Pea Shoots Research Articles

Effects of Protein Synthesis Inhibitors on ent-Kaurene Biosynthesis during Photomorphogenesis of Etiolated Pea Seedlings

Excised shoot tips from 10-day-old etiolated pea (Pisum sativum L. cv. Alaska) seedlings were incubated in solutions of chloramphenicol, cycloheximide, and lincomycin at different concentrations during periods of 0, 4, 8, and 12 hours of irradiation with high intensity white light. Enzyme extracts were prepared from the whole shoot tips and compared with extracts from nontreated shoot tips for their capacity to synthesize ent-kaurene from mevalonate. In control samples, kaurene synthesis increased during the first 8 hours of irradiation and decreased after 12 hours. Chlorophyll content increased steadily up to 12 hours of irradiation. Chloramphenicol and cycloheximide reduced both kaurene synthesis and chlorophyll formation to a similar extent during all periods of irradiation, the reduction being greatest after 8 hours of irradiation. Lincomycin, a specific inhibitor of the formation of chloroplast ribosomes in detached pea shoot tips, did not significantly affect kaurene synthesis activity but strongly inhibited chlorophyll formation. It is tentatively concluded that the increase in kaurene synthesis activity during normal photomorphogenesis in pea seedlings is due to photoinduction of de novo synthesis of one or more proteins involved in the biosynthetic pathway from mevalonate to kaurene.

Light-dependent Reduction of Oxidized Glutathione by Ruptured Chloroplasts

Crude extracts of pea shoots (Pisum sativum) catalyzed oxidized glutathione (GSSG)-dependent oxidation of NADPH which was attributed to NADPH-specific glutathione reductase. The pH optimum was 8 and the K(m) values for GSSG and NADPH were 23 mum and 4.9 mum, respectively. Reduced glutathione (GSH) inhibited the reaction. Crude extracts also catalyzed NADPH-dependent reduction of GSSG; the ratio of the rate of NADPH oxidized to GSH formed was 0.49. NADH and various substituted mono- and disulfides would not substitute for NADPH and GSSG respectively. Per mg of chlorophyll, enzyme activity of isolated chloroplasts was 69% of the activity of crude extracts.Illuminated sonicated pea chloroplasts, in the presence of catalytic amounts of NADPH, catalyzed GSSG-dependent O(2) evolution (mean of 10 determinations, 10.4 mumol per mg chlorophyll per hour, sd 1.4) with the concomitant production of GSH. The molar ratio of GSH produced to O(2) evolved was 3.8 and the highest ratios for O(2) evolved to GSSG added were 0.46 and 0.44. The K(m) value for GSSG was 26 mum. GSH inhibited the reaction. The reaction was attributed to photosynthetically coupled glutathione reductase.Ruptured chloroplasts, in the presence of catalytic amounts of GSSG and NADPH, did not catalyze sustained O(2) evolution in the presence of substrate amounts of hydrogen peroxide, dehydroascorbate, l-cystine, sulfite, or sulfate.

The quantitative ultrastructure of the pea shoot apex in relation to leaf initiation

The ultrastructure of the pea shoot apical meristem was examined quantitatively in longitudinal sections. Photographs were taken at eleven defined positions in the apex, at six developmental stages within a single plastochron. The only change in ultrastructure during the period of a single plastochron was the increase in the proportion of plastids with starch in the central regions of the apex and in the young leaf axils. This increase occurred midway in time between the emergence of successive leaves, at precisely the time that the orientation of growth changes in the region where a new leaf is to emerge. There were quantitative changes in ultrastructure associated with cell differentiation. In the sequence of cell development from the summit of the apex (central zone) to the incipient pith, cell enlargement was accompanied by an increase in the volume of endoplasmic reticulum, dictyosomes, microbodies and vacuoles per cell, an increase in the number of mitochondria, microbodies and vacuoles per cell, and an increase in the volume, but not the number, of plastids per cell. In the sequence of axillary development (before the axillary bud begins to grow) the number of mitochondria per cell decreased as cell volume decreased but the number of plastids per cell remained constant. The number of plastids per cell increased only in the developmental sequence leading to leaf development, in which the number of mitochondria and dictyosomes per cell also increased. There appeared to be no features of ultrastructure, qualitative or quantitative, which could be correlated with the different rates of cell division in different regions of the meristem. The differences in ultrastructure throughout the apex were mainly quantitative and seemed to be associated with cellular differentiation rather than with the plastochronic functioning of the apex during leaf initiation.

Inhibition of ent-Kaurene Oxidation and Growth by α-Cyclopropyl-α-(p-methoxyphenyl)-5-pyrimidine Methyl Alcohol

Growth of Alaska peas (Pisum sativum) is inhibited more than 60% by alpha-cyclopropyl-alpha-(p-methoxyphenyl)-5-pyrimidine methyl alcohol (ancymidol) treatment. This growth inhibition can be reversed completely by gibberellic acid application. Cell-free enzyme preparations from pea shoot tips and wild cucumber (Marah oreganus) endosperm were used to test the effects of this substituted pyrimidine on the incorporation of mevalonic acid-(14)C into ent-kaurene and ent-kaurenol, respectively. Ancyidol (10(-6)m) completely blocks the conversion of ent-kaurene to ent-kaurenol. This result was confirmed with the wild cucumber endosperm system by testing the direct conversion of labeled ent-kaurene to ent-kaurenol. Ancymidol at higher concentrations (10(-3)m) inhibits the incorporation of mevalonic acid-(14)C into ent-kaurene to a lesser extent. It is concluded that one mode of action of this growth regulator is the inhibition of gibberellin biosynthesis.

Some Regulatory Properties of Pea Leaf Carbamoyl Phosphate Synthetase

Carbamoyl phosphate synthetase of pea shoots (Pisum sativum L.) was purified 101-fold. Its stability was greatly increased by the addition of substrates and activators. The enzyme was strongly inhibited by micromolar amounts of UMP (Ki less than 2 mum). UDP, UTP, TMP, and ADP were also inhibitory. AMP caused either slight activation (under certain conditions) or was inhibitory. Uridine nucleotides were competitive inhibitors, as was AMP, while ADP was a noncompetitive inhibitor. Enzyme activity was increased manyfold by the activator ornithine. Ornithine acted by increasing the affinity for Mg.ATP by a factor of 8 or more. Other activators were IMP, GMP, ITP, and GTP, IMP, like ornithine, increased the Michaelis constant for Mg.ATP. The activators ornithine, GMP, and IMP (but not GTP and ITP) completely reversed inhibition caused by pyrimidine nucleotides while increasing the inhibition caused by ADP and AMP.

Inhibition of chloroplast development by tentoxin

Light-dependent chloroplast development in detached pea shoots was measured in terms of chlorophyll synthesis and the synthesis of Fraction 1 protein. Both synthetic processes were inhibited more than 90% by the fungal metabolite, tentoxin (1 or 10 μg/ml). These results place Pisum sativum in the class of tentoxin-sensitive higher plants. Tentoxin, actinomycin D, lincomycin, D- threo-chloramphenicol and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) were compared in their ability to inhibit RNA and protein synthesis by isolated pea chloroplasts. Energy for the synthetic reactions was supplied either by light or by added ATP. Only CCCP gave the same pattern of inhibition as tentoxin, i.e. inhibition of both RNA and protein synthesis in the light-driven system but no inhibition in the ATP-driven system. It is concluded that chloroplast developmental processes are inhibited by tentoxin through the inhibition of photophosphorylation.

Conversion of geranylgeranyl pyrophosphate to ent-kaurene in enzyme extracts of sonicated chloroplasts

Farnesyl pyrophosphate-[ 14C] and geranylgeranyl pyrophosphate-[ 14C] were biosynthesized from mevalonic acid-[2- 14C] by cell-free enzyme extracts of pea ( Pisum sativum) cotyledons containing MgCl 2, MnCl 2, ATP and AMO-1618. Maximum yields of farnesyl pyrophosphate were obtained after 30 min incubation while geranylgeranyl pyrophosphate was the primary product after 180 min. Biosynthesized geranylgeranyl pyrophosphate-[ 14C] served as an efficient substrate for ent-kaurene biosynthesis in reaction mixtures containing cotyledon enzymes when AMO-1618 was omitted. Enzyme extracts from green pea shoot tips and chloroplasts also converted geranylgeranyl pyrophosphate to ent-kaurene in very low yields. Ent-kaurene production from mevalonic acid-[2- 14C] in extracts of pea shoot tips was also enhanced by addition of chloroplast enzymes. This evidence indicates that kaurene synthetase is present in pea chloroplasts and adds to the possibility that some gibberellin biosynthesis may be compartmentalized in those organelles.

Kaurene synthetase from plastids of developing plant tissues

Summary Kaurene synthesis from copalyl pyrophosphate is present in the plastid fraction from all three sources of developing plant tissue studied. Isolated plastids from Echinocystis macrocarpa endosperm have kaurene synthetic capacity from either geranylgeranyl pyrophosphate or copalyl pyrophosphate. However, organelle preparations from etiolated pea shoot tips or developing castor bean endosperm have either no or a very limited ability to synthesize kaurene from geranylgeranyl pyrophosphate. These results suggest that the first committed step in gibberellin biosynthesis, geranylgeranyl pyrophosphate conversion to copalyl pyrophosphate, may be limited under certain stages of plant development.

Einfluß der Kulturmethode auf die Ausscheidung von Phytohormonen durch Erbsenwurzeln

Earlier experiments (Tietz 1971 and 1974) had shown that pea roots contain very different amounts of abscisic acid (ABA) according to their growth in a mixture of vermiculite and pumice stones (20.1 μ g ABA/kg) or in water culture (1.31 μ g ABA/kg). Hence it was interesting to test whether roots are able to excrete hormones into the culture media. 5 days old pea seedlings were grown on a water culture for two weeks. The roots were well aerated and in some cases illuminated with fluorescent lamps. An analysis of the culture media using a biotest revealed ABA and auxin activity. A quantitative determination of ABA using gas liquid chromatography yielded about two times more ABA in illuminated vessels in comparison to dark grown controls. The ABA content of the culture media increased towards the end of the experiment whereas the ABA content of roots decreased. The IAA concentration in roots is also dependent on the culture media. Roots growing in sand contain 11.0 μ g/kg fresh weight, pea roots from a water culture only 4.1 μ g/kg. [2- 14 C]-ABA applied to 5 days old pea shoots is transported to the roots only in very small amounts. An illumination of the roots reduces the concentration of [2- 14 C]-ABA in the culture media as well as in the roots Therefore it is concluded that ABA which accumulated after illumination in the roots and in the culture media is mainly synthesized in the roots

Experimentally induced binding of phytochrome to mitochondrial and microsomal fractions in etiolated pea shoots

A brief irradiation with red light of pea (Pisum sativum L.) shoot segments kept at 0° resulted in very rapid binding of both Pr and Pfr to mitochondrial and microsomal fractions. The effect was not far-red reversible. The amount of phytochrome bound to the mitochondrial fraction was proportional to the percentage of Pfr of the fraction, and the ratio of Pr and Pfr in the bound form was the same as that in 12,000 x g supernatant. After a brief exposure of the segments to red light at 0° and a subsequent dark incubation at 30° in Tris-HCL buffer containing dithiothreitol or EDTA, which bot inhibit Pfr decay, the contents of phytochrome in the mitochondrial and microsomal fractions were significantly enhanced with time. The red-light effect was reversed by far-red light. The increase of the phytochrome content in the particulate fractions continued for at least 2 h, reaching a ca. 3 times higher level in terms of Δ (ΔA) per mg protein.

Inhibition of chloroplast protein synthesis by lincomycin and 2-(4-methyl-2,6- dinitroanilino)- N-methylpropionamide

Selective effects of lincomysin and cycloheximide in detached shoots of Pisum sativum on the synthesis of photosystem I and II proteins, and a chloroplast membrane protein of molecular weight 32000, confirm results obtained from studies of protein synthesis by isolated chloroplasts. A model is proposed in which one role of chloroplast ribosomes is to synthesize membrane proteins required for the immobilization of chloroplast components, such as photosystem I protein, which are synthesized by cytoplasmic ribosomes. 2-(4-Methyl-2,6-dinitroanilino)- N-methylpropionamide rapidly inhibits the synthesis of both the large and small subunits of Fraction I protein in greening detached pea shoots. This observation can be reconciled with the site of synthesis of the large subunit being in the chloroplast by a model which proposes that the small subunit is a positive initiation factor for the synthesis or translation of the messenger RNA for the large subunit.

Studies on gibberellins in shoots of light grown peas III. Interconversion of [ 3H]Ga 9 and [ 3H]GA 20 to other gibberellins by an in vitro system derived from chloroplasts of Pisum sativum

Isolated pea chloroplasts interconverted [ 3H]GA 9 into two zones of radio-activity, both of which exhibited gibberellin (GA)-like activity in the Tanginbozu dwarf rice assay. [ 3H]GA 20 was converted by isolated pea chloroplasts into a single acidic metabolite, chromatographically identical to GA 29. It is suggested that pea chloroplasts are a major site of gibberellin interconversion in the pea shoot.

Studies of gibberellins in shoots of light-grown peas II. The metabolism of tritiated gibberellin A 9 and gibberellin A 20 by light-and dark-grown shoots of dwarf Pisum sativum var. meteor

Etiolated shoots of dwarf “Meteor” pea converted tritiated gibberellin A 9 ([ 3H]GA 9) into GA 20 and 2,3-dihydro GA 31 (2,3-DH-GA 31) more readily than did shoots of light-grown pea seedlings. The conversion of [ 3H]GA 9 into very polar acidic gibberellins was unaffected by light. The inter-conversion of [ 3H]GA 20 into GA 29 was markedly reduced by light treatment; These results are discussed in relation to GA-controlled internode growth of light grown pea shoots.

Metabolism of Tritiated Gibberellin A9 by Shoots of Dark-grown Dwarf Pea, cv. Meteor

Tritium-labeled gibberellin A(9) ((3)H-GA(9)) was metabolized by etiolated shoots of dwarf pea (Pisum sativum cv. Meteor) to GA(20), GA(10), 2,3-dihydro-GA(31), and a number of highly polar, acidic GA-like substances. Identifications were made by gasliquid radiochromatography and combined gas chromatography-mass spectrometry. Kinetic studies showed that GA(30) and 2,3-dihydro-GA(31) were produced within 5 hours following (3)H-GA(9) application to pea shoots. The polar GA-like substances were produced between 5 and 10 hours after (3)H-GA(9) application. Levels of GA(10) increased with time, and since no GA(10) was produced during the purification procedures, GA(10) was, in all probability, produced from (3)H-GA(9) within the plant tissue. The radioactive interconversion products produced by pea from (3)H-GA(9) have chromatographic properties similar to biologically active GA-like substances present in etiolated shoots of dwarf pea. Large scale applications of (3)H-GA(9) with very low specific activity to etiolated pea shoots showed that the radioactivity of the interconversion products was correlated exactly with biological activity as assayed by dwarf rice (Oryza sativa cv. Tan-ginbozu).

Protein Kinase Activity Associated with Isolated Ribosomes from Peas and Lemna

Protein kinase (ATP:protein phosphokinase) has been found associated with ribosomes prepared from 5-day-old etiolated pea shoots and sterile cultures of Lemna minor. The enzyme catalyzes the phosphorylation of ribosomal proteins in vitro and may be involved in the formation of phosphoproteins in the ribosomes in vivo. Protein kinase sediments with ribosomes during sucrose density gradient centrifugation through a buffer containing 0.02 m KCl. Seventy per cent of the enzymic activity dissociates from the ribosomes in 0.3 m KCl, but the remaining protein kinase remains firmly bound in 0.7 m KCl. Cyclic AMP does not modify the activity of these protein kinases in vitro. Benzyladenine inhibits the protein kinase, but only at high concentrations of this cytokinin.

Studies on gibberellins in shoots of light grown peas I. A re-evaluation of the data

Light-grown shoots of ‘Alaska’ pea seedlings were found to contain at least 6 gibberellin-like substances. Chloroplasts isolated from young, fully expanded leaves exhibited a similar spectrum of gibberellin-like activity to the shoots but the absence of a very polar gibberellin, normally present in total leaf extracts, was apparent. Centrifugation studies indicated the association of a gibberellin A 5 20 like (GA 5 20 ) substance with chloroplasts and its absence in the cytoplasm. Tritium-labelled GA 5 ([ 3H]GA 5) and GA 1 ([ 3H]GA 1) applied to light-grown pea shoots were both found to diffuse into agar gel. Preliminary evidence suggests that pea chloroplasts exert a compartmentalization effect on gibberellins in the pea shoot.

Intracellular site of sucrose synthesis in leaves

Improved conditions for extraction and assay increased rates of sucrose synthesis from uridine diphosphate glucose (UDPglucose) plus fructose 6-phosphate (F.6.P) catalysed by leaf extracts 20-fold. Rates of 17.9, 25·0, 9·2 and 27·7 μmol/hr/g fr. wt respectively were obtained from pea shoots, spinach, wheat and bean leaves. Chloroplasts isolated from pea shoots, in which half the plastids were intact, contained less than 4% of the total UDPglucose-fructosephosphate glucosyltransferase, more than 30% of the ribulose diphosphate (RuDP) carboxylase, and more than 40% of the total chlorophyll of the leaf. Although some of the UDPglucose-fructose-phosphate glucosyltransferase was associated with particles smaller than chloroplasts at least 85% of the enzyme was not precipitated at 38 000 g. UDPglucose pyrophosphorylase, also thought to be essential for sucrose synthesis, was distributed between the cell fractions in a similar manner to UDPglucose-fructosephosphate glucosyltransferase. It is concluded that sucrose synthesis in pea shoots and spinach leaves occurs mainly, in the cytoplasm.

Einfluß von Morphaktinen auf den Stoffwechsel höherer Pflanzen: 1. Die Wirkung auf die Atmung uud auf einige glykolytische Enzyme

Investigations were made dealing with the influence of the inhibitor Chloroflurenol on the respiration of pea seedlings, corn seedlings and Lemna . Respiration rate, the activity of aldolase and malate dehydrogenase of dark grown pea roots increase similarly at 5 × 10 −5 M. Morphactins decrease O 2 - consumption of pea shoots; there is no effect on light grown pea roots. The multiplication of Lemna and the respiration of Lemna and corn is decreased. Mitochondria of various pea tissues are modified by Chloroflurenol. They show intramitochondrial inclusions of parallel layers of thin lamellae.

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

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 μg ABA were detected in chloroplasts isolated from 1 kg fresh weight of pea shoots.

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-(14)C from mevalonic acid-2-(14)C 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.