Abscisic Acid Biosynthesis in Leaves and Roots of Xanthium strumarium

Research on the biosynthesis of abscisic acid (ABA) has focused primarily on two pathways: (a) the direct pathway from farnesyl pyrophosphate, and (b) the indirect pathway involving a carotenoid precursor. We have investigated which biosynthetic pathway is operating in turgid and stressed Xanthium leaves, and in stressed Xanthium roots using long-term incubations in ¹⁸O₂. It was found that in stressed leaves three atoms of ¹⁸O from ¹⁸O₂ are incorporated into the ABA molecule, and that the amount of ¹⁸O incorporated increases with time. One ¹⁸O atom is incorporated rapidly into the carboxyl group of ABA, whereas the other two atoms are very slowly incorporated into the ring oxygens. The fourth oxygen atom in the carboxyl group of ABA is derived from water. ABA from stressed roots of Xanthium incubated in ¹⁸O₂ shows a labeling pattern similar to that of ABA in stressed leaves, but with incorporation of more ¹⁸O into the tertiary hydroxyl group at C-1′ after 6 and 12 hours than found in ABA from stressed leaves. It is proposed that the precursors to stress-induced ABA are xanthophylls, and that a xanthophyll lacking an oxygen function at C-6 (carotenoid numbering scheme) plays a crucial role in ABA biosynthesis in Xanthium roots. In turgid Xanthium leaves, ¹⁸O is incorporated into ABA to a much lesser extent than it is in stressed leaves, whereas exogenously applied ¹⁴C-ABA is completely catabolized within 48 hours. This suggests that ABA in turgid leaves is either (a) made via a biosynthetic pathway which is different from the one in stressed leaves, or (b) has a half-life on the order of days as compared with a half-life of 15.5 hours in water-stressed Xanthium leaves. Phaseic acid showed a labeling pattern similar to that of ABA, but with an additional ¹⁸O incorporated during 8′-hydroxylation of ABA to phaseic acid.     

Evidence for a universal pathway of abscisic Acid biosynthesis in higher plants from o incorporation patterns

Previous labeling studies of abscisic acid (ABA) with ¹⁸O₂ have been mainly conducted with water-stressed leaves. In this study, ¹⁸O incorporation into ABA of stressed leaves of various species was compared with ¹⁸O labeling of ABA of turgid leaves and of fruit tissue in different stages of ripening. In stressed leaves of all six species investigated, avocado (Persea americana), barley (Hordeum vulgare), bean (Phaseolus vulgaris), cocklebur (Xanthium strumarium), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum), ¹⁸O was most abundant in the carboxyl group, whereas incorporation of a second and third ¹⁸O in the oxygen atoms on the ring of ABA was much less prominent after 24 h in ¹⁸O₂. ABA from turgid bean leaves showed significant ¹⁸O incorporation, again with highest ¹⁸O enrichment in the carboxyl group. The ¹⁸O-labeling pattern of ABA from unripe avocado mesocarp was similar to that of stressed leaves, but in ripe fruits there was, besides high ¹⁸O enrichment in the carboxyl group, also much additional ¹⁸O incorporation in the ring. In ripening apple fruit tissue (Malus domestica), singly labeled ABA was most abundant with more ¹⁸O incorporated in the tertiary hydroxyl group than in the carboxyl group of ABA. Smaller quantities of this monolabeled product (C-1′-¹⁸OH) were also detected in the stressed leaves of barley, bean, and tobacco, and in avocado fruits. It is postulated that a large precursor molecule yields an aldehyde cleavage product that is, in some tissues, rapidly converted to ABA with retention of ¹⁸O in the carboxyl group, whereas in ripening fruits and in the stressed leaves of some species the biosynthesis of ABA occurs at a slower rate, allowing this intermediate to exchange ¹⁸O with water. On the basis of ¹⁸O-labeling patterns observed in ABA from different tissues it is concluded that, despite variations in precursor pool sizes and intermediate turnover rates, there is a universal pathway of ABA biosynthesis in higher plants which involves cleavage of a larger precursor molecule, presumably an oxygenated carotenoid.     

Xanthophylls and abscisic Acid biosynthesis in water-stressed bean leaves

Experiments were designed to obtain evidence about the possible role of xanthophylls as abscisic acid (ABA) precursors in water-stressed leaves of Phaseolus vularis L. Leaves were exposed to ¹⁴CO₂ and the specific activities of several major leaf xanthophylls and stress-induced ABA were determined after a chase in ¹²CO₂ for varying periods of time. The ABA specific radioactivities were about 30 to 70% of that of lutein and violaxanthin regardless of the chase period. The specific activity of neoxanthin, however, was only about 15% of that of ABA. The effects of fluridone on xanthophyll and ABA levels and the extent of labeling of both from ¹⁴CO₂ were determined. Fluridone did not inhibit the accumulation of ABA when leaves were stressed once, although subsequent stresses in the presence of fluridone did lead to a reduced ABA accumulation. The incorporation of ¹⁴C from ¹⁴CO₂ into ABA and the xanthophylls was inhibited by fluridone and to about the same extent. The incorporation of ¹⁸O into ABA from violaxanthin which had been labeled in situ by means of the violaxanthin cycle was measured. The results indicated that a portion of the ABA accumulated during stress was formed from violaxanthin which had been labeled with ¹⁸O. The results of these experiments are consistent with a preformed xanthophyll(s) as the major ABA precursor in water-stressed bean leaves.     

Abscisic alcohol is an intermediate in abscisic Acid biosynthesis in a shunt pathway from abscisic aldehyde

It has previously been shown that the abscisic acid (ABA)-deficient flacca and sitiens mutants of tomato are impaired in ABA-aldehyde oxidation and accumulate trans-ABA-alcohol as a result of the biosynthetic block (IB Taylor, RST Linforth, RJ Al-Naieb, WR Bowman, BA Marples [1988] Plant Cell Environ 11: 739-745). Here we report that the flacca and sitiens mutants accumulate trans-ABA and trans-ABA glucose ester and that this accumulation is due to trans-ABA biosynthesis. ¹⁸O labeling of water-stressed wild-type and mutant tomato leaves and analysis of [¹⁸O]ABA by tandem mass spectrometry show that the tomato mutants synthesize a significant percentage of their ABA and trans-ABA as [¹⁸O]ABA with two ¹⁸O atoms in the carboxyl group. We further show, by feeding experiments with [²H₆]ABA-alcohol and ¹⁸O₂, that this doubly-carboxyl-labeled ABA is synthesized from [¹⁸O]ABA-alcohol with incorporation of molecular oxygen. In vivo inhibition of [²H₆]ABA-alcohol oxidation by carbon monoxide establishes the involvement of a P-450 monooxygenase. Likewise, carbon monoxide inhibits the synthesis of doubly-carboxyl-labeled ABA in ¹⁸O-labeling experiments. This minor shunt pathway from ABA-aldehyde to ABA-alcohol to ABA operates in all plants examined. For the ABA-deficient mutants impaired in ABA-aldehyde oxidation, this shunt pathway is an important source of ABA and is physiologically significant.     

Violaxanthin is an abscisic Acid precursor in water-stressed dark-grown bean leaves

The leaves of dark-grown bean (Phaseolus vulgaris L.) seedlings accumulate considerably lower quantities of xanthophylls and carotenes than do leaves of light-grown seedlings, but they synthesize at least comparable amounts of abscisic acid (ABA) and its metabolites when water stressed. We observed a 1:1 relationship on a molar basis between the reduction in levels of violaxanthin, 9′-cis-neoxanthin, and 9-cis-violaxanthin and the accumulation of ABA, phaseic acid, and dihydrophaseic acid, when leaves from dark-grown plants were stressed for 7 hours. Early in the stress period, reductions in xanthophylls were greater than the accumulation of ABA and its metabolites, suggesting the accumulation of an intermediate which was subsequently converted to ABA. Leaves which were detached, but not stressed, did not accumulate ABA nor were their xanthophyll levels reduced. Leaves from plants that had been sprayed with cycloheximide did not accumulate ABA when stressed, nor were their xanthophyll levels reduced significantly. Incubation of dark-grown stressed leaves in an ¹⁸O₂-containing atmosphere resulted in the synthesis of ABA with levels of ¹⁸O in the carboxyl group that were virtually identical to those observed in light-grown leaves. The results of these experiments indicate that violaxanthin is an ABA precursor in stressed dark-grown leaves, and they are used to suggest several possible pathways from violaxanthin to ABA.     

18O Labeling of Chlorophyll d in Acaryochloris marina Reveals That Chlorophyll a and Molecular Oxygen Are Precursors

The cyanobacterium Acaryochloris marina was cultured in the presence of either H₂¹⁸O or ¹⁸O₂, and the newly synthesized chlorophylls (Chl a and Chl d) were isolated using high performance liquid chromatography and analyzed by mass spectroscopy. In the presence of H₂¹⁸O, newly synthesized Chl a and d, both incorporated up to four isotopic ¹⁸O atoms. Time course H₂¹⁸O labeling experiments showed incorporation of isotopic ¹⁸O atoms originating from H₂¹⁸O into Chl a, with over 90% of Chl a ¹⁸O-labeled at 48 h. The incorporation of isotopic ¹⁸O atoms into Chl d upon incubation in H₂¹⁸O was slower compared with Chl a with ∼50% ¹⁸O-labeled Chl d at 115 h. The rapid turnover of newly synthesized Chl a suggested that Chl a is the direct biosynthetic precursor of Chl d. In the presence of ¹⁸O₂ gas, one isotopic ¹⁸O atom was incorporated into Chl a with approximately the same kinetic incorporation rate observed in the H₂¹⁸O labeling experiment, reaching over 90% labeling intensity at 48 h. The incorporation of two isotopic ¹⁸O atoms derived from molecular oxygen (¹⁸O₂) was observed in the extracted Chl d, and the percentage of double isotopic ¹⁸O-labeled Chl d increased in parallel with the decrease of non-isotopic-labeled Chl d. This clearly indicated that the oxygen atom in the C3¹-formyl group of Chl d is derived from dioxygen via an oxygenase-type reaction mechanism.     

Incorporation of Oxygen into Glycolate, Glycine, and Serine during Photorespiration in Maize Leaves

Glycolate, glycine, and serine extracted from excised Zea mays L. leaves which had been allowed to photosynthesize in the presence of ¹⁸O₂ were analyzed by gas chromatography-mass spectrometry. In each case, only one of the oxygen atoms of the carboxyl group had become labeled. The maximum enrichment observed in glycine and serine was attained after 5 minutes and 15 minutes of exposure to ¹⁸O₂ at the CO₂ compensation point; the labeling was very high, reaching 70 to 73% of that in the applied O₂. Thus, it appears that all or nearly all of the glycine and serine are synthesized in maize leaves via fixation of O₂. In the presence of CO₂ (380 or 800 microliters per liter), ¹⁸O-labeling was markedly slower.

Glycolate enrichment was variable and much lower than that in glycine and serine. It is possible that there are additional pathways of glycolate synthesis which do not result in the incorporation of ¹⁸O from molecular oxygen. An estimation of the metabolic flow of O₂ through the photorespiratory cycle was made. It appeared that less than 75% of the O₂ taken up by maize leaves is involved in this pathway. Therefore, other processes of O₂ metabolism must occur in the light.

     

Role of oxygenases in pisatin biosynthesis and in the fungal degradation of maackiain

Some isolates of the plant pathogen Nectria haematococca detoxify the isoflavonoid phytoalexin (−)maackiain by hydroxylation at carbon 6a. Precursor feeding studies strongly suggest that the penultimate step in (+)pisatin biosynthesis by Pisum sativum is 6a-hydroxylation of (+)maackiain. We have used ¹⁸O labeling to test the involvement of oxygenases in these two reactions. When fungal metabolism of maackiain took place under ¹⁸O₂, the product was labeled with 99% efficiency; no label was incorporated by metabolism in H₂¹⁸O. Pisatin synthesized by pea pods in the presence of ¹⁸O₂ or H₂¹⁸O was a mixture of molecules containing up to three labeled oxygen atoms. Primary mass spectra of such mixtures were complex but were greatly simplified by tandem MS. This analysis indicated that the 6a oxygen of pisatin was derived from H₂O and not from O₂. Labeling patterns for the other five oxygen atoms were consistent with the proposed pathway for biosynthesis of pisatin and related isoflavonoids. We conclude that the fungal hydroxylation of maackiain is catalyzed by an oxygenase, but the biosynthetic route to the 6a hydroxyl of pisatin is unknown.     

Characterization of five CIPK genes expressions in maize under water stress

ABA, H₂O₂ and Ca²⁺ play critical roles as signals in the adaptive responses of plants to water and other stresses. They accumulate in plant cells under water and other stresses and induce changes in stress-related gene expressions. CIPKs, protein kinases associated with a calcineurin B-like calcium sensor, play a role in the regulation of stress gene expression in plants. However, it is still unclear whether ABA and H₂O₂ are key inducers that regulate the changes in CIPK expressions under water stress. In this study, five stress-inducible CIPKs in maize were retrieved from Database. They were designated as ZmCIPK1, 3, 8, 17 and 18, based on their homologies with known CIPK sequences. The expressions of the five ZmCIPKs in maize leaves and roots were analyzed and found to be regulated by PEG, CaCl₂, ABA and H₂O₂ to different extents. Moreover, the effect of ABA and H₂O₂ on the expressions of ZmCIPKs under water stress was in an organ-dependent manner.     

Photorespiratory rates in wheat and maize as determined by o-labeling

A method was devised to quantify short-term photorespiratory rates in terrestrial plants using ¹⁸O-intermediates of the glycolate pathway, specifically glycolate, glycine, and serine. The pathway intermediates were isolated and analyzed on a GC/MS to determine molecular percent ¹⁸O-enrichment. Rates of glycolate synthesis were determined from ¹⁸O-labeling kinetics of the intermediates, derived rate equations, and nonlinear regression techniques. Glycolate synthesis in wheat (Triticum aestivum L.), a C₃ plant, and maize (Zea mays L.), a C₄ plant, was stimulated by high O₂ concentrations and inhibited by high CO₂ concentrations. The synthesis rates were 7.3, 2.1, and 0.7 micromoles per square decimeter per minute under a 21% O₂ and 0.035% CO₂ atmosphere for leaf tissue of wheat, maize seedlings, and 3-month-old maize, respectively. Photorespiratory CO₂ evolution rates were estimated to be 27, 6, and 2%, respectively, of net photosynthesis for the three groups of plants under the above atmosphere. The results from maize tissue support the hypothesis that C₄ plants photorespire, albeit at a reduced rate in comparison to C₃ plants, and that the CO₂/O₂ ratio in the bundle sheath of maize is higher in mature tissue than in seedling tissue. The pool size of the three photorespiratory intermediates remained constant and were unaffected by changes in either CO₂ or O₂ concentrations throughout the 10-minute labeling period. This suggests that photorespiratory metabolism is regulated by other mechanism besides phosphoglycolate synthesis by ribulose-1,5-bisphosphate carboxylase/oxygenase, at least under short-term conditions. Other mechanisms could be alternate modes of synthesis of the intermediates, regulation of some of the enzymes of the photorespiratory pathway, or regulation of carbon flow between organelles involved in photorespiration. The glycolate pool became nearly 100% ¹⁸O-labeled under an atmosphere of 40% O₂. This pool failed to become 100% ¹⁸O-enriched under lower O₂ concentrations.     

Study on the properties of the S3-state by mass spectrometry in the filamentous cyanobacterium Oscillatoria chalybea

In the present paper we analyzed the properties of the S₃-state in the filamentous cyanobacterium Oscillatoria chalybea by mass spectrometry. In this organism a substantial O₂-signal due to a single flash is observed even after extensive dark adaptation (20 min). This signal can be measured by mass spectrometry as well as amperometrically on an oxygen electrode and is not due to an interference of respiratory and photosynthetic electron transport in the prokaryotic membrane. The mass spectrometric analysis shows that, if S₃ is generated by two flashes in a medium containing only H₂¹⁶O, addition of H₂¹⁸O and subsequent firing of a third flash yields O₂ evolution labelled with ¹⁸O. It appears that the isotopic composition of the O₂ evolved corresponds to the isotopic composition of the water in the suspension. This experiment shows that water oxidation does not proceed via an oxygen precursor or water derivatives bound to the S₃-state. This conclusion has been reached shortly before ours by Radmer and Ollinger [15] in the reverse marker experiment. From our study with O. chalybea it appears that freshly generated S₃ can be distinguished from metastable S₃ by the mass spectrometric method. It looks as if in contrast to freshly generated S₃ metastable S₃ contained bound unexchangeable water or an oxidized water derivative.     

Abscisic (ABA)-Aldehyde Is a Precursor to, and 1',4'-trans-ABA-Diol a Catabolite of, ABA in Apple

Previous ¹⁸O labeling studies of abscisic acid (ABA) have shown that apple (Malus domestica Borkh. cv Granny Smith) fruits synthesize a majority of [¹⁸O]ABA with the label incorporated in the 1′-hydroxyl position and unlabeled in the carboxyl group (JAD Zeevaart, TG Heath, DA Gage [1989] Plant Physiol 91: 1594-1601). It was proposed that exchange of ¹⁸O in the side chain with the medium occurred at an aldehyde intermediate stage of ABA biosynthesis. We have isolated ABA-aldehyde and 1′-4′-trans-ABA-diol (ABA-trans-diol) from ¹⁸O-labeled apple fruit tissue and measured the extent and position of ¹⁸O incorporation by tandem mass spectrometry. ¹⁸O-Labeling patterns of ABA-aldehyde, ABA-trans-diol, and ABA indicate that ABA-aldehyde is a precursor to, and ABA-trans-diol a catabolite of, ABA. Exchange of ¹⁸O in the carbonyl of ABA-aldehyde can be the cause of loss of ¹⁸O from the side chain of [¹⁸O]ABA. Results of feeding experiments with deuterated substrates provide further support for the precursor-product relationship of ABA-aldehyde → ABA → ABA-trans-diol. The ABA-aldehyde and ABA-trans-diol contents of fruits and leaves were low, approximately 1 and 0.02 nanograms per gram fresh weight for ABA-aldehyde and ABA-trans-diol, respectively, while ABA levels in fruits ranged from 10 to 200 nanograms per gram fresh weight. ABA biosynthesis was about 10-fold lower in fruits than in leaves. In fruits, the majority of ABA was conjugated to β-d-glucopyranosyl abscisate, whereas in leaves ABA was mainly hydroxylated to phaseic acid. Parallel pathways for ABA and trans-ABA biosynthesis and conjugation in fruits and leaves are proposed.     

Evidence for 18O labeling of photorespiratory CO2 in photoautotrophic cell cultures of higher plants illuminated in the presence of 18O2

The ¹⁸O-enrichment of CO₂ produced in the light or during the post-illumination burst was measured by mass spectrometry when a photoautotrophic cell suspension of Euphorbia characias L. was placed in photorespiratory conditions in the presence of molecular ¹⁸O₂. The only ¹⁸O-labeled species produced was C¹⁸O¹⁶O; no C¹⁸O¹⁸O could be detected. Production of C¹⁸O¹⁶O ceased after addition of two inhibitors of the photosynthetic carbon-oxidation cycle, aminooxyacetate or aminoacetonitrile, and was inhibited by high levels of CO₂. The average enrichment during the post-illumination burst was estimated to be 46 ± 15% of the enrichment of the O₂ present during the preceding light period. Addition of exogenous carbonic anhydrase, by catalyzing the exchange between CO₂ and H₂O, drastically diminished the ¹⁸O-enrichment of the produced CO₂. The very low carbonio-anhydrase level of the photoautotrophic cell suspension probably explains why the ¹⁸O labeling of photorespiratory CO₂ could be observed for the first time. These data allow the establishment of a direct link between O₂ consumption and CO₂ production in the light, and the conclusion that CO₂ produced in the light results, at least partially, from the mitochondrial decarboxylation of the glycine pool synthesized through the photosynthetic carbon-oxidation cycle. Analysis of the C¹⁸O¹⁶O and CO₂ kinetics provides a direct and reliable way to assess in vivo the real contribution of photorespiratory metabolism to CO₂ production in the light.     

Pathway-specific metabolome analysis with <Superscript>18</Superscript>O<Subscript>2</Subscript>-labeled <Emphasis Type="Italic">Medicago truncatula</Emphasis> via a mass spectrometry-based approach

Introduction

Oxygen from carbon dioxide, water or molecular oxygen, depending on the responsible enzyme, can lead to a large variety of metabolites through chemical modification.

Objectives

Pathway-specific labeling using isotopic molecular oxygen (¹⁸O₂) makes it possible to determine the origin of oxygen atoms in metabolites and the presence of biosynthetic enzymes (e.g., oxygenases). In this study, we established the basis of ¹⁸O₂-metabolome analysis.

Methods

¹⁸O₂ labeled whole Medicago truncatula seedlings were prepared using ¹⁸O₂-air and an economical sealed-glass bottle system. Metabolites were analyzed using high-accuracy and high-resolution mass spectrometry. Identification of the metabolite was confirmed by NMR following UHPLC–solid-phase extraction (SPE).

Results

A total of 511 peaks labeled by ¹⁸O₂ from shoot and 343 peaks from root were annotated by untargeted metabolome analysis. Additionally, we identified a new flavonoid, apigenin 4′-O-[2′-O-coumaroyl-glucuronopyranosyl-(1–2)-O-glucuronopyranoside], that was labeled by ¹⁸O₂. To the best of our knowledge, this is the first report of apigenin 4′-glucuronide in M. truncatula. Using MSⁿ analysis, we estimated that ¹⁸O atoms were specifically incorporated in apigenin, the coumaroyl group, and glucuronic acid. For apigenin, an ¹⁸O atom was incorporated in the 4′-hydroxy group. Thus, non-specific incorporation of an ¹⁸O atom by recycling during one month of labeling is unlikely compared with the more specific oxygenase-catalyzing reaction.

Conclusion

Our finding indicated that ¹⁸O₂ labeling was effective not only for the mining of unknown metabolites which were biosynthesized by oxygenase-related pathway but also for the identification of metabolites whose oxygen atoms were derived from oxygenase activity.

     

Oxygen-18 Content of Atmospheric Oxygen Does Not Affect the Oxygen Isotope Relationship between Environmental Water and Cellulose in a Submerged Aquatic Plant, Egeria densa Planch

We determined that the oxygen isotopic composition of cellulose synthesized by a submerged plant, Egeria densa Planch., is related to the isotopic composition of environmental water by a linear function, δ¹⁸O cellulose = 0.48 δ¹⁸O water + 24.1%‰. The observation of a slope of less than 1 indicates that a portion of cellulose oxygen is derived from an isotopically constant source other than water. We tested whether this source might be molecular oxygen by growing plants in the presence of high concentrations of ¹⁸O in the form of O₂ bubbled into the bottom of an aquarium. Cellulose synthesized during this experiment did not have significantly different oxygen isotope ratios than that synthesized by control plants exposed to O₂ of normal ¹⁸O abundance. We propose that oxygen in organic matter recycled from senescent portions of the plant is incorporated into cellulose. Our findings indicate that paleoclimatic models linking the oxygen isotope composition of environmental water to cellulose from fossil plants will have to be modified to account for contributions of oxygen from this or other sources besides water.     

Oxygen-18 and deuterium labeling studies of choline oxidation by spinach and sugar beet

Chenopods synthesize betaine by a two-step oxidation of choline: choline → betaine aldehyde → betaine. The pathway is chloroplastic; the first step has been shown in isolated spinach (Spinacia oleracea L.) chloroplasts to be O₂- and light-dependent, the role of light being to provide reducing power (P Weigel, EA Weretilnyk, AD Hanson 1988 Plant Physiol 86: 54-60). Here, we report use of in vivo¹⁸O- and ²H-labeling in conjunction with fast atom bombardment mass spectrometry to test for two hypothetical choline-oxidizing reactions that would explain the observed requirements for O₂ and reductant: a desaturase or an oxygenase. Simple syntheses for ²H₃-choline, ²H₃, ¹⁸O-choline, and ²H₃, ¹⁸O-betaine are given. A desaturase mechanism was sought by giving choline deuterated at the 2-carbon, or choline unlabeled at this position together with ²H₂O and by analyzing newly synthesized betaine. About 15% of the ²H at C-2 was lost during oxidation of choline to betaine, and about 10% of the betaine made in the presence of 50% ²H₂O was monodeuterated. These small effects are more consistent with chemical exchange than with a desaturase, because 10 to 15% losses of ²H from the C-2 position also occurred if choline was converted to betaine by a purified bacterial choline oxidase. To test for an oxygenase, the incorporation of ¹⁸O from ¹⁸O₂ into newly synthesized betaine was compared with that from ¹⁸O-labeled choline, in light and darkness. Incorporation of ¹⁸O from ¹⁸O-choline was readily detectable and varied from about 15 to 50% of the theoretical maximum value; the ¹⁸O losses were attributable to exchange of the intermediate betaine aldehyde with water. In darkness, incorporation of ¹⁸O from ¹⁸O₂ approached that from ¹⁸O-choline, but in the light was severalfold lower, presumably due to isotopic dilution by photosynthetic ¹⁶O₂. These data indicate that the chloroplast choline-oxidizing enzyme is an oxygenase.     

A Novel Pathway for the Partial Oxidation of Propionyl-CoA to Pyruvate via Seven-carbon Tricarboxylic Acids in Yeasts

We examined the biosynthetic pathway of abscisic acid (ABA) after isopentenyl diphosphate in a fungus, Cercospora cruenta. All oxygen atoms at C-1, -1, -1′, and -4′ of ABA produced by this fungus were labeled with ¹⁸O from ¹⁸O₂. The fungus did not produce the 9Z-carotenoid possessing γ-ring that is likely a precursor for the carotenoid pathway, but produced new sesquiterpenoids, 2E,4E-γ-ionylideneethane and 2Z,4E-γ-ionylideneethane, along with 2E,4E,6E-allofarnesene. The fungus converted these sesquiterpenoids labeled with ¹³C to ABA, and the incorporation ratio of 2Z,4E-γ-ionylideneethane was higher than that of 2E,4E-γ-ionylideneethane. From these results, we concluded that C. cruenta biosynthesized ABA by the direct pathway via oxidation of ionylideneethane with molecular oxygen following cyclization of allofarnesene. This direct pathway via ionylideneethane in the fungus is consistent with that in Botrytis cinerea, except for the positions of double bonds in the rings of biosynthetic intermediates, suggesting that the pathway is common among ABA-producing fungi.     

Microfluidic technology: an economical and versatile approach for the synthesis of O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET)

A new synthesis of O-(2-[¹⁸F]fluoroethyl)-l-tyrosine [¹⁸F]FET was developed using a NanoTek® microfluidic synthesis system (Advion BioSciences, Inc.). Optimal reaction conditions were studied through screening different reaction parameters like temperature, flow rate, reaction time, concentration of the labeling precursor, and the applied volume ratio between the labeling precursor and [¹⁸F]fluoride. [¹⁸F]FET was obtained after HPLC purification with 50% decay-corrected radiochemical yield starting from as little as 40 μg of labeling precursor. Small animal PET studies in EMT-6 tumor bearing mice showed radioactivity accumulation in the tumor (SUV_60min 1.21 ± 0.2) resulting in an slightly increasing tumor-to-muscle ratio over time.     

Evidence for abscisic acid biosynthesis in Cuscuta reflexa, a parasitic plant lacking neoxanthin

Abscisic acid (ABA) is a plant hormone found in all higher plants; it plays an important role in seed dormancy, embryo development, and adaptation to environmental stresses, most notably drought. The regulatory step in ABA synthesis is the cleavage reaction of a 9-cis-epoxy-carotenoid catalyzed by the 9-cis-epoxy-carotenoid dioxygenases (NCEDs). The parasitic angiosperm Cuscuta reflexa lacks neoxanthin, one of the common precursors of ABA in all higher plants. Thus, is C. reflexa capable of synthesizing ABA, or does it acquire ABA from its host plants? Stem tips of C. reflexa were cultured in vitro and found to accumulate ABA in the absence of host plants. This demonstrates that this parasitic plant is capable of synthesizing ABA. Dehydration of detached stem tips caused a big rise in ABA content. During dehydration, ¹⁸O was incorporated into ABA from ¹⁸O₂, indicating that ABA was synthesized de novo in C. reflexa. Two NCED genes, CrNCED1 and CrNCED2, were cloned from C. reflexa. Expression of CrNCEDs was up-regulated significantly by dehydration. In vitro enzyme assays with recombinant CrNCED1 protein showed that the protein is able to cleave both 9-cis-violaxanthin and 9′-cis-neoxanthin to give xanthoxin. Thus, despite the absence of neoxanthin in C. reflexa, the biochemical activity of CrNCED1 is similar to that of NCEDs from other higher plants. These results provide evidence for conservation of the ABA biosynthesis pathway among members of the plant kingdom.Abscisic acid (ABA) is found in all higher plants and algae and is also produced by some fungi (Oritani and Kiyota, 2003; Schwartz and Zeevaart, 2004; Nambara and Marion-Poll, 2005). In higher plants, ABA is involved in seed dormancy, embryo development, and adaptation to various abiotic stresses. ABA is a sesquiterpenoid (C₁₅). In some fungi, there is a direct pathway from isopentenyl pyrophosphate (C₅) via farnesyl pyrophosphate (C₁₅; Oritani and Kiyota, 2003). Higher plants synthesize ABA via the C₄₀ indirect pathway. A C₄₀ carotenoid is oxidatively cleaved to form a C₂₅ byproduct and the C₁₅ precursor of ABA, xanthoxin. Biochemical and molecular evidence has shown that the cleavage reaction is the rate-limiting step in the ABA biosynthetic pathway.Although the pathway of ABA biosynthesis in higher plants has been well established, there are still a few unresolved questions. One is the endogenous substrate of the cleavage reaction. The biochemical evidence has indicated that the C₄₀ substrate for production of biologically active ABA is an epoxy-carotenoid in the 9-cis configuration in order for biologically active ABA to be produced. In higher plants, the major 9-cis-epoxy-carotenoids are 9′-cis-neoxanthin and 9-cis-violaxanthin. Because 9′-cis-neoxanthin is the most abundant 9-cis-epoxy-carotenoid in higher plants, it has been speculated that 9′-cis-neoxanthin is the main endogenous substrate of ABA. However, in vitro enzyme assays with recombinant 9-cis-epoxy-carotenoid dioxygenase (NCED) proteins from several plant species have shown that NCEDs are capable of cleaving both 9′-cis-neoxanthin and 9-cis-violaxanthin (Schwartz et al., 1997; Qin and Zeevaart, 1999; Chernys and Zeevaart, 2000), with a higher activity when 9-cis-violaxanthin is used as substrate (Schwartz et al., 2003b). This raised the question whether one or both are the endogenous substrates of ABA biosynthesis.Cuscuta reflexa is of interest in addressing this question, because it lacks 9′-cis-neoxanthin, one of the major 9-cis-epoxy-carotenoids found in green plants. A study by Bungard et al. (1999) investigated the xanthophyll-carotenoid complement of the main light-harvesting complex involved in light capture of photosynthesis. They found that in most higher plants, the complex is highly conserved and includes, in addition to chlorophyll, the carotenoids neoxanthin, violaxanthin, and lutein. In C. reflexa, neoxanthin is replaced by another xanthophyll, lutein-5,6-epoxide. Therefore, C. reflexa lacks the step that converts violaxanthin to neoxanthin. There are two xanthophyll cycles in C. reflexa: under excess irradiance violaxanthin and lutein-5,6-epoxide are converted to zeaxanthin and lutein, respectively, by de-epoxidation; the cycle is reversed under low irradiance (Bungard et al., 1999).C. reflexa is parasitic on the above-ground parts of other plants. The leafless stems coil around the host stems and petioles. C. reflexa contains only a small amount of chlorophyll and relies on obtaining photosynthetic assimilates from the phloem of its host. It forms specialized structures, called haustoria, which are used to acquire nutrients from its host plant (Hibberd et al., 1998).The lack of 9′-cis-neoxanthin in C. reflexa may result in an inability of the plant to produce ABA, and the parasite may acquire it from its host, thereby eliminating the need to synthesize ABA on its own (Bungard et al., 1999). The objective of this study was to determine whether C. reflexa is able to synthesize ABA or acquires it from its host. Increased ABA in C. reflexa grown independently of a host would indicate that it is capable of ABA synthesis. We also searched for the presence of an NCED gene in the Cuscuta genome. Presence of an NCED gene would be evidence for the 9-cis-epoxy-carotenoid cleavage reaction, the regulatory step for ABA synthesis. Our results show that despite the lack of 9′-cis-neoxanthin, C. reflexa is capable of ABA synthesis in a manner similar to that in other higher plants.     

Heme-Dependent Rubber Oxygenase RoxA of Xanthomonas sp. Cleaves the Carbon Backbone of Poly(cis-1,4-Isoprene) by a Dioxygenase Mechanism

Oxidative cleavage of poly(cis-1,4-isoprene) by rubber oxygenase RoxA purified from Xanthomonas sp. was investigated in the presence of different combinations of ¹⁶O₂, ¹⁸O₂, H₂¹⁶O, and H₂¹⁸O. 12-Oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD; m/z 236) was the main cleavage product in the absence of ¹⁸O-compounds. Incorporation of one ¹⁸O atom in ODTD was found if the cleavage reaction was performed in the presence of ¹⁸O₂ and H₂¹⁶O. Incubation of poly(cis-1,4-isoprene) (with RoxA) or of isolated unlabeled ODTD (without RoxA) with H₂¹⁸O in the presence of ¹⁶O₂ indicated that the carbonyl oxygen atoms of ODTD significantly exchanged with oxygen atoms derived from water. The isotope exchange was avoided by simultaneous enzymatic reduction of both carbonyl functions of ODTD to the corresponding dialcohol (12-hydroxy-4,8-dimethyl-trideca-4,8-diene-1-ol (HDTD; m/z 240) during RoxA-mediated in vitro cleavage of poly(cis-1,4-isoprene). In the presence of ¹⁸O₂, H₂¹⁶O, and alcohol dehydrogenase/NADH, incorporation of two atoms of ¹⁸O into the reduced metabolite HDTD was found (m/z 244), revealing that RoxA cleaves rubber by a dioxygenase mechanism. Based on the labeling results and the presence of two hemes in RoxA, a model of the enzymatic cleavage mechanism of poly(cis-1,4-isoprene) is proposed.