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Seasonal changes in the level of abscisic acid in xylem sap of peach
Plant Science Letters, 2 (1974) 79--82 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
SEASONAL CHANGES IN THE LEVEL OF ABSCISIC ACID IN XYLEM SAP OF PEACH
R.M. DAVISON and H. YOUNG
Plant Diseases Division, Department of Scientific and Industrial Research, Auckland (New Zealand)
(Received July 19th, 1973)
SUMMARY
Concentrations of abscisic acid (ABA) in xylem sap of peach were determined by gas chromatographic methods throughout one year. A marked increase in levels occurred in autumn coinciding with leaf fall, with winter values (50--60 ng/ml) some 10 times those found during summer. Levels decreased when the first visible signs of bud swell were seen, suggesting a possible relationship with dormancy.
INTRODUCTION
A strong, plant-growth inhibitor demonstrated to be present in xylem sap of woody species by bioassay means ~ has since been shown by gas chromatography to be ABA :,3. The amounts present in different species sampled during the dormant period varied widely. In apples, the only species where comparisons with other growth substances were possible, amounts of ABA seemed similar to levels of gibberellins rather than the much higher levels of auxin and cytokinin 4. Previous studies s using bioassay methods suggested that an increase in total acidic inhibitors of willow xylem sap occurred during winter. Bowen and Hoad 6 reported an increase in apparent ABA with the onset of d o r m a n c y A further study has now been made of seasonal changes in the level of ABA in peach xylem sap using gas-liquid chromatographic procedures to measure this hormone specifically. It is of particular interest to consider the relationship between ABA level and the dormant period because of the many past suggestions that this inhibitor may be intimately involved with bud dormancy in deciduous trees T. The possibility of ABA occurring in bound form in this sap has also been investigated. Abbreviations: ABA, abscisic acid.
79
METHODS
At intervals throughout the year branches of peach (Prunus persica cv. Golden Queen) of 1 . 2 5 - 2 . 5 0 cm basal diameter, from which twigs of 1-yearold w o o d were removed, were sampled from a block of trees. On each occasion xylem sap was collected from 6 to 8 such branches using a vacuum technique s and combined to give a composite sample. Collected sap was held at --15 ° until used. Aliquots of sap were adjusted to pH 3 and shaken three times with peroxide-free ether to extract the ABA. Residue from these ether shakings was redissolved in a small volume of methanol before methylating with an ethereal solution of diazomethane in the usual way. Levels of methyl ABA were determined by gas-liquid chromatography using a Varian Aerograph (Model 1525C) instrument equipped with an electron-capture detector 9. A 90 X 0.31 cm stainless steel column packed with 5% FFAP on Chromosorb W (AW-DMCS) was employed. The column was conditioned at 230 ° overnight without flow and then with flow in the usual way. The temperature was maintained at 210 ° and Lhe flow rate through the column was 30 ml per min. With strict attention to cleanliness and the use of purified reagents, clear peaks of methyl ABA were obtained with this procedure. Hydrolysi~ of the non-acidic aqueous fraction remaining after the initial ether shakings were carried out at pH 11.0 at 60 ° for 30 min. The solution was then readjusted to pH 3, shaken a further three times with ether and the foregoing procedure again used to measure ABA by gas.liquid chromatography. R E S U L T S A N D DISCUSSION
Amounts of ABA in xylem sap of peach throughout a year are shown in Fig. 1. A marked increase in level occurs in winter with concentrations building to as much as 10 times those determined for the summer months. There is a dramatic rise in the level in early autumn with peak values being attained in June--July. There is an equally marked drop in levels in late July--August after which time the levels remain low throughout the summer months. There is, however, some suggestion of a small rise in concentration of ABA in midsummer, January--February. This general pattern of high winter and low summer levels of inhibitor is similar to that previously shown in willow by bioassay means s although in willow the spring fall in inhibitor level occurred much later after bud break. Hydrolysis of the residual aqueous sap fraction after extraction of free ABA gave only a minor recovery of ABA. Between monthly samples the amount varied from 1--7% of the total. No seasonal pattern was detected. It is not certain whether this small, relatively insignificant fraction resulted from hydrolysis of a conjugated form of ABA or simply represented residual ABA which was not fully extracted by the first ether shakings. The most likely explanation for the sharp rise in level of ABA which occurs during leaf fall is that it results from movement from ageing and senescing
80
701BU~well lil ,oom it
July
:/°
sept
nov
Jan
mar
may
July
TIME
Fig. 1. Levels of A B A in xylem sap of peach throughout one year. Closed circles represent the mean of duplicate determinations (open circles).
leaves into the transport stream. It has been well established that there is a considerable increase of ABA in leaf tissue in this state. Movement of ABA from such leaves into buds under short day conditions has long been proposed as a mechanism for the induction of bud dormancy 7. The decrease in ABA level in the sap in early spring coincided with the first visible signs of the end of the dormancy -- the beginnings of bud swell and the initiation of new root growth. Also at this time a renewal o f stem cambial activity would have occurred. It is notable that the early spring fall in levels occurs well before blossoming and the presence of any leaves on the trees. This decrease is thus not associated with the large change in transpiration rate which takes place when buds open and the first leaves expand. Decrease in ABA concentration, well before any significant dilution effect due to transpiration is likely, implies either a decrease in the rate of ABA production or transport into the sap, or else an increased loss from the sap. The mechanism for such changes may involve not only movement from roots but also the participation of living cells of the xylem, cambium and phloem resuming a higher level of activity after the dormant period. A considerable metabolism of xylem sap constituents is known to occur in isolated w o o d y stem segments Jo. A balance between promoters and inhibitors, particularly ABA, has been suggested as a main factor in the regulation of bud dormancy :. The present demonstration by specific measurements of a correlation between changing
81
ABA sap levels and the release of the tree from dormancy provides evidence that this p o t e n t inhibitor present in the sap could well be concerned in the regulation of this process. The maximum concentration of ABA in the sap 50--60 ng/ml is generally less than levels found in shoot and bud tissue (e.g. ref. 11). Nevertheless ff sap ABA is accumulated into bud tissue it seems feasible that the inhibitor from this source could play a significant role. Clearly, the pattern of change of other growth-promoting hormones known to be present in xylem sap also needs to be determined before further consideration of this problem. ACKNOWLEDGEMENTS
Thanks are due to Mrs. L. Connor for skilled assistance, and to Shell Research Ltd., Sittingbourne, England for a gift o f ABA. REFERENCES
1 R.M. Davison, Nature (Lond.), 197 (1963) 620. 2 J.R. Lenton, M.R. Bowen and P.F. Saunders, Nature (Lond.), 220 (1968) 86. 3 R.M. Davison and H. Young, Planta (Berl.), 109 (1973) 95. 4 L.C. Luckwill and P. ~ y t e , Soc. Chem. Ind. Monograph, 31 (1968) 87. 5 R.M. Davison, Aust. J. Biol. Sci., 18 (1965) 475. 6 M.R. Bowen and G.V. Hoad, Planta (Bed.), 81 (1968) 64. 7 P.F. Wareing and P.F. Saunders, Ann. Rev. Plant Physiol., 22 (1971) 261. 8 E.G. Boll0rd, J. Exptl. Bot., 4 (1953)363. 9 S.D. Seeley and L.E. Poweil, Analyt. Biochem., 35 (1970) 530. 10 C.P. Lloyd-Jones and D.G. Hill-Cottingham, Rept. Agric. and Hort. Res. star., University of Bristol, Long Ashton, 1970, p. 78. 11 J.R. Lenton, V.M. Pe~y and P.F. Saunders, Planta (Berl.), 106 (1972) 13.
82
SEASONAL CHANGES IN THE LEVEL OF ABSCISIC ACID IN XYLEM SAP OF PEACH
R.M. DAVISON and H. YOUNG
Plant Diseases Division, Department of Scientific and Industrial Research, Auckland (New Zealand)
(Received July 19th, 1973)
SUMMARY
Concentrations of abscisic acid (ABA) in xylem sap of peach were determined by gas chromatographic methods throughout one year. A marked increase in levels occurred in autumn coinciding with leaf fall, with winter values (50--60 ng/ml) some 10 times those found during summer. Levels decreased when the first visible signs of bud swell were seen, suggesting a possible relationship with dormancy.
INTRODUCTION
A strong, plant-growth inhibitor demonstrated to be present in xylem sap of woody species by bioassay means ~ has since been shown by gas chromatography to be ABA :,3. The amounts present in different species sampled during the dormant period varied widely. In apples, the only species where comparisons with other growth substances were possible, amounts of ABA seemed similar to levels of gibberellins rather than the much higher levels of auxin and cytokinin 4. Previous studies s using bioassay methods suggested that an increase in total acidic inhibitors of willow xylem sap occurred during winter. Bowen and Hoad 6 reported an increase in apparent ABA with the onset of d o r m a n c y A further study has now been made of seasonal changes in the level of ABA in peach xylem sap using gas-liquid chromatographic procedures to measure this hormone specifically. It is of particular interest to consider the relationship between ABA level and the dormant period because of the many past suggestions that this inhibitor may be intimately involved with bud dormancy in deciduous trees T. The possibility of ABA occurring in bound form in this sap has also been investigated. Abbreviations: ABA, abscisic acid.
79
METHODS
At intervals throughout the year branches of peach (Prunus persica cv. Golden Queen) of 1 . 2 5 - 2 . 5 0 cm basal diameter, from which twigs of 1-yearold w o o d were removed, were sampled from a block of trees. On each occasion xylem sap was collected from 6 to 8 such branches using a vacuum technique s and combined to give a composite sample. Collected sap was held at --15 ° until used. Aliquots of sap were adjusted to pH 3 and shaken three times with peroxide-free ether to extract the ABA. Residue from these ether shakings was redissolved in a small volume of methanol before methylating with an ethereal solution of diazomethane in the usual way. Levels of methyl ABA were determined by gas-liquid chromatography using a Varian Aerograph (Model 1525C) instrument equipped with an electron-capture detector 9. A 90 X 0.31 cm stainless steel column packed with 5% FFAP on Chromosorb W (AW-DMCS) was employed. The column was conditioned at 230 ° overnight without flow and then with flow in the usual way. The temperature was maintained at 210 ° and Lhe flow rate through the column was 30 ml per min. With strict attention to cleanliness and the use of purified reagents, clear peaks of methyl ABA were obtained with this procedure. Hydrolysi~ of the non-acidic aqueous fraction remaining after the initial ether shakings were carried out at pH 11.0 at 60 ° for 30 min. The solution was then readjusted to pH 3, shaken a further three times with ether and the foregoing procedure again used to measure ABA by gas.liquid chromatography. R E S U L T S A N D DISCUSSION
Amounts of ABA in xylem sap of peach throughout a year are shown in Fig. 1. A marked increase in level occurs in winter with concentrations building to as much as 10 times those determined for the summer months. There is a dramatic rise in the level in early autumn with peak values being attained in June--July. There is an equally marked drop in levels in late July--August after which time the levels remain low throughout the summer months. There is, however, some suggestion of a small rise in concentration of ABA in midsummer, January--February. This general pattern of high winter and low summer levels of inhibitor is similar to that previously shown in willow by bioassay means s although in willow the spring fall in inhibitor level occurred much later after bud break. Hydrolysis of the residual aqueous sap fraction after extraction of free ABA gave only a minor recovery of ABA. Between monthly samples the amount varied from 1--7% of the total. No seasonal pattern was detected. It is not certain whether this small, relatively insignificant fraction resulted from hydrolysis of a conjugated form of ABA or simply represented residual ABA which was not fully extracted by the first ether shakings. The most likely explanation for the sharp rise in level of ABA which occurs during leaf fall is that it results from movement from ageing and senescing
80
701BU~well lil ,oom it
July
:/°
sept
nov
Jan
mar
may
July
TIME
Fig. 1. Levels of A B A in xylem sap of peach throughout one year. Closed circles represent the mean of duplicate determinations (open circles).
leaves into the transport stream. It has been well established that there is a considerable increase of ABA in leaf tissue in this state. Movement of ABA from such leaves into buds under short day conditions has long been proposed as a mechanism for the induction of bud dormancy 7. The decrease in ABA level in the sap in early spring coincided with the first visible signs of the end of the dormancy -- the beginnings of bud swell and the initiation of new root growth. Also at this time a renewal o f stem cambial activity would have occurred. It is notable that the early spring fall in levels occurs well before blossoming and the presence of any leaves on the trees. This decrease is thus not associated with the large change in transpiration rate which takes place when buds open and the first leaves expand. Decrease in ABA concentration, well before any significant dilution effect due to transpiration is likely, implies either a decrease in the rate of ABA production or transport into the sap, or else an increased loss from the sap. The mechanism for such changes may involve not only movement from roots but also the participation of living cells of the xylem, cambium and phloem resuming a higher level of activity after the dormant period. A considerable metabolism of xylem sap constituents is known to occur in isolated w o o d y stem segments Jo. A balance between promoters and inhibitors, particularly ABA, has been suggested as a main factor in the regulation of bud dormancy :. The present demonstration by specific measurements of a correlation between changing
81
ABA sap levels and the release of the tree from dormancy provides evidence that this p o t e n t inhibitor present in the sap could well be concerned in the regulation of this process. The maximum concentration of ABA in the sap 50--60 ng/ml is generally less than levels found in shoot and bud tissue (e.g. ref. 11). Nevertheless ff sap ABA is accumulated into bud tissue it seems feasible that the inhibitor from this source could play a significant role. Clearly, the pattern of change of other growth-promoting hormones known to be present in xylem sap also needs to be determined before further consideration of this problem. ACKNOWLEDGEMENTS
Thanks are due to Mrs. L. Connor for skilled assistance, and to Shell Research Ltd., Sittingbourne, England for a gift o f ABA. REFERENCES
1 R.M. Davison, Nature (Lond.), 197 (1963) 620. 2 J.R. Lenton, M.R. Bowen and P.F. Saunders, Nature (Lond.), 220 (1968) 86. 3 R.M. Davison and H. Young, Planta (Berl.), 109 (1973) 95. 4 L.C. Luckwill and P. ~ y t e , Soc. Chem. Ind. Monograph, 31 (1968) 87. 5 R.M. Davison, Aust. J. Biol. Sci., 18 (1965) 475. 6 M.R. Bowen and G.V. Hoad, Planta (Bed.), 81 (1968) 64. 7 P.F. Wareing and P.F. Saunders, Ann. Rev. Plant Physiol., 22 (1971) 261. 8 E.G. Boll0rd, J. Exptl. Bot., 4 (1953)363. 9 S.D. Seeley and L.E. Poweil, Analyt. Biochem., 35 (1970) 530. 10 C.P. Lloyd-Jones and D.G. Hill-Cottingham, Rept. Agric. and Hort. Res. star., University of Bristol, Long Ashton, 1970, p. 78. 11 J.R. Lenton, V.M. Pe~y and P.F. Saunders, Planta (Berl.), 106 (1972) 13.
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