Planta

Planta 146, 591-596 (1979)

9 by Springer-Verlag 1979

Abscisic Acid, Abscisic Acid-Esters and Phaseic Acid in Vegetative Terminal Buds of Acer pseudoplatanus during Emergence from Winter Dormancy I.D.J. Phillips and A. Hofmann Department of BiologicalSciences,Washington-SingerLaboratories,Universityof Exeter, Exeter, Devon EX4 4QG, U.K.

Abstract. Levels of free-abscisic acid and "boundabscisic acid" (alkaline hydrolyzable abscisic acidesters) in replicated samples of terminal vegetative buds of sycamore trees were measured during naturaI emergence from winter dormancy by gas chromatographic methods together with isotope dilution estimation of recovery rates. Not until after the buds had been released from true dormancy in January by winter chilling did any clear change occur in either abscisic acid (ABA) fraction, or in total ABA, on any basis of comparison. The percentage of total ABA present as the free acid declined at the end of true dormancy to approximately two-thirds of its value in the earlier winter months. It is concluded that glucosylation of ABA is unlikely to play a major part in the mechanism of release from dormancy in vegetative sycamore buds. At the end of true dormancy there was a large transient increase in what appeared to be phaseic acid, but this was not accompanied by any marked decrease in either free- or bound-ABA. Key words: Abscisic acid Dormancy (bud).

Acer - Bud dormancy

Introduction

A number of earlier studies indicated that levels of an endogenous growth-inhibitory fraction ("fi-inhibitor") in buds of several species decreased during the course of the winter, to reach a low level by the time innate (true) dormancy was removed by chilling temperatures (see Wareing and Saunders, 1971). On the other hand, emergence of buds from dormancy in some plants was found not to be clearly associated with a decline in endogenous inhibitor (Bachelard Abbreviations: ABA=abscisic acid; TLC=thin layer chromatography; GLC= gas chromatography

and Wightman, 1974; Taylor and Dumbroff, 1975). These data were, in the main, based upon bioassays of crudely fractionated extracts and it is probable that in many or alI instances the measared growth responses in assays represented a net effect of promoting and inhibiting substances in extracts. The isolation and chemical characterisation of abscisic acid (ABA) from the/~-inhibitor fraction (Ohkuma et al., 1965; Cornforth et al., 1965), and the development of gas chromatographic (GLC) techniques for its qualitative and quantitative analysis in plant extracts (see Saunders, 1978), made it possible to follow changes in ABA levels in relation to dormancy of plant tissues. Harrison and Saunders (1975) found fluctuating levels of free-ABA during the winter in buds of Betula pubescens, but they did observe a steady decrease in the ratio, free/free + bound (esterified) ABA. Wright (1975), on the other hand, found a gradual decline in free-ABA during winter in buds of Ribes nigrum and Fagus sylvatica together with a generally constant level of bound-ABA, resulting in a gradual decline in the free/bound ABA ratio during emergence from dormancy. Seeley (1971) found that during winter the level of bound-ABA increased as the level of free-ABA decreased. Such results led to the suggestions that emergence from dormancy is associated with an increased capacity of tissues to esterify free-ABA to a bound form (Harrison and Saunders, 1975), and that bound-ABA accumulates in buds because it cannot be translocated or further metabolised during the winter months (Wright, 1975). Both free and bound-ABA levels of Prunus eerasus flower buds declined between November and late January, but no obvious trend was found in the relative amounts of the free and bound forms (Mielke and Dennis, 1975). In buds of almond trees no significant changes were found in either free or esterified ABA during the winter (Lesham et al., 1974).

0032-0935/79/0146/0591/$01.20

I.D.J. Phillips and A. Hofmann : ABA in Buds of Acer

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The data of Harrison and Saunders (1975) were obtained by use of GLC and isotope dilution recovery estimation, but only a single bud sample was analysed each month, and those of Wright (1975) although based on replicated samples involved analysis of ABA by bioassay and with no method of estimating losses of ABA during extraction and purification. The values for Prunus cerasus flower buds (Mielke and Dennis, 1975) were obtained from unreplicated samples and with no measured recovery rates for extracted ABA. Thus, we have in this study analysed free and bound-ABA in replicated samples of vegetative terminal buds (flower buds were eliminated as these commonly emerge from dormancy earlier than do vegetative buds) by GLC and with an isotopedilution method to estimate losses of ABA. Acer pseudoplatanus was selected as the experimental subject as earlier work on this species revealed a positive correlation between depth of dormancy and/J-inhibitor content of buds (Phillips and Wareing, 1958, 1959).

(.a)

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c~-~BA

s-ABA

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Materials and Methods

R t (h~in)

Fig. I a and h. GLC traces of a methylated fraction from a purified extract of vegetative terminal buds in October. a before, and b after exposure to U.V. light

Plant Material Twigs were collected monthly during 1976 1977 from naturallyoccurring juvenile specimens of Acer pseudoplatanus in Exeter. At each harvest a random sample of 10 buds from each tree were dissected to ensure that flower-buds were absent. Harvested buds were weighed, immediately frozen in liquid NE and stored at - 2 0 ~ C until extracted. Four replicate samples, each of 50 buds, were separately extracted and processed on each occasion.

silica gel GF254 TLC plate that had been activated (100~ for 0.5 h). The plate was developed to a distance of 20 cm three times in toluene:ethyl acetate:acetic acid (40:5:2, by vol.). The zone corresponding to the 2, cis-ABA marker was eluted four times with acetone :methanol (9:1, v/v), reduced to dryness under vacuum at 30 ~ C and subsequently dissolved in an appropriate small volume of ethanol for GLC analysis and scintillation counting.

Collection of Xylem Sap Quantitative Estimation of ABA and ABA-Esters Two batches of 50 twigs were collected monthly and sap obtained from them by the air displacement method (Bollard, 1953). The sap was filtered and stored at - 2 0 ~ C.

Extraction and Purification of ABA and ABA-Esters Methods used were essentially identical to those of Harrison and Saunders (1975) and Saunders (1978). Briefly, buds were macerated and extracted three times in darkness for 18 h at 4 ~ C with 80 per cent alkaline methanol (5 g N a H C O 3 . d m a). The extract was filtered, reduced to aqueous, adjusted to pH 7.0 and passed through a 18. 100 mm column of PVP (Polyclar AT, mesh No. 30-100), then adjusted to pH 2.5 and extracted four times with diethyl ether. The combined ether extract, containing free ABA, was dried over anhydrous sodium sulphate and evaporated to dryness prior to thin-layer chromatography (TLC). The aqueous residue remaining after acidic ether extraction was adjusted to pH 11.0 with KOH, shaken for 1 h at 60 ~ C, then readjusted to p H 2.5 and partitioned with ether to obtain previously " b o u n d '~ ABA. Each dried ether extract was taken up in a small volume of MeOH, strip-loaded alongside a spot of authentic mixed isomers of ABA on a pre-run (ethanol:acetic acid, 98:2, v/v) 0.5 mm

Samples for GLC were methylated with ethereal diazomethane prior to being taken into ethanol. 1 pl aliquots of each extract were injected 3 4 times into the GLC (Pye-Unicam 104 series ECD ; 1 . 5 m . 6 m m internal diameter column of 3% OV17 on 80-100 mesh gas chrom Q; column temperature 250 ~ C; electroncapture detector temperature 300 ~ C; 30 m l . m i n - 1 Nz carrier gas through both column and detector). One methylated aliquot was injected to determine the appropriate quantity of 2, trans-ABA to add as an internal standard to a second aliquot (see below). The identity of presumed methyl-ABA peaks were confirmed by both co-chromatography and U.V.-isomerisation (Fig. 1). Recovery rates were estimated by an isotope dilution method using t4C-labelled ABA (Saunders, 1978). A measured quantity of [a4C]ABA (mixed isomers; specific activity 1.8.108Bq.mmol - 1) was added to the plant extract after maceration (or t o the xylem sap after collection). The absolute amount of [14C]ABA added was determined by GLC and by liquid scintillation counting. The extract was purified as described above, and recovered radioactivity measured on duplicate aliquots. GLC peak areas corresponding to 2, cis-ABA (ABA) and 2, trans-ABA were determined by weighing, and the amount of ABA present calculated by proportion from the added 2, trans-ABA internal standard. In those extracts

I.D.J. Phillips and A. Hofmann : ABA in Buds of Acer that contained large amounts of putative phaseic acid (see Results section) internal standardisation with 2, trans-ABA was not practicable, for methyl phaseic acid effectively co-chromatographed with methyl 2, trans-ABA. In these cases, therefore, amounts of 2, cisABA were calculated by reference to a frequently checked calibration graph for ABA and with multiple injections of each sample. The amount of free-ABA released from esterified ABA by alkaline hydrolysis was calculated after taking into account the presence of any natural free-ABA remaining in the aqueous residue (usually about 15 per cent of total endogenous free-ABA) after initial partitioning with ether at pH 2.5. Recovery of ABA-esters from the hydrolysed extract was measured by adding to it a known amount of [~4C]ABA prior to acidification and ether extraction, followed by scintillation counting of the purified extract as described previously. Mean recovery rates recorded (per cent + standard error) for all extracts were 43.7_+5.69 for free-ABA and 47.1 _+6.25 for ABAesters.

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Results

At the time of each collection of buds from outdoors, a sample of 20 twigs was also brought indoors to follow the progress of emergence from dormancy in the vegetative terminal buds. The lower ends of the twigs were sterilized fn 2% sodium hypochlorite for 0.5 h followed by thorough washing. The twigs were then kept in jars of water at 20~5~ C under 16 h photoperiods. Lateral buds were removed as it was found that some of these often commenced growth indoors even when the apical bud was completely dormant, and it was considered possible that growing lateral buds may exert a correlative growth influence upon the terminal bud (Phillips, 1975). Bud burst was recorded as having occurred when the first leaf of the terminal bud was visible. The terminal buds on the September sample of twigs did not start growth within 4 months indoors. Fifty per cent bud burst had occurred in the October sample after 53 days indoors, but the remaining buds never grew. By December, buds on the twig samples were visibly swollen after having been indoors for 19 days, and after 50 days all the buds had burst. In January, all buds were swollen after 14 days indoors and after 33 days they had burst. Thus, from December onwards the buds could be regarded as having emerged from winter dormancy and entered the stage of post-dormancy (or quiescence) where growth is held in check by environmental conditions. Natural bud break on the trees outdoors occurred by mid-March. During the course of the winter and early spring the dry weight of the terminal buds gradually fell. The fresh weight also declined from September to February, but increased in March as the young leaves of the inner primordial tissues expanded. The fresh weight/dry weight ratio fell by March to about 50% of its value in the previous September.

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Fig. 2. ABA contents of whole vegetative terminal buds between October and March on three bases of comparison (fresh wt., dry wt., per bud). ~ ~ mean + S.E. for free-ABA. ~ =bound-ABA

All free-ABA fractions from buds showed on GLC a clear peak which corresponded to methyl-2, cisABA and a much smaller second peak corresponding to Me-2, trans-ABA. In January, the peak at the retention time (Rt) of Me-2, trans-ABA was much larger than at other times (see below). The "boundABA" fraction never showed a peak at the Rt of Me-2, trans-ABA. The results of GLC analysis of the free- and bound-ABA contents of buds, expressed on fresh weight, dry weight and per bud bases are shown in Fig. 2. The free-ABA content of the buds remained approximately constant from October through to January, and then declined to about a third of the October value by March. The bound-ABA levels closely followed the same pattern. None of the free- or bound-ABA contents between October and January shown in Fig. 2 were significantly different from one harvest date to another. Similarly, total ABA (free+ bound) in the buds showed no obvious change during the winter on any basis of comparison (Fig. 3). The ratio, free-ABA/free-ABA+bound-ABA (i.e. percentage ABA present as the free acid) fell by January to about one-half to two-thirds of its value in October (Fig. 4). As mentioned earlier, all free-ABA fractions obtained from buds in January showed a much larger than usual peak on GLC with a Rt similar to that of Me-2, trans-ABA (Fig. 5A). As it was unlikely that

594

I.D.J. Phillips and A. Hofmann: ABA in Buds of Acer

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cis-PA?

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Fig. 3. Total ABA levels in buds between October and March. = mean _+S.E. gg- g 1drywt. ; 9 = gg- g- 1freshwt. ; x = gg. bud 1

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Fig. 4. Percentage ABA present as free acid between October and March. 9 9 and x = based on fresh wt., dry wt. and per-bud bases of comparison respectively I

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o1 this was attributable to the natural occurrence of large amounts of 2, trans-ABA in the buds (Milborrow, 1970), further extractions of January buds were made taking even greater precautions than usual to prevent exposure of extracts to conditions conducive to isomerisation of ABA. Again the large second peak was obtained, whereas extracts from buds of the other harvest dates showed no or only a very small peak with the Rt of 2, trans-ABA. After the January extracts were isomerised by exposure to U.V. light, instead of two equal peaks corresponding to Me-2, cisABA and Me-2, trans-ABA appearing on G L C traces, a larger peak with the Rt (3.3 rain) of the Me-2, trans-ABA was obtained (Fig. 5 B), indicating that some other substance was co-chromatographing with Me-2, trans-ABA. Furthermore, a third peak appeared after isomerization of the extract, with a slightly longer Rt (3.6 rain) than that of Me-2, transABA (Fig. 5B). It is known that Me-2, trans-ABA and Me-2, cis-phaseic acid show very similar retention times on an OV 17 column (Zeevaart and Milborrow, 1975). A sample of authentic Me-2, cis-PA was therefore injected and found to co-chromatograph with Me-2, trans-ABA. Isomerization of the Me-2, cis-PA with U.V. light (as with the unknown peak it look longer to isomerize than did ABA) resulted in the appearance of a second peak of Rt 3.6 min. Thus,

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Fig. 5A. GLC trace of a methylated fraction from a purified extract of vegetative terminal buds in January. B the same extract after exposure to U.V. light. PA phaseic acid

it seems highly likely that the large peak of Rt 3.3 min in the January extracts was attributable t o phaseic acid. Analysis of xylem sap involved two replicate samples, each obtained from 50 twigs, each month, commencing in September and ending in mid-January. N o collections were made in February and March as the buds were no longer truly dormant at those times, but a final collection of sap was made in midApril when the buds had burst and young leaves were rapidly expanding. Each sample of xylem sap was diluted to 20 cm 3 and partitioned four times at p H 2.5 with an equal volume of ether. The ether extract was dried over anhydrous sodium sulphate and evaporated to dryness at 30 ~ C. All further purification and quantitative methods were as described for bud extracts. The highest level of free-ABA in the sap occurred in the September sample (0.455 ~tg.cm -3) but the level had fallen to 0.04 gg. c m - 3 in October, then remained constant during the winter followed by an increase to 0.152 g g - c m - 3 when active leaf growth was taking place in mid-April (Fig. 6). Confir-

I.D.J. Phillips and A. H o f m a n n : A B A in Buds of Acer

595 Table 1. Changes in the free-ABA/bound-ABA ratio on 3 bases of comparison : gg A B A .g 1 fresh weight; gg ABA- g 1 dry weight and ; gg A B A . b u d - 1

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ABA- g - 1 fresh wt.

ABA. g 1 dry wt.

ABA. b u d -

October November December January M arch

1.45 0.97 1.13 0.66 0.74

1.43 1.00 1.11 0.47 0.52

1.42 1.00 1.13 0.68 0.67

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Fig. 6. Free-ABA in xylem sap between September and March

mation of the identity of ABA in xylem sap was obtained by isomerization with U.V. light which in all cases resulted in the appearance of two equal sized peaks with Rts corresponding to those of 2, cis-ABA and 2, trans-ABA. None of the xylem sap exudates contained any detectable ABA-esters.

Discussion

It is clear from these results that neither total ABA nor either free- or bound:ABA contents of whole vegetative buds of sycamore were obviously correlated with emergence from dormancy (Figs. 2 and 3). There did appear to be a slight fall in both ABA fractions between January and March, but measurements of bud growth revealed that innate (true) dormancy had already ended by January, after which time the buds were in the condition of post-dormancy (quiescence). Thus, the disappearance of dormancy in these buds cannot be ascribed to a reduction in overall levels of ABA. Since ABA contents of whole sycamore buds did not change significantly during their release from dormancy, the previously reported gradual decline in /Jinhibitor content of sycamore buds (Phillips and Wareing, 1958) must be accounted for by either a decrease in other unidentified inhibitors or by an increase in co-chromatographing growth promoters such as gibberellins which would antagonise ABA in bioassays. Although Harrisonand Saunders (1975) found no marked decrease in either free- or bound-ABA levels in buds of Betula pubescens during winter, they did observe a decrease between September and March in the percentage of total ABA present as the free acid. However, their data also show that the percentage fell from approximately 55% in October to approximately 45% in January. Rather similarly, we found that the percentage of ABA present as free acid in sycamore buds declined over the same period from about 58% to 40% (on a fresh weight or per-bud

basis) or 32.5% (on a dry weight basis) (Fig. 4). As it is during the months October-January that natural winter chilling releases buds of sycamore from innate dormancy, the smallness of the measured decline in free-ABA relative to total ABA suggests that glucosylation of free-ABA does not play a major part in the release from dormancy of vegetative buds in Acer pseudoplatanus. Similarly, although Wright (1975) did not express his data as percentages of ABA present as free acid in the samples, examination of his results for buds of Ribes nigrum and Fagus sylvatica reveals that the situation was essentially the same as in Betula pubescens (Harrison and Saunders, 1975) and in Acer pseudoplatanus: in F. sylvatica the value fell from approximately 70% in October to 52% in February just prior to bud expansion, and in R. nigrurn the value fell sharply from approximately 70% in October to 30% in November and then remained constant until bud swelling occurred in January. Wright (1975) compared free and bound-ABA levels on the basis of the free-ABA/bound-ABA ratios at each harvest date, and found in both F. sylvatica and R. nigrurn an annual cycle with the highest ratio in the autumn and lowest at the time of bud burst. Our data for sycamore may be similarly displayed, and when this is done it is seen that there was indeed a gentle downward drift in the free-ABA/bound-ABA ratio between October and January (Table 1), but to a much smaller extent than that recorded for R. nigrurn and f'. sylvatica. However, it must be said that in the absence of any clear changes in level of the biologically active free acid form of ABA, the significance of alterations in the relative levels of free-ABA and ABA-esters remains very unclear. The large and transient increase in what appeared to be phaseic acid in the sycamore buds in January (Fig. 5) is certainly suggestive of degradation of ABA at that time. The absence of any substantial amounts of phaseic acid at earlier harvest dates indicates that active degradation of ABA occurred only at or after the end of the true dormancy, which could explain why there was no decrease in either free or bound

596

ABA between October and January (Figs. 2 and 3). However, phaseic acid accumulation was not accompanied by a noticeable decrease in total ABA in January, nor even by March. The reason for this is not apparent. One possibility is that degradation of ABA in January could be balanced by translocation of ABA into the buds; certainly the xylem sap contained free-ABA throughout the winter (Fig. 6) which was presumably available to the buds. On the other hand, ABA synthesis is generally regarded to occur primarily in mature leaves (Hoad, 1973, 1975, 1978) which makes it rather unlikely that significant amounts of ABA are translocated into the buds of leafless shoots in late winter. Another possible explanation for the maintenance of ABA levels after January is that it is synthesised in the developing buds after their emergence from dormancy to an extent that balances losses by degradation. These results therefore underline the previously stated (Harrison and Saunders, 1975) desirability of studies of ABA metabolism in excised dormant buds. Doubts which have been expressed (Mielke and Dennis, 1978) on the involvement of ABA in the control of bud dormancy would seem justified on the basis of results such as those we have presented, but it remains possible that antagonisms between varying levels of endogenous growth promoters, such as gibberellins, and a constant level of ABA could be important in bud growth. The Dr. Duisberg Gesellschaft, F.D.R., is thanked for financial support of this work. Phaseic acid was a gift from Dr. J. MacMillan, F.R.S., University of Bristol, U.K.

References Bachelard, E.P., Wightman, F. : Biochemical and physiological studies on dormancy release in tree buds. III. Changes in endogenous growth substances and a possible mechanism of dormancy release. Can. J. Bot. 52, 1483-1489 (1974) Bollard, E.G.: The use of tracheal sap in the study of apple tree nutrition. J. Exp. Bot. 4, 363 368 (1953) Cornforth, J.W., Milborrow, B.V,, Ryback, G., Wareing, P.F,: Identity of sycamore 'dormin' with abscisin II. Nature (London) 205, 1269 (1965) Harrison, M.A., Saunders, P.F.: The abscisic acid content of dormant birch buds. Planta 123, 291-298 (1975)

I.D.J. Phillips and A. Hofmann: ABA in Buds of Acer Hoad, G.V. : Effect of moisture stress on abscisic acid levels in Ricinus communis L., with particular reference to phloem exudate. Planta 113, 367-372 (1973) Hoad, G.V. : Effect of osmotic stress on abscisic acid levels in xylem sap of sunflower (Helianthus annuus L.). Planta 124, 25-29 (1975) Hoad, G.V. : Effect of water stress on abscisic acid levels in white lupin (Lupinus albus L.) fruit, leaves and phloem exudate. Planta 142, 287-290 (1978) Lesham, Y., Philosoph, S., Wurzburger, J. : Glucosylation of free trans-abscisic acid as a contributing factor in bud dormancy break. Biochem. Biophys. Res. Commun. 57, 526-531 (1974) Mielke, E.A., Dennis, F.G., Jnr.: Hormonal control of flower bud dormancy in sour cherry (Prunus cerasus L.). II. Levels of abscisic acid and its water soluble complex. J. Am. Soc. Hortic. Sci. 100, 287-290 (1975) Mielke, E.A., Dennis, F.G., Jnr.: Hormonal control of flower bud dormancy in sour cherry (Prunus cerasus L.). III. Effects of leaves, defoliation and temperature on levels of abscisic acid in flower primorida. J. Am. Soc. Hortic. Sci. 103, 446-449 (1978) Milborrow, B.V.: The metabolism of abscisic acid. J. Exp. Bot. 21, 1%29 (1970) Ohkuma, K., Addicott, F.T., Smith, O.E. Thiessen, W.C.: The structure of abscisin iI. Tetrahedron Lett. 29, 2529-2535 (1965) Phillips, I.D.J., Wareing, P.F.: Studies in dormancy of sycamore I. Seasonal changes in growth substance content of the shoot. J. Exp. Bot. 9, 350-364 (1958) Phillips, t.D.J., Wareing, P.F. : Studies in dormancy of sycamore II. The effect of daylength on the natural growth-inhibitor content of the shoot. J. Exp. Bot. 10, 504-514 (1959) Phillips, I.D.J. : Apical dominance. Annu. Rev. Plant Physiol. 26, 341-367 (1975) Saunders, P.F. : The identification and quantitative analysis of abscisic acid in plant extracts. In: Isolation of Plant Growth Substances, pp. 115-134, Hillman, J.R., ed. Cambridge: Cambridge Univ. Press 1978 Seeley, S.D.: Electron capture gas chromatography of plant hormones with special reference to abscisic acid in apple bud dormancy. P h . D . thesis, Cornell Univ., Ithaca, N.Y. 1971 Taylor, J.S., Dumbroff, E.B.: Bud, root, and growth regulator activity in Acer saccharum during the dormant season. Can. J. Bot. 53, 321-331 (1975) Wareing, P.F., Sauders, P.F.: Hormones and dormancy. Annu. Rev. Plant Physiol. 22, 261 288 (1971) Wright, S.T.C. : Seasonal changes in the levels of free and bound abscisic acid in blackcurrant (Ribes nigrum) buds and beech (Fagus syIvatica) buds. J. Exp. Bot. 26, 161 174 (I975) Zeevaart, J.A.D., Milborrow, B.V.: Metabolism of (_+) abscisic acid and the occurrence of epi-dihydro-phaseic acid in Phaseolus vulgaris. Plant Research '74, Rep. of Michigan State Univ./AEC Plant Res. Lab~, pp. 50-53 (1975)

Received 5 April; accepted 4 June 1979

Abscisic acid, abscisic acid-esters and phaseic acid in vegetative terminal buds of Acer pseudoplatanus during emergence from winter dormancy.

Levels of free-abscisic acid and "boundabscisic acid" (alkaline hydrolyzable abscisic acidesters) in replicated samples of terminal vegetative buds of...
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