DNA Strand-Transfer Activity in Pea (Pisum sativum L.) Chloroplasts

The occurrence of DNA recombination in plastids of higher plants is well documented. However, little is known at the enzymic level. To begin dissecting the biochemical mechanism(s) involved we focused on a key step: strand transfer between homologous parental DNAs. We detected a RecA-like strand transfer activity in stromal extracts from pea (Pisum sativum L.) chloroplasts. Formation of joint molecules requires Mg2+, ATP, and homologous substrates. This activity is inhibited by excess single-stranded DNA (ssDNA), suggesting a necessary stoichiometric relation between enzyme and ssDNA. In a novel assay with Triton X-100-permeabilized chloroplasts, we also detected strand invasion of the endogenous chloroplast DNA by 32P-labeled ssDNA complementary to the 16S rRNA gene. Joint molecules, analyzed by electron microscopy, contained the expected displacement loops.     

Distribution of Secondary Plant Metabolites and Their Biosynthetic Enzymes in Pea (Pisum sativum L.) Leaves : Anthocyanins and Flavonol Glycosides

Leaves of a novel strain of peas (Pisum sativum L.) were used to determine the distribution of secondary metabolites and their biosynthetic enzymes. Leaf epidermal layers in this strain are easily separated from the parenchyma. Anthocyanins and flavonol glycosides were localized in epidermal vacuoles only. Among the biosynthetic enzymes studied, phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), S-adenosyl-1-methionine (SAM):caffeic acid and SAM:quercetin methyltransferases (o-dihydric phenol methyltransferase, EC 2.1.1.42) and a flavonoid 7-O-glucosyltransferase (EC 2.4.1.91) were chiefly localized in the parenchyma, whereas trans-cinnamate 4-monooxygenase (EC 1.14.13.11), hydroxycinnamate:CoA ligases (EC 6.2.1.12) and a flavonoid 3-O-glucosyltransferase (EC 2.4.1.91) were found mainly in the epidermis. Flavanone (chalcone) synthase activity was found only in the epidermis, whereas chalcone isomerase (EC 5.5.1.6) was evenly distributed in epidermal and parenchyma tissues.     

Properties of an Aminotransferase of Pea (Pisum sativum L.)

A transaminase (aminotransferase, EC 2.6.1) fraction was partially purified from shoot tips of pea (Pisum sativum L. cv. Alaska) seedlings. With α-ketoglutarate as co-substrate, the enzyme transaminated the following aromatic amino acids: d,l-tryptophan, d,l-tyrosine, and d,l-phenylalanine, as well as the following aliphatic amino acids: d,l-alanine, d,l-methionine, and d,l-leucine. Of other α-keto acids tested, pyruvate and oxalacetate were more active than α-ketoglutarate with d,l-tryptophan. Stoichiometric yields of indolepyruvate and glutamate were obtained with d,l-tryptophan and α-ketoglutarate as co-substrates. The specific activity was three times higher with d-tryptophan than with l-tryptophan.     

Isozymes of beta-N-Acetylhexosaminidase from Pea Seeds (Pisum sativum L.)

Four isozymes of β-N-acetylhexosaminidase (β-NAHA) from pea seeds (Pisum sativum L.) have been separated, with one, designated β-NAHA-II, purified to apparent homogeneity by means of an affinity column constructed by ligating p-aminophenyl-N-acetyl-β-d-thioglucosaminide to Affi-Gel 202. The other three isozymes have been separated and purified 500- to 1750-fold by chromatography on Concanavalin A-Sepharose, Zn²⁺ charged immobilized metal affinity chromatography, hydrophobic chromatography, and ion exchange chromatography on CM-Sephadex. All four isozymes are located in the protein bodies of the cotyledons. The molecular weight of each isozyme is 210,000. β-NAHA-II is composed of two heterogenous subunits. The subunits are not held together by disulfide bonds, but sulfhydryl groups are important for catalysis. All four isozymes release p-nitrophenol from both p-nitrophenyl-N-acetyl-β-d-glucosaminide and p-nitrophenyl-N-acetyl-β-d-galactosaminide. The ratio of activity for hydrolysis of the two substrates is pH dependent. The K_m value for the two substrates and pH optima of the isozymes are comparable to β-NAHAs from other plant sources.     

Kinetin Transport to Axillary Buds of Dwarf Pea (Pisum sativum L.)

Decapitation resulted in the transport of significant amountsof ¹⁴C to the axillary buds from either point of application,but pretreatment of the cut internode surface of decapitatedplants with IAA (alone or in combination with unlabelled kinetin)inhibited the transport of label to the axillary buds and resultedin its accumulation in the IAA-treated region of the stem. Inintact plants to which labelled kinetin was applied to the apicalbud there was little movement of ¹⁴C beyond the internode subtendingthis bud; when labelled kinetin was applied to the roots ofintact plants, ¹⁴C accumulated in the stem and apical bud butwas not transported to the axillary buds. A considerable proportionof the applied radioactivity became incorporated into ethanol-insoluble/NaOH-solublecompounds in the apical bud of intact plants, in internodestreated with IAA, and in axillary buds released from dominanceby removal of the apical bud. The results are discussed in relation to the possible role ofhormone-directed transport of cytokinins m the regulation ofaxillary bud growth.     

Seed and Pod Wall Development in Pisum sativum, L. in Relation to Extracted and Applied Hormones

Developing fruits of Pisum sativum, L., cv. ‘Alaska’,contain relatively large amounts of hormones, mainly concentratedin the embryos and liquid endosperm. A close relationship canbe demonstrated between changes in extractable amounts of gibberellins(mainly GA₂₀), auxins (methyl 4-chloroindol-3yl acetate andprobably 4-chloroindol-3yl acetic acid), and abscisic acid,and changes in growth rates of both the pod wall and seeds.Growth of the pod wall appears to depend largely on hormonessupplied by the seeds. Marked changes in the germination capacity of the maturing seedsare closely associated with changes in extractable amounts ofmethyl-4-chloroindol-3yl acetate and abscisic acid. It is believedthat high concentrations of these substances in the embryo,rather than any restriction imposed by the testa, may preventprecocious germination of the seeds     

Pectolytic and Cellulolytic Enzymes of Two Populations of Ditylenchus dipsaci on 'Wando' Pea (Pisum sativum L.)

''Wando'' pea is susceptible to Ditylenchus dipsaci from Raleigh, N. C. (RNC) but resistant to the same species from Waynesville, N. C. (WNC). Homogenates of RNC and WNC were analyzed for pectolytic and cellulolytic enzyme activity; both had high C_x activity with WNC two to three times more active than RNC. Polymethylglacturonase activity was three to five times higher in RNC, but polygalacturonase was up to 100 times higher in WNC. Polygalacturonate-trans-eliminase was not detected although a Ca⁺⁺-stimulated pectin methyl-trans-eliminase was present. Enzyme analyses of healthy and infected pea tissue showed only slight enzyme activity unrelated to that in nematode homogenates. No correlation between enzyme activity and the differing pathogenicities could be detected.     

Respiratory and Carbohydrate Changes in Developing Pea (Pisum sativum L.) Seeds in Relation to their Ability to Withstand Desiccation

In the development of garden pea seeds (P. sativum L., cv. ‘KelvedonWonder’) the ability to withstand desiccation was foundto be preceded by a fall in respiration rate and seed moisturecontent, both of which followed a sharp decline in ethanol-solublesugars. When harvested seeds were kept in high humidity conditionsrespiration was found to decline even though the moisture contentwas maintained. The fall in respiration was always associatedwith a fall in the level of sugars. Further experiments showedthat the seeds whose respiration had fallen in humid storagecould be induced to respire more rapidly by the addition ofsucrose. It is suggested that seeds can only withstand rapid desiccationafter a decrease in physiological activity following a fallin the supply of respiratory substrate in the form of sugars.     

Transformation and Regeneration of Two Cultivars of Pea (Pisum sativum L.)

A reproducible transformation system was developed for pea (Pisum sativum L.) using as explants sections from the embryonic axis of immature seeds. A construct containing two chimeric genes, nopaline synthase-phosphinothricin acetyl transferase (bar) and cauliflower mosaic virus 35S-neomycin phosphotransferase (nptII), was introduced into two pea cultivars using Agrobacterium tumefaciens-mediated transformation procedures. Regeneration was via organogenesis, and transformed plants were selected on medium containing 15 mg/L of phosphinothricin. Transgenic peas were raised in the glasshouse to produce flowers and viable seeds. The bar and nptII genes were expressed in both the primary transgenic pea plants and in the next generation progeny, in which they showed a typical 3:1 Mendelian inheritance pattern. Transformation of regenerated plants was confirmed by assays for neomycin phosphotransferase and phosphinothricin acetyl transferase activity and by northern blot analyses. Transformed plants were resistant to the herbicide Basta when sprayed at rates used in field practice.     

Purification and Characterization of Ornithine Transcarbamylase from Pea (Pisum sativum L.)

Pea (Pisum sativum) ornithine transcarbamylase (OTC) was purified to homogeneity from leaf homogenates in a single-step procedure, using δ-N-(phosphonacetyl)-l-ornithine-Sepharose 6B affinity chromatography. The 1581-fold purified OTC enzyme exhibited a specific activity of 139 micromoles citrulline per minute per milligram of protein at 37°C, pH 8.5. Pea OTC represents approximately 0.05% of the total soluble protein in the leaf. The molecular weight of the native enzyme was approximately 108,200, as estimated by Sephacryl S-200 gel filtration chromatography. The purified protein ran as a single molecular weight band of 36,500 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the pea OTC is a trimer of identical subunits. The overall amino acid composition of pea OTC is similar to that found in other eukaryotic and prokaryotic OTCs, but the number of arginine residues is approximately twofold higher. The increased number of arginine residues probably accounts for the observed isoelectric point of 7.6 for the pea enzyme, which is considerably more basic than isoelectric point values that have been reported for other OTCs.     

Leaf and Flower Development in Pea (Pisum sativum L.): Mutants cochleata andunifoliata

The stipule mutant cochleata(coch) and the simple-leaf mutantunifoliata(uni) are utilized to increase understanding of the controlof compound leaf and flower development in pea. The phenotypeof the coch mutant, which affects the basal stipules of thepea leaf, is described in detail. Mutant coch flowers have supernumeraryorgans, abnormal fusing of flower parts, mosaic organs and partialmale and female sterility. The wild-type Coch gene is shownto have a role in inflorescence development, floral organ identityand in the positioning of leaf parts. Changes in meristem sizemay be related to changes in leaf morphology. In the coch mutant,stipule primordia are small and their development is retardedin comparison with that of the first leaflet primordia. Thediameter of the shoot apical meristem of the uni mutant is approx.25% less than that of its wild-type siblings. This is the firsttime that a significant difference in apical meristem size hasbeen observed in a pea leaf mutant. Genetic controls in thebasal part of the leaf are illustrated by interactions betweencoch and other mutants. The mutantcoch gene is shown to changestipules into a more ‘compound leaf-like’ identitywhich is not affected by thestipules reduced mutation. The interactionof coch and tendril-less(tl) genes reveals that the expressionof the wild-type Tl gene is reduced at the base of the leaf,supporting the theories of gradients of gene action. Copyright2001 Annals of Botany Company Pisum sativum, garden pea, leaf morphogenesis, compound leaf, leaf mutants, flower morphology     

Localization of alpha-Amylase in the Apoplast of Pea (Pisum sativum L.) Stems

Most of the activity of an α-amylase present in crude pea (Pisum sativum L. cv Laxton's Progress No. 9) leaf preparations cannot be found in isolated pea leaf protoplasts. The same extrachloroplastic α-amylase is present in pea stems, representing approximately 6% of total stem amylolytic activity and virtually all of the α-amylase activity. By a simple infiltration-extraction procedure, the majority (87%) of this α-amylase activity was recovered from the pea stem apoplast without significantly disrupting the symplastic component of the tissue. Only 3% of the β-amylase activity and less than 2% of other cellular marker enzymes were removed during infiltration-extraction.     

Effects of Soil Structure on Pea (Pisum sativum L.) Root Development According to Sowing Date and Cultivar

Spring peas are known to be very sensitive to compaction, particularly when sowing takes place soon after winter. Winter peas, which are sown in autumn, should present an opportunity to sow the crop in better soil structural conditions than for spring peas, because of more favourable moisture conditions at that time. As environmental conditions have a big influence on root systems, it is important to determine the effects of soil structure on pea root systems for different cultivars and sowing dates. A spring pea cultivar and a winter pea cultivar were both sown at two dates (one in autumn and one in spring) on soils with different plough-layer structures (compacted and uncompacted) at two sites in 2002 and one site in 2003. Soil structure was characterised by bulk density and the percentage of highly compacted zones in the ploughed layer. Root distribution maps were produced every month, from February to maturity. Root development was described in terms of general root dynamics, root elongation rate (RER) in the subsoil, final maximum root depth (Dmax) and root distribution at maturity. Root depth dynamics depended on compaction and its interaction with climatic conditions. The effects of compaction on RER in the subsoil depended on the experimental conditions. Dmax was reduced by 0.10 m by compaction. Compaction also reduced root distribution between 10 and 40% in the ploughed layer only. Pea cultivars differed in sensitivity to soil compaction, with a direct effect on the final depth explored by roots. These results are discussed in terms of their relevance to water and nutrient uptake.     

Photosynthetic and Respiratory Studies During Pod and Seed Development in Pisum sativum L.

The photosynthetic and respiratory potential of Pisum sativumfruit depends on a combination of factors such as fruit age,light intensity and the atmospheric CO₂ concentration. Fruitwere capable of a net CO₂ uptake from the atmosphere only duringthe period of pod extension growth and at light intensitiesgreater than 12 klx. During subsequent development the respiratoryCO₂ evolution attributable to seed growth exceeded the photosyntheticcapacity of the pod. Despite this the extent of fruit CO₂ losswas consistently less in the light than in the dark. An increase in the CO₂ concentration beyond 300 p.p.m. markedlyreduced fruit CO₂ loss and in some instances effected a transitionfrom a net CO₂ output to a net CO₂ uptake. Conversely, a decreasein the CO₂ concentration substantially increased the extentof fruit CO₂ loss. The CO₂ compensation point concentrationincreased with fruit age from 150 p.p.m. to possibly more than550 p.p.m., whereas the corresponding value for the pod (minusseed) remained between 120 and 175 p.p.m. throughout fruit development. A proportion of the CO₂ respired by the seed and pod accumulatedwithin the pod cavity. The CO₂ concentration attained dependedon fruit age and nodal location. In the course of fruit developmentconcentrations within the range 0•1 to 4{dot}3 per centCO₂ occurred during mid-photoperiod. The CO₂ concentration washighest when the enclosed seed had attained approximately 50per cent of their final dry weight.     

Pyrroline-5-Carboxylate Reductase Is in Pea (Pisum sativum L.) Leaf Chloroplasts

Proline accumulation is a well-known response to water deficits in leaves. The primary cause of accumulation is proline synthesis. Δ¹-Pyrroline-5-carboxylate reductase (PCR) catalyzes the final reaction of proline synthesis. To determine the subcellular location of PCR, protoplasts were made from leaves of Pisum sativum L., lysed, and fractionated by differential and Percoll density gradient centrifugation. PCR activity comigrated on the gradient with the activity of the chloroplast stromal marker NADPH-dependent triose phosphate dehydrogenase. We conclude that PCR is located in chloroplasts, and therefore that chloroplasts can synthesize proline. PCR activities from chloroplasts and etiolated shoots were compared. PCR activity from both extracts is stimulated at least twofold by 100 millimolar KCl or 10 millimolar MgCl₂. The pH profiles of PCR activity from both extracts reveal two separate optima at pH 6.5 and 7.5. Native isoelectric focusing gels of sampies from etiolated tissue reveal a single band of PCR activity with a pl of 7.8.     

Leaf Developmental Age Controls Expression of Genes Encoding Enzymes of Chlorophyll and Heme Biosynthesis in Pea (Pisum sativum L.)

The effects of leaf developmental age on the expression of three nuclear gene families in pea (Pisum sativum L.) coding for enzymes of chlorophyll and heme biosynthesis have been examined. The steady-state levels of mRNAs encoding aminolevulinic acid (ALA) dehydratase, porphobilinogen (PBG) deaminase, and NADPH:protochlorophyllide reductase were measured by RNA gel blot and quantitative slot-blot analyses in the foliar leaves of embryos that had imbibed for 12 to 18 h and leaves of developing seedlings grown either in total darkness or under continuous white light for up to 14 d after imbibition. Both ALA dehydratase and PBG deaminase mRNAs were detectable in embryonic leaves, whereas mRNA encoding the NADPH:protochlorophyllide reductase was not observed at this early developmental stage. All three gene products were found to increase to approximately the same extent in the primary leaves of pea seedlings during the first 6 to 8 d after imbibition (postgermination) regardless of whether the plants were grown in darkness or under continuous white-light illumination. In the leaves of dark-grown seedlings, the highest levels of message accumulation were observed at approximately 8 to 10 d postgermination, and, thereafter, a steady decline in mRNA levels was observed. In the leaves of light-grown seedlings, steady-state levels of mRNA encoding the three chlorophyll biosynthetic enzymes were inversely correlated with leaf age, with youngest, rapidly expanding leaves containing the highest message levels. A corresponding increase in the three enzyme protein levels was also found during the early stages of development in the light or darkness; however, maximal accumulation of protein was delayed relative to peak levels of mRNA accumulation. We also found that although protochlorophyllide was detectable in the leaves immediately after imbibition, the time course of accumulation of the phototransformable form of the molecule coincided with NADPH:protochlorophyllide reductase expression. In studies in which dark-grown seedlings of various ages were subsequently transferred to light for 24 and 48 h, the effect of light on changes in steady-state mRNA levels was found to be more pronounced at later developmental stages. These results suggest that the expression of these three genes and likely those genes encoding other chlorophyll biosynthetic pathway enzymes are under the control of a common regulatory mechanism. Furthermore, it appears that not light, but rather as yet unidentified endogenous factors, are the primary regulatory factors controlling gene expression early in leaf development.     

The Effect of Cotyledon Excision on Reproductive Development in Pea (Pisum sativum L.)

The excision of cotyledons from two cultivars of peas soon aftergermination resulted in a lowering of the node of first floweringas well as a delay in both flower initiation and flowering,especially in the late cultivar Greenfeast. This is contraryto the usual view that, when cotyledon excision reduces nodeof first flowering, flower initiation and flowering are hastened,and it seems irreconcilable with the colysanthin theory of Barber(1959). An alternative explanation is proposed.