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 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     

Identification of Cytokinins of Root Nodules of the Garden Pea, Pisum sativum L

Five cytokinin activities which induced soybean callus proliferation were detected in ethanol extracts of root nodules of the garden pea (Pisum sativum L., cv. Little Marvel). The most active factors among them were identified as zeatin and its riboside on the basis of their mobility on thin layer chromatography in three solvent systems. Smaller activities of zeatin ribotide, isopentenyladenine and its riboside were also detected. Cytokinin activity gradually decreased with the cultivation period, but no qualitative change in the active compounds was found.     

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.     

Deteriorative Changes in Pea Seeds (Pisum sativum L.) Stored in Humid or Dry Conditions

Seeds stored under various conditions showed deteriorative changesin extremely dry (1% r.h. at 10 °C) or humid (93% r.h. at25 °C) conditions after 6 weeks storage, when little orno loss of viability had taken place; no changes were detectedin intermediate conditions (45% r.h. at 10 °C). The lossof electrolytes from seeds into water increased after 3 weeksof humid storage, and subsequently dead areas developed on thecotyledons of seeds held in either humid or dry conditions.With time in storage some of the seeds in dry conditions showeda reduction in the rate of imbibition, and consequently a lowlevel of electrolyte leakage. Other seeds showed an increasein leakage following dry storage. No change in solute content(sugars, potassium, and electrolytes) was detected in seedsstored in humid conditions, suggesting that the increased electrolyteleakage was caused by an impaired ability to retain solutes.Thus increased leakage was recorded in seeds whose cotyledonscontained no dead areas as revealed by vital staining, and wastherefore attributable to changes in living cells, possiblydeterioration in cell membranes. Viability began to declineafter 6 weeks in humid storage at 25 °C and after 2 d in94% r.h. at 45 °C, but was maintained in both dry and intermediateconditions. The rate at which viability fell in humid storagewas greatly influenced by the initial condition of the seed.     

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.     

Changes in Chloroplast DNA Levels during Development of Pea (Pisum sativum)

Determinations were made of the percentage of chloroplast DNA (ct DNA) in total cell DNA isolated from shoots of pea at different stages of development. Labeled pea ct DNA was reassociated with a high concentration of total DNA; the percentage of ct DNA was estimated by comparing the rate of reassociation of this reaction with that of a model reaction containing a known concentration of unlabeled ct DNA. The maximum change in ct DNA content was from 1.3% of total DNA in young shoots to 7.3% in fully greened shoots. Analyses were also performed on DNA from embryos, etiolated tissue, roots, and leaves. The first leaf set to develop in pea was excised over a growth period of 8 days during which leaf length increased from 4 to 12 millimeters. Young leaves contained about 8% ct DNA; in fully greened leaves the level of ct DNA approached 12%, equivalent to as many as 9,575 copies of ct DNA per cell. Root tissue contained only 0.4% ct DNA.     

Matromorphy in Pisum sativum L.

Summary The possibility of obtaining instant pure breeding lines by matromorph seed development in Pisum sativum L. has been investigated. Two types of maternal parents, namely, homozygous for the recessive marker genes and heterozygous for the dominant marker genes were pollinated with Lathyrus odoratus and the P174 variety of Pisum sativum L. carrying dominant markers. For both pollinators, induction of matromorphy by prickle pollination, irradiated pollen and IAA treatment was examined. Promising matromorphs were identified in the M₁ generation which were studied in the M₂ generation for assessing their genetic status with respect to homozygosis. The success of pod set varied from zero to 28% with a varying number of matromorphic seeds following different treatments. The possible mechanisms for matromorphic origin have been discussed. The evidence presented herein favours induction of matromorphy in peas for the production of homozygous stocks. In addition, the recovery of double recessive seed markers of the maternal parents along with plant markers from the paternals has prospective implications in plant breeding as an alternative tool to recurrent back crossing.     

Signal Integration by ABA in the Blue Light-Induced Acidification of Leaf Pavement Cells in Pea (Pisum sativum L. var. Argenteum)

Leaf pavement cell expansion in light depends on apoplastic acidification by a plasma membrane proton-pumping ATPase, modifying cell wall extensibility and providing the driving force for uptake of osmotically active solutes generating turgor. This paper shows that the plant hormone ABA inhibits light-induced leaf disk growth as well as the blue light-induced pavement cell growth in pea (Pisum sativum L.). In the phytochrome chromophore-deficient mutant pcd2, the effect of ABA on the blue light-induced apoplastic acidification response, which exhibits a high fluence phase via phytochrome and a low fluence phase via an unknown blue light receptor, is still present, indicating an interaction of ABA with the blue light receptor pathway. Furthermore, it is shown that ABA inhibits the blue light-induced apoplastic acidification reversibly. These results indicate that the effect of ABA on apoplastic acidification can provide a mechanism for short term, reversible adjustment of leaf growth rate to environmental change.Key Words: ABA, apoplastic acidification, blue light, epidermal pavement cell growth, leaf growth, pea (Pisum sativum L.), signal integration     

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.     

Correlations of Growth Rate and De-etiolation with Rate of Ent-Kaurene Biosynthesis in Pea (Pisum sativum L.)

Biosynthesis of the gibberellin precursor ent-kaurene-¹⁴C from mevalonic acid-2-¹⁴C was assayed in cell-free extracts of shoot tips of etiolated and light-grown Alaska (normal) and Progress No. 9 (dwarf) peas (Pisum sativum L.). During ontogeny of light-grown Alaska peas, kaurene-synthesizing activity increased from an undectectable level in 3-day-old epicotyls to a maximum in shoot tips of 9-day-old plants and remained relatively constant thereafter until postanthesis. The capacity for kaurene synthesis in extracts from shoot tips of 10-day-old etiolated Alaska seedlings increased approximately exponentially during the first 12 hr of de-etiolation in continuous high intensity white light and remained relatively constant during the succeeding 24 hr of irradiation. Extracts from light-grown Alaska (normal) shoot tips possessed greater capacity for kaurene synthesis than did extracts from light-grown Progress No. 9 (dwarf) shoot tips. Extracts from shoot tips of either light-grown cultivar displayed greater kaurene-synthesizing capacity than was observed in extracts from their dark-grown counterparts. It is concluded that gibberellin biosynthesis in pea shoot tips is subject to partial regulation by factors controlling the rate of biosynthesis of kaurene.     

Biochemical and Physiological Changes During Maturation of Fruit of the Field Pea (Pisum arvense L.)

Quantitative changes in the carbohydrates, soluble nitrogenouscompounds, and specific proteins of seeds and pods of fieldpea have been followed during two seasons and related to theoverall pattern of growth of the embryo and other parts of thefruit. Reserves of solutes are established in the pod and inthe seed coat and endosperm of the seed some time before theembryo commences its exponential phase of growth. Later, thesereserves disappear and it is estimated that their mobilizationmight provide, at the most, only one-fifth of the embryo's requirementsfor carbon and nitrogen. Starch and certain albumin-type proteins accumulate relativelyearly in the life of the embryo. Globulin-type proteins, bycontrast, are laid down only after the cotyledons are almostfull size and after most of the reserve of starch has been accumulated.The content of hemicellulose, the principal carbohydrate ofthe embryo, increases steadily as the embryo grows. Neitherhemicellulose nor globulin is found in appreciable amounts inorgans other than the cotyledons. Each organ of the fruit has its own highly characteristic poolof free amino acids. Orderly and reproducible changes occurin the composition of these pools during fruit development,suggesting that variations in the amounts of specific compoundsmay have special significance in the programme of developmentand synthesis in the fruit.     

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.     

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.     

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.     

Gibberellin translocation in Pisum sativum L.

The physiological and biochemical consequences of treating Le (tall) and le (dwarf) pea seedlings with varying quantities of the gibberellins [³H]GA₂₀ and GA₁ have been investigated. Although the percentage uptake of these compounds from the site of application on the 3 stipules was low and most of the applied GA remained unmetabolised in situ, the quantitative relationship between GA translocation and GA dosage was found to be linear for GA₁ but saturating for GA₂₀. The movement of the GAs and their subsequently produced metabolites was mainly acropetal. They accumulated in greatest quantity in the apical extremities of the shoot. Overall, the extent to which GA₂₀ was metabolished in le seedlings was considerably less than in Le pea seedlings. Although all le tissues contained significantly less [³H]GA₁ than their Le counterparts, phenotypic effects of the le mutation were apparent only on internode and tendril development. Increased tissue growth, consequent upon GA treatment, was also apparent only in the internodes and tendrils of le plants. For internodes, GA₁ content determined the mid-logarithmic-phase growth rate and, consequently, final length. For tendrils, GA₂₀ rather than GA₁ may be the primary stimulatory agent.Abbreviations GA gibberellin - HPLC high-performance liquid chromatography - 1–6 consecutive developmental numbering system for plant tissues/organs as shown in Fig. 1 The author gratefully acknowledges financial support from Imperial Chemical Industries, Plant Protection, Jealott's Hill, Bracknell, Berks., UK and the Science and Engineering Research Council.