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Rhythmic Nitrate Reductase Activity in Leaves of Capsicum annuum L. and the Influence of Kinetin.

In the expanding leaves of Capsicum annuum L. cv. California Wonder, two of the three peaks of nitrate reductase activity associated with the light period exhibit a circadian rhythm that persists in continuous light.The spray application of kinetin to the whole shoot or to leaves other than the ones used for nitrate reductase assay causes a phase shift in the activity peaks and this has been used in preliminary investigations of the character of the mechanisms controlling the timing of the peaks.There was some indication that the rate of translocation of nitrate from the roots might be involved. The levels of nitrate moving up the stem after kinetin treatment were more dependent upon the rate of sap flow than on the concentration of nitrate in the sap. For this reason, transpiration rates in whole plants were measured after kinetin treatment but no change in pattern was seen that would correlate with the phase shift in nitrate reductase activity.The occurrence of nitrate reductase peaks in excised leaves suggested a leaf-based in addition to a root-or stem-based mechanism in the timing of nitrate reductase activity in the leaves.

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Within-day Changes in the Polyribosome Content and in Synthesis of Proteins in Leaves of Capsicum annuum L.

Capsicum annuum cv. California Wonder was grown in controlled environment with a 12-hour photoperiod. The polyribosome content of leaves varied from 60 to 72% of total ribosomes with the highest level occurring in the middle of the photoperiod and the lowest in the middle of the dark period. The variation was accounted for by changes in the content of large polyribosomes (hexamers and larger). There was no indication of an immediate effect on polyribosome content of light-on or light-off.The synthesis of proteins at two times in the 24-hour cycle was compared using a dual isotope technique. Statistically significant results were obtained that suggested that protein(s) with molecular weights of 26,000 daltons were preferentially synthesized in the photoperiod compared to the dark period. No evidence was found for the differential synthesis of proteins within the photoperiod.

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Terpenoid Metabolism in Plastids : Localization of alpha-Tocopherol Synthesis in Capsicum Chromoplasts.

The synthesis of alpha-tocopherol from 2,3-dimethylphytylquinol and S-adenosyl-l-methionine was achieved using Capsicum annuum fruit chromoplasts. The enzymes involved in the cyclization (2,3-dimethyl-phytylquinol cyclase) and methylation (S-adenosyl methionine:gamma-tocopherol methyl-transferase) are both localized in the chromoplast membrane fraction (envelopes and/or a-chlorophyll lamellae), in contrast to the stroma fraction.

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Mechanical Resistance of the Seed Coat and Endosperm during Germination of Capsicum annuum at Low Temperature.

Decoated pepper (Capsicum annuum L. cv Early Calwonder) seeds germinated earlier at 25 degrees C, but not at 15 degrees C, compared to coated seeds. The seed coat did not appear to impose a mechanical restriction on pepper seed germination. Scarification of the endosperm material directly in front of the radicle reduced the time to germination at both 15 degrees C and 25 degrees C.The amount of mechanical resistance imposed by the endosperm on radicle emergence before germination was measured using the Instron Universal Testing Machine. Endosperm strength decreased as imbibition time increased. The puncture force decreased faster when seeds were imbibed at 25 degrees C than at 15 degrees C. The reduction in puncture force corresponded with the ability of pepper seeds to germinate. Most radicle emergence occurred at 15 degrees C and 25 degrees C after the puncture force was reduced to between 0.3 and 0.4 newtons.Application of gibberellic acid(4+7) (100 microliters per liter) resulted in earlier germination at 15 degrees C and 25 degrees C and decreased endosperm strength sooner than in untreated seeds. Similarly, high O(2) concentrations had similar effects on germination earliness and endosperm strength decline as did gibberellic acid(4+7), but only at 25 degrees C. At 15 degrees C, high O(2) concentrations slowed germination and endosperm strength decline.

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Leaf Starch Metabolism during the Growth of Pepper (Capsicum annuum) Plants.

Levels of starch and sugars, and the activities of amylase and starch phosphorylase were measured in expanding leaves harvested in early morning and early evening during the growth of pepper (Capsicum annuum L.; cv ;Bellboy') plants. The differences between starch levels 1 hour after dawn and 1 hour after dusk increased during the period of initial fruit expansion. This diurnal starch difference was strongly correlated with post-dusk amylase activities in leaves at both stages of expansion. There was also a strong correlation between levels of amylase in immature and those in mature leaves throughout the experiment. Phosphorylase activity showed no direct relationship to leaf starch levels, and there was no similarity between activities in immature and mature leaves. An increase in photosynthesis during plant development was observed which could account for the increased starch synthesis at initial fruit expansion.

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Terpenoid Metabolism in Plastids : Isolation and Biochemical Characteristics of Capsicum annuum Chromoplasts.

A technique for the isolation and the purification of Capsicum annum L. var. Yolo Wonder chromoplasts is described. The degree of purity of the isolated chromoplasts is greatly improved by the absence of MgCl(2) in the extraction medium and in the gradient purification system, as shown by electron micrographs and the near absence of antimycin-insensitive NADH:cytochrome c reductase activity and succinate:cytochrome c reductase activity. Furthermore, phosphatidylserine was absent and the phosphatidylethanolamine content was reduced by a factor of 5 in these preparations, which were active in the synthesis of galactolipids, prenylquinones, and carotenoids.

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Function of Rhizodermal Transfer Cells in the Fe Stress Response Mechanism of Capsicum annuum L.

A variety of red pepper (Capsicum annuum L., cv Yaglik) responds to Fe deficiency stress with simultaneously enhanced H(+) extrusion, reduction of ferric ions and synthesis of malic and citric acid in a swollen subapical root zone densely covered with root hairs. It is demonstrated that these stress responses temporally coincide with the development of rhizodermal and hypodermal transfer cells in this root zone. During stress response the transfer cells show a marked autofluorescence which could arise from endogenous iron chelators of the phenolic acid type. The presence of organelle-rich cytoplasm which often exhibits rotational cytoplasmic streaming points to high physiological activity and makes these cells, with their increased plasmalemma surface, particularly well suited for the entire stress response mechanism. Since Fe stress-induced acidification is diminished by vanadate and erythrosin B, both specific inhibitors of plasmalemma ATPases, it seems reasonable to suppose that H(+) pumping from transfer cells is activated by an ATPase located in their plasmamembrane. H(+) extrusion is also shown to be inhibited by abscisic acid. Raised phosphoenolpyruvate carboxylase activity and simultaneous accumulation of malate in the swollen root zone point to the action of a pH stat preventing a detrimental rise in cytoplasmic pH of transfer cells during enhanced H(+) extrusion. The simultaneous increase in citric acid concentration favors chelation of iron at the site of its uptake and thus ensures long distance transport to the areas of metabolic demand. A direct link between citrate accumulation and ferric ion reduction as proposed in recent literature further supports the crucial role of transfer cells in the response to Fe deficiency stress.

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Chromoplast-Specific Proteins in Capsicum annuum.

Chromoplasts are a common differentiation state of plastids in which the photosynthetic apparatus is absent and carotenoids accumulate to high levels. As a first step toward the isolation of chromoplast-specific genes, we have examined plastids of the bell pepper, Capsicum annuum L., for the presence of chromoplast-specific proteins. Intact chromoplasts were isolated from mature fruits of C. annuum var Emerald Giant, Golden Cal Wonder, and DNAP VS-12 by differential centrifugation followed by isopycnic sedimentation in gradients of silica sols. The plastids were then fractionated into soluble and membrane components and the proteins analyzed by one- and two-dimensional gel electrophoresis using isoelectric focusing, sodium dodecyl sulfate, and sodium dodecyl sulfate-urea gels. Two polypeptides with M(r) of 35,000 and 58,000 accumulate to high levels in membrane fractions of chromoplasts of var Emerald Giant. These polypeptides are either not detectable or barely detectable in chloroplasts from immature fruits. Both polypeptides have been purified to near homogeneity. Yellow chromoplasts from var Golden Cal Wonder and red chromoplasts from var DNAP VS-12 contained the 35-kilodalton polypeptide, but not the 58-kilodalton species.

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Depression of Photosynthesis, Growth, and Yield in Field-Grown Green Pepper (Capsicum annuum L.) Exposed to Acidic Fog and Ambient Ozone.

The relationship among physiological, injury, growth, and yield responses was examined in field-grown green pepper (Capsicum annuum L. ;California Wonder') subjected to two airborne environmental stresses. The primary objectives were to determine if the stresses could cause alterations in the plant responses, and to determine if any stress induced alterations in physiological or injury responses were correlated with effects on growth or yield. Responses were monitored in green pepper exposed to simulated acidic fog alone, or in combination with ambient concentrations of ozone in open-top field chambers. Both highly acidic fog and ambient ozone depressed green pepper growth and yield responses via the inhibition of photosynthesis. Applications of highly acidic fog (i.e. two exposures of pH 1.68 fog per week for 11 weeks) caused a significant depression of net photosynthesis, reduction in leaf buffering capacity, and an extensive amount of leaf injury. These alterations closely paralleled decreases in growth and yield on a percentage basis. In contrast, ambient ozone had similar impacts on net photosynthesis, growth and yield, but enhanced leaf buffering capacity, and caused no visible injury. The pollutant-specific differences in plant response are discussed with respect to whole-plant carbon metabolism and physiological compensation.

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Changes in Fructose 2,6-Bisphosphate Levels in Green Pepper (Capsicum annuum L.) Fruit in Response to Temperature.

The regulatory metabolite, fructose 2,6-bisphosphate (Fru 2,6-P(2)) was found in green pepper (Capsicum annuum L.). The Fru 2,6-P(2) level was found to: (a) rise rapidly in response to heat; (b) drop rapidly, followed by recovery, in response to cold storage of fruit and, (c) oscillate during cold storage of fruit. The possible existence of a relationship between chilling injury and Fru 2,6-P(2) is considered.

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Synthesis of Two Chromoplast-Specific Proteins During Fruit Development in Capsicum annuum.

The time-course of accumuiation of two membrane proteins during fruit ripening was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blots in tissue extracts of Capsicum annuum L., vars Emerald Giant, Albino, and DNAP VS-12. The proteins, named ChrA and ChrB, were previously shown to occur specifically in chromoplasts. Fruit development was divided into five stages based on changes in color. ChrA was not detectable in the first three stages, but accumulated to a high level in the fully mature, red fruit. ChrB was not detectable in the first, mature-green stage of fruit maturation, but was found in the second stage, when carotenoid accumulation first appeared, and in all later stages. The patterns of accumulation in chromoplasts that develop from proplastids or leucoplasts are similar to those in chromoplasts that develop from chloroplasts. We conclude that ChrA and ChrB are probably synthesized de novo during chromoplast development.

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Solubilization of covalently bound extensin from capsicum cell walls.

Acidified sodium chlorite cleaves isodityrosine and solubilizes covalently bound hydroxyproline-rich material from cell walls. This has been taken as evidence that isodityrosine acts as a cross-link holding the hydroxyproline-rich glycoprotein extensin in the cell wall. However, acidified chlorite was found to cleave peptide bonds in salt-soluble extensin and in bovine serum albumin (BSA). This invalidates the use of conventional acidified chlorite treatment to provide evidence for isodityrosine cross-links. The ratio of BSA:chlorite was important in determining peptidyl cleavage. At a ratio of 0.75:1.00 (mole amino acid residues/mole chlorite), or higher, peptidyl cleavage was not detected. Furthermore, in samples where a low concentration of radioactive extensin was present, BSA substantially protected the peptide bonds of the extensin against peptidyl cleavage during treatment with acidified chlorite, while not preventing the cleavage of isodityrosine. Therefore, acidified sodium chlorite plus BSA was a more specific reagent for the cleavage of isodityrosine than was acidified chlorite alone. This modified treatment solubilized in intact form the ;covalently bound' extensin from cell walls of Capsicum frutescens (chili pepper) suspension cultures, providing new evidence compatible with the view that extensin molecules are held in the cell wall by isodityrosine cross-links.

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ChrA Is a Carotenoid-Binding Protein in Chromoplasts of Capsicum annuum.

Chromoplasts of Capsicum annuum var Albino contain a carotenoid-protein complex, which migrates as a brilliant orange band in gels under conditions of nondenaturing electrophoresis. In a second, denaturing separation, the complex resolves into a principal protein (ChrA) of 58 kilodaltons and several minor proteins of 20 to 55 kilodaltons, which may be adventitiously associated. Analysis of Western blots of both one- and two-dimensional gels showed that the principal protein component of the carotenoid complex is ChrA, a protein previously shown to be located specifically in chromoplast membranes. The identification of ChrA as a carotenoid-binding protein appears to be the first instance of a nonthylakoid, carotenoid-binding protein in higher plants.

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Regulation of Carbon Partitioning in Source and Sink Leaf Parts in Sweet Pepper (Capsicum annuum L.) Plants : Role of Fructose 2,6-Bisphosphate.

Area expansion rate, partitioning of photosynthetically fixed carbon, and levels of fructose 2,6-bisphosphate (fru-2,6-P(2)) were determined in individual parts of developing leaves of sweet pepper (Capsicum annuum L.). The base was rapidly expanding and allocated less carbon to sucrose synthesis in comparison to the leaf tip, where expansion had almost stopped. The change in leaf expansion rate and carbon partitioning happened gradually. During day time levels of fru-2,6-P(2) were consistently higher in the leaf base than in the leaf tip. Leaf expansion rate and carbon partitioning were closely related to day time levels of fru-2,6-P(2), suggesting that fru-2,6-P(2) is an important factor in adjustment of metabolism during sink-to-source transition of leaf tissue. The levels of fru-2,6-P(2) changed markedly after a dark-to-light transition in the leaf base, but not in the leaf tip, suggesting that regulatory systems based on fru-2,6-P(2) are different in sink and source leaf tissue. During the period upon dark-to-light transition the variations in level of fru-2,6-P(2) did not show a close correlation to changes in the carbon partitioning, until the metabolism had reached a steady state.

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Stomatal and Nonstomatal Components to Inhibition of Photosynthesis in Leaves of Capsicum annuum during Progressive Exposure to NaCl Salinity.

Young bell pepper (Capsicum annuum L.) plants grown in nutrient solution were gradually acclimated to 50, 100, or 150 moles per cubic meter NaCl, and photosynthetic rates of individual attached leaves were measured on several occasions during the salinization period at external CO(2) concentrations ranging from approximately 70 to 1900 micromoles per mole air. Net CO(2) assimilation (A) was plotted against computed leaf internal CO(2) concentration (C(i)), and the initial slope of this A-C(i) curve was used as a measure of photosynthetic ability. During the 10 to 14 days after salinization began, leaves from plants exposed to 50 moles per cubic meter NaCl showed little change in photosynthetic ability, whereas those treated to 100 or 150 moles per cubic meter NaCl had up to 85% inhibition, with increase in CO(2) compensation point. Leaves appeared healthy, and leaf chlorophyll content showed only a 14% reduction at the highest salinity levels. Partial stomatal closure occurred with salinization, but reductions in photosynthesis were primarily nonstomatal in origin. Photosynthetic ability was inversely related to the concentration of either Na(+) or Cl(-) in the leaf laminas sampled at the end of the experimental period. However, the concentration of Cl(-) expressed on a tissue water basis was greater, exceeding 300 moles per cubic meter, and Cl(-) was more closely associated (R(2) = 0.926) with the inhibition of photosynthetic ability. Leaf turgor was not reduced by salinization and leaf osmotic potential decreased to a slightly greater extent than the osmotic potential decreases of the nutrient solutions. Concentration of accumulated Na(+) and Cl(-) (on a tissue water basis) accounted quantitatively for maintenance of leaf osmotic balance, assuming that these ions were sequestered in the vacuoles.

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The carotenoid 7,8-dihydro-psi end group can be cyclized by the lycopene cyclases from the bacterium Erwinia uredovora and the higher plant Capsicum annuum.

The genes for geranylgeranyl diphosphate synthase (crtE) and phytoene synthase (crtB) from the epiphytic bacterium Erwinia uredovora and the phytoene desaturase gene from the photosynthetic bacterium Rhodobacter capsulatus (Rc-crtI) were introduced into Escherichia coli, which resulted in the accumulation of the acyclic carotenoid, neurosporene. Further introduction of the lycopene cyclase gene from E. uredovora (crtY) or the higher plant Capsicum annuum (Icy) resulted in the production of a bicyclic carotenoid, 7,8-dihydro-beta-carotene, via monocyclic beta-zeacarotene. zeta-Carotene was also found to be cyclized to bicyclic 7,8,7',8'-tetrahydro-beta-carotene by the Erwinia cyclase. These results indicate that both lycopene cyclases can cyclize a 7,8-dihydro-psi end group to a 7,8-dihydro-beta end group, in addition to the usual cyclization of the psi end group to the beta end group. Furthermore, beta-carotene hydroxylase from Erwinia (CrtZ) was able to add a hydroxyl group to the 7,8-dihydro-beta end group and the beta end group.

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Effects of oleoresin capsicum pepper spray on human corneal morphology and sensitivity.

PURPOSE: To examine the potential harmful effects on corneal structure, innervation, and sensitivity of a spray containing the neurotoxin capsaicin (oleoresin capsicum, OC). METHODS: Ten police officers who volunteered for the study were exposed to OC. Clinical signs were assessed. Corneal sensitivity was measured using a Cochet-Bonnet or a noncontact esthesiometer that provides separate measurements of mechanical, chemical, and thermal sensitivity. Tear fluid nerve growth factor (NGF) was measured. Corneal cell layers and subbasal nerves were examined by in vivo confocal microscopy. The subjects were examined before application and 30 minutes, 1 day, 1 week, and 1 month after OC exposure. RESULTS: OC spray produced occasional areas of focal epithelial cell damage that healed within 1 day. Each eye showed conjunctival hyperemia and in two subjects, mild chemosis. All except one eye had unchanged best corrected visual acuity (BCVA). A transient decrease (day 1) of mechanical sensitivity was observed with the Cochet-Bonnet esthesiometer. With the gas esthesiometer, mechanical sensitivity remained below normal values for 7 days. Chemical sensitivity to CO2 was high for as much as 1 day and decreased below normal 1 week later, whereas sensitivity to cold was unaffected. Two subjects had measurable tear NGF that increased after exposure. Basal epithelial cell morphology suggested temporary corneal epithelial swelling, whereas keratocytes, endothelial cells, and subbasal nerves remained unchanged. CONCLUSIONS: Although OC causes immediate changes in mechanical and chemical sensitivity that may persist for a week, a single exposure to OC appears harmless to corneal tissues. The changes are possibly associated with damage of corneal nerve terminals of mainly unmyelinated polymodal nociceptor fibers.

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