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Growth of epicotyls, turgor maintenance and osmotic adjustment in pea plants (Pisum sativum L.) subjected to water stress
Field Crops Research 86 (2004) 81–90
Growth of epicotyls, turgor maintenance and osmotic adjustment in pea plants (Pisum sativum L.) subjected to water stress F.J. Sa´ncheza,*, E.F. de Andre´sb, J.L. Tenoriob, L. Ayerbec a
Dpto. Investigacio´n Agraria, Instituto Madrilen˜o de Investigacio´n Agraria y Alimentaria (IMIA), Apartado de Correos 127, 28800 Alcala´ de Henares, Madrid, Spain b Dpto. de Medio Ambiente, Instituto Nacional de Investigacio´n y Tecnologı´a Agraria y Alimentaria (INIA), Apartado de Correos 1045, 28800 Alcala´ de Henares, Madrid, Spain c Centro de Recursos Fitogene´ticos (CRF-INIA), Apartado de Correos 1045, 28800 Alcala´ de Henares, Madrid, Spain Received 30 November 2002; received in revised form 10 May 2003; accepted 23 May 2003
Abstract The growth of pea epicotyls was dramatically reduced when subjected to water stress induced by PEG 6000. The degree of inhibition was proportional to the concentration of PEG, although variability among cultivars was observed. Intraspecific variability in growth under water stress could be due to differences in the osmotic adjustment or turgor maintenance capability of each variety. To test this hypothesis, osmotic adjustment (the difference in Cs at saturation in watered epicotyls and Cs at saturation when epicotyls were at 70% relative water content (RWC), as measured from log Cs against log RWC plots) and turgor maintenance (measured from Cs versus Cw plots as Cw at the point of turgor loss) were calculated in epicotyls. All cultivars were capable of osmotic adjustment from 0.30 to 0.65 MPa, while turgor maintenance varied between 2.436 and 3.906 MPa. A significant correlation between growth and osmotic adjustment, and turgor maintenance was observed, but only at the highest concentrations of PEG assayed. The coefficient of correlation was at 30 mM PEG, r ¼ 0:70 (P < 0:01) and r ¼ 0:79 (P < 0:01), and at 46 mM PEG, r ¼ 0:64 (P < 0:05) and r ¼ 0:89 (P < 0:01) for osmotic adjustment and turgor maintenance, respectively. Water stress induced the accumulation of soluble sugars in epicotyls between 2.8- and 5.1-fold. Their contribution to osmotic adjustment was very important, varying from 34 to 46% depending on cultivar. Free proline in the epicotyls increased between 5- and 50-fold. Its contribution to osmotic adjustment varied from 3 to 5% depending on cultivar. To determine whether osmotic adjustment and turgor maintenance were related in epicotyl and adult stages, a comparison was made between them, and a significant correlation found for turgor maintenance (r ¼ 0:78; P < 0:01). The results obtained indicate that measurements made at early stages of development could be used to identify drought-tolerant genotypes. # 2003 Elsevier B.V. All rights reserved. Keywords: Drought tolerance; Soluble sugars; Proline; Water potential; Osmotic potential
1. Introduction
*
Corresponding author. Tel.: þ34-918-892-943; fax: þ34-918-828-124. E-mail address: [email protected] (F.J. Sa´nchez).
Drought is a multi-factorial syndrome in which water deficit, heat stress and oxidative stress all interact to reinforce one another. Tolerance to drought is therefore a complex phenomenon in which different
0378-4290/$ – see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-4290(03)00121-7
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traits are involved. Among the characteristics that putatively confer drought tolerance, osmotic adjustment has received increasing interest during recent years. Associations between osmotic adjustment and grain yield under water stress in wheat (Morgan et al., 1986; Morgan, 1995; Moustafa et al., 1996), sorgum (Santamaria et al., 1990), chickpea (Morgan et al., 1991) and pea (Rodrı´guez Maribona et al., 1992) have been reported. In this last crop an association between turgor maintenance and harvest index under water stress has been reported (Sa´ nchez et al., 1998). However, the utility of osmotic adjustment as a mechanism of drought tolerance is open to debate. Some authors have suggested that osmotic adjustment is the consequence of reduced growth induced by drought (Munns, 1988). Other authors have pointed out that osmotic adjustment only operates under severe water deficit when crop survival is threatened and the gain in yield is extremely low (Serraj and Sinclair, 2002). On the other hand, it has been reported that osmotic adjustment is an important factor for root elongation in dry soil (Voetberg and Sharp, 1991). Also, it has been suggested that osmotic adjustment decreases lethal water potential and postpones dehydration (Basnayake et al., 1993). A delay in time to death allows a plant to accumulate more photo-assimilates in the grain, thereby increasing its harvest index (Ludlow and Muchow, 1990). Osmotic adjustment involves the active accumulation of inorganic and organic solutes in a cell in response to a fall in the water potential (Cw) of the cell’s environment. As a consequence, the osmotic potential (Cs) of the cell is lowered, which in turn attracts water into the cell, tending to maintain turgor pressure. Generally, osmotic adjustment contributes to turgor maintenance in both shoots and roots when plants experience water deficit. This allows turgor dependent processes such as growth and stomatal activity to continue to progressively lower leaf Cw (Babu et al., 1999). Other factors affecting turgor maintenance in water stress conditions are greater tissue elasticity and smaller cell volume. The relationship between turgor and growth is complex. In agreement with more recent ideas about the mechanism of cell wall extensibility, cell enlargement begins with a reduction, or relaxation of wall stress. As a consequence, turgor pressure and water potential are reduced (turgor is the Newtonian counterforce
to wall stress and is a major component of water potential), and water is drawn into the cell. The result is that the cell enlarges by uptake of water (a reversible process), initiated by a yielding of the wall (an irreversible process). Synthesis and deposition of new wall materials is needed during or after cell enlargement to prevent wall rupture in subsequent growth. Note that wall synthesis per se is insufficient to cause cell enlargement since it does not induce water uptake. A weakening of the load-bearing network in the wall is needed to reduce turgor and thereby cause water uptake (Cosgrove, 1993). Several types of wall polymer rearrangements could plausibly induce wall relaxation and lead to turgor-driven wall expansion. These include weakening of non-covalent bonding between polysaccharides (as postulated for expansions), cleavage of the backbone of major matrix polymers (by endoglucanases, pectinases, transglycosylases and hydroxyl radicals), and breakage of crosslinks between matrix polymers (by esterases) (Cosgrove, 1999). The transcription and activity of all these enzymes (wall loosening agents) is regulated by hormonal, environmental and developmental factors. Thus, it is possible that wall loosening agents may be inhibited in cells with enough turgor potential (Ct) to grow. In this case, no correlation is found between turgor and growth, as is often observed (Munns, 1988), but the lack of a correlation is not sufficient to disprove the role of turgor in growth (Feng et al., 1994). Taking measurements of osmotic adjustment and turgor maintenance is laborious and time consuming. The associated technical difficulties have rendered these characteristics of little use as selection criteria when screening a large number of lines. For this reason, some workers have proposed other drought tolerance traits as selection criteria. The conservation of a high relative water content (RWC) in water stress conditions is usually well correlated with biomass production and grain yield (Matin et al., 1989; Teulat et al., 1997). Conservation of RWC may be attained though osmotic adjustment, more efficient soil water extraction by roots (increase in root length), or reduced transpiration. Selecting for this last character is of limited interest since it implies reduction in stomatal conductance and leaf area, which would have a negative effect on carbon balance (Teulat et al., 1997).
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induced by PEG 6000. Seeds were surface sterilised with 5% commercial bleach for 20 min, washed several times with water, and germinated on moist filter paper in the dark at 25 8C. After 72 h, epicotyl length was measured using an image analyser. Groups of 10 seedlings were transferred to containers with distilled water or four different dilutions of PEG 6000 (15, 25, 30 and 46 mM) and incubated in the dark at 25 8C for 96 h. At the end of this period, the length of the epicotyls was measured again. The seedlings were washed with water and left in containers with distilled water in the dark at 25 8C. After 72 h, the growth of the epicotyls during the recovery from water stress was recorded.
Cultivars with a lower canopy temperature in water stress conditions have a higher grain yield (Blum and Pnuel, 1990; Sa´ nchez et al., 2001). Canopy temperature depends on the stomatal transpiration and light reflecting properties of the leaf. However, as drought progresses, stomata are closed and water loss to the atmosphere occurs though the leaf cuticle without CO2 fixation. Epicuticular wax deposition decreases cuticular permeability and increases crop albedo (Blum, 1996). This paper reports growth, osmotic adjustment and turgor maintenance in epicotyls of 12 pea cultivars under water stress conditions. Osmotic adjustment and turgor maintenance was also examined in adult plants of the same cultivars. The aim of the work was: (i) to study the relationship between growth, turgor maintenance and osmotic adjustment in water stress conditions; (ii) to determine whether the ability of maintaining turgor in water stress conditions is conserved throughout development; (iii) to analyse if the growth of epicotyls may be used as an indirect measure of turgor maintenance and osmotic adjustment.
2.2. Measurement of water and osmotic potential in epicotyls Seeds were germinated as stated above. Three dayold seedlings were placed on dry filter paper in Petri dishes (10 per dish) in the dark at 25 8C. At different times, usually, 0, 3, 7, 10 and 14 days, the epicotyl was cut 0.5 cm from the tip. Five tips were used to determine RWC and five to measure Cw and Cs. RWC was calculated according to the formula RWC ¼ ðFW DWÞ=ðTW DWÞ, where FW is the fresh weight, DW the dry weight and TW the turgid weight. Turgid weight was determined after floating the sample on distilled water for 24 h at 4 8C in darkness. Dry weight was measured after oven drying the sample for 48 h at 80 8C.
2. Materials and methods 2.1. Plant growth An assay was carried out comparing the growth of 12 pea cultivars (cited in Table 1) during water stress
Table 1 Relative growth of epicotyls from 12 pea cultivars subjected to water stressa Cultivar
4 Frisson 6 Desso 9 Fride 12 Desso Filby 16 Cea 40 Ballet 42 Gloton 44 Ascona 49 HR-1 50 Azur 51 Ibiza 53 Moron
PEG 6000 (mM) 0
15 (Cw ¼ 0:15 MPa)
100 100 100 100 100 100 100 100 100 100 100 100
38.31 30.96 29.15 61.62 36.10 16.54 61.34 53.00 43.34 57.86 74.08 50.73
3.82 8.71 10.37 9.46 2.75 1.44 5.22 4.72 4.51 6.43 17.89 6.02
25 (Cw ¼ 0:3 MPa) 6.90 11.15 12.54 20.29 14.87 11.82 20.15 15.94 17.26 14.57 22.36 21.52
0.34 0.08 0.34 2.85 0.51 0.71 1.16 1.43 2.72 3.64 4.60 2.70
30 (Cw ¼ 0:52 MPa) 5.72 6.72 6.64 5.13 9.12 12.17 7.63 11.25 16.39 5.87 5.25 14.79
0.68 3.20 2.09 2.17 0.69 2.29 0.88 1,47 2.50 0.84 0.41 3.82
46 (Cw ¼ 1:5 MPa) 2.42 1.27 1.91 1.97 2.97 6.97 0.43 4.38 7.59 0.26 0.96 3.71
1.30 0.71 0.46 0.29 0.58 0.77 0.06 0.34 1.07 0.16 0.11 0.70
a Different dilutions of PEG 6000 were used to adjust the stress intensity. The results are the mean S:D: of three assays (10 plants per assay).
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Cw was measured in psychrometric chambers connected to a CR7 measurement and control system (Campbell Sci. Ltd., UK), using the isopiestic method (Boyer and Knipling, 1965). Briefly, the thermocouple wire is bent to make a spiral loop containing the measurement junction. A droplet of a NaCl solution of known Cw is placed in the spiral, and the thermocouple introduced into the chamber with the sample. When the voltage output is steady the thermocouple is removed, the drop is cleaned-off, a new droplet is loaded in the spiral, and the thermocouple is reinserted into the chamber. The Cw of the second droplet is chosen in such way that the Cw of the sample is between that of the two droplets. The readings of the dry thermocouple and the thermocouple loaded with the two solutions are used to calculate the Cw of the sample. Cs is measured in the same way but the sample is previously frozen and then thawed. 2.3. Measurement of water and osmotic potential in leaves Plants were grown in 15 cm diameter pots containing vermiculite (three plants per pot) in a controlled environment chamber (23/16 8C day/night, 16 h photoperiod). The plants were watered every 2 or 3 days with tap water and 0.03% Bayfolan (Bayer). When the plants were 24 days old, drought was imposed by withholding water. At the onset and at various times during the water stress period (usually after 4, 7, 10 and 14 days) two opposing leaflets from the first fully expanded leaf were cut (three replicates on each sampling date) and wrapped in aluminium foil to prevent dehydration. When cultivars lacked leaves, stipules were taken. One leaflet or stipule was used for RWC determination and the other for Cw and Cs measurements as described above. 2.4. Compatible solute determination Three day-old seedlings were treated as described for the measurement of water and osmotic potential in epicotyls. At 0, 3, 7 and 14 days after initiating water stress, 0.02 g dry weight of powdered tissue was extracted in 6 ml of distilled water at 70 8C. The suspension was centrifuged at 2000 g for 10 min.
The supernatant was collected for the determination of free proline and sugars. Proline was determined by the ninhydrin method (Troll and Lindsley, 1955), omitting phosphoric acid to avoid interference with concentrated sugars (Magne´ and Larher, 1992). In a total volume of 1.5 ml, the reaction mixture contained 200 ml tissue extract, 11.6 M acetic acid and 98 mM ninhydrin. The test tubes were boiled in a water bath for 45 min and then cooled. The coloured product formed was extracted with 3 ml toluene by vigorous shaking, and the absorbance of the resulting organic layer was measured at 520 nm. A calibration curve was made with L-proline as a standard. Soluble carbohydrates were determined by the anthrone method (Spiro, 1966). An amount of 100 ml tissue extract were added to 3 ml (final volume) assay media containing 1.08 M H2SO4, 1.09 mM thiourea and 2.1 mM anthrone. The mixture was heated at 100 8C for 10 min and absorbance read at 620 nm. A calibration curve was plotted with D-glucose as a standard.
3. Results 3.1. Growth of epicotyls The growth of the epicotyls from 3 day-old pea plantlets was dramatically reduced in the presence of PEG. Control plants grew linearly during the 7 days of the experiment (Fig. 1) but plants exposed to both 30 and 46 mM PEG almost stopped growing. The inhibition of growth was not due to a toxic effect of the PEG: when the plants were removed from the PEG solution and transferred to containers with water, growth continued at a rate similar to that of control plants. The amount of inhibition was proportional to the concentration of PEG (Table 1). Approximately 50% of growth was inhibited by 15 mM PEG (Cw ¼ 0:15 MPa), and continued to decrease as PEG concentration increased, reaching 3% at 46 mM PEG (Cw ¼ 1:5 MPa). Differences in growth were observed among cultivars over the whole range of PEG concentrations used (Table 1). However, this variability was greatest at the highest PEG concentration, in which growth was 30-fold greater in some varieties than in others.
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21 Water PEG 30mM PEG 46 mM
Growth (cm)
16
11
6 H2O
1 0
2
4
6
8
Days Fig. 1. Growth of pea epicotyls in the presence of PEG 6000; 30 and 46 mM PEG are equivalent to a Cs of 0.52 and 1.5 MPa, respectively. After 3 days (arrow) the epicotyls were transferred to water. Each point represents the average of 10 measurements S.D.
3.2. Turgor maintenance in epicotyls Differences in growth under water stress conditions may be due to the turgor maintaining capability of each cultivar. Cw and Cs were measured in epicotyls and turgor maintenance was calculated from plots of Cs against Cw, as Cw at the point of turgor loss. Fig. 2 shows that no correlation was observed between growth and turgor maintenance in the presence of low concentrations of PEG, but when PEG concentration was increased a significant correlation was seen. Probably, at mild stress, all cultivars had enough turgor for growth and differences in growth were caused by other factors. However, when water stress was severe, turgor became the most important factor controlling growth. Solute accumulation in response to drought—osmotic adjustment—is one way to maintain turgor. Osmotic adjustment was measured in epicotyls (from log Cs against log RWC plots) as the difference between Cs at saturation in watered epicotyls and Cs at saturation in epicotyls with 70% RWC. All varieties were capable of osmotic adjustment, which ranged from 0.30 to 0.65 MPa (Table 2). Osmotic adjustment was significantly correlated with growth but with a lower
Table 2 Turgor maintenance (Cw at the point of turgor loss) and osmotic adjustment (Cs at saturation in watered samples minus Cs at saturation when samples had 70% RWC) in epicotyls and leaves of 12 pea cultivarsa Cultivar
Leaves
Epicotyls
Cw tl (MPa)
DCs100 at 70% RWC (MPa)
Cw tl (MPa)
DCs100 at 70% RWC (MPa)
4 6 9 12 16 40 42 44 49 50 51 53
2.385 2.166 1.820 1.917 1.691 2.804 1.557 2.520 3.003 1.662 2.044 2.033
0.225 0.362 0.202 0.569 0.070 0.371 0.125 0.827 0.568 0.206 0.335 0.527
2.949 2.650 2.726 3.317 2.824 3.501 2.795 3.395 3.906 2.436 2.763 3.338
0.488 0.591 0.488 0.490 0.446 0.571 0.467 0.472 0.641 0.300 0.403 0.653
Mean S.D.
2.133 0.461
0.365 0.222
3.050 0.433
0.501 0.101
a
The values of the parameters for each cultivar were calculated from 16 measurements of Cw, Cs and RWC made throughout the dehydration cycle.
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86
PEG 15 mM
PEG 25 mM 51
70
12
60 50
42
53
40
4 16 6
30
9
40
20 10 -4,5
50
44 49
Relative Growth (%)
Relative Growth (%)
80
-4
-3,5
-3
-2,5
-2
24 22 20 18 16 14 12 10 8 6 -4,5
53 49
44
4 -4
-3,5
40 44
10 8
4 -4,5
Relative Growth (%)
Relative Growth (%)
53
12
6 -4
-3,5
-2,5
-2
PEG 46 mM 8
12
-3
Ψ w tl
49
14
50
9 6
PEG 30 mM 16
51
16
40
Ψ w tl
18
42
12
4
-3
16 42 9
6 51 -2,5
50 -2
Ψ w tl
7
49
40
6 5
44
4
53
3
12
2
16 9 51
1 0 -4,5
4
42 -4
-3,5
-3
6 50 -2,5
-2
Ψ w tl
Fig. 2. Relationship between relative growth at different concentrations of PEG and turgor maintenance of epicotyls of 12 pea cultivars. The regression equations at 30 and 46 mM PEG are: y ¼ 7:04x 12:57; r ¼ 0:79; P < 0:01 and y ¼ 4:94x 12:16; r ¼ 0:89; P < 0:01. At 15 and 25 mM PEG, no significant correlations were observed.
correlation coefficient than turgor maintenance (r ¼ 0:70, P < 0:01 at 30 mM PEG and r ¼ 0:64, P < 0:05 at 46 mM PEG). Free proline and soluble sugars were determined to see if these compatible solutes were accumulated during water stress. Fig. 3 shows the soluble sugars and free proline concentration as a function of Cw of one of the cultivars. The other cultivars gave similar results. Total and non-reducing sugars increased proportionally with decreasing Cw over the entire experiment. Reducing sugar concentration increased following the reduction of Cw, but at the end of the experiment, when water stress was more pronounced, it decreased. When total soluble sugar concentration was estimated at the same Cw for all cultivars (2 MPa), a drought-induced stimulation of the sugar level was seen (D½sugar ¼ ½sugar at 2 MPa =½sugar irrigated plants ) from 2.8- to 5.1-fold depending on the cultivar. This stimulation was sig-
nificantly correlated with the osmotic adjustment capability of each variety (y ¼ 11:21 2:13; r ¼ 0:91; P < 0:001). Free proline concentration increased exponentially following the decrease in Cw over the entire duration of the assay. The proline concentration was calculated at the same Cw for all lines (2 MPa). A water stress induced stimulation of proline levels between 5- and 50-fold was seen, depending on variety. This stimulation was significantly correlated with the osmotic adjustment capability of each cultivar (y ¼ 207:66x 89:46; r ¼ 0:86; P < 0:001). 3.3. Turgor maintenance in leaves As shown in Table 2, the average Cw tl was greater in leaves than in epicotyls, which means that the leaves were poorer maintainers of turgor than the epicotyls. Osmotic adjustment was also greater in
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4. Discussion
600 Totals
Sugars (mM)
500
Non reducing Reducing
400 300 200 100 0 -0,5
-1
-1,5
-2
-2,5
-3
Ψw (MPa) 120
Proline (mM)
100 80 60 40 20 0 -0,5
-1
-1,5
-2
-2,5
-3
Ψw (MPa) Fig. 3. Soluble sugars and proline accumulation during water stress in epicotyls of pea variety 40 Ballet. Other cultivars gave similar results. Each point is the average of two observations, where symbols have no associated bar, the error bar is smaller than the symbol size.
epicotyls than leaves, but the difference was not significant (Fisher test). The better performance of epicotyls than leaves in maintaining turgor under water stress conditions was probably due to their higher tissue elasticity. When epicotyl turgor maintenance was plotted against that of the adult stage, a correlation was observed (Fig. 4). The best cultivars at maintaining turgor in the epicotyl stage were also the best turgor maintainers at the adult stage. However, the ability of each cultivar to make osmotic adjustments was not conserved throughout development. No significant correlation was observed between osmotic adjustment in the epicotyl and adult stages in the cultivars studied.
The exposure of 3 day-old plantlets to water stress induced by PEG dramatically reduced their growth (Fig. 1). This inhibition was proportional to PEG concentration and growth was recovered when plants were removed and placed on filter paper moistened with water. Growth inhibition is a general response of plants to soil water deficit. Reduction in leaf growth rate is an early phenomenon which occurs before decreases in leaf Cw or Ct can be detected. It has been suggested that a signal produced by roots in drying soil, probably ABA, is transmitted via the xylem to the leaves, thus affecting their growth rate (Davies and Zhang, 1991; Ali et al., 1999). Apart from non-hydraulic signals, osmotic adjustment is also involved in leaf growth rate under water stress conditions. Lines with high osmoregulatory capacity show more growth than lines with low osmoregulatory capacity (Blum, 1989; Morgan, 1995). A positive relationship between osmotic adjustment, turgor and shoot growth under water stress conditions has been reported in Indian mustard (Wright et al., 1996), especially in early development. Feng et al. (1994) concluded that the overall growth response to water stress is the combined result of its effects on turgor potential and the biological processes controlling yielding stress and extensibility, which may also respond to a biochemical signal from the roots. The results shown in Table 1 indicate that some cultivars were less sensitive to growth inhibition than others when subjected to water stress. Variability in growth under water stress conditions has been reported by other authors. Blum (1989) found differences in growth reduction caused by drought stress in different cultivars of barley. Similar results have been obtained with varieties of sorghum (Premachandra et al., 1992), canola, Indian mustard (Wright et al., 1996), and amaranth (Liu and Stutzel, 2002). An assay was undertaken to determine whether the observed variability in growth inhibition might be due to the differences in osmotic adjustment or turgor maintenance of each cultivar. As shown in Fig. 2, a significant correlation was found between growth and turgor maintenance, but only at the highest levels of water stress (30 and 46 mM PEG 6000). Probably, at low stress intensity (15 and 25 mM PEG) most of the cultivars conserved enough turgor to grow, and the
F.J. Sa´ nchez et al. / Field Crops Research 86 (2004) 81–90
88 -2
50 -2,5
6
9
51
Epicotyls
4 16
-3 12
53
44
42
40 -3,5
49 -4
-4,5 -3,25
-3
-2,75
-2,5
-2,25
-2
-1,75
-1,5
Leaves Fig. 4. Correlation between turgor maintenance (Cw at the point of turgor loss) in epicotyls and adult leaves of 12 pea cultivars. Each value of turgor maintenance was calculated from 16 measurements of Cw and Cs made throughout the dehydration cycle. The regression equation is y ¼ 0:74x 1:48; r ¼ 0:78; P < 0:01.
behaviour of the cultivars depended on cell wall extensibility properties or sensitivity to ABA. However, when the intensity of water stress was increased, most cultivars lost turgor; only those with the best osmotic adjustment or turgor maintaining characteristics were able to grow. The accumulation of compatible solutes was observed in plants subjected to water stress. Soluble sugars increased between 2.8- and 5.1-fold in epicotyls, indicating reserve products accumulated in the seed during the water stress period. Soluble sugars played an important role in osmotic adjustment. According to the van’t Hoff equation, the measured sugar concentration represented between 34 and 46% of the osmotic adjustment observed, depending on cultivar. This result agrees with those of other authors (Turner, 1979; Premachandra et al., 1995), showing that sugar is a major contributor to the Cs of cell sap in many plant species. The free proline concentration also underwent a remarkable increase in water stress conditions by between 5- and 50-fold, depending on cultivar.
However, even after the imposition of stress the proline pool was insufficient to account for a pronounced contribution to the Cs (from 3 to 5% of the osmotic adjustment observed), unless the accumulation was compartmentalised in either the cytosol or the organelles. Some authors have found proline concentration to be higher in the cytoplasm than in the vacuoles (Leigh et al., 1981; Fricke and Pahlich, 1990). In this case, proline could make a more important contribution to the osmotic balance of the cytoplasm. Besides its possible function as compatible solute, proline accumulation during water stress could be involved in the recycling of NADPH (Hare and Cress, 1997). Inhibition of carbon fixation during water stress leads to an increase in the ratio NADPH/NADPþ. Synthesis of proline from glutamate requires the oxidation of two NADPH to NADPþ, which may ameliorate the effects of the reduction of NADPþ caused by stress. By restoring the pool of the terminal electron acceptor of the photosynthetic electron transport chain, proline synthesis may provide some protection against photoinhibition under adverse conditions. Also, proline
F.J. Sa´ nchez et al. / Field Crops Research 86 (2004) 81–90
synthesis may enhance the activity of redox sensitive pathways such as the oxidative pentose phosphate pathway, which is dependent on NADPþ availability and inhibited by NADPH. Table 2 shows that epicotyls were better osmotic adjusters than adult plants, but differences were not significant. In order to compare the osmotic adjustment from the two different experiments, the rate of development of water deficit must be taken into account. The rapid imposition of stress may hinder the synthesis or translocation of osmotic solutes, and the maximum expression of osmotic adjustment will, therefore, not be observed (Basnayake et al., 1996). In this study, the rate of development of water deficit in epicotyls and leaves was 0:13 0:02 and 0:10 0:03 MPa per day, respectively. The rate was therefore very similar in the two assays, and the differences seen in osmotic adjustment were probably not caused by this factor. Although there was no significant difference in osmotic adjustment, turgor maintenance was significantly better in epicotyls than leaves (Table 2). Probably, the greater elasticity of the epicotyl tissue helps maintain turgor. However, a correlation was found between the two stages (Fig. 4). The best turgor maintaining cultivars at the epicotyl stage were also the best turgor maintainers as adults (the same held for the worse turgor maintainers). To date, no other reports have shown this relationship between such distant developmental stages. Moustafa et al. (1996) recorded the preservation of the maintenance of turgor in barley between the tillering and heading stages. These authors distinguished two groups of barley cultivars according to their turgor maintenance at tillering stage. At heading, the leaf Cw at turgor lost was lower than at tillering, but there were no crossovers of cultivars between groups. Unlike turgor maintenance, there was no correlation of osmotic adjustment capability between epicotyls and adult leaves. Chimenti and Hall (1993) reported, however, that osmotic adjustment between different ontogenetic stages is correlated in sunflower, although they did not study such widely separated stages as this study. These authors found a correlation between the eight-leaf and pre-anthesis or post-anthesis stages of r ¼ 0:84 (P ¼ 0:05) and r ¼ 0:88 (P ¼ 0:05), respectively, among seven sunflower cultivars. The maintenance of turgor under water stress is the result of
89
osmotic and elastic adjustment. It is possible that epicotyls and leaves use these two adjustments differentially in response to drought. Thus, it is possible that turgor maintenance can be correlated between epicotyls and leaves, but not osmotic adjustment. The results obtained in this study indicate that measurements of turgor maintenance under water stress conditions made at early stages of development could be used to identify genotypes that can maintain this ability throughout the crop cycle. Screening at the epicotyl stage allows the use of controlled environments where selection can be more efficient and permits the handling of a greater number of lines. The relationship between epicotyl growth at 46 mM PEG and turgor maintenance in epicotyls (r ¼ 0:89, P ¼ 0:01) and adult leaves (r ¼ 0:86, P ¼ 0:01) facilitates the use of this measure as an easy, rapid, cheap and non-destructive screening tool for selecting genotypes with high turgor maintenance capability.
Acknowledgements The authors are grateful to the Instituto Nacional de Investigacio´ n y Tecnologı´a Agraria y Alimentaria (INIA) for financial support of this work (Project No. SC98-019-C2-2) and for providing postdoctoral grants to FJS and EFA.
References Ali, M., Jensen, C.R., Mogensen, V.O., Andersen, M.N., Henson, I.E., 1999. Root signalling and osmotic adjustment during intermittent soil drying sustain grain yield of field grown wheat. Field Crops Res. 62, 35–52. Babu, R.C., Pathan, M.S., Blum, A., Nguyen, H.T., 1999. Comparison of measurement methods of osmotic adjustment in rice cultivars. Crop Sci. 39, 150–158. Basnayake, J., Ludlow, M.M., Cooper, M., Henzell, R.G., 1993. Genotypic variation of osmotic adjustment and desiccation tolerance in contrasting sorghum inbred lines. Field Crops Res. 35, 51–62. Basnayake, J., Cooper, M., Henzell, R.G., Ludlow, M.M., 1996. Influence of rate of development of water deficit on the expression of maximum osmotic adjustment and desiccation tolerance in three grain sorghum lines. Field Crops Res. 49, 65–76. Blum, A., 1989. Osmotic adjustment and growth of barley genotypes under drought stress. Crop Sci. 29, 230–233. Blum, A., 1996. Crop responses to drought and the interpretation of adaptation. Plant Growth Regul. 20, 135–148.
90
F.J. Sa´ nchez et al. / Field Crops Research 86 (2004) 81–90
Blum, A., Pnuel, Y., 1990. Physiological attributes associated with drought resistance of wheat cultivars in a Mediterranean environment. Aust. J. Agric. Res. 41, 799–810. Boyer, J.S., Knipling, E.B., 1965. Isopiestic technique for measuring leaf water potentials with a thermocouple psychrometer. Proc. Natl. Acad. Sci. USA 54, 1044–1051. Cosgrove, D.J., 1993. Wall extensibility: its nature, measurement and relationship to plant cell growth. New Phytol. 124, 1–23. Cosgrove, D.J., 1999. Enzymes and other agents that enhance cell wall extensibility. Ann. Rev. Plant Physiol. Plant Mol. Biol. 50, 391–417. Davies, W.J., Zhang, J., 1991. Root signal and the regulation of growth and development of plants in drying soil. Ann. Rev. Plant Physiol. Plant Mol. Biol. 42, 55–76. Feng, Y., Li, X., Boersma, L., 1994. Roles of osmotic adjustment and turgor potential in leaf area expansion. Physiol. Plant. 90, 1–8. Fricke, W., Pahlich, E., 1990. The effect of water stress on the vacuole–extravacuole compartmentation of proline in potato cell suspension cultures. Physiol. Plant 78, 374–378. Hare, P.D., Cress, W.A., 1997. Metabolic implications of stress induced proline accumulation in plants. Plant Growth Regul. 21, 79–102. Leigh, R.A., Ahmad, N., Wyn Jones, R.G., 1981. Assessment of glycinebetaine and proline compartmentation by analysis of isolated beet vacuoles. Planta 153, 34–41. Liu, F., Stutzel, H., 2002. Leaf water relations of vegetable amaranth (Amaranthus spp.) in response to soil drying. Eur. J. Agron. 16, 137–150. Ludlow, M.M., Muchow, R.C., 1990. A critical evaluation of traits for improving crop yield in water limited environments. Adv. Agron. 43, 107–153. Magne´ , C., Larher, F., 1992. High sugar content of extracts interferes with colorimetric determination of amino acids and free proline. Anal. Biochem. 200, 115–118. Matin, M.A., Brown, J.H., Ferguson, H., 1989. Leaf water potential, relative water content, and diffusive resistance as screening techniques for drought resistance in barley. Agron. J. 81, 100–105. Morgan, J.M., 1995. Growth and yield of wheat lines with differing osmoregulative capacity at high soil water deficit in seasons of varying evaporative demand. Field Crops Res. 40, 143–152. Morgan, J.M., Hare, R.A., Fletcher, R.J., 1986. Genetic variation in osmoregulation in bread and durum wheat and its relationship to grain yield in a range of field environments. Aust. J. Agric. Res. 37, 449–457. Morgan, J.M., Rodrı´guez Maribona, B., Knights, E.J., 1991. Adaptation to water deficit in chickpea breeding lines by osmoregulation: relationship to grain yield in the field. Field Crops Res. 27, 61–70. Moustafa, M.A., Boersma, L., Kronstad, W.E., 1996. Response of four spring wheat cultivars to drought stress. Crop Sci. 36, 982–986.
Munns, R., 1988. Why measure osmotic adjustment? Aust. J. Plant Physiol. 15, 717–726. Premachandra, G.S., Saneoka, H., Fujita, K., Ogata, S., 1992. Leaf water relations, osmotic adjustment, cell membrane stability, epicuticular wax load and growth as affected by increasing water deficits in sorghum. J. Exp. Bot. 43, 1569–1576. Premachandra, G.S., Hahn, D.T., Rhodes, D., Joly, R.J., 1995. Leaf water relations and solute accumulation in two grain sorghum lines exhibiting contrasting drought tolerance. J. Exp. Bot. 46, 1833–1841. Rodrı´guez Maribona, B., Tenorio, J.L., Conde, J.R., Ayerbe, L., 1992. Correlation between yield and osmotic adjustment of peas (Pisum sativum L.) under drought stress. Field Crops Res. 29, 15–22. Sa´ nchez, F.J., Manzanares, M., de Andre´ s, E.F., Tenorio, J.L., Ayerbe, L., 1998. Turgor maintenance, osmotic adjustment and soluble sugar and proline accumulation in 49 pea cultivars in response to water stress. Field Crops Res. 59, 225–235. Sa´ nchez, F.J., Manzanares, M., de Andre´ s, E.F., Tenorio, J.L., Ayerbe, L., 2001. Residual transpiration rate, epicuticular wax load and leaf colour of pea plants in drought conditions. Eur. J. Agron. 15, 57–70. Santamaria, J.M., Ludlow, M.M., Fukai, S., 1990. Contribution of osmotic adjustment to grain yield in Shorgum bicolor (L.) Moench under water limited conditions. I. Water stress before anthesis. Aust. J. Agric. Res. 41, 51–65. Serraj, R., Sinclair, T.R., 2002. Osmolyte accumulation: can it really help increase crop yield under drought conditions? Plant Cell Environ. 25, 333–341. Spiro, R.G., 1966. Analysis of sugars found in glycoproteins. In: Neufeld, E.S., Ginsburg, V. (Eds.), Methods in Enzymology, vol. VIII, Complex Carbohydrates. Academic Press, New York, pp. 3–26. Teulat, B., Monneveux, P., Wery, J., Borries, C., Souyris, I., Charrier, A., This, D., 1997. Relationships between relative water content and growth parameters under water stress in barley: a QTL study. New Phytol. 137, 99–107. Troll, W., Lindsley, J., 1955. A photometric method for the determination of proline. J. Biol. Chem. 215, 655–660. Turner, N.C., 1979. Drought resistance and adaptation to water deficits in crop plants. In: Mussell, H., Staples, R.C. (Eds.), Stress Physiology in Crop Plants. Wiley/Interscience, New York, pp. 343–372. Voetberg, G.S., Sharp, R.E., 1991. Growth of the maize primary root at low water potentials. III. Role of increased proline deposition in osmotic adjustment. Plant Physiol. 96, 1125–1130. Wright, P.R., Morgan, J.M., Jessop, R.S., 1996. Comparative adaptation of canola (Brassica napus) and Indian mustard (B. juncea) to soil water deficits: plant water relations and growth. Field Crops Res. 49, 51–64.
Growth of epicotyls, turgor maintenance and osmotic adjustment in pea plants (Pisum sativum L.) subjected to water stress F.J. Sa´ncheza,*, E.F. de Andre´sb, J.L. Tenoriob, L. Ayerbec a
Dpto. Investigacio´n Agraria, Instituto Madrilen˜o de Investigacio´n Agraria y Alimentaria (IMIA), Apartado de Correos 127, 28800 Alcala´ de Henares, Madrid, Spain b Dpto. de Medio Ambiente, Instituto Nacional de Investigacio´n y Tecnologı´a Agraria y Alimentaria (INIA), Apartado de Correos 1045, 28800 Alcala´ de Henares, Madrid, Spain c Centro de Recursos Fitogene´ticos (CRF-INIA), Apartado de Correos 1045, 28800 Alcala´ de Henares, Madrid, Spain Received 30 November 2002; received in revised form 10 May 2003; accepted 23 May 2003
Abstract The growth of pea epicotyls was dramatically reduced when subjected to water stress induced by PEG 6000. The degree of inhibition was proportional to the concentration of PEG, although variability among cultivars was observed. Intraspecific variability in growth under water stress could be due to differences in the osmotic adjustment or turgor maintenance capability of each variety. To test this hypothesis, osmotic adjustment (the difference in Cs at saturation in watered epicotyls and Cs at saturation when epicotyls were at 70% relative water content (RWC), as measured from log Cs against log RWC plots) and turgor maintenance (measured from Cs versus Cw plots as Cw at the point of turgor loss) were calculated in epicotyls. All cultivars were capable of osmotic adjustment from 0.30 to 0.65 MPa, while turgor maintenance varied between 2.436 and 3.906 MPa. A significant correlation between growth and osmotic adjustment, and turgor maintenance was observed, but only at the highest concentrations of PEG assayed. The coefficient of correlation was at 30 mM PEG, r ¼ 0:70 (P < 0:01) and r ¼ 0:79 (P < 0:01), and at 46 mM PEG, r ¼ 0:64 (P < 0:05) and r ¼ 0:89 (P < 0:01) for osmotic adjustment and turgor maintenance, respectively. Water stress induced the accumulation of soluble sugars in epicotyls between 2.8- and 5.1-fold. Their contribution to osmotic adjustment was very important, varying from 34 to 46% depending on cultivar. Free proline in the epicotyls increased between 5- and 50-fold. Its contribution to osmotic adjustment varied from 3 to 5% depending on cultivar. To determine whether osmotic adjustment and turgor maintenance were related in epicotyl and adult stages, a comparison was made between them, and a significant correlation found for turgor maintenance (r ¼ 0:78; P < 0:01). The results obtained indicate that measurements made at early stages of development could be used to identify drought-tolerant genotypes. # 2003 Elsevier B.V. All rights reserved. Keywords: Drought tolerance; Soluble sugars; Proline; Water potential; Osmotic potential
1. Introduction
*
Corresponding author. Tel.: þ34-918-892-943; fax: þ34-918-828-124. E-mail address: [email protected] (F.J. Sa´nchez).
Drought is a multi-factorial syndrome in which water deficit, heat stress and oxidative stress all interact to reinforce one another. Tolerance to drought is therefore a complex phenomenon in which different
0378-4290/$ – see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-4290(03)00121-7
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traits are involved. Among the characteristics that putatively confer drought tolerance, osmotic adjustment has received increasing interest during recent years. Associations between osmotic adjustment and grain yield under water stress in wheat (Morgan et al., 1986; Morgan, 1995; Moustafa et al., 1996), sorgum (Santamaria et al., 1990), chickpea (Morgan et al., 1991) and pea (Rodrı´guez Maribona et al., 1992) have been reported. In this last crop an association between turgor maintenance and harvest index under water stress has been reported (Sa´ nchez et al., 1998). However, the utility of osmotic adjustment as a mechanism of drought tolerance is open to debate. Some authors have suggested that osmotic adjustment is the consequence of reduced growth induced by drought (Munns, 1988). Other authors have pointed out that osmotic adjustment only operates under severe water deficit when crop survival is threatened and the gain in yield is extremely low (Serraj and Sinclair, 2002). On the other hand, it has been reported that osmotic adjustment is an important factor for root elongation in dry soil (Voetberg and Sharp, 1991). Also, it has been suggested that osmotic adjustment decreases lethal water potential and postpones dehydration (Basnayake et al., 1993). A delay in time to death allows a plant to accumulate more photo-assimilates in the grain, thereby increasing its harvest index (Ludlow and Muchow, 1990). Osmotic adjustment involves the active accumulation of inorganic and organic solutes in a cell in response to a fall in the water potential (Cw) of the cell’s environment. As a consequence, the osmotic potential (Cs) of the cell is lowered, which in turn attracts water into the cell, tending to maintain turgor pressure. Generally, osmotic adjustment contributes to turgor maintenance in both shoots and roots when plants experience water deficit. This allows turgor dependent processes such as growth and stomatal activity to continue to progressively lower leaf Cw (Babu et al., 1999). Other factors affecting turgor maintenance in water stress conditions are greater tissue elasticity and smaller cell volume. The relationship between turgor and growth is complex. In agreement with more recent ideas about the mechanism of cell wall extensibility, cell enlargement begins with a reduction, or relaxation of wall stress. As a consequence, turgor pressure and water potential are reduced (turgor is the Newtonian counterforce
to wall stress and is a major component of water potential), and water is drawn into the cell. The result is that the cell enlarges by uptake of water (a reversible process), initiated by a yielding of the wall (an irreversible process). Synthesis and deposition of new wall materials is needed during or after cell enlargement to prevent wall rupture in subsequent growth. Note that wall synthesis per se is insufficient to cause cell enlargement since it does not induce water uptake. A weakening of the load-bearing network in the wall is needed to reduce turgor and thereby cause water uptake (Cosgrove, 1993). Several types of wall polymer rearrangements could plausibly induce wall relaxation and lead to turgor-driven wall expansion. These include weakening of non-covalent bonding between polysaccharides (as postulated for expansions), cleavage of the backbone of major matrix polymers (by endoglucanases, pectinases, transglycosylases and hydroxyl radicals), and breakage of crosslinks between matrix polymers (by esterases) (Cosgrove, 1999). The transcription and activity of all these enzymes (wall loosening agents) is regulated by hormonal, environmental and developmental factors. Thus, it is possible that wall loosening agents may be inhibited in cells with enough turgor potential (Ct) to grow. In this case, no correlation is found between turgor and growth, as is often observed (Munns, 1988), but the lack of a correlation is not sufficient to disprove the role of turgor in growth (Feng et al., 1994). Taking measurements of osmotic adjustment and turgor maintenance is laborious and time consuming. The associated technical difficulties have rendered these characteristics of little use as selection criteria when screening a large number of lines. For this reason, some workers have proposed other drought tolerance traits as selection criteria. The conservation of a high relative water content (RWC) in water stress conditions is usually well correlated with biomass production and grain yield (Matin et al., 1989; Teulat et al., 1997). Conservation of RWC may be attained though osmotic adjustment, more efficient soil water extraction by roots (increase in root length), or reduced transpiration. Selecting for this last character is of limited interest since it implies reduction in stomatal conductance and leaf area, which would have a negative effect on carbon balance (Teulat et al., 1997).
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induced by PEG 6000. Seeds were surface sterilised with 5% commercial bleach for 20 min, washed several times with water, and germinated on moist filter paper in the dark at 25 8C. After 72 h, epicotyl length was measured using an image analyser. Groups of 10 seedlings were transferred to containers with distilled water or four different dilutions of PEG 6000 (15, 25, 30 and 46 mM) and incubated in the dark at 25 8C for 96 h. At the end of this period, the length of the epicotyls was measured again. The seedlings were washed with water and left in containers with distilled water in the dark at 25 8C. After 72 h, the growth of the epicotyls during the recovery from water stress was recorded.
Cultivars with a lower canopy temperature in water stress conditions have a higher grain yield (Blum and Pnuel, 1990; Sa´ nchez et al., 2001). Canopy temperature depends on the stomatal transpiration and light reflecting properties of the leaf. However, as drought progresses, stomata are closed and water loss to the atmosphere occurs though the leaf cuticle without CO2 fixation. Epicuticular wax deposition decreases cuticular permeability and increases crop albedo (Blum, 1996). This paper reports growth, osmotic adjustment and turgor maintenance in epicotyls of 12 pea cultivars under water stress conditions. Osmotic adjustment and turgor maintenance was also examined in adult plants of the same cultivars. The aim of the work was: (i) to study the relationship between growth, turgor maintenance and osmotic adjustment in water stress conditions; (ii) to determine whether the ability of maintaining turgor in water stress conditions is conserved throughout development; (iii) to analyse if the growth of epicotyls may be used as an indirect measure of turgor maintenance and osmotic adjustment.
2.2. Measurement of water and osmotic potential in epicotyls Seeds were germinated as stated above. Three dayold seedlings were placed on dry filter paper in Petri dishes (10 per dish) in the dark at 25 8C. At different times, usually, 0, 3, 7, 10 and 14 days, the epicotyl was cut 0.5 cm from the tip. Five tips were used to determine RWC and five to measure Cw and Cs. RWC was calculated according to the formula RWC ¼ ðFW DWÞ=ðTW DWÞ, where FW is the fresh weight, DW the dry weight and TW the turgid weight. Turgid weight was determined after floating the sample on distilled water for 24 h at 4 8C in darkness. Dry weight was measured after oven drying the sample for 48 h at 80 8C.
2. Materials and methods 2.1. Plant growth An assay was carried out comparing the growth of 12 pea cultivars (cited in Table 1) during water stress
Table 1 Relative growth of epicotyls from 12 pea cultivars subjected to water stressa Cultivar
4 Frisson 6 Desso 9 Fride 12 Desso Filby 16 Cea 40 Ballet 42 Gloton 44 Ascona 49 HR-1 50 Azur 51 Ibiza 53 Moron
PEG 6000 (mM) 0
15 (Cw ¼ 0:15 MPa)
100 100 100 100 100 100 100 100 100 100 100 100
38.31 30.96 29.15 61.62 36.10 16.54 61.34 53.00 43.34 57.86 74.08 50.73
3.82 8.71 10.37 9.46 2.75 1.44 5.22 4.72 4.51 6.43 17.89 6.02
25 (Cw ¼ 0:3 MPa) 6.90 11.15 12.54 20.29 14.87 11.82 20.15 15.94 17.26 14.57 22.36 21.52
0.34 0.08 0.34 2.85 0.51 0.71 1.16 1.43 2.72 3.64 4.60 2.70
30 (Cw ¼ 0:52 MPa) 5.72 6.72 6.64 5.13 9.12 12.17 7.63 11.25 16.39 5.87 5.25 14.79
0.68 3.20 2.09 2.17 0.69 2.29 0.88 1,47 2.50 0.84 0.41 3.82
46 (Cw ¼ 1:5 MPa) 2.42 1.27 1.91 1.97 2.97 6.97 0.43 4.38 7.59 0.26 0.96 3.71
1.30 0.71 0.46 0.29 0.58 0.77 0.06 0.34 1.07 0.16 0.11 0.70
a Different dilutions of PEG 6000 were used to adjust the stress intensity. The results are the mean S:D: of three assays (10 plants per assay).
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Cw was measured in psychrometric chambers connected to a CR7 measurement and control system (Campbell Sci. Ltd., UK), using the isopiestic method (Boyer and Knipling, 1965). Briefly, the thermocouple wire is bent to make a spiral loop containing the measurement junction. A droplet of a NaCl solution of known Cw is placed in the spiral, and the thermocouple introduced into the chamber with the sample. When the voltage output is steady the thermocouple is removed, the drop is cleaned-off, a new droplet is loaded in the spiral, and the thermocouple is reinserted into the chamber. The Cw of the second droplet is chosen in such way that the Cw of the sample is between that of the two droplets. The readings of the dry thermocouple and the thermocouple loaded with the two solutions are used to calculate the Cw of the sample. Cs is measured in the same way but the sample is previously frozen and then thawed. 2.3. Measurement of water and osmotic potential in leaves Plants were grown in 15 cm diameter pots containing vermiculite (three plants per pot) in a controlled environment chamber (23/16 8C day/night, 16 h photoperiod). The plants were watered every 2 or 3 days with tap water and 0.03% Bayfolan (Bayer). When the plants were 24 days old, drought was imposed by withholding water. At the onset and at various times during the water stress period (usually after 4, 7, 10 and 14 days) two opposing leaflets from the first fully expanded leaf were cut (three replicates on each sampling date) and wrapped in aluminium foil to prevent dehydration. When cultivars lacked leaves, stipules were taken. One leaflet or stipule was used for RWC determination and the other for Cw and Cs measurements as described above. 2.4. Compatible solute determination Three day-old seedlings were treated as described for the measurement of water and osmotic potential in epicotyls. At 0, 3, 7 and 14 days after initiating water stress, 0.02 g dry weight of powdered tissue was extracted in 6 ml of distilled water at 70 8C. The suspension was centrifuged at 2000 g for 10 min.
The supernatant was collected for the determination of free proline and sugars. Proline was determined by the ninhydrin method (Troll and Lindsley, 1955), omitting phosphoric acid to avoid interference with concentrated sugars (Magne´ and Larher, 1992). In a total volume of 1.5 ml, the reaction mixture contained 200 ml tissue extract, 11.6 M acetic acid and 98 mM ninhydrin. The test tubes were boiled in a water bath for 45 min and then cooled. The coloured product formed was extracted with 3 ml toluene by vigorous shaking, and the absorbance of the resulting organic layer was measured at 520 nm. A calibration curve was made with L-proline as a standard. Soluble carbohydrates were determined by the anthrone method (Spiro, 1966). An amount of 100 ml tissue extract were added to 3 ml (final volume) assay media containing 1.08 M H2SO4, 1.09 mM thiourea and 2.1 mM anthrone. The mixture was heated at 100 8C for 10 min and absorbance read at 620 nm. A calibration curve was plotted with D-glucose as a standard.
3. Results 3.1. Growth of epicotyls The growth of the epicotyls from 3 day-old pea plantlets was dramatically reduced in the presence of PEG. Control plants grew linearly during the 7 days of the experiment (Fig. 1) but plants exposed to both 30 and 46 mM PEG almost stopped growing. The inhibition of growth was not due to a toxic effect of the PEG: when the plants were removed from the PEG solution and transferred to containers with water, growth continued at a rate similar to that of control plants. The amount of inhibition was proportional to the concentration of PEG (Table 1). Approximately 50% of growth was inhibited by 15 mM PEG (Cw ¼ 0:15 MPa), and continued to decrease as PEG concentration increased, reaching 3% at 46 mM PEG (Cw ¼ 1:5 MPa). Differences in growth were observed among cultivars over the whole range of PEG concentrations used (Table 1). However, this variability was greatest at the highest PEG concentration, in which growth was 30-fold greater in some varieties than in others.
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21 Water PEG 30mM PEG 46 mM
Growth (cm)
16
11
6 H2O
1 0
2
4
6
8
Days Fig. 1. Growth of pea epicotyls in the presence of PEG 6000; 30 and 46 mM PEG are equivalent to a Cs of 0.52 and 1.5 MPa, respectively. After 3 days (arrow) the epicotyls were transferred to water. Each point represents the average of 10 measurements S.D.
3.2. Turgor maintenance in epicotyls Differences in growth under water stress conditions may be due to the turgor maintaining capability of each cultivar. Cw and Cs were measured in epicotyls and turgor maintenance was calculated from plots of Cs against Cw, as Cw at the point of turgor loss. Fig. 2 shows that no correlation was observed between growth and turgor maintenance in the presence of low concentrations of PEG, but when PEG concentration was increased a significant correlation was seen. Probably, at mild stress, all cultivars had enough turgor for growth and differences in growth were caused by other factors. However, when water stress was severe, turgor became the most important factor controlling growth. Solute accumulation in response to drought—osmotic adjustment—is one way to maintain turgor. Osmotic adjustment was measured in epicotyls (from log Cs against log RWC plots) as the difference between Cs at saturation in watered epicotyls and Cs at saturation in epicotyls with 70% RWC. All varieties were capable of osmotic adjustment, which ranged from 0.30 to 0.65 MPa (Table 2). Osmotic adjustment was significantly correlated with growth but with a lower
Table 2 Turgor maintenance (Cw at the point of turgor loss) and osmotic adjustment (Cs at saturation in watered samples minus Cs at saturation when samples had 70% RWC) in epicotyls and leaves of 12 pea cultivarsa Cultivar
Leaves
Epicotyls
Cw tl (MPa)
DCs100 at 70% RWC (MPa)
Cw tl (MPa)
DCs100 at 70% RWC (MPa)
4 6 9 12 16 40 42 44 49 50 51 53
2.385 2.166 1.820 1.917 1.691 2.804 1.557 2.520 3.003 1.662 2.044 2.033
0.225 0.362 0.202 0.569 0.070 0.371 0.125 0.827 0.568 0.206 0.335 0.527
2.949 2.650 2.726 3.317 2.824 3.501 2.795 3.395 3.906 2.436 2.763 3.338
0.488 0.591 0.488 0.490 0.446 0.571 0.467 0.472 0.641 0.300 0.403 0.653
Mean S.D.
2.133 0.461
0.365 0.222
3.050 0.433
0.501 0.101
a
The values of the parameters for each cultivar were calculated from 16 measurements of Cw, Cs and RWC made throughout the dehydration cycle.
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PEG 15 mM
PEG 25 mM 51
70
12
60 50
42
53
40
4 16 6
30
9
40
20 10 -4,5
50
44 49
Relative Growth (%)
Relative Growth (%)
80
-4
-3,5
-3
-2,5
-2
24 22 20 18 16 14 12 10 8 6 -4,5
53 49
44
4 -4
-3,5
40 44
10 8
4 -4,5
Relative Growth (%)
Relative Growth (%)
53
12
6 -4
-3,5
-2,5
-2
PEG 46 mM 8
12
-3
Ψ w tl
49
14
50
9 6
PEG 30 mM 16
51
16
40
Ψ w tl
18
42
12
4
-3
16 42 9
6 51 -2,5
50 -2
Ψ w tl
7
49
40
6 5
44
4
53
3
12
2
16 9 51
1 0 -4,5
4
42 -4
-3,5
-3
6 50 -2,5
-2
Ψ w tl
Fig. 2. Relationship between relative growth at different concentrations of PEG and turgor maintenance of epicotyls of 12 pea cultivars. The regression equations at 30 and 46 mM PEG are: y ¼ 7:04x 12:57; r ¼ 0:79; P < 0:01 and y ¼ 4:94x 12:16; r ¼ 0:89; P < 0:01. At 15 and 25 mM PEG, no significant correlations were observed.
correlation coefficient than turgor maintenance (r ¼ 0:70, P < 0:01 at 30 mM PEG and r ¼ 0:64, P < 0:05 at 46 mM PEG). Free proline and soluble sugars were determined to see if these compatible solutes were accumulated during water stress. Fig. 3 shows the soluble sugars and free proline concentration as a function of Cw of one of the cultivars. The other cultivars gave similar results. Total and non-reducing sugars increased proportionally with decreasing Cw over the entire experiment. Reducing sugar concentration increased following the reduction of Cw, but at the end of the experiment, when water stress was more pronounced, it decreased. When total soluble sugar concentration was estimated at the same Cw for all cultivars (2 MPa), a drought-induced stimulation of the sugar level was seen (D½sugar ¼ ½sugar at 2 MPa =½sugar irrigated plants ) from 2.8- to 5.1-fold depending on the cultivar. This stimulation was sig-
nificantly correlated with the osmotic adjustment capability of each variety (y ¼ 11:21 2:13; r ¼ 0:91; P < 0:001). Free proline concentration increased exponentially following the decrease in Cw over the entire duration of the assay. The proline concentration was calculated at the same Cw for all lines (2 MPa). A water stress induced stimulation of proline levels between 5- and 50-fold was seen, depending on variety. This stimulation was significantly correlated with the osmotic adjustment capability of each cultivar (y ¼ 207:66x 89:46; r ¼ 0:86; P < 0:001). 3.3. Turgor maintenance in leaves As shown in Table 2, the average Cw tl was greater in leaves than in epicotyls, which means that the leaves were poorer maintainers of turgor than the epicotyls. Osmotic adjustment was also greater in
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4. Discussion
600 Totals
Sugars (mM)
500
Non reducing Reducing
400 300 200 100 0 -0,5
-1
-1,5
-2
-2,5
-3
Ψw (MPa) 120
Proline (mM)
100 80 60 40 20 0 -0,5
-1
-1,5
-2
-2,5
-3
Ψw (MPa) Fig. 3. Soluble sugars and proline accumulation during water stress in epicotyls of pea variety 40 Ballet. Other cultivars gave similar results. Each point is the average of two observations, where symbols have no associated bar, the error bar is smaller than the symbol size.
epicotyls than leaves, but the difference was not significant (Fisher test). The better performance of epicotyls than leaves in maintaining turgor under water stress conditions was probably due to their higher tissue elasticity. When epicotyl turgor maintenance was plotted against that of the adult stage, a correlation was observed (Fig. 4). The best cultivars at maintaining turgor in the epicotyl stage were also the best turgor maintainers at the adult stage. However, the ability of each cultivar to make osmotic adjustments was not conserved throughout development. No significant correlation was observed between osmotic adjustment in the epicotyl and adult stages in the cultivars studied.
The exposure of 3 day-old plantlets to water stress induced by PEG dramatically reduced their growth (Fig. 1). This inhibition was proportional to PEG concentration and growth was recovered when plants were removed and placed on filter paper moistened with water. Growth inhibition is a general response of plants to soil water deficit. Reduction in leaf growth rate is an early phenomenon which occurs before decreases in leaf Cw or Ct can be detected. It has been suggested that a signal produced by roots in drying soil, probably ABA, is transmitted via the xylem to the leaves, thus affecting their growth rate (Davies and Zhang, 1991; Ali et al., 1999). Apart from non-hydraulic signals, osmotic adjustment is also involved in leaf growth rate under water stress conditions. Lines with high osmoregulatory capacity show more growth than lines with low osmoregulatory capacity (Blum, 1989; Morgan, 1995). A positive relationship between osmotic adjustment, turgor and shoot growth under water stress conditions has been reported in Indian mustard (Wright et al., 1996), especially in early development. Feng et al. (1994) concluded that the overall growth response to water stress is the combined result of its effects on turgor potential and the biological processes controlling yielding stress and extensibility, which may also respond to a biochemical signal from the roots. The results shown in Table 1 indicate that some cultivars were less sensitive to growth inhibition than others when subjected to water stress. Variability in growth under water stress conditions has been reported by other authors. Blum (1989) found differences in growth reduction caused by drought stress in different cultivars of barley. Similar results have been obtained with varieties of sorghum (Premachandra et al., 1992), canola, Indian mustard (Wright et al., 1996), and amaranth (Liu and Stutzel, 2002). An assay was undertaken to determine whether the observed variability in growth inhibition might be due to the differences in osmotic adjustment or turgor maintenance of each cultivar. As shown in Fig. 2, a significant correlation was found between growth and turgor maintenance, but only at the highest levels of water stress (30 and 46 mM PEG 6000). Probably, at low stress intensity (15 and 25 mM PEG) most of the cultivars conserved enough turgor to grow, and the
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88 -2
50 -2,5
6
9
51
Epicotyls
4 16
-3 12
53
44
42
40 -3,5
49 -4
-4,5 -3,25
-3
-2,75
-2,5
-2,25
-2
-1,75
-1,5
Leaves Fig. 4. Correlation between turgor maintenance (Cw at the point of turgor loss) in epicotyls and adult leaves of 12 pea cultivars. Each value of turgor maintenance was calculated from 16 measurements of Cw and Cs made throughout the dehydration cycle. The regression equation is y ¼ 0:74x 1:48; r ¼ 0:78; P < 0:01.
behaviour of the cultivars depended on cell wall extensibility properties or sensitivity to ABA. However, when the intensity of water stress was increased, most cultivars lost turgor; only those with the best osmotic adjustment or turgor maintaining characteristics were able to grow. The accumulation of compatible solutes was observed in plants subjected to water stress. Soluble sugars increased between 2.8- and 5.1-fold in epicotyls, indicating reserve products accumulated in the seed during the water stress period. Soluble sugars played an important role in osmotic adjustment. According to the van’t Hoff equation, the measured sugar concentration represented between 34 and 46% of the osmotic adjustment observed, depending on cultivar. This result agrees with those of other authors (Turner, 1979; Premachandra et al., 1995), showing that sugar is a major contributor to the Cs of cell sap in many plant species. The free proline concentration also underwent a remarkable increase in water stress conditions by between 5- and 50-fold, depending on cultivar.
However, even after the imposition of stress the proline pool was insufficient to account for a pronounced contribution to the Cs (from 3 to 5% of the osmotic adjustment observed), unless the accumulation was compartmentalised in either the cytosol or the organelles. Some authors have found proline concentration to be higher in the cytoplasm than in the vacuoles (Leigh et al., 1981; Fricke and Pahlich, 1990). In this case, proline could make a more important contribution to the osmotic balance of the cytoplasm. Besides its possible function as compatible solute, proline accumulation during water stress could be involved in the recycling of NADPH (Hare and Cress, 1997). Inhibition of carbon fixation during water stress leads to an increase in the ratio NADPH/NADPþ. Synthesis of proline from glutamate requires the oxidation of two NADPH to NADPþ, which may ameliorate the effects of the reduction of NADPþ caused by stress. By restoring the pool of the terminal electron acceptor of the photosynthetic electron transport chain, proline synthesis may provide some protection against photoinhibition under adverse conditions. Also, proline
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synthesis may enhance the activity of redox sensitive pathways such as the oxidative pentose phosphate pathway, which is dependent on NADPþ availability and inhibited by NADPH. Table 2 shows that epicotyls were better osmotic adjusters than adult plants, but differences were not significant. In order to compare the osmotic adjustment from the two different experiments, the rate of development of water deficit must be taken into account. The rapid imposition of stress may hinder the synthesis or translocation of osmotic solutes, and the maximum expression of osmotic adjustment will, therefore, not be observed (Basnayake et al., 1996). In this study, the rate of development of water deficit in epicotyls and leaves was 0:13 0:02 and 0:10 0:03 MPa per day, respectively. The rate was therefore very similar in the two assays, and the differences seen in osmotic adjustment were probably not caused by this factor. Although there was no significant difference in osmotic adjustment, turgor maintenance was significantly better in epicotyls than leaves (Table 2). Probably, the greater elasticity of the epicotyl tissue helps maintain turgor. However, a correlation was found between the two stages (Fig. 4). The best turgor maintaining cultivars at the epicotyl stage were also the best turgor maintainers as adults (the same held for the worse turgor maintainers). To date, no other reports have shown this relationship between such distant developmental stages. Moustafa et al. (1996) recorded the preservation of the maintenance of turgor in barley between the tillering and heading stages. These authors distinguished two groups of barley cultivars according to their turgor maintenance at tillering stage. At heading, the leaf Cw at turgor lost was lower than at tillering, but there were no crossovers of cultivars between groups. Unlike turgor maintenance, there was no correlation of osmotic adjustment capability between epicotyls and adult leaves. Chimenti and Hall (1993) reported, however, that osmotic adjustment between different ontogenetic stages is correlated in sunflower, although they did not study such widely separated stages as this study. These authors found a correlation between the eight-leaf and pre-anthesis or post-anthesis stages of r ¼ 0:84 (P ¼ 0:05) and r ¼ 0:88 (P ¼ 0:05), respectively, among seven sunflower cultivars. The maintenance of turgor under water stress is the result of
89
osmotic and elastic adjustment. It is possible that epicotyls and leaves use these two adjustments differentially in response to drought. Thus, it is possible that turgor maintenance can be correlated between epicotyls and leaves, but not osmotic adjustment. The results obtained in this study indicate that measurements of turgor maintenance under water stress conditions made at early stages of development could be used to identify genotypes that can maintain this ability throughout the crop cycle. Screening at the epicotyl stage allows the use of controlled environments where selection can be more efficient and permits the handling of a greater number of lines. The relationship between epicotyl growth at 46 mM PEG and turgor maintenance in epicotyls (r ¼ 0:89, P ¼ 0:01) and adult leaves (r ¼ 0:86, P ¼ 0:01) facilitates the use of this measure as an easy, rapid, cheap and non-destructive screening tool for selecting genotypes with high turgor maintenance capability.
Acknowledgements The authors are grateful to the Instituto Nacional de Investigacio´ n y Tecnologı´a Agraria y Alimentaria (INIA) for financial support of this work (Project No. SC98-019-C2-2) and for providing postdoctoral grants to FJS and EFA.
References Ali, M., Jensen, C.R., Mogensen, V.O., Andersen, M.N., Henson, I.E., 1999. Root signalling and osmotic adjustment during intermittent soil drying sustain grain yield of field grown wheat. Field Crops Res. 62, 35–52. Babu, R.C., Pathan, M.S., Blum, A., Nguyen, H.T., 1999. Comparison of measurement methods of osmotic adjustment in rice cultivars. Crop Sci. 39, 150–158. Basnayake, J., Ludlow, M.M., Cooper, M., Henzell, R.G., 1993. Genotypic variation of osmotic adjustment and desiccation tolerance in contrasting sorghum inbred lines. Field Crops Res. 35, 51–62. Basnayake, J., Cooper, M., Henzell, R.G., Ludlow, M.M., 1996. Influence of rate of development of water deficit on the expression of maximum osmotic adjustment and desiccation tolerance in three grain sorghum lines. Field Crops Res. 49, 65–76. Blum, A., 1989. Osmotic adjustment and growth of barley genotypes under drought stress. Crop Sci. 29, 230–233. Blum, A., 1996. Crop responses to drought and the interpretation of adaptation. Plant Growth Regul. 20, 135–148.
90
F.J. Sa´ nchez et al. / Field Crops Research 86 (2004) 81–90
Blum, A., Pnuel, Y., 1990. Physiological attributes associated with drought resistance of wheat cultivars in a Mediterranean environment. Aust. J. Agric. Res. 41, 799–810. Boyer, J.S., Knipling, E.B., 1965. Isopiestic technique for measuring leaf water potentials with a thermocouple psychrometer. Proc. Natl. Acad. Sci. USA 54, 1044–1051. Cosgrove, D.J., 1993. Wall extensibility: its nature, measurement and relationship to plant cell growth. New Phytol. 124, 1–23. Cosgrove, D.J., 1999. Enzymes and other agents that enhance cell wall extensibility. Ann. Rev. Plant Physiol. Plant Mol. Biol. 50, 391–417. Davies, W.J., Zhang, J., 1991. Root signal and the regulation of growth and development of plants in drying soil. Ann. Rev. Plant Physiol. Plant Mol. Biol. 42, 55–76. Feng, Y., Li, X., Boersma, L., 1994. Roles of osmotic adjustment and turgor potential in leaf area expansion. Physiol. Plant. 90, 1–8. Fricke, W., Pahlich, E., 1990. The effect of water stress on the vacuole–extravacuole compartmentation of proline in potato cell suspension cultures. Physiol. Plant 78, 374–378. Hare, P.D., Cress, W.A., 1997. Metabolic implications of stress induced proline accumulation in plants. Plant Growth Regul. 21, 79–102. Leigh, R.A., Ahmad, N., Wyn Jones, R.G., 1981. Assessment of glycinebetaine and proline compartmentation by analysis of isolated beet vacuoles. Planta 153, 34–41. Liu, F., Stutzel, H., 2002. Leaf water relations of vegetable amaranth (Amaranthus spp.) in response to soil drying. Eur. J. Agron. 16, 137–150. Ludlow, M.M., Muchow, R.C., 1990. A critical evaluation of traits for improving crop yield in water limited environments. Adv. Agron. 43, 107–153. Magne´ , C., Larher, F., 1992. High sugar content of extracts interferes with colorimetric determination of amino acids and free proline. Anal. Biochem. 200, 115–118. Matin, M.A., Brown, J.H., Ferguson, H., 1989. Leaf water potential, relative water content, and diffusive resistance as screening techniques for drought resistance in barley. Agron. J. 81, 100–105. Morgan, J.M., 1995. Growth and yield of wheat lines with differing osmoregulative capacity at high soil water deficit in seasons of varying evaporative demand. Field Crops Res. 40, 143–152. Morgan, J.M., Hare, R.A., Fletcher, R.J., 1986. Genetic variation in osmoregulation in bread and durum wheat and its relationship to grain yield in a range of field environments. Aust. J. Agric. Res. 37, 449–457. Morgan, J.M., Rodrı´guez Maribona, B., Knights, E.J., 1991. Adaptation to water deficit in chickpea breeding lines by osmoregulation: relationship to grain yield in the field. Field Crops Res. 27, 61–70. Moustafa, M.A., Boersma, L., Kronstad, W.E., 1996. Response of four spring wheat cultivars to drought stress. Crop Sci. 36, 982–986.
Munns, R., 1988. Why measure osmotic adjustment? Aust. J. Plant Physiol. 15, 717–726. Premachandra, G.S., Saneoka, H., Fujita, K., Ogata, S., 1992. Leaf water relations, osmotic adjustment, cell membrane stability, epicuticular wax load and growth as affected by increasing water deficits in sorghum. J. Exp. Bot. 43, 1569–1576. Premachandra, G.S., Hahn, D.T., Rhodes, D., Joly, R.J., 1995. Leaf water relations and solute accumulation in two grain sorghum lines exhibiting contrasting drought tolerance. J. Exp. Bot. 46, 1833–1841. Rodrı´guez Maribona, B., Tenorio, J.L., Conde, J.R., Ayerbe, L., 1992. Correlation between yield and osmotic adjustment of peas (Pisum sativum L.) under drought stress. Field Crops Res. 29, 15–22. Sa´ nchez, F.J., Manzanares, M., de Andre´ s, E.F., Tenorio, J.L., Ayerbe, L., 1998. Turgor maintenance, osmotic adjustment and soluble sugar and proline accumulation in 49 pea cultivars in response to water stress. Field Crops Res. 59, 225–235. Sa´ nchez, F.J., Manzanares, M., de Andre´ s, E.F., Tenorio, J.L., Ayerbe, L., 2001. Residual transpiration rate, epicuticular wax load and leaf colour of pea plants in drought conditions. Eur. J. Agron. 15, 57–70. Santamaria, J.M., Ludlow, M.M., Fukai, S., 1990. Contribution of osmotic adjustment to grain yield in Shorgum bicolor (L.) Moench under water limited conditions. I. Water stress before anthesis. Aust. J. Agric. Res. 41, 51–65. Serraj, R., Sinclair, T.R., 2002. Osmolyte accumulation: can it really help increase crop yield under drought conditions? Plant Cell Environ. 25, 333–341. Spiro, R.G., 1966. Analysis of sugars found in glycoproteins. In: Neufeld, E.S., Ginsburg, V. (Eds.), Methods in Enzymology, vol. VIII, Complex Carbohydrates. Academic Press, New York, pp. 3–26. Teulat, B., Monneveux, P., Wery, J., Borries, C., Souyris, I., Charrier, A., This, D., 1997. Relationships between relative water content and growth parameters under water stress in barley: a QTL study. New Phytol. 137, 99–107. Troll, W., Lindsley, J., 1955. A photometric method for the determination of proline. J. Biol. Chem. 215, 655–660. Turner, N.C., 1979. Drought resistance and adaptation to water deficits in crop plants. In: Mussell, H., Staples, R.C. (Eds.), Stress Physiology in Crop Plants. Wiley/Interscience, New York, pp. 343–372. Voetberg, G.S., Sharp, R.E., 1991. Growth of the maize primary root at low water potentials. III. Role of increased proline deposition in osmotic adjustment. Plant Physiol. 96, 1125–1130. Wright, P.R., Morgan, J.M., Jessop, R.S., 1996. Comparative adaptation of canola (Brassica napus) and Indian mustard (B. juncea) to soil water deficits: plant water relations and growth. Field Crops Res. 49, 51–64.