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.     

A temperature-sensitive nodulation mutant (sym 5) of Pisum sativum L.

Abstract. In peas ( Pisum sativum L.) homozygous for sym 5, nodulation has an unusual temperature dependence. These sym 5 mutants nodulate poorly at a root temperature of 20°C but nodulate better at 12°C. By lowering the root temperature of the sym 5 mutants from a lightroom temperature of 20/15°C to a constant 12°C, 8d after planting, the number of nodules can be further increased. A cool period (12°C) as short as 6h, early in the infection process, is sufficient to significantly increase nodulation of plants otherwise growing at 20/15°C. This temperature-sensitivity of nodulation is not due to a temperature induced change of a sym 5-related, 66-kD peptide but may involve accumulation of a gas in the rhizosphere.     

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.     

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 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.     

豌豆Sparkle及其单基因突变体E107的铁锰吸收及转移效率

用豌豆Ｓｐａｒｋｌｅ及其单基因突变体Ｅ１０７进行的水培的试验表明,－Ｆｅ和＋Ｆｅ处理的Ｅ１０７幼苗以及－Ｆｅ处理的Ｓｐａｒｋｌｅ幼苗均表现出根系Ｈ＋分泌量大、根系Ｆｅ（Ⅲ）还原力强等特点,其中尤以＋Ｆｅ处理的Ｅ１０７最为突出;而十Ｆｅ处理的Ｓｐａｒｋｌｅ则无以上特点。与Ｓｐａｒｋｌｅ相比,Ｅ１０７各处理的地上部Ｆｅ、Ｍｎ合量均很高,但根部含量则相反。与Ｓｐｅｋｌｅ相比,Ｅ１０７—Ｆｅ处理表现为Ｆｅ高效,即使在＋Ｆｅ处理下,Ｅ１０７仍表现出－Ｆｅ条件下的根系生理特性,活化并还原了根际大量Ｆｅ（Ⅲ）和Ｍｎ,因而它对Ｆｅ、Ｍｎ具有较高的吸收效率,但是这些元素并不在根系中贮存,而是源源不断地运输到地上部,并在叶片中累积乃至使叶片中毒坏死,充分表现了Ｅ１０７单基因突变体对Ｆｅ、Ｍｎ也具有较高的转移效率。     

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.     

Mapping of the nodulation loci sym9 and sym10 of pea ( Pisum sativum L.)

Several mutants defective in the nodulation process during rhizobial or endomycorrhizal endosymbiosis of pea have been identified previously. We have integrated the map positions of two such nodulation mutations, sym9 and sym10, into the molecular map of pea by applying molecular-marker techniques combined with bulked segregant analysis (BSA). Lines P2 and P54 were found to carry alleles of sym9, line P56 carried an allele of sym10. F2 populations were derived from crosses of P2, P54 and P56, to JI281 and JI15, two of the parental lines that have been used previously to generate a molecular map of pea. sym9 was located on linkage group IV by AFLP-BSA analysis and subsequently mapped by RFLP in both F2 populations, P2 2 JI281 and P54 2 JI281. RFLP-BSA analysis was applied to assign sym10 to linkage group I. The RFLP marker locus, chs2, co-segregates with sym10 in the F2 population of P56 2 JI15.     

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.     

Morphological alterations of pea (Pisum sativum cv. Sparkle) arbuscular mycorrhizas as a result of exogenous ethylene treatment

Little is known about the role of phytohormones in the formation of arbuscular mycorrhizas (AM). Although the involvement of ethylene in AM formation is unclear, it is considered very important for several aspects of plant growth and development. The effect of a suspected inhibitory level of ethylene was investigated to help elucidate its role in regulating the formation of AM. In particular, the morphology of AM fungal structures at various stages of the colonization process was documented. Exogenous application of 5.5 ppm ethylene to the substrate resulted in typical morphological changes to Pisum sativum and a significant reduction in the colonization of roots by the AM fungus Glomus aggregatum. Elevated substrate-ethylene did not affect the number of appressoria formed; however, it did result in the formation of abnormal appressoria, which appeared swollen and highly branched. Deformation of appressoria was correlated with a reduction of AM fungal entry into the root tissue, resulting in less colonization by intraradical hyphae and arbuscules. Colonization generally proceeded normally provided the fungal hyphae breached the epidermis, although the extension of colonization units was restricted.     

Separation of mitochondria from microbodies of Pisum sativum (L. cv. Alaska) cotyledons

A method is described for separating mitochondria from microbodies in cotyledon preparations of Pisum sativum L. cv. Alaska. Pure and intact mitochondria were obtained on a continuous: discontinuous sucrose density gradient as shown by marke-enzyme assay and electron microscopy. Manipulation of sucrose-gradient construction to widen the distance between organelles provided a quick method for the separation of the mitochondria from the microbodies. The shorter time of exposure of mitochondria to centrifugation and osmotic stress produces mitochondria free of contamination.     

Induction of Flavonoid Synthesizing Enzymes by Light in Etiolated Pea (Pisum sativum cv. Midfreezer) Seedlings

Etiolated pea (Pisum sativum cv. Midfreezer) seedlings respond to illumination with white light by changes in the activity of phenylpropanoid and flavonoid synthesizing enzymes. Unlike in cell cultures, changes in enzyme activity in pea seedlings are not concerted. Phenylalanine ammonia-lyase (EC 4.3.1.5) activity peaked approximately 18 hours after onset of illumination. The phenylacetate path did not interfere with the measurement of phenylalanine ammonia-lyase activity. Activity of cinnamic acid 4-hydroxylase (EC 1.14.13.11) showed an early peak after 8 hours illumination, declined thereafter sharply, then gradually increased during the remainder of the experiment. Activities of chalcone synthase and UDP glucose:flavonol 3-O-glucosyltransferase (EC 2.4.1.91) increased steadily and reached a plateau after approximately 70 hours illumination time. Activity of 4-hydroxycinnamate:coenzyme A ligase (EC 6.2.1.12) remained relatively unchanged, whereas that of chalcone isomerase (EC 5.5.1.6) declined steadily during the course of the experiment. The relative in vitro enzyme activities suggest that the rate-limiting step for the phenylpropanoid path is the cinnamic acid 4-hydroxylase, that of the flavonoid pathway is the chalcone synthase. Integration of enzyme activity curves, however, show that only the curve deriving from phenylanine ammonia-lyase activity matches closely the production of the flavonol glycosides.     

Carbon Dioxide Photoassimilation in Normal-leaved and Mutant Forms of Pisum sativum L.

The reproducible steady-state carbon dioxide (CO₂) photoassimilationpotentials of three mutants and a normal form of pea (Pisumsativum L.) have been compared. The three mutants studied differed markedly in foliar morphology:genotype af af Tl Tl had leaflets converted to tendrils; AfAf tl tl had tendrils converted to leaflets; af af tl tl hadrelatively minute leaflets on a branched petiole. Interest layprimarily in the phenotype with only tendrils since it provideda potential means of reducing the volume of haulm that has tobe rapidly processed in the case of vining peas, and dried inthe case of harvest peas. These mutants had been derived from relatively unimproved cultivars.Before completion of the lengthy backcrossing required to makea full assessment of the value of such mutants an interim studyusing infra red gas analysis indicated that, in terms of CO₂ photoassimilation perunit area of youngest expanded attached leaf of glasshouse-grownplants, the mutants were comparable to normal. The phenotypewith only tendrils was the least efficient of those assayedat utilizing light of an intensity below 100 J m² sec¹ (400–700nm), and on a unit dry-weight basis it was only 18 per centas efficient as a normal-leaved pea. The other mutants werecomparable to normal in this respect. Comparison of CO₂ photoassimilation of glasshouse-grown andfield-grown plants showed them to be similar though they differedin dry weight, transpiration, and dark respiration.     

The pea (Pisum sativum L.) genes sym33 and sym40 control infection thread formation and root nodule function

Two novel non-allelic mutants that were unable to fix nitrogen (Fix^?) were obtained after EMS (ethyl methyl sulfonate) mutagenesis of pea (Pisum sativum L.). Both mutants, SGEFix^?–1 and SGEFix^?–2, form two types of nodules: SGEFix^?–1 forms numerous white and some pink nodules, while mutant SGEFix^?–2 forms white nodules with a dark pit at the distal end and also some pinkish nodules. Both mutations are monogenic and recessive. In both lines the manifestation of the mutant phenotype is associated with the root genotype. White nodules of SGEFix^?–1 are characterised by hypertrophied infection threads and infection droplets, mass endocytosis of bacteria, abnormal morphological differentiation of bacteroids, and premature degradation of nodule symbiotic structures. The structure of the pink nodules of SGEFix^?–1 does not differ from that of the parental line, SGE. White nodules of SGEFix^?–2 are characterised by “locked” infection threads surrounded with abnormally thick plant cell walls. In these nodules there is no endocytosis of bacteria into host-cell cytoplasm. The pinkish nodules of SGEFix^?–2 are characterised by virtually undifferentiated bacteroids and premature degradation of nodule tissues. Thus, the novel pea symbiotic genes, sym40 and sym33, identified after complementation analysis in SGEFix^?–1 and SGEFix^?–2 lines, respectively, control early nodule developmental stages connected with infection thread formation and function.     

Gibberellin A20 in seed of Pisum sativum L., cv. Alaska

Summary The main gibberellin in immature seed of Pisum sativum L., cv. Alaska, is identified as GA₂₀ by GC-MS. GA₉ may also be present.     

Induction of Root Nodule Senescence by Combined Nitrogen in Pisum sativum L

Root nodule senescence induced by nitrate and ammonium in Pisum sativum L. was defined by determining nitrogenase activity and leghemoglobin content with the acetylene reduction and pyridine hemochrome assays. Root systems supplied with 100 mm KNO(3) or 100 mm NH(4)Cl exhibited a decrease in nitrogenase activity followed by a decline in leghemoglobin content. Increasing the CO(2) concentration from 0.000320 atm to 0.00120 atm had no effect on the time course of root nodule senescence when 20 mm KNO(3) was supplied to the roots; in vitro nitrate reductase activity was detected in leaves and roots, but not bacteroids. Nitrate appeared in leaves, roots, and the nodule cytosol fraction but not bacteroids when 20 mm KNO(3) was supplied to roots. When nitrate entered through the shoots, however, no root nodule senescence was observed, and no nitrate was detected in root or nodule cytosol fractions although nitrate and nitrate reductase were found in leaves. The results suggest that nitrate does not induce root nodule senescence through competition between nitrate reductase and nitrogenase for products of photosynthesis.     

Isoenzymes of Glutamine Synthetase in Roots of Pea (Pisum sativum L. cv Little Marvel) and Alfalfa (Medicago media Pers. cv Saranac)

Cell organelles have been isolated from protoplast lysates and total homogenates obtained from root tips of Pisum sativum L. (cv Little Marvel) and Medicago media Pers. (cv Saranac) grown in hydroponics with nitrate nutrient solutions. Density-gradient and differential centrifugation procedures have been used to prepare mitochondria-and plastid-enriched fractions in which glutamine synthetase (GS) activity was estimated. Even when purified protoplasts were gently ruptured, significant breakage of plastids occurred during preparation as shown by the high proportion of nitrite reductase recovered in the soluble fraction. Of the total GS activity recovered, up to 20% was associated with the plastid fraction, depending on the source of plant material and the GS assay utilized; when corrected for recovery of the plastid marker nitrite reductase, it was calculated that 15 to 57% of alfalfa and 14 to 64% of pea root GS was located in the plastids. A true biosynthetic assay in which glutamine production was monitored by high performance liquid chromatography was devised to estimate the physiological significance of the transferase and the semibiosynthetic assays currently used for activity measurements. When compared with the true and semibiosynthetic assays, the transferase assay for GS appeared to underestimate the root plastid enzyme. Root plastid GS was partially purified by ion-exchange chromatography, and results show that the isoenzyme found in root plastids is different from chloroplastic or cytosolic GS.