Dark fixation of CO2 during gluconeogenesis by the cotyledons of Cucurbita pepo L.

We did this work to discover the pathway of CO₂ fixation into sugars in the dark during gluconeogenesis by the cotyledons of 5-day-old seedlings of Cucurbita pepo L. We paid particular attention to the possibility of a contribution from ribulosebisphosphate carboxylase. The detailed distribution of ¹⁴C after exposure of excised cotyledons to ¹⁴CO₂ in the dark was determined in a series of pulse and chase experiments. After 4s in ¹⁴CO₂, 89% of the ¹⁴C fixed was in malate and aspartate. In longer exposures, and in chases in ¹²CO₂, label appeared in alanine, phosphoenolpyruvate, 3-phosphoglycerate and sugar phosphates, and accumulated in sugars. The transfer of label from C-4 acids to sugars was restricted by inhibition of phosphoenolpyruvate carboxykinase in vivo by 3-mercaptopicolinic acid. We conclude as follows. Initial fixation of CO₂ in the dark is almost entirely into phosphoenolpyruvate, probably via phosphoenolpyruvate carboxylase (EC 4.1.1.31) which we showed to be present in appreciable amounts. Incorporation into sugars occurs chiefly, if not completely, as a result of randomization of the carboxyl groups of the C-4 acids and subsequent conversion of the oxaloacetate to sugars via the accepted sequence for gluconeogenesis. Ribulosebisphosphate carboxylase appears to make very little contribution to sugar synthesis from fat.     

Phytochrome and phosphotungstate-chromate-positive vesicles from Cucurbita pepo L.

The phosphotungstic acid-chromic acid (PTA-CrO₃) stain, putatively specific for the plasma membrane of plants, has been used in an attempt to monitor the distribution of this membrane in a 20,000 x g particulate fraction from Cucurbita hypocotyl hooks. On discontinuous sucrose gradients, the relative distributions of the phytochrome and PTA-CrO₃-positive vesicles present in this fraction appear to be correlated. When intact tissue is stained, however, other components, in addition to the plasma membrane, react positively to the stain. These components include prolamellar-body membranes, lipid droplets, and ribosomes. This lack of specificity calls into question the reliability of the technique for the unequivocal identification and accurate quantitation of plasma-membrane fragments in isolated particulate fractions. The present data do not, therefore, provide unambiguous evidence that phytochrome is associated with plasma membrane in tissue homogenates from Cucurbita.Abbreviations PTA-CrO₃ phosphotungstate-chromate - RNP ribonucleoprotein     

Quality of a stress-sensitive Cucurbita pepo L. pollen

The quality of Cucurbita pepo L. pollen was studied using field pollinations and the fluorochromatic-reaction test. The extreme sensitivity of this pollen to dehydration and ageing is demonstrated. Controlled stress applied to mature pollen leads to the development of seedless fruits. Molecular signals seem to be involved in the induction of this parthenocarpy. These results indicate the existence of distinct sequences involved in the completion of the fertilization program of pollen. With pollen altered by stress, the fertilization process may be stopped at different stages of its completion. We bring evidence that Cucurbita pepo plants have developed special adaptations in order to compensate for the poor viability of their pollen.Abbreviation FCR fluorochromatic reaction     

An endogenous RNA-synthesis-promoting oligopeptide from Cucurbita pepto var. patissonina

The effect of CPPTI (Cucurbita pepo patissonina trypsin inhibitor) on the growth of Cucurbita pepo var patissonina (White bush) was examined. Plants treated with CPPTI grew faster than untreated plants and similarly to those treated with gibberellic acid. Isolated cell nuclei from plants treated with CPPTI showed that of the three DNA-dependent RNA polymerases assayed, RNA polymerase I (EC2.7.7.6) activity was preferentially elevated.Abbreviations CPPTI Cucurbita pepo patissonina trypsin inhibitor - GA₃ gibberellic acid - IAA indole-3-acetic acid - kDa kilodalton - RP I DNA-dependent RNA polymerase I     

Genetic control of plastid carotenoids and transformation in the skin of Cucurbita pepo L. fruit

Summary The influence of allelic state of gene B on skin pigmentation in two cultivars of Cucurbita pepo L. has been studied. Total carotenoids were lower at early stages of fruit development in cultivar (cv.) Early Prolific (EP) BB YY fruit skin, than in EP B ⁺ B ⁺ YY fruit skin, but no differences were observed in total skin carotenoids twenty days after anthesis. Total carotenoids were lower in cv. Fordhook Zucchini (FZ) BB yy fruit skin, than in FZ B ⁺ B ⁺ yy fruit skin at all developmental stages from anthesis to maturity. Both green and yellow tissues contained typical foliar carotenoids. The carotenoids from yellow fruit skin of both EP genotypes and of FZ BB were characterized by a low carotene: xanthophyll ratio, with a high proportion of the xanthophylls esterified to fatty acids. The xanthophylls of the yellow tissues were esterified with 120, 140, 160 fatty acids. The carotenoids from the green fruit skin of FZ B ⁺ B ⁺ had a higher percentage of carotenes (primarily -carotene) and a lower percentage of esterified xanthophylls. Spectral shapes of carotenoid fractions from all yellow tissues were similar and distinguishable from those of green FZ B ⁺ B ⁺ tissue. The results of these studies are discussed in terms of the genetic control of plastid transformation in Cucurbita pepo L.New Jersey Agricultural Experiment Station No. D99201 (NE-9) 32-83, supported by state funds and funds from the Rutgers University Research Council     

Control of gluconeogenesis by phosphoenolpyruvate carboxykinase in cotyledons ofCucurbita pepo L.

3-Mercaptopicolinic acid, a non-competitive inhibitor of phosphoenolpyruvate carboxykinase (EC 4.1.1.19) was used to study the control of gluconeogenesis by this enzyme in germinating marrow (Cucurbita pepo) cotyledons. In vitro, phosphoenolpyruvate carboxykinase was inhibited by 3-mercaptopicolinic acid, with aKi of 5.9 M. At 25°C the inhibitor caused an increase in the label incorporated from [2-¹⁴C]acetate into CO₂, and a decrease in the label incorporated into the insoluble and neutral fractions. Phosphoenolpyruvate carboxykinase had a flux control coefficient for gluconeogenesis (C _PEPCK ^J ) of between 0.7 and 1.0. 3-Mercaptopicolinic acid was a less effective inhibitor of phosphoenolpyruvate carboxykinase at lower temperatures (Ki = 8.6 M at 17°C, 13.3 M at 10°C) and had similar effects on the metabolism of [2-¹⁴C]acetate by marrow cotyledons when the temperature was reduced to 17°C and 10°C. The control coefficient for this enzyme did not change with temperature, indicating that phosphoenolpyruvate carboxykinase exerts a high degree of control over gluconeogenesis at all temperatures examined.Abbreviations PEP Phosphoenolpyruvate - PEPCK PEP carboxykinase The authors thank Dr. Ian Woodrow (University of Melbourne, Australia) for helpful discussions. This work was supported by a grant from the Science and Engineering Research Council, U.K. (GR/F 50978).     

Secondary plasmodesmata formation in the minor-vein phloem of Cucumis melo L. and Cucurbita pepo L.

Numerous branched plasmodesmata (pd) are present between bundle-sheath cells (BSCs) and specialized companion cells known as intermediary cells (ICs) in the minor-vein phloem of melon (Cucumis melo L.) and squash (Cucurbita pepo L.). These pd were found to be secondary, i.e., they form across existing walls. Sink, sink-source transition, and source tissues were sampled from developing and mature leaves. In sink tissue, IC precursors divide to produce the two to four ICs and associated sieve elements which are present by the time of the sink-source transition. Plasmodesmata along the interface between the IC precursor and adjacent BSCs in sink tissue are unbranched and few in number. Before the leaf tissue undergoes the sink-source transition, the number of pd channels (individual branches of pd) becomes more numerous. This increase in number of pd channels occurs at least in part and perhaps entirely by branching, resulting in more channels on the IC-side than on the BSC-side. In melon there is a 12-fold increase in the number of pd channels within the IC-side of the interface and a corresponding 9-fold increase in pd channels within the BSC-side. Thus, secondary pd form by the time of the sink-source transition and may be involved in phloem loading and photoassimilate export. The system described is well-defined and amenable to experimental manipulation: secondary pd form in large numbers, at a particular interface, over a short period of time, and in a highly predictable manner.Abbreviations BSC bundle-sheath cell - DAP days after planting - IC intermediary cell - LPI leaf plastochron index - pd plasmodesmata - PI plastochron interval We thank Edith Haritatos, Rich Medville, Esther Gowan, and Nancy Dussault for expert technical assistance. This research was supported by an NSF/DOE/USDA Cornell Plant Science Center fellowship (G.M.V.), Natural Sciences and Engineering Research Council Grant GP0138401 and Université de Montréal, Fonds internes de recherche (D.U.B.), and NSF grant IBN-9419703 (R.T.).     

The effects of calcium deficiency on Cucurbita pepo L. hypocotyl cells: A 31P nuclear-magnetic-resonance study

Calcium deficiency in zucchini (Cucurbita pepo L.) is associated with reduced growth and a reduced ability to transport auxin (Allan and Rubery, 1991, Planta 183, 604–612). An investigation of the effects of calcium-deficiency on zucchini hypocotyl cells was made using weak-acid uptake and ³¹P-nuclear-magneticresonance (³¹P-NMR) spectroscopy in vivo and in tissue extracts. Calcium-deficient tissue had the same cytoplasmic and vacuolar pHs as normal tissue when extracellular pH was near neutral. At acidic external pH the vacuolar pH was lower in deficient tissue. Adenine nucleotides were present predominantly as ATP in both control and calcium-deficient tissues. Addition of calcium to calcium-deficient tissue, under conditions which cause recovery of auxin transport induced no changes in the ³¹P-NMR spectra of deficient tissue. The content of mobile, phosphorylated metabolites was reduced in calcium-deficient tissue in comparison to control tissue. However, a substantial increase in the content of phosphorylcholine occurs in calcium-deficient tissues compared with controls; this may reflect changes in lipid turnover in calcium-stressed cells. We wish to thank Drs. Terry Moore and Jamie Vandenberg for technical assistance and Professor Peter Morris for providing the gated oxygen device. A.C.A. thanks the Cambridge Commonwealth Trust for a Prince of Wales Scholarship and the O.R.S. Awards Scheme for an award.     

Chromatin-bound DNA-dependent RNA polymarase in developing pea cotyledons

The chromatin-bound DNA-dependent RNA polymerase activity, and template availability to added homologous RNA polymerase, were determined for pea cotyledons which were allowed to develop in different environments. Both the maximum polymerase activity and the template availability were higher in cotyledons that were developing at the faster rate. The template availability was approximately constant within an environment throughout most of the development, but differed between environments. The extra DNA sythesised, that is that above the 2C level, during cytyledon development appeared to be more utilised for RNA synthesis in slowly developing cotyledons than in more rapidly developing ones.     

Effect of benzyladenine on the development of plastids and microbodies in excised watermelon cotyledons

Excised watermelon (Citrullus vulgaris Schrad.) cotyledons were grown in the dark in the presence of 0.1 mM benzyladenine (BA). Under these conditions reserve breakdown and organelle differentiation progress very slowly. Treatment with BA accelerates, breakdown of reserves and stimulates development of organelles. Electron micrographs of cells from treated cotyledons show a larger number of plastids with a more developed inner membrane system. The levels of plastid pigments and enzymes are increased while starch content is reduced. Glyoxysomal enzyme levels are increased by BA during the first three days of development and their decline is accelerated thereafter. Also the activity of hydroxypyruvate reductase (EC 1.1.1.81.), a peroxisomal enzyme, is increased, but this increase is not followed by a decay phase. In water controls, hydroxypyruvate reductase bands together with glyoxysomal enzymes after equilibrium centrifugation in a sucrose gradient. In treated cotyledons the equilibrium position of glyoxysomal enzymes is uchanged while that of hydroxypyruvate reductase is shifted to a lower density.Abbreviations BA benzyladenine - RuDP ribulose-1,5-diphosphate - HPR hydroxypyruvate reductase     

Targeting of auxin carriers to the plasma membrane: effects of monensin on transmembrane auxin transport in Cucurbita pepo L. tissue

Treatment of etiolated zucchini (Cucurbita pepo L.) hypocotyl tissue with sub-micromolar concentrations of the cationophore monensin rapidly (<20 min) inhibited the transport catalytic activity of the specific auxin-anion efflux carrier and reduced the inhibition of this carrier by the phytotropin N-1-naphthylphthalamic acid (NPA). Monensin inhibited the basipetal polar transport of indol-3yl-acetic acid (IAA) in long (30 mm) zucchini segments. At concentrations lower than 10^–5 mol·dm^–3 monensin did not affect uptake of the pH probe [2-¹⁴C]5,5-dimethyloxazolidine-2,4-dione (DMO) or that of the membrane-potential probe tetra[¹⁴C-phenyl]phosphonium bromide (TPP⁺), did not affect the response of IAA net uptake to external Ca²⁺ concentration and did not alter the metabolism of IAA. It was concluded that low concentrations of monensin inhibit transport through the Golgi apparatus of auxin efflux carrier protein and that the efflux carriers turn over very rapidly in the plasma membrane. Monensin pretreatment did not affect the saturable binding of [³H]NPA to microsomal membranes, indicating that the auxin-efflux catalytic sites and the NPA-binding sites are located on separate proteins. At higher concentrations (10^–5 mol·dm^–3) monensin inhibited both mediated uptake and mediated efflux components of IAA transport. This effect was at least in part attributable to perturbation by monensin of the driving forces for mediated uptake since high concentrations of monensin also reduced the uptake of DMO and TPP⁺.Abbreviations CH cycloheximide - DMO 5,5-dimethyloxazolidine-2,4-dione - MDMP 2-(4-methyl-2,6-dinitroanlilino)N-methyl-propionamide - NPA N-1-naphthylphthalamic acid - TPP⁺ tetraphenylphosphonium ion We thank Mrs. R.P. Bell for technical assistance and Drs. G.F. Katekar and M.A. Venis for generous gifts of NPA. S.W. was supported by the U.K. Science and Engineering Research Council.     

Pollen gene expression analysed by micro-isoelectric focusing of proteins from isolated pollen grains in Cucurbita pepo L.

Summary Isoelectric focusing of proteins from single pollen grains of Cucurbita pepo L. has been developed for large scale study of pollen grain populations' heterogeneity. Forty to forty-five protein bands from one pollen grain are revealed after silver staining. Applications of this technique to pollen grain populations from different genotypes are described in this paper. Possible applications and limits of this technique are discussed with respect to plant breeding especially for the measure of gene frequencies in pollen grain populations.The work was partly supported by a grant from LIMAGRAIN and AGPM     

Efficient protein expression from the endogenous RNA polymerase I promoter using a human ribosomal DNA targeting vector

Vector systems to deliver, integrate and express therapeutic genes in host cells are essential for gene therapy. In the present study, we investigated a novel vector system for integration and expression of a transgene. In this system, the transgene expression was driven by an endogenous RNA polymerase I (Pol I) promoter after being integrated into the ribosomal DNA (rDNA) locus. Human coagulation factor IX coding sequence (FIX), with an internal ribosome entry sites element at its leader region, was targeted into the 18S rDNA locus via homologous recombination. FIX protein expression, which was under the control of the endogenous Pol I promoter, was found to be similar to that of a moderate Pol II promoter. The average FIX expression level of the rDNA recombinants was additionally enhanced to that from a strong Pol II promoter as a result of elimination of position effects. Our data suggest the possibility of applying this system in gene therapy for hereditary diseases.     

Phylogenetic relationship of Alexandrium tamiyavanichii (Dinophyceae) to other Alexandrium species based on ribosomal RNA gene sequences

The phylogenetic relationship of the thecate PSP-toxin producing dinoflagellate Alexandrium tamiyavanichii Balech to other species of Alexandrium was studied based on nucleotide sequences of the ITS1, ITS2, 5.8S, 18S and 28S subunits of the ribosomal RNA gene. These are the first such sequences available for A. tamiyavanichii, which is one of the producers of paralytic shellfish poisoning toxins in tropical waters. Based on the nucleotide sequences of the 28S, 18S and 5.8S subunits of the rRNA gene, A. tamiyavanichii grouped together with A. tamarense, A. catenella and A. fundyense. More interestingly, A. tamiyavanichii was most closely affiliated to A. tamarense isolates from Thailand. This result reaffirmed conclusions from previous studies that, for the A. tamarense/fundyense/catenella species complex, geographical origin rather than morphology seems to determine genetic relatedness. Results of this study also suggest that A. tamiyavanichii most probably belongs to the same species complex. Ribosomal RNA gene sequences do not separate the PSP toxin producing from the non-producing species of Alexandrium.     

Purification and restriction endonuclease mapping of soybean 18 S and 25 S ribosomal RNA genes

The restriction endonuclease map of the 25 S and 18 S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifugation in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm^–3 by analytical centrifugation in CsCl. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Bam H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9·10⁶ or approximately 9,000 kb. The 25 S and 18 S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [¹²⁵I]25 S or [¹²⁵I]18 S rRNA. It was demonstrated that there is no heterogeneity even in the spacer region of the soybean rDNA.