Conversion of N6-isopentenyladenine to zeatin by Actinidia tissues

Tissue cultures grown from stem explants of three $\text{Actinidia}$ species and a hybrid species rapidly converted N6-isopentenyladenine (i⁶Ade) to zeatin (io⁶Ade), a potent hydroxylated cytokinin. Within 24 h on 50 uM i⁶Ade, callus tissues of $\text{A.}\text{chinensis}\text{×}\text{arguta}$ accumulated 83 ± 6 nmol/g io⁶Ade which was purified using HPLC and identified by its characteristic UV and mass spectra. Activity converting i⁶Ade to io⁶Ade was also demonstrated in stem segments from intact plants where it was low in the tip (3 cm), highest in the region corresponding to rapid leaf growth and very low in the mature stem. Root segments converted i⁶Ade to io⁶Ade almost as rapidly as the most active region of the stem while leaf petioles produced little io⁶Ade. Fruits of $\text{A.}\text{arguta}$ and $\text{A.}\text{chinensis}$ produced little or no io⁶Ade, respectively.     

Alteration of thiol pools in roots and shoots of maize seedlings exposed to cadmium : adaptation and developmental cost

Roots of intact 5-day-old maize (Zea mays L.) seedlings were exposed to 3 micromolar Cd during a 7-day period. Cysteine, γ-glutamylcysteine, glutathione (GSH), and Cd-induced acid-soluble thiols (ASTs), including phytochelatins, were quantified in roots and shoots. Adaptation to Cd and its cost to seedling development were evaluated by measuring Cd content, tissue fresh weight, and rate of root elongation. Roots contained 60 to 67% of the Cd in the seedlings between 4 and 7 days of exposure. Exposure to Cd decreased the fresh weight gain in roots from day 4 onward without affecting the shoots. Between days 1.5 and 3.5 of Cd treatment, roots elongated more slowly than controls; however, their growth rate recovered thereafter and exceeded that of controls. Exposure to Cd did not appreciably affect the concentration of cysteine in the seedlings. However, the initial low concentration of γ-glutamylcysteine increased (after a lag of 6 hours in roots and 2 days in shoots), reaching a plateau by day 6 at 28.5 nanomoles per gram of fresh weight in roots and by day 5 at 19.1 nanomoles per gram of fresh weight in shoots. During the first 9 hours of Cd exposure, the concentration of GSH in roots decreased dramatically (at 31.6 nanomoles per gram of fresh weight per hour) and thereafter decreased more slowly than in controls. The depletion of GSH in the roots (366 nanomoles per gram of fresh weight) matched the synthesis of ASTs (349 nanomoles per gram of fresh weight) during the first 48 hours. The concentration of ASTs in roots increased steadily thereafter to reach 662.2 nanomoles per gram of fresh weight by 6 days of Cd exposure. In shoots, Cd had little influence on the concentration of GSH, but ASTs still accumulated to 173.3 nanomoles per gram fresh weight after 5 days. The molar ratio of thiols in ASTs to Cd increased to a maximum of 10.24 in roots after 4 hours and of 4.25 in shoots after 2 days of Cd exposure. After 4 days, the ratio reached a plateau of approximately 2 in roots and between 2 and 3 in shoots, as if a steady state of Cd chelation had been achieved in both organs. The plateau coincided with recovered root elongation or an adaptation to Cd. The reduced fresh weight gain of the roots during this time, however, indicated that the synthesis of Cd-induced thiols was at a cost to root development.     

Stimulation of Isopentenyl Pyrophosphate Incorporation into Polyisoprene in Extracts from Guayule Plants (Parthenium argentatum Gray) by Low Temperature and 2-(3,4-Dichlorophenoxy) Triethylamine

Rubber particles isolated from guayule (Parthenium argentatum Gray) stem homogenates contain a polyprenyl transferase which catalyzes the polymerization of isopentenyl pyrophosphate into polyisoprene. The polymerization reaction is stimulated with the addition of an allylic pyrophosphate initiator and forms a polymer of polyisoprene with a molecular weight distribution from 10³ to 10⁷. The polymerization reaction in crude stem homogenates is not affected by the addition of an initiator probably due to the high activity of isopentenyl pyrophosphate isomerase furnishing saturating levels of dimethylallyl pyrophosphate. Polyisoprene formation in stems of guayule plants exposed to cold winter temperatures increased from 15.4 milligrams per gram dry weight in October to 24.5 milligrams per gram dry weight in January and increased from 16.2 to 38.1 milligrams per gram dry weight in the same period by additionally treating the plants with 5000 ppm of 2-(3,4-dichlorophenoxy)triethylamine. The rate of polymerization of isopentenyl pyrophosphate into polyisoprene in stem homogenates of the cold treated plants increased from 12.1 nanomoles per hour per gram fresh weight in October to 144.3 nanomoles per hour per gram fresh weight in January and increased from 17.7 to 446.8 nanomoles per hour per gram fresh weight in the same period by additionally treating the plants with 5000 ppm of 2-(3,4-dichlorophenoxy)triethylamine. These results show that the increase in polyprenyl transferase activity partially accounts for the increase in polyisoprene synthesis in guayule plants exposed to low temperature and treated with 2-(3,4-dichlorophenoxy)triethylamine.     

Localization of enzymes of ureide biosynthesis in peroxisomes and microsomes of nodules

The intracellular location of enzymes involved in the synthesis of the ureides, allantoin and allantoic acid, was investigated in nodules of Glycine max L. Merr. Cellular organelles were separated on isopycnic sucrose density gradients. Xanthine dehydrogenase activity (270 nanomoles per min per gram fresh weight) was totally soluble, whereas approximately 15% of the total uricase and catalase activities (1 and 2000 micromoles per minute per gram fresh weight, respectively) was in the fraction containing intact peroxisomes. Allantoinase activity (680 nanomoles per minute per gram fresh weight) was associated with the microsomal fraction, which apparently originates from the endoplasmic reticulum.     

Cytokinin metabolism in autonomous and crown gall tissue cultures

When tissues ofCatharanthus roseus A6 crown gall were incubated on medium supplemented with 50 (μM N6-isopentenyladenine (i⁶Ade), endogenous i⁶Ade, N6-isopentenyladenosine (i⁶A) and i⁶A nucleotide (i⁶AXP) increased to. 6, 5 and 12 nmol g^-1, respectively, during 100 h. Whereas i⁶Ade and i⁶AXP increased rapidly during the initial 4 h and then remained relatively constant, the level of i⁶A continued to increase to 25 nmol g^-1 by 16 h and then decreased; Ribosylzeatin (io⁶A) and its nucleotide (io⁶AXP) remained constant at 1.5 and 1.7 nmol g^-1, respectively. Upon transfer to cytokininless medium, i⁶Ade and i⁶AXP declined rapidly but i⁶A increased to 10 nmol g^-1 after 4 h and then declined. Again, io⁶A and io⁶AXP were unchanged. Prolonged incubation of crown gall tissue on i⁶Ade completely inhibited growth. By contrast, nonrtransformed, autonomous tissue lines fromCalycanthus fertilis andActinidia chinensis Xarguta continued to proliferate on this medium. TheActinidia Une was shown to metabolize i⁶Ade to zeatin and to accumulate this cytokinin to levels in excess of 70 nmol g^-1.     

Tissue Distribution of beta-Cyanoalanine Synthase in Leaves

β-Cyanoalanine synthase, which catalyzes the reaction between cysteine and HCN to form β-cyanoalanine and H₂S, was assayed in leaf tissues from cyanogenic (Sorghum bicolor × Sorghum sudanense [sorghum]) and noncyanogenic (Pisum sativum [pea], Zea mays [maize], and Allium porrum [leek]) plants. The activity in whole leaf extracts ranged from 33 nanomoles per gram fresh weight per minute in leeks, to 1940 nanomoles per gram fresh weight per minute in sorghum. The specific activities of β-cyanoalanine synthase in epidermal protoplasts from maize and sorghum and in epidermal tissues from peas were in each case greater than the corresponding values for mesophyll protoplasts or tissues, or for strands of bundle sheath cells.

The tissue distributions for this enzyme were determined for pea, leek, and sorghum: the mesophyll protoplasts and tissues in these three plants contained 65% to 78% of the enzyme, while epidermal protoplasts and tissues contained 10% to 35% of the total leaf activity. In sorghum, the bundle sheath strands contained 13% of the leaf activity. The presence of β-cyanoalanine synthase in all tissues and species studied suggests a fundamental role for this enzyme in plant metabolism.

     

Stimulation of glutathione synthesis in photorespiring plants by catalase inhibitors

The effect of various herbicides on glutathione levels in barley (Hordeum vulgare L.), tobacco (Nicotiana tabacum L.), soybean (Glycine max [L.] Merr.), and corn (Zea mays L.) was examined. Illumination of excised barley, tobacco, and soybean plants for 8 hours in solution containing 2 millimolar aminotriazole (a catalase inhibitor) resulted in an increase in leaf glutathione from 250 to 400 nanomoles per gram fresh weight to 600 to 1800 nanomoles per gram fresh weight, depending on the species tested. All of this increase could be accounted for as oxidized glutathione. Between 25 and 50% of this oxidized glutathione was reduced when plants were darkened for 16 hours, but there was no significant decline in total glutathione. Another catalase inhibitor, thiosemicarbazide, was as effective as aminotriazole in elevating glutathione in soybean but was less effective in barley and tobacco. Glyphosate, an inhibitor of aromatic amino acid biosynthesis, had no significant effect on glutathione levels in any of the plants examined. Whereas methyl viologen (paraquat), which is a sink for photosystem I electrons, caused oxidation of leaf glutathione in all of the plants but did not increase the total amount of glutathione present.     

Osmosensitivity of Sucrose Uptake by Immature Pea Cotyledons Disappears during Development

Sucrose uptake was studied in isolated, immature pea cotyledons (Pisum sativum L. cv Marzia) in relation to their developmental stage. During the developmental period examined the water content of the cotyledons decreased from ≈80% “stage 1” to ≈55% “stage 2”. When assayed in an isotonic medium (400 osmoles per cubic meter) the influx capacity per gram fresh weight for sucrose was almost constant during this developmental period. The influx could be analyzed into a saturable component (K_m ≈ 9 moles per cubic meter; V_max ≈ 150 nanomoles per minute per gram fresh weight) and an unsaturable component (k_i ≈ 0.5 nanomoles per minute per gram fresh weight [per mole per cubic meter]). Incubation in a hypotonic medium reduced the sucrose influx in stage 1 cotyledons, up to 80% reduction at 0 milliosmole (medium without mannitol), but had no effect on sucrose uptake by stage 2 cotyledons. Reduced uptake in a hypotonic medium (100 osmoles per cubic meter) could be attributed to a lowering of the V_max from 150 to 36 nanomoles per minute per gram fresh weight. During incubation of stage 1 cotyledons and stage 2-cotyledons in a hypotonic medium (200 osmoles per cubic meter) their volume increased by 16% and 5.6%, respectively, while the calculated turgor pressure increased from 0.2 to 0.6 megapascal for cotyledons of both developmental stages. Reduced sucrose influx in hypotonic medium, therefore, seems to be related to cell swelling (membrane stretching) rather than to increased turgor pressure.     

Protein Metabolism in Cultured Plant Tissues: III. Changes in the Rate of Protein Synthesis, Accumulation, and Degradation in Cultured Pith Tissue

During the transition of tobacco (Nicotiana tabacum) pith tissue to callus tissue, there were changes in the composition of the soluble amino acid pools, in the distribution of amino acids between pool and protein, and in the synthesis, accumulation, and degradation of proteins. The size of the leucine pool decreased from 90 nanomoles per gram fresh weight in fresh pith to 20 nanomoles in 24-hour cultured pith, followed by a return to 90 nmoles in pith cultured longer than 5 days. The latter value is the same as that reported for exponentially growing callus cells. Many other pool amino acids changed as dramatically. However, they always approached callus levels after 5 days of culturing. The total amino acid content of pith tissue (the sum of both pool and protein) remained unchanged during culturing. The value for total amino acid content (34 to 42 nanomoles per gram fresh weight) was also similar to that found in callus. The distribution of amino acids between pool and protein did change during culturing. The transition of pith tissue with 88% of its total amino acids free in the soluble pool to callus with 92% of its amino acids in protein was further characterized by changes in protein metabolism. Both protein synthesis and accumulation increased over the first 50 hours in culture to a maximum rate of 45 milligrams protein synthesized gram protein⁻¹ hour⁻¹. After 50 hours in culture, the rate of protein accumulation decreased to equal the rate of fresh weight accumulation (10 mg g⁻¹ hour⁻¹). However, protein synthesis continued at a high rate for several days, suggesting protein degradation was turned on by this time. By 5 days protein synthesis had decreased to a rate similar to that of callus.     

Euphorbia escula L. Root and Root Bud Indole-3-Acetic Acid Levels at Three Phenologic Stages

Endogenous indoleacetic acid (IAA) levels of Euphorbia esula L. primary root and root buds were examined at three phenologic stages. High performance liquid chromatography coupled with fluorescence detection and gas chromatography-mass spectrometry, using ¹³C₆[benzene ring]-indole-3-acetic acid as internal standard, were used to measure root bud free and bound IAA levels in vegetative, full flower, and post-flower plants. Highest levels of free IAA (103 nanograms per gram fresh weight) were found in root buds during full flower. Esterified and amide IAA increased significantly in root buds of full flower and post-flower plants, but were not detectable in root buds of vegetative plants. Primary rootfree IAA was highest in vegetative and full flower plants (34.5 nanograms per gram fresh weight) and decreased by 50% in post-flower plants.     

Cysteine, gamma-Glutamylcysteine, and Glutathione Levels in Maize Seedlings : Distribution and Translocation in Normal and Cadmium-Exposed Plants

The levels of cysteine (Cys), γ-glutamylcysteine (γEC), and glutathione (GSH) were measured in the endosperms, scutella, roots, and shoots of maize (Zea mays L.) seedlings. GSH was the major thiol in roots, shoots, and scutella, Cys predominated in endosperms. The endosperm, scutellum, and functional phloem translocation were required for maintenance of GSH pools in roots and shoots of 6-day-old seedlings. Exposure of roots to 3 micromolar Cd, besides causing a decline in GSH, caused an accumulation of γEC, as if the activity of GSH synthetase was reduced in vivo. [³⁵S]Cys injected into endosperms of seedlings was partly metabolized to [³⁵S]sulfate. The scutella absorbed both [³⁵S]sulfate and [³⁵S]Cys and transformed 68 to 87% of the radioactivity into [³⁵S]GSH. [³⁵S]GSH was translocated to roots and shoots in proportion to the tissue fresh weight. Taken together, the data supported the hypothesis that Cys from the endosperm is absorbed by the scutellum and used to synthesize GSH for transfer through the phloem to the root and shoot. The estimated flux of GSH to the roots was 35 to 60 nanomoles per gram per hour, which totally accounted for the small gain in GSH in roots between days 6 and 7. For Cd-treated roots the GSH influx was similar, yet the GSH pool did not recover to control levels within 24 hours. The estimated flux of GSH to the entire shoot was like that to the roots; however, it was low (11-13 nanomoles per gram per hour) to the first leaf and high (76-135 nanomoles per gram per hour) to the second and younger leaves.     

Protein Folding: Grand Challenge of Nature

Abstract

Conformational preferences of the hypermodified nucleic acid bases N6-(Δ² -cis-hydroxyisopentenyl)adenine, cis-io⁶Ade also known as cis-zeatin, and N⁶-(Δ² -trans-hydroxyisopentenyl)adenine, trans-io⁶ade or trans-zeatin, and 2-methylthio derivatives of these cis-ms²io⁶Ade or cis-ms²zeatin, and trans-ms²io⁶Ade or trans-ms²zeatin have been investigated theoretically by the quantum chemical Perturbative Configuration Interaction with Localized Orbitals (PCILO) method. Automated geometry optimization using quantum chemical MNDO, AMI and PM3 methods has also been made to compare the salient features. The predicted most stable conformation of cis-io⁶Ade, trans-io⁶Ade, cis-ms²io⁶Ade and trans-ms²io⁶Ade are such that in each of these molecules the isopentenyl substituent spreads away (has “dista” conformation) from the five membered ring imidazole moiety of the adenine. The atoms N(6), C(10) and C(11) remain coplanar with the adenine ring in the predicted preferred conformation for each of these molecules. In cis-io⁶Ade as well as cis- ms²io⁶Ade the hydroxyl oxygen may participate in intramolecular hydrogen bonding with the H-C(10)-H group. In trans-io⁶Ade the hydroxyl group is oriented towards the H-C(2) instead. This orientation is retained in trans-ms²io⁶Ade, possible O-H…S hydrogen bonding may be a stabilizing factor. In all these four modified adenines C(11)-H is favourably placed to participate in intramolecular hydrogen bonding with N(1). In cis-ms²io⁶Ade as well as trans-ms²io⁶Ade the 2-methylthio group preferentially orients on the same side as C(2)-N(3) bond, due to this nonobstrusive placing, orientation of the hydroxyisopentenyl substituent remains unaffected by 2-methylthiolation. Thus the N(1) site remains shielded irrespective of the 2-methylthiolation status in these various cis-and trans-zeatin analogs alike. Firmly held orientation of hydroxyisopentenyl substituent in zeatin isomers and derivatives, in contrast to adaptable orientation of isopentenyl substituent in i⁶Ade and ms²i⁶Ade, may account for the increased efficiency of suppressor tRNA and reduced codon context sensitivity accompanied with the occurrence of ms²-zeatin (ms²io⁶Ade) modification.     

Free space iron pools in roots: generation and mobilization

A rapid and simple method for the determination of a ferric iron pool in the free space of roots is described. Formation of this pool depended on the source of iron in the nutrient solution. During growth in water culture at pH 5 to 6 with Fe-ethylenediaminetetraacetate, a free space pool of 500 to 1000 nanomoles Fe per gram fresh weight was formed in the roots of bean (Phaseolus vulgaris L. var. Prélude), maize (Zea mays L. var. Capella), and chlorophytum (Chlorophytum comosum [Thunb.] Jacques). No significant pool (less than 100 nanomoles per gram fresh weight) was formed with ferrioxamine. Upon impending Fe deficiency, bean and chlorophytum were able to mobilize this pool. Fe-deficient bean plants mobilized iron from the free space iron pool of another plant in the same vessel.     

Determination of plant growth substances in liquid endosperm of immature walnut (Juglans nigra) nuts by an ELISA technique

Plant growth substances (PGSs) were analysed in liquid endosperm of black walnut using HPLC and an ELISA procedure. Of all the PGSs studied, we show no GA₃, low levels of cytokinins (io⁶A, i⁶Ade, i⁶Ado) and ABA, and very high level of IAAAbbreviations ABA Abscisic acid: - Ade Adenine: - GA₃ Gibberellic acid: - IAA Indole-3-acetic acid: - i⁶Ade N⁶(²-1) adenine: - i⁶Ado N⁶(²-isopentenyl adenosine: - io⁶A Zeatin riboside:     

Accumulation of Putrescine during Chilling Injury of Fruits

Putrescine (Put) increased 68% in lemon (Citrus limon (L.) Burm. f. cv Bearss) flavedo, 39% in grapefruit (C. paradisi Macf. cv Marsh) flavedo, 49% in grapefruit juice, and 149% in pepper (Capsicum annuum L. cv Early Calwonder) pericarp when fruits were stored at chilling temperatures. In lemon flavedo, the coefficient of correlation (r²) between Put concentration with severity of chilling was 0.90 and Put levels almost doubled; the injury index going from 1 to 2 units. Pepper pericarp, which was the most chilling-sensitive tissue tested (injury index going from 1 to 3.8 units), showed the greatest difference in Put accumulation (166 to 413 nanomoles per gram fresh weight) between storage temperatures of 7.2 and 1°C. The least difference (338 to 470 nanomoles per gram fresh weight) was found in grapefruit flavedo between storage temperatures of 15.5 and 4.4°C; the injury index going from 1 to 1.3 units.     

Activities of arginine and ornithine decarboxylases in various plant species

In extracts from the youngest leaves of Avena sativa, Hordeum vulgare, Zea Mays, Pisum sativum, Phaseolus vulgaris, Lactuca sativa, and four pyrrolizidine alkaloid-bearing species of Heliotropium, the activities of ornithine decarboxylase, close to V_max, ranged between traces and 1.5 nanomoles per hour per gram fresh weight when based on putrescine formed during incubation with labeled ornithine. The arginine decarboxylase activities in the same extracts ranged between 8 and 8000 nanomoles per hour per gram fresh weight being lowest in the borages and highest in oat and barley. α-Difluoromethylornithine and α-difluoromethylarginine inhibited ornithine and arginine decarboxylases, respectively, in all species. Agmatine, putrescine, spermidine, and spermine were found in all, diaminopropane in eight, and cadaverine in three species.

No correlation was observed between arginine or ornithine decarboxylase level and the levels of total polyamines. The in vitro decarboxylase activities found in the borages cannot explain the high accumulation of putrescine-derived pyrrolizidines in their youngest leaves if the pyrrolizidines are produced in situ from arginine and/or ornithine as precursors; other possibilities are discussed.

In assays of ornithine decarboxylase, an interference of decarboxylation not due to this enzyme was observed in extracts from all species. In arginine decarboxylase assays, the interfering decarboxylation as well as the interference of arginase were apparent in two species. Addition of aminoguanidine was needed to suppress oxidative degradation of putrescine and agmatine during incubation of extracts from pea, bean, lettuce, Heliotropium angiospermum, and Heliotropium indicum.

     

Nicotinate, nicotinamide, and the reactivity of leghemoglobin in soybean root nodules

Nicotinate has been postulated to interfere with the binding of O₂ to ferrous leghemoglobin in soybean (Glycine max) root nodules. For such a function, the levels of nicotinate in nodules must be sufficiently high to bind a significant amount of leghemoglobin. We have measured levels of nicotinate, nicotinamide, and leghemoglobin in soybean nodules from plants 34 to 73 days after planting in a glasshouse. On a per gram nodule fresh weight basis, levels between 10.4 and 21 nanomoles for nicotinate, 19.2 and 37.8 nanomoles for nicotinamide, and 170 to 280 nanomoles for leghemoglobin were measured. Even if all the nicotinate were bound to ferrous leghemoglobin, only 11% or less of the total leghemoglobin would be unavailable for binding O₂. Using the measured levels of nicotinate and a pH of 6.8 in the cytosol of presenescent soybean nodules, we estimate that the proportion of ferrous leghemoglobin bound to nicotinate in such nodules would be less than 1%. These levels of nicotinate are too low to interfere with the reaction between ferrous leghemoglobin and O₂ in soybean root nodules.     

Isolation and Characterization of a Mutant of Arabidopsis thaliana Resistant to alpha-Methyltryptophan

Mutants of Arabidopsis thaliana have been selected for resistance to growth inhibition at the seedling stage by α-methyltryptophan (aMT). One mutant, amt-1 has been characterized in detail. The appearance and growth rate of the mutant in the absence of the inhibitor are similar to wild type, both as plants and callus. However, mutant plant growth is unaffected by 25 micromolar aMT and mutant callus growth by 50 micromolar aMT, concentrations that completely inhibit the growth of wild-type plants and callus, respectively. Tryptophan levels in mutant and wild-type plants are 24.3 ± 2.7 and 4.7 ± 1.2 micrograms per gram fresh weight, respectively, and in the corresponding callus 64.0 ± 2.6 and 31.8 ± 8.4 micrograms per gram fresh weight, respectively. Anthranilate synthase (AS) activity levels in crude extracts from whole plants are 3.09 ± 0.54 nanomoles per milligram protein per hour in amt-1 and 1.32 ± 0.21 nanomoles per milligram protein per hour in wild-type plants. In crude extracts from callus, anthranilate synthase levels are 11.54 ± 2.05 nanomoles per milligram protein per hour and 7.74 ± 1.58 in amt-1 and wild type, respectively. Enzyme extracts are inhibited by l-tryptophan; the concentrations required for 50% inhibition (I₅₀) are 3.9 and 1.9 micromolar for amt-1 and for wild type, respectively. The mutation segregates as a single nuclear allele and shows incomplete dominance. The concomitant increases in both AS activity and its I₅₀ for tryptophan suggest that the mutation amt-1 either resides in one of the AS structural genes or causes increased expression of an AS isoform with an I₅₀ greater than the average for the entire extract.     

Stabilization of nitrate reductase in maize roots by chymostatin

Nitrate reductase (NR) in maize (Zea mays cv W64A × W182E) roots has been stabilized in vitro by the addition of chymostatin to extraction buffer. Contrary to previous observations, levels of NR were higher in the mature root than in root tip sections when chymostatin was included in the extraction buffer. Two forms of NR were identified, an NADH monospecific NR found mainly in the 1cm root tip and an NAD(P)H bispecific NR found predominantly in mature regions of the root. During the first 10 days of seedling growth, NR activity in the root ranged from 50 to 80% of the activities found in the leaf (a maximum of 2.4 micromoles NO⁻₂ produced per hour per gram fresh weight was measured at 4 days).     

Levels of Free and Conjugated Abscisic Acid in Developing Floral Organs of the Navel Orange (Citrus sinensi [L.] Osbeck cv Washington)

The levels of abscisic acid (ABA) and alkaline-hydrolyzable ABA-conjugate (putatively identified as the glucosyl ester, abscisyl-β-d-glucopyranoside) were determined by enzyme immunoassay in the organs of developing navel orange (Citrus sinensis [L.] Osbeck cv Washington) flowers. Although both compounds were detected in every tissue, developmentally related differences between organs in the total and relative contents were observed. The highest ABA levels were observed in the stigma/style shortly after anthesis (11.5 ± 0.6 nanomoles ABA per gram fresh weight and 4.8 ± 0.6 nanomoles ABA-conjugate per gram fresh weight); whereas, the highest ABA-conjugate levels were observed at the same time in the floral disc (hypogynous disc plus calyx; 3.5 ± 0.1 nmol nanomols ABA per gram fresh weight and 11.8 ± 0.9 nanomoles ABA-conjugate per gram fresh weight). These results suggest that differences in ABA content reflect tissue-specific variation in the facility for ABA conjugation. Increased ABA levels were observed in the stigma/style near anthesis; however, a relationship with pollination is discounted, since `Washington' navel orange flowers are male sterile and devoid of pollen.