Journal of Experimental Botany, Vol. 53, No. 378, pp. 2201-2206,
November 1, 2002
© 2002 Oxford University Press
Salt-stress-induced ABA accumulation is more sensitively triggered in roots than in shoots
Received 24 June 2002; Accepted 3 July 2002
1 College of Horticulture, China Agricultural University, Beijing, China
2 College of Biology, China Agricultural University, Beijing, China
3 Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong
4 To whom correspondence should be addressed. Fax: +852 3411 5995. E-mail: jzhang{at}hkbu.edu.hk
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Abstract |
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Salt-stress-induced ABA accumulation in maize root tissues was compared with that in leaf tissues. While salt stress with NaCl resulted in a significant ABA accumulation in root tissues (up to 10-fold), the same stress only led to a small ABA accumulation in leaf tissues (about 1-fold). Pretreatment with ethylene glycol (EG), a permeable and inert monomer of PEG, could prevent the shrinkage of cell volume and completely block the ABA accumulation in leaf tissues under salt stress, but substantial salt-induced ABA accumulation was still observed in root tissues following such pretreatment. Hypotonic salt solutions, i.e. below 100 mM NaCl, still induced a significant ABA accumulation (more than 3-fold) in roots, but showed no effect on that in leaf tissues. Results suggest that the salt-stress-induced ABA accumulation in roots may also be triggered by an osmosensing mechanism, which is in addition to the perception of the changes in reduced cellular volume or plasmalemma tension that leads to ABA accumulation in leaves. When leaf and root tissues were immersed into salt solutions, salt entered into the cells as a function of time and salt concentrations. Such entrance apparently led to a loss of sensitivity of leaf tissues to accumulate ABA under the salt stress, and also prevented the leaf tissues from responding to further air-drying in terms of ABA accumulation. Roots showed no such responses. Results suggest that the entrance of salt into leaf cells brought about some toxic effect that might have reduced the capability of leaf cells to produce ABA under dehydration.
Key words: Key words: Osmosensor, signal perception, tissue dehydration, water deficit, Zea mays.
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Introduction |
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Salt stress and water deficit are perhaps the two most important soil stresses that limit plant growth and development. To cope with them, plants must be able to sense and respond to them rapidly. The mechanisms involved include a series of cellular signalling processes from the stress signal perception to the expression of many genes. During the last two decades, it has been well established that abscisic acid (ABA) is a vital cellular signal that mediates the expression of a number of salt and water deficit-responsive genes. The basis for ABA as an important signal is that both salt stress and water deficit can induce a rapid and massive accumulation of ABA in plant tissues. This process itself is a cellular signalling cascade, in which the perception of salt- or water deficit-signal or the initial triggering for ABA accumulation is the most important step. While many studies have tried to explore the triggering mechanism for water deficit-induced ABA accumulation (Pierce and Raschke, 1980; Ackerson and Radin, 1981; Jia et al., 2001), much less is known about the triggering mechanism for the salt-stress-induced ABA accumulation.
Possibly because salt stress can lead to cell dehydration and/or the loss of cell turgor, it has been proposed that the perception of salt stress and water deficit may share a common mechanism (Jensen et al., 1996; Shinozaki and Yamaguchi-Shinozaki, 1997). Earlier studies have related the triggering mechanism to the changes in cellular water relations parameters and/or cell volume (Zabadal, 1974; Pierce and Raschke, 1980; Ackerson and Radin, 1981; Jia and Zhang, 2000). However, salt stress has actually more than its dehydration effect on plant cells. Many salt-induced plant responses suggest that roots in soil must have evolved some mechanisms to detect a developing salt stress at its initial stage before a serious dehydration occurs. An osmosensor is perhaps one of these mechanisms.
An osmosensor is known to exist in some bacteria and yeast. For example, in Escherichia coli, EnvZ functions as an osmosensor and is involved in osmotic responses (Chang and Stewart, 1998). In yeast, Sln1P is suggested to act as a sensor protein transducing a signal of high osmotic stimulus to downstream signal molecules (Wurgler-Murphy and Saito, 1997). Importantly, an osmosensor has recently been identified in a higher plant (Urao et al., 1999). An Arabidopsis cDNA encoding a histidine kinase ATHK1 was shown to complement the yeast sln1 mutant and functioned as an osmosensor in yeast. Furthermore, the study by Urao et al. (1999) suggested that the ATHK1 transcript is more abundant in roots than in other tissues. A recent study suggested that there might be different signalling cascades for the stress-induced ABA between leaf and root cells (Jia and Zhang, 2000). Seo et al. (2000) also found that one of the key enzymes that are regulated under water stress for ABA accumulation, the ABA aldehyde oxidase, has different isoforms and therefore different genes in the roots and leaves of Arabidopsis. In view of this, the hypothesis here is that the salt-stress-induced ABA accumulation may be triggered in a different way from that of water deficit, moreover, root and leaf tissues may have different triggering mechanisms for salt and water deficit-induced ABA accumulation. The present study has compared leaf and root tissues in their responses to salt stress and tried to identify their differences in terms of salt-induced ABA accumulation.
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Materials and methods |
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Plant materials
Maize (Zea mays cv. Zhongyou 1) seeds were sown in trays of sands and watered with Hoagland nutrition solution. The seedlings were grown for about 6 d at 28 °C in a greenhouse. When the second leaf appeared, the fully expanded first leaf was used for experiments. For root samples, the primary roots were carefully washed out when the coleoptiles were about 2 cm long and excised about 10–12 mm long from the tips.
Measurement of NaCl-induced weight loss and EG pretreatment
To investigate whether salt-stress-induced ABA accumulation was related to changes of cellular volume, NaCl-induced changes in tissue weight were measured. Leaves were cut into 0.5 cm2 squares (about 0.3 g fresh weight), vacuum-infiltrated with and incubated in various concentrations of NaCl solutions at 25 °C for 15 min. For root samples, 0.3 g fresh weight of root tips were put directly into NaCl solutions and then incubated at 25 °C for 15 min. After the incubation, any solution on the sample surface was rapidly absorbed with tissue papers and the samples were immediately weighed.
Ethylene glycol (EG) is an inert monomer of PEG and can freely permeate cell membranes with no adverse effects on cell physiology (Kiyosawa, 1993; Saftner, 1992). In this study, EG pretreatment was adopted to increase the cellular osmotic concentration of the root or leaf samples and to prevent weight loss or a change in cellular volume when the samples were incubated in NaCl solutions. For EG pretreatment, leaf or root samples were incubated in 1 M EG for 1 h at 25 °C, following which the samples were treated with NaCl as described in other specific experiments.
Measurement of NaCl entrance into cells
Maize leaves were cut into 0.5 cm2 squares (0.3 g), vacuum-infiltrated with and incubated in various concentrations of NaCl solutions at 25 °C for different times. For root samples, 0.3 g of root tips were immersed in NaCl solutions and then incubated at 25 °C for different times. After incubation, solutions attached to the sample surface were absorbed with tissue paper, and the 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 pressing the tissues inside the vials. The osmotic concentration of the cell sap was measured using a vapour pressure osmometer (Model 5500, Wescor Inc., USA).
NaCl-induced ABA accumulation
Preliminary experiments showed that salt-induced ABA accumulated in leaf and root samples could be released into NaCl solutions. To prevent the possible loss of ABA from root or leaf tissues, NaCl treatment were performed in two stages. Leaf or root samples were first infiltrated with and incubated in NaCl solution for a relatively short period, i.e. from 15 min to 1 h, then removed with solutions attached and incubated in a sealed moisture chamber for 6 h at 25 °C. After incubation, the samples were immediately frozen in liquid nitrogen and used for ABA assay as described below.
Water-deficit-induced ABA accumulation
Leaf squares or root tips were allowed to lose water under gently moving air at 25 °C in the dark. After about 30 min when the weight of leaf and root tissue samples was about 85% and 65% of their original fresh weights, respectively, the samples were sealed with aluminium foil and incubated in a sealed moisture chamber at 25 °C for 6 h. In the experiment where salt-stressed samples were used for the study of dehydration-induced ABA accumulation, the samples were first infiltrated with and incubated in 300 mM NaCl for 1 h at room temperature, and then dehydrated as described above.
ABA analysis
After various treatments the samples were immediately frozen with liquid nitrogen, and then homogenized in water at ice-cold temperature. The samples were then centrifuged for 25 min at 20 000 g and the supernatants were used for the ABA assay. ABA analyses were carried out using the radioimmumoassay (RIA) method as described by Quarrie et al. (1988). The highly specific monoclonal antibody (Mac 252) was provided by Dr SA Quarrie (John Innes Centre, UK). Aqueous extracts of root tissues were used for the assay without purification. 50 µl of crude extracts was mixed with 200 µl phosphate-buffered saline (pH 6.0), 100 µl diluted antibody solution, and 100 µl 3H-ABA (about 8000 cpm) solution. The reaction mixture was incubated at 4 °C for 45 min and the bound radioactivity was measured in 50%-saturated (NH4)2SO4 precipitated pellets with a liquid scintillation counter. The extraction efficiency and immunoreactive contamination in crude extracts of roots were tested earlier and the method shown to be reliable (Zhang and Davies, 1990).
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Results |
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When the NaCl concentration was above 100 mM and its osmotic concentration was higher than that of root or leaf cells, NaCl treatment caused a visible loss of fresh weight for both leaf (Fig. 1A) and root tissues (Fig. 1B). EG pretreatment, 1 M for 1 h, completely prevented such weight loss (Fig. 1), indicating that EG indeed entered into the cells and reduced the cell osmotic and water potentials.
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While NaCl treatment only induced a small ABA accumulation (less than 1-fold) in leaf tissues (Fig. 2A), the same treatment of NaCl caused a significant ABA accumulation (near 10-fold) in root tissues (Fig. 2B). Importantly, EG pretreatment completely blocked the salt-stress-induced ABA accumulation in leaf tissues (Fig. 2A). By contrast, it did not block the salt-stress-induced ABA accumulation in root tissues (Fig. 2B). It should be noted that the salt-stress-induced ABA accumulation in leaf tissues was much less sensitive than that in root tissues in responding to the salt treatment. Figure 3 shows that, even when NaCl concentration was less than 100 mM and caused no visible tissue volume changes (Fig. 1), it was the roots, rather than the leaves, that responded more sensitively to the salt-stress in terms of ABA accumulation.
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Did the salt get into the incubated tissues and have any effect on the tissues’ responses to salt? Figure 4 shows that such inflow is basically a function of salt concentrations and the duration of the salt treatment. Even 15 min could lead to a substantial increase of cell osmolality if the salt concentration reached to 300 mM (Fig. 4). Such an inflow of salt abolished the leaf tissue’s capability to produce ABA under prolonged salt stress (Fig. 5A). The longer the salt treatment, possibly the greater the inflow of salt into the cells, and the less ABA was produced in the leaf tissues. By contrast, root tissues showed no such response (Fig. 5B). The root tissues accumulated about 5-fold more ABA compared to the base ABA level and maintained more or less the same amount of ABA in its tissues when the duration of salt treatment was extended.
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The salt entrance into the cells might also have reduced the leaf tissue’s ability to produce ABA when subjected to further air-drying (Fig. 6). After the leaf tissues were treated with 300 mM NaCl for 1 h, they only weakly responded to the following air-drying-induced dehydration in terms of ABA production (Fig. 6A). The roots, however, showed no significant difference following the salt treatment and produced the same amount of ABA in response to air-drying as the control (the non-salt pretreatment) (Fig. 6B). Such results suggest that the inflow of salt into cells can have inhibitory effects on the leaf tissues, but not on the root tissues.
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Discussion |
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It is well known that many different environmental stresses such as drought, salt and cold can lead to plant water deficit or dehydration. Earlier studies suggested that the expressions of the environmental stress-responsive genes might share a common triggering or stress-perception mechanism (Hanson and Hitz, 1982; Chapin, 1991; Jensen et al., 1996; Shinozaki and Yamaguchi-Shinozaki, 1997). It has recently been shown that such dehydration-induced ABA accumulation in maize plants is triggered by the perception of a plasmalemma-tension (Jia et al., 2001). To prove whether salt-stress-induced ABA accumulation is also triggered through the ‘dehydration-mechanism’, leaf and root samples pretreated with ethylene glycol (EG) were studied. The results showed that the EG-pretreated leaf tissues, which did not get dehydrated when treated with salt (Fig. 1), did not show any ABA accumulation, but the EG-pretreated root tissues still accumulated substantial amounts of ABA in response to salt stress (Fig. 2). Obviously, such salt-induced ABA accumulation in roots cannot be entirely attributed to dehydration or a shrinkage of volume as suggested earlier with leaf tissues (Ackerson and Radin, 1981).
More difference between leaf and root tissues appeared when they were treated with hypotonic salt solutions that could not induce visible cell volume reduction. Roots accumulated substantial amounts of ABA under such salt treatments while the leaf tissues showed no detectable increase in ABA content (Fig. 3). These results strongly suggest that salt-stress-induced ABA accumulation is differently triggered in leaf and root tissues. Roots seem able to sense the salt in the environment sensitively, with or without a reduction in cellular volume, and trigger their ABA production, the so-called osmosensing mechanism. By contrast, leaf tissues may produce ABA in response to salt stress, but it is triggered by the ‘dehydration mechanism’, i.e. the perception of plasmalemma-tension, with much less sensitivity.
It is well established that ABA accumulation is very sensitive to dehydration in leaf tissues. In fact, dehydration-induced ABA accumulation is usually more sensitively triggered in leaves than in roots. For example, while dehydration may induce an ABA accumulation of about 20-fold in maize leaves, a similar dehydration will only lead to an ABA accumulation of about 5-fold in roots (Jia et al., 2001). Therefore, substantial ABA accumulation in leaves should still be induced by salt stress, with or without an osmosensing mechanism, because salt stress could also lead to significant dehydration as shown in Fig. 1. Unexpectedly, while salt stress induced a significant ABA accumulation in roots (about 10-fold), it only induced a very small ABA accumulation (just about 1-fold) in leaves (Fig. 2). Interestingly, by extending the incubation in salt solution, the salt-induced ABA accumulation in leaves rapidly decreased to zero (Fig. 5). These results suggested that dehydration-induced ABA accumulation might be inhibited by the salt that entered into the leaf cells, because the cellular osmolality significantly decreased with the longer incubation in salt solution (Fig. 4). To prove such a possibility, leaf and root tissues that were pretreated with salt were used and then subjected to further air-drying. ABA accumulation in leaf tissues that were pretreated with salt was greatly inhibited under further air-drying. By contrast, roots showed no such difference (Fig. 6).
It is possible that such inhibition was due to the toxic effect of salt on leaf cells. No matter what reasons, this result provided an explanation as to why salt stress could not induce substantial ABA accumulation in leaf tissues even though dehydration occurred during salt treatment. By comparison, the high sensitivity of ABA accumulation to salt stress in root tissues should be attributed not only to its osmosensing mechanism, but also to its tolerance to the toxic effect of salt, a possible result of evolution if it is considered that it is the roots that are exposed to salt in soil and under the pressure of natural selection.
Many earlier studies showed that leaf can accumulate much ABA in response to salt stress (Zhao et al., 1991; Nagy and Galiba, 1995; He and Cramer, 1996; Montero et al., 1997; Sibole et al., 1998; Gomez et al., 1998). Many of these experiments were performed with whole intact plants and it is possible that such salt treatment in the roots leads to the development of shoot water deficit that, in turn, triggers the dehydration-induced ABA accumulation in the shoots. These results have provided evidence that roots may produce substantial ABA when exposed to even mild salt stress. Such ABA should be able to be transported to shoots to regulate shoot physiological responses with or without the development of shoot water deficit. Earlier experiments showed that it was possible that shoot stomatal opening and leaf expansion rate can be regulated by such root-sourced chemical signals before a visible shoot deficit develops (Passioura and Gardner, 1990; Davies and Zhang, 1991).
These results suggested the existence of an osmosensing mechanism and also the organ-specific nature of such a response. This suggestion is supported by many earlier studies that many gene expressions in response to salt stress were organ- or tissue-specific. For example, root-specific gene expression has been shown in several plants and plant responses to salt stress (Winicov and Deutch, 1994; Nakashima et al., 2000; Strizhov et al., 1997; Xu et al., 1998; Urao et al., 1999). The genes that are responsible for stress-induced ABA production also seem different between leaves and roots (Seo et al., 2000). The fact that histidine kinase ATHK1 transcript from Arabidopsis, which may function as an osmosensor (Urao et al., 1999), is more abundant in roots than in other tissues, coincides with the present observation that the osmosensing mechanism triggering ABA accumulation occurs only in roots. However, it is not known whether the salt-stress-induced ABA accumulation in roots is related to this ATHK1-like osmosensor. Further molecular approaches should test this hypothesis.
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Acknowledgements |
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We are grateful for the grants from the Research Grants Council of Hong Kong University Grants Council (HKBU 2041/01M) and research fund of Area of Excellence for Plant and Fungal Biotechnology. WJ and SZ are also grateful for grants from the Foundation of National Natural Science of China (30070071) and the ‘State Key Basic Research and Development Plan’ (G 199901700).
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