Planta
Planta 149, 118-122 (1980)
9 by Springer-Verlag 1980
Effects of Light and Photoperiodie Conditions on Abseisic Acid in Leaves and Roots of Acer pseudoplatanus L. I.D.J. Phillips, J. Miners, and J.G. Roddick Department of Biological Sciences, Washington-Singer Laboratories, University of Exeter, Exeter EX4 4QG, U.K.
Abstract. Roots of Acer pseudopIatanus seedlings grown in liquid nutrient medium contained much lower levels of both free and bound abscisic acid than did leaves. The levels of free abscisic acid were similar in young expanding and of mature leaves, but lower in older senescing leaves. Growing plants under long days or short days did not influence the levels of free and bound abscisic acid in leaves. However, under both long days and short days, levels of bound abscisic acid were lower at the end of the dark period than 8 h later during the light period. Phaseic acid was also detected during the light period but never at the end of the dark period.
Key words: Abscisic acid Acer - Leaves - Light and ABA - Roots - Photoperiodism.
Introduction
To date, studies of abscisic acid (ABA) levels in relation to bud dormancy have yielded rather inconclusive and conflicting results. Some workers found that removal of bud dormancy was correlated with a decrease in total free ABA (Corgan and Martin 1971; Wright 1975; Mielke and Dennis 1975) whereas others reported no significant change in free ABA (Leshem etal. 1974; Harrison and Saunders 1975; Phillips and Hofmann 1979). Some evidence has been obtained indicating that the level of esterified ABA increases during breaking of bud dormancy (Harrison and Saunders 1975; Wright 1975) but no such change was found in buds of Acer pseudoplatanus (Phillips and Hofmann 1979). Another approach to the problem has been to Abbreviations: ABA = abscisic acid; PA = phaseic acid; SD = short day; GLC = gas-liquid chromatography; LD = long day
0032-0935/80/0149/0118/$01.00
examine the effects of photoperiodic conditions on the onset of shoot dormancy and associated changes in endogenous growth substances. Bioassay of leaf extracts from several woody species indicated that leaves contain higher levels of 'fi-inhibitor' under short days (SD) than under long days (LD) (see Wareing and Saunders 1971). On the other hand, it has been reported recently that photoperiodic conditions have no effect on levels of fl-inhibitor in Salix viminalis (Alvim et al. 1979). Lenton et al. (1971), using GLC methods, found that ABA levels in leaves and shoot apices of several tree species tended to be higher under LD than under SD and Alvim et al. (1979), using similar methods, found no effect of photoperiodic treatments on ABA levels in Salix viminalis. Possible explanations for the discrepancies in observations have been discussed by Wareing and Saunders (1971), and particularly that; a) SD may reduce the level of growth promoters (e.g. gibberellins) that antagonise ABA in bioassays, b) SD enhance transport of ABA from leaves to the shoot apex, and c) that lack of control of water stress in experimental plants could lead to variations in ABA levels large enough to mask any influence of photoperiodic conditions on levels of this hormone. This last possibility seems particularly important in view of well-substantiated instances of even slight water stress inducing large increases in endogenous ABA (see Milborrow 1974). A number of workers (see Torrey 1976) have described effects of photoperiodic conditions around the shoot on root development and it seems likely that some of these effects involve endogenous growth substances such as ABA. However, roots of many temperate-zone trees such as A. pseudoplatanus and Acer saccharum show two periods of maximum growth activity each year, one occurring in spring some weeks prior to the onset of renewed shoot development and the second in late autumn/early winter (Romberger 1963 ; Taylor and Dumbroff 1975 ; Dum-
I.D.J. Phillips et al. : Abscisic Acid in Acer
broff and Brown 1976), suggesting that root growth in such trees is, to a certain extent, independent of dormancy in the shoot. It is therefore uncertain to what extent annual cycles of root development in trees are determined by water stress and/or photoperiodic conditions around the shoot. To help clarify the situation regarding possible effects of photoperiodic conditions on endogenous ABA, our studies have been conducted on seedlings of A. pseudoplatanus grown in aerated liquid nutrient medium in order to reduce the variations in water stress that usually occur in soil-grown plants. Measurements have been made of free and bound ABA in roots and leaves of various ages under either SD or LD and at various times in the daily light/dark cycle. Phaseic acid (PA) was also measured in leaf extracts.
Material and Methods Plant Material Seeds of A. pseudoplatanus were collected in the Exeter area and stratified in the dark at 8 ~ in damp vermiculite for approx. 4 months by which time they had germinated. Seedlings were transferred, still in vermiculite, to warmer LD conditions (25 ~ C, 18 h photoperiods). When the cotyledons had expanded the roots were surface-sterilized for 5 rain in 2% (w/v) sodium hypochlorite followed by thorough washing in sterile distilled water prior to transferring the seedlings to aerated mineral nutrient solution (Johnson et al. 1957 modified by Epstein 1972) where they were grown for a further 7 weeks under LD (460 pmol photons I l l - 2 S- 1 fi'om a mixed fluorescent/incandescent source for 18/24 h) at 20 ~ C in the light periods and 15~ during the 6 h dark periods. At the end of this time, plants were divided between SD and LD regimes. Plants exposed to SD received 8 h irradiance (as above) per 24 h while those under LD received an additional 10 h of low intensity illumination (100 Ixmol photons m -2 s 1) from incandescent lamps. After various numbers of SD or LD cycles, plants were harvested for extraction.
i19 taken up in a small volume of ethanol for GLC and scintillation counting. After methylation, 1 ~tl aliquots were injected into the GLC (1.5 m x 6 mm column containing 3% OV-17 on 80-100 mesh Gas Chrom Q; column temp. 250 ~ C, ECD temp. 300 ~ C; N2 carrier at 30 cm s min- 1). ABA was quantified by reference to a calibration graph for authentic 2-cis-ABA with 2-trans-ABA as internal standard. Losses during extraction were estimated from the recovery of [14C]ABA (Harrison and Saunders 1975). The identity of the ABA peak was confirmed by both co-chromatography and u.v.-isomerization. All extraction and purification procedures were conducted in darkness or dim light.
Statistical Analysis Each treatment was replicated at least three times and results analysed by t-test and analysis of variance.
Results In the first experiment, leaves of all ages and roots were harvested at the end of the third 6 h (LD) or 16 h (SD) dark period. As shown in Fig. 1 there was no effect of day length on free ABA contents of either leaves or roots, but leaves contained approximately thirty times more ABA per unit weight of tissue than did roots. Because in A. pseudoplatanus photoperiodic perception occurs in the mature, rather than young, growing leaves (Wareing 1956), a second experiment examined free ABA in leaves of different ages. After three photoperiodic cycles, leaves were divided into three groups: 'young' leaves, consisting of small expanding leaves from near the apex; 'mature' leaves which were fully expanded but showed no signs of senescence, and; 'old' leaves being the four lowermost leaves which were yellowing. There was no effect of photoperiodic conditions on levels of free ABA
100
500
Extraction, Purification and Assay Procedures
Leaves 1
Methods have been detailed previously (Phillips and Hofmann 1979) and were essentially the same as those of Harrison and Saunders (1975). Macerated tissues were extracted three times with 80% (v/v) alkaline methanol and a measured quantity of (_+)-cis, trans-[2-14C]ABA (specific activity i73.9 MBq retool-t) added to the extract. The extract was filtered, reduced to aqueous residue, adjusted to pH 7.0, passed through a polyvinylpyrrolidone column, adjusted to pH 3.0, partitioned four times with diethyl ether and the organic phase (containing free ABA) dried over anhydrous sodium sulphate and evaporated to dryness. The aqueous phase was adjusted to p H 10.0 and agitated for 1 h at 60 ~ C to hydrolyse ABA esters, re-adjusted to pH 3.0 and partitioned with diethyl ether, dried and evaporated as before. Each dried ether extract was taken up in a small volume of methanol and subjected to TLC (plates coated with silica gel GF254 and pre-run in ethanol: acetic acid, 98:2, v/v and activated at 100 ~ C for 0.5 h), developing to 200 mm three times in toluene : ethyl acetate: acetic acid (40 : 5 : 2, v/v). The zone corresponding to a 2, cis-ABA marker spot was eluted with acetone:methanol (9:1, v/v), reduced to dryness and
8O
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300
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t~ 40
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Roots
LD
SO
LD
SD
Fig. 1. Effect of photoperiodic conditions on levels of free ABA in leaves (of all stages of development) and roots of 7-week old A. pseudoplatanus. All extracts were made at the end of the third experimental dark period (6 h under LD and 16 h under SD). Values are means from four replicate extractions
I.D.J. Phillips et al. : Abscisic Acid in Acer
120
150
'Young' 'Mature'
.t-,
~100
m
3" '01d' r
LD SD
LD SD
LD SD
Fig. 2. Effect of photoperiodic conditions on levels of free A B A in leaves of different ages from 7-week old A. pseudoplatanus. All extractions were conducted at the end of the third experimental dark period (6 h under LD and 16 h under SD). ' Y o u n g ' leaves were small, not fully expanded; ' m a t u r e ' leaves were fully expanded with no visible senescence; ' o l d ' leaves comprised the four lowermost yellowing leaves. Values are m e a n s from three, four, or six replicate extractions
in leaves of the different ages but senescent leaves contained significantly less ABA than mature or young leaves (Fig. 2). In the next experiment, mature leaves and root systems were harvested separately, both at the end of the seventh experimental dark period and after 8 h of light following the seventh dark period. Both free and bound ABA were measured. In the roots, levels of free and bound ABA were again much lower than in leaves but no differences were found in either
free ABA or bound ABA under L D or SD, or between extracts made at the end of the dark period and after 8 h light (Table 1). Similarly, in the mature leaves no significant differences were observed in levels of free ABA in the four treatments (Table 2). However, in both the L D and SD treatments levels of bound ABA were much higher 8 h into the light period than at the end of the preceding dark period (Table 2). Extraction of leaves at the end of either a long or a short dark period yielded no G L C peak corresponding to 2, trans-ABA. However, all leaf extracts (under LD and SD, free and bound fractions) made after 8 h light showed a large peak with what initially appeared to be 2, trans-ABA. Closer examination revealed that the new peak had a retention time (Rt) of 3.4 min whereas 2, trans-ABA had a Rt of 3.3 min (2, cis-ABA had a Rt of 2.8 min). Nevertheless, this interfering peak meant that 2, trans-ABA could not be used as an internal standard, and quantification had to be carried out solely by reference to a calibration graph for 2, cis-ABA. In all cases it was found that exposing extracts that contained the peak of Rt 3.4 min to u.v.-light resulted in; a) decreases in the peaks at 2.8 and 3.4 min, and b) the appearance of peaks at 3.3 min (2, trans-ABA) and 3.6 min (2, transPA?). It has been reported that 2, trans-ABA and 2, cis-PA have very similar retention times on an OV-17 column (Zeevaart and Milborrow 1975). Authentic 2, cis-PA was found to have a Rt of 3.4 min. Thus, it was possible that the extract peak of Rt 3.4 min was 2, cis-PA. Exposure of authentic 2, cisPA to UV light resulted in peaks of equal areas with Rts of 3.4 and 3.6 rain, further suggesting that the unknown peak present in extracts from illuminated leaves was 2, cis-PA. This was confirmed by observations that both the unknown peak and authentic PA took longer to attain equilibrium during isomeriza-
Table 1. Levels of free and b o u n d A B A in roots of A. pseudoplatanus grown under LD (18 h photoperiods) and SD (8 h photoperiods). Approx. 8-week old plants were harvested at the end of the seventh dark period and after 8 h light following the seventh dark period. Values represent the means of four replicates •
Compound
Treatment
ng g 1 F W
ng g - 1 D W
F W basis
D W basis
396.8• 263.0 • 26.9 328.0_+3.1 342.0 • 9.1
-
-
11.3 14.2 17.5 9.5
16.5 17.7 16.8 I2.7
13.1•
15.9•
Free A B A
LD, LD, SD, SD,
end of dark after light end of dark after light
26.0_+2.2 20.0 _+ 1.2 20.3• 19.0 • 1.4
Bound A B A
LD, LD, SD, SD,
end of dark after light end of dark after light
3.3• 3.3 _+0.3 4.3 +_ 1.5 2.0 • 0.9
Mean
Bound A B A as % of total
ABA
78.3• 56.7 • 66.3 • 49.7 •
17.9 21.0 28.5 24.3
I.D.J. Phillips et al. : Abscisic Acid in Acer
121
Table 2. Levels of free and b o u n d A B A in mature leaves ofA. pseudoplatanus grown under LD (18 h photoperiods) and SD (8 h photoperiods). Approx. 8-week old plants were harvested at the end of the seventh dark period and after 8 h light following the seventh dark period. Values for free A B A are means of four replicates _+S.E., and values for b o u n d A B A means of three replicates _+S.E,
Compound
Free A B A
Bound A B A
Treatment
LD, LD, SD, SD,
end of dark after light end of dark after light
LD, LD, SD, SD,
end of dark after light end of dark after light
ABA
Bound A B A as % of total
ng g - 1 F W
ng g - * D W
153.8_+20.9 138.3 _+26.1 148.8_+ 6.1 171.8_+16.7
925.5_+120.9 728.0 _+ 167.8 849.8_+ 37.5 838.8_+ 81.5
5.0_+ 35.3-+ 16.4-t37.3_+
2.6 4.0 b 0.3 2.2 a
28.0_+ 14.8 181.0_+ 18.1 u 91.3_+ 3.3 182.3_+ 11.0 a
Mean " b
F W basis
D W basis
-
-
3.1 20.3 9.9 17.8
2.9 19.9 9.7 17.9
12.8_+3.4
12.6_+3.4
Significantly different from dark control at 5% level At 1% level
Table 3. Relative a m o u n t s of putative PA in mature leaves of A.
pseudoplatanus exposed to light for 8 h after the end of either the seventh 16 h (SD) or seventh 6 h (LD) dark period. No PA was detected in any of the fourteen extracts at the end of the dark period Treatment
Relative a m o u n t of P A (units g - 1 FW) Free
Bound
LD, end of dark LD, after light extract 1 extract 2 extract 3
ND
ND
6.1 4.6 3.1
2.7 3.0 3.7
Mean _+S.E.
4.6+0.7
3.1 _+0.2
SD, end of dark SD, after light extract 1 extract 2 extract 3 extract 4
ND
ND
2.3 3.2 4.4 5.3
0.3 0.8 1.3 2.0
Mean _+ S.E.
3.8_+0.6
1.1+_0.3 a
N D = not detectable Significantly different from LD control at 5% level
tion by UV light than did ABA, and that authentic 2, cis-PA co-chromatographed with the unknown peak. Estimation of the relative amount of PA in each extract was made by correcting peak (Rt 3.4 min) area for dilution factors and applying the same recovery rate value as determined for ABA. In none of the extracts made at the end of the dark period was
any PA detectable, whereas it was present in all extracts made after 8 h light (Table 3). The level of free PA was similar in L D and SD leaves but bound PA was more abundant under LD. Under LD, amounts of free and bound PA were not significantly different, but SD leaves contained less bound PA than free PA (Table 3).
Discussion
The use of liquid nutrient medium in these experiments largely removed the possibility of water-stressinduced variations in endogenous ABA levels, thus permitting a more critical examination of ABA levels in relation to photoperiodic conditions. Unpublished data showed that leaf water saturation deficits (%) per plant ranged from 1.79 to 11.68 (mean 7.06 _+0.97 S.E.) in soil, but only from 6.34 to 7.00 (mean 6.65_+ 0.08 S.E.) in plants in liquid nutrient medium. In all experiments, levels of ABA on the roots were considerably lower than in the leaves. Since the xylem sap of woody plants, including A. pseudoplatanus (Phillips and Hofmann 1979), contains considerable amounts of ABA, and mature leaves appear to be the principal sources of this ABA (Hoad 1973, 1975, 1978), it seems reasonable to assume that the direction of ABA transport in the stem is primarily upwards and that the root system is normally exposed to very much lower levels of shoot-synthesised ABA than are shoot tissues. The levels of free ABA found in the roots (approx. 13-26 ng kg-1 fresh weight; Fig. 1 and Table 1) are almost identical with those recorded for A. saccharum roots (Cohen et al. 1978).
122
However, whereas these workers found no alkaline hydrolyzable ABA in roots of A. saccharum, we did detect small amounts of bound ABA in roots of A. pseudoplatanus (Table 1). In fact, the proportion of total ABA present in the bound form was very similar in roots and leaves (Tables 1 and 2). Our results show that there was no effect of the length of the dark period upon levels of free or bound ABA in leaves or in roots (Figs. 1 and 2, Tables 1 and 2). However, light (mixed white fluorescent and tungsten lamps) did enhance glucosylation of ABA (Table 2) and induce the appearance of both free and bound PA (Table 3). It has been reported recently (Loveys 1979) that a far-red-enriched light source, similar to that employed by us, greatly enhanced the amount of [14C]PA detectable in extracts of [l~C]ABA-treated tomato plants. Conversion of exogenous [14C]ABA to [lgC]PA in tomato extracts was complete within 8 h under far-red-enriched light (Loveys 1979). In our experiments, PA was present in extracts of A. pseudoplatanus made after 8 h light whereas it was not detectable at the end of the preceding dark period (Table 3). In a previous paper (Phillips and Hofmann 1979) it was noted that emergence of vegetative buds of A. pseudoplatanus from winter dormancy was found to be associated with the appearance of large amounts of PA but not with a reduction in ABA level. All these observations indicate that photoperiodic induction of dormancy in A. pseudoplatanus involves more than a simple effect of daylength on levels of extractable free ABA and underline the need for more detailed studies of ABA metabolism and compartmentation.
References Alvim, R., Saunders, P.F., Barros, R.S.: Abscisic acid and the photoperiodic induction of dormancy in Salix viminalis L. Plant Physiol. 63, 774-777 (1979) Cohen, D.B., Dumbroff, E.B., Webb, D.P. : Seasonal patterns of abscisic acid in roots of Acer saccharum. Plant Sci. Lett. 11, 35-39 (1978) Corgan, J.N., Martin, G.C.: Abscisic acid levels in peach floral cups. Hortic. Sci. 6, 405406 (1971) Dumbroff, E.B., Brown, D.C.U. : Cytokinins and inhibitor activity in roots and stem of sugar maple seedlings through the dormant season. Can. J. Bot. 54, 191-197 (1976)
I.D.J. Phillips et al. : Abscisic Acid in Acer Epstein, E.: Mineral nutrition of plants. Principles and perspectives. New York: Wiley 1972 Harrison, M.A., Saunders, P.F. : The abscisic acid content of dormant birch buds. Planta 123, 291-298 (1975) Hoad, G.C. : 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.C. : Effect of osmotic stress on abscisic acid levels in xylem sap of sunflower (Helianthus annuus L.). Planta 124, 25-29 (1975) Hoad, G.C. : Effect of water stress on abscisic acid levels in whitelupin (Lupinus alba L.) fruit, leaves and phloem exudate. Planta 142, 287-290 (1978) Johnson, C.M., Stout, P.R., Broyer, T.C., Carlton, A.B. : Comparative chlorine requirements of different plant species. Plant Soil 8, 337-353 (1957) Lenton, J.R., Perry, V.M., Saunders, P.F. : The identification and qualitative analysis of abscisic acid in plant extracts by gasliquid chromatography. Planta 96, 271480 (1971) Leshem, Y., Philosoph, S., Wurzburger, J.: Glycosylation of free trans-abscisic acid as a contributing factor in bud dormancy break. Biochem. Biophys. Res. Commun. 57, 526 531 (1974) Loveys, B.R. : The influence of light quality on levels of abscisic acid in tomato plants, and evidence for a novel abscisic acid metabotite. Physiol. Plant. 44, 79-84 (1979) Mielke, E.A., Dennis, F.G., Jr.: Hormonal control of flower bud dormancy in sour cherry. II. Levels of abscisic acid and its water-soluble complex. J. Am. Soc. Hort. Sci. 100, 287-290 (1975) Milborrow, B.V.: The chemistry and physiology of abscisic acid. Annu. Rev. Plant Physiol. 25, 259-307 (1974) Phillips, I.D.J., Hofmann, A.: Abscisic acid (ABA), ABA esters and phaseic acid in vegetative terminal buds of Acer pseudoplatanus during emergence from winter dormancy. Planta 146, 591-596 (1979) Romberger, J.A. : Meristems, growth, and development in woody plants. USDA, Forest Service, Tech. Bull. 1293 (1963) 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) Torrey, J.G. : Root hormones and plant growth. Annu. Rev. Plant Physiol. 27, 435-459 (1976) Wareing, P.F. : Photoperiodism in woody plants. Annu. Rev. Plant Physiol. 7, 191-214 (1956) Wareing, P.F., Saunders, 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 sylvatica) buds. J. Exp. Bot. 26, 161-174 (1975) Zeevaart, J.A.D., Milborrow, B.V.: Metabolism of (_+) abscisic acid and the occurrence of epi-dihydrophaseic acid in Phaseolus vulgar#. Plant Research '74. Report of Michigan State University/AEC Plant Research Laboratory, pp. 50-53 (1975)
Received 12 August; accepted 8 October 1979
Planta 149, 118-122 (1980)
9 by Springer-Verlag 1980
Effects of Light and Photoperiodie Conditions on Abseisic Acid in Leaves and Roots of Acer pseudoplatanus L. I.D.J. Phillips, J. Miners, and J.G. Roddick Department of Biological Sciences, Washington-Singer Laboratories, University of Exeter, Exeter EX4 4QG, U.K.
Abstract. Roots of Acer pseudopIatanus seedlings grown in liquid nutrient medium contained much lower levels of both free and bound abscisic acid than did leaves. The levels of free abscisic acid were similar in young expanding and of mature leaves, but lower in older senescing leaves. Growing plants under long days or short days did not influence the levels of free and bound abscisic acid in leaves. However, under both long days and short days, levels of bound abscisic acid were lower at the end of the dark period than 8 h later during the light period. Phaseic acid was also detected during the light period but never at the end of the dark period.
Key words: Abscisic acid Acer - Leaves - Light and ABA - Roots - Photoperiodism.
Introduction
To date, studies of abscisic acid (ABA) levels in relation to bud dormancy have yielded rather inconclusive and conflicting results. Some workers found that removal of bud dormancy was correlated with a decrease in total free ABA (Corgan and Martin 1971; Wright 1975; Mielke and Dennis 1975) whereas others reported no significant change in free ABA (Leshem etal. 1974; Harrison and Saunders 1975; Phillips and Hofmann 1979). Some evidence has been obtained indicating that the level of esterified ABA increases during breaking of bud dormancy (Harrison and Saunders 1975; Wright 1975) but no such change was found in buds of Acer pseudoplatanus (Phillips and Hofmann 1979). Another approach to the problem has been to Abbreviations: ABA = abscisic acid; PA = phaseic acid; SD = short day; GLC = gas-liquid chromatography; LD = long day
0032-0935/80/0149/0118/$01.00
examine the effects of photoperiodic conditions on the onset of shoot dormancy and associated changes in endogenous growth substances. Bioassay of leaf extracts from several woody species indicated that leaves contain higher levels of 'fi-inhibitor' under short days (SD) than under long days (LD) (see Wareing and Saunders 1971). On the other hand, it has been reported recently that photoperiodic conditions have no effect on levels of fl-inhibitor in Salix viminalis (Alvim et al. 1979). Lenton et al. (1971), using GLC methods, found that ABA levels in leaves and shoot apices of several tree species tended to be higher under LD than under SD and Alvim et al. (1979), using similar methods, found no effect of photoperiodic treatments on ABA levels in Salix viminalis. Possible explanations for the discrepancies in observations have been discussed by Wareing and Saunders (1971), and particularly that; a) SD may reduce the level of growth promoters (e.g. gibberellins) that antagonise ABA in bioassays, b) SD enhance transport of ABA from leaves to the shoot apex, and c) that lack of control of water stress in experimental plants could lead to variations in ABA levels large enough to mask any influence of photoperiodic conditions on levels of this hormone. This last possibility seems particularly important in view of well-substantiated instances of even slight water stress inducing large increases in endogenous ABA (see Milborrow 1974). A number of workers (see Torrey 1976) have described effects of photoperiodic conditions around the shoot on root development and it seems likely that some of these effects involve endogenous growth substances such as ABA. However, roots of many temperate-zone trees such as A. pseudoplatanus and Acer saccharum show two periods of maximum growth activity each year, one occurring in spring some weeks prior to the onset of renewed shoot development and the second in late autumn/early winter (Romberger 1963 ; Taylor and Dumbroff 1975 ; Dum-
I.D.J. Phillips et al. : Abscisic Acid in Acer
broff and Brown 1976), suggesting that root growth in such trees is, to a certain extent, independent of dormancy in the shoot. It is therefore uncertain to what extent annual cycles of root development in trees are determined by water stress and/or photoperiodic conditions around the shoot. To help clarify the situation regarding possible effects of photoperiodic conditions on endogenous ABA, our studies have been conducted on seedlings of A. pseudoplatanus grown in aerated liquid nutrient medium in order to reduce the variations in water stress that usually occur in soil-grown plants. Measurements have been made of free and bound ABA in roots and leaves of various ages under either SD or LD and at various times in the daily light/dark cycle. Phaseic acid (PA) was also measured in leaf extracts.
Material and Methods Plant Material Seeds of A. pseudoplatanus were collected in the Exeter area and stratified in the dark at 8 ~ in damp vermiculite for approx. 4 months by which time they had germinated. Seedlings were transferred, still in vermiculite, to warmer LD conditions (25 ~ C, 18 h photoperiods). When the cotyledons had expanded the roots were surface-sterilized for 5 rain in 2% (w/v) sodium hypochlorite followed by thorough washing in sterile distilled water prior to transferring the seedlings to aerated mineral nutrient solution (Johnson et al. 1957 modified by Epstein 1972) where they were grown for a further 7 weeks under LD (460 pmol photons I l l - 2 S- 1 fi'om a mixed fluorescent/incandescent source for 18/24 h) at 20 ~ C in the light periods and 15~ during the 6 h dark periods. At the end of this time, plants were divided between SD and LD regimes. Plants exposed to SD received 8 h irradiance (as above) per 24 h while those under LD received an additional 10 h of low intensity illumination (100 Ixmol photons m -2 s 1) from incandescent lamps. After various numbers of SD or LD cycles, plants were harvested for extraction.
i19 taken up in a small volume of ethanol for GLC and scintillation counting. After methylation, 1 ~tl aliquots were injected into the GLC (1.5 m x 6 mm column containing 3% OV-17 on 80-100 mesh Gas Chrom Q; column temp. 250 ~ C, ECD temp. 300 ~ C; N2 carrier at 30 cm s min- 1). ABA was quantified by reference to a calibration graph for authentic 2-cis-ABA with 2-trans-ABA as internal standard. Losses during extraction were estimated from the recovery of [14C]ABA (Harrison and Saunders 1975). The identity of the ABA peak was confirmed by both co-chromatography and u.v.-isomerization. All extraction and purification procedures were conducted in darkness or dim light.
Statistical Analysis Each treatment was replicated at least three times and results analysed by t-test and analysis of variance.
Results In the first experiment, leaves of all ages and roots were harvested at the end of the third 6 h (LD) or 16 h (SD) dark period. As shown in Fig. 1 there was no effect of day length on free ABA contents of either leaves or roots, but leaves contained approximately thirty times more ABA per unit weight of tissue than did roots. Because in A. pseudoplatanus photoperiodic perception occurs in the mature, rather than young, growing leaves (Wareing 1956), a second experiment examined free ABA in leaves of different ages. After three photoperiodic cycles, leaves were divided into three groups: 'young' leaves, consisting of small expanding leaves from near the apex; 'mature' leaves which were fully expanded but showed no signs of senescence, and; 'old' leaves being the four lowermost leaves which were yellowing. There was no effect of photoperiodic conditions on levels of free ABA
100
500
Extraction, Purification and Assay Procedures
Leaves 1
Methods have been detailed previously (Phillips and Hofmann 1979) and were essentially the same as those of Harrison and Saunders (1975). Macerated tissues were extracted three times with 80% (v/v) alkaline methanol and a measured quantity of (_+)-cis, trans-[2-14C]ABA (specific activity i73.9 MBq retool-t) added to the extract. The extract was filtered, reduced to aqueous residue, adjusted to pH 7.0, passed through a polyvinylpyrrolidone column, adjusted to pH 3.0, partitioned four times with diethyl ether and the organic phase (containing free ABA) dried over anhydrous sodium sulphate and evaporated to dryness. The aqueous phase was adjusted to p H 10.0 and agitated for 1 h at 60 ~ C to hydrolyse ABA esters, re-adjusted to pH 3.0 and partitioned with diethyl ether, dried and evaporated as before. Each dried ether extract was taken up in a small volume of methanol and subjected to TLC (plates coated with silica gel GF254 and pre-run in ethanol: acetic acid, 98:2, v/v and activated at 100 ~ C for 0.5 h), developing to 200 mm three times in toluene : ethyl acetate: acetic acid (40 : 5 : 2, v/v). The zone corresponding to a 2, cis-ABA marker spot was eluted with acetone:methanol (9:1, v/v), reduced to dryness and
8O
400!
6o
300
J i
"O
"T
"7
O~
200
t~ 40
"~ 20
100 ,,~
Roots
LD
SO
LD
SD
Fig. 1. Effect of photoperiodic conditions on levels of free ABA in leaves (of all stages of development) and roots of 7-week old A. pseudoplatanus. All extracts were made at the end of the third experimental dark period (6 h under LD and 16 h under SD). Values are means from four replicate extractions
I.D.J. Phillips et al. : Abscisic Acid in Acer
120
150
'Young' 'Mature'
.t-,
~100
m
3" '01d' r
LD SD
LD SD
LD SD
Fig. 2. Effect of photoperiodic conditions on levels of free A B A in leaves of different ages from 7-week old A. pseudoplatanus. All extractions were conducted at the end of the third experimental dark period (6 h under LD and 16 h under SD). ' Y o u n g ' leaves were small, not fully expanded; ' m a t u r e ' leaves were fully expanded with no visible senescence; ' o l d ' leaves comprised the four lowermost yellowing leaves. Values are m e a n s from three, four, or six replicate extractions
in leaves of the different ages but senescent leaves contained significantly less ABA than mature or young leaves (Fig. 2). In the next experiment, mature leaves and root systems were harvested separately, both at the end of the seventh experimental dark period and after 8 h of light following the seventh dark period. Both free and bound ABA were measured. In the roots, levels of free and bound ABA were again much lower than in leaves but no differences were found in either
free ABA or bound ABA under L D or SD, or between extracts made at the end of the dark period and after 8 h light (Table 1). Similarly, in the mature leaves no significant differences were observed in levels of free ABA in the four treatments (Table 2). However, in both the L D and SD treatments levels of bound ABA were much higher 8 h into the light period than at the end of the preceding dark period (Table 2). Extraction of leaves at the end of either a long or a short dark period yielded no G L C peak corresponding to 2, trans-ABA. However, all leaf extracts (under LD and SD, free and bound fractions) made after 8 h light showed a large peak with what initially appeared to be 2, trans-ABA. Closer examination revealed that the new peak had a retention time (Rt) of 3.4 min whereas 2, trans-ABA had a Rt of 3.3 min (2, cis-ABA had a Rt of 2.8 min). Nevertheless, this interfering peak meant that 2, trans-ABA could not be used as an internal standard, and quantification had to be carried out solely by reference to a calibration graph for 2, cis-ABA. In all cases it was found that exposing extracts that contained the peak of Rt 3.4 min to u.v.-light resulted in; a) decreases in the peaks at 2.8 and 3.4 min, and b) the appearance of peaks at 3.3 min (2, trans-ABA) and 3.6 min (2, transPA?). It has been reported that 2, trans-ABA and 2, cis-PA have very similar retention times on an OV-17 column (Zeevaart and Milborrow 1975). Authentic 2, cis-PA was found to have a Rt of 3.4 min. Thus, it was possible that the extract peak of Rt 3.4 min was 2, cis-PA. Exposure of authentic 2, cisPA to UV light resulted in peaks of equal areas with Rts of 3.4 and 3.6 rain, further suggesting that the unknown peak present in extracts from illuminated leaves was 2, cis-PA. This was confirmed by observations that both the unknown peak and authentic PA took longer to attain equilibrium during isomeriza-
Table 1. Levels of free and b o u n d A B A in roots of A. pseudoplatanus grown under LD (18 h photoperiods) and SD (8 h photoperiods). Approx. 8-week old plants were harvested at the end of the seventh dark period and after 8 h light following the seventh dark period. Values represent the means of four replicates •
Compound
Treatment
ng g 1 F W
ng g - 1 D W
F W basis
D W basis
396.8• 263.0 • 26.9 328.0_+3.1 342.0 • 9.1
-
-
11.3 14.2 17.5 9.5
16.5 17.7 16.8 I2.7
13.1•
15.9•
Free A B A
LD, LD, SD, SD,
end of dark after light end of dark after light
26.0_+2.2 20.0 _+ 1.2 20.3• 19.0 • 1.4
Bound A B A
LD, LD, SD, SD,
end of dark after light end of dark after light
3.3• 3.3 _+0.3 4.3 +_ 1.5 2.0 • 0.9
Mean
Bound A B A as % of total
ABA
78.3• 56.7 • 66.3 • 49.7 •
17.9 21.0 28.5 24.3
I.D.J. Phillips et al. : Abscisic Acid in Acer
121
Table 2. Levels of free and b o u n d A B A in mature leaves ofA. pseudoplatanus grown under LD (18 h photoperiods) and SD (8 h photoperiods). Approx. 8-week old plants were harvested at the end of the seventh dark period and after 8 h light following the seventh dark period. Values for free A B A are means of four replicates _+S.E., and values for b o u n d A B A means of three replicates _+S.E,
Compound
Free A B A
Bound A B A
Treatment
LD, LD, SD, SD,
end of dark after light end of dark after light
LD, LD, SD, SD,
end of dark after light end of dark after light
ABA
Bound A B A as % of total
ng g - 1 F W
ng g - * D W
153.8_+20.9 138.3 _+26.1 148.8_+ 6.1 171.8_+16.7
925.5_+120.9 728.0 _+ 167.8 849.8_+ 37.5 838.8_+ 81.5
5.0_+ 35.3-+ 16.4-t37.3_+
2.6 4.0 b 0.3 2.2 a
28.0_+ 14.8 181.0_+ 18.1 u 91.3_+ 3.3 182.3_+ 11.0 a
Mean " b
F W basis
D W basis
-
-
3.1 20.3 9.9 17.8
2.9 19.9 9.7 17.9
12.8_+3.4
12.6_+3.4
Significantly different from dark control at 5% level At 1% level
Table 3. Relative a m o u n t s of putative PA in mature leaves of A.
pseudoplatanus exposed to light for 8 h after the end of either the seventh 16 h (SD) or seventh 6 h (LD) dark period. No PA was detected in any of the fourteen extracts at the end of the dark period Treatment
Relative a m o u n t of P A (units g - 1 FW) Free
Bound
LD, end of dark LD, after light extract 1 extract 2 extract 3
ND
ND
6.1 4.6 3.1
2.7 3.0 3.7
Mean _+S.E.
4.6+0.7
3.1 _+0.2
SD, end of dark SD, after light extract 1 extract 2 extract 3 extract 4
ND
ND
2.3 3.2 4.4 5.3
0.3 0.8 1.3 2.0
Mean _+ S.E.
3.8_+0.6
1.1+_0.3 a
N D = not detectable Significantly different from LD control at 5% level
tion by UV light than did ABA, and that authentic 2, cis-PA co-chromatographed with the unknown peak. Estimation of the relative amount of PA in each extract was made by correcting peak (Rt 3.4 min) area for dilution factors and applying the same recovery rate value as determined for ABA. In none of the extracts made at the end of the dark period was
any PA detectable, whereas it was present in all extracts made after 8 h light (Table 3). The level of free PA was similar in L D and SD leaves but bound PA was more abundant under LD. Under LD, amounts of free and bound PA were not significantly different, but SD leaves contained less bound PA than free PA (Table 3).
Discussion
The use of liquid nutrient medium in these experiments largely removed the possibility of water-stressinduced variations in endogenous ABA levels, thus permitting a more critical examination of ABA levels in relation to photoperiodic conditions. Unpublished data showed that leaf water saturation deficits (%) per plant ranged from 1.79 to 11.68 (mean 7.06 _+0.97 S.E.) in soil, but only from 6.34 to 7.00 (mean 6.65_+ 0.08 S.E.) in plants in liquid nutrient medium. In all experiments, levels of ABA on the roots were considerably lower than in the leaves. Since the xylem sap of woody plants, including A. pseudoplatanus (Phillips and Hofmann 1979), contains considerable amounts of ABA, and mature leaves appear to be the principal sources of this ABA (Hoad 1973, 1975, 1978), it seems reasonable to assume that the direction of ABA transport in the stem is primarily upwards and that the root system is normally exposed to very much lower levels of shoot-synthesised ABA than are shoot tissues. The levels of free ABA found in the roots (approx. 13-26 ng kg-1 fresh weight; Fig. 1 and Table 1) are almost identical with those recorded for A. saccharum roots (Cohen et al. 1978).
122
However, whereas these workers found no alkaline hydrolyzable ABA in roots of A. saccharum, we did detect small amounts of bound ABA in roots of A. pseudoplatanus (Table 1). In fact, the proportion of total ABA present in the bound form was very similar in roots and leaves (Tables 1 and 2). Our results show that there was no effect of the length of the dark period upon levels of free or bound ABA in leaves or in roots (Figs. 1 and 2, Tables 1 and 2). However, light (mixed white fluorescent and tungsten lamps) did enhance glucosylation of ABA (Table 2) and induce the appearance of both free and bound PA (Table 3). It has been reported recently (Loveys 1979) that a far-red-enriched light source, similar to that employed by us, greatly enhanced the amount of [14C]PA detectable in extracts of [l~C]ABA-treated tomato plants. Conversion of exogenous [14C]ABA to [lgC]PA in tomato extracts was complete within 8 h under far-red-enriched light (Loveys 1979). In our experiments, PA was present in extracts of A. pseudoplatanus made after 8 h light whereas it was not detectable at the end of the preceding dark period (Table 3). In a previous paper (Phillips and Hofmann 1979) it was noted that emergence of vegetative buds of A. pseudoplatanus from winter dormancy was found to be associated with the appearance of large amounts of PA but not with a reduction in ABA level. All these observations indicate that photoperiodic induction of dormancy in A. pseudoplatanus involves more than a simple effect of daylength on levels of extractable free ABA and underline the need for more detailed studies of ABA metabolism and compartmentation.
References Alvim, R., Saunders, P.F., Barros, R.S.: Abscisic acid and the photoperiodic induction of dormancy in Salix viminalis L. Plant Physiol. 63, 774-777 (1979) Cohen, D.B., Dumbroff, E.B., Webb, D.P. : Seasonal patterns of abscisic acid in roots of Acer saccharum. Plant Sci. Lett. 11, 35-39 (1978) Corgan, J.N., Martin, G.C.: Abscisic acid levels in peach floral cups. Hortic. Sci. 6, 405406 (1971) Dumbroff, E.B., Brown, D.C.U. : Cytokinins and inhibitor activity in roots and stem of sugar maple seedlings through the dormant season. Can. J. Bot. 54, 191-197 (1976)
I.D.J. Phillips et al. : Abscisic Acid in Acer Epstein, E.: Mineral nutrition of plants. Principles and perspectives. New York: Wiley 1972 Harrison, M.A., Saunders, P.F. : The abscisic acid content of dormant birch buds. Planta 123, 291-298 (1975) Hoad, G.C. : 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.C. : Effect of osmotic stress on abscisic acid levels in xylem sap of sunflower (Helianthus annuus L.). Planta 124, 25-29 (1975) Hoad, G.C. : Effect of water stress on abscisic acid levels in whitelupin (Lupinus alba L.) fruit, leaves and phloem exudate. Planta 142, 287-290 (1978) Johnson, C.M., Stout, P.R., Broyer, T.C., Carlton, A.B. : Comparative chlorine requirements of different plant species. Plant Soil 8, 337-353 (1957) Lenton, J.R., Perry, V.M., Saunders, P.F. : The identification and qualitative analysis of abscisic acid in plant extracts by gasliquid chromatography. Planta 96, 271480 (1971) Leshem, Y., Philosoph, S., Wurzburger, J.: Glycosylation of free trans-abscisic acid as a contributing factor in bud dormancy break. Biochem. Biophys. Res. Commun. 57, 526 531 (1974) Loveys, B.R. : The influence of light quality on levels of abscisic acid in tomato plants, and evidence for a novel abscisic acid metabotite. Physiol. Plant. 44, 79-84 (1979) Mielke, E.A., Dennis, F.G., Jr.: Hormonal control of flower bud dormancy in sour cherry. II. Levels of abscisic acid and its water-soluble complex. J. Am. Soc. Hort. Sci. 100, 287-290 (1975) Milborrow, B.V.: The chemistry and physiology of abscisic acid. Annu. Rev. Plant Physiol. 25, 259-307 (1974) Phillips, I.D.J., Hofmann, A.: Abscisic acid (ABA), ABA esters and phaseic acid in vegetative terminal buds of Acer pseudoplatanus during emergence from winter dormancy. Planta 146, 591-596 (1979) Romberger, J.A. : Meristems, growth, and development in woody plants. USDA, Forest Service, Tech. Bull. 1293 (1963) 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) Torrey, J.G. : Root hormones and plant growth. Annu. Rev. Plant Physiol. 27, 435-459 (1976) Wareing, P.F. : Photoperiodism in woody plants. Annu. Rev. Plant Physiol. 7, 191-214 (1956) Wareing, P.F., Saunders, 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 sylvatica) buds. J. Exp. Bot. 26, 161-174 (1975) Zeevaart, J.A.D., Milborrow, B.V.: Metabolism of (_+) abscisic acid and the occurrence of epi-dihydrophaseic acid in Phaseolus vulgar#. Plant Research '74. Report of Michigan State University/AEC Plant Research Laboratory, pp. 50-53 (1975)
Received 12 August; accepted 8 October 1979
