Inheritance of Resistance to Crown Gall in Pisum sativum

We screened a total of 1365 pea (Pisum sativum) lines for response to inoculation with Agrobacterium tumefaciens, strain B6, and characterized resistance in one cultivar, Sweet Snap. Sweet Snap seedlings were highly resistant to tumorigenesis under most conditions. Resistance was overcome at inoculum concentrations of greater than 10⁹ bacteria per milliliter. At such high concentrations, very small tumors developed on Sweet Snap in response to four wide-host-range Agrobacterium strains, but tumors on other cultivars were two-to sevenfold larger than those that formed on Sweet Snap. The hypervirulent strain A281 induced larger tumors on Sweet Snap than did other Agrobacterium strains, but tumors on other genotypes were more than 100% larger than those on Sweet Snap. Physiological experiments suggested that tumorigenesis in Sweet Snap is not blocked in early stages of infection, and genetic analysis indicated that inheritance of resistance to crown gall is a quantitative trait. In addition to the observed resistance in Sweet Snap, three `supersusceptible' genotypes, which developed very large tumors, also were identified.     

Difference between uracilylalanine synthases and cysteine synthases in Pisum sativum

Purification of cysteine synthase from seedlings of pea (Pisum sativum) reveals the presence of three forms of this enzyme, separated by chromatography on DEAE-Sephadex A-50, and also differences between the cysteine- and uracilylalanine-synthases. Isoenzymes A and B of pea cysteine synthase were purified about 1200-fold and had specific activities of 933 U/mg protein and 892 U/mg protein, respectively. Both isoenzymes were found to have the same M_r (52 000) and to dissociate into two identical subunits (M_r 26 000). The K_m value of isoenzyme A is 2.1 mM for O-acetyl-l-serine (OAS) and 36 μM for sulphide, while that of isoenzyme B is 2.3 mM for OAS and 38 μM for sulphide. None of the three isoenzymes from pea seedlings catalyses the formation of the uracilylalanines l-willardiine and l-isowillardiine from OAS and uracil, although isoenzyme A catalyses the formation of β-cyano-l-alanine, and isoenzyme C catalyses the formation of l-quisqualic acid and l-mimosine. Other significant differences occur in the substrate specificity of the three isoenzymes. Several properties, including the amino acid composition of the purified cysteine synthase isoenzymes, are also described.     

Relationship between gibberellin, ethylene and nodulation in Pisum sativum

? Gibberellin (GA) deficiency resulting from the na mutation in pea (Pisum sativum) causes a reduction in nodulation. Nodules that do form are aberrant, having poorly developed meristems and a lack of enlarged cells. Studies using additional GA-biosynthesis double mutants indicate that this results from severe GA deficiency of the roots rather than simply dwarf shoot stature. ? Double mutants isolated from crosses between na and three supernodulating pea mutants exhibit a supernodulation phenotype, but the nodule structures are aberrant. This suggests that severely reduced GA concentrations are not entirely inhibitory to nodule initiation, but that higher GA concentrations are required for proper nodule development. ? na mutants evolve more than double the amount of ethylene produced by wild-type plants, indicating that low GA concentrations can promote ethylene production. The excess ethylene may contribute to the reduced nodulation of na plants, as application of an ethylene biosynthesis inhibitor increased na nodule numbers. However, these nodules were still aberrant in structure. ? Constitutive GA signalling mutants also form significantly fewer nodules than wild-type plants. This suggests that there is an optimum degree of GA signalling required for nodule formation and that the GA signal, and not the concentration of bioactive GA per se, is important for nodulation.     

The same allele of translation initiation factor 4E mediates resistance against two Potyvirus spp. in Pisum sativum

Pathogenicity of two sequenced isolates of Bean yellow mosaic virus (BYMV) was established on genotypes of Pisum sativum L. reported to carry resistance genes to BYMV and other potyviruses. Resistance to the white lupin strain of BYMV (BYMV-W) is inherited as a recessive gene named wlv that maps to linkage group VI together with other Potyvirus resistances. One of these, sbm1, confers resistance to strains of Pea seedborne mosaic virus and previously has been identified as a mutant allele of the eukaryotic translation initiation factor 4E gene (eIF4E). Sequence comparison of eIF4E from BYMV-W-susceptible and -resistant P. sativum genotypes revealed a polymorphism correlating with the resistance profile. Expression of eIF4E from susceptible plants in resistant plants facilitated BYMV-W infection in inoculated leaves. When cDNA of BYMV-W was agroinoculated, resistance mediated by the wlv gene frequently was overcome, and virus from these plants had a codon change causing an Arg to His change at position 116 of the predicted viral genome-linked protein (VPg). Accordingly, plants carrying the wlv resistance gene were infected upon inoculation with BYMV-W derived from cDNA with a His codon at position 116 of the VPg coding region. These results suggested that VPg determined pathogenicity on plants carrying the wlv resistance gene and that wlv corresponded to the sbm1 allele of eIF4E.     

Growth substances and the relation between phenotype and genotype in Pisum sativum

Summary Reciprocal grafts between plants of the tall variety Alaska and the dwarf Progress No. 9 show that neither roots nor mature leaves determine shoot phenotype. It is demonstrated that differences in stem growth between the two varieties are essentially controlled by a single Mendelian factor, and the effect of this Le locus is not graft transmissible. Combined with published data for gibberellin content this confirms that the Le locus does not control shoot phenotype by regulating gibberellin synthesis. Growth of slender plants (Le la cry ^s ) and early growth of microcryptodwarfs (le la cry ^c lm) is not inhibited by AMO-1618 at concentrations which greatly reduce growth of tall plants. This is consistent with the suggestion that rapid growth in these varieties, in the absence of the inhibitory effect of La and Cry, is not dependent on endogenous gibberellin.The work was supported by United States Atomic Energy Commission Contract No AT (11-1)-1338 wilhe Dr. A.J.McComb was on leave from the University of Western Australia.     

Carbon Transfer and Partitioning between Vegetative and Reproductive Organs in Pisum sativum L

Assimilate partitioning was studied in the common pea (Pisum sativum L.) by feeding ¹⁴CO₂ to whole plants and measuring radioactivity in different organs 48 hours after labeling. Two experimental protocols were used. For the first, one reproductive node was darkened with an aluminum foil, to prevent photosynthesis during labeling. The aim was to study assimilate translocation among nodes. The second was carried out to assess any priority among sinks. Whole plants were shaded, during labeling, to reduce carbon assimilation. Various developmental stages between the onset of flowering and the final stage in seed abortion of the last pod were chosen for labeling. When all photosynthetic structures at the first reproductive node were darkened at any stage of development after the formation of the first flower, the first pod was supplied with assimilates from other nodes. In contrast, later developed pods, when photosynthetic structures at their node were darkened, received assimilates from other nodes only when they were beyond their final stage in seed abortion. Reducing illumination to 30% did not change distribution of assimilated carbon between vegetative and reproductive structures, nor among pods. It appears that the relative proportion of ¹⁴C allocated to any one pod, compared to other pods, depends on the dry weight of that pod as a proportion of the total reproductive dry weight. When the plant was growing actively, following the start of the reproductive phase until a few days before the end of flowering, the top of the plant (i.e., all the organs above the last opened flower) had a higher sink strength and a higher relative specific activity than pods, suggesting that it was a more competitive sink for assimilates. The pattern of assimilate distribution described here provides an explanation for pod and seed abortion.     

Morphological and cytological abnormalities in a homozygous interchange in Pisum sativum

Plants homozygous for an interchange in the long arms of chromosomes 3 and 5 were selected from the progeny of translocated heterozygotes through mitotic chromosome analysis. While a deficit of translocated homozygotes was observed in these segregating families, an increase in the proportion of normal plants was obtained. The translocated homozygotes showed a bushy, dwarfed and weak form coupled with nearly complete sterility. Meiosis turned out to be normal except for the presence of anaphase bridges that occurred in about 10 percent of the pollen mother cells. However, vegetative and floral buds degenerated in the majority of cases before pods developed. In mitosis, anaphase bridges were observed. Their frequency was 30 percent and they were formed by only one of the exchanged chromosomes. Such bridges are considered a consequence of the so-called point errors, which are suggested as the main cause of developmental disturbances.The investigations were supported by the Ministry of Education and Sciences of the Federal Republic of Germany and by the Association Euratom-Ital at Wageningen, The Netherland.     

Correlation between callus growth and nodule number in pea (Pisum sativum)

A comparative study of nodulation and callusing in seven (Pisum sativum) varieties has been carried out. The nodulation was characterized by number of nodules and their mass per plant. The frequency of various weight nodules was described by the Lorentz equation. Growth of callus was described using an S-shaped dependent equation. It was shown that plants can produce a maximal (optimum for nitrogen fixation) mass of nodules via an increase in either the nodule number or each nodule mass. This feature is specific to the pea variety. It was also found that the varieties the plants of which have a large number of nodules produce calluses of maximum size. Small calluses are characteristic for varieties having plants with a small amount of large nodules. The data we obtained are interpreted on the assumption that both differentiation and proliferation underlie the nodulation, whereas callusing is based only on the proliferation process.     

A relationship between irradiation, carbohydrates and rooting in cuttings of Pisum sativum

Rooting ability was studied for cuttings derived from pea plants ( Pisum sativum , L. cv. Alaska) grown in controlled environment rooms. When the cuttings were rooted at 70 μmol m⁻² s⁻, ¹ (photosynthetic photon flux density) or more, a stock plant irradiance at 100 μmol m⁻² s⁻¹ decreased rooting ability in cuttings compared to 5 μmol m⁻², s⁻¹, However, cuttings rooted at 160 μmol m⁻² s⁻¹ formed more roots compared to 5 (μmol m⁻² s⁻¹. Although a high irradiance increased the number of roots formed, it could not overcome a decreased potential for root formation in stock plants grown at high irradiance. Light compensation point and dark respiration of cuttings decreased by 70% during the rooting period, and the final levels were strongly influenced by the irradiance to the cuttings. Respiratory O₂ uptake decreased in the apex and the base of the cutting from day 2 onwards, whereas a constant level was found in the leaves. Only the content of extractable fructose, glucose, sucrose and starch varied during the early part of the rooting period. We conclude that the observed changes in the cuttings are initiated by excision of the root system, and are not involved in the initiation of adventitious roots.     

Predicted sequence and structure of a vegetative lectin in Pisum sativum

We report the predicted sequence of four vegetative homologues (Blec1,2,3 and 4) of the pea seed lectin. This study indicates that, in contrast to the single-copy pea seed lectin (Kaminski et al., Plant Mol Biol 9: 497–507, 1987), the pea vegetative lectin is transcribed by at least four members of a highly conserved multigene family whose members are only distantly related to the pea seed lectin at the primary amino-acid sequence level. For example, Blec1 shares only 38% amino-acid identity with the pea seed lectin. However, molecular homology modelling predicts that Blec1 probably forms a similar tertiary structure to the pea seed lectin.     

A Comparison between Geotropism and Geoelectric Effect in Pisum sativum and Its Mutant ageotropum

The auxin concentration in roots of Pisum sativum ageotropum was examined by three indirect methods:

• 1) Supply of auxin before geotropic stimulation;
• 2) Lateral placing of the root tip;
• 3) Inhibiting the auxin transport in half of the root.

All the results indicated supraoptimal auxin concentration. When decapitated ageotropum roots were supplied with 1.5 mm long tips from normal Pisum roots their geotropic reactivity was almost restored. The geoelectric potential of stems of Pisum sativum and its mutant ageotropum was measured. In ageotropum stems the geoelectric potential was less and the geotropic reaction appeared later than in the normal stems.     

Interactions between Symbiotic Nitrogen Fixation, Combined-N Application, and Photosynthesis in Pisum sativum

The effect of photosynthetic photon flux density (PPFD) on nitrogen utilization was determined in peas (Pisum sativum L. cv. Alaska) inoculated with Rhizobium leguminosarum and treated with nutrient solutions containing no combined nitrogen, 16 mM NO₃^?, or 16 mM NH₄⁺. Plants were grown under controlled conditions at three PPFD values ranging from severely limiting to nearly saturating. Carboxylation efficiencies and CO₂-exchange rates were highest in the N₂-fixing plants and lowest in plants supplied with NH₄⁺, and they generally increased with increasing PPFD. Photoefficiencies increased with PPFD but did not differ appreciably with the form of nitrogen applied. Nitrogen fixation, calculated from C₂H₂-reduction and H₂-evolution data, was inhibited more by NH₄⁺ than by NO₃^?application. Inhibition was counteracted by increasing PPFD. Percentage nitrogen decreased with increasing PPFD in plants treated with combined nitrogen and increased in the plants dependent on N₂ fixation. The data reveal that photosynthetic efficiency and the capacity to fix N₂ in peas are functions of PPFD and the availability of combined nitrogen and that these two factors are interrelated.     

Resistance to Vanilla Necrosis Potyvirus in Transgenic Nicotiana benthamiana Plants Containing the Virus Coat Protein Gene

The full-length vanilla necrosis potyvirus (VNV) coat protein (CP) gene was introduced into Nicotiana benthamiana plants via Agrobacterium tumefaciens-mediated transformation. Four constructs contained either: sense (+) CP sequence, antisense (-) CP sequence, sense CP sequence with a Kozak's consensus ATG resulting in a change in the first amino acid, or antisense CP sequence with the Kozak's modification. When mechanically inoculated with a high concentration of VNV, one of the plant lines containing the full-length sense CP gene was highly resistant to virus infection. Plants from the resistant lines expressed the CP at a relatively low level compared to susceptible lines containing the same construct. Plants containing the other three constructs were either susceptible or showed delayed symptom expression.     

Synthesis of Phytochelatins and Homo-Phytochelatins in Pisum sativum L

In the roots of pea plants (Pisum sativum L.) cultivated with 20 [mu]M CdCl2 for 3 d, synthesis of phytochelatins [PCs or ([gamma]EC)nG, where [gamma]EC is [gamma]glutamylcysteine and G is glycine] and homophytochelatins [h-PCs, ([gamma]EC)n[beta]-alanine] is accompanied by a drastic decrease in glutathione (GSH) content, but an increase in homoglutathione (h-GSH) content. In contrast, the in vitro activity of GSH synthetase increases 5-fold, whereas h-GSH synthetase activity increases regardless of Cd exposure. The consititutive enzyme PC synthase, which catalyzes the transfer of the [gamma]-EC moiety of GSH to an acceptor GSH molecule thus producing ([gamma]EC)2G, is activated by heavy metals, with Cd and Cu being strong activators and Zn being a very poor activator. Using h-GSH or hm-GSH for substrate, the synthesis rate of([gamma]EC)2[beta]-alanine and [gamma]EC)2-serine is only 2.4 and 0.3%, respectively, of the sythesis rate of ([gamma]EC)2G with GSH as substrate. However, in the presence of a constant GSH level, increasing the concentration of h-GSH or hm-GSH results in increased synthesis of ([gamma]EC)2[beta]-alanine or ([gamma]EC)2-serine, respecively; simultaneously, the synthesis of ([gamma]EC)2G is inhibited. [gamma]EC is not a substrate of PC synthase. These results are best explained by assuming that PC synthase has a [gamma]EC donor binding site, which is very specific for GSH, and a [gamma]EC acceptor binding site, which is less specific and accepts several tripeptides, namely GSH, h-GSH, and hm-GSH.     

UTILLdb, a Pisum sativum in silico forward and reverse genetics tool

The systematic characterization of gene functions in species recalcitrant to Agrobacterium-based transformation, like Pisum sativum, remains a challenge. To develop a high throughput forward and reverse genetics tool in pea, we have constructed a reference ethylmethane sulfonate mutant population and developed a database, UTILLdb, that contains phenotypic as well as sequence information on mutant genes. UTILLdb can be searched online for TILLING alleles, through the BLAST tool, or for phenotypic information about mutants by keywords.     

Virus Resistance Induced with Mannan Sulphates in Hypersensitive Host Plants

Mannan sulphates (MS), obtained by sulphatation of extracellular linear mannan (LM) from Rhodotorula rubra, induced resistance to tobacco mosaic virus (TMV) in hypersensitive plants, retained in isolated protoplasts. MS-induced resistance in tobacco is accompanied by a marked increase of lytic processes in cells and a decrease of total (acidic and alkaline) protein content. In addition to this, new protein components, including PR-proteins and antiviral substances, of the inhibitor of virus replication (IVR) type, appeared in treated plant tissues. MS-induced resistance resembles localized resistance induced by TMV in hypersensitive tobacco plants and could serve as an experimental model for study of the latter.     

Distinct CG and CNG DNA methyltransferases in Pisum sativum

DNA methyltransferase activity, present in low salt extracts of nuclei from young pea shoot apices, has been fractionated into two different species by assaying with model substrates. The CG methyltransferase (an unstable enzyme believed to be of 140 kDa) methylates cytosine only in oligonucleotides with CG and CI dinucleotide targets while an enzyme of 110 kDa (the CNG methyltransferase) methylates the cytosines in 5′-CAG-3′ and 5′-CTG-3′ target sequences, especially when hemimethylated, but not in 5′-CCG-3′ nor in 5′-CGG-3′ target sequences present in oligonucleotides.     

Ethylene formation in Pisum sativum and Vicia faba protoplasts

Protoplasts isolated from leaves of peas (Pisum sativum L.) and of Vicia faba L. produced 1-aminocyclopropane-1-carboxylic acid (ACC) from endogenous substrate. Synthesis of ACC and conversion of ACC to ethylene was promoted by light and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and carbonyl cyanide m-chlorophenylhydrazone. Aminoethoxyvinylglycine inhibited ethylene synthesis to a minor extent when given during incubation of the protoplasts but was very effective when added both to the medium in which the protoplasts were isolated and to the incubation medium as well. Radioactivity from [U-¹⁴C]methionine was incorporated into ACC and ethylene. However, the specific radioactivity of the C-2 and C-3 atoms of ACC, from which ethylene is formed, increased much faster than the specific radioactivity of ethylene. It appears that ACC and ethylene are synthesized in different compartments of the cell and that protoplasts constitute a suitable system to study this compartmentation.Abbreviations ACC 1-Aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CCCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea