Biosynthesis of Cutin omega-Hydroxylation of Fatty Acids by a Microsomal Preparation from Germinating Vicia faba

omega-Hydroxylation of fatty acids, which is a key reaction in the biosynthesis of cutin and suberin, has been demonstrated for the first time in a cell-free preparation from a higher plant. A crude microsomal fraction (105,000g pellet) from germinating embryonic shoots of Vicia faba catalyzed the conversion of palmitic acid to omega-hydroxypalmitic acid. As the crude cell-free preparation also catalyzes the formation of other hydroxy acids such as alpha- and beta-hydroxy acids, the omega-hydroxylation product was identified by gas chromatography on a polyester column and reverse phase, high performance liquid chromatography, two techniques which were shown to resolve the positional isomers. Gas chromatographic analysis of the dicarboxylic acid obtained by CrO(3) oxidation of the enzymic product also confirmed the identity of the enzymic omega-hydroxylation product. This enzymic hydroxylation required O(2) and NADPH, but substitution of NADH resulted in nearly half the reaction rate obtained with NADPH. Maximal rates of omega-hydroxylation occurred at pH 8 and the rate increased in a sigmoidal manner with increasing concentrations of palmitic acid. This omega-hydroxylation was inhibited by the classical mixed function oxidase inhibitors such as metal chelators (o-phenanthroline, 8-hydroxyquinoline, and alpha,alpha-dipyridyl), NaN(3) and thiol reagents (N-ethylmaleimide and p-chloromercuribenzoate). As expected of a hydroxylase, involving cytochrome P(450), the present omega-hydroxylase was inhibited by CO and this enzyme system showed unusually high sensitivity to this inhibition; 10% CO caused inhibition and 30% CO completely inhibited the reaction. Another unusual feature was that the inhibition caused by any level of CO could not be reversed by light (420-460 nm).     

gamma-Guanidinobutyraldehyde Dehydrogenase of Vicia faba Leaves

γ-Guanidinobutyraldehyde dehydrogenase was purified 27-fold in 40% yield from extracts of Vicia faba leaves. High specificity exist only for γ-guanidinobutyraldehyde and γ-aminobutyraldehyde; the K_m value was 3.4 micromolar for γ-guanidinobutyraldehyde, 25 micromolar for γ-aminobutyraldehyde, and 84 micromolar (case of γ-guanidinobutyraldehyde) for NAD, respectively. The enzyme had a molecular weight of approximately 83,000. Optimal pH and temperature for activity were 9.5 and 45°C, respectively. The enzyme was inhibited strongly by p-chloromercuribenzoate, N-ethylmaleimide, and zincon (2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene).     

In Vitro Biosynthesis of Vicia faba Polyphenoloxidase

Poly A⁺ mRNA was isolated from Vicia faba leaves and translated in vitro using a rabbit reticulocyte translation system. From analysis of the total translation products, the major proteins synthesized in vitro were 32 kilodaltons and 20 kilodaltons. When antibodies to Vicia faba polyphenoloxidase were added, a specific immunoprecipitable protein was observed. This protein's molecular weight was shown to be similar to that of the isolated enzyme (45 kilodaltons). The isolated enzyme successfully competed with the in vitro synthesized product for antipolyphenoloxidase. In addition, the in vitro synthesized product was not immunoprecipitated with antitomato peroxidase and comigrated with isolated and/or iodinated enzyme in sodium dodecylsulfate-polyacrylamide gel electrophoresis. Using in vitro translation and specific immunoprecipitation, a primary translation product corresponding to Vicia faba polyphenoloxidase was identified as a 45 kilodaltons protein.     

Biosynthesis of the phytoalexin wyerone in Vicia faba

In contrast to earlier results [1-¹⁴C] acetate, [2-¹⁴C] malonate and [n 9,10-³H] oleate show significant incorporations into wyerone and related Vicia faba phytoalexins, following infection by Botrytis cinerea.     

Wilting of Leaves in Vicia faba L.

Wilting of leaves of Vicia faba L., which occurs when the pressurepotential (_p) is zero, and the leaf-water potential () at wiltingboth depend entirely upon the solute potential at incipientplasmolysis (_so) and not on soil-water status. Wilting in V.faba is acropetal; this is consistent with the hypothesis thatthere is a gradient of decreasing _so up the plant and that wateris transferred from the lower to the upper leaves, hasteningthe overall water loss from the lower leaves to the point when_p is zero. The gradient in _so up the plant is of the order of3–8 bar. It is proposed that wilting when _p>0 (i.e. > _so) shouldbe ‘apparent wilting’ and that when _p0 (i.e. _so),‘true wilting’.     

Some Properties of the Arginine Decarboxylase in Vicia faba Leaves

Growth of Vicia faba seedlings is accompanied by a rapid increasein arginine decarboxylase (EC 4.1.1.19 [EC] ) in the leaves and epicotyl.Increased enzyme activity was observed under saline conditionsin the presence of NaCl and with osmotic stress by mannitol.The partially purified enzyme (about 86-fold) readily decarboxylatedL-arginine, while D-arginine, L-homoarginine, L-ornithine andL-lysine were decarboxylated very slowly, and L-citrulline andL-glutamic acid were not decarboxylated. The K_m value was 5.8?10^–4M for L-arginine. The optimal pH and temperature for activitywere 8.5 and 45?C, respectively. p-Chloromercuribenzoate andN-ethylmaleimide were effective inhibitors of the enzyme. Inhibitionby spermidine, putrescine and agmatine suggested a possiblefeed-back mechanism in the pathway of polyamine biosynthesis. (Received October 11, 1983; Accepted February 24, 1984)     

Changes with Age in the Status of Water and Tissue Characteristics in Individual Leaves of Vicia faba L.

Leaf-water potential () and relative water content (R) of leavesat different position on the plant were different on any oneday. This was because the effect of environment on and R andof age on physical and chemical properties of tissues variedwith the position of the leaf. Changes with age in the physicaland chemical properties of a leaf depended on the time it emergedin the life of the plant.     

The Effect of Temperature Changes on the Expansion of Individual Leaves of Vicia faba L

The growth in area of the sixth leaf of field bean plants wasinvestigated in growth room experiments. Temperatures were variedduring the periods from appearance to unfolding and from unfoldingto full expansion. The effects on the duration of growth weregreater than those on absolute growth rates. Counts of epidermalcell number showed that the changes in final leaf area couldbe explained by changes in epidermal cell size for these temperaturesand these times of treatment. Epidermal cell number was notaffected by the treatments. Vicia faba, leaf expansion, temperature, growth, cell division     

Purification and Properties of the Diamine Oxidase from Vicia faba Leaves

Diamine oxidase (EC 1.4.3.6 [EC] ) from the leaves of Vicia faba waspurified to homogeneity by polyacrylamide gel electrophoresis.The molecular weight estimated by Sephadex G-200 gel filtrationwas about 126,000. Sodium dodecyl sulfate gel electrophoresisgave a single band at the molecular weight of 74,000. The isoelectricpoint was at pH 7.2. The enzyme contained two copper atoms permole of enzyme. Inhibition with phenylhydrazine showed thatthe Vicia enzyme contains one mole of the carbonyl group permole of the enzyme. The amino acid composition of the enzymealso is described. (Received February 23, 1981; Accepted April 7, 1981)     

Regulation of Apoplastic pH in Source Leaves of Vicia faba by Gibberellic Acid

Leaf discs of broad bean (Vicia faba L.), peeled on the spongy mesophyll side, rapidly altered the pH of the surrounding medium (apoplast). Using pH indicator paper appressed against the leaf, immediately after peeling, initial apoplastic pH was estimated to be 4.5. Changes in the apoplastic pH were measured with a microelectrode placed into a 100-microliter drop of an unbuffered solution (2 millimolar KCl, 0.5 millimolar CaCl₂, and 200 millimolar mannitol) on the peeled surface. Discs acidified the medium until the pH stabilized at about 5.0 (about 10 minutes). Acidification was inhibited by 50 micromolar sodium vanadate, an inhibitor of the plasmalemma H⁺-ATPase and attenuated by omitting the osmoticum or potassium ions from the medium. Fusicoccin (10 micromolar) greatly enhanced the rate of acidification. The presence of 0.1 to 1 micromolar gibberellic acid resulted in a slower rate of medium acidification. Gibberellic acid appeared to modulate the activity of the H⁺-translocating ATPase located at the plasma membrane of the mesophyll cells.     

Biosynthesis of Cutin: Enzymatic Conversion of omega-Hydroxy Fatty Acids to Dicarboxylic Acids by Cell-free Extracts of Vicia Faba Epidermis

Long chain dicarboxylic acids are constituents of the protective biopolymers cutin and suberin of plants. Cell-free extracts from the excised epidermis of Vicia faba leaves catalyzed conversion of 16-hydroxy[G-(3)H]hexadecanoic acid to the corresponding dicarboxylic acid with nicotinamide-adenine dinucleotide phosphate as the preferred cofactor. This enzymatic activity, located largely in the 100,000g supernatant fraction, had a pH optimum near 8. This dehydrogenase showed an apparent Km of 1.25 x 10(-5)m and 3.6 x 10(-4)m for 16-hydroxyhexadecanoic acid and NADP, respectively. Modification of the substrate, either by esterification of the carboxyl group or by introduction of another hydroxyl group at C-10, resulted in a substantial (two-thirds) decrease in the rate of reaction, and hexadecanol was not a good substrate. The enzyme was inhibited by thiol reagents such as N-ethylmaleimide and p-chloromercuribenzoate. The aldehyde intermediate was trapped by the inclusion of dinitrophenyl hydrazine in the reaction mixture, and the 16-oxo compound was regenerated and identified. Furthermore, synthetic 16-oxo-[G-(3)H] hexadecanoic acid was readily converted to the dicarboxylic acid by the cell-free preparation. These results demonstrate that epidermis of Vicia faba contains an omega-hydroxyacid dehydrogenase and an omega-oxoacid dehydrogenase.     

Effect of Photoperiod on Stomatal Opening in Vicia faba

Stomatal apertures in darkness and subsequent average opening rates in light were measured in Vicia faba leaf discs throughout the nyctoperiods for plants grown on three light:dark cycles (8:16, 12:12, and 16:8). The time course of opening in darkness depended on the specific light:dark cycle with the maximum aperture always occurring at the time the lights normally went on. The light-induced opening rate was also maximum at the end of the nyctoperiod.     

The Effect of Temperature on the Growth of Individual Leaves of Vicia faba L. in the Field

The generalized logistic curve was used to describe the growthof individual leaves in crops of Vicia faba L. Durations of.expansionand mean absolute growth rates were derived from these curves.The duration of expansion was inversely related to temperatureaveraged over four days from unfolding. This relationship wasindependent of leaf position except for the lowest leaves. Theduration of expansion of a leaf was related to the rate of productionof new leaves, the number of expanding leaves remaining relativelyconstant. Absolute growth rates varied with leaf position upto leaf 10. At higher leaves, in the absence of water stress,absolute growth rate was a function of temperature and radiation. Vicia faba L., field bean, leaf growth, temperature     

Involvement of Nitric Oxide in the Mechanism for Stomatal Opening in Vicia faba Leaves

Nitrite, as well as the nitric oxide (NO) donor S-nitroso-N-acethylpenisilamine (SNAP), was found to increase the aperture of stoma on Vicia faba leaf peels. The results demonstrated here suggest that the nitrite-dependent NO production pathway would be involved in the signal transduction for stomatal movements.     

Isolation of Guard Cell Protoplasts from Mechanically Prepared Epidermis of Vicia faba Leaves

A method for isolating guard cell protoplasts (GCP) from mechanically prepared epidermis of Vicia faba is described. Epidermis was prepared by homogenizing leaves in a Waring blender in a solution of 10% Ficoll, 5 millimolar CaCl₂, and 0.1% polyvinylpyrrolidone 40 (PVP). Attached mesophyll and epidermal cells were removed by shaking epidermis in a solution of Cellulysin, mannitol, CaCl₂, PVP, and pepstatin A. Cleaned epidermis was transferred to a solution of mannitol, CaCl₂, PVP, pepstatin A, cellulase “Onozuka” RS, and pectolyase Y-23 for the isolation of GCP. Preparations made by this method included both adaxial and abaxial GCP and contained ≤0.017% mesophyll protoplasts, ≤0.6% mesophyll fragments, and no epidermal cell contaminants. Yields averaged 9 × 10⁴ protoplasts/leaflet and 98 to 100% of the GCP excluded trypan blue, concentrated neutral red, and hydrolyzed fluorescein diacetate. Isolated GCP increased in diameter by 2.2 micrometers after incubation in darkness in 10 micromolar fusicoccin, 0.4 molar mannitol, 5 millimolar KCl, and 1 millimolar CaCl₂. Illumination of GCP with 800 micromoles per square meter per second of red light resulted in alkalinization of their suspension medium. When 10 micromolar per square meter per second of blue light was superimposed onto the red light background, the medium acidified. Measurements of chlorophyll a fast fluorescence transients from isolated GCP indicated that GCP were capable of electron transport, and slow transients contained the “M” peak usually associated with a functional photosynthetic carbon reduction pathway.     

Apical Dominance in Vicia faba

Apical dominance phenomena have been studied in seedlings ofVicia faba particularly in relation to the movement about theplant of uracil-2-¹⁴C applied to the cotyledons. Decapitationjust below the second node releases the growth of the lowermostlateral bud and inhibition is completely reimposed by applicationof indole-3-acetic acid (IAA) to the cut surface. Uracil-2-¹⁴Capplied in solution to the cotyledons is distributed in thestems of all experimental seedlings with no consistent differencesdue to decapitation or IAA application. On the other hand, decapitationresults in a rapid increase in uracil-2-¹⁴C content in the lateralbuds which far exceeds their promoted growth. This uptake iscompletely suppressed by IAA application. A ring of tri-iodobenzoicacid (TIBA)-lanolin paste around the stem above the bud suppressesIAA action both in bud growth and on uracil-2-¹⁴C uptake, andalso on the movement of IAA-1-¹⁴C down the stem. TIBA-application to the base of the bud does not prevent IAAaction on bud growth, but also does not prevent the movementof IAA-1-¹⁴C (or a water soluble product of its metabolism)into the bud. Direct application of kinetin to the lateral bud of intact plantscauses a short-lived release of growth. Gibberellic acid producesa smaller and scarcely significant increase which is additiveto the kinetin effect. Neither has any effect on uracil-2-¹⁴Cmovement into the bud. The implications of these findings are discussed in relationto various existing theories of the mode of auxin action inapical dominance and it is concluded that their strongest supportis for a mechanism involving the suppression of phloem differentiationin the vascular supply to the bud.     

Compartmentation in Vicia faba Leaves: I. Kinetics of C in the Tissues following Pulse Labeling

Leaflets of Vicia faba were pulse-labeled with ¹⁴CO₂ to follow the subsequent movement of photosynthate between leaf tissues. Samples were taken during a ¹²CO₂ chase, quick frozen, freeze-substituted, and embedded in methacrylate. Paradermal sections provided tissue samples consisting only of upper epidermis, palisade parenchyma, spongy parenchyma and veins, spongy parenchyma, or lower epidermis. Most CO₂ fixation occurred in the palisade parenchyma, but its ¹⁴C content declined rapidly after labeling. Concomitant with the decline of activity in the palisade parenchyma, there was an increase in activity in the spongy parenchyma and upper epidermis and a slow increase in the lower epidermis. Activity in the palisade parenchyma and spongy parenchyma eventually reached similar levels and remained constant. Tissue samples containing veins were consistently the most radioactive, and activity in those samples showed a decline. Very little change occurred in the insoluble fraction from any tissue. The results support previous assumptions regarding the pathway of assimilate transport to the veins, and demonstrate the rapidity of such transport. Sucrose is apparently the principal mobile compound.     

Galactolipid Synthesis in Vicia faba Leaves: III. Site(s) of Galactosyl Transferase Activity

Leaves of Vicia faba were fed ¹⁴CO₂ in light for periods of up to 6 hours. At intervals, leaf samples were homogenized and separated into fractions which contained “broken” and “intact” chloroplasts, and three other high speed centrifugal fractions containing other cell membranes and chloroplast envelopes. Analyses of the radioactive labeling of galactose from the galactolipids in these fractions and in purified chloroplast envelopes indicated that the major site of galactosyl transferase enzyme activity was in the chloroplast envelope. The data suggest that in time much of the radioactive galactolipid was transferred from the envelope to the thylakoid-containing fractions. The major site of galactolipid synthesis appears to be in the envelope but there is some evidence of another site in the thylakoids.     

Nitrogen Metabolism of Vicia faba

Vicia faba plants were grown for four and six weeks without externally supplied nitrogen. Some nitrogen was transported to the plant axis from the cotyledons throughout this period, but the amount available was insufficient to support maximum shoot growth. During this period the protein content of the shoot declined whilst the free amino acids, especially aspartic acid, glutamic acid, histamine and the combined pool for threonine, serine, asparagine and glutamine and ammonia, increased in amount. In contrast to the shoot the protein content of the root increased as did their free amino acid content, but the increase in the latter was less than in the shoot and only the combined value for threonine, serine, asparagines and glutamine increased significantly. During tbe last two weeks growth, some soluble non-amino acid compound appeared to donate nitrogen to the pool of free amino acids in the root and shoot.