The Resolution by HPLC of RS-[2-14C]Me 1', 4'-cis-Diol of Abscisic acid and the Metabolism of (-)-R- and (+)-S-Abscisic Acid

The R- and S-enantiomers of racemic [2-¹⁴C]Me 1', 4'-cis-diolof abscisic acid have been separated by high performance liquidchromatography on an optically-active Pirkle column. R-[2-¹⁴C]-and S-[2-¹⁴C]abscisic acids, formed from the Me 1', 4'-cis-diolby oxidation and alkyline hydrolysis were fed to tomato shootsand the extracts analysed by reversed phase high performanceliquid chromatography. R-[2-¹⁴C]abscisic acid formed mainlythe abscisic acid glucose ester (ABAGE), abscisic acid l'-glucoside(ABAGS) and an uncharacterized conjugate. Dihydrophaseic acid4'-B-D-glucoside, the major metabolite of RS-abscisic acid intomato shoots, was found to be derived virtually exclusivelyfrom the natural, S-abscisic acid. Phaseic acid and conjugatesof abscisic acid were also found as products of the naturallyoccurring enantiomer. The resolution method was used to measurethe relative proportions of R and S enantiomers in the freeacid liberated from conjugates formed from RS-[2-¹⁴C]ABA fedto shoots. The ratios show an excess of the R-enantiomer: 5.8:1, ABAGE; 29.4: 1, ABAGE; 8.3: 1 for an uncharacterized conjugateand 6.1: 1 for the residual free [2-¹⁴C]ABA. Key words: ABA, HPLC, Tomato     

2,6-Dichlorobenzonitrile and Boron Deficiency

Symptoms of 2,6-dichlorobenzonitrile poisoning in plants aredescribed and compared with the symptoms produced by phenylboronicacid and boron deficiency. The effects on the macroscopic andmicroscopic appearance of plants and on their ability to translocategrowth regulators are so alike that it is thought that both2,6-dichlorobenzonitrile and boron deficiency affect the samebasic process.     

Physiological Properties of Abscission Accelerator from Senescent Leaves

A substance which is not abscisic acid accelerates leaf abscission by regulating ethylene production.     

The Reduction of ({+/-})-(2-14C) Abscisic Acid to the 1', 4'-Trans-Diol by Pea Seedlings and the Formation of 4'-Desoxy ABA as an Artefact

(±)(2-¹⁴C]ABA is metabolized by pea seedlings to phaseicand dihydrophaseic acids and conjugates and it is also reducedto the 1', 4'-trans-diol of ABA. This compound runs at the sameR_F as the putative metabolite identified as 4'-desoxy ABA byTietz et al (1979). The 1', m4'-/trans-diol is dehydrated duringgas/liquid chromatography to 4'-desoxy ABA. No labelled zoneon the autoradiogram of the plant extract could be attributedto 4'-desoxy ABA so it is presumably an artefact formed by thedehydration of the 1', 4'-trans-diol of ABA. This probably occursin the injection port of the G. L.C. Key words: Plsum sativum, A. B.A., Metabolism, Phaseic/dihydrophaseic acids     

The Specificity of the Carrier-Mediated Uptake of ABA by Root Segments of Phaseolus coccineus L.

Earlier work has established that the saturable component ofuptake of RS-[2¹⁴C]ABA by bean (Phaseolus coccineus L. cv. Prizewinner)root segments can be attributed to the action of a carrier.We now show that the carrier-mediated uptake is unaffected byRS-2-trans-ABA and lunularic acid and the unnatural R-ABA alsoappears to be ineffective. The specificity for S-ABA requiresthe halving of the K_m value for ABA determined previously (2.6mmol m_-3 for RS-; 1.3 mmol m_-3 for S-ABA). The RS-1', 4'-cis-dioland RS-1'-deoxy ABA reduce the uptake of RS-[2-₁₄C]ABA aboutas strongly as does unlabelled ABA, the K¹ for 1'-deoxy ABAwas similar to the K_m for ABA. The K₁ for RS-1', 4'-trans-diolwas 15.7 mmol m_-3. Consideration of the stereochemistry of thesecompounds suggests that the face of the ring of ABA away fromthe 1'-hydroxyl group interacts with the carrier site. Labelled material diffused out of undamaged root surfaces whichhad absorbed RS-[³H]ABA through an apical cut, suggesting thatABA is present in the apoplast. A simplified hypothesis is presented that can account for polartransport of ABA based on a gradient of a carrier in a tissuebut where the carrier is distributed uniformly on the apicaland basal ends of each cell. Key words: Uptake carrier, Abscisic acid, 1', 4'-Diol, Lunularic acid, Phaseolus coccineus, Polar-transport, Deoxyabscisic acid     

The Stability of Conjugated Abscisic Acid during Wilting

Tomato and silverbeet shoots that had been fed (±)-[2-¹⁴C]abscisicacid (ABA) some days previously did not hydrolyse their alkali-saponifiable[¹⁴C]ABA fraction when they wilted. The content of alkali-saponifiableABA in untreated plants did not fall on wilting so the endogenousconjugate is not hydrolysed either. In tomato and silverbeet,therefore, conjugation of ABA is irreversible.     

The Long Term Metabolism of ({+/-})-(2-14C)Abscisic Acid by Apple Seeds

Apple seeds soaked in a solution of [±]-[2-¹⁴C]abscisicacid for up to 70 d formed phaseate and the epimeric dihydrophaseatesand all four acids were converted into alkali-hydrolysable conjugates.This metabolism occurred in the husks and in the embryos. Bothparts of untreated control seeds contained the four acids andtheir conjugates. In addition to these characterized metabolites,a number of other, labelled, acidic products were isolated fromthe seeds. ¹⁴CO₂ was evolved from unsterilized seeds but notfrom surface-sterilized ones. Bacterial cultures isolated fromsoil and rotting fruit metabolized [¹⁴C]ABA to a range of compounds,but not to phaseic or the dihydro-phaseic acids. A new, acidicconjugate of ABA is described.     

The Metabolism of Abscisic Acid

The light-catalysed isomerization of (+)-abscisic acid (ABA)to its trans isomer during isolation from leaves was monitoredby the addition of (±)-[2-¹⁴C]ABA to the extraction medium.(+)Trans-abscisic acid (t-ABA) was found to occur naturallyin rose (Rosa arvensis) leaves at 20µg/kg fresh weight;(+)-ABA was present at 594µg/kg. (±)-[2-¹⁴D]Trans-abscisicacid was not isomerized enzymically to ABA in tomato shoots. (±)-Abscisic acid was converted by tomato shoots to awater-soluble neutral product, ‘Metabolite B’, whichwas identified as abscisyl-ß-D-glucopyranoside. When(±)-[2-¹⁴C]trans-abscisic acid in an equimolar mixturewith (±)-[2-¹⁴C}ABA was fed to tomato shoots it was convertedto its glucose ester 10 times faster than was ABA. Trans-abscisyl-ß-D-glucopyrano8ide only was formedfrom (±)-[2-¹⁴C]t-ABA in experiments lasting up to 30h. Glucosyl abscisate was formed slowly from ABA and the freeacid fraction contained an excess of the unnatural (–).ABAas did the ABA released from abscisyl-ß-D-glucopyranosideby alkaline hydrolysis. The (+).ABA appeared to be the solesource of the acidic ‘Metabolite C" previously noted. The concentrations of endogenous (+)-, (+)-[2-¹⁴C]-, and (–)-[2-¹⁴C]ABAremaining as free acid, and also in the hydrolysate of abscisyl-ß-D-glucopyranoside,were measured by the ORD, UV absorption, and scintillation spectrometryof highly purified extracts of ABA from tomato shoots whichhad been supplied with racemic [2-^l4C]ABA.     

Factors Affecting the Biosynthesis of Abscisic Acid

Incorporation of labelled mevalonate into abscisic acid (ABA)has been demonstrated in the cotyledons of mature avocado seeds,embryos and endosperms of developing wheat seeds, and avocadostems. The increase in ABA concentration on wilting parallelsthe increased incorporation of [2^–14C)mevalonate intoABA in avocado leaves and stems, suggesting that the increasein ABA content occurs by synthesis rather than by release froma stored precursor. Incorporation of [2^–14C]mevalonateby avocado mesocarp segments is unaffected by an 18 per centwater loss. The ABA content of roots was hardly affected bya 30 per cent water loss, indicating that the wilt-activatedmechanism is not fully operative in these tissues. Submerged Ceratophyllum plants and submerged parts of Callitricheshoots show a twofold increase in ABA content on wilting whereasthe aerial rosettes of the latter plant show a sixfold increase.This suggests that the occurrence of the wilt-induced mechanismis affected by previous growth conditions as well as by themorphology of the tissue.     

Stored xylem sap from wheat and barley in drying soil contains a transpiration inhibitor with a large molecular size

Xylem sap was collected from wheat and barley growing in a drying soil, and the effect of the sap on transpiration was detected by a bioassay with detached wheat leaves. The inhibitory activity of fresh sap was small, and could be largely accounted for by the abscisic acid content (about 2×10^-5mol m^-3). When fresh sap was stored at -20°C for several days, the activity increased. Maximum activity developed after a week. This increase in activity was due to a compound that increased in size with storage at -20°C. When fresh sap was fractionated with filters of different molecular size exclusion characteristics, and the separated fractions stored at -20°C for a week, activity developed only in the fraction containing compounds smaller than 0·3 kDa. However, when sap already stored at -20°C was fractionated, activity was only in fractions containing compounds larger than 0·3 kDa. The increase in activity and in size did not occur with storage in liquid nitrogen (-196°C) or at -80°C. These results suggest that storage at -20°C causes the aggregation or polymerization of a small compound with low activity to form a large compound with high activity. This change is not catalysed by an enzyme because it can occur in a fraction from which molecules larger than 0·3 kDa are removed. It is probably promoted by high solute concentrations when ice crystals form. Sap collected from plants in soils of high water potential had little or no activity after storage at -20°C.