Conversion of 5-(1,2-epoxy-2,6,6-trimethylcyclohexyl) -3-methylpenta-cis-2-trans-4-dienoic acid into abscisic acid in plants

(±)-5-(1,2-Epoxy-2,6,6-trimethylcyclohexyl) -3-methyl[2-¹⁴C]penta-cis-2-trans-4-dienoic acid is converted into abscisic acid by tomato fruit in 1.8% yield (or 3.6% of one enantiomer if only one is utilized) and 15% of the abscisic acid is derived from the precursor. The 2-trans-isomer is not converted. The amounts of [2-³H]mevalonate incorporated into abscisic acid have shown that the 40-times higher concentration of (+)-abscisic acid in wilted wheat leaves in comparison with unwilted ones reported by Wright & Hiron (1969) arises by synthesis. The conversion of (±)-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl) -3-methyl-[2-¹⁴C]penta-cis-2-trans-4-dienoic acid into abscisic acid by wheat leaves is also affected in the same way by wilting and it is concluded from this that the epoxide or a closely related compound derived from it is on the biosynthetic pathway leading to abscisic acid. The oxygen of the epoxy group was shown, by ¹⁸O-labelling, to become the oxygen of the tertiary hydroxyl group of abscisic acid.     

Activity and metabolism of C-(+/-)-abscisic Acid derivatives

Several radioactive analogues of abscisic acid have been tested for their growth-inhibitory effects and their metabolism in excised embryonic axes of Phaseolus vulgaris. The compounds tested were the methyl and ethyl esters of 2-¹⁴C-abscisic acid and the cis- and trans-1′,4′-diols of 2-¹⁴C-abscisic acid. All four compounds cause less growth inhibition than abscisic acid, and all four compounds are converted to abscisic acid in the axes at rates which are sufficient to account for most, if not all, of the observed growth-inhibitory activity. None of the four compounds is metabolized to the extent that abscisic acid is metabolized in the axes, suggesting that the structural requirements for growth-inhibitory activity and metabolism may be similar.     

Incorporation of oxygen into abscisic Acid and phaseic Acid from molecular oxygen

Abscisic acid accumulates in detached, wilted leaves of Xanthium strumarium. When these leaves are subsequently rehydrated, phaseic acid, a catabolite of abscisic acid, accumulates. Analysis by gas chromatography-mass spectrometry of phaseic acid isolated from stressed and subsequently rehydrated leaves placed in an atmosphere containing 20% ¹⁸O₂ and 80% N₂ indicates that one atom of ¹⁸O is incorporated in the 6′-hydroxymethyl group of phaseic acid. This suggests that the enzyme that converts abscisic acid to phaseic acid is an oxygenase.

Analysis by gas chromatography-mass spectrometry of abscisic acid isolated from stressed leaves kept in an atmosphere containing ¹⁸O₂ indicates that one atom of ¹⁸O is present in the carboxyl group of abscisic acid. Thus, when abscisic acid accumulates in water-stressed leaves, only one of the four oxygens present in the abscisic acid molecule is derived from molecular oxygen. This suggests that either (a) the oxygen present in the 1′-, 4′-, and one of the two oxygens at the 1-position of abscisic acid arise from water, or (b) there exists a stored precursor with oxygen atoms already present in the 1′- and 4′-positions of abscisic acid which is converted to abscisic acid under conditions of water stress.

     

Biosynthesis of abscisic acid from farnesol derivatives in Cercospora rosicola

(E,E)?[1?¹⁴C]Farnesyl phosphate and (E,E)?[1?¹⁴C]farnesyl pyrophosphate were both converted to abscisic acid by Cercospora rosicola resuspensions. (E,E)?[1?¹⁴C]Farnesol, (E,Z)?[1?¹⁴C]farnesol, (E,Z)?[1?¹⁴C]farnesyl pyrophosphate, (E,E)?[1?¹⁴C]farnesic acid, and (E,Z)?[1?¹⁴C]farnesic acid were not converted to abscisic acid by the fungus. These findings provide information on the sequence of the reactions involved in converting farnesyl pyrophosphate to abscisic acid. Specifically, they suggest that the transformations involving the three terminal carbons in the side chain occur after one or more changes elsewhere in the molecule.     

Abscisic Acid and stomatal regulation

The closure of stomata by abscisic acid was examined in several species of plants through measurements of CO₂ and H₂O exchange by the leaf. The onset of closure was very rapid, beginning at 3 minutes from the time of abscisic acid application to the cut base of the leaf of corn, or at 8 or 9 minutes for bean, Rumex and sugarbeet; rose leaves were relatively slow at 32 minutes. The timing and the concentration of abscisic acid needed to cause closure were related to the amounts of endogenous abscisic acid in the leaf. Closure was obtained in bean leaves with 8.9 picomoles/cm². (+)-Abscisic acid had approximately twice the activity of the racemic material. The methyl ester of abscisic acid was inactive, and trans-abscisic acid was likewise inactive. The effects of stress on levels of endogenous abscisic acid, and the ability of very small amounts of abscisic acid to cause rapid closure suggests that stomatal control is a regulatory function of this hormone.     

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

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

Growth Regulators Have Rapid Effects on Photosynthate Unloading from Seed Coats of Phaseolus vulgaris L

Of nine plant growth regulators (indoleacetic acid, 1-naphthalene acetic acid, gibberellic acid, giberellin 4/7, 6-benzylaminopurine, 6-furfurylaminopurine, abscisic acid, and 1-aminocyclopropane carboxylic acid) tested, only 6-benzylaminopurine and abscisic acid affected ¹⁴C-photosynthate unloading from excised seed coats of Phaseolus vulgaris L. Unloading, in the presence of KCl, was stimulated by 25 to 40%. Stimulation occurred immediately for 6-benzylaminopurine and for abscisic acid within 10 to 12 minutes of application.     

Occurrence and metabolism of 1′,4′-trans-diol of abscisic acid

The 1′,4′-trans-diol of abscisic acid was first identified in higher plants with GC-ECD and GC-SIM. The ²H-labelled derivative was converted into abscisic acid (ABA) in pea and avocado, but ²H-labelled ABA was not converted into the diol. These results suggest that the diol is one of the precursors of ABA in higher plants.     

The effect of plant hormone abscisic acid on model membranes - differential scanning calorimetry and planar bilayer membranes investigations

The effect of abscisic acid on the thermotropic properties of dipalmitoylphosphatidylcholine (DPPC) and on phosphatidylethanolamines (natural (PE) and dipalmitoylphosphatidylethanolamine (DPPE)) bilayers was investigated by differential scanning calorimetry (DSC). Abscisic acid eliminates the pretransition of DPPC, causes a downward shift of its temperature of melting (T_m) and broadens the melting peak without changing the enthalpy of melting. In natural PE bilayers interacting with abscisic acid a small decrease in the enthalpy of melting almost without change of T_m was detected, whereas in synthetic DPPE abscisic acid caused a small shift of T_m and small broadening of the melting peak without changing the enthalpy of melting. Abscisic acid increases the conductance to Na⁺ or K⁺ by three orders of magnitude in planar lipid membranes formed from PE monolayers and by less than two orders of magnitude in membranes formed from PC monolayers.     

Metabolism of 2-C-(+/-)-abscisic Acid in excised bean axes

Excised embryonic bean axes (Phaseolus vulgaris, var. White Marrowfat) rapidly metabolize 2-¹⁴C-(±)-abscisic acid to two compounds, M-1 and M-2, which have very low growth-inhibitory activity. Chemical tests indicate the M-1 and M-2 are not previously described abscisic acid metabolites. M-2 accumulates in the axes and evidence is presented for the hypothesis that abscisic acid → M-1 → M-2. Zeatin, which partially reverses the abscisic acid-mediated growth inhibition of axes, neither decreases abscisic acid uptake nor causes any major changes in its metabolism. It was observed that axes transferred from abscisic acid-containing solutions to buffer resume control rates of fresh weight increase while still containing considerable quantities of abscisic acid.     

The Metabolism of Hormones during Seed Germination and Dormancy: IV. The Metabolism of (S)-2-C-Abscisic Acid in Ash Seed

Embryos from dormant and stratified Fraxinus americana seed were incubated with (S)-2-¹⁴C-abscisic acid (ABA) under a variety of conditions. Both dormant and stratified embryos rapidly metabolize abscisic acid to phaseic acid, dihydrophaseic acid, and an unidentified polar metabolite apparently derived from dihydrophaseic acid. Although the stratified embryos may have an increased capacity to metabolize abscisic acid, our calculations suggest that such an increased capacity would probably not be physiologically significant.     

Effects of fluridone and abscisic acid on lateral root initiation and root elongation of excised tomato roots cultured in vitro

Roots of tomato (Lycopersicon esculentum Mill. cv. Bonny Best) were excised and cultured in the presence of the abscisic acid synthesis inhibitor fluridone, and with concentrations of exogenous abscisic acid ranging from 10⁻¹⁰to 10⁻⁴M to determine the effects of abscisic acid and its synthesis inhibition on the development of lateral roots in in vitro cultured tomato roots. Exogenous abscisic acid inhibited lateral root initiation and emergence at concentrations of 10⁻⁶M and greater. Fluridone (10⁻⁶M) enhanced the formation of lateral roots even in the presence of abscisic acid, at all concentrations tested except 10⁻⁴M. Abscisic acid increased apical distance, and fluridone reduced it up to 10⁻⁵M abscisic acid. Both fluridone and abscisic acid reduced lateral and primary root lengths. It was concluded the endogenous abscisic acid is probably involved in the regulation of lateral root initiation and root apical dominance, and that abscisic acid may affect lateral root initiation differently than lateral root emergence. This revised version was published online in June 2006 with corrections to the Cover Date.     

Alteration of Gene Expression during the Induction of Freezing Tolerance in Brassica napus Suspension Cultures

Brassica napus suspension-cultured cells can be hardened to a lethal temperature for 50% of the sample of −20°C in eight days at room temperature with abscisic acid. During the induction of freezing tolerance, changes were observed in the electrophoretic pattern of [³⁵S]methionine labeled polypeptides. In hardening cells, a 20 kilodalton polypeptide was induced on day 2 and its level increased during hardening. The induction of freezing tolerance with nonmaximal hardening regimens also resulted in increases in the 20 kilodalton polypeptide. The 20 kilodalton polypeptide was associated with a membrane fraction enriched in endoplasmic reticulum and was resolved as a single spot by two-dimensional electrophoresis. In vitro translation of mRNA indicate alteration of gene expression during abscisic acid induction of freezing tolerance. The new mRNA encodes a 20 kilodalton polypeptide associated with increased freezing tolerance induced by either abscisic acid or high sucrose. A 20 kilodalton polypeptide was also translated by mRNA isolated from cold-hardened B. napus plants.     

Abscisic Aldehyde Is an Intermediate in the Enzymatic Conversion of Xanthoxin to Abscisic Acid in Phaseolus vulgaris L. Leaves

The enzymatic conversion of xanthoxin to abscisic acid by cell-free extracts of Phaseolus vulgaris L. leaves has been found to be a two-step reaction catalyzed by two different enzymes. Xanthoxin was first converted to abscisic aldehyde followed by conversion of the latter to abscisic acid. The enzyme activity catalyzing the synthesis of abscisic aldehyde from xanthoxin (xanthoxin oxidase) was present in cell-free leaf extracts from both wild type and the abscisic acid-deficient molybdopterin cofactor mutant, Az34 (nar2a) of Hordeum vulgare L. However, the enzyme activity catalyzing the synthesis of abscisic acid from abscisic aldehyde (abscisic aldehyde oxidase) was present only in extracts of the wild type and no activity could be detected in either turgid or water stressed leaf extracts of the Az34 mutant. Furthermore, the wilty tomato mutants, sitiens and flacca, which do not accumulate abscisic acid in response to water stress, have been shown to lack abscisic aldehyde oxidase activity. When this enzyme fraction was isolated from leaf extracts of P. vulgaris L. and added to extracts prepared from sitiens and flacca, xanthoxin was converted to abscisic acid. Abscisic aldehyde oxidase has been purified about 145-fold from P. vulgaris L. leaves. It exhibited optimum catalytic activity at pH 7.25 in potassium phosphate buffer.     

The role of abscisic Acid in cross-adaptation of tobacco plants

Tobacco plants (Nicotiana rustica L.) pre-exposed to leaf dehydration, mineral deprivation, salination, or BO₃³⁻ toxicity exhibited increased resistance to subzero temperature and to reduced oxygen in the root medium. The stressed plants all showed an elevated content of leaf abscisic acid. Upon transfer of mineral deprived and salinated plants to prestress conditions, a decline in leaf abscisic acid content to prestress levels took place together with a loss of the increased resistance to subzero temperature and to deprivation of root oxygen. Treatment with abscisic acid by direct application to the leaves or by addition to the root medium improved leaf resistance to subzero temperature and to deprivation of root oxygen. A common hormone-regulation mechanism involving abscisic acid is suggested for this phenomenon of “cross-adaptation” by which a given stress confers increased resistance to other, apparently unrelated stresses.     

Biosynthesis of abscisic acid from [1,2-13C2]acetate in Cercospora rosicola

[1,2-¹³C₂]Sodium acetate was converted to abscisic acid (ABA) by Cerospora rosicola. The labelling pattern, determined by NMR spectroscopy,     

Formation of (-⊃-1′,2′-epi-2-cis-xanthoxin acid from a precursor of abscisic acid

Tomato shoots and avocado mesocarp supplied with (±)-[2-¹⁴C]-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl)-3-methylpenta-cis-2-trans-4-dienoic acid metabolize it into (+)-abscisic acid and a more polar material that was isolated and identified as (?)-epi-1′(R),2′(R)-4′(S)-2-cis-xanthoxin acid. The (+)-1′(S),2′(S)-4′(S)-2-cis-xanthoxin acid recently synthesized from natural violaxanthin, has the 1′,2′-epoxy group on the opposite side of the ring to that of the 4′(S)-hydroxyl group and the compound is rapidly converted into (+)-abscisic acid. The 1′,2′-epoxy group of (?)-1′,2′-epi-2-cis-xanthoxin acid is on the same side of the ring as the 4′(S) hydroxyl group: the compound is not metabolized into abscisic acid. The configuration of the 1′,2′-epoxy group probably controls whether or not the 4′(S) hydroxyl group can be oxidized. (+)-2-cis-Xanthoxin acid is probably not a naturally occurring intermediate because a ‘cold trap’, added to avocado fruit forming [¹⁴C]-labelled abscisic acid from [2-¹⁴C]mevalonate, failed to retain [¹⁴C] label.     

Cytokinin Reversal of Abscisic Acid Inhibition of Growth and α-Amylase Synthesis in Barley Seed

The effect of cytokinin, kinetin, on abscisic acid (dormin) inhibition of α-amylase synthesis and growth in intact barley seed was investigated. Abscisic acid at 5 × 10^?5M nearly completely inhibited growth response and α-amylase synthesis in barley seed. Kinetin reversed to a large extent abscisic acid inhibition of α-aniylase synthesis and coleoptile growth. The response curves of α-amylase synthesis and coleoptile growth in presence of a fixed amount of abscisic acid (6 × l0^?6M) and increasing concentrations of kinetin (from 5 × l0^?7M to 5 × 10^?5 M) showed remarkable similarity. Kinetin and abscisic acid caused synergistic inhibition of root growth. Gibberellic acid was far less effective than kinetin in reversing abscisic acid inhibition of α-amylase synthesis and coleoptile growth. A combination of kinetin and gibberellic acid caused nearly complete reversal of abscisic acid inhibition of α-amylase synthesis but not the abscisic acid inhibition of growth. The results suggest that factors controlling α-amylase synthesis may not have a dominant role in all growth responses of the seed. Kinetin possibly acts by removing the abscisic acid inhibition of enzyme specific sites thereby allowing gibberellic acid to function to produce α-amylase.     

Abnormal Stomatal Behavior and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: V. Effect of Abscisic Acid on Indoleacetic Acid Metabolism and Ethylene Evolution

The wilty tomato mutant flacca, the normal cultivar Lycopersicon esculentum Mill. Rheinlands Ruhm, and abscisic acid-induced phenotypic revertants were compared with respect to ethylene evolution, activity of tryptophan aminotransferase, and [1-¹⁴C]indoleacetic acid decarboxylation.     

Oilbody Proteins in Microspore-Derived Embryos of Brassica napus: Hormonal, Osmotic, and Developmental Regulation of Synthesis

A number of treatments were tested for their ability to affect the synthesis of oilbody proteins in microspore-derived embryos of rapeseed (Brassica napus). Synthesis of the oilbody proteins was determined by [³⁵S]methionine incorporation in vivo and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of washed oilbody fractions. Oilbody proteins of approximately 19, 23, and 32 kilodaltons were found to be prominent. These proteins showed differential patterns of regulation. The 19 and 23 kilodalton proteins (oleosins) were greatly enhanced by treatments with abscisic acid, jasmonic acid, and osmotic stress imposed using sorbitol (12.5%). Synthesis of the 32 kilodalton protein was inhibited by abscisic acid and by sorbitol (12.5%), but unaffected by jasmonates. The strong promotion of synthesis of the 19 and 23 kilodalton oilbody proteins appeared to be specific as they are not seen with gibberellic acid treatment or with a stress such as heat shock. Time course experiments revealed that the abscisic acid stimulation of oleosin synthesis is quite rapid (less than 2 hours), reaching a maximum at 6 to 8 hours. The response of the oleosins to abscisic acid is found in all stages of embryogenesis, with a major increase in synthetic rates even in globular embryos on abscisic acid treatment. This suggests that these proteins may accumulate much earlier in embryogenesis than has previously been believed. The 32 kilodalton oilbody-associated protein appears different from the oleosins in several ways, including its distinct pattern of regulation and its unique property, among the oilbody proteins, of undergoing phosphorylation.