Study of morphological and symbiotic traits during the ontogeny of supernodular and hypernodular pea mutants

Morphological (plant height and vegetative biomass amount) and symbiotic (number of nodules and nitrogenase activity) traits of six symbiotic pea mutants and the original cultivar Rondo were studied at different vegetation periods. Of the mutants studied, one (K10a) was supemodular and the remaining five (K1a, K2a, K5a, K7a, and K27a) were hypemodular. Essential distinctions in the absolute values and time course of the changes in individual morphological and symbiotic traits of different pea mutants were demonstrated. The supemodular type is inferior to the original cultivar in plant height and production of vegetative biomass, but exceeds it in nodulation and nitrogen fixation. The hypemodular mutants either surpass the original cultivar with respect to the production capacity or display similar results. The symbiotic traits-number of nodules and nitrogen fixation activity--of these mutants are higher compared with the Rondo cultivar. The mutants K1a, K2a, and K27a were demonstrated to be useful in breeding pea for an increase in nitrogen fixation.     

Study of dominant symbiotic mutants of pea <Emphasis Type="Italic">Pisum sativum</Emphasis> L.

Phenogenetic studies of four symbiotic hypernodulating mutants of pea (Pisum sativum L.) induced from seeds of cultivar Rodno by chemical mutagen EMS were conducted. All mutants have improved symbiotic traits, i.e., an increased number of root nitrogen fixating nodules and high activity of nitrogenase. Symbiotic traits were shown to be inherited dominantly. Mutants grown in the field or in a greenhouse showed superiority over the original cultivar in productivity. An important feature of hypernodulating mutants was found that is responsible for the appearance of high-height productive plants in F₂ after crossing mutants and the original cultivar. Constant lines retaining the ability for high-level production up to the F₅ generation were created based on individual plants.     

Physiological and agrochemical properties of different symbiotic genotypes of pea (Pisum sativum L.)

Physiological characters of symbiotic mutants of pea were studied: nodulation, activities of nitrogenase and nitrate reductase, chlorophyll content in leaves and their water-holding capacity, biomass accumulation, and nitrogen forms. The parameters reflecting the genotype state of the macrosymbiont under soil conditions considerably varied. Supernodulation mutants stood out against symbiotic pea genotypes by high contents of chlorophyll and nonprotein nitrogen compounds, high nitrogenase activity, and low nitrate reductase activity. The efficiency of the legume-rhizobium symbiosis was largely mediated by the macrosymbiont genotype. The highest atmospheric nitrogen fixation (50–80%) was observed in the parental pea varieties. Despite the highest nitrogenase activity in the nodules, the supernodulation mutants were inferior to the parental varieties by the nitrogen fixation capacity (40–60%), which was due to their low productivity.     

Phytohormone Levels in Different Types of Symbiotic Mutants of Pea

The levels of the phytohormones auxin and gibberellin were studied in the original pea (Pisum sativumL.) cultivars Rondo and Ramonskii 77 and in different types of symbiotic mutants (non-nodulating, with single nodules, and supernodulating) induced from them. The results obtained indicated that the levels of the phytohormones in the symbiotic mutants depend on the plant's genotype, developmental phase, and infection with rhizobia. Two mutants were isolated whose phytohormonal status markedly differed from the original forms. These mutants may be used for identification of the genes that determine the auxin and gibberellin statuses.     

Level of phytohormones in various types of symbiotic pea mutants]

The levels of the phytohormones auxin and gibberellin were studied in the original pea (Pisum sativum L.) cultivars Rondo and Ramonskii 77 and in different types of symbiotic mutants (non-nodulating, with single nodules, and supernodulating) induced from them. The results obtained indicated that the levels of the phytohormones in the symbiotic mutants depend on the plant's genotype, developmental phase, and infection with rhizobia. Two mutants were isolated whose phytohormonal statuses markedly differed from the original forms. These mutants may be used for identification of the genes that determine the auxin and gibberellin statuses.     

Symbiotic Nitrogen Fixation and Ammonium Nitrogen Assimilation in rrrbrb-, rrRbRb-, and RRrbrb-Pea Mutants

Wrinkled-seeded pea mutants (Pisum sativum L., genotypes rrrbrb-, rrRbRb-, and RRrbrb-) have seeds with reduced, but different, starch content and modified starch properties. Analysis of these mutants revealed an enhanced capacity of root nodules for symbiotic nitrogen fixation and of host plant organs for assimilation of ammonium nitrogen. This observation was confirmed by morphological data on organization of symbiotic system, by elevated nitrogenase activity, high protein accumulation in plants due to nitrogen fixation, and by enhanced activity of glutamine synthase in leaves and glutamate dehydrogenase in roots of mutants, as compared with the organs of wild-type pea. It is supposed that the aforementioned advantages of mutants are related to accumulation in seeds of elevated protein reserves that satisfy their demand for nitrogen during formation of symbiotic systems.     

Genetic potential of local endemic forms of the pea (Pisum sativum L.) on the basis of nitrogen fixation and productivity

Morphological and symbiotic traits were studied in local endemic forms of the pea originating from Egypt, Syria, Afghanistan, and Palestine. A number of endemic forms exceeded the regionalized Druzhnaya cultivar of the fodder pea in productivity of the seeds in field and greenhouse experiments. In order to improve nodulation and nitrogen fixation, endemic forms were crossed with the supernodulating K301 mutant (marked by the nod4 gene). Recurrent selection of lines up to F_5,6 generations was conducted with an estimation of productivity, nodulation, and nitrogen fixation. The most promising recurrent lines with a high productivity, active nodulation, and high nitrogen fixation were obtained during the crossing of endemic forms with the recurrent line marked by the nod4 gene. The line was previously created during the crossing between the Druzhnaya cultivar and the supernodulating K301 mutant marked by the nod4 gene.     

Nod factors stimulate seed germination and promote growth and nodulation of pea and vetch under competitive conditions

Nod factors are lipochitooligosaccharide (LCO) produced by soil bacteria commonly known as rhizobia acting as signals for the legume plants to initiate symbiosis. Nod factors trigger early symbiotic responses in plant roots and initiate the development of specialized plant organs called nodules, where biological nitrogen fixation takes place. Here, the effect of specific LCO originating from flavonoid induced Rhizobium leguminosarum bv. viciae GR09 culture was studied on germination, plant growth and nodulation of pea and vetch. A crude preparation of GR09 LCO significantly enhanced symbiotic performance of pea and vetch grown under laboratory conditions and in the soil. Moreover, the effect of GR09 LCOs seed treatments on the genetic diversity of rhizobia recovered from vetch and pea nodules was presented.     

Gene centres,a source for genetic variants in symbiotic nitrogen fixation: The symbiotic response of the cultivated pea toRhizobium leguminosarum strains from Europe and the Middle East

Summary Soil samples from several European countries; Sweden, the Netherlands, Spain, Italy and Greece, contained rhizobial populations capable of forming an effective symbiosis with the cultivated pea cv. Rondo from the Netherlands. The range of variation among the European Rhizobium strains, as expressed on pea cv. Rondo, was not so large and almost the same variation could be found within the rhizobial population within each country. Superior Rhizobium strains for the Dutch pea were not restricted to soils from the Netherlands but were also found in those from Sweden and Italy.Soils from Turkey and Israel also contained Rhizobium strains capable of nodulating pea cv. Rondo. However, the genetic variation among these Middle East Rhizobium strains was much larger than that of the European strains. When tested on pea cv. Rondo the majority of the Middle East strains belonged to the medium or low effective classes and only a few strains were comparable with European Rhizobium strains.Dutch Rhizobium strains induced effective nodules on both the Dutch pea cv. Rondo and the Swedish cv. L 110. However, in association with a Turkish Rhizobium strain effective nodules were formed on pea cv. Rondo and ineffective nodules on cv. L 110.We suggest that the genetic uniformity of EuropeanR. leguminosarum strains is the result of selection and domestication of Rhizobium strains originally derived from the gene centres of the pea plant.     

Effetto della rimozione dei fiori sulla crescita delle piante e sul contenuto in leghemoglobina dei tubercoli radicali di pisello e fava

Abstract

Observations on vegetative growth and leghemoglobin contents of root nodules of pea and bean plants after flower bud removal. — These studies found their origin in the papers by MATTIROLO (1899) on the effect of the removal of flowers as they formed in bean plants; he observed that deflowering resulted in extraordinary plant growth, stem branching and flower buds formation as well as in a delayed root nodule senescence. In the light of modern knowledge of the leghemoglobin role in symbiotic nitrogen fixation, the aim of the present research was to ascertain any possible relation between flower bud removal and haeme pigment contents in the root nodules. The experiments were carried out during two growing seasons (1966 and 1970) using Vicia faba L. cv. Regina and Pisum sativum L. cv. Senatore during 1966 and cv. Vittoria in 1970. In both control and test plants the seasonal trends of average plant height, fresh and dry weight of vegetative portions, fresh and dry weight of root system, fresh weight of nodules, root nodule leghemoglobin concentration and total leghemoglobin content per plant, were determined. The data obtained are quoted in Table 2 and reported in Figures 1, 2, and 3. The removal of flower buds caused in both species: an increased plant growth, a marked stem branching, a longer blooming period, an increased flower number, an increased root nodule number and a certain delay in root nodule reabsorption. Deflowering did not significantly extend — at least in the species studied — life span (senescence was delayed only of one week). On the basis of these and of other Authors' results, we conclude that deflowering may actually delay senescence; the size of this delay, however, depends on the plant species considered and is fairly negligible both in pea and bean. The different effects of deflowering and of preventing floral induction on life span extension, are discussed, and these facts lead to consider floral induction as the onset of a chain of processes leading annual plants toward senescence in a more or less delayable, but definitive way. After having stressed the generally accepted importance of leghemoglobin concentration as an index of nodule nitrogen fixing ability, a correlation between biomass increase of test plants and number and total weight increase of root nodules, is put in evidence. No correlation between test plant biomass and the leghemoglobin concentration in root nodules, was however observed. Leghemoglobin concentration in root nodules is known to change in connection with various factors depending either on host plants and on Rhizobium strains and also in connection with several environmental conditions. Any prevented flower onset (ROPONEN and VIRTANEN, 1968) and deflowering (our data) however exerted no significant influence. The effects of flower bud removal were therefore the following: increased stem, leaf and root weights and increased root nodule number; no difference between control and test plants was however observed as regards size and leghemoglobin concentration of root nodules and hence probably no difference as regards their nitrogen fixing ability.     

Isolation and symbiotic characterization of aromatic amino acid auxotrophs of Sinorhizobium meliloti

Ten aromatic amino acid auxotrophs of Sinorhizobium meliloti (previously called Rhizobium meliloti) Rmd201 were generated by random mutagenesis with transposon Tn5 and their symbiotic properties were studied. Normal symbiotic activity, as indicated by morphological features, was observed in the tryptophan synthase mutants and the lone tyrosine mutant. The trpE and aro mutants fixed trace amounts of nitrogen whereas the phe mutant was completely ineffective in nitrogen fixation. Histology of the nodules induced by trpE and aro mutants exhibited striking similarities. Each of these nodules contained an extended infection zone and a poorly developed nitrogen fixation zone. Transmission electron microscopic studies revealed that the bacteroids in the extended infection zone of these nodules did not show maturation tendency. A leaky mutant, which has a mutation in trpC, trpD, or trpF gene, was partially effective in nitrogen fixation. The histology of the nodules induced by this strain was like that of the nodules induced by the parental strain but the inoculated plants were stunted. These studies demonstrated the involvement of anthranilic acid and at least one more intermediate of tryptophan biosynthetic pathway in bacteroidal maturation and nitrogen fixation in S. meliloti. The alfalfa plant host seems to provide tryptophan and tyrosine but not phenylalanine to bacteroids in nodules.     

Cultivar differences in assimilate partitioning and capacity to maintain N2 fixation rate in pea during pod-filling

Effects of plant development on the rate of N₂ fixation and assimilate partitioning in pea were investigated. In growth cabinets, N₂ fixation declined with the onset of pod-filling in a small, determinant cultivar of field pea (cv. Express). In contrast, in a larger, indeterminant variety (cv. Century) N₂ fixation rate did not peak until several weeks into the pod-filling period. The smaller cultivar, Express, fixed 66% less nitrogen than the cultivar Century. Dry matter and nitrogen content increased during pod-filling in nodules but declined, or held steady in leaves, stems, and roots for Century. This indicates that nodules could compete successfully with pods for assimilates during pod-filling. In contrast, dry matter and nitrogen content did not increase in all non-reproductive plant parts (including nodules) for the smaller cultivar, Express. Under field conditions, rates of N₂ fixation declined severely for cv. Century with the onset of pod-filling. It is proposed that maintenance of the rate of N₂ fixation with the onset of pod-filling is dependent on genetic and environmental factors which influence the source-to-sink ratio of carbon in the plant at the start of pod-filling. This hypothesis is incorporated into a proposed scheme of how to maximize nitrogen accumulation by a legume in a growing season.     

On the efficiency of legume supernodulating mutants

The supernodulating mutants of legumes lack the internal regulation of the number of symbiotic root nodules that harbour N₂-fixing nodule bacteria. On one hand, these mutants represent an efficient tool for dramatic increase in the degree of rhizobial symbiosis development. The trait of released nodulation is often associated with the desirable resistance of nodule initiation and functioning to the inhibition by ambient nitrate. On the other hand, the more intense and stable atmospheric nitrogen fixation of supernodulated plants is devalued by plant growth depression that results from the disproportion between the photosynthetic capacity of the shoot and the catabolic demands of symbiotic nodules. The deleterious effects of excessive nodulation can be neutralised or alleviated by a breeding strategy aimed at creating an ideotype of N₂-fixing legume. The growth depression can be diminished by the reduction in the nodule number typical for supernodulators, that is, 6–10-fold of the wild type, to the level found permissive for the particular crop. This shift should be accompanied with breeding aimed at the increased photosynthetic capacity of the shoot. Forage varieties of legumes represent a reserve of high photosynthetic and shoot growth capacity, thanks to a long-term breeding history for green biomass accumulation. Moreover, the deleterious effects of supernodulation are less perceived after introgression into the background of forage varieties in view of different criteria in their evaluation, such as nitrogen accumulation and biomass production per crop area unit. The growth of supernodulators can be further corrected by breeding for auxiliary traits such as long-vine shoot architecture, a longer vegetation period and late flowering. The same strategy is applicable to the compensation for inherent pleiotropic changes in plant development, which are often associated with primarily symbiotic mutations. Supporting evidence for the efficiency of the described approach has already been reported.     

Symbiotic N2 fixation in pea and field bean estimated by15N fertilizer dilution in field experiments with barley as a reference crop

Summary The total amount of nitrogen derived from symbiotic nitrogen fixation in two pea and one field bean cultivar, supplied with 50 kg N ha⁻¹ at sowing (‘starter’-N), was estimated to 165, 136, and 186 kg N ha⁻¹, respectively (three-year means). However, estimates varied considerably between the three years. At the full bloom/flat pod growth stage from 30 to 59 per cent of total N₂ fixation had taken place. The proportion of total N derived from N₂ fixation at maturity was higher in seeds than in vegetative plant parts and amounted to 59.5, 51.3 and 66.3 per cent of total above-ground plant N in the two pea cultivars and field bean, respectively (three-year means). The recovery of fertilizer N was 62.2, 70.2, 52.1, and 69.5 per cent in the two pea cultivars, field bean and barley, respectively. Growth analysis indicated that barley did not meet the claims for an ideal reference crop in the¹⁵N fertilizer dilution technique for estimating N₂ fixation in pea and field bean. ‘Starter’-N neither increased the seed yield nor the N content of the grain legumes.     

A Medicago truncatula Tobacco Retrotransposon Insertion Mutant Collection with Defects in Nodule Development and Symbiotic Nitrogen Fixation

A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation. Primary screening of 9,300 mutant lines yielded 317 lines with putative defects in nodule development and/or nitrogen fixation. Of these, 230 lines were rescreened, and 156 lines were confirmed with defective symbiotic nitrogen fixation. Mutants were sorted into six distinct phenotypic categories: 72 nonnodulating mutants (Nod-), 51 mutants with totally ineffective nodules (Nod+ Fix-), 17 mutants with partially ineffective nodules (Nod+ Fix+/-), 27 mutants defective in nodule emergence, elongation, and nitrogen fixation (Nod+/- Fix-), one mutant with delayed and reduced nodulation but effective in nitrogen fixation (dNod+/- Fix+), and 11 supernodulating mutants (Nod++Fix+/-). A total of 2,801 flanking sequence tags were generated from the 156 symbiotic mutant lines. Analysis of flanking sequence tags revealed 14 insertion alleles of the following known symbiotic genes: NODULE INCEPTION (NIN), DOESN'T MAKE INFECTIONS3 (DMI3/CCaMK), ERF REQUIRED FOR NODULATION, and SUPERNUMERARY NODULES (SUNN). In parallel, a polymerase chain reaction-based strategy was used to identify Tnt1 insertions in known symbiotic genes, which revealed 25 additional insertion alleles in the following genes: DMI1, DMI2, DMI3, NIN, NODULATION SIGNALING PATHWAY1 (NSP1), NSP2, SUNN, and SICKLE. Thirty-nine Nod- lines were also screened for arbuscular mycorrhizal symbiosis phenotypes, and 30 mutants exhibited defects in arbuscular mycorrhizal symbiosis. Morphological and developmental features of several new symbiotic mutants are reported. The collection of mutants described here is a source of novel alleles of known symbiotic genes and a resource for cloning novel symbiotic genes via Tnt1 tagging.     

Gene centres,a source for genetic variants in symbiotic nitrogen fixation: Host-induced ineffectivity inPisum sativum ecotype fulvum

Summary Pisum sativum ecotype fulvum forms ineffective nodules with Rhizobium strains, isolated from effective nodules of the cultivated pea in Europe. Rhizobium strains isolated from nodules of fulvum peas in Israel are fully effective on this host plant, but in association with the cultivated pea they induce nodules of poor N₂-fixing activity. The distribution of these fulvum-specific Rhizobium strains is restricted to regions where the fulvum pea occurs naturally. Rhizobium strains from other geographical regions induce mainly ineffective, or partially effective nodules on fulvum plants.A wide genetic variation, with regard to symbiotic response to a standard set of Rhizobium strains, was demonstrated in the fulvum plants collected in Israel. Based on variation in N₂-fixation three groups of plants can be distinguished. These plants offer the possibility for the study of host-genetic control on symbiotic nitrogen fixation.     

Influence of boron and calcium on the tolerance to salinity of nitrogen-fixing pea plants

The effects of different levels of B (from 9.3 to 93 M B) and Ca (from 0.68 to 5.44 mM Ca) on plant development, nitrogen fixation, and mineral composition of pea (Pisum sativum L. cv. Argona) grown in symbiosis with Rhizobium leguminosarum bv. viciae 3841 and under salt stress, were analysed. The addition of extra B and extra Ca to the nutrient solution prevented the reduction caused by 75 mM NaCl of plant growth and the inhibition of nodulation and nitrogen fixation. The number of nodules recovered by the increase of Ca concentration at any B level, but only nodules developed at high B and high Ca concentrations could fix nitrogen. Addition of extra B and Ca during plant growth restored nodule organogenesis and structure, which was absolutely damaged by high salt. The increase in salt tolerance of symbiotic plants mediated by B and Ca can be co-related with the recovery of the contents of some nutrients. Salinity produced a decrease of B and Ca contents both in shoots and in nodulated roots, being increased by the supplement of both elements in the nutrient solution. Salinity also reduced the content in plants of other nutrients important for plant development and particularly for symbiotic nitrogen fixation, as K and Fe. A balanced nutrition of B and Ca (55.8 M B, 2.72 mM Ca) was able to counter-act the deficiency of these nutrients in salt-stressed plants, leading to a huge increase in salinity tolerance of symbiotic pea plants. The necessity of nutritional studies to successfully cultivate legumes in saline soils is discussed and proposed.     

Effect of mutations in the pea genes <Emphasis Type="Italic">Sym33</Emphasis> and <Emphasis Type="Italic">Sym40</Emphasis>

Two pea (Pisum sativum L.) symbiotic mutants SGEFix(-)-1 (sym40) and SGEFix(-)-2 (sym33) with abnormalities in infection thread development and function in symbiotic root nodules have been characterised in terms of mycorrhizal colonisation of roots, shoot and root biomass accumulation and shoot and root phosphorus (P) content. The mutation in gene sym33 decreased mycorrhizal colonisation of roots (except arbuscule abundance in mycorrhizal root fragments, which increased) but did not change the effectiveness of mycorrhiza function. The mutation in sym40 did not affect either of these processes. Both mutants showed differences in plant development compared with the wild-type line SGE. The mutants had delayed flowering and pod ripening, and shoot/root biomass ratios and P accumulation also differed from those of SGE. These observations suggest that the gene mutations cause systemic changes in plant development.