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 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.     

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 μL/L ethylene applied continually to the roots for 3 weeks. The inhibition was overcome by treating roots with 1 μm 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.     

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

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.     

Rhizobium leguminosarum Biovar viciae 1-Aminocyclopropane-1-Carboxylate Deaminase Promotes Nodulation of Pea Plants

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in R. leguminosarum bv. viciae 128C53K enhances the nodulation of P. sativum L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes.     

Induced symbiosis mutants of pea (Pisum sativum) and sweetclover (Melilotus alba annua)

Treatment of Pisum sativum (L.) cv. ‘Sparkle’ with ethylmethane sulfonic acid or γ or neutron radiation induced stable mutants which do not form nodules, or form few nodules. Six mutants were at the previously described sym 5 locus. Non-nodulating mutants of Melilotus alba annua (Desr.) cv. U389 were obtained by treatment with ethylmethane sulfonic acid (EMS) or neutron radiation, but not by γ radiation or azide.     

Phenotypic characterization of sym 21, a gene conditioning shoot-controlled inhibition of nodulation in Pisum sativum cv. Sparkle

E132 ( sym 21) is a stable pleiotropic mutant of Pisum sativum cv. Sparkle obtained by mutagenesis with ethyl methane sulfonic acid. The line forms few nodules and short, highly branched roots. Microscopy studies revealed that infection by rhizobia is normal, and low nodulation is mainly due to a low rate of emergence of the nodule meristems. E132 shoots depressed nodulation on Sparkle stocks, whereas in reciprocal grafts more nodules formed on E132 stocks than on control roots or self-grafted Sparkle plants. Nodule number on the mutant was slightly increased by exogenous ethylene inhibitors, which, however, did not alter the root phenotype.     

Nitrate-induced ethylene biosynthesis and the control of nodulation in alfalfa

We previously reported that inhibition of ethylene biosynthesis with aminoethoxyvinylglycine (AVG) eliminated the inhibitory effect of NO₃^– on nodulation of alfalfa (Medicago sativa L. cv. Aragon) plants grown aeroponically. In this work, the effect of Ag⁺, as an inhibitor of ethylene action, has been studied in plants growing aeroponically or in darkened tubes with vermiculite, and low-nitrate or high-nitrate solution. Vermiculite-grown plants developed up to 3 times as many nodules as did those growing aeroponically. Nodule formation was mirrored by dry-matter accumulation. High (10 mol m^–3) NO₃^– applied from planting inhibited nodulation to an equal extent (c. 50%) in the two growth conditions. In contrast, Ag⁺ treatment increased nodule formation at all NO₃^– concentrations assayed under the two growth conditions, with the stimulation being higher in plants grown aeroponically. Finally, no effect of Ag⁺ (10 mmol m^–3) on plant growth was observed in either of the growth conditions. The effectiveness of NO₃^– as a nodulation inhibitor and enhancer of ethylene biosynthesis in roots of alfalfa was also studied. Within 24 h after inoculation, 10 mol m^–3 NO₃^– exerted most of its inhibitory effect on nodulation. At the same time, both 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase activity and ethylene evolution rates markedly increased in inoculated and uninoculated alfalfa roots treated with NO₃^–. Support for a role of endogenous ethylene in the control of nodule formation in legumes is discussed.     

Effects of cytokinin on ethylene production and nodulation in pea (Pisum sativum) cv. Sparkle

In this study, we were interested in learning if cytokinins play a role in the developmental process that leads to nodulation in the pea cv. Sparkle. We demonstrate that the application of the synthetic cytokinin BAP (6-benzyl-amino-purine) results in a number of nodulation-related changes. BAP stimulates the production of ethylene, a known inhibitor of nodulation. At low levels (up to 1 μ M ), BAP also stimulates nodulation but as its concentration is increased (up to 25 μ M ), nodule number decreases. In BAP-treated roots, the infection threads are abnormal; they are twisted, very knotty, and generally grow in a direction parallel to the root surface. In addition, the centers of cell division in the inner cortex are very few. Thus, BAP-treated Sparkle appears to phenocopy the low-nodulating pea mutant R50 [Guinel FC, Sloetjes LL (2000) Ethylene is involved in the nodulation phenotype of Pisum sativum R50 ( sym 16 ), a pleiotropic mutant that nodulates poorly and has pale green leaves. J Exp Bot 51: 885–894]. However, it appears doubtful that there is a direct correlation between the actions of cytokinin and ethylene in causing a reduction in nodule organogenesis because nodulation is not restored by treating BAP-treated Sparkle with ethylene inhibitors.     

Regulation of soybean nodulation independent of ethylene signaling

Leguminous plants regulate the number of Bradyrhizobium- or Rhizobium-infected sites that develop into nitrogen-fixing root nodules. Ethylene has been implicated in the regulation of nodule formation in some species, but this role has remained in question for soybean (Glycine max). The present study used soybean mutants with decreased responsiveness to ethylene, soybean mutants with defective regulation of nodule number, and Ag⁺ inhibition of ethylene perception to examine the role of ethylene in the regulation of nodule number. Nodule numbers on ethylene-insensitive mutants and plants treated with Ag⁺ were similar to those on wild-type plants and untreated plants, respectively. Hypernodulating mutants displayed wild-type ethylene sensitivity. Suppression of nodule numbers by high nitrate was also similar between ethylene-insensitive plants, wild-type plants, and plants treated with Ag⁺. Ethylene insensitivity of the roots of etr1-1 mutants was confirmed using assays for sensitivity to 1-aminocyclopropane-1-carboxylic acid and for ethylene-stimulated root-hair formation. Additional phenotypes of etr1-1 roots were also characterized. Ethylene-dependent pathways regulate the number of nodules that form on species such as pea and Medicago truncatula, but our data indicate that ethylene is less significant in regulating the number of nodules that form on soybean.     

Molecular mechanism of lipopolysaccharide (LPS) in promoting biomineralization on bacterial surface

Biomineralization on bacterial surface is affected by biomolecules of bacterial cell surface. Lipopolysaccharide (LPS) is the main and outermost component on the extracellular membrane of Gram-negative bacteria. In the present study, the molecular mechanism of LPS in affecting biomineralization of Ag⁺/Cl^? colloids was investigated by taking advantages of two LPS structural deficient mutants of Escherichia coli. The two mutants were generated by impairing the expression of waaP or wbbH genes with CRISPR/Cas9 technology and it induced deficient polysaccharide chain of O-antigen (ΔwbbH) or phosphate groups of core oligosaccharide (ΔwaaP) in LPS structures. There were significant changes of the cell morphology and surface charge of the two mutants in comparing with that of wild type cells. LPS from ΔwaaP mutant showed increased ΔH_ITC upon interacting with free Ag⁺ ions than LPS from wild type cells or ΔwbbH mutant, implying the binding affinity of LPS to Ag⁺ ions is affected by the phosphate groups in core oligosaccharide. LPS from ΔwbbH mutant showed decreased endotherm (ΔQ) upon interacting with Ag⁺/Cl^? colloids than LPS from wild type or ΔwaaP mutant cells, implying LPS polysaccharide chain structure is critical for stabilizing Ag⁺/Cl^? colloids. Biomineralization of Ag⁺/Cl^? colloids on ΔwbbH mutant cell surface showed distinctive morphology in comparison with that of wild type or ΔwaaP mutant cells, which confirmed the critical role of O-antigen of LPS in biomineralization. The present work provided molecular evidence of the relationship between LPS structure, ions, and ionic colloids in biomineralization on bacterial cell surface.     

Rhizobium leguminosarum biovar viciae 1-aminocyclopropane-1-carboxylate deaminase promotes nodulation of pea plants

Ethylene inhibits nodulation in various legumes. In order to investigate strategies employed by Rhizobium to regulate nodulation, the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene was isolated and characterized from one of the ACC deaminase-producing rhizobia, Rhizobium leguminosarum bv. viciae 128C53K. ACC deaminase degrades ACC, the immediate precursor of ethylene in higher plants. Through the action of this enzyme, ACC deaminase-containing bacteria can reduce ethylene biosynthesis in plants. Insertion mutants with mutations in the rhizobial ACC deaminase gene (acdS) and its regulatory gene, a leucine-responsive regulatory protein-like gene (lrpL), were constructed and tested to determine their abilities to nodulate Pisum sativum L. cv. Sparkle (pea). Both mutants, neither of which synthesized ACC deaminase, showed decreased nodulation efficiency compared to that of the parental strain. Our results suggest that ACC deaminase in R. leguminosarum bv. viciae 128C53K enhances the nodulation of P. sativum L. cv. Sparkle, likely by modulating ethylene levels in the plant roots during the early stages of nodule development. ACC deaminase might be the second described strategy utilized by Rhizobium to promote nodulation by adjusting ethylene levels in legumes.     

Promotion ofin vitro shoot formation from excised roots of silktree (Albizzia julibrissin) by an oxime ether derivative and other ethylene inhibitors

Summary This report describes the regeneration response of excised seedling roots of silktree (Albizzia julibrissin) to added ethylene precursors/generators (1-amino-cyclopropane-1-carboxylic acid [ACC], 2-chloroethylphosphonic acid [CEPA]), biosynthesis inhibitors (aminoethoxyvinylglycine [AVG], an oxime ether derivative [OED={[(ispropylidene)-amino]oxy}-acetic acid-2-(methoxy)-2-oxoethyl ester], CoCl² [Co⁺⁺]), and an ethylene action inhibitor (AgNO₃ [Ag⁺]). When placed on B5 medium, about 50% of the control explants formed shoot buds within 15 days. Addition of ACC or CEPA (1–10 µM) to the culture medium decreased both the percentage of cultures forming shoots and the number of shoots formed per culture. In contrast, AVG and OED (1–10 µM) increased shoot formation to almost 100% and increased the number of shoots formed per culture. Likewise, both Co⁺⁺ and Ag⁺ (1–10 µM) increased shoot regeneration, but the number of shoots produced after 30 days was less than with AVG or OED. The inhibitors of ethylene biosynthesis were partially effective in counteracting the inhibitory effect of ACC on shoot formation. These results suggest that modulation of ethylene biosynthesis and/or action can strongly influence the formation of adventitious shoots from excised roots of silktree.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CEPA 2-chloroethylphosphonic acid - OED oxime ether derivative     

Effect of silver ion, carbon dioxide, and oxygen on ethylene action and metabolism

The relationship between ethylene action and metabolism was investigated in the etiolated pea seedling (Pisum sativum L. cv. Alaska) by inhibiting ethylene action with Ag⁺, high CO₂, and low O₂ and then determining if ethylene metabolism was inhibited in a similar manner. Ag⁺ (100 milligrams per liter) was clearly the most potent antiethylene treatment. Ag⁺ pretreatment inhibited the growth retarding action of 0.2 microliters per liter ethylene by 48% and it also inhibited the incorporation of 0.2 microliters per liter ¹⁴C₂H₄ into pea tips by the same amount. As the ethylene concentration was increased from 0.2 to 30 microliters per liter, the effectiveness of Ag⁺ in reducing ethylene action and metabolism declined in a similar fashion. Although Ag⁺ significantly inhibited the incorporation of ¹⁴C₂H₄ into tissue metabolites, the oxidation of ¹⁴C₂H₄ to ¹⁴CO₂ was unaffected in the same tissue.     

Physiological Characterization of a Single-Gene Mutant of Pisum sativum Exhibiting Excess Iron Accumulation: I. Root Iron Reduction and Iron Uptake

Root systems of mutant (E107) and parental (cv `Sparkle') Pisum sativum genotypes were studied to determine the basis for excess Fe accumulation in E107. Plants were grown with (+Fe-treated) or without (−Fe-treated) added Fe(III)-N,N'-ethylenebis[2-(2-hydroxyphenyl)glycine] in aerated nutrient solutions. Daily measurements of Fe(III) reduction indicated a four-to seven-fold higher reduction rate in +Fe- or −Fe-treated E107, and −Fe-treated Sparkle, when compared with +Fe-treated Sparkle. An agarose-based staining technique used to localize Fe(III) reduction, revealed Fe(III) reduction over most of the length of the roots (but not at the root apices) in both E107 treatments and −Fe-treated Sparkle. In +Fe-treated Sparkle, Fe(III) reduction was either nonexistent or localized to central regions of the roots. Measurements of short-term Fe influx (with 0.1 millimolar ⁵⁹Fe(III)-ethylenediaminetetraacetic acid) was also enhanced (threefold) in +Fe- or −Fe-treated E107 and −Fe-treated Sparkle, relative to +Fe-treated Sparkle. The physiological characteristics of E107 root systems, which are similar to those seen in Fe-deficient Sparkle, have led us to conclude that the mutation causes E107 to act functionally as an Fe-deficient plant, and appears to explain the excess Fe accumulation in E107.     

The effect of ethylene on adventitious root formation in mung bean (Vigna radiata) cuttings

A promotive effect of ethylene on the formation of adventitious roots by mung bean cuttings was demonstrated using a recirculating solution culture system to apply dissolved ethylene. The number of roots increased in proportion to the length of exposure to the gas. Mean root numbers per cutting for a 4-day exposure to ethylene and an air control were 45 and 19, respectively. The tissue was most sensitive to a 24-h ethylene “pulse” 2–3 days after taking cuttings. Rooting was maximal at a concentration of 13 μl 1^?1 ethylene. The ethylene treatment inhibited the growth of roots and terminal buds. Application of Ag⁺, as silver thiosulfate, reversed the effect of ethylene on the two growth responses but had no effect on root numbers. Norbornadiene, another inhibitor of ethylene action, reversed all three ethylene responses.     

Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii

Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into Nod⁻R. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod⁺, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod⁺, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod⁻ mutants. All seven mutants were restored to Nod⁺ on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.     

Nitrate inhibition of nodulation can be overcome by the ethylene inhibitor aminoethoxyvinylglycine

Previously, we reported (a) a positive correlation between the nitrate concentrations in growth medium and ethylene evolved from uninoculated and inoculated alfalfa (Medicago sativa) roots and (b) a negative correlation between ethylene evolution and nodulation. Here, we report that the inhibitory effect of NO₃⁻ on nodulation of alfalfa can be eliminated by the ethylene inhibitor aminoethoxyvinylglycine (AVG). This effect was probably related to the strong inhibition (90%) of ethylene biosynthesis caused by AVG in these inoculated and NO₃⁻-treated roots. These results support our hypothesis that the inhibitory effect of NO₃⁻ is mediated through the phytohormone ethylene. A possible role of endogenous ethylene in the autoregulation of nodulation also is discussed. AVG at 10 micromolar significantly (P < 0.05) increased total nitrogenase activity (acetylene reduction) in 2.5 and 5 millimolar NO₃⁻-fed plants probably as a result of the very high stimulation of nodulation.     

Ethylene as a regulator of senescence in tobacco leaf discs

The regulatory role of ethylene in leaf senescence was studied with excised tobacco leaf discs which were allowed to senesce in darkness. Exogenous ethylene, applied during the first 24 hours of senescence, enhanced chlorophyll loss without accelerating the climacteric-like pattern of rise in both ethylene and CO₂, which occurred in the advanced stage of leaf senescence. Rates of both ethylene and CO₂ evolution increased in the ethylene-treated leaf discs, especially during the first 3 days of senescence. The rhizobitoxine analog, aminoethoxy vinyl glycine, markedly inhibited ethylene production and reduced respiration and chlorophyll loss. Pretreatment of leaf discs with Ag⁺ or enrichment of the atmosphere with 5 to 10% CO₂ reduced chlorophyll loss, reduced rate of respiration, and delayed the climacteric-like rise in both ethylene and respiration. Ag⁺ was much more effective than CO₂ in retarding leaf senescence. Despite their senescence-retarding effect, Ag⁺ and CO₂, which are known to block ethylene action, stimulated ethylene production by the leaf discs during the first 3 days of the senescing period; Ag⁺ was more effective than CO₂. The results suggest that although ethylene production decreases prior to the climacteric-like rise during the later stages of senescence, endogenous ethylene plays a considerable role throughout the senescence process, presumably by interacting with other hormones participating in leaf senescence.