Phytochrome Controlled RNA Changes in Terminal Buds of Dark Grown Peas (Pisum sativum cv. Alaska)

The terminal buds of intact dark grown Alaska pea plants (Pisum sativum) respond to low intensity red light by an enhanced rate of expansion of their leaflets. Twenty four hours after irradiation, red light was found to have increased the ribosomal fraction of the RNA content of the terminal buds. The increase in total RNA liter did not occur if the red light was immediately followed by irradiance with low intensity far red light.     

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.     

Comparative Effects of Indoleacetic Acid and Gibberellic Acid on Growth of Decapitated Etiolated Epicotyls of Pisum sativum cv. Alaska

Measurements were made of the growthof the sub-apical region of decapitated, etiolated epicotyls of Pisum sativum L. cv. Alaska after treatments with indoleacetic acid (IAA), gibberellic acid (GA) and triiodobenzoic acid (TIBA). Growth was measured either at the end of a 2-day period, at short intervals during growth, or was monitored continuously for 2–3 h using a position-sensing transducer. In experiments measuring growth after 2 days, high levels (0.1–10 μg/plnat) of IAA caused expansion, whereas similar levels of GA caused elongation. When both hormones were applied together, the effects of IAA were dominant and expansion ensued, even when GA was present at 100 times the amount of IAA. Very low amounts of IAA (0.5–5 ng/plant), however, caused elongation. The elongation elicited by high GA or low IAa was inhibited to a similar extent by TIBA and this inhibition of elongation was associated with an increased expansion at the extreme tip. When application of the hormones was delayed, GA-induced elongation was reduced considerably, IAA-induced elongation was lessened somewhat and IAA-induced expansion was partially converted into elongation. In experiments measuring elongation at short intervals, high levels of IAA caused rapid elongation followed after 3 to 6 h by expnasion. Both GA and low levels of IAA extended the duration of elongation with little apparent effect on the rate of growth. In fast-growth experiments, low, intermediate and high levels of IAA doubled the rate of elongation with a lag period of about 20 min, whereas GA had at most a very slight stimulatory effect on the growth rate. It is concluded that the main role of GA in this system is to maintain physiological levels of IAA in the growing zone and that the level of IAA present determines whether elongation or expansion will take place.     

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.     

Rapidly Induced Wound Ethylene from Excised Segments of Etiolated Pisum sativum L., cv. Alaska: I. Characterization of the Response

A rapidly induced, transitory increase in the rate of ethylene synthesis occurred in wounded tissue excised from actively growing regions of etiolated barley, cucumber, maize, oat, pea, tomato, and wheat seedlings. Cutting intact stems or excising 9-mm segments of tissue from near the apex of 7-day-old etiolated Pisum sativum L., cv. Alaska seedlings induced a remarkably consistent pattern of ethylene production. At 25 C, wound-induced ethylene production by segments excised 9 mm below the apical hook increased linearly after a lag of 26 minutes from 2.7 nanoliters per g per hour to the first maxium of 11.3 nanoliters per g per hour at 56 minutes. The rate of production then decreased to a minimum at 90 minutes, increased to a lower second maximum at 131 minutes, and subsequently declined over a period of about 100 minutes to about 4 nanoliters per g per hour. Removal of endogenous ethylene, before the wound response commenced, had no effect on the kinetics of ethylene production. Tissue containing large amounts of dissolved ethylene released it as an exponential decay with no lag period. Rapidly induced wound ethylene is synthesized by the tissue and is not merely the result of facilitated diffusion of ethylene already present in the tissue through the newly exposed cut surfaces. Previously wounded apical sections did not exhibit a second response when rewounded. No significant correlation was found between wound-induced ethylene synthesis and either CO₂ or ethane production.     

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.     

Rapidly Induced Wound Ethylene from Excised Segments of Etiolated Pisum sativum L., cv. Alaska: II. Oxygen and Temperature Dependency

Wound-induced ethylene synthesis by subapical stem sections of etiolated Pisum sativum L., cv. Alaska seedlings, as described by Saltveit and Dilley (Plant Physiol 1978 61: 447-450), was half-saturated at 3.6% (v/v) O₂ and saturated at about 10% O₂. Corresponding values for CO₂ production during the same period were 1.1% and 10% O₂, respectively. Anaerobiosis stopped all ethylene evolution and delayed the characteristic pattern of wound ethylene synthesis. Exposing tissue to 3.5% CO₂ in air in a flow-through system reduced wound ethylene synthesis by 30%. Enhancing gas diffusivity by reducing the total pressure to 130 mm Hg almost doubled the rate of wound ethylene synthesis and this effect was negated by exposure to 250 μl liter⁻¹ propylene. Applied ethylene or propylene stopped wound ethylene synthesis during the period of application, but unlike N₂, no lag period was observed upon flushing with air. It is concluded that the characteristic pattern of wound-induced ethylene synthesis resulted from negative feedback control by endogenous ethylene.

No wound ethylene was produced for 2 hours after excision at 10 or 38 C. Low temperatures prolonged the lag period, but did not prevent induction of the wound response, since tissue held for 2 hours at 10 C produced wound ethylene immediately when warmed to 30 C. In contrast, temperatures above 36 C prevented induction of wound ethylene synthesis, since tissue cooled to 30 C after 1 hour at 40 C required 2 hours before ethylene production returned to normal levels. The activation energy between 15 and 36 C was 12.1 mole kilocalories degree⁻¹.

     

Paraquat-resistant lines in Pisum sativum cv. Alaska: biochemical and phenotypic characterization

In plants, the oxygen generated by photosynthesis can be excited to form reactive oxygen species (ROS) under excessive sunlight. Excess ROS including singlet oxygen (¹O₂) inhibit the growth, development and photosynthesis of plants. To isolate ROS-resistant crop plants, we used paraquat (PQ), a generator of O₂ ^·− as a source of screening and mutagen, and obtained two PQ-resistant lines in Pisum sativum, namely R3-1 and R3-2. Both lines showed greater resistance to PQ than their wild type (WT) siblings with respect to germination, root growth, and shoot growth. Biochemical analysis showed differences in these lines, in which ROS-scavenging enzymes undergo changes with a distinguishable increase in Mn-SOD. We further observed that the cytosolic catalases (CATs) in leaves in both lines were shifted in a native-PAGE analysis compared with that of the WT, indicating that the release of bound ¹O₂ was enhanced. Phenotypic analysis revealed distinguishable differences in leaf development, and in flowering time and position. In addition, R3-1 and R3-2 showed shorter individual internode lengths, dwarf plant height, and stronger branching compared with the WT. These results suggested that PQ-induced ROS-resistant Pisum have the potential pleiotropic effects on flowering time and stem branching, and that ROS including ¹O₂ plays not only important roles in plant growth and development as a signal transducer, but also appears as a strong inhibitor for crop yield. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Studies of Rapidly Induced Wound Ethylene Synthesis by Excised Sections of Etiolated Pisum sativum L., cv. Alaska: IV. Requirement of a Water-soluble, Heat-stable Factor

The rate of wound ethylene synthesis was reduced by more than 85% when 9-millimeter subapical sections of etiolated 7-day-old Pisum sativum L., cv. Alaska seedlings were incubated in water during the 26-minute induction period prior to wound ethylene synthesis, but the rate of synthesis was unaffected if sections were incubated in water during the actual synthesis of wound ethylene. The characteristic timing of the wound response was unaffected by either treatment. The ability of various chemical solutions and aqueous plant extracts to alter the rate of wound ethylene synthesis was studied by first incubating subapical pea stem sections in solutions under anaerobic conditions (anaerobiosis delays the induction and synthesis of wound ethylene; Plant Physiol 61: 675-679), and then measuring wound ethylene synthesis after the tissue was transferred to air. Solutions of several reported precursors of ethylene synthesis, such as methionine, homoserine, or propanal, did not reverse the water-caused reduction of wound ethylene synthesis. A water-soluble, heat-stable factor in extracts from pea seedlings, and solutions of 23 nanomolar triacontanol, 10 micromolar kinetin, or 10 micromolar benzyladenine prevented the reduction of wound ethylene synthesis, but were ineffective if administered after an initial 15-minute anaerobic water incubation. This suggested that the active solutions may have only prevented the loss of some ephemeral, though necessary factor, rather than actually containing the substrate or inducer of wound ethylene synthesis. Attempts to isolate and characterize the active fraction from aqueous tissue extracts were unsuccessful. Free radical quenchers, inhibitors of protein synthesis, and rhizobitoxine, an inhibitor of ethylene synthesis from methionine, all reduced wound ethylene synthesis when administered in solutions which previously had maintained wound ethylene synthesis.     

GTP-binding proteins in etiolated epicotyls of Pisum sativum (Alaska) seedlings

Seven fractions of GTP-binding proteins separated by gel filtration of an extract of epicotyls of Pisum sativum seedlings were partially characterized. Seven fractions of GTP-binding proteins tentatively designated GP1 to GP7 had the capacity to be ADP-ribosylated by pertussis toxin. Pooled fractions of GP2 to GP7 showed Km values 2, 20, 50, 10, 3 and 1 nM, respectively. The binding of [35S]GTP gamma S to GTP-binding proteins was prevented competitively in the presence of 0.1 mM GTP and also prevented in the presence of 0.1 mM ATP. Binding of [35S]GTP gamma S to the proteins produced a decrease in their molecular weights.     

Rapidly Induced Wound Ethylene from Excised Segments of Etiolated Pisum sativum L., cv. Alaska: III. Induction and Transmission of the Response

Increased ethylene synthesis was rapidly induced throughout the apical meristematic region of etiolated seedlings of Pisum sativum L., cv. Alaska by cuts made 1 centimeter from the apical hook. The wound signal was transmitted at about 2 millimeters per minute. Accumulation of substance(s) at the cut surfaces of excised sections, as the result of interrupted translocation, did not initiate or significantly contribute to wound-induced ethylene synthesis, nor was the cut surface the site of enhanced ethylene synthesis. Cutting subapical sections into shorter pieces showed that cells less than 2 millimeters from a cut surface produced about 30% less ethylene than cells greater than 2 millimeters from a cut surface.     

Development of the quiescent center in maturing embryonic radicles of pea (Pisum sativum L. cv. Alaska)

Maturing embryos of pea (Pisum sativum L. cv. Alaska) were treated with an aqueous solution of tritiated thymidine for 1 h, sectioned, and processed for autoradiography. An analysis of the distribution of labelled nuclei and mitotic figures demonstrated the presence of a quiescent center (QC) in the radicles of developing embryos. The QC developed in the radicle during the growth of the embryo. Immature radicles that did not contain a well-formed zone of root-cap initials did not show a QC. In the latter stages of seed ripening, the pattern of arrest of DNA synthesis and mitosis was tissue-specific. Cells within the QC remained inactive. The region lacking labelled nuclei and mitotic figures progressively expanded to include the root cap initials and then the provascular cylinder. Mitosis was arrested before DNA synthesis in the embryonic cortex. Cells within the QC synthesized DNA during the first stages of seed germination.Abbreviations [³H]TdR tritiated thymidine - QC quiescent center     

Non-Specific Association of Phytochrome to Nuclei during Isolation from Dark-Grown Pea (Pisum sativum cv. Alaska) Plumules.

Although phytochrome is known to regulate gene expression inplants, the location of the phytochrome molecules involved inthe response remains unclear. Thus the aim of this investigationwas to test the possibility that nuclei contain phytochrome.Nuclei were isolated from dark-grown pea (Pisum sativum cv.Alaska) plumules in the dark and the nuclear proteins were resolvedon SDS polyacrylamide gels and transferred to nitrocelluloseby western blotting. A significant amount of phytochrome wasdetected in the nuclear proteins by immunostaining using monoclonaland polyclonal anti-phytochrome antibodies. Most of the phytochromeassociated with the nuclei could not be removed by treatmentwith high salt and low magnesium, indicating that the bindingwas rather stable. However, the isolation of nuclei in the presenceof exogenously added [¹²⁵I]phytochrome of P_R form in the darksuggested that significant amount of the phytochrome detectedin the nuclei was derived from contamination by the solublefraction during isolation. It is an open question as to whether,besides this contaminated phytochrome, isolated nuclei endogenouslycontain very small amount of phytochrome. ⁴Present Address: Plant Molecular Biology and Biochemistry ResearchGroup, Departments of Biochemistry and Botany, University ofGlasgow, Glasgow G12 8QQ, U.K. (Received February 8, 1988; Accepted July 27, 1988)     

Characterization of abscisic acid in chloroplasts of Pisum sativum L. cv. Alaska by combined gas-chromatography-mass spectrometry

Summary Abscisic acid was characterized from extracts of isolated pea chloroplasts by combined gas-chromatography-mass spectrometry. By single ion monitoring of the base peak (m/e 190) in the mass spectrum, 8.6 g ABA were detected in chloroplasts isolated from 1 kg fresh weight of pea shoots.GC-MS combined gas chromatography-mass spectrometry     

Production and Characterization of Monoclonal Antibodies Which Distinguish Different Surface Structures of Pea (Pisum sativum cv. Alaska) Phytochrome

Eight monoclonal antibodies to pea 114,000 dalton phytochrome(mAP-1 to mAP-8) were produced by fusing spleen cells from immunizedBALB/c mice with NS-1 myeloma cells. Antibody production bymAP-1 to mAP-7 was detected by cellular radioimmunoassay usingsheep red blood cells coated with pea phytochrome. Relativebinding positions of these mAPs were determined using a competitivebinding assay, and at least 6 different antigenic regions weredefined on the 114,000 dalton chromopeptide. Location of theregions in relation to the chromophore was studied by examiningthe cross-reactions of the relevant mAPs with chromophore-containingproteolytic fragments (62,000, 37,000 and 24,000 daltons) ofphytochrome. The following cross-reactions were observed; mAP-1with 62,000, mAP-6 and mAP-7 with 62,000, 37,000 and 24,000.Only mAP-7 caused "spectral denaturation" of phytochrome. AllmAPs reacted with P_R and P_FR with similar affinity. These mAPswill be useful probes in future studies for detecting each siteof the apoprotein of phytochrome. By using another screeningmethod, namely radioimmunoassay with antigen-coupled Sepharose4B, mAP-8 was also obtained. (Received May 4, 1984; Accepted June 25, 1984)     

Changes in growth and structure of pea primary roots (Pisum sativum L. cv. Alaska) as a result of sudden flooding

Pea (Pisum sativum L. cv. Alaska) primary roots were exposed to flooding after growth for 4 or 5 d at 25 degrees C under relatively dry conditions. Flooding after 4 d growth reduced, but did not stop, primary root growth, and cavities caused by degradation of central vascular cells were typically found from 10-60 mm from the tips. Flooding after 5 d stopped primary root growth and caused cell death in the tips, and vascular cavities formed that typically were 20-60 mm from the tips of the roots. Degradation of root tip cells in 5-day-roots was very rapid and began in the elongation zone and later in the apical zone. Root tips discolored, narrowed or curled before growth arrest. The mitotic indices of 5-day-root tips were suppressed by the flooding treatment. A few mitotic figures were observed in roots treated with flooding after 4 d growth. Affected cells had condensed nuclei, but cytoplasms appeared to be normal in the early stages of cell degradation. Later these cells became very vacuolated. The relationship of flooding to root growth, vascular cavity formation, and the morphology of pea primary roots is described with regard to the ability to resist flooding stress.     

Influence of Bud Position and Plant Ontogeny on the Morphology of Branch Shoots in Pea (Pisum sativum L. cv. Alaska)

The morphology of axillary shoots of pea plants (Pisum sativumL. cv. Alaska) was analysed as a function of the position ofthe bud on the plant axis and the stage of plant developmentwhen the buds began to grow. Buds from the three most basalnodes were stimulated to develop by decapitating the main shootwhen buds were still growing (4 d plants), shortly after budsbecame dormant (7 d plants) or after the initiation of floweringon the main shoot (post-flowering plants, about 21 d after sowing).Branch shoots were scored for node of floral initiation (NFI),shoot length, and node of multiple leaflets (NML), a measureof leaf complexity. Shoots that developed spontaneously fromupper nodes (nodes 5-9) on intact post-flowering plants werescored for NFI. NFI for basal buds on 4 and 7 d plants variedas a function of nodal position and ranged from 5 to 6·7nodes. NFI on these plants was not influenced by bud size orwhether a bud was growing or dormant when the plant was decapitated.NFI for shoots derived from basal buds on decapitated post-floweringplants and upper nodes on intact post-flowering plants was about4. Reduced NFI on post-flowering plants may be due to depletionof a cotyledon-derived floral inhibitor. Basal axillary shootson 4 d plants were about 20% longer than those on 7 d plantsand about five times longer than those on post-flowering plants.These differences may be due to depletion of gibberellic acidsfrom the cotyledons. NFI and NML for the main shoot and forbasal axillary shoots were similar under some experimental conditionsbut different under other conditions, so it is likely that eachdevelopmental transition is regulated independently.Copyright1995, 1999 Academic Press Apical dominance, bud development, garden pea, initiation of flowering, Pisum sativum L., shoot morphology     

The solubilization of a glucuronyltransferase involved in pea (Pisum sativum var. Alaska) glucuronoxylan synthesis.

A glucuronyltransferase involved in glucuronoxylan biosynthesis was obtained from the epicotyls of 1-week-old etiolated pea (Pisum sativum var. Alaska) seedlings and was solubilized in Triton X-100, a non-ionic detergent. The enzyme was inactivated by SDS and inhibited by Derriphat 160 and cholic acid. The enzyme was active in the presence of NN-dimethyldodecylanium-N-oxide, but was not solubilized by it. The stimulatory effect of UDP-D-xylose on the particulate and solubilized enzymes was the same, but the optimum Mn2+ concentration was lower for the solubilized enzyme, and the product formed by the solubilized enzyme has altered structure and solubility properties. Gel filtration of the solubilized enzyme on Sepharose CL-6B permitted partial separation of the stimulatory effect of UDP-D-xylose from the activity in the absence of UDP-D-xylose. The solubilized enzyme was more stable than the particulate enzyme and could be stored for 2 weeks at -20 degrees C without loss of activity.