JXB Advance Access originally published online on July 2, 2004
Journal of Experimental Botany 2004 55(403):1751-1760; doi:10.1093/jxb/erh215
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RESEARCH PAPER |
Chilling responses of maize (Zea mays L.) seedlings: root hydraulic conductance, abscisic acid, and stomatal conductance
Department of Crop and Soil Sciences, Bradfield Hall, Cornell University, Ithaca, New York 14853-1901, USA
* To whom correspondence should be addressed. Fax: +1 607 255 2106. E-mail: jjm11{at}cornell.edu
Received 24 February 2004; Accepted 28 May 2004
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Abstract |
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Maize seedling water relations and abscisic acid (ABA) levels were measured over 24 h of root chilling (5.5 °C). At 2.5 h into chilling, leaf ABA levels increased by 40x and stomatal conductance (gs) decreased to 20% compared with prechill levels. Despite a rapid gs response to root chilling, leaf water potential (
L) of chilled seedlings decreased to –2.2 MPa resulting in a complete loss of turgor potential (
p). Ineffective gs control early in chilling resulted from decreased root hydraulic conductance (Lr) due to increased water viscosity and factor(s) intrinsic to the roots. After 24 h chilling, L and p of chilled seedlings recovered to control levels due to stomatal control of transpiration and increased Lr. The impact of the temporal changes in gs and Lr on maize seedling water relations during chilling was analysed using a simple, quantitative hydraulic model. It was determined that gs is critical to stabilizing L at non-lethal levels in chilled seedlings at 2.5 h and 24 h chilling. However, there was also a significant contribution due to increased Lr at 24 h chilling so that p increased to control levels. As a first step in determining the factor(s) responsible for the increase in Lr, cDNA microarrays were used to quantify the transcript levels of eight aquaporins obtained from mature root tissue at 24 h chilling. None of these were significantly up-regulated, suggesting that the increase in Lr was not due to regulation of these aquaporins at the transcriptional level. Key words: Abscisic acid, aquaporin, leaf turgor potential, leaf water potential, model, recovery, steady-state, stomatal conductance, up-regulation
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Introduction |
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Chilling-sensitive plants exposed to low temperature often exhibit water-stress symptoms (low leaf water and turgor potential) described as chilling-induced water stress. Chilling-induced water stress is initiated by decreased root hydraulic conductance (Lr) followed by a large decrease in leaf water and turgor potential (Markhart et al., 1979
; Bagnall et al., 1983; Fennell and Markhart, 1998; Aroca et al., 2001). In studies where shoots and roots are chilled, stomata do not close for up to 24–48 h after chilling despite low leaf water and turgor potentials. This loss of stomatal control of leaf transpiration in addition to decreased Lr can further exacerbate chilling-induced water stress (Pardossi et al., 1992; Lee et al., 1993). Leaf relative water contents can decline to lethal levels, whether roots alone or roots and shoots are chilled (Bagnall et al., 1983; Pardossi et al., 1992; Lee et al., 1993). Increased water viscosity accounts for part of the decrease in Lr early in chilling (Levan, 1985; Matzner and Comstock, 2001). However, in those studies where the viscosity component of Lr is accounted for, there is an additional decrease in Lr unrelated to changes in viscosity (Bolger et al., 1992; Fennell and Markhart, 1998; Vernieri et al., 2001; Bigot and Boucaud, 1996). Little is known about the factor(s) responsible for this component of the decrease in Lr. It has been suggested that it is the result of low-temperature-induced alteration of membrane properties that lowers the hydraulic conductance of the symplastic component of radial root water flux (Markhart et al., 1979; Sanders and Markhart, 2001). The root endodermis and, to a lesser extent, exodermis have been identified as possible membrane control points for radial water flux in roots and, therefore, potential sites where chill-induced membrane changes may affect Lr. This is especially true in more mature roots where there may be a large symplastic component to radial root water flux because of the presence of water-impermeable substances in the apoplast of exodermal and endodermal tissues (Steudle, 2000). With time into chilling, some chilling-sensitive plants can exhibit partial or complete recovery from chilling-induced water stress (Bagnall et al., 1983; Vernieri et al., 1991, 2001; Pardossi et al., 1992; Fennell and Markhart, 1998). Increased Lr and lower stomatal conductance (gs), which re-established some stomatal control of leaf transpiration, contributed to recovery from chilling-induced water stress although the relative contributions of gs and Lr to this recovery were not determined (Fennell and Markhart, 1998; Vernieri et al., 2001).
There is no consensus on what factor(s) may be responsible for recovery of Lr with time into chilling. Suggested factors include root growth during chilling (Sanders and Markhart, 2001), changes in the lipid composition of membranes in the symplastic pathway for radial root water flux (Murata, 1994), abscisic acid (ABA) induced increase in Lr by an unknown mechanism (Bagnall et al., 1983; Markhart, 1984), and water potential-driven changes in the water flux pathway in roots (Steudle, 2000; Vernieri et al., 2001), although there is no definitive experimental evidence for any one of these factors. There are an increasing number of reports suggesting that aquaporins (major intrinsic proteins that act as selective water channels) may play an important role in the regulation of the symplastic component of root water flux (Tyerman et al., 2002). For example, Matre et al. (2002) transformed Arabidopsis plants to reduce aquaporin expression and demonstrated that aquaporins significantly contributed to Lr at relatively high transpirational fluxes. This indicates that, when symplastic water flux makes up a significant portion of total root water flux, it can be regulated by aquaporin activity or abundance (and possibly other factors that alter membrane conductance to water). Low temperature may act to regulate aquaporin abundance or activity and Lr via a ‘cold stimulus’ signal transduction (Bigot and Boucaud, 2000).
Decreased gs in response to chilling can ameliorate chilling-induced water stress (Lee et al., 1993; Capell and Dörffling, 1993; Ristic et al., 1998). This decrease in gs is correlated with increased leaf ABA and, for a given evapourative demand, can prevent the development of severe leaf water deficits that can result in leaf death (Lee et al., 1993; Vernieri et al., 1991). However, the decrease in gs is not always closely correlated with recovery from chilling-induced water stress. For example, McWilliam et al. (1982) reported that gs of bean plants began to decrease within 1 h after exposing the bean roots to 5 °C. Despite this relatively rapid stomatal response to root chilling, recovery from chilling-induced water stress did not begin until 8–10 h into chilling. Lr was not measured in this study, but was cited by the authors as a factor that could have delayed recovery from chilling-induced water stress. In experiments where roots and shoots are chilled, decreased gs, partial recovery of Lr and recovery from chilling-induced water stress all occur within the same measurement interval, and at about 24–48 h into chilling (Vernieri et al., 2001).
These studies establish that both gs and Lr can change during chilling and contribute to recovery from chilling-induced water stress. However, there has been no assessment of the relative contribution of gs and Lr to this recovery, nor has the timing of recovery relative to changes in gs and Lr been precisely determined. It remains unclear, therefore, whether the reported increase in Lr is a significant factor in the recovery from chilling-induced water stress in chilling-sensitive plants, particularly when the magnitude of Lr recovery during chilling is small (Vernieri et al., 2001).
The objectives of the current study were to document changes in maize seedling leaf water relations, gs, Lr, and leaf and root ABA during chilling, and to determine the relative contributions of gs and Lr to changes in leaf water relations early (2.5 h) in chilling and at 24 h chill. In particular, there was a desire to determine if Lr recovery later in chilling (24 h) contributed to the recovery of leaf water relations from chilling-induced water stress. It was found that, early in chilling, reduced gs in response to decreased Lr was critical to preventing severe leaf water deficits. Later in chilling, partial recovery of Lr significantly contributed to the stabilization of leaf water relations of chilled plants to control levels. Using cDNA microarrays, no evidence was found of an up-regulation of aquaporins in mature root tissue at 24 h chilling. This suggests that aquaporin regulation at the transcriptional level did not contribute to the partial recovery of Lr that was observed at 24 h chilling.
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Materials and methods |
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Plant material
Maize (Zea mays L.) cv. HL2020, Hyland Seeds, Blenheim, Ontario) was seeded in 0.26 L cylindrical containers (D16 Deepot Cells, Stuewe and Sons, Inc., Corvallis, OR) containing peat, vermiculite, and perlite (1:1:1 by vol.) with 6 g of pulverized limestone, 35 g of CaSO4, 42 g of powdered FeSO4, 1 g of fritted trace elements (Peters FTE 555, Scotts Co., Marysville OH), and 3 g of wetting agent (AquaGro G, Aquatrols, Pennauken, NJ). Plants were maintained well-watered at all times with a nutrient solution containing 0.6 g l–1 of Peters 15-16-17 (Scotts Co., Marysville OH). Watering was sufficient to leach excess nutrient salts. The plants were grown in a greenhouse where ambient light was supplemented from 06.00 h to 20.00 h solar time with 400 W high pressure sodium lights to provide a minimum of 650 µmol m–2 s–1 of photosynthetically active radiation (PAR, 400–700 nm wavelengths) at plant height. Day/night temperatures averaged 24±1 °C (day) and 13±1 °C (night), and daytime vapour pressure deficits averaged 2.0±0.1 kPa.
Plants were sown on four dates, creating four replicate batches. For each batch, 48 plants of uniform size were selected at approximately 21 d after seeding and were randomly assigned to control and chill treatments.
Chilling treatment
The 24 seedlings of each treatment were placed in one of two custom-designed root boxes capable of controlling root temperatures, while the shoots were exposed to the ambient greenhouse environment. Root temperatures of three randomly selected seedlings of each treatment were monitored as a check on the temperature treatments. Seedlings were maintained in the root boxes at a root temperature of 25±1 °C for at least 3 d prior to the start of the chilling treatment.
The chilling treatment was imposed instantaneously by thoroughly watering seedlings in one of the root boxes with chilled water (approximately 6 °C). At that time, root box temperature was reduced and a root temperature of 5.5±1 °C was achieved within 15 min after applying the chilled water. The chilling treatment was maintained for 24 h.
All measurements, and root and leaf samples were collected 1 d prior to chilling (prechill) and at 2.5 h and 24 h into the chilling treatment in order to provide a time-course for the parameters of interest. These sampling times were selected based on preliminary measurements that indicated 2.5 h and 24 h represented critical time points (maximum chilling stress at about 2.5 h chill and long-term (at least 4 d) stabilization of plant water relations in the chill treatment by 24 h chill). Measurements and sampling were done in the middle of the photoperiod at all sampling times.
Water relations measurements
Abaxial stomatal conductance (gs) was measured on the third leaf of five randomly selected control and chill treatment seedlings at each sampling time with a steady-state porometer (Li-1600, Li-Cor Inc., Lincoln, NE).
Leaf samples for leaf water potential (L) determinations, measured with a thermocouple psychrometer (Decagon Devices, Inc., Pullman, WA), were obtained from the same location as the gs measurements immediately following those measurements. L was determined after a 4 h equilibration. The thermocouple psychrometer was calibrated before each use with five potassium chloride (KCl) solutions of known water potential in the expected range of L. In addition, two KCl solutions in the expected L range were also included with each sample set as a check on the calibration. Leaf osmotic potentials (o) were measured on the L samples by freezing in liquid N2 and storing them at –20 °C for 24 h, thawing, and equilibrating the samples for 4 h in the sample chamber. Leaf turgor potential (p) was calculated from the difference between L and o.
Immediately following the gs, water relations, and leaf abscisic acid (ABA) sampling (see below), root system water flux rate (Jv) and root hydraulic conductance (Lr) were determined on detopped seedlings in a temperature-controlled pressure chamber with the cut end of the seedlings protruding from the chamber. Using compressed air, pressure in the chamber was slowly increased to 0.25 MPa and steady-state Jv was measured at 0.25, 0.35, and 0.45 MPa. Preliminary tests of this method for determining Lr in both unchilled seedling root systems, and chilled seedling root systems at both 2.5 h and 24 h chilling consistently produced a linear relationship between Jv and applied pressure over this pressure range. Root hydraulic conductance was determined as the slope of the linear relationship between Jv and pressure. All Lr values were normalized to root fresh weight determined immediately after the Lr measurement was concluded. It was noted that all Lr were determined from Jv: hydrostatic pressure curves at pressures that were large enough so that the relationship between Jv and hydrostatic pressure was linear. This is good evidence that the differences in Jv between control and chilled seedling root systems over the hydrostatic pressure range were due to chill-induced effects on Lr and not due to possible changes in the osmotic component of the total driving force for radial water flux in the root systems (Fiscus, 1977; Markhart et al., 1979).
Water relations model parameterization
A simple steady-state hydraulic model was used based on an Ohm's law analogy in order to analyse control and chill treatment water relations data:
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S is the soil water potential; Jw is the transpirational flux; and Lp is the whole plant conductance to liquid water flux (Campbell, 1985). Since the seedlings in these experiments were maintained well-watered, soil water potential (S) was assumed to be near zero and hence, –L=Jw/Lp. It was necessary to partition Lp into root and shoot components in order to weight properly the contribution of changes in Lp to changes in L during chilling. The same shoot conductance for control and chill treatments was assumed over the 24 h of the experiment, since the shoots of both treatments were maintained at warm temperatures and the experiment was of short duration. Shoot conductance of the control treatment at each sampling time was calculated from the measured root conductance based on the assumption that 70% of the total plant resistance to liquid water flow resided in the roots, as reported for maize seedlings (Neumann et al., 1974). Control and chill treatment Lp were then calculated at each sampling time using the calculated control treatment shoot conductance, and measured Lr for each treatment. It should be noted that, at later growth stages, the contribution of the root system to total plant resistance may be as low as 50% (Tiekstra et al., 2000; Matzner and Comstock, 2001).
The model was parameterized by first calculating Jw of the control treatment at each sampling time. Prechill Jw was calculated from the measured prechill L and Lp. The calculated prechill Jw referenced to total root fresh weight (RFW), 0.73 1x10–3 kg kg–1 RFW s–1, compared well with mean prechill gravimetric measurement of Jw (0.60 1x10–3 kg kg–1 RFW s–1, standard error: 0.01 1x10–3 kg kg–1 RFW s–1) of six randomly selected seedlings. Control Jw was calculated similarly at 2.5 h chill and 24 h chill. The effect of chill-induced reductions in gs on chill treatment Jw at 2.5 h and 24 h chilling were estimated by multiplying control treatment Jw by the fractional gs of the chill treatment (chill treatment gs/control treatment gs) at each sampling time. This assumes gs and Jw were tightly coupled during the experiments and was achieved by maintaining a constant light airflow over the seedlings. Model estimates of L for the different scenarios (shown in Table 2) were calculated using the control and chill treatment Jw and Lp determined as described above.
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Leaf and root sampling
Immediately following the gs measurements and sampling for leaf water relations parameters, 1 cm2 samples of leaf tissue for ABA determination were obtained from the same region of the leaf where the water relations measurements were made. The samples were weighed and ABA was extracted in a 20:1 ratio (v/w) of chilled extraction solvent (0.8 m3 methanol m–3 aqueous solution) to tissue fresh weight. Following addition of the extraction solvent, the samples were maintained at –20 °C for ABA determination.
Immediately following the Lr measurements for each seedling, total root fresh weight was determined. Root samples for ABA analysis and RNA extraction were collected from primary, seminal, and adventitious roots between 4 cm and 10 cm from the root tip and frozen in liquid N2. This portion of the root system was selected for sampling because the pathway of water uptake in this region was expected to have a significant symplastic component and, therefore, potentially greater control of radial root water flux by aquaporins (Perumalla and Peterson, 1985).
Abscisic acid (ABA) chromatography
A 200 µl aliquot of each extract was dried in vacuo then redissolved in 130 µl of Solvent I ([0.3 m3 methanol+0.01 m3 glacial acetic acid] m–3 aqueous solution). ABA was separated with a reverse-phase chromatography on columns packed with 25 mg of 40 µm diameter C18-silica material (JT Baker Chemicals, Phillipsburg, NJ). Solvents were eluted by vacuum suction into collection plates. Columns were pre-equilibrated with Solvent I, then samples were loaded in 180 µl, contaminants were eluted with 200 µl of Solvent I, then, in a separate collection plate, ABA was eluted with 200 µl of Solvent II ([0.65 m3 methanol+0.01 m3 glacial acetic acid] m–3 aqueous solution). ABA fractions were dried in vacuo.
ABA assay
ABA fractions from C18 chromatography were assayed for ABA by indirect enzyme linked immunosorbent assay (ELISA), as outlined by Setter et al. (2001) based on the methodology similar to that described by Walker-Simmons (1987) and Ober et al. (1991). Plates were prepared with ABA-BSA (4‘-bovine serum albumin) as described by Setter et al. (2001). The remaining ELISA procedure was modified from Setter et al. (2001) as follows: HBSA was substituted for TBSA where HBSA contained HEPES-buffered saline (HBS: 50 mM N-[2-hydroxyethyl]piperazine-N’-[2-ethanesulphonic acid], pH 7.5, 1 mM MgCl2, 100 mM NaCl, and 0.2 g l–1 NaN3) to which 1 g l–1 bovine serum albumin (no. A-8022, Sigma Chemical Co.) was added. ABA eluates from C18 chromatography were redissolved in 100 µl of HBSA. Samples were then incubated with primary antibody with the following in each well: 90 µl of HBSA, 10 µl of C18 eluate, and 100 µl of HBSA containing 0.2 µg of anti-ABA monoclonal antibody (Phytodetek clone no. 15-I-C5; currently available from Agdia Inc., Elkhart, IN). On each plate, a duplicate set of (+)ABA standards (Sigma Chemical Co.) containing a 1:2 dilution series of 12 values from 12.3 to 0.006 pmol per well served as a calibration curve. The antibody was added to all wells of the plate. After incubation overnight at 5 °C, plates were washed four times with TBST and 200 µl of secondary antibody solution containing 15 nl of anti-mouse IgG-alkaline phosphatase conjugate (no. A-3562, Sigma Chemical Co.) in HBSA were added per well. After incubation overnight at 5 °C, plates were washed four times with TBST and 0.2 mg p-nitrophenyl phosphate (PNPP) was added in 200 µl buffer containing 0.9 M diethanolamine, pH 9.8, and 3 mM MgCl2. Plates were incubated for about 1 h at 24 °C and absorbance at 405 nm was read with a plate reader (model 750, Cambridge Technology, Watertown, MA). (+)ABA content was determined by calculations based on (+)ABA calibration standards and a logit transformation of data.
RNA analysis
Analysis of RNA transcript abundance was performed as described by Yu and Setter (2003), except cDNA microarray slides were Unigene 1-01-02 (Galbraith Laboratory, University of Arizona, http://www.zmdb.iastate.edu/zmdb/microarray/index.html). Total RNA was extracted from root segments with RNeasy buffer containing guanidine isothiocyanate to inhibit RNase (Qiagen, Valencia, CA, USA), and were separated with silica-gel based membrane according to the manufacturer's procedure (RNeasy, Qiagen, CA). RNA targets were labelled with aminoallyl dUTP via first strand cDNA synthesis followed by coupling of the aminoallyl groups to either Cyanine 3 or Cyanine 5 fluorescent molecules. Labelled cDNA was hybridized to slides, washed, scanned for fluorescence, background signal was subtracted, and signals were normalized as described previously (Yu and Setter, 2003). Normalized data from duplicate spots within each slide were first averaged to obtain each gene's fluorescence value, and then values from four replicates of each treatment/tissue combination from four replicate plant samples were analysed by SAM (Significance Analysis of Microarrays), a statistical analysis tool (Tusher et al., 2001).
Statistical analysis
Leaf water relations and ABA data presented are means and standard errors of the means of four complete repetitions of the experiment. Independent t-tests were performed on control and chill treatment means for each of the parameters shown in Figs 1 and 2 at each sampling time to determine if the treatment means were significantly different at P<0.05, P<0.01, or were not significantly different. Paired t-tests were performed for within treatment means between sampling times where indicated.
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Water relations
Chilling maize seedling root systems at 5.5 °C resulted in an 80% decrease in stomatal conductance (gs) by 2.5 h into the chilling stress (Fig. 1a). At this time, chill treatment seedlings were severely wilted with extensive leaf rolling characteristic of water-stressed maize. In preliminary experiments, a significant decrease in gs was measured within 0.5 h after imposing the root chilling treatment and minimum gs at approximately 2–3 h into chilling (data not shown).
At 24 h into chilling (approximately 6 h into the photoperiod following a 10 h dark period), chill treatment gs partially recovered to approximately 40% of control treatment gs (Fig. 1a).
Chill treatment leaf water potential (L) decreased significantly below control treatment L by 2.5 h into chilling (Fig. 1b). The large decrease in L and the complete loss of leaf turgor (leaf turgor potential [p] = 0; Fig. 1c) at this time is consistent with the water-stressed appearance (glaucous coloration, partially rolled) of the chill treatment seedlings. The decrease in chill treatment L at 2.5 h occurred despite the large decrease in gs at that time, indicative of a disequilibrium between stomatal control of transpiration and changes in conductance of the pathway for water flux from the soil to transpirational surfaces in the stomatal cavities of the leaves.
By 24 h into the chilling treatment, chill treatment L recovered to near control treatment L (approximately –1.0 MPa). This was accompanied by the complete recovery of p to control p and the disappearance of visible symptoms of water stress in the leaves of chilled plants (Fig. 1c).
At 2.5 h chilling, chill treatment root hydraulic conductance (Lr) decreased 75% compared with control treatment Lr (Fig. 1d). Increased water viscosity accounted for 57% of this decrease in Lr (Table 1). Based on the calculated change in water viscosity from control soil temperature of 25 °C to 5.5 °C (the average chill treatment root temperature at 2.5 h chilling) it was estimated that Lr would have decreased from 0.940 to 0.527 1x10–3 kg kg–1 RFW s–1 MPa–1 due to increased water viscosity at 5.5 °C. The difference between 0.527 1x10–3 kg kg–1 RFW s–1 MPa–1 and the measured chill treatment Lr at 2.5 h chilling, 0.217 1x10–3 kg kg–1 RFW s–1 MPa–1, represents the effect of chilling on one or more components of the pathway for water movement within the root system.
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Between 2.5 h and 24 h into chilling, chill treatment Lr significantly increased (P<0.05), from 25% to 45% of control treatment Lr. This increase in chill treatment Lr represents an internal adjustment in the pathway for water flow within root systems of chilled plants and is not due to changes in water viscosity (root temperatures of chill and control treatment plants remained unchanged from 2.5 h to 24 h into chilling). Because of this upward adjustment of Lr at 24 h chill in the chill treatment, the percentage of the decrease in Lr accounted for by viscosity effects alone increased from 57% to 80%, and the percentage of measured Lr accounted for by the root systems of the chill plants decreased to 20% from 43% (Table 1).
Abscisic acid
Chill treatment mean leaf [ABA] increased almost 40x above control treatment leaf [ABA] by 2.5 h into chilling (Fig. 2a). This increase in leaf [ABA] is consistent with the large decrease in L and complete loss of p in the chill treatment at that time (Fig. 1b, c). At 24 h chill, chill treatment leaf [ABA] had decreased by two-thirds from the concentration at 2.5 h, but was still approximately 12x higher than control treatment leaf [ABA] at that time. Note that at 24 h, chill treatment L stabilized at control treatment L and chill treatment p recovered to prechill p (Fig. 1b, c).
By contrast with leaf ABA concentration, chill treatment root [ABA] increased from 2.5 h to 24 h compared with control treatment root [ABA] (Fig. 2b). At 2.5 h chill, there was a slight, non-significant increase in chill treatment root [ABA] compared with the control treatment. However, at 24 h, chill treatment root [ABA] increased to 6.5 times that of the control treatment.
ABA levels were expressed per unit fresh weight (leaf or root). By doing so, leaf and, possibly, root dehydration may have artificially increased chill treatment [ABA], particularly at 2.5 h chill. To check this, leaf and root [ABA] were expressed on a residual dry weight basis in one of the repetitions of the experiment to eliminate any differences in [ABA] due to dehydration in the chilling treatment. (Residual dry weight is the dry weight of material after extraction with 80% methanol: primarily cell walls, polysaccharides and proteins. This dry weight is unlikely to change during the experiment.) It was found that expressing leaf [ABA] on a residual dry weight basis slightly narrowed treatment differences compared with [ABA] expressed on a fresh weight basis. At 2.5 h, chill treatment leaf [ABA] was 40x greater than the controls on a fresh weight basis, and 16x greater on a residual dry weight basis. The fold increase of chill treatment root [ABA] compared with control treatment root [ABA] was similar whether expressed on a fresh weight or residual dry weight basis. Furthermore, chill effects were similar when expressed on a leaf area basis as on a residual dry weight basis. Hence, tissue dehydration did not alter overall conclusions regarding chill effects on [ABA].
Modelling the water relations
Model predictions of changes in L for different levels of Lr and gs are compared with observed L in Table 2. The model estimates that, without the decrease in gs (gs assumed equal to control values) and at the measured Lr chill treatment L would have reached lethal levels (–3.2 MPa) at 2.5 h chill and severe water stress levels at 24 h chill (L= –1.8 MPa). This demonstrates the critical role that gs plays in moderating decreases in L when there is a large decrease in Lr due to chilling.
L was then modelled based on measured gs at 2.5 h and 24 h chill and Lr at 2.5 h chill (fourth column, Table 2). In this way it was possible to calculate the stomatal contribution to the control of L but without the contribution of partial recovery of Lr measured at 24 h chill. Model predictions of L increased from –3.2 MPa without stomatal control to –0.6 MPa with stomatal control at 2.5 h chill and from –1.8 MPa to –1.3 MPa at 24 h chill. The additional impact on L of the partial recovery of Lr at 24 h (fifth column, Table 2) was estimated. When both measured gs and Lr at 24 h were included in the model calculations, L increased from –1.3 MPa to –0.8 MPa. According to these model calculations, therefore, stomatal control and the recovery of Lr each contributed approximately 50% to the increase in chill treatment L at 24 h. There are some reports that the roots contribute only 50% to whole plant resistance rather than the 70% assumed in the calculations above (Tiekstra et al., 2000). As a check on this assumption, the gs and Lr contributions to L recovery were recalculated assuming equal root and shoot contribution to whole plant resistance. It was found that this had little impact on the model predictions that both gs and increased Lr were critical for the recovery of L in the chill treatment at 24 h. Assuming a 50% contribution of the root system to total plant resistance to water flow, model predictions are that the increase in Lr at 24 h contributed 44% to the recovery of L at that time while stomatal control contributed 56%.
Thus, model results indicated that stomata exerted substantial control over L throughout the 24 h chilling event, without which L would decrease to potentially lethal levels (
–2.2 MPa, 2.5 h chill) or to levels associated with severe water stress (–1.8 MPa, 24 h chill). The increase between 2.5 h and 24 h in the apparent biological component of Lr not only permitted L and turgor to recover, it also led to partial stomatal opening, and hence, recovery of leaf gas exchange.
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Discussion |
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Chilling root systems of maize seedlings to 5.5 ºC rapidly and substantially decreased Lr after just 2.5 h. Part of the chilling-induced lowering of Lr was due to water viscosity, but a significant fraction of the decrease in Lr was due to chilling-induced changes intrinsic to the root system (Fig. 1d; Table 1). Changes in shoot factors followed the same time course as Lr. After 2.5 h of chilling,
L and p decreased (Fig. 1b, c), leaf [ABA] increased (Fig. 2a), and gs decreased (Fig. 1a). Between 2.5 h and 24 h after root zone chilling, Lr partially recovered, L returned to near control L, p returned to control values, and leaf [ABA] declined. Stomatal conductance also increased over this same time period. However, the precise time-course of changes in water relations parameters and leaf [ABA] cannot be determined from just two sampling times.
This pattern of rapid leaf dehydration early in chilling followed by partial or complete recovery of leaf water relations in the ensuing hours has been documented in several chilling-sensitive crop species including maize (Aroca et al., 2001), bean (Phaseolus vulgaris) (Fennel and Markhart, 1998; Pardossi et al., 1992; Vernieri et al., 1991, 2001), and rice (Oryza sativa) (Lee et al., 1993). However, in these studies, both roots and shoots were chilled, so it was not clear whether L decline and subsequent recovery was due to low shoot temperature, low root temperature, or both. Consistent with the idea that shoot chilling alone could explain the effects, Vernieri et al. (1991) showed that chilling delayed ABA accumulation in osmoticum-treated leaf discs, and suggested that such delay may slow stomatal response and, in turn, the ability to maintain L following a decrease in hydraulic conductance due to chilling. However, McWilliam et al. (1982) showed that even when root zones only of bean and cotton were chilled, L decreased to stressful levels within 1–2 h after treatment. The data here suggest that, in maize, when shoots are held at a normal temperature while root zones are chilled, both physical and biological components of Lr rapidly decrease to such an extent that stomata do not respond sufficiently to avoid development of water stress. However, subsequently, decreases in gs together with an increase in the biological component of Lr permit partial recovery of leaf water relations. Although these data suggest that recovery of leaf water relations was largely the result of these changes in gs and Lr, osmotic adjustment in the chill treatment may also have occurred and contributed to the partial recovery of leaf water relations in that treatment at 24 h chill (Premachandra et al., 1992). Chill treatment o at 24 h chill (–1.36 MPa) were slightly though non-significantly lower than control treatment o at that time (–1.23 MPa). The data to correct these measurements to a standard leaf water content for a valid estimate of osmotic adjustment were not available (Campbell et al., 1979). In addition, it was not possible to compare o of the chill and control treatments at a common L at 24 h chill as a check for osmotic adjustment since chill treatment L was also slightly, though non-significantly, lower than control treatment L at that time (Fig. 1b).
In the long term, partial reopening of stomata such that photosynthetic CO2 fixation can resume, may require an increase in Lr, as observed in the present study. Consistent with this, Aroca et al. (2001) reported that a chilling-tolerant maize genotype partially recovered its Lr after 30–54 h of whole-plant chilling, but a chilling-sensitive genotype did not.
Quantitative model
Plant hydraulic conductance and water potential were modelled to provide a quantitative assessment of the role of Lr and gs to changes in water relations during chilling. An implicit assumption of this model is that the hydraulic conductance of the medium surrounding the roots is high. This assumption was valid because a peat moss soilless medium was used that was thoroughly wet at all times.
The model used assumes steady-state liquid water transport from the soil to the leaves. However, under conditions where sap flow and/or L are rapidly changing, this assumption may be invalid and substantial discrepancies between observed water relations parameters and model predictions of those parameters can occur (Li et al., 2002). This was the case at 2.5 h chill where a L of –0.6 MPa was predicted with measured Lr and gs, while the actual L was –2.07 MPa (Table 2). At 2.5 h chill, therefore, it was concluded that the water relations of the chilled plants were not in steady state. This is not surprising because of the rapid and severe decrease in Lr: over 50% of the total decrease in Lr from prechill to 2.5 h chill could be attributed to the immediate increase in viscosity upon chilling (Table 1). Although it was known from preliminary studies that gs of maize seedlings decreases rapidly upon root chilling (0.5 h into chilling, data not shown), the decrease in gs was not sufficiently rapid or large enough to prevent non-steady-state water relations, a large decrease in L, and loss of p that was measured at 2.5 h chill. McWilliam et al. (1982) reported similar data for bean where gs of bean plants with roots chilled to 5 °C rapidly declined to almost zero, but L did not begin to recover for approximately another 10 h into chilling. Despite the non-steady-state conditions in plants at 2.5 h chilling, the large decrease in gs at that time was effective in preventing severe dehydration and leaf death.
Recovery of root hydraulic conductance
Several hypotheses have been proposed to explain Lr recovery with time after chilling. Sanders and Markhart (2001) suggested that, over a period of several days, continued root growth could increase Lr. This is unlikely to account for the recovery of Lr reported here given the short time scale of the experiments (24 h). Increasing the proportion of polyunsaturated fatty acids in membrane lipids can confer tolerance to chilling (Kodama et al., 1995). However, this also requires several days of acclimation at low temperature and is not always effective at conferring chilling tolerance, at least to short-term chilling events (Wu and Browse, 1995; Wu et al., 1997).
Recent reports suggest that aquaporins (major intrinsic proteins that function as water channels) can exert significant control over root water flux in transpiring plants (Kaldenhoff et al., 1998; Martre et al., 2001). If so, changes in aquaporin abundance or activity are plausible mechanisms for Lr recovery with time after chilling. For example, Matre et al. (2002) demonstrated that down-regulation of plasma membrane aquaporin expression in Arabidopsis reduced Lr up to 68%. They also showed that recovery from plant water deficit was more rapid and complete in wild-type plants compared with plants that were modified to reduce aquaporin expression. In conjunction with the present study, cDNA microarrays were used to analyse the transcriptional expression of genes in mature root segments that were sampled from the present experimental material. The transcript levels of eight aquaporins were quantified, among them six plasma membrane intrinsic proteins (GenBank accession numbers AI770766, AI770450, AI833978, AI622338, AI738281, and AI740155). With the false discovery rate set at 16% (Tusher et al., 2001), chilling for 24 h did not significantly up-regulate any of these aquaporins; instead, transcript levels of each of them were marginally lower in the chill treatment compared with the control treatment (not significantly different). These data are consistent with a recent report that maize genotypes differing in chilling sensitivity and in osmotic water permeability of isolated protoplasts did not differ in transcript abundance of the aquaporin ZmPIP1 (Aroca and Chrispeels, 2003). Thus, it appears that the increase in Lr that is induced by chilling does not involve the regulation of these aquaporins at the transcriptional level.
Alternatively, Bigot and Boucaud (2000) proposed that the Lr recovery they observed in chilled Brassica rapa after several h of chilling (Bigot and Boucaud, 1996) may have occurred as a result of increased activity of existing aquaporins. Consistent with this, studies have indicated that water transport activity of a plasma membrane aquaporin is regulated by changes in its phosphorylation state (Johansson et al., 1998). Based on experimental evidence, Bigot and Boucaud (2000) suggested that such an increase in aquaporin activity could occur via a signal cascade that starts with a direct effect of temperature on the plasma membranes of root cells. Other studies suggest that calcium signalling, which is involved in numerous chilling responses, could be involved in regulating aquaporin activity (Javot and Maurel, 2002).
Role of ABA in root and leaf responses
There is conflicting data regarding the role, if any, of ABA on Lr recovery. Several studies have reported increased Lr during chilling when physiologically realistic levels of exogenous ABA are applied to root systems of chilling-sensitive plants (Markhart, 1984; Bigot and Boucaud, 1998; Quintero et al., 1999; Hose et al., 2000). Vernieri et al. (2001), however, reported no relationship between endogenous ABA levels in roots and Lr recovery in chilled bean plants. It has recently been suggested that ABA may increase Lr by increasing the density or activity of aquaporins in membranes within the symplastic pathway for water movement in roots. However, Johansson et al. (1998) reported that ABA did not activate PM28A, an aquaporin found in spinach leaf plasma membranes that is responsive to apoplastic water potential.
It has been reported that changes in gs of maize seedlings during chilling are correlated with xylem [ABA], suggesting that root-sourced ABA is a root-to-shoot signal for stomatal closure in chilled maize seedlings (Janowiak and Doerffling, 1996). Similar observations have been reported for rice, another chilling-sensitive species (Lee et al., 1993). By contrast, Vernieri et al. (2001) reported that changes in gs of bean during chilling were correlated with leaf [ABA] that originated in the leaves and not roots. Root [ABA] increased, but, using techniques to block phloem transport, they demonstrated that the rise in root [ABA] resulted from transport to the root of leaf ABA, or some ‘message’ from the leaves that resulted in ABA synthesis in roots. The data from this study are consistent with the observations of Vernieri et al. (2001). Leaf ABA levels of chilled seedlings rose to high levels at 2.5 h into chilling, coincident with decreased gs (Figs 1a, 2a), while chill and control treatment root [ABA] were similar at 2.5 h chill (Fig. 2b). Leaf turgor potential of chilled seedlings had decreased to zero at this time and it has been shown that low leaf p promotes de novo ABA synthesis (Pierce and Rashke, 1980).
By 24 h into chilling, an increase in chill treatment root [ABA] was measured compared with the control treatment (Fig. 2b). Increased root [ABA] during chilling can occur via direct transport of leaf ABA or indirect transfer of a promoter of root ABA synthesis (Vernieri et al., 2001; Smith and Dale, 1988). Root ABA synthesis during chilling has been reported in rice (Lee et al., 1993) and maize (Janowiak and Doerffling, 1996) although it is delayed by low temperatures and, once initiated, proceeds at a slower rate compared with ABA synthesis at warmer temperatures (Vernieri et al., 1991; Pardossi et al., 1992).
In summary, the present investigation showed that chilling root zones of maize initially decreased Lr which, in turn, led to loss of leaf water status, elevated leaf [ABA], and stomatal closure. Between 2.5 h and 24 h after chilling, root [ABA] increased and the biological component of Lr partially recovered. Stomatal closure early in chilling followed by partial recovery of Lr resulted in improved leaf water status and decreased leaf ABA levels. This permitted partial stomatal opening while maintaining leaf water relations at control levels. A quantitative model provided evidence that temporal changes in both the biological component of root hydraulic conductance and partial stomatal closure were necessary to explain the initial loss of water potential, and its subsequent recovery. No significant up-regulation occurred for several plasma membrane intrinsic aquaporins, indicating that the partial recovery of Lr did not appear to involve regulation of these aquaporins at the transcriptional level. This work points to the need for further research to identify the cellular mechanisms by which Lr recovery is achieved.
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Acknowledgements |
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This research was supported by the US Department of Agriculture Special Grants Agricultural Ecosystems Program. We also gratefully acknowledge the expert technical assistance of Brian Flannigan in this research.
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Footnotes |
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Abbreviations: gs, stomatal conductance to water vapour; Lr, root hydraulic conductance;
L, leaf water potential; p, leaf turgor potential; ABA, abscisic acid. |
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