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Applied abscisic acid, root growth and turgor pressure responses of roots of wild-type and the ABA-deficient mutant, Notabilis, of tomato
PI""" ••'''' • IOW •••LOF.
© 1997 by Gustav Fischer Verlag. Jena
Applied abscisic acid, root growth and turgor pressure responses of roots of wild-type and the ABA-deficient mutant, Notabilis, of tomato A.
12 GRIFFITHS • .*,
H.
4 G. JONES 3 • ,
and A. D. TOMOS 1
I
Ysgol Gwyddorau Biolegol, Prifysgol Cymru Bangor. Bangor. Gwynedd, LL57 2uw, Wales, UK
2
Present address: Department of Physiology and Environmental Science, Plant Sciences Section, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, England, UK
3
Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, England, UK
4
Present address: Department of Biological Sciences, University of Dundee, Dundee. DDI4MN, Scotland, UK
Received April 19. 1996 · Accepted December 5, 1996
Summary
Abscisic acid (ABA) applied to the rooting medium of intact, hydroponically-grown tomato (Lycopersicon esculentum) seedlings severely inhibited root growth at concentrations ~0.5 mmol m- 3 • At 1.0 mmol m -3 the effect was more apparent in the ABA deficient mutant notabiiis (not) than in Ailsa Craig (wild-type) seedlings. Application of 1.0 mmol m -3 ABA to wild-type roots resulted in reductions in turgor pressure within 72 h. In contrast, little if any effect on turgor pressure was observed with not roots at the same concentration. That turgor pressure was more sttongly influenced in the wild-type, but that growth was more strongly influenced in not, suggests that 1 mmol m -3 ABA has independent effects on turgor pressure and growth rate.
Key words: Lycopersicon esculentum, notabiiis, abscisic acid, roots, turgor pressure, growth. Abbreviations: ABA = abscisic acid; P hydraulic conductivity; not= notabiiis.
= turgor
Introduction
Contradictory reports appear in the literature regarding the effects of applied ABA on plant tissue. Recent work on sunflower seedlings appear to indicate an inhibitory effect of applied ABA on shoot and root growth (Lenzi et a1., 1995). In contrast, ABA promoted the growth of excised soya bean roots (Yamaguchi and Street, 1977). Measurements of singlecell water relations, using the pressure probe, have also shown ABA to have differing effects. Applied ABA increased Lp but had no apparent effect on P or £i in leaf epidermal cells of Rhoeo discolor (Commelinaceae) (Eamus and Tomos, 1983). In contrast, wheat roots treated for 48 h with 25 mmol m- 3 ABA exhibited increased turgor pressures in cells located * To whom correspondence should be addressed.
J Plant Physiol. Wll.
151. pp. 60-62 (1997)
pressure; £i
= instantaneous
elastic modulus; Lp
=
within 1 cm of the root apex, Oones et a1., 1987). Further away from the root apex (4 - 5 cm) turgor pressure was found to have declined to zero in the outer cells, but had increased in the inner cells. Growth was inhibited at ABA concentrations which brought about these turgor pressure changes. In the present study the effect of applied ABA was approached by comparing two tomato genotypes; Ailsa Craig (wild-type) and the ABA-deficient mutant notabilis (not). Although not has been well characterised biochemically (see Reid, 1990) relatively few studies have been concerned with the cellular water relations of this mutant. Quantifiable differences in the growth and water relations characteristics might be expected due to the lower endogenous concentration of ABA in not compared with wild-type (Griffiths et a1., 1996; Parry et a1., 1992). The present study had two objectives; first, to determine the effect of ABA in the nutrient
61
ABA, growth and root turgor pressures
medium on root growth of the two genotypes; second, to determine the effect of ABA on the turgor pressures of the two genotypes;
Materials and Methods Plant material: Following overnight soaking in aerated distilled water, seeds of Lycopersicon esculentum Mill. cv. Ailsa Craig (wildtype) and not were covered and germinated on moistened filter paper for 2 days in a growth cabinet. Growth conditions and nutrient solutions were as previously described (Griffiths et al., 1996). For pressure-probe experiments seedlings were transferred onto 1.5 mm gauge polyvinyl-mesh, attached to polystyrene floats, in the appropriate continuously-aerated experimental solution in 500 mL pots. For growth studies, 10-16 seedlings of approximately similar length were placed on 1.5 mm gauge polyvinyl mesh in purposebuilt Perspex growth containers in the appropriate experimental solutions. The containers were approximately 20 cm in height, 2.5 cm in width and 24 cm in length. A front panel of clear Perspex allowed for observations of the roots. Following transfer to the appropriate experimental solution seedlings in both pots and growth containers remained covered for a further 2 days. Root growth: Seedlings in the growth containers were uncovered in the afternoon, 2 days after being transferred to the appropriate experimental solution. The roots were photographed [using a 35 mm lens (f5.6)] in the mornings and evenings for the following 3 days. Growth rates were calculated from photographs, which included scales of relevant length. Abscisic acid solutions: Stock solutions of ABA were prepared by dissolving synthetic ± cis-tram-ABA (Sigma Chemical Co., UK) in 1 mL of ethanol (100 %) and then made up to a final volume with distilled water. Final ABA solutions were prepared by dissolving in the nutrient solution. Turgor pressures: Cell turgor pressures were measured in the region at 2 mm from the root apex using the pressure probe (Hiisken et al., 1978), with the roots placed in a purpose-built Perspex probing container (Griffiths et al., 1996). Statistical analysis: Where required a three factor analysis of variance was applied for statistical analysis.
Table 1: Growth rates (mm/h) of wild-type and not seedlings following 5 days ABA treatment (0.05-1.0 mmol m- 3). Measurements commenced when the seedlings were 4-days-old and were completed by the time seedlings were 7-days-old. The results from the ABA treatments were based on two replicate experiments for each genotype, with the results of controls based on ten replicates for each genotype. Each experiment consisted of 10-16 roots. Analysis of variance was used in order to compare treatments. Growth rate (mm/h)
Treatment
Control 0.05 mmol m- 3 0.10 mmol m- 3 0.25 mmol m- 3 0.50 mmol m- 3 1.00 mmol m- 3
Wild-type
not
0.53 0.50 0.50 0.53 0.11 0.15
0.40 0.44 0.42 0.44 0.12 0.01
Least significant differences (L. S. D.) (P<0.05). L. S. D. between controls, based on ten replicates = 0.040. L. S. D. between treatments, based on two replicates = 0.089. L. S. D. between treatments and controls = 0.069. 0.8 ....---,--....,...--r---r---r--.,---r--.----, (A) Wild-type
0.6
0.4
0.2
.-... . ~
:::s
... ... 0-
l;~
0.0 0.8
~
IB) Notabilis
:::s l-
0.6
Results and Discussion
Control growth rates were slower in not than in the wildtype, and ABA concentrations of ~0.5 mmol m- 3 inhibited root growth by some 70-80 % in both genotypes. At 1.0 mmol m -3 ABA growth in not was completely inhibited (Table 1). Treatment with 1.0mmolm- 3 ABA caused a slow decline in mean turgor pressure, which was especially marked in the wild-type (Figs. 1 A and 1 B). The decline in mean turgor pressure was related to increasing numbers of cells having zero turgor. For both wild-type and not, epidermal and cortical cells tended to behave in a similar manner, therefore only the data from one cell type are presented. No increases in turgor pressure were detected at the ABA concentrations used in the present work. In contrast, turgor pressure increases were reported for wheat roots following treatment with the higher 25 mmol m -3 ABA treatments 00nes et al., 1987). In the wheat experiments, for cells at
0.4
0.2
a
a
24
48
72
96
120
144
168
192
Time (Hours) Figs.1A and 1 B: Timecourse of the effect of 1.0 mmol m -3 ABA on cortical cell turgor pressures at 2 mm from the root apex of (A) wildtype; and (B) not seedlings. Closed symbols represent the untreated controls. Roots were immersed in the experimental solutions following 2 days germination and remained in the experimental solutions for up to 7 days. Mean values were based on measurements of 3-8 individual cells and the associated standard errors are shown.
62
A. GRIFFITHS, H. G. JONES, and A. D. TOMOS
4- 5 cm from the root apex, high turgor pressure was only de-
tected in the inner cells (4-5 cells deep) with those near the epidermis otten being close to zero. It was suggested that the outer cells had collapsed under high initial pressures. In the present experiments with 1.0 mmol m -3 ABA there was no evidence for enhanced turgor pressures at any point in the timecourse, so that the increasing number of cells with P = 0 were unlikely to have resulted from bursting under excess pressure. The greatest growth inhibition was in not, the genotype with the lower endogenous ABA concentration, this contrasts with results from sunflower and maize ABA mutants (Lenzi et al., 1995; Pilet and Chanson, 1981). The fact that 1.0 mmol m -3 ABA inhibited growth rate more strongly in not, but influenced turgor pressure more strongly in wildtype implies that either cell elongation or cell division are influenced independently from turgor pressure. Several studies using the pressure probe have concluded that changes in wall rheology result in changes in growth rate with no change in turgor pressure (e.g. mustard stem, Rich and Tomos, 1988; wheat and maize, Tomos and Pritchard, 1994; Begonia argenteneogutta L. leaves, Serpe and Matthews, 1992). In droughted and ABA-treated sunflower plants reduced root elongation was primarily due to the inhibition of mitotic activity (Robertson et al., 1990). Further studies on the developmental responses of these two tomato genotypes to applied ABA would be required to fully understand the current findings. However, the differing responses of wild-type and not to 1 mmol m- 3 ABA, appear to indicate that growth rate and turgor pressure are influenced independently in tomato roots. Acknowledgements
A.G. acknowledges support from the Science and Engineering Research Council and Horticulture Research International for the SERC-CASE award studentship.
References
&Mus, D. and A. D. TOMOS: The influence of abscisic acid on the water relations of leaf epidermal cells of Rhoeo discolor. Plant Sci. Lett. 31, 253-259 (1983). GRIFFITHS, A., A. D. PARRY, H. G. JONES, and A. D. TOMOS: Abscisic acid and turgor pressure regulation in tomato roots. J. Plant Physiol. 149, 372-376 (1996). HUSKEN, D., E. STEUDLE, and U. ZIMMERMANN: The pressure probe technique for measuring water relations of cells in higher plants. Plant Physiol. 61, 158-163 (1978). JONES, H., R. A. LEIGH, A. D. TOMOS, and R. G. WYN JONES: The effect of abscisic acid on cell turgor pressures, solute content and growth of wheat roots. Planta 170,257-262 (1987). LENZI, A., M. FAMBRINI, S. BAROTTI, C. PUGUESI, and P. VERNIERI: Seed germination and seedling growth in a wilty mutant of sunflower (Helianthus annus L.): Effect of abscisic acid and osmotic potential. Environ. Exp. Bot. 35,427-434 (1995). PARRY, A. D., A. GRIFFITHS, and R. HORGAN: Abscisic acid (ABA) biosynthesis in roots. II. The effects of water-stress on ABA biosynthesis in roots of wild-type and ABA-deficient mutant (notabilis) plants of Lycopersicon esculentum Mill. Planta 187, 192-197 (1992). PILET, P.-E. and A. CHANSON: Effect of abscisic acid on maize root growth. A critical examination. Plant Sci. Lett. 21, 99-106 (1981). REID, J. B.: Phytohormone mutants in plant research. J. Plant. Growth Regul. 9,97-111 (1990). RICH, T. c. G. and A. D. TOMOS: Turgor pressure and phototropism in Sinapis alba L. seedlings. J. Exp. Bot. 39, 291-299 (1988). ROBERTSON, J. M., E. C. YEUNG, D. M. REID, and K. T. HUBICK: Developmental responses to drought and abscisic acid in sunflower. J. Exp. Bot. 41,339-350 (1990). SERPE, M. D. and M. A: Matthews: Rapid changes in cell wall yielding of elongating Begonia argenteo-gutta L. leaves in response to changes in plant water status. Plant Physiol. 100, 1852-1857 (1992). TOMOS, A. D. and J. PRITCHARD: Biophysical and biochemical control of cell expansion in roots and leaves. J. Exp. Bot. 45, 1721-1731 (1994). "YAMAGUCHI. T. and H. E. STREET: Stimulation of the growth of excised cultured roots of soya bean by abscisic acid. Ann. Bot. 41, 1129-1133 (1977).
© 1997 by Gustav Fischer Verlag. Jena
Applied abscisic acid, root growth and turgor pressure responses of roots of wild-type and the ABA-deficient mutant, Notabilis, of tomato A.
12 GRIFFITHS • .*,
H.
4 G. JONES 3 • ,
and A. D. TOMOS 1
I
Ysgol Gwyddorau Biolegol, Prifysgol Cymru Bangor. Bangor. Gwynedd, LL57 2uw, Wales, UK
2
Present address: Department of Physiology and Environmental Science, Plant Sciences Section, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, England, UK
3
Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, England, UK
4
Present address: Department of Biological Sciences, University of Dundee, Dundee. DDI4MN, Scotland, UK
Received April 19. 1996 · Accepted December 5, 1996
Summary
Abscisic acid (ABA) applied to the rooting medium of intact, hydroponically-grown tomato (Lycopersicon esculentum) seedlings severely inhibited root growth at concentrations ~0.5 mmol m- 3 • At 1.0 mmol m -3 the effect was more apparent in the ABA deficient mutant notabiiis (not) than in Ailsa Craig (wild-type) seedlings. Application of 1.0 mmol m -3 ABA to wild-type roots resulted in reductions in turgor pressure within 72 h. In contrast, little if any effect on turgor pressure was observed with not roots at the same concentration. That turgor pressure was more sttongly influenced in the wild-type, but that growth was more strongly influenced in not, suggests that 1 mmol m -3 ABA has independent effects on turgor pressure and growth rate.
Key words: Lycopersicon esculentum, notabiiis, abscisic acid, roots, turgor pressure, growth. Abbreviations: ABA = abscisic acid; P hydraulic conductivity; not= notabiiis.
= turgor
Introduction
Contradictory reports appear in the literature regarding the effects of applied ABA on plant tissue. Recent work on sunflower seedlings appear to indicate an inhibitory effect of applied ABA on shoot and root growth (Lenzi et a1., 1995). In contrast, ABA promoted the growth of excised soya bean roots (Yamaguchi and Street, 1977). Measurements of singlecell water relations, using the pressure probe, have also shown ABA to have differing effects. Applied ABA increased Lp but had no apparent effect on P or £i in leaf epidermal cells of Rhoeo discolor (Commelinaceae) (Eamus and Tomos, 1983). In contrast, wheat roots treated for 48 h with 25 mmol m- 3 ABA exhibited increased turgor pressures in cells located * To whom correspondence should be addressed.
J Plant Physiol. Wll.
151. pp. 60-62 (1997)
pressure; £i
= instantaneous
elastic modulus; Lp
=
within 1 cm of the root apex, Oones et a1., 1987). Further away from the root apex (4 - 5 cm) turgor pressure was found to have declined to zero in the outer cells, but had increased in the inner cells. Growth was inhibited at ABA concentrations which brought about these turgor pressure changes. In the present study the effect of applied ABA was approached by comparing two tomato genotypes; Ailsa Craig (wild-type) and the ABA-deficient mutant notabilis (not). Although not has been well characterised biochemically (see Reid, 1990) relatively few studies have been concerned with the cellular water relations of this mutant. Quantifiable differences in the growth and water relations characteristics might be expected due to the lower endogenous concentration of ABA in not compared with wild-type (Griffiths et a1., 1996; Parry et a1., 1992). The present study had two objectives; first, to determine the effect of ABA in the nutrient
61
ABA, growth and root turgor pressures
medium on root growth of the two genotypes; second, to determine the effect of ABA on the turgor pressures of the two genotypes;
Materials and Methods Plant material: Following overnight soaking in aerated distilled water, seeds of Lycopersicon esculentum Mill. cv. Ailsa Craig (wildtype) and not were covered and germinated on moistened filter paper for 2 days in a growth cabinet. Growth conditions and nutrient solutions were as previously described (Griffiths et al., 1996). For pressure-probe experiments seedlings were transferred onto 1.5 mm gauge polyvinyl-mesh, attached to polystyrene floats, in the appropriate continuously-aerated experimental solution in 500 mL pots. For growth studies, 10-16 seedlings of approximately similar length were placed on 1.5 mm gauge polyvinyl mesh in purposebuilt Perspex growth containers in the appropriate experimental solutions. The containers were approximately 20 cm in height, 2.5 cm in width and 24 cm in length. A front panel of clear Perspex allowed for observations of the roots. Following transfer to the appropriate experimental solution seedlings in both pots and growth containers remained covered for a further 2 days. Root growth: Seedlings in the growth containers were uncovered in the afternoon, 2 days after being transferred to the appropriate experimental solution. The roots were photographed [using a 35 mm lens (f5.6)] in the mornings and evenings for the following 3 days. Growth rates were calculated from photographs, which included scales of relevant length. Abscisic acid solutions: Stock solutions of ABA were prepared by dissolving synthetic ± cis-tram-ABA (Sigma Chemical Co., UK) in 1 mL of ethanol (100 %) and then made up to a final volume with distilled water. Final ABA solutions were prepared by dissolving in the nutrient solution. Turgor pressures: Cell turgor pressures were measured in the region at 2 mm from the root apex using the pressure probe (Hiisken et al., 1978), with the roots placed in a purpose-built Perspex probing container (Griffiths et al., 1996). Statistical analysis: Where required a three factor analysis of variance was applied for statistical analysis.
Table 1: Growth rates (mm/h) of wild-type and not seedlings following 5 days ABA treatment (0.05-1.0 mmol m- 3). Measurements commenced when the seedlings were 4-days-old and were completed by the time seedlings were 7-days-old. The results from the ABA treatments were based on two replicate experiments for each genotype, with the results of controls based on ten replicates for each genotype. Each experiment consisted of 10-16 roots. Analysis of variance was used in order to compare treatments. Growth rate (mm/h)
Treatment
Control 0.05 mmol m- 3 0.10 mmol m- 3 0.25 mmol m- 3 0.50 mmol m- 3 1.00 mmol m- 3
Wild-type
not
0.53 0.50 0.50 0.53 0.11 0.15
0.40 0.44 0.42 0.44 0.12 0.01
Least significant differences (L. S. D.) (P<0.05). L. S. D. between controls, based on ten replicates = 0.040. L. S. D. between treatments, based on two replicates = 0.089. L. S. D. between treatments and controls = 0.069. 0.8 ....---,--....,...--r---r---r--.,---r--.----, (A) Wild-type
0.6
0.4
0.2
.-... . ~
:::s
... ... 0-
l;~
0.0 0.8
~
IB) Notabilis
:::s l-
0.6
Results and Discussion
Control growth rates were slower in not than in the wildtype, and ABA concentrations of ~0.5 mmol m- 3 inhibited root growth by some 70-80 % in both genotypes. At 1.0 mmol m -3 ABA growth in not was completely inhibited (Table 1). Treatment with 1.0mmolm- 3 ABA caused a slow decline in mean turgor pressure, which was especially marked in the wild-type (Figs. 1 A and 1 B). The decline in mean turgor pressure was related to increasing numbers of cells having zero turgor. For both wild-type and not, epidermal and cortical cells tended to behave in a similar manner, therefore only the data from one cell type are presented. No increases in turgor pressure were detected at the ABA concentrations used in the present work. In contrast, turgor pressure increases were reported for wheat roots following treatment with the higher 25 mmol m -3 ABA treatments 00nes et al., 1987). In the wheat experiments, for cells at
0.4
0.2
a
a
24
48
72
96
120
144
168
192
Time (Hours) Figs.1A and 1 B: Timecourse of the effect of 1.0 mmol m -3 ABA on cortical cell turgor pressures at 2 mm from the root apex of (A) wildtype; and (B) not seedlings. Closed symbols represent the untreated controls. Roots were immersed in the experimental solutions following 2 days germination and remained in the experimental solutions for up to 7 days. Mean values were based on measurements of 3-8 individual cells and the associated standard errors are shown.
62
A. GRIFFITHS, H. G. JONES, and A. D. TOMOS
4- 5 cm from the root apex, high turgor pressure was only de-
tected in the inner cells (4-5 cells deep) with those near the epidermis otten being close to zero. It was suggested that the outer cells had collapsed under high initial pressures. In the present experiments with 1.0 mmol m -3 ABA there was no evidence for enhanced turgor pressures at any point in the timecourse, so that the increasing number of cells with P = 0 were unlikely to have resulted from bursting under excess pressure. The greatest growth inhibition was in not, the genotype with the lower endogenous ABA concentration, this contrasts with results from sunflower and maize ABA mutants (Lenzi et al., 1995; Pilet and Chanson, 1981). The fact that 1.0 mmol m -3 ABA inhibited growth rate more strongly in not, but influenced turgor pressure more strongly in wildtype implies that either cell elongation or cell division are influenced independently from turgor pressure. Several studies using the pressure probe have concluded that changes in wall rheology result in changes in growth rate with no change in turgor pressure (e.g. mustard stem, Rich and Tomos, 1988; wheat and maize, Tomos and Pritchard, 1994; Begonia argenteneogutta L. leaves, Serpe and Matthews, 1992). In droughted and ABA-treated sunflower plants reduced root elongation was primarily due to the inhibition of mitotic activity (Robertson et al., 1990). Further studies on the developmental responses of these two tomato genotypes to applied ABA would be required to fully understand the current findings. However, the differing responses of wild-type and not to 1 mmol m- 3 ABA, appear to indicate that growth rate and turgor pressure are influenced independently in tomato roots. Acknowledgements
A.G. acknowledges support from the Science and Engineering Research Council and Horticulture Research International for the SERC-CASE award studentship.
References
&Mus, D. and A. D. TOMOS: The influence of abscisic acid on the water relations of leaf epidermal cells of Rhoeo discolor. Plant Sci. Lett. 31, 253-259 (1983). GRIFFITHS, A., A. D. PARRY, H. G. JONES, and A. D. TOMOS: Abscisic acid and turgor pressure regulation in tomato roots. J. Plant Physiol. 149, 372-376 (1996). HUSKEN, D., E. STEUDLE, and U. ZIMMERMANN: The pressure probe technique for measuring water relations of cells in higher plants. Plant Physiol. 61, 158-163 (1978). JONES, H., R. A. LEIGH, A. D. TOMOS, and R. G. WYN JONES: The effect of abscisic acid on cell turgor pressures, solute content and growth of wheat roots. Planta 170,257-262 (1987). LENZI, A., M. FAMBRINI, S. BAROTTI, C. PUGUESI, and P. VERNIERI: Seed germination and seedling growth in a wilty mutant of sunflower (Helianthus annus L.): Effect of abscisic acid and osmotic potential. Environ. Exp. Bot. 35,427-434 (1995). PARRY, A. D., A. GRIFFITHS, and R. HORGAN: Abscisic acid (ABA) biosynthesis in roots. II. The effects of water-stress on ABA biosynthesis in roots of wild-type and ABA-deficient mutant (notabilis) plants of Lycopersicon esculentum Mill. Planta 187, 192-197 (1992). PILET, P.-E. and A. CHANSON: Effect of abscisic acid on maize root growth. A critical examination. Plant Sci. Lett. 21, 99-106 (1981). REID, J. B.: Phytohormone mutants in plant research. J. Plant. Growth Regul. 9,97-111 (1990). RICH, T. c. G. and A. D. TOMOS: Turgor pressure and phototropism in Sinapis alba L. seedlings. J. Exp. Bot. 39, 291-299 (1988). ROBERTSON, J. M., E. C. YEUNG, D. M. REID, and K. T. HUBICK: Developmental responses to drought and abscisic acid in sunflower. J. Exp. Bot. 41,339-350 (1990). SERPE, M. D. and M. A: Matthews: Rapid changes in cell wall yielding of elongating Begonia argenteo-gutta L. leaves in response to changes in plant water status. Plant Physiol. 100, 1852-1857 (1992). TOMOS, A. D. and J. PRITCHARD: Biophysical and biochemical control of cell expansion in roots and leaves. J. Exp. Bot. 45, 1721-1731 (1994). "YAMAGUCHI. T. and H. E. STREET: Stimulation of the growth of excised cultured roots of soya bean by abscisic acid. Ann. Bot. 41, 1129-1133 (1977).