Changes in abscisic acid and its glucose ester in Phaseolus vulgaris L. during chilling and water stress

Levels of free and conjugated abscisic acid (ABA) were determined in leaves and roots of intact bean (Phaseolus vulgaris L., cv. Mondragone) seedlings under chilling (3C) and drought as well as during recovery from stress. Abscisic acid-glucose ester (ABAGE) was the only conjugate releasing free ABA after alkaline hydrolysis of the crude aqueous extracts. During the first 20–30 h chilled plants rapidly dehydrated and wilted without any change in ABA and ABAGE levels. Subsequently, leaf and root ABA levels increased and plants regained turgor. ABAGE concentration showed a slight increase in leaves but not in roots. Upon recovery from chilling a transient, but significant, rise in leaf ABA content was observed, while no appreciable change in ABAGE was found. Drought triggered ABA accumulation in leaves and roots, while a rise in ABAGE content was detected only in leaf tissues. Recovery from stress caused a drop in ABA levels without a correspondent increase in ABAGE concentration. We conclude that ABAGE is not a source of free ABA during either chilling or water stress and that only a small proportion of the ABA produced under stress is metabolised to ABAGE during recovery.Abbreviations ABA = abscisic acid - ABAGE = abscisic acid-glucose ester - DW = dry weight - FW = fresh weight - RIA = radioimmunoassay - RWC = relative water content - w = water potential - o = osmotic potential - p = turgor potential     

Changes in abscisic acid and its β-D-glucopyranosyl ester levels during tomato (Lycopersicon esculentum Mill.) seed development

Summary The role of abscisic acid (ABA) in tomato (Lycopersicon esculentum Mill.) zygotic embryogenesis was analysed. ABA and ABA ß-D-glucopyranosyl ester (ABA-GE) changes were determined in seeds and fruit tissues — placenta and mesocarp — during seed development, which was defined with eight embryo stages: from globular (stage 1) to mature embryo (stage 8). In whole seeds, ABA changes paralleled fresh and dry weight pattern curves and could be characterized by a high increase during embryo growth followed by a decrease as the seed matured and dehydrated. Moreover this dehydration phase led, at stage 8, to a new ABA distribution within the seed, preferentially into integument and embryo. Fruit tissue analyses provided new information about the ABA origin in seeds. ABA-GE levels were also measured and the results suggested different ABA metabolism in seed and fruit tissues.Abbreviations ABA abscisic acid - ABA-GE abscisic acid ß-D-glucopyranosyl ester - ABTS 2,2 — azino — bis (3 — ethylben-zthiazoline — 6 — sulfonic acid) - BHT butylhydroxytoluene - DW dry weight - ELISA Ezyme linked immunosorbent assay - HPLC high performance liquid chromatography     

Potato cold hardiness development and abscisic acid. I. Conjugated abscisic acid is not the source of the increase in free abscisic acid during potato (Solanum commersonii) cold acclimation

Free and conjugated abscisic acid (ABA) levels in stem-cultured plantlets of potato ( Solanum commersonii Dun, PI 458317) during cold acclimation were measured. The levels of free and conjugated ABA were measured by an enzyme immunoassay (EIA) with rabbit anti-ABA-serum. The use of immunoglobulin G fraction purified from rabbit antiserum and the methylated form of ABA resulted in an improved measuring range (0.01 to 10 pmol ABA) and precision (slope of logit-log plot, −1.35) of EIA, compared to the use of antiserum and free ABA. Estimates of the EIA were consistent with those resulting from a commercial EIA. Under a 4/2°C (day/night) temperature regime, the potato plantlets increased cold hardiness from −5°C (warm-grown control) to −10°C by the 7th day. During the same period, there were two transitory increases in free ABA, the first one three-fold from 1.5 to 5.3 nmol (g dry weight)⁻¹ on the 2nd day and the second one five-fold from 1.5 to 7.6 nmol (g dry weight)⁻¹ on the 6th day. Each increase in ABA concentration was followed by an increase in cold hardiness. There was no significant change in conjugated ABA content (4.2±0.6 nmol [g dry weight]⁻¹) throughout the cold acclimation period. The lack of an interrelationship between levels of free and conjugated ABA suggested that the transitory increase in free ABA during cold acclimation was not a result of the conversion of conjugated ABA. The increase in free ABA due to biosynthesis of ABA during potato cold acclimation is discussed.     

The Effects of Mechanical Impedance to Growth on the Levels of ABA and IAA in Root Tips of Zea mays L.

Extraction and analytical methods have been refined and newones devised to allow precise determinations by GC-EC of thelevels of abscisic acid (ABA) and indol-3ylacetic acid (IAA)in samples of maize root tips as small as 1.0 g fr. wt. Seminalroots of 5-d-old maize seedlings grown in normal (bulk density1200 kg m^–3) and compacted (bulk density 1600 kg m^–3)sand/garden loam mixtures have been examined. Seminal rootsfrom compacted soil had an average length of about 40% of thatof control roots and were much thicker. The ABA levels in 10mm tips of impeded roots (c. 25–35 ng g^–1 fr.wt.)did not differ significantly from those of normal root tipson both a fresh and dry weight basis. The levels in 0–1mm tips were approximately double those in the remaining 1–10mm zones. IAA levels were increased by about 3 times in impededroots (176.3 as compared with 52.4 ng g^–1 fr.wt) and itis concluded that this response is likely to be the main causeof the morphological and growth changes brought about by soilcompaction.     

Effect of Fruit Load on Partitioning of Dry Matter and Energy in Cantaloupe (Cucumis melo L.)

Cantaloupe (Cucumis melo L.) plants set groups of fruits whichgenerate large variations in the reproductive:vegetative dryweight balance. We studied the influence of fruit number onthe partitioning of dry matter and energy between the vegetativeand reproductive organs and among the seeds and the variousfruit tissues during the development of the first fruits. Over2 years and on two Charentais cantaloupe cultivars, fruit numberwas either limited to one or left unrestricted, which led tothe setting of two to six fruits. Because of the high lipidcontent in seeds, the distribution of assimilates was studiedin terms of energy equivalent as well as dry weight. Measureddry weights were converted into energy equivalents by calculatingthe construction cost of tissues from their elemental composition.Seeds differed from other tissues in showing an increase inconstruction cost, from 1.1 to 1.8 g CH₂O g^-1d. wt between 10and 30 d after pollination. For this reason, during the secondhalf of fruit development on plants with unrestricted fruitload, they made up to 31% of the fruit and 12% of the aerialpart of the whole plant in terms of dry weight, but 39 and 18%in terms of energy (glucose equivalents). The fraction of assimilatesallocated to the fruits showed a saturation-type response tothe number of fruits per plant. It did not increase in cultivarTalma above two fruits per plant, which could be due to a decreasingsink strength with fruit rank, whereas cultivar Galoubet maintaineda more homogeneous fruit size within plants. At a similar fruitload, the reproductive:vegetative dry weight balance differedbetween the 2 years of the experiment, probably because of variationin the fruit sink strength. Copyright 1999 Annals of BotanyCompany Charentais cantaloupe, Cucumis melo L., assimilate distribution, construction cost, development, dry matter partitioning, fruit load, seeds, sink strength.     

Changes in indole-3-acetic acid levels during tomato (Lycopersicon esculentum Mill.) seed development

Summary The changes in the level of indole-3-acetic acid (IAA) were investigated in seeds and fruit tissues-placenta and mesocarp-during tomato (Lycopersicon esculentum Mill.) zygotic embryogenesis, which was characterized through eight morphological embryo stages [from globular (stage 1) to mature embryo (stage 8)]. In whole seeds, IAA levels increased mainly at stage 3 (young torpedo) and at stage 5 (late torpedo stage). As the seed matured and dehydrated, IAA levels decreased and showed a new distribution pattern within seed structures, preferentially in endosperm tissue. IAA contents in fruit tissues were lower but followed the same pattern as those of seeds. These data support the hypothesis of IAA biosynthesis in seeds with a transient role of the endosperm at the end of embryo development and suggest a role of IAA in fruit and seed growth. Moreover a comparison of IAA and ABA changes suggests that IAA could be especially necessary for the beginning of embryo growth, whereas ABA could act mainly at the end of the growth phase.Abbreviations ABA abscisic acid - ABTS 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) - BHT butylhydroxytoluene - DW dry weight - ELISA enzyme linked immunosorbent assay - HPLC high performance liquid chromatography - IAA indole-3-acetic acid. PGRs: plant growth regulators     

Radioimmunoassays for the differential and direct analysis of free and conjugated abscisic acid in plant extracts

Two radioimmunoassays have been developed which allow the parallel quantitation of free as well as conjugated natural (+)-abscisic acid (ABA) directly and separately, in unpurified plant extracts. The differential specificity of antisera has been achieved by coupling ABA through C₁ (for total ABA determination) or C₄ (for free ABA determination), respectively, to proteins to obtain the immunogenic conjugates. Compounds structurally related to ABA, such as, dihydrophaseic acid or phaseic acid, do not interfere with either of the assays, even when present in more than ten-fold excess. Other related compounds, such as, violaxanthin or xanthoxin, do not cross react at all. Both antisera respond to (+)-ABA but show very low immunoreactivity with (-)-ABA. As little as 27 pg of ABA (serum for free ABA) or 47 pg (serum for total ABA) may be detected and the measuring ranges are from 0.2–8 and 0.2–30 pmol, respectively. Average recoveries are greater than 99%. Using these assays, more than 100 samples can be assayed for free and conjugated ABA per day. Levels of free ABA, as determined by radioimmunoassay (RIA), correlated well with those reported in the literature. Levels of conjugated ABA were found to be generally higher than previously reported for ABA after alkaline hydrolysis of the extracts. Conjugated ABA accumulates during aging of leaves and levels of conjugated ABA up to 17-fold higher than those of free ABA have been detected in senescent leaves of Hyoscyamus niger L. Evidence was obtained for the presence of ABA conjugates other than the glucose ester in some plants.Abbreviations ABA abscisic acid - BHT 2,6-di-t-4-methyl phenol - BSA bovine serum albumin - HSA human serum albumin - RIA radioimmunoassay - TLC thin-layer chromatography - EDC 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide · HCl Part 11 in the series: Use of Immunoassay in Plant Science     

Influence of growth regulator treatments on dry matter production,fruit abscission,and14C-assimilate partitioning in citrus

Experiments were performed to monitor (1) uptake and translocation of foliar-applied microdroplets of¹⁴C hormones and (2) effects of multiple growth regulator sprays on foliar and fruit growth variables and photosynthate partitioning in Valencia orange trees (Citrus sinensis (L.) Osbeck). The uptake of¹⁴C-sucrose,¹⁴C-paclobutrazol (PP333), and¹⁴C-napthaleneacetic acid (NAA) in 6-month-old greenhouse-grown trees exceeded that of¹⁴C-abscisic acid (ABA) and¹⁴C-benzyladenine (BA) 48 h after microdroplet application.¹⁴C-sucrose transport from the application site was much greater than any other source, especially¹⁴C-BA. In a second study, 2-year-old Valencia orange trees were maintained under field conditions and were sprayed to foliar runoff (3 × /week for 3 weeks) with BA, NAA, ABA, PP333, and gibberellic acid (GA₃) at 100 M during flowering and early fruit set. Select branches were then briefly exposed to¹⁴CO₂ and harvested 24 h later. Both GA₃ and BA sprays promoted foliar growth. BA also stimulated fruit growth, whereas GA₃ sharply increased fruit dry weight while fruit number decreased. BA and GA₃ enhanced¹⁴C assimilate export by the foliage to the developing fruit, and GA₃ was especially active in promoting fruit sink intensity (¹⁴C/dry wt). The other compounds (NAA, ABA, PP333) restricted foliar and fruit growth. They also inhibited transport of¹⁴C assimilate from the leaves to the fruit. Results indicate that foliar-applied growth regulators can influence source-sink relations in citrus early in reproductive development by manipulating photoassimilate production and partitioning.     

Phytohormones and structure of cells ofAcer saccharinum seed embryo

Endogenous phytohormone levels and cell structure ofAcer saccharinum embryos were studied during seed development. Mature seeds had high water content (50%) and were able to germinate immediately after fruit abscission. The submicroscopic cell structure was similar to the structure of functionally active cells. Free and conjugated indole-3-acetic acid (IAA), abscisic acid (ABA), zeatin riboside and dihydrozeatin content and gibberellin-like substances (GLS) activity were determined during embryo maturation. Decrease in ABA, free IAA and cytokinin levels was observed at the end of maturation. Mature seeds contained considerable amounts of conjugated IAA and had high GLS activity.     

Abscisic acid,sucrose, and auxin coordinately regulate berry ripening process of the Fujiminori grape

The aim of this study was to examine the effect of abscisic acid (ABA), sucrose, and auxin on grape fruit development and to assess the mechanism of these three factors on the grape fruit ripening process. Different concentrations of ABA, sucrose, and auxin were used to treat the grape fruit, and the ripening-related indices, such as physiological and molecular level parameters, were analyzed. The activity of BG protein activity was analyzed during the fruit development. Sucrose, ABA, and auxin influenced the grape fruit sugar accumulation in different ways, as well as the volatile compounds, anthocyanin content, and fruit firmness. ABA and sucrose induced, but auxin blocked, the ripening-related gene expression levels, such as softening genes PE, PG, PL, and CELL, anthocyanin genes DFR, CHI, F3H, GST, CHS, and UFGT, and aroma genes Ecar, QR, and EGS. ABA, sucrose, and glucose induced the fruit dry weight accumulation, and auxin mainly enhanced fruit dry weight through seed weight accumulation. In the early development of grape, starch was the main energy storage; in the later, it was glucose and fructose. Sucrose metabolism pathway-related gene expression levels were significant for glucose and fructose accumulation. BG protein activity was important in the regulation of grape ABA content levels. ABA plays a core role in the grape fruit development; sucrose functions in fruit development through two pathways: one was ABA dependent, the other ABA independent. Auxin blocked ABA accumulation to regulate the fruit development process.     

The Coordination of Organ Growth in Developing Citrus Flowers: A Possibility for Sink Type Regulation

The interrelationships between simultaneously developing organsof citrus flowers were investigated. Examination of flower organgrowth kinetics shows that petals grow mainly through enhancedwater absorption whereas ovaries accumulate a high percentageof dry matter. Using excised flowers implanted in an agar—sucrose mediumand supplied with [¹⁴C]-sucrose, [³H]-acetate, and [¹⁴C]-acetate,a characteristic distribution of label among organs could beestablished for each isotope. Wounding or application of -naphthaleneacetic acid (NAA) toa single petal completely changed the normal distribution patterns,shifting the bulk of [¹⁴C]-sucrose towards the treated organs. The findings are interpreted in the light of the ‘sink’hypothesis. It is proposed that each flower organ meristem createsa sink of its own which acts in a typical manner according toits specific endogenous hormonal balance. The sink activityof all meristems in concert results in a sensitive regulatorymechanism which is responsible for the coordination observedin flower development.     

The biochemical and energetic composition of somatic body components of echinoderms from the northern Gulf of Mexico

• 1.1. The biochemical and energetic compositions of the somatic body components of seven species of asteroids, one ophiuroid, and four echinoids from the northern Gulf of Mexico (30–95 m depth) were ascertained.
• 2.2. Levels of ash were high (68.5–90.8% dry wt) in all body-wall tissues, with the exception of the asteroid Echinaster modestus (51.6% dry wt). Levels of ash were low in the pyloric cecae (nutrient storage organ) of asteroids (4.6–30.8% dry wt).
• 3.3. Levels of lipid (8.1–34.5% dry wt), soluble protein (15.9–28.7% dry wt), and insoluble protein (18.1–48.6%, dry wt) were high in the pyloric cecae of all asteroids, but generally low in ophiuroid and echinoid body-wall tissues. High protein levels (28.5–44.5% dry wt) in the body-wall of the asteroids Echinaster modestus and Anthenoides pierceisuggest it may play a role in nutrient storage.
• 4.4. All somatic tissues contained low levels of carbohydrate (0.2–1.4% dry wt).
• 5.5. Levels of energy in pyloric cecal tissues (12.99–26.05 kJ/g dry wt) were 4–8 times higher than in echinoderm body-wall tissues (2.92–11.91 kJ/g dry wt).
• 6.6. The biochemical and energetic compositions of echinoderms from the northern Gulf of Mexico are similar to those of species from other latitudes and depths.

     

Metabolism of Abscisic Acid in Developing Seeds of Phaseolus vulgaris L. and its Correlation to Germination and {alpha}-Amylase Activity

Bean seeds were unable to germinate during their developmentwhich was characterized either by an increasing ABA content(expressed as ng ABA/seed) or by a fairly high and constantABA concentration (expressed as ng ABA g^–1 fr. wt). During seed maturation the mother plant induces a dormant stateby depressing the ABA catabolism and keeping the endogenousABA at a high level to prevent both the premature hydrolysisof starch (-amylase activity) and the germination of the morphogeneticallyadult yet immature seeds. The depth of this induced dormancyis positively correlated with endogenous ABA concentration. Application of exogenous ABA to fully mature seeds, which containno endogenous ABA and show a very active ABA catabolism, onlyprolonged the lag phase but had no influence on the furtherdevelopment of the -amylase activity.     

The isolation of an abscisic-acid metabolite, 4′-dihydrophaseic acid,from non-imbibed Phaseolus vulgaris seed

Summary Naturally occurring 4-dihydrophaseic acid (DPA) has been isolated from mature, non-imbibed bean seed. The concentrations of abscisic acid (ABA), phaseic acid (PA) and DPA in the seed were estimated to be 0.06, 0.11 and 5.95 mg/kg dry wt., respectively. The results suggest that DPA is a major inactivation product of ABA in this tissue. The possible pathway from ABA to DPA is discussed.Abbreviations ABA abscisic acid - DPA 4-dihydrophaseic acid - PA phaseic acid     

A label-free differential quantitative mass spectrometry method for the characterization and identification of protein changes during citrus fruit development

Background

Citrus is one of the most important and widely grown commodity fruit crops. In this study a label-free LC-MS/MS based shot-gun proteomics approach was taken to explore three main stages of citrus fruit development. These approaches were used to identify and evaluate changes occurring in juice sac cells in various metabolic pathways affecting citrus fruit development and quality.

Results

Protein changes in citrus juice sac cells were identified and quantified using label-free shotgun methodologies. Two alternative methods, differential mass-spectrometry (dMS) and spectral counting (SC) were used to analyze protein changes occurring during earlier and late stages of fruit development. Both methods were compared in order to develop a proteomics workflow that could be used in a non-model plant lacking a sequenced genome. In order to resolve the bioinformatics limitations of EST databases from species that lack a full sequenced genome, we established iCitrus. iCitrus is a comprehensive sequence database created by merging three major sources of sequences (HarvEST:citrus, NCBI/citrus/unigenes, NCBI/citrus/proteins) and improving the annotation of existing unigenes. iCitrus provided a useful bioinformatics tool for the high-throughput identification of citrus proteins. We have identified approximately 1500 citrus proteins expressed in fruit juice sac cells and quantified the changes of their expression during fruit development. Our results showed that both dMS and SC provided significant information on protein changes, with dMS providing a higher accuracy.

Conclusion

Our data supports the notion of the complementary use of dMS and SC for label-free comparative proteomics, broadening the identification spectrum and strengthening the identification of trends in protein expression changes during the particular processes being compared.     

Hormonal changes associated with fruit set and development in mandarins differing in their parthenocarpic ability

Satsuma [Citrus unshiu (Mak) Marc.] and Clementine [Citrus reticulata (Hort.) Ex. Tanaka, cv. Oroval] are two related species of seedless mandarins which differ in their tendency to set parthenocarpic fruits. Satsuma fruits naturally set parthenocarpically whereas Clementine mandarins show very low ability to set fruit in the absence of cross-pollination. The endogenous levels of gibberellins (GAs) and free and conjugated indole-acetic acid (IAA) and abscisic acid (ABA) throughout early stages of fruit development were investigated in seedless cultivars of both species. Analyses performed by full-scan combined gas chromatography-mass spectrometry (GC-MS) of extracts from ovaries at anthesis demonstrated the presence of GA₁₉, GA₂₀, GA₂₉, GA₁, GA₈, GA₃ and iso-GA₃ in Satsuma mandarin, whereas only GA₂₉, GA₃ and trace levels of GA₈ were detected in Clementine. At this developmental stage GA-like substances, as estimated by bioassay, reached their highest levels in Satsuma, while Clementine mandarins contained relatively lower levels. In both species the highest levels of free IAA were found at petal-fall stage at which time free ABA levels also peaked. Developing fruits of Clementine had higher amounts of both free IAA and ABA. In Satsuma, levels of conjugated IAA remained low throughout reproductive development whereas in Clementine they increased as the free form declined. In contrast, conjugated ABA was at low levels in Clementine but reached higher concentrations in Satsuma. These results suggest that in these mandarins the potential for setting parthenocarpic fruits is mainly influenced by the hormonal status of the fruit during the later stages of cell division and early stages of cell enlargement. Thus, the condition of low ability to set parthenocarpic fruits appears to be associated with lower levels of active GAs, lower capability to catabolize ABA to conjugated ABA and higher ability to conjugate IAA during this period.     

Changing Responses of Stomata to Abscisic Acid and CO2 as Leaves and Plants Age

Willmer, C. M., Wilson, A. B. and Jones, H. G. 1988. Changingresponses of stomata to abscisic acid and CO₂ as leaves andplants age.—J. exp. Bot. 39: 401–410. Stomatal conductances were measured in ageing leaves of Commelinacommunis L. as plants developed; stomatal responses to CO₂ andabscisic acid (ABA) in epidermal strips of C. communis takenfrom ageing leaves of developing plants and in epidermal stripsfrom the same-aged leaves (the first fully-expanded leaf) ofdeveloping plants were also monitored. Stomatal behaviour wascorrelated with parallel measurements of photosynthesis andleaf ABA concentrations. Stomatal conductance in intact leavesdecreased from a maximum of 0-9 cm s^{– 1} at full leaf expansionto zero about 30 d later when leaves were very senescent. Conductancesdeclined more slowly with age in unshaded leaves. Photosynthesisof leaf slices also declined with age from a maximum at fullleaf expansion until about 30 d later when no O₂ exchange wasdetectable. Exogenously applied ABA (0.1 mol m^{– 3}) didnot affect respiration or photosynthesis. In epidermal stripstaken from ageing leaves the widest stomatal apertures occurredabout 10 d after full leaf expansion (just before floweringbegan) and then decreased with age; this decrease was less dramaticin unshaded leaves. The inhibitory effects of ABA on stomatalopening in epidermal strips decreased as leaves aged and wasgreater in the presence of CO₂ than in its absence. When leaveswere almost fully-senescent stomata were still able to open.At this stage, guard cells remained healthy-looking with greenchloroplasts while mesophyll cells were senescing and theirchloroplasts were yellow. Similar data were obtained for stomatain epidermal strips taken from the same-aged leaves of ageingplants. The inhibitory effects of ABA on stomatal opening alsodecreased with plant age. In ageing leaves both free and conjugated ABA concentrationsremained low before increasing dramatically about 30 d afterfull leaf expansion when senescence was well advanced. Concentrationsof free and conjugated ABA remained similar to each other atall times. It is concluded that the restriction of stomatal movements inintact leaves as the leaves and plants age is due mainly toa fall in photosynthetic capacity of the leaves which affectsintracellular CO₂ levels rather than to an inherent inabilityof the stomata to function normally. Since stomatal aperturein epidermal strips declines with plant and leaf age and stomatabecome less responsive to ABA (while endogenous leaf ABA levelsremain fairly constant until leaf senescence) it is suggestedthat some signal, other than ABA, is transmitted from the leafor other parts of the plant to the stomata and influences theirbehaviour. Key words: Abscisic acid, CO₂, Commelina, leaf age, senescence, stomatal sensitivity     

Synthesis and metabolism of abscisic acid in detached leaves of Phaseolus vulgaris L. after loss and recovery of turgor

Metabolism of abscisic acid (ABA) was studied after wilting and upon recovery from water stress in individual, detached leaves of Phaseolus vulgaris L. (red kidney bean). Loss of turgor was correlated with accumulation of ABA and its metabolites, resulting in a 10-fold increase in the level of phaseic acid (PA) and a doubling of the level of conjugated ABA. The level of conjugated ABA in turgid leaves was no higher than that of the free acid. These results indicate that accumulation of ABA in wilted leaves resulted from a stimulation of ABA synthesis, rather than from a release from a conjugated form or from inhibition of the metabolism of ABA. The rate of synthesis of ABA was at its maximum between 2.5 and 5 h after turgor was lost, and slackened there-after. In wilted leaves, the rate of conversion of ABA to PA climbed steadly until it matched the rate of synthesis, after about 7.5 h. Upon rehydration of sections from wilted leaves, the rate of synthesis of ABA dropped close to zero within about 3 h, while the rate of conversion to PA accelerated. Formation of PA was two to four times faster than in sections maintained in the wilted condition; it reached a rate sufficient to convert almost one-half of the ABA present in the tissue to PA within 1 h. In contrast, the alternate route of metabolism of ABA, synthesis of conjugated ABA, was not stimulated by rehydration. The role of turgor in the stimulation of the conversion of ABA to PA was investigated. When leaves that had been wilted for 5 h were rehydrated to different degrees, the amount of ABA which disappeared, or that of PA which accumulated during the next 3 h, did not depend linearly on the water potential of the rehydrated leaf. Rather, re-establishment of the slightest positive turgor was sufficient to result in maximum stimulation of conversion of ABA to PA.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - PA phaseic acid - _leaf leaf water potential - osmotic pressure     

Transgenic livers expressing mitochondrial and cytosolic CK: mitochondrial CK modulates free ADP levels

The function of creatinekinase (CK) and its effect on phosphorus metabolites was studied inlivers of transgenic mice expressing human ubiquitous mitochondrial CK(CK-Mit) and rat brain CK (CK-B) isoenzymes and their combination.³¹P NMR spectroscopy and saturation transfer were recordedin livers of anesthetized mice to measure high-energy phosphates andhepatic CK activity. CK reaction velocity was related to total enzyme activity irrespective of the isoenzyme expressed, and it increased with increasing concentrations of creatine (Cr). The fluxesmediated by both isoenzymes in both directions (phosphocreatine or ATP synthesis) were equal. Over a 20-fold increase in CK-Mit activity (28-560 µmol · g wetwt¹ · min¹), the fraction ofphosphorylated Cr increased 1.6-fold. Hepatic free ADP concentrationscalculated by assuming equilibrium of the CK-catalyzed reaction in vivodecreased from 84 ± 9 to 38 ± 4 nmol/g wet wt. Calculatedfree ADP levels in mice expressing high levels of CK-B (920-1,635µmol · g wet wt¹ · min¹)were 52 ± 6 nmol/g wet wt. Mice expressing both isoenzymes had calculated free ADP levels of 36 ± 4 nmol/g wet wt. Thesefindings indicate that CK-Mit catalyzes its reaction equally well inboth directions and can lower hepatic apparent free ADP concentrations.