Inhibition of selected plant systems by stereoisomers of chloramphenicol

The stereospecificity of chloramphenicol isomers on the inhibition of several plant systems was investigated. l-Threo, d-erythro, l-erythro and the antibiotic d-threo-chloramphenicol were effective inhibitors of auxin-induced elongation, ¹⁴C-leucine uptake and ¹⁴C-leucine incorporation into the protein fraction of coleoptiles from Avena sativa and Triticum vulgare. The isomers also inhibited Avena coleoptile uptake of ¹⁴C-α-aminoisobutyric acid and the de novo synthesis of α-amylase by aleurone layers from Hordeum vulgare seeds. All four compounds inhibited these processes to about the same extent and over a similar high concentration range (5 × 10⁻⁴ to 5 × 10⁻³ M). Bioassay of extracts from Avena coleoptiles treated with the non-antibiotic isomers revealed no tissue conversion into d-threo-chloramphenicol.     

Abscisic Acid metabolism by source and sink tissues of sugar beet

The fate of exogenously applied, labeled abscisic acid (±)-(ABA) was followed in source leaves and taproot sink tissues of sugar beet (Beta vulgaris cv AH-11). The objective was to determine if differential pathways for ABA metabolism exist in source and sink tissues. Tissue discs were incubated for up to 13 hours in a medium containing 1 micromolar labeled ABA. At various time intervals, samples were taken for metabolite determination by reverse-phase high performance liquid chromatography. The labeled metabolites were identified by retention times using an online scintillation counter.

Dihydrophaseic acid (DPA) aldopyranoside, DPA, phaseic acid (PA), ABA glucose ester (ABA-GE), and two unidentified compounds were recovered from both tissues. An additional unidentified metabolite was also present in root tissue. Leaf tissue discs exhibited a higher capacity for ABA conjugation, and root discs showed a greater preference for ABA catabolism to PA and DPA. After 4 to 5 hours, ABA incorporation into the various metabolites was proportional to the external ABA concentration in both tissues. But the internal ABA pool size was independent of external concentrations below 10⁻⁶ molar. These results suggested that rates of ABA metabolism was proportional to the rates of uptake in both tissues.

     

Gibberellic Acid enhancement of DNA turnover in barley aleurone cells

When imbibed, deembryonated halfseeds from barley (Hordeum vulgare L., var. Himalaya) are incubated in buffer, the DNA content of the aleurone layer increases 25 to 40% over a 24-hour period. In contrast, the DNA of isolated aleurone layers declines by 20% over the same time period. Gibberellic acid (GA) causes a reduction in DNA levels in both halfseed aleurone layers and isolated aleurone layers. GA also increases the specific radioactivity of [³H]thymidine-labeled halfseed aleurone layer DNA during the first 12 hours of treatment. Pulse-chase studies demonstrated that the newly synthesized DNA is metabolically labile.     

Impact of Irrigation Strategies on Abscisic Acid and its Catabolites Profiles in Leaves and Berries of Baco noir Grapes

To understand the relationship among soil and plant water status, plant physiology, and the hormonal profiles associated with it, abscisic acid (ABA) and its catabolites [phaseic acid (PA), dihydrophaseic acid (DPA), 7-hydroxy-ABA, 8′-hydroxy-ABA, neophaseic acid, and abscisic acid glucose ester (ABA-GE)] in leaves and berries from wine grape cultivar Baco noir (Folle blanche × Vitis riparia) were analyzed. The experiment was conducted during the growing seasons 2006 and 2007 in an irrigation trial set up in a commercial vineyard located in Niagara-on-the-Lake, ON, Canada. ABA and its metabolites were quantified using liquid chromatography with ion trap combined with electrospray ionization-mass spectrometry. The hormonal profile indicated a direct relationship between the amount of ABA and climatic factors. The ABA varied between 582 and 4,026 ng g^?1 dry matter (DM), DPA between 417 and 562 ng g^?1, and ABA-GE between 337 and 2,764 ng g^?1 DM. At many sampling times PA in the leaves was undetectable, and its highest concentration (260 ng g^?1 DM) was at beginning of July 2007. ABA followed different catabolic pathways depending on the plant water status. ABA was likely catabolized by conjugation to form ABA-GE in treatments at higher water deficit levels, whereas in treatments with high water status, the oxidation pathway leading to DPA or PA was likely preferred. The ABA and ABA-GE concentrations in the berries at harvest showed high correlation with soil and plant water status.     

The relationship between leaf growth and ABA accumulation in the grass leaf elongation zone

Detached barley (Hordeum vulgare L.) shoots, maintained at different air temperatures and VPDs, were fed ABA via the sub-crown internode in a leaf elongation assay. Analysis of variance of leaf elongation rate (LER) showed significant effects of temperature (T), fed [ABA] and the interaction T × [ABA]. However, the interaction became non-significant when LER was modelled against the [ABA] of the elongation zone, [EZ-ABA] When detached barley shoots were fed sap from droughted maize (Zea mays L.) plants, sap [ABA] could not explain the growth inhibitory activity. Measurement of [EZ-ABA] accounted for this ‘unexplained’ growth inhibition. The detached shoot experiments indicated that [EZ-ABA], and not xylem sap [ABA], was an appropriate explanatory variable to measure in droughted plants. However, ABA accumulation in the elongation zone could not explain a 35% growth reduction in intact droughted plants; thus we considered an interaction of water status and ABA. Using a coleoptile growth assay, we applied mild osmotic stresses (ψ=0 to ?0.06 MPa) and 10^?4 mol m^?3 ABA. Individually, these treatments did not inhibit growth. However, osmotic stress and ABA applied together significantly reduced growth. This interaction may be an important mechanism in explaining leaf growth inhibition of droughted plants.     

Control of lactate dehydrogenase, lactate glycolysis, and alpha-amylase by o(2) deficit in barley aleurone layers

After 4 days in an atmosphere of N₂, aleurone layers of barley (Hordeum vulgare L. cv Himalaya) remained viable as judged by their ability to produce near normal amounts of α-amylases when incubated with gibberellic acid (GA₃) in air. However, layers did not produce α-amylase when GA₃ was supplied under N₂, apparently because α-amylase mRNA failed to accumulate.     

Metabolism of Abscisic Acid in Guard Cells of Vicia faba L. and Commelina communis L

Metabolism of abscisic acid (ABA) was investigated in isolated guard cells and in mesophyll tissue of Vicia faba L. and Commelina communis L. After incubation in buffer containing [G-³H]±ABA, the tissue was extracted by grinding and the metabolites separated by thin layer chromatography. Guard cells of Commelina metabolized ABA to phaseic acid (PA), dihydrophaseic acid (DPA), and alkali labile conjugates. Guard cells of Vicia formed only the conjugates. Mesophyll cells of Commelina accumulated DPA while mesophyll cells of Vicia accumulated PA. Controls showed that the observed metabolism was not due to extracellular enzyme contaminants nor to bacterial action.

Metabolism of ABA in guard cells suggests a mechanism for removal of ABA, which causes stomatal closure of both species, from the stomatal complex. Conversion to metabolites which are inactive in stomatal regulation, within the cells controlling stomatal opening, might precede detectable changes in levels of ABA in bulk leaf tissue. The differences observed between Commelina and Vicia in metabolism of ABA in guard cells, and in the accumulation product in the mesophyll, may be related to differences in stomatal sensitivity to PA which have been reported for these species.

     

Hormonal Response and Seed Viabil ity of Parteresia coarctata,a Brackish Water Species

Stimulation of α-amylase activity was observed in Porteresia coarctata immature seeds (20-day-old) when de-embryonated prewashed half seeds were incubated in media containing gibberellic acid (GA₃, 10^?5M). No such activity was observed in mature seeds even when GA₃ concentration was increased up to five fold. ABA suppressed the GA₃ enhanced α-amylase synthesis up to nearly 70% in the immature seeds. Absence of this enzyme activity in mature seeds may be due to high levels of ABA. The immature aleurone showed a 23 kD polypeptide induced by ABA.     

Control of alpha-amylase mRNA accumulation by gibberellic Acid and calcium in barley aleurone layers

Pulse-labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA₃) with or without 5 millimolar CaCl₂ shows that α-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca²⁺. A cDNA clone for α-amylase was isolated and used to measure α-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca²⁺. No difference was observed in α-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA₃ with 5 millimolar CaCl₂ and layers incubated in GA₃ alone. RNA isolated from layers incubated for 12 hours in GA₃ with and without Ca²⁺ was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca²⁺ in the incubation medium. Immunoprecipitation of translation products showed that the RNA for α-amylase synthesized in Ca²⁺-deprived aleurone layers was translatable. Ca²⁺ is required for the synthesis of α-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing.     

The Catabolism of (+/-)-Abscisic Acid by Excised Leaves of Hordeum vulgare L. cv Dyan and Its Modification by Chemical and Environmental Factors

Excised light-grown leaves and etiolated leaves of Hordeum vulgare L. cv Dyan catabolized applied (±)-[2-¹⁴C]abscisic acid ([±]-[2-¹⁴C]ABA) to phaseic acid (PA), dihydrophaseic acid (DPA), and 2′-hydroxymethyl ABA (2′-HMABA). Identification of these catabolites was made by microchemical methods and by combined capillary gas chromatographymass spectrometry (GC-MS) following high dose feeds of nonlabeled substrate to leaves. Circular dichroism analysis revealed that 2′-HMABA was derived from the (−) enantiomer of ABA. By selecting tissue samples in which endogenous catabolites were undetectable by gas chromatography, it was possible to identify unequivocally ABA catabolites by GC-MS without the need to employ deuteriated substrate to distinguish the (±)-ABA catabolites from the same endogenous compounds. Refeeding studies were used to confirm the catabolic route. The methyl ester of (±)-[2¹⁴C]-ABA was hydrolyzed efficiently by light-grown leaves of H. vulgare. Leaf age played a significant role in (±)-ABA catabolism, with younger leaves being less able than their older counterparts to catabolize this compound. The catabolism of (±)-ABA was inhibited markedly in water-stressed Hordeum leaves which was characterized by a decreased incorporation of label into 2′-HMABA, DPA, and conjugates. The specific, mixed function oxidase inhibitor, ancymidol, did not inhibit, dramatically, (±)-ABA catabolism in light-grown leaves of Hordeum whereas the 80s ribosome, translational inhibitor, cycloheximide, inhibited this process markedly. The 70s ribosome translational inhibitors, lincomycin and chloramphenicol, were less effective than cycloheximide in inhibiting (±)-ABA catabolism, implying that cytoplasmic protein synthesis is necessary for the catabolism of (±)-ABA in Hordeum leaves whereas chloroplast protein synthesis plays only a minor role. This further suggests that the enzymes involved in (±)-ABA catabolism in this plant are cytoplasmically synthesized and are `turned-over' rapidly, although the enzyme responsible for glycosylating (±)-ABA itself appeared to be stable.     

Profile of Plant Hormones and their Metabolites in Germinated and Ungerminated Canola (Brassica napus) Seeds Imbibed at 8°C in either GA4+7, ABA, or a Saline Solution

Abscisic acid (ABA) and gibberellins (GAs) are two major phytohormones that regulate seed germination in response to internal and external factors. In this study we used HPLC-ESI/MS/MS to investigate hormone profiles in canola (Brassica napus) seeds that were 25, 50, and 75% germinated and their ungerminated counterparts imbibed at 8°C in either water, 25 μM GA₄₊₇, a 80 mM saline solution, or 50 μM ABA, respectively. During germination, ABA levels declined while GA₄ levels increased. Higher ABA levels appeared in ungerminated seeds compared to germinated seeds. GA₄ levels were lower in seeds imbibed in the saline solution compared to seeds imbibed in water. Ungerminated seeds imbibed in ABA had lower GA₄ levels compared to ungerminated seeds imbibed in water; however, the levels of GA₄ were similar for germinated seeds imbibed in either water or ABA. The ABA metabolites PA and DPA increased in seeds imbibed in either water, the saline solution, or ABA, but decreased in GA₄₊₇-imbibed seeds. In addition, ABA inhibited GA₄ accumulation, whereas GA had no effect on ABA accumulation but altered the ABA catabolism pathway. Information from our studies strongly supports the concept that the balance of ABA and GA is a major factor controlling germination.     

Characterization of the increased lysophospholipase activity in gibberellic Acid-treated barley aleurone layers

A lysophospholipase (LPL) activity appears in the aleurone of barley (Hordeum vulgare L. cv Himalaya) half seeds during imbibition on moist agar. Secretion of LPL by half seeds is promoted by GA₃; the increase in secretory rate is almost linear from 10⁻¹⁰ to 10⁻⁶ molar GA₃. LPL activity is likewise promoted in isolated aleurone layers by GA₃. Its secretion into the incubation medium requires the continued presence of GA₃ and commences after a 10 to 14 hour lag period when 10 millimolar Ca²⁺ is present. In the absence of Ca²⁺, the lag period remains unchanged but attainment of the maximum secretory rate is delayed. Ca²⁺ alone has very little effect either on LPL activity accumulated in the aleurone layer or in the surrounding medium. However, 50 millimolar Ca²⁺ together with GA₃ dramatically increase the level of secreted activity and of total (accumulated and secreted) activity.

The metabolic inhibitors cycloheximide and actinomycin D inhibit the accumulation of LPL activity in the aleurone and also the secreted activity. Actinomycin D added after the lag period results in a much lower inhibition. The increase in LPL activity in response to GA₃ occurs as a result of de novo synthesis; LPL activity from barley half seeds incubated in 80% D₂O in the presence of GA₃ undergoes a shift to higher density compared with the activity from similar controls incubated in H₂O. The characteristics of the GA₃ enhancement of LPL activity are compared specifically with α-amylase and generally with other GA₃-controlled hydrolases.

     

Proanthocyanidins of barley and sorghum; composition as a function of maturity of barley ears

Sorghum vulgare seeds contain a proanthocyanidin polymer consisting largely of 2,3-cis procyanidin units with M?_n, 2500. Hordeum vulgare ears contain low levels of proanthocyanidin oligomers containing 2–4 units, and composed largely of 2,3-trans procyanidin and prodelphinidin units with catechin as the terminal unit. The concentration of the oligomers in barley ears was virtually constant throughout the 33 day growth and ripening period.     

Nitric oxide accelerates seed germination in warm-season grasses

The nitric oxide (NO) donor sodium nitroprusside (SNP) significantly promoted germination of switchgrass (Panicum virgatum L. cv Kanlow) in the light and in the dark at 25°C, across a broad range of concentrations. SNP also promoted seed germination in two other warm-season grasses. A chemical scavenger of NO inhibited germination and blocked SNP stimulation of seed germination. The phenolic (+)-catechin acted synergistically with SNP and nitrite in promoting seed germination. Acidified nitrite, an alternate NO donor also significantly stimulated seed germination. Interestingly, sodium cyanide, potassium ferricyanide and potassium ferrocyanide at 200 μM strongly enhanced seed germination as well, whereas potassium chloride was without effect. Ferrocyanide and cyanide stimulation of seed germination was blocked by an NO scavenger. Incubation of seeds with a fluorescent NO-specific probe provided evidence for NO production in germinating switchgrass seeds. Abscisic acid (ABA) at 10 μM depressed germination, inhibited root elongation and essentially abolished coleoptile emergence. SNP partially overcame ABA effects on radicle emergence but did not overcome the effects of ABA on coleoptile elongation. Light microscopy indicated extension of the radicle and coleoptiles in seeds maintained on water or on SNP after 2 days. In contrast, there was minimal growth of the radicle and coleoptile in ABA-treated seeds even after 3–4 days. These data indicate that seed germination of warm-season grasses is significantly influenced by NO signaling pathways and document that NO could be an endogenous trigger for release from dormancy in these species.     

Effects of phaseic Acid and dihydrophaseic Acid on stomata and the photosynthetic apparatus

Plant extracts containing phaseic acid (PA), as well as solutions of purified PA and dihydrophaseic acid (DPA) were applied to leaves, isolated mesophyll cells, and isolated epidermal strips. In Commelina communis, stomatal closure began 4 minutes after the addition of either 20 micromolar (±)-abscisic acid or 10 micromolar PA. Stomata closed less rapidly after treatment with 10 micromolar PA than after treatment with 10 micromolar (±)-abscisic acid in Amaranthus powelli, Hordeum vulgare, Xanthium strumarium, and Zea mays and did not respond at all to PA in Vicia faba. DPA (10 micromolar) did not cause stomatal closure in any species.     

Dormancy termination of western white pine (<Emphasis Type="Italic">Pinus monticola</Emphasis> Dougl. Ex D. Don) seeds is associated with changes in abscisic acid metabolism

Western white pine (Pinus monticola) seeds exhibit deep dormancy at maturity and seed populations require several months of moist chilling to reach their uppermost germination capacities. Abscisic acid (ABA) and its metabolites, phaseic acid (PA), dihydrophaseic acid (DPA), 7-hydroxy ABA (7OH ABA) and ABA-glucose ester (ABA-GE), were quantified in western white pine seeds during dormancy breakage (moist chilling) and germination using an HPLC–tandem mass spectrometry method with multiple reaction monitoring and internal standards incorporating deuterium-labeled analogs. In the seed coat, ABA and metabolite levels were high in dry seeds, but declined precipitously during the pre-moist-chilling water soak to relatively low levels thereafter. In the embryo and megagametophyte, ABA levels decreased significantly during moist chilling, coincident with an increase in the germination capacity of seeds. ABA catabolism occurred via several routes, depending on the stage and the seed tissue. Moist chilling of seeds led to increases in PA and DPA levels in both the embryo and megagametophyte. Within the embryo, 7OH ABA and ABA-GE also accumulated during moist chilling; however, 7OH ABA peaked early in germination. Changes in ABA flux, i.e. shifts in the ratio between biosynthesis and catabolism, occurred at three distinct stages during the transition from dormant seed to seedling. During moist chilling, the relative rate of ABA catabolism exceeded ABA biosynthesis. This trend became even more pronounced during germination, and germination was also accompanied by a decrease in the ABA catabolites DPA and PA, presumably as a result of their further metabolism and/or leaching/transport. The transition from germination to post-germinative growth was accompanied by a shift toward ABA biosynthesis. Dormant imbibed seeds, kept in warm moist conditions for 30 days (after an initial 13 days of soaking), maintained high ABA levels, while the amounts of PA, 7OH ABA, and DPA decreased or remained at steady-state levels. Thus, in the absence of conditions required to break dormancy there were no net changes in ABA biosynthesis and catabolism.Abbreviations ABA abscisic acid - ABA-GE abscisic acid glucose ester - DPA dihydrophaseic acid - 7OH ABA 7-hydroxy abscisic acid - 8OH ABA 8-hydroxy abscisic acid - MRM multiple reaction monitoring - PA phaseic acid     

Effect of Abscisic Acid on Uptake and Metabolism of [H]Gibberellin A(1) and [H]Pseudogibberellin A(1) by Barley Half-seeds

Uptake and metabolism of 1,2-[³H]gibberellin A₁ ([³H]GA₁, I) and its 3-hydroxy epimer ([³H]pseudoGA₁, II) by barley (Hordeum vulgare L.) half-seeds were measured after 24 hours of incubation, in the presence or absence of abscisic acid in the media. Uptake of both compounds was enhanced by abscisic acid, and abscisic acid enhanced the extent of metabolism of [³H]GA₁. However, [³H]pseudoGA₁ was not metabolized, even in the presence of abscisic acid. The significance of the stereo-chemistry of the 3-hydroxyl position is discussed.     

Effects of Exogenously Applied Jasmonates on Growth and Intracellular pH in Maize Coleoptile Segments

The effects of jasmonic acid (JA) on elongation growth of coleoptile segments from etiolated maize (Zea mays L.) were investigated in the presence and absence of auxin. When supplied alone, at physiological concentrations (10⁻⁹, 10⁻⁸, and 10⁻⁵ m), JA (or methyl-JA) inhibited growth. JA at a similar range of concentrations also inhibited auxin-induced elongation growth. To determine whether this effect on growth depended on endogenous abscisic acid (ABA), we grew maize coleoptiles in the presence of norflurazon (an inhibitor of carotenoid biosynthesis) that results in reduced endogenous ABA levels. Growth of etiolated coleoptile segments from these plants was inhibited by JA (or methyl-JA) in both the absence and presence of auxin. Previously, we have observed a correlation between elongation growth and cytosolic pH (pH_i), in which auxin lowers pH_i, and growth inhibitors such as ABA raise pH_i. We examined the effect of low concentrations of methyl-JA on pH_i with dual emission dye, carboxy seminaphthorhodafluor-1, and confocal microscopy. To confirm these studies, we also used in vivo ³¹P NMR spectrometry to ascertain the changes in pH_i after addition of jasmonate to maize coleoptiles. Coleoptiles grown in either the absence or presence of norflurazon responded to methyl-JA or JA by increases in pH_i of approximately 0.2 pH unit. This response occurs over a period of 15–20 min and appears to be independent of endogenous ABA. This alkalization induced by JA is likely to form a permissive environment for JA signal transduction pathway(s). Received February 5, 1999; accepted August 25, 1999