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°C. If inhibitors of ethylene formation or action (Co²⁺, 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.     

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.     

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.     

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.     

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.     

An explanation for the light requirement of AgNO3 to inhibit ethylene-induced abscission

The loss of the antiethylene activity of Ag⁺ on leaf abscission by incubation in the dark was investigated. When primary leaves were removed from cuttings of Vigna radiata previously sprayed with AgNO₃, dark-induced abscission of the petioles was inhibited, compared to untreated leafless controls, in the presence or absence of ethephon, an ethylene-releasing compound. Malformin did not negate inhibition of petiole abscission induced by Ag⁺. Although leaf removal restored the antiethylene activity of Ag⁺ in the dark, macerates of leaves from dark-aged cuttings did not negate the ability of Ag⁺ to inhibit petiole abscission in the dark. Abscisic acid completely abolished the ability of Ag⁺ to counteract ethephon-induced leaf abscission in the light, and almost completely abolished the Ag⁺-induced inhibition of petiole abscission from explants in the dark. It is proposed that the phytochrome requirement for the antiethylene activity of Ag⁺ on ethephon-induced leaf abscission involves prevention of the formation, accumulation, or transport of a substance in leaves in the dark which negates Ag⁺ activity. This substance may be abscisic acid or another substance with similar biological activity.     

Gravitropism in Higher Plant Shoots: I. A ROLE FOR ETHYLENE

It has long been known that applied ethylene can redirect the gravitropic response, but only occasionally has it been suggested that ethylene normally plays a role in gravitropism. Two inhibitors of ethylene synthesis [Co²⁺ and aminoethoxyvinylglycine (AVG)] and two inhibitors of ethylene action (Ag⁺ and CO₂) were shown to delay the gravitropic response of cocklebur (Xanthium strumarium L.), tomato (Lycopersicon esculentum Mill.), and castor bean (Ricinus communis L.) stems. Gentle shaking on a mechanical shaker does not inhibit the gravitropic response, but vigorous hand shaking for 120 seconds delays the response somewhat. AVG and Ag⁺ further delay the response of mechanically stimulated plants. AVG delays the response of defoliated and of decapitated plants. Plants laid on their side and restricted so that they cannot bend upward store both bending energy and gravitropic stimulus; they bend immediately when released from restriction (stored energy) and continue to bend for some hours after (stored stimulus). AVG retards the storage of bending energy but not of stimulus. In gravitropism, graviperception may first stimulate ethylene evolution, which may then influence bending directly, or responses involving ethylene could be more indirect.     

Excessive aluminium accumulation in the pea mutant E107 (brz)

E107 is a pleiotropic mutant of peaPisum sativum cv. ‘Sparkle’, characterized by forming few nodules and developing bronze necrotic spots on older leaves. The mutant accumulates Al and has symptoms typical of Al toxicity. The lateral roots of E107 are fewer (40%) and shorter (50%) than those of its parent. High concentrations of Al accumulate in E107 shoots (1000 mg kg^-1) and roots (3000 mg kg^-1), and three-week old E107 plants extrude 2.5 times more protons than ‘Sparkle’ plants of similar age. Al concentrations of the roots of the mutant and of its parent ‘Sparkle’ are similar for the first two weeks of growth. Thereafter they differ. In 2 week old plants Al continues to accumulate in excessive amounts in E107 primary and lateral roots whereas in ‘Sparkle’ roots, it reaches a plateau. In E107, Al is erratically distributed in the walls of root hairs and epidermal cells in both primary and lateral roots. Some of these cells have also Al in their nucleus.     

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.     

SPATIAL AND TEMPORAL DEVELOPMENT OF IRON(III) REDUCTASE ACTIVITY IN ROOT SYSTEMS OF PISUM SATIVUM (FABACEAE) CHALLENGED WITH IRON-DEFICIENCY STRESS

The development of plasma membrane-associated iron(III) reductase activity was characterized in root systems of Pisum sativum during the first 2 wk of growth, as plants were challenged with iron-deficiency stress. Plants of a parental genotype (cv. Sparkle) and a functional iron-deficiency mutant genotype (E107) were grown hydroponically with or without supplemental iron. Iron(III) reductase activity was visualized by placing the roots in an agarose matrix containing 0.2 idm Fe(III)-ethylenediaminetetraacetic acid and 0.3 mM Na₂-bathophenanthrolinedisulfonic acid (BPDS). Red staining patterns, resulting from the formation of Fe(II)-BPDS, were used to identify iron(III)-reducing regions. Iron(III) reduction was extensive on roots of E107 as early as d 7, but not until d 11 for -Fe-treated Sparkle. Roots of +Fe-treated Sparkle showed limited regions of reductase activity throughout the period of study. For secondary lateral roots, iron(III) reduction was found for all growth types except + Fe-treated Sparkle. Treating Sparkle plants alternately to a cycle of iron deficiency, iron sufficiency, and iron deficiency revealed that reductase activity at a given root zone could be alternatively present, absent, and again present. Our results suggest that for Pisum roots grown under the present conditions, iron-deficiency stress induces the activation of iron(III) reductase capacity within 2 d.     

Lateral Bud Growth in Phaseolus vulgaris L. and the Levels of Ethylene in the Bud and Adjacent Tissue

Ethephon and the ethylene inhibitors Ag⁺ and aminoethoxyvinylglycine (AVG) inhibited outgrowth of the axillary bud of thefirst trifoliate leaf in decapitated plants of Phaseolus vulgaris.Endogenous ethylene levels decreased in the stem upon decapitationalthough it is not conclusive that a causal relationship existsbetween this decrease and the release of axillary buds frominhibition. The proposition that auxin-induced ethylene is responsiblefor the suppression of axillary bud growth in the decapitatedplant when the apical shoot is replaced by auxin is not borneout in this study. Application of IAA directly to the axillarybud of intact plants gave rise to a transient increase in budgrowth. This growth increment was annulled when AVG was suppliedwith IAA to the bud despite the fact that the dosage of AVGused did not affect the normal slow growth rate of the bud ofthe intact plant or bud outgrowth resulting from shoot decapitation.     

Does Iron Deficiency in Pisum sativum Enhance the Activity of the Root Plasmalemma Iron Transport Protein?

Roots of Fe-sufficient and Fe-Deficient pea (Pisum sativum L.) were studied to determine the effect of Fe-deficiency on the activity of the root-cell plasmalemma Fe²⁺ transport protein. Rates of Fe(III) reduction and short-term Fe²⁺ influx were sequentially determined in excised primary lateral roots using Fe(III)-ethylene-diaminetetraacetic acid (Fe[III]-EDTA). Since the extracellular Fe²⁺ for membrane transport was generated by root Fe(III) reduction, rates of Fe²⁺ influx for each root system were normalized on the basis of Fe(III) reducing activity. Ratios of Fe²⁺ influx to Fe(III) reduction (micromole Fe²⁺ absorbed/micromole Fe[III] reduced) revealed no enhanced Fe²⁺ transport capacity in roots of Fe-deficient peas (from the parental genotype, Sparkle) or the functional Fe-deficiency pea mutant, E107 (derived from Sparkle), relative to roots of Fe-sufficient Sparkle plants. Data from studies using 30 to 100 micromolar Fe(III)-EDTA indicated a linear relationship between Fe²⁺ influx and Fe(III) reduction (Fe²⁺ generation), while Fe²⁺ influx saturated at higher concentrations of Fe(III)-EDTA. Estimations based on current data suggest the Fe²⁺ transport protein may saturate in the range of 10^−4.8 to 10⁻⁴ molar Fe²⁺. These results imply that for peas, the physiological rate limitation to Fe acquisition in most well-aerated soils would be the root system's ability to reduce soluble Fe(III)-compounds.     

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 μm 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 <sup>59</sup>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.     

Effect of inhibitors of ethylene biosynthesis and action, as well as calcium and magnesium on rose shoot rooting, shoot-tip necrosis and leaf senescence in vitro

Microcuttings of easy-to-root dwarf rose cv. Starina, showing early symptoms of leaf senescence and shoot-tip necrosis in rooting stage, were chosen for the study. The effects of inhibitors of ethylene biosynthesis (AOA, AIB) and action (AgNO₃), and Ca²⁺ and Mg²⁺ were studied in relation to rooting, leaf senescence and shoot-tip necrosis. The effects of these substances were examined with respect to IAA presence in a medium, which stimulated leaf yellowing and shoot-tip necrosis. AOA strongly inhibited rooting of microcuttings, but did not affect ethylene biosynthesis. AIB at 250 mg·l⁻¹ and AgNO₃ 2.5 mg·l⁻¹ in the presence of IAA did not affect rooting but effectively prevented leaf senescence. Ca²⁺ alone or combined with Mg²⁺ at raised concentration, or an ethylene action inhibitor Ag⁺, reduced shoot-tip necrosis in microcuttings treated with IAA. Addition of Ag⁺ to IAA medium drastically increased ethylene production by the shoots. Interaction between endogenous levels of auxin, ethylene and calcium in relation to rooting, shoot-tip necrosis and leaf senescence was discussed. Ethylene could enhance tissue sensitivity to auxin. Moreover, the tissue of rose shoots is very sensitive in the in vitro condition on standard medium because of the calcium deficiency. Thus, the raised Ca/Mg level counteracted shoot-tip necrosis through enhancing cell membrane and wall resistance to ethylene and IAA.     

Ethylene is involved in the nodulation phenotype of Pisum sativum R50 (sym 16), a pleiotropic mutant that nodulates poorly and has pale green leaves

R50 is characterized as a pleiotropic pea mutant; it forms few nodules and has short lateral roots, short stature and pale leaves. Using grafting techniques, R50 paleness was found to be controlled by the shoot of the mutant whereas the nodulation phenotype was regulated by its root. The paleness of R50 is due to a lower than normal total chlorophyll content in its young leaves. The defect appears to be overcome with age because, as the plant matures, the chlorophyll levels increase in the older leaves. The reduction in stature is attributed to shorter internodes, and the oldest internodes are thicker than those of the parent Sparkle. Upon rhizobial inoculation, R50 forms as many infection threads as Sparkle. However, most of these are arrested in the inner cortex. The threads appear to have lost their directional growth towards the stele, and they coil around within enlarged cortical cells. In addition, very few infection threads are associated with divisions of the inner cortical cells. These aborted nodule primordia are abnormal, flat and mainly composed of cells which have divided anticlinally only. Nodulation of R50 was restored by treating the roots with ethylene inhibitors. The R50 mutant further supports the postulated role of ethylene in regulating rhizobial infection with a probable role in the control of the primordium development.     

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.     

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.     

Enhanced Nodulation and Nitrogen Fixation by a Revertant of a Nodulation-Defective Bradyrhizobium japonicum Tryptophan Auxotroph

In greenhouse studies, the symbiotic properties of a prototrophic revertant (TA11 NOD⁺) of a nodulation defective tryptophan auxotroph of Bradyrhizobium japonicum were compared with those of the normally nodulating wild-type strain, B. japonicum I-110 ARS. Strain I-110 ARS was the parent of auxotrophic mutant TA11. Plants inoculated with TA11 NOD⁺ contained significantly more nitrogen per plant than did plants inoculated with wild-type bacteria (275.9 ± 35 versus 184 ± 18 mg). Also, plants that received the revertant were larger, averaging 8.4 ± 0.9 g (dry weight) versus 6.4 ± 0.6 g for those that received the wild-type bacterial strain. Additionally, plants that received the NOD⁺ strain had 56% more nodules and 41% more nodule mass than did control plants. With both inocula, average nodule size and amount of nitrogen fixed per gram of nodule were about the same. These data indicated that the improvement in nitrogen fixation observed with the TA11 NOD⁺ resulted from an increase in the overall nodule number. The physiological basis for this increase in nodulation is not known, but enhanced tryptophan catabolism does not appear to be involved.