JXB Advance Access originally published online on March 26, 2004
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Journal of Experimental Botany, Vol. 55, No. 399, pp. 1115-1123, May 1, 2004
© 2004 Oxford University Press
Plants and the Environment |
Rapid and tissue-specific changes in ABA and in growth rate in response to salinity in barley leaves
Received 7 January 2004; Accepted 21 January 2004
1 Division of Biological Sciences, University of Paisley, Paisley PA1 2BE, Scotland, UK
2 Ufa Research Centre, Russian Academy of Sciences, 450054 Ufa, Russia
* To whom correspondence should be addressed. Fax: +44 (0)141 848 3663. E-mail: fric-bs0{at}wpmail.paisley.ac.uk
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Abstract |
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The addition of 100 mM NaCl to the root medium of barley plants caused the rapid cessation of elongation of the growing leaf three, followed by a sudden resumption of growth during the following hour. The idea that resumption of growth is preceded and mediated by rapid and tissue-specific changes in ABA concentration and by changes in transpiration was tested. Leaf elongation velocity was recorded continuously using linear variable displacement transducers (LVDT), ABA was determined by immunoassay, and transpiration and stomatal conductivity were measured gravimetrically and by porometry, respectively. Within 10 min following addition of salt, ABA increased 6-fold in the distal portion of the leaf elongation zone; in the proximal portion, ABA accumulated with a delay. In the portion of the growing blade that had emerged ABA increased 3-fold and remained elevated during the following 20 min. This preceded a decrease in transpiration and stomatal conductivity, which, in turn, coincided with growth resumption. Twenty hours following the addition of salt, the ABA concentrations had returned to the level before stress. Leaf elongation velocity was still reduced. It is concluded that NaCl causes a rapid increase in ABA in the transpiring portion of the growing leaf. This leads to a decrease in transpiration. As a result, xylem water potential is expected to rise. The moment that the water potential gradient between the xylem and the peripheral cells in the growth zone favours water uptake again into the latter, leaf elongation resumes. The results suggest that ABA causes different responses in different leaf regions, all aimed at promoting the resumption of leaf growth.
Key words: Abscisic acid, cell elongation, Hordeum vulgare, leaf growth, salinity, water relations.
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Introduction |
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Salinity affects leaf growth in the short- and long-term. The causes of growth reduction differ (Munns, 1993; Munns et al., 1995). The immediate effect of the addition of salt to the root medium is a lowering in the external water potential and a reduction in water uptake or water content of the plants. As salinity continues, Na and Cl are taken up and increasingly displace mineral nutrients such as K, Ca, and nitrate. Although accumulation of Na and Cl aids the osmotic and water potential adjustment of the cells, it increases the risk of long-term ion toxicity, if not compartmentalized appropriately, exported, or secreted.
Leaf elongation velocity (LEV) in grasses can be measured continuously and at high resolution using linear variable displacement transducers (LVDT). Many studies have shown that LEV responds within seconds to changes in external water potential or evaporative demand (Acevedo et al., 1971; Cramer and Bowman, 1991; Cutler et al., 1980; Hsiao et al., 1970; Passioura and Munns, 2000). Concerning salinity, LEV may be reduced to zero or close to zero, depending on the stress level (Thiel et al., 1988; Yeo et al., 1991). The most obvious explanation for the rapid cessation in leaf growth is that salinity reduces xylem water potential in the elongation zone below the water potential of growing, peripheral cells. Since water cannot be pumped actively against a gradient in water potential, growth stops. Similarly, if the change in external and xylem water potential is smaller than the gradient in water potential between xylem and growing cells before stress application, growth does not stop, but is merely reduced, due to a reduction of the driving force (water potential gradient).
To resume water uptake into growing cells and, therefore, leaf elongation, the gradient in water potential between the xylem (higher) and the peripheral cells (lower water potential) has to be restored. This can be achieved either through the lowering of the water potential of peripheral cells by increasing osmolality or decreasing turgor, or an increase in xylem water potential by a decrease in transpiration (reduced water loss), or increase in root hydraulic conductance (increased water supply for a given driving force).
Abscisic acid (ABA) has been shown to influence both tissue hydraulic and stomatal conductivity (Collins and Kerrigan, 1974; Davies and Zhang, 1991; Freundl et al., 2000; Hose et al., 2000), and has been proposed to influence growth in response to drought or salinity through changes in cell wall extensibility (Bacon, 1999; Cramer et al., 1998; Dodd and Davies, 1996; Thompson et al., 1997) or apoplastic pH (Bacon et al., 1998). Much attention has been paid to the growth-inhibiting role of ABA in leaves (Cramer et al., 1998; Cramer and Bowman, 1991; Dodd and Davies, 1996; He and Cramer, 1996; Thompson et al., 1997). Surprisingly little attention has been paid to a potential role of ABA in facilitating growth resumption following the application of stress (Thompson et al., 1997).
In the present study, the hypothesis was tested that ABA levels in the growing grass leaf change rapidly, within minutes, in response to salinity and precede growth resumption. Leaf three of barley was studied during the period of maximum and steady elongation velocity. Plants were exposed to 100 mM NaCl, and elongation velocity, transpiration, and ABA content was followed with time. To distinguish between tissue-specific responses, ABA was determined in (i) the proximal and (ii) the distal portion of the leaf elongation zone, (iii) the adjacent, non-elongating leaf tissue still enclosed by sheaths of older leaves, and (iv) the emerged, transpiring portion of the growing blade.
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Materials and methods |
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Plant material and growth conditions
The present study was part of a collaborative project between Paisley University, UK, and Ufa Scientific Research Centre, Russia. Most of the growth analyses and the preparation of leaf samples for ABA analysis were carried out at Paisley (for specification of which experiments were carried out where, see the Results section and figure legends). In Paisley, plants were grown in a growth chamber, while no such facilities were available at Ufa. Although this can render interpretation of combined results difficult, it has the advantage of providing additional information on environmental variables that potentially affect the growth response to salinity. All experiments were carried out with barley (Hordeum vulgare L. cv. Golf (Svalöf Weibull AB, Svalöv, Sweden)), on the elongating leaf three. Plants were grown hydroponically. At Paisley, plants received modified Hoagland solution as detailed previously (Fricke et al., 1997; Fricke and Peters, 2002) and were grown in a growth chamber (Microclima MC1000HE, CEC Technology, Glasgow, UK). Photosynthetically active radiation at the third-leaf level was 350–400 µmol photons m–2 s–1, relative humidity was 70%, temperature was 21 °C, and the light/dark period lasted for 16/8 h. At Ufa, plants received 0.1 strength Hoagland–Arnon nutrient solution (Kudoyarova et al., 1997) under illumination of 400 µmol photons m–2 s–1 from ZN and DNAT-400 fluorescent lamps, with a 14 h photoperiod at 24 °C.
Salinity treatments
Salinity was applied and plants were analysed at the time that leaf three was elongating at a steady and maximum velocity. At this developmental stage, between 4–10 cm of leaf three had emerged from the sheath of leaf two. Plants were exposed to 100 mM NaCl for 10 min to 1 d. Salinity was applied by mixing 800 ml of nutrient solution from plant pots with 200 ml of 0.5 M NaCl. Plants were kept in the growth chamber (Paisley) during this process and harvested at the times specified in the figure legends and text. The determination of the ABA content of leaf zones required successive harvesting and pooling of segments from three to four leaves. For the two shortest salinity treatments, referred to as ‘10 min’ and ‘30 min’ in the figure legends and text, this meant that plants were actually harvested between 10–16 min and 30–36 min, respectively.
Leaf elongation measurements
Leaf elongation was measured either in increments, with a ruler and over a period of 3 h to 1 d, or continuously, with a linear variable differential transducer (at Paisley: model DFG 2.5, RS-components Ltd, Corby, UK; at Ufa: custom-built device) during the first 2 h following the addition of NaCl. Measurements with the LVDT were also performed in the growth chamber (Paisley). The tip of leaf three was attached with Sellotape to fishing line connected to the metal rod of the LVDT, and a counterweight of 2.4 g (Ufa: 1.8 g) was applied. Connecting leaf three to the LVDT caused an increase in elongation velocity to almost 200% of the original value. This was probably due to the elastic extension of the leaf and added pulling force for plastic extension caused by the counterweight and the LVDT rod. After 2 h, elongation velocity had decreased and stabilized for more than 1 h at the level before attachment to the LVDT (98.4% the velocity obtained by measurement with a ruler; average from five determinations). At this stage, NaCl was added, and elongation was recorded for up to 2 h using a chart recorder. Millivolt outputs were converted in mm by calibrating the LVDT with a micrometer.
Transpiration and stomatal conductivity measurements
Transpiration was measured gravimetrically, as the loss of water by 6–10 plants which were placed in a container with 80–100 ml of nutrient solution. The container was sealed with aluminium foil and tape to prevent the evaporative loss of water from the nutrient solution. The sealed container was placed onto a digital balance with an accuracy of 1–10 mg and loss of weight measured with time. Salt stock solution (0.5 M NaCl) was added through a tube connected to a syringe to reach a final concentration of 100 mM. At Paisley, plants grew in a controlled environment (growth chamber), and transpiration rates of salinized plants were compared with the transpiration rates of the same plants shortly before the addition of NaCl. At Ufa, where growth conditions were less controlled, the transpiration rates of salinized plants were compared with rates of non-salinized plants analysed in parallel.
Water loss rates had to be related to leaf areas. After completion of transpiration measurements, plants were removed from pots. The fully-expanded blades of leaf one and two were cut off together with the emerged portion of the blade of the growing leaf three. The blades were taped onto paper, photocopied, cut out, and weighed. A 40 cm2 piece of paper was also weighed and used to convert grams of cut-out leaf sections into cm2 leaf area. This value was multiplied by two to account for upper and lower leaf surfaces. Blade areas of leaves one, two, and three (emerged portion) were 11.54±0.65, 24.21±1.63, and 8.24±0.51 cm2, respectively (means ±SD of 8 pots).
Stomatal conductivity was determined using a porometer (Mk3, Delta-T Devices, UK). Leaves two and three were analysed from plants grown at Ufa.
Determination of relative water content (RWC)
Leaf pieces were weighed (fresh weight) and floated on distilled water for 3–4 h at 22 °C in darkness. After blotting, the turgid weight of the leaf pieces was determined; dry weight was determined after drying samples for 5 h at 80 °C. Relative water content was calculated as the difference between fresh and dry weight, divided by the difference between turgid and dry weight.
Plant harvest and ABA determination
ABA was determined in root and leaf samples. Root samples were prepared from the tip 1 cm of all seminal roots of a particular plant. For preparation of leaf samples, 2 cm long sections were cut from four positions along the elongating leaf three: from the proximal and distal half of the elongation zone (0–20 mm and 20–40 mm from the point of leaf insertion), from the adjacent, enclosed non-elongation zone (45–65 mm), and from the emerged portion of the blade (mature zone). Younger leaves, emerging inside leaf three, were pulled out prior to sectioning. Root and leaf sections were immediately transferred into 1.5 ml centrifuge tubes stored in liquid nitrogen. Between three to four plants were harvested in quick succession and the respective segments pooled. Each pooled sample was treated as one root sample or as one leaf sample of a particular leaf region. Samples were freeze-dried, either immediately or after being kept overnight at –78 °C. Freeze-dried samples were stored in a dessiccator over silica gel in the dark.
For ABA extraction, freeze-dried samples were homogenized in 80% ethanol and incubated overnight at 4 °C. After filtration and vacuum evaporation of extracts to remove all traces of ethanol, the aqueous residue was diluted with distilled water, acidified with HCl to pH 2.5, and partitioned twice with peroxide-free diethyl ether (ratio of organic to aqueous phases was 1:3). Subsequently, ABA was transferred from the organic phase into 1% sodium hydrogen carbonate (pH 7–8, ratio of organic to aqueous phases was 3:1), re-extracted with diethyl ether, methylated with diazomethane, and immunoassayed using antibodies to ABA (Mustafina et al., 1998). Recovery of added ABA was about 80%. The reduction of the amount of extractant, based on the calculated distribution of ABA in organic solvents, increases the selectivity of ABA recovery and reliability of the immunoassay (Veselov et al., 1992). This was confirmed by analysing ABA standards by immunoassay and by gas liquid chromatography with electron-capture detection.
Statistics
Statistical significance of differences between treatments was assessed by t-test (Excel). The number of replicates and the level of significance is given in the figure legends and figures.
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Results |
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Leaf elongation velocity
Addition of NaCl to a final concentration of 100 mM to the root medium of barley plants caused an immediate reduction in elongation velocity of the growing leaf three. A typical experiment is shown in Fig. 1A. In most plants, leaf elongation velocity was zero or so close to zero that it was difficult to distinguish between ‘no growth’ and residual extension caused by the counterweight of the LVDT. Of a total of 16 plants analysed at Paisley, eight plants showed zero LEV, six plants elongated at up to 6%, while two plants elongated at 17–18% original LEV at 10 min following salt addition. In the experiment shown in Fig. 1A (five plants), LEV averaged 0.11 mm h–1, or 6% of the pre-stress level, at 10 min after salt addition. Between 20 min and 30 min, elongation velocity recovered suddenly, to 1.13 mm h–1. This corresponded to 46% of the pre-stress level. Elongation velocity increased only slightly during the following day. Figure 1B shows traces of LVDT recordings. The oscillatory pattern of traces was only observed when experiments were carried out in the growth chamber and could reflect either periodic changes in relative humidity and evaporative demand (plant response) or in temperature (LVDT response–temperature-dependence of LVDT output; Hsiao et al., 1970). Leaf elongation velocity recovered suddenly, in an ‘on–off’ manner. Some threshold, either in terms of resistance (mechanical, hydraulic) or driving force (direction of water potential gradient) must have been overcome.
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Recordings obtained at Ufa, where plants grew under different environmental conditions, showed the same pattern of response (Fig. 1C). There were two differences compared with experiments at Paisley: (i) leaf elongation velocity took longer, about 60–70 min to recover suddenly; and (ii) addition of salt caused transient shrinkage of the leaf, which lasted about 3 min and amounted to about 90 µm.
When plants were kept inside a humid polyethylene bag to reduce transpiration for 20 h prior to the addition of salt, leaf elongation took longer to resume and at greatly reduced velocities. It took 52±15 min for growth to resume in plants stressed under conditions of reduced transpiration (humid polyethylene bag), as compared with 27±4 min in plants stressed under ‘ambient’ (standard growth) conditions. Once growth resumed, it recovered to 48±8% of the pre-stress level in ‘ambient’ plants (Fig. 1A), but to only 17±4% in plants kept inside a humid polyethylene bag (not shown; means ±SD of five plants analysed at Paisley). A similar response was observed for plants that had been kept in polyethylene bags for 40 min, which increased RWC from 92% to 98%, prior to salt treatment (not shown, experiments at Ufa).
ABA
In non-salinized plants, concentrations of ABA in different developmental zones of the elongating leaf three of barley were in the same range. Average concentrations ranged from 25–58 ng g–1 DW. ABA levels in non-salinized plants changed little during the experimental period (Fig. 2, values at 0 min, and 2 h and 20 h control). Addition of NaCl to the root medium caused rapid and tissue-specific changes in ABA. In all cases, ABA increased in response to salt. Within 10 min, ABA increased 6-fold in the distal, but hardly changed in the proximal half of the leaf elongation zone. In the adjacent, enclosed non-elongation zone, ABA almost doubled, while in the emerged portion of the blade ABA increased almost 3-fold. Twenty minutes later, at 30 min following addition of salt, ABA was still highest in the distal half of the elongation zone, but had doubled in the proximal half. In the emerged blade, ABA had decreased by about one-third, yet was still twice as high as before stress. Between 30 min and 1 h, ABA in the emerged portion of the blade continued to decrease towards the pre-stress level. In the elongation and adjacent non-elongation zone, ABA concentrations levelled at about 300% of the concentration before stress. Between 1 h and 2 h following the addition of salt, ABA in the enclosed leaf tissue decreased. In some experiments, ABA decreased considerably and matched levels in non-salinized plants, whereas in other experiments, ABA decreased little. This explains the large standard deviation of means for plants stressed for 2 h. Twenty hours after the addition of salt, ABA levels in salinized plants were similar to those in non-salinized plants.
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Root ABA was measured too, but only for some treatments (not shown; tip 1 cm of seminal roots). In non-salinized plants, root ABA averaged 12–14 ng g–1 DW, which was similar to levels in plants salinized for 20 h. The highest levels of root ABA were observed in plants 10 min after salt addition, averaging 61±13 ng g–1 DW (means ±SD, n=3 samples). At 30 min, levels had decreased already by more than 50%, to 22 ng g–1 DW.
Transpiration
Changes in transpiration in response to salinity were followed at Paisley and Ufa (Fig. 3). Transpiration was measured for the whole plant, and the results represent the sum of the transpirational water loss from the mature leaves one and two and the growing leaf three. Ideally, transpirational water loss would have been measured separately for the growing leaf three to get the closest indication of (qualitative) changes in xylem water potential in the elongation zone. This would have required either removing or covering the blades of older leaves. The former reduces LEV of leaf three considerably (Fricke, 2002a); the latter will alter xylem water potential and, most likely, LEV response to the addition of salt. Therefore, no attempts were made to distinguish between transpirational water loss of growing and mature leaves.
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The basic pattern of response was similar at the two locations, although time-scales differed slightly. At Paisley, the addition of salt caused a rapid increase in transpiration, which lasted 1–1.5 min (Fig. 3A). Thereafter, transpiration returned to the control level, until about 15 min when it decreased by almost 20% below the rate in the control plants (Fig. 3B). This preceded growth resumption by 5–15 min (compare Fig. 1A, B). At Ufa, the addition of salt to the root medium caused a considerable decrease in transpiration from about 35– 40 min onwards (Fig. 3C). A decrease in transpiration preceded growth resumption by 20–25 min (Fig. 1C). Transpiration in salinized plants remained below the level in control plants for the remaining experimental period.
Stomatal conductivity was determined only for plants grown at Ufa. Stomatal conductivity of the growing leaf three was higher than that of the fully-expanded leaf two in non-salinized plants (Fig. 4). The addition of salt to the root medium caused a decrease in stomatal conductivity of leaf three. This was already apparent after 10 min of salt treatment. By contrast, leaf two responded to salinity first with a slight increase in stomatal conductivity, and it took 40 min until stomatal conductivity decreased below the level in non-salinized plants.
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Discussion |
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The present study shows that ABA accumulates in leaf tissue more than 6-fold within 10 min in response to an environmental stress, salinity. The time-course of ABA accumulation in transpiring leaf tissue, together with its known effect on stomatal physiology suggests that ABA causes xylem water potential to rise and leaf growth to recover. However, ABA accumulates even more and faster in non-transpiring, growing tissue, and there must be other mechanisms through which ABA affects cell and leaf expansion. This is not surprising, considering that ABA has been implicated in the control of every biophysical variable relevant to growth at cell and tissue level: solute transport (Roberts and Snowman, 2000) and, by implication, turgor (Jones et al., 1987), hydraulic conductivity (Collins and Kerrigan, 1974; Freundl et al., 2000), wall properties (Bacon, 1999; Cramer et al., 1998), and photosynthate import (Jones et al., 1987; for discussion see Munns and Cramer, 1996).
The present results support a role of ABA in stimulation of leaf growth in stressed plants (Sharp and LeNoble, 2002). This contrasts with the general focus on ABA as an inhibitor of leaf growth (Cramer et al., 1998; Dodd and Davies, 1996; He and Cramer 1996; Munns and Cramer 1996; Thompson et al., 1997). ABA has been proposed to stimulate leaf growth in water-stressed maize by preventing excess production of the growth-inhibitory hormone ethylene (Sharp and LeNoble, 2002). The rapidity of ABA accumulation in response to salinity in the present study points to a more direct role of ABA in promoting growth.
Figure 5 summarizes biophysical scenarios of what might have happened before and after the addition of salt to the root medium. It is hypothesized that the growing barley leaf employs different mechanisms to recover and maintain residual elongation growth. Four stages are distinguished.
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e) and mesophyll cells (Ms) of the elongation zone (EZ) is lower than the water potential of the xylem (Before stress: Before the application of salinity, cells were elongating and at maximum velocity. Water moved into cells along a gradient in water potential between leaf xylem (less negative) and peripheral growing cells (more negative water potential). It is debatable how large the water potential gradient was (for discussion, see Fricke, 2002b), but there must have been a gradient. About 98–99% of water entering the growing leaf via the xylem was lost through transpiration, the rest was consumed (or stored) through cell expansion in the basal growth zone (Fricke, 2002b; recycling between shoot and root not considered).
First 1–1.5 min following addition of salt: The addition of 100 mM NaCl to the root medium caused two immediate effects: it lowered the water potential of the root medium by 0.46 MPa and it caused a transient burst in transpirational water loss; this lasted (at Paisley) for 1– 1.5 min. Previous studies suggest that the change in external water potential caused an equivalent change in water potential in mature leaf tissue and, by implication, the xylem (reflection coefficient approaching 1.0; Fricke, 1997; Thiel et al., 1988). It is unlikely that the water potential gradient between the xylem and growing leaf cells exceeded 0.3 MPa prior to salt application (Fricke, 2002b). Therefore, the addition of 100 mM NaCl levelled or reversed this gradient, stopping water supply to peripheral cells. As previously growing cells underwent their last expansion event, walls relaxed and turgor decreased to the yield threshold (Matyssek et al., 1988) without being regenerated by water entry. As a result, the water potential of peripheral growing cells decreased too; the possibility that the leaf epidermis is hydraulically isolated, as recently concluded for the epidermis of growing hypocotyl tissue of soybean (Passioura and Boyer, 2003), does not contradict this idea. It is possible that the reversal in water potential gradient between the xylem and peripheral cells was sufficient and the initial loss of turgor through wall relaxation in peripheral cells so small, that water exited the elongation zone and was lost as part of the transpiration burst. This could explain why, at Ufa, leaves shrunk immediately following the addition of salt (see also Thiel et al., 1988). Leaf shrinkage was not observed at Paisley, nor was the water content of the growth and mature zone of leaf three altered in response to salinity (measured at 0 min, 10 min, and 1 h; W Fricke, unpublished results). The difference in shrinkage between Ufa and Paisley either reflects differences in the magnitude of growth-induced water potential gradients before the application of stress or results from lower counterweight and friction during LVDT recordings at Ufa.
A sudden burst in transpiration in response to osmotic shock has been reported previously for grasses (Falk, 1966). The epidermal turgor in mature barley leaves decreases within seconds to minutes following the addition of salt (Fricke 1997), and release of back-pressure from guard cells and transient stomatal opening could easily explain the observed increase in transpiration (Meidner, 1990; Raschke, 1970). Such a response of guard cells could be viewed as design fault of stomata, increased water loss in response to reduced water availability, but it has also two advantages: it amplifies the original signal (change in water potential) and it aids the recovery of water supply from root to shoot following the application of osmotic stress (Falk, 1966). The latter promotes the delivery of signalling substances, including ABA from root to shoot tissues.
Between 10–60 min following addition of salt: As salinity continued, increased ABA in the emerged portion of the growing leaf blade caused stomatal aperture and transpiration to decrease. It appears justified to assume that this must have resulted in xylem water potential becoming less negative. At some point (20–30 min), the gradient in water potential between xylem and peripheral cells in the growth zone again favoured water uptake into the latter. As a result, leaf growth resumed, and in an ‘on-off’ manner. This idea is further supported by the observation that the resumption of growth took longer in plants kept under conditions of no or reduced transpiration: the less plants were transpiring, and the less negative xylem water potential was before the addition of salt, the more difficult it was to recover xylem water potential sufficiently through a reduction in transpiration. The observation that plants kept under low- or non-transpiring conditions recovered leaf elongation at greatly reduced velocities suggests that other growth variables, such as hydraulic conductivity were also affected by the transpiration environment of plants.
An ‘on–off’ response of resumption of growth can be explained too by a lowering of the yield threshold of walls below cell turgor, as proposed for roots of osmotically-stressed maize plants (Frensch and Hsiao, 1994). It is possible that leaves respond in the same way (Serpe and Matthews, 1992), but at a risk: turgor in growing grass leaf cells is generally between 0.4–0.5 MPa (Fricke, 2002b; Tang and Boyer, 2002). The addition of 100 mM NaCl to the root medium lowers water potential by 0.46 MPa. If turgor is lost by the equivalent, and cells continue to expand, they must do so at turgor just above zero. Any additional change in external water potential would cause plasmolysis and possibly cell death. In addition, ABA is thought to cause an increase, not decrease in yield threshold of leaf tissues in response to salt (Cramer and Bowman 1991; Munns and Cramer 1996), yet, in the present study, ABA accumulated throughout the growth zone. This was observed even in the proximal portion of the growth zone, where growth was expected to resume first to keep the duration of displacement of cells and leaf development short (Fricke, 2002b).
In the distal portion, where ABA accumulated the most initially, ABA might have suppressed growth resumption through an increase in peroxidase activity (Bacon, 1999; Bacon et al., 1997). This would have aided the channelling of resources (water, solutes) to the proximal portion of the elongation zone and assured that cell elongation resumed here first. If this was the case, proximal and distal tissues responded to the same stimulus (ABA) differentially.
Not all plants (leaves) stopped to elongate during the period immediately following salt addition. Some plants continued to elongate at reduced rates, and two plants elongated at 17–18% of the velocity before salt addition. This could be explained by reduced water potential gradients, the driving force for water transport, between xylem and growing leaf tissues. If so, why did plants which had shown a complete cessation of growth resume leaf elongation at substantially higher velocities? Assuming that growth resumed the moment that water potential gradients favoured elongation growth, these gradients must have been at least as small as in plants which never ceased elongation. There exist two explanations: the idea that the restoration of water potential gradients causes growth to resume is wrong; or, during the initial 20–30 min when water potential gradients were gradually restored to drive water uptake into growing tissues, additional changes in growth variables occurred, for example, an increase in tissue hydraulic conductivity in response to ABA (Freundl et al., 2000; Hose et al., 2000).
The reduction in evaporative loss of water from leaf surfaces is not the only means through which xylem water potential can be raised. The same can be achieved through an increased supply of water from the root, at unchanged transpirational water loss rates. ABA has been shown to increase hydraulic conductance of roots both at cell and organ level and at concentrations in the range reported here (Freundl et al., 2000; Hose et al., 2000; Jones et al., 1987). Such a response would keep the reduction in transpiration and gas exchange, which is required for growth resumption, at a minimum.
Between 1–20 h following addition of salt: A related study has shown that a significant accumulation of solutes in leaf tissue is detectable first after 1 h of stress (Fricke, 2004). Early solute accumulation is confined to the proximal portion of the leaf elongation zone. As stress continues, solute accumulation proceeds towards the tip and becomes significant successively in the distal portion of the elongation zone, the adjacent (enclosed) non-elongation zone, and the emerged portion of the blade. By 8 h of stress, solute accumulation in the leaf elongation zone is large enough to account for the entire recovery of water potential gradient and growth. This could explain why ABA, which affected growth through other variables, returned to near control levels between 2 h and 20 h of stress, yet elongation continued at the same velocity.
One day after the application of stress, elongation velocity was still reduced and only half that in non-salinized plants. It is possible that the rate of solute accumulation to achieve osmotic adjustment in expanding leaf cells became the growth-limiting factor (Delane et al., 1982; Fricke and Peters, 2002; however, see Michelena and Boyer, 1982).
Dodd and Davies (1996) measured ABA along the leaf growth zone of maize plants, which had not received water for 7–9 d and did not detect any gradient in ABA. He and Cramer (1996) followed changes in ABA in response to NaCl in rapid cycling Brassica species and observed after 1 h, their first sampling point, a significant increase in ABA in leaf tissue. Cramer et al. (1998) observed an increase in ABA in the elongation zone of barley plants exposed to salt for 4 h. These studies, as others, were concerned with an inhibitory role of ABA in leaf growth. The present data suggest that within the first hour following the stress event, ABA has a growth-promoting function. Different developmental zones of the growing barley leaf respond differentially to the same signal, ABA.
ABA increased in response to salinity in all plant tissues analysed. This rules out the possibility that an increase in one pool, for example, the leaf growth zone, was at the expense of another pool, for example, the root-tip region. Increases in ABA were considerable and fast. This implies that ABA was either synthesized new or released at high rates from glucose-ester conjugates of the hormone (Hartung et al., 2002; Sauter et al., 2002).
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Acknowledgements |
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This work was supported by the Russian Foundation for Basic Research (RFFR; grant No 03-04-49780 to GK) and the Biotechnology and Biological Sciences Research Council (BBSRC, UK; International Scientific Interchange Scheme, ISIS, grant no 1070 to WF).
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