic conditions. The mitochondria were immediately active when incubated aerobically with substrates, and their respiration rates were higher than mitochondria prepared in air. The mitochondria lacked fumarate reductase and were not inhibited by 5% CO(2). When incubated under microaerobic conditions, their respiration could be measured by leghemoglobin spectroscopy. Microaerobic respiration was inhibited approximately 50% by 1 millimolar malonate, and was completely inhibited by cyanide. O(2) uptake and the ATP/O ratio declined under microaerobic conditions, and therefore ATP production may be low in the environment of infected nodule cells.

Preparation and properties of mitochondria from cowpea nodules.

Mitochondria were isolated from nodules of cowpea (Vigna unguiculata (L). Walp.) and purified on a Percoll gradient. They were only slightly contaminated by bacteroids (an average of 3.5%), and had low lipoxygenase activity. Compared to mitochondria from hypocotyls the nodule mitochondria had similar O(2) uptake rates and respiratory control ratios. The ADP/O ratios for both preparations were 1.4 to 1.7 and 2.3 to 2.6 with succinate and malate, respectively. Whereas mitochondria isolated from etiolated cowpea hypocotyls had 14 to 18% of their respiration insensitive to KCN, the respiration of nodule mitochondria was completely inhibited by KCN. Enzyme activities of nodule mitochondria were similar to those found in hypocotyl mitochondria, except for NAD(+)-malic enzyme which was 12-fold lower in the mitochondria from nodules.

Root respiration associated with nitrate assimilation by cowpea.

Nitrate uptake by roots of cowpea (Vigna unguiculata) was measured using (15)NO(3) (-), and the energy cost to the root was estimated by respirometry. Roots of 8-day-old cowpea seedlings respired 0.6 to 0.8 milligram CO(2) per plant per hour for growth and maintenance. Adding 10 millimolar NO(3) (-) to the root medium increased respiration by 20 to 30% during the following 6 hours. This increase was not observed if the shoots were in the dark. Removal of NO(3) (-) from the root medium slowed the increase of root respiration. The ratios of additional respiration to the total nitrogen uptake and reduced nitrogen content in roots were 0.4 gram C per gram N and 2.3 grams C per gram N, respectively. The latter value is close to theoretical estimates of nitrate assimilation, and is similar to estimates of 1 to 4 grams C per gram N for the respiratory cost of symbiotic N(2) fixation.

Legume agglutinins that bind to Rhizobium meliloti.

A protein found in seeds and roots of alfalfa (Medicago sativa) was implicated in the specificity of the infection process, based on its binding to the symbiont Rhizobium meliloti. We found an agglutinin with similar properties in seeds and roots of sweet clover (Melilotis alba). The sweet clover differed from alfalfa in nodulation by a mutant strain of R. meliloti, but the agglutinins were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Rhizobium agglutination, and cross-reactivity to antibodies. Similar agglutinins binding R. meliloti were found in seeds of legumes from different cross-inoculation groups, including soybean (Glycine max), cowpea (Vigna unguiculata), pea (Pisum sativum L), and mung bean (Vigna mungo). The agglutinins from these legumes were recognized by antibodies raised against the agglutinins of alfalfa and sweet clover. Seeds of corn (Zea mays) and tomato (Lycopersicon esculentum) contained a protein similar to the legume agglutinin, but it did not react with the antibodies. We conclude that the alfalfa agglutinin is representative of a common legume protein and that there is no evidence for its role in specificity or nodule initiation.

Effect of nitrogen source on ureides in soybean.

In field-grown soybeans (Glycine max L. Merr. cv Harosoy), the percentage of N in the xylem as ureides increased with increasing N(2) fixation. During a 9-week collection period, the ureide content varied from 9.0 to 69.2% of the xylary N. Between 9 and 11 weeks (early pod fill), there was a good correlation (r = 0.93) between C(2)H(2) reduction and the per cent N in xylem as ureides. The per cent N as ureides, however, does not always indicate the reliance of the plant on symbiotic N(2) fixation. This ureide content also depended on the level of NO(3) (-) available to the roots. Non-nodulated soybeans given from 0 to 200 kilogram N per hectare produced xylem sap which averaged from 31.8% to 9.0% N, respectively, in the xylem as ureides over the 9-week period.Feeding of (15)N(2), (15)NH(4), or (15)NO(3) to greenhouse-grown soybeans indicated substantial differences in the initial distribution of N by the xylem stream, but the ultimate distribution of N between plant parts and grain did not vary with available N or percentage of xylary N as ureides. Amino acids, not ureides, were the major source of N in the phloem. The soybeans maintained a similar composition in phloem irrespective of the xylem sap constituents, with N derived from N(2), NH(4), or NO(3) being equally accessible to the phloem stream.

Effect of supra-ambient oxygen on nitrogenase activity (c(2)h(2)) and root respiration of soybeans and isolated soybean bacteroids.

Isolated soybean (Glycine max [L.] Merr. cv Wilkin) bacteroids have O(2)-dependent nitrogenase activity which is strongly inhibited by supraoptimal O(2) concentrations. Oxygen-inhibited nitrogenase activity is recovered by addition of 10 millimolar sodium succinate or by lowering the O(2) concentration.Brief treatment of roots of intact soybean plants with 1.0 atmosphere O(2) reduces nitrogenase activity (C(2)H(2)). There is a rapid partial recovery of activity within 2 to 3 hours, and a slower return to near normal levels by 36 hours. The drop and recovery of nitrogenase activity is accompanied by a parallel drop and increase in root respiration. There is a direct relationship between the change in respiration and the change in acetylene reduction following O(2) treatment. The O(2)-mediated changes in nitrogenase activity and root respiration are not affected by the planting medium. The ratio of the change in respiration to the change in nitrogenase activity was the same in 13 soybean cultivars.

Root respiration associated with nitrogenase activity (c(2)h(2)) of soybean, and a comparison of estimates.

Root respiration associated with symbiotic fixation in soybean (Glycine max [L.] Merr.) was estimated by four methods.Averaged over the life of the plant, the root respires 5.8 milligrams C per milligram N accumulated from fixation. When nitrogenase (C(2)H(2)) activity and root respiration were decreased by treating roots briefly with 1.0 atmosphere O(2), the respiration associated with nitrogenase was estimated as 2.10 micromoles CO(2) per micromole C(2)H(4).When nitrogenase activity and respiration were decreased by addition of nitrate, the respiration associated with fixation was calculated as 2.90 micromoles CO(2) per micromole C(2)H(4). Removing nodules from roots decreased fixation and root respiration, and the ratio was 4.08 micromoles CO(2) per micromole C(2)H(4). When soybean plants were kept in prolonged darkness, then returned to light, the associated drop and recovery of respiration and nitrogenase activity had a ratio of 4.36 micromoles CO(2) per micromole C(2)H(2).