Journal of Experimental Botany, Vol. 52, No. 355, pp. 295-300,
February 2001
© 2001 Oxford University Press
Original Papers |
Initiation and regulation of water deficit-induced abscisic acid accumulation in maize leaves and roots: cellular volume and water relations
1 Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong
2 College of Horticulture, China Agricultural University, Beijing, China
3 College of BioSciences and BioTechnology, Yangzhou University, Jiangsu, China
Received 15 May 2000; Accepted 14 September 2000
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Water deficit-induced ABA accumulation in relation to cellular water relations was investigated in maize root and leaf tissues. While polyethylene glycol (PEG) treatment led to a significant increase of ABA content in both root and leaf tissues, ethylene glycol (EG), a permeable monomer of PEG, had no effect on ABA accumulation at similar or much lower osmotic potentials. A rapid and massive accumulation of ABA in leaf tissues occurred at a specific threshold of PEG 6000 concentration, about 20% (w/v), and closely coincided with the start of the tissue weight loss and the obvious decrease of cellular osmotic potential. Pretreatment with EG lowered the cell sap osmotic potential and also lowered the capability of both root and leaf tissues to accumulate ABA in response to further air-drying or PEG treatment. When samples were dehydrated and incubated under pressure, a method to maintain high water potential and pressure potential during dehydration, ABA accumulation was similar to those dehydrated and incubated under atmospheric pressure. Such results suggest that both the absolute water potential and pressure potential per se had no direct effects on the dehydration-induced ABA accumulation. The results have provided evidence that the initiation of ABA accumulation is related to the weight loss of tissues or changes in cellular volume rather than the cell water relation parameters, and the capability of ABA accumulation can be regulated by cellular osmotic potential.
Key words: Polyethylene glycol (PEG), ethylene glycol, abscisic acid, ABA, water relations, maize, Zea mays.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
ABA is well known as a stress hormone and plays an important role in the plant response to water stress at both a whole-plant level (Davies and Zhang, 1991
; Zhang and Davies, 1990) and at a cellular level (Gosti et al., 1995; Pla et al., 1993; Straub et al., 1994). The basis of ABA as a stress hormone is its rapid and massive accumulation under water deficit conditions. Thus, water deficit-induced ABA accumulation is a key question for revealing the mechanisms of cellular stress signalling.
When the plant cell experiences water stress, the earliest events induced are possibly the changes in cellular water relations parameters, i.e. cellular water potential, osmotic potential and pressure potential. Although several studies have been carried out to study the relationships between the initiation of ABA accumulation and water relations parameters, it is still not clear which parameter determines the initiation of water deficit-induced ABA accumulation. Earlier research (Zabadal, 1974) suggested that ABA accumulation was a function of the decrease in total water potential. Further studies suggested that such an accumulation was a function of the loss of leaf turgor or the initiation of zero turgor (Beardsell and Cohen, 1975; Pierce and Raschke, 1980). There were other reports suggesting that ABA accumulation might not be initiated at zero turgor under all conditions (Ackerson and Radin, 1983; Henson, 1982; Aghofack et al., 1991; Creelman and Mullet, 1991; Dingkuhn et al., 1991).
One of the reasons for this controversy may be due to the fact that some studies investigated one parameter of the water relations while overlooking the interactions among the others. It is obvious that the cellular water relations parameters are interrelated. In order to investigate the accumulation of ABA in relation to all the cellular water relations parameters, one approach is to break the interactions among the parameters and thereby directly observe the effect of each parameter on ABA accumulation. This approach was tried here and the influence of changes in osmotic and pressure potentials on stress-induced ABA accumulation investigated separately.
The experiments applied osmotic stresses with polyethylene glycol (PEG) and ethylene glycol (EG). EG is commonly used to compare the effects of water potential and cellular turgor potential because EG can freely permeate cell membranes with adverse effects on the cell physiology (Kiyosawa, 1993; Saftner, 1992). Hence, using EG treatment the changes in osmotic potential can be directly manipulated while not affecting the pressure potential and, therefore, the effects of osmotic or pressure potential on ABA accumulation can be directly observed. In addition, using the pressure chamber, the connection between water stress and the decrease in water or pressure potential can be broken and the effects of water or pressure potentials on the capability of ABA accumulation can be observed directly.
Furthermore, earlier studies mainly focused on the initiation of water deficit-induced ABA accumulation. It is not known, however, whether the water relations parameters can modify ABA accumulation after initiation. There might be the possibility that ABA accumulation could be controlled or regulated by some of the water relations parameters even though these parameters may not necessarily be directly related to the initiation of ABA accumulation. This study investigated this hypothesis.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Plant materials
Maize seeds were sown in sand and supplied with Hoaglands nutrition solution. The seeds were allowed to germinate and grow at 28 °C in a growth chamber. When the second leaf emerged, the fully expanded first leaf was used for experiments. Root samples were collected when the coleoptiles were about 2 cm long. Roots were carefully washed with tap water and the root tips (about 10–12 mm) were cut and used for the experiments.
Measurement of osmotic potential (
s)
To investigate the effect of EG or PEG treatment on the cellular osmotic potential of leaves, maize leaves were cut into 0.25 cm2 squares (0.25 g), vacuum-infiltrated with and incubated in various concentrations of EG or PEG solutions at 25 °C for 1 h. After incubation, solutions adhering to the leaf surface were absorbed with tissue paper and the leaf samples were sealed in cap vials and frozen immediately in liquid nitrogen. The samples were then allowed to thaw at room temperature and the cell sap was collected by gently pressing the tissues inside the vials. The s of the cell sap was measured using a vapour pressure osmometer (Model 5500, Wescor Inc., USA).
Water deficit treatments
Water deficit was induced by air dehydration, EG or PEG 6000 treatments. For air dehydration treatment the root tips or leaves were allowed to lose water under gently moving air. After about 30 min when the weight of the root tips was about 60% or the leaves about 80% of their fresh weight, the samples were sealed with aluminium foil and incubated in moist chamber at 25 °C for specific times. The sealing and incubation of the root or leaf samples in a moist chamber prevented further water loss from the samples and allowed the samples to accumulate enough ABA at the specific level of water deficit. For PEG or EG treatments, the root tips were directly put into PEG 6000 or EG solutions at various concentrations. The leaves were cut into 0.25 cm2 squares, vacuum-infiltrated with PEG or EG solutions and then incubated at 25 °C for specific times.
In order to investigate the effect of EG or PEG treatment on the weight of leaf samples, the fresh leaf samples were weighed before EG or PEG treatment. After vacuum-infiltration with EG or PEG solutions, the surface of the samples was blotted dry with tissue paper and the samples were then weighed again. In order to investigate the effect of cellular osmotic potential on water deficit-induced ABA accumulation, the root or leaf samples were first infiltrated with 0.6 M EG for 1 h at room temperature and then either air dehydrated or treated with PEG 6000 as described above.
Dehydration under pressurization
Leaves with the whole sheath attached were detached from the plant seedlings. The leaf was then placed in a pressure chamber with its sheath protruding from the pressure chamber. A pressure of 0.5 MPa was gradually applied and maintained for about 5 min, and then a further pressure of about 0.8–1.0 MPa was applied and maintained for about another 10 min. A balance pressure was reached when no further water was pressed out from the leaf sheath. At the balance pressure of about 0.8–1.0 MPa the leaf would be dehydrated to about 80–85% of its original weight.
To investigate the accumulation of ABA in relation to cellular water potential and pressure potential, three experiments were performed. The first compared the accumulation of ABA induced by pressure dehydration following incubation under pressure with that induced by air dehydration following incubation in the air. The leaf was dehydrated to about 85% of the original weight either under pressure or air dehydration and when the dehydration processes were completed the leaves were incubated either under the balance pressure or in the air at room temperature. The second compared the accumulation of ABA induced by air dehydration, but following two different incubation conditions, i.e. in the air or under pressure. The leaves were dehydrated by air dehydration to about 85% of the original weight, then one batch of samples was placed inside a pressure chamber, a pressure of 0.8 MPa applied (a balance pressure for such water loss) and another was directly incubated in the air. The third experiment compared the accumulation of ABA induced by pressure dehydration following two different incubation conditions. The leaves were dehydrated by pressure to 85% of their original weight and then one batch of samples was incubated in the air and another remained inside the pressure chamber under the balance pressure of 0.8 MPa.
ABA analysis
ABA analysis was carried out using the radioimmumoassay (RIA) method as described previously (Quarrie et al., 1988). After various treatments the materials were immediately frozen in liquid nitrogen. They were then homogenized in water at ice-cold temperature. After shaking extraction for 24 h at 4 °C, the samples were centrifuged for 25 min at 20 000 g. The supernatants were used for the ABA assay. The highly specific monoclonal antibody (Mac 252) was provided by Dr SA Quarrie (Cambridge Laboratory, IPSR, John Innes Centre, UK). 50 µl crude extracts were mixed with 200 µl phosphate-buffered saline (pH 6.0), 100 µl diluted antibody solution, and 100 µl 3H-ABA (8000 cpm) solution. The reaction mixture was incubated at 4 °C for 45 min. The bound radioactivity was measured in 50% saturated (NH4)2SO4-precipitated pellets with a liquid scintillation counter. The immunoreactive contamination in crude extracts was tested earlier and showed no significant interference (Zhang and Davies, 1990).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The changes in cellular osmotic concentration in maize leaf tissues during the treatment with EG and PEG are shown in Fig. 1
. The cellular osmotic concentration increased almost linearly with the increase in EG concentration. The PEG treatment had no effect on the cellular osmolality when the PEG concentration was below 20% (w/v), mainly because the cells were substantially dehydrated and their sap was concentrated when the PEG concentration was above 20%.
|
) treatment. Points are means±SD of four samples.PEG treatment led to significant accumulation of ABA in both root (Fig. 2A
) and leaf tissues (Fig. 2B). In comparison, EG treatment had no effect on ABA contents in either roots or leaf tissues under all the concentrations tested. PEG-induced ABA accumulation in leaf tissues was different from that in root tissues. PEG treatment led to a massive ABA accumulation in leaves when the PEG concentration was above 20%, suggesting a clear threshold of PEG concentration. However, such a threshold was not so apparent in the root tissues.
|
EG did not dehydrate leaf tissues and might even have increased the tissue weight at high concentrations through the uptake of EG (Fig. 3
). When the PEG concentration reached 20% or above, tissue began to be substantially dehydrated. It should be noted that the threshold for the PEG-induced ABA accumulation coincided with PEG-induced weight loss here.
|
Pretreatment with 0.6 M EG, which could substantially increase the osmotic concentration, also reduced the PEG-induced ABA accumulation in leaf tissues (Fig. 4
). Such pretreatment also led to the decrease in air dehydration-induced ABA accumulation in both root (Fig. 5A) and leaf tissues (Fig. 5B).
|
|
When leaves were dehydrated and incubated either in the pressure chamber or in air, the presence or absence of a pressure to the dehydrated leaf tissue had no effect on the dehydration-induced ABA accumulation (Fig. 6
), suggesting that it is not the loss of absolute pressure inside the cells, as a consequence of turgor loss, that controls the ABA accumulation.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In this study it has been demonstrated that the water deficit-induced ABA accumulation is not related to the cellular osmotic potential. As high as 0.8 mol l-1 of EG could not induce ABA accumulation, irrespective of how low the cellular osmotic potential was. Contrary to EG treatment, PEG induced a significant accumulation of ABA although it did not induce much change in the cellular osmotic potential. It should be noted that the PEG-induced ABA accumulation occurred just when the weight of leaf tissues and also the cellular osmotic potential began to decrease. It is understandable that the PEG-induced decrease in osmotic potential must be due to a decrease in cellular volume, because PEG is not permeable, therefore the initiation of water deficit-induced ABA accumulation was in fact brought about by the loss of weight of the leaf tissues (Figs 2B
, 3). Because the loss of weight reflected the changes in cellular volume, these results indicate that the initiation of ABA accumulation was related to the changes in cellular volume, rather than the water relations parameters.
If the water relations parameters are not related to the initiation of ABA accumulation, how can it be explained that the initiation of ABA accumulation occurs at a specific water potential or at zero turgor? According to the pressure–volume curves, at the start of water deficit, changes in cellular volume induced by a unit decrease of water potential is very small. When turgor approaches zero, the decrease in cellular volume rapidly becomes substantial, such that zero turgor may reflect the threshold for the significant changes in cellular volume. Hence, it generally seems that zero turgor is the threshold for ABA accumulation. Because the turgor–volume relationship is also dependent on the cell wall properties (e.g. elasticity or rigidity), the larger the cell wall elasticity, the less that zero turgor is related to the threshold change in volume. This may explain why, in some cases, there is no relationship between the pressure potential and water deficit-induced ABA accumulation (Henson, 1982; Aghofack et al., 1991; Creelman and Mullet, 1991; Dingkuhn et al., 1991).
Apart from the initiation of water deficit-induced ABA accumulation, another important question is whether the capability of ABA accumulation is regulated. Although these studies and others (Ackerson and Radin, 1983; Creelman and Mullet, 1991) suggest that none of the water relations parameters is directly involved in the initiation of water deficit-induced ABA accumulation, it is necessary to know whether they modify the capability of the water deficit-induced ABA accumulation. EG permeation into root and leaf cells reduced cellular osmotic potential and also the water stress-induced ABA accumulation (Fig. 5), suggesting that the cellular osmotic potential may be related. It is understandable that the increased solute concentration will lead to less volume shrinkage as a result of PEG or air dehydration and thus a lower accumulation of ABA. These results proved this prediction and showed that water deficit-induced ABA accumulation was lower in EG-permeated tissues than in non-EG-permeated tissues when they were dehydrated to the same degree (Fig. 5). On the other hand, the presence and absence of a balance pressure during dehydration and/or incubation period had no influence on the capacity of water deficit-induced ABA accumulation although the tissue's water potential was maintained at high values (Fig. 6).
Although EG pretreatment inhibited both PEG and air dehydration-induced ABA accumulation, the inhibition on air dehydration-induced ABA accumulation is much stronger than that on PEG-induced ABA accumulation (Figs 4, 5). The reason is possibly due to the fact that during PEG treatment, EG could diffuse out of the tissue, which thus reduced the cellular EG concentration and also the cellular osmotic potential. But in the case of air dehydration, no loss of EG occurred. Therefore the effect of inhibition was entirely due to the increased EG concentration.
It is not known why the water deficit-induced ABA accumulation is directly regulated by osmotic potential. Possibly some cellular biochemical reactions are affected by cellular osmotic concentration. In comparison, the water and pressure potentials are unlikely to affect biochemical reactions because their implication is only to reflect the water's energy status. No matter what the mechanism is, the relationship between ABA accumulation and cellular osmotic potential should have important significance in plant response to water stress, because osmoregulation is a very important adaptation response of plants to water deficit (Ackerson and Hebert, 1981; Dingkuhn et al., 1991). These results suggest that a ‘hardened’ plant, with more osmotic solutes in their cells as a result of previous water deficit, may produce less ABA with repeated water deficit and therefore have less inhibition to processes such as stomatal opening.
The mechanism of the water deficit-induced ABA accumulation is very complex and includes a series of cellular signalling processes. Although the changes in cellular volume seem to be the reason for the initiation of ABA accumulation, its triggering mechanisms are still unknown. The cell membrane is well known as the site for most signal recognition. It is relatively difficult to study water deficit signal perception because the water deficit signal can not be labelled to find its receptors as other signals, for example, hormones. Some evidence suggests that changes in membrane tension may lead to the changes in activity of some ion channels or membrane proteins, which in turn may start the signal cascades (Altendorf and Epstein, 1994; Curti et al., 1993; Erdos et al., 1991; Heath, 1995; Walderhaug et al., 1992). Understandably, changes in cellular volume, as a result of turgor relaxation, should bring about the changes of membrane tension. In this case, the changes in the tension of cell membrane might be more likely the real trigger. In addition, there might be other mechanisms, for example, the action of the cell cytoskeleton, which are also related to the changes in cellular volume (Cantiello, 1997; Money and Hill, 1997; Moran et al., 1996; Rudenko et al., 1997).
|
Acknowledgments |
|---|
We are grateful for grants from FRG (Faculty Research Grant) of the Hong Kong Baptist University and from the Croucher Foundation in support of this research. JW and JL are grateful for the financial support from the Foundation of National Natural Science of China (30070071) and the ‘State Key Basic Research and Development Plan (G1999011700)’.
|
Notes |
|---|
4 To whom correspondence should be addressed. Fax: +852 2339 5995. E-mail: jzhang{at}net1.hkbu.edu.hk
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Ackerson RC, Hebert RR.1981. Osmoregulation in cotton in response to water stress. 1. Alterations in photosynthesis, leaf conductance, translocation and ultrastructure. Plant Physiology 67, 484–488.
Ackerson RC, Radin JW.1983. Abscisic acid accumulation in cotton leaves in response to dehydration at high pressure. Plant Physiology 71, 432–433.
Aghofack NJ, Christmann A, Frosch S, Tremolieres A, Wager E.1991. Contrasting photo- and thermo-period-induced changes in abscisic acid and lipid contents in leaves of mungbean seedlings. Physiologia Plantarum 83, 346–352.
Altendorf K, Epstein W.1994. Kdp-ATPase of Escherichia coli. Cellular Physiology and Biochemistry 4, 160–168.
Beardsell MF, Cohen D.1975. Relationships between leaf water status, abscisic acid levels, and stomatal resistance in maize and sorghum. Plant Physiology 56, 207–212.
Cantiello HF.1997. Role of actin filament organization in cell volume and ion channel regulation. Journal of Experimental Zoology 279, 425–435.
Creelman RA, Mullet JE.1991. Abscisic acid accumulates at positive turgor potential in excised soybean seedling growing zones. Plant Physiology 95, 1209–1213.
Curti G, Massardi F, Lado P.1993. Synergistic activation of plasma membrane proton-ATPase in Arabidopsis thaliana cells by turgor decrease and by fusicoccin. Physiologia Plantarum 87, 592–600.
Davies WJ, Zhang J.1991. Root signals and the regulation of growth and development of plants in drying soil. Annual Review of Plant Physiology and Plant Molecular Biology 42, 55–76.[ISI]
Dingkuhn M, Cruz RT, O'Toole JC, Turner NC, Döerffling K.1991. Responses of seven diverse rice cultivars to water deficits. III. Accumulation of abscisic acid and proline in relation to leaf water-potential and osmotic adjustment. Field Crops Research 27, 103–118.
Erdos T, Butler BGS, Rappaport L.1991. Mechanogenetic regulation of transcription. Biochimie 73, 1219–1232.[Medline]
Gosti F, Bertauche N, Vartanian N, Giraudat J.1995. Abscisic acid-dependent and -independent regulation of gene expression by progressive drought in Arabidopsis thaliana. Molecular and General Genetics 246, 10–18.
Heath IB.1995. Integration and regulation of hyphal tip growth. Canadian Journal of Botany 73, S131–S139.
Henson IE.1982. Abscisic acid and water relations of rice (Oryza sativa L.): sequential responses to water stress in the leaf. Annals of Botany 50, 9–24.
Kiyosawa K.1993. Permeability of the Chara cell membrane for ethylene glycol, glycerol, mesoerythritol, xylitol, and mannitol. Physiologia Plantarum 88, 366–371.
Money NP, Hill TW.1997. Correlation between endoglucanase secretion and cell wall strength in oomycete hyphae: implications for growth and morphogenesis. Mycologia 89, 777–785.
Moran J, Sabanero M, Meza I, Pasantes MH.1996. Changes of actin cytoskeleton during swelling and regulatory volume decrease in cultured astrocytes. American Journal of Physiology 271, C1901–C1907.
Pierce M, Raschke K.1980. Correlation between loss of turgor and accumulation of abscisic acid in detached leaves. Planta 148, 174–182.
Pla M, Vilardell J, Guiltinan MJ, Marocoite WR, Niogret MF, Quatrano RS, Pagès M.1993. The cis-regulatory element CCACGTGG is involved in ABA and water-stress responses of the maize gene rab28. Plant Molecular Biology 21, 259–266.[ISI][Medline]
Quarrie SA, Whitford PN, Appleford NEJ, Wang TL, Cook SK, Henson IE, Loveys BR.1988. A monoclonal antibody to (S)-abscisic acid: its characterization and use in a radio immunoassay for measuring abscisic acid in crude extracts of cereal and lupin leaves. Planta 173, 330–339.
Rudenko SV, Bojok GA, Nipot EE.1997. Bee venom-induced shrinkage of erythrocyte ghosts. Biochemistry 62, 104–109.[Medline]
Saftner RA.1992. Effects of turgor potential on 1-aminocyclopropane-1-carboxylic acid uptake into the vacuolar compartment and ethylene production in tomato pericarp slices. Physiologia Plantarum 85, 157–166.
Straub PF, Shen-Q, Ho THJ.1994. Structure and promoter analysis of an ABA- and stress-regulated barley gene, HVA1. Plant Molecular Biology 26, 617–630.[ISI][Medline]
Walderhaug MO, Polarek JW, Voelkner P, Daniel JM, Hesse JE, Altendorf K, Epstein W.1992. KdpD and KdpE, proteins that control expression of the kdpABC operon, are members of the two-component sensor-effector class of regulators. Journal of Bacteriology 174, 2152–2159.
Zabadal TJ.1974. A water potential threshold for the increase of abscisic acid in leave. Plant Physiology 53, 125–127.
Zhang J, Davies WJ.1990. Does ABA in the xylem control the rate of leaf growth in soil-dried maize and sunflower plants. Journal of Experimental Botany 41, 1125–1132.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  B. Lu, Z. Gong, J. Wang, J. Zhang, and J. Liang Microtubule dynamics in relation to osmotic stress-induced ABA accumulation in Zea mays roots J. Exp. Bot., July 1, 2007; 58(10): 2565 - 2572. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zhu, D. Schraut, W. Hartung, and A. R. Schaffner Differential responses of maize MIP genes to salt stress and ABA J. Exp. Bot., November 1, 2005; 56(421): 2971 - 2981. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Gilliham and M. Tester The Regulation of Anion Loading to the Maize Root Xylem Plant Physiology, March 1, 2005; 137(3): 819 - 828. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Jia, Y. Wang, S. Zhang, and J. Zhang Salt-stress-induced ABA accumulation is more sensitively triggered in roots than in shoots J. Exp. Bot., November 1, 2002; 53(378): 2201 - 2206. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||