Water deficit effects on osmotic adjustment and solute accumulation in leaves of annual clovers

European Journal of Agronomy 16 (2002) 111– 122 www.elsevier.com/locate/eja

Water deficit effects on osmotic adjustment and solute accumulation in leaves of annual clovers Anna Iannucci a,*, Mario Russo b, Lucia Arena b, Natale Di Fonzo b, Pasquale Martiniello a a

Istituto Sperimentale per le Colture Foraggere, Via Napoli, 52, 71100 Foggia, Italy Istituto Sperimentale per la Cerealicoltura, s.s. 16, Km. 675, 71100 Foggia, Italy

b

Received 6 November 2000; received in revised form 29 May 2001; accepted 13 July 2001

Abstract Annual clovers are very popular forage crops in the Mediterranean areas. However, their herbage and seed yields are often reduced by high temperatures and water stress occurring during spring and summer. Field experiments using the four most widely grown forage legumes, berseem (Trifolium alexandrinum L.), crimson clover (T. incarnatum L.), Persian clover (T. resupinatum L.) and squarrosum clover (T. squarrosum L.), were conducted in 1992 and 1993 at Foggia (Italy) to evaluate the osmotic adjustment capability and the relevant contribution of inorganic and organic solutes in response to water deficits. Soil water depletion reduced leaf water potential (cw) and leaf osmotic potential at full turgor (c 100 p ) in both years. In particular, berseem and squarrosum clovers showed lower values ( − 1.46 and −1.51 MPa on average, for cw and c 100 p , respectively). Furthermore, water-stressed plants showed an increase in potassium, reducing sugars and proline concentrations and a decrease in non-reducing sugar contents. Generally, about 80% of the measured cellular osmotic potential was attributable to assayed osmotically active solutes; the inorganic ions represented the major contributors (about 59%). However, only proline levels appeared to change clearly in terms of relative contribution to c 100 p , showing an increase of about 2.2%, under water-stressed conditions. Statistically significant differences (P 50.05) in the osmotic adjustment trait were not found for either species or year. Because clover species showed the same value for solute accumulation capability (0.34 MPa), degree of tolerance to dehydration and turgor maintenance but differed in leaf water parameters, other physiological and/or morphological traits will have to be investigated to better explain the performance of each species under water-stress conditions. However, the tendency of crimson and Persian clovers to maintain high leaf water potentials could be an example of a stress-avoidance mechanism. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Clover species; Leaf osmotic potential at full turgor; Leaf water potential; Osmotic adjustment; Solute accumulation; Water stress

1. Introduction * Corresponding author. Tel./fax: + 39-881-741632. E-mail address: [email protected] (A. Iannucci).

Several annual forage legumes, such as berseem (Trifolium alexandrinum L.), crimson clover (T.

1161-0301/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 1 6 1 - 0 3 0 1 ( 0 1 ) 0 0 1 2 1 - 6

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incarnatum L.), Persian clover (T. resupinatum L.) and squarrosum clover (T. squarrosum L.), with early spring growth habits, are grown as mixtures in the Mediterranean warm-temperate zones since they can utilize the winter rainfall. Forage crops in these environments are not irrigated and are often subjected to the detrimental effects of high temperatures and water deficits which occur during the spring–summer period that can seriously reduce herbage and seed production. Water stress has been identified as a factor which negatively affects the ratio of reproductive to vegetative growth, growth parameters, seed yield and its components in some annual clovers (Iannucci and Martiniello, 1998; Martiniello and Ciola, 1993). However, little information is available concerning the physiological responses and the resistance mechanisms to soil drying for these species. As reported by Hsiao (1973), leaf water deficit developing as a consequence of soil water depletion, affects many physiological processes with eventual consequences for biomass and seed yield. Many of these changes represent adaptive responses by which plants cope with water stress. The mechanisms developed as survival strategies include tolerance and avoidance of tissue water stress (Lewitt, 1972). Generally, stress avoidance involves stomatal closure, hydraulic conductance and root growth patterns. Stress tolerance usually includes osmotic adjustment and changes in tissue elasticity (Jones et al., 1981). In particular, osmotic adjustment, that is the lowering of osmotic potential by net solute accumulation in response to dehydration, assists the maintenance of turgor at lower water potentials, and it has been considered a beneficial drought tolerance mechanism in both the vegetative and reproductive phases of crop growth (Ludlow and Muchow, 1990; Rascio et al., 1994). In fact, many physiological processes, such as cell expansion, photosynthesis, gas exchanges or enzyme activity, are dependent upon cell turgor. As reported by Morgan (1984) a number of osmotically active substances, both organic and inorganic solutes, play a role in the osmotic adjustment phenomenon. However, conflicting results have emerged regarding the nature and quantitative contribution of solutes and ions such

as sugars, amino acids and K+, mainly because of the method used for the stress induction. Indeed, as reported by Radin (1983), osmotic adjustment may be absent or very weak in pot-grown plants for laboratory experimentation (as opposed to field plants), probably because they have severely restricted root volumes and may become stressed too rapidly precluding the adaptive response. Thus, information gathered in the laboratory may be difficult to extrapolate to field conditions. Osmoregulation and the role of osmolytes in the physiology of stressed plants have been investigated in a number of crop species (Bittman and Simpson, 1989; Handa et al., 1983; Martin et al., 1993; Turner and Jones, 1980). Because there are large differences among and within species in the degree of adaptation to water deficit, it is important to investigate the metabolic changes involved. In fact, knowledge of plant adaptive strategies to water stress and their physiological basis can serve to formulate plant breeding and management strategies adapted to semi-arid environmental conditions. This study was conducted to investigate: (i) the interspecific variation in osmotic adjustment capability, and (ii) the relevant contribution of inorganic and organic solutes in response to water stress in leaves of four annual clovers under field conditions.

2. Materials and methods

2.1. Plant materials and experimental design Field experiments were conducted at the Forage Crop Institute farm located in Foggia (South Italy: 15°33%E; 41°31%N; 76 m above sea level) during the 1991–1992 and 1992–1993 growing seasons on a black clay loam soil (mixed, mesic, Typic Cromoxererts) with the following characteristics: clay 21%, silt 43%, sand 36%, pH 8, available P 15 mg kg − 1 (Olsen method), available K 800 mg kg − 1 (NH4Ac) and organic matter 21 g kg − 1 DM. The climate variables (rainfall and average temperature) were recorded daily in a meteorological station approximately 1000 m from the experiment sites and are shown in Fig. 1.

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Each experiment was conducted on a different site from the previous year’s trial. Four annual clovers: berseem cv. Sacromonte, crimson clover ecotype Campano, Persian clover cv. Accadia and squarrosum clover ecotype Toscano-Laziale, were grown under two water regimes from late vegetative to late flowering stages: well-watered as control and water-stressed. Cultivars and ecotypes were chosen because they are widely distributed and considered representative of each species in the area in which trials were conducted. The experimental design was a split-plot with four replications, with the irrigation treatments in the main plot and the clover species in the subplot. Each plot consisted of eight rows, 5 m long and 0.18 m apart. Seeds were planted on the third week of October in both years of evaluation, using a seed drill at 1100, 1200, 3100 and 700 germinated seeds per m2 for berseem, crimson, Persian and squarrosum clovers, respectively. Before sowing, the soil was chemically fertilized (N and P at the rate of 16 and 70 kg ha − 1, respec-

113

tively) and disinfested (Diazinone:[0,0-diethyl-0(isopropyl-4-methyl-6-pyrimidil)thiophosphate] at rate of 35 kg product ha − 1. Water requirements for the crops were estimated on the basis of the evaporated water, calculated from a Class A water pan, the useful rainfall and the FAO’s crop coefficient (Doorenbos and Pruitt, 1977). Irrigation was applied when evapotranspiration reached a volume of 80 mm. The plots were watered by means of a horizontal bar, with low nozzle pressure, 16 m long, 1.25 m from ground level, with 270 and 213 mm being applied in 1992 and 1993, respectively. Plants were regularly watered to field capacity until late vegetative stage (about 175 days after sowing). At this stage water stress was imposed by withholding water and the effect of water regimes was monitored during the growth cycle by measuring leaf water potential (cw) of the uppermost fully expanded leaflets. The cw was estimated on three different single plants from each species and treatment, near midday (11.00–14.00 h) at approximately weekly intervals from April to May. At late flowering (216 and 220 days from sowing for 1992 and 1993, respectively), when the vegetative development of the four species was complete, the youngest fully expanded leaf samples (about 80– 100 g of fresh weight) were randomly removed from within the canopy for chemical analysis. On the same sampling data, cw, leaf osmotic potential at full turgor (c 100 p ) and relative water content (RWC) were measured.

2.2. Leaf water parameters

Fig. 1. Monthly average temperature (°C) and rainfall (mm) from October to June in 1991 –1992 and 1992 – 1993.

cw was determined by estimating xylem-pressure potential in a pressure chamber (PMS Instrument Co., Corvallis, OR, USA). RWC was determined gravimetrically. The excised leaf was weighed immediately to provide the fresh weight (FW). Excised leaves were then rehydrated in distilled water for 4 h at 20 °C to provide the turgid weight (TW), and then dried for 48 h at 60 °C to provide the dry weight (DW). RWC was calculated by the ratio: RWC= (FW − DW)/ (TW − DW) ×100. Tissue water content was determined as (TW−DW). c 100 was obtained using p the cryoscopic method. Leaves after rehydration

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A. Iannucci et al. / Europ. J. Agronomy 16 (2002) 111–122

were frozen in liquid N2 and stored at −20 °C overnight, the cell sap was obtained by pressing leaf tissue in a syringe using a hydraulic press at 5 atm for 1 min. Osmotic potential at full turgor of the expressed sap was measured with a micro-osmometer (Ro¨ ebling 16R.1, Berlin, Germany). RWC was used with the measured c 100 to calcup late the leaf osmotic potential (cp) (Wilson et al., 1979) as cp =c 100 p ×(100 − B)/(RWC −B) where B is the apoplastic water content, assumed to be 10% (Kramer, 1983). Osmotic adjustment was calculated as the difference between c 100 of p the irrigated and non-irrigated plants. Leaf turgor potentials (cp) were obtained from the relationship: cw = cp + cp. All leaf water relation parameters were measured at the middle of the photoperiod on three uppermost fully expanded leaves from three randomly selected plants for each plot.

2.3. Solute analysis Leaf samples for inorganic solute analyses were oven-dried at 60 °C for 4 days, whereas an aliquot of fresh material was liophylized for organic solute determinations. Both samples were ground to pass through a 40-mesh screen and stored at − 20 °C until solutes were determined. Cl− was extracted from 100 mg of samples, which were incubated twice in 10 ml of deionized water at 40 °C for 30 min. After centrifugation (4500g, 10 min) the decanted supernatants were pooled and estimated by potentiometric titration with Hg(NO3)2 using chloride as a standard (Schales and Schales, 1941). The cations Na+, K+, Ca2 + and Mg2 + were determined from 200 mg of samples by atomic absorption spectroscopy (Perkin Elmer 370A) after wet digestion in HNO3 (90%):HClO4 (54%) (1:1). Sugar analysis was conducted after extracting a 100 mg sample for 30 min with 10 ml solution containing 4.5 g sodium acetate, 3 ml acetic acid and 4.5 ml sulfuric acid (96%) in 1 1 H2O. The extract was filtered through fiberglass filter paper and divided into two aliquots. Reducing sugars (primarily glucose and fructose) in an extract aliquot were assayed directly, while total water

soluble carbohydrates were measured, on the other aliquot, following the acid hydrolysis at 100 °C for 30 min. The determinations were based on the colorimetric method using a glucose standard (Wolf and Ellmore, 1975). Non-reducing sugars, such as sucrose, were calculated as the difference between total water-soluble carbohydrates and reducing sugars. For proline determinations, leaf samples were extracted with 3% sulfosalicyclic acid and filtered through Whatman paper. Ninhydrin solution (2 ml) and 2 ml of acetic acid were added to the aqueous phase and incubated for 45 min at 100 °C. Toluene (4 ml) was added and the absorbance was read at 520 nm (Bates et al., 1973).

2.4. Osmotic contribution of solutes To determine the contribution of solutes to c 100 their concentrations were calculated dividing p the contents (mmol g − 1 DW) by leaf water volume measured as the ratio between leaf water content (turgid weight− dry weight) and leaf dry weight. Using the assumption that solutes behaved as ideal osmolytes, with an ionization constant of 1, a 1 M solution at 20 °C would lower the osmotic potential by 2.4 MPa. For molecular weights, reducing sugars were assumed to be glucose (180 g mol − 1) and non-reducing sugars sucrose (342 g mol − 1).

2.5. Statistical analysis Analysis of variance of the combined data over the years showed several significant interactions between years and main effects (treatments and species). Therefore, experimental values for leaf water parameters and solute contents were analyzed for each year separately. The LSD test at the 0.05 probability level was performed to determine the mean separations. Linear regression analyses were performed to determine differences among species for the relationships between cw and RWC. Non-linear regressions were calculated using the least-squares method for determining the relationship between cw and cp.

A. Iannucci et al. / Europ. J. Agronomy 16 (2002) 111–122

Fig. 2. Leaf water potential at various times after April 1st under well-watered and water-stressed conditions in 1992 and 1993. Means 9SDs (n =16) represent pooled data of four clover species. Vertical bars show rainfall, arrows indicate irrigation date.

3. Results As shown in Fig. 1, during October through February of 1991–1992 rainfall was greater and the temperature was lower than in 1992– 1993 (mean values of 148.2 and 92.5 mm and 9.9 and 11.2 °C, respectively). However, from April to June rainfall was greater in 1993 than in 1992 (92.6 and 83.5 mm, respectively); as a consequence, the amount of total water applied by irrigation was greater in 1992 than 1993. As evidenced by cw values, water stress occurred during the experiment in both years when water was withheld (Fig. 2). At the last observation (late May) leaf cw of the water-stressed treatment reached minimum values, averaged over the species, of −1.41 MPa in 1992 and −2.15 MPa in 1993 (−0.75 and −1.29 MPa lower than those of the control, respectively).

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The differences among species and between watering treatments, for cw and c 100 p , measured at late flowering, were significant in 1992 (Table 1) and 1993 (Table 2). Reduced water supply resulted in a decrease of these traits in all species. Because there was no statistically significant genotype × environment (i.e. wet, dry) interaction in both years, the species which were lowest in cw and c 100 under well-watered conditions, were alp ways the lowest under water-stressed conditions. In particular, berseem and squarrosum clovers showed the lowest values in both years (− 1.31 and − 1.61 MPa for cw and − 1.46 and − 1.55 MPa for c 100 p , over year average, respectively). The lowering of osmotic potential in waterstressed leaves indicated that solute accumulation occurred. However, statistically significant differences (P5 0.05) in osmotic adjustment (0.34 MPa, mean over the species and years), were not found among species and between years (Tables 1 and 2). In all species water stress significantly increased the leaf content of K+ (58 and 10% on average in 1992 and 1993, respectively), proline (138 and 134% on average in the first and second year, respectively) and reducing sugars (18 and 31% on average in 1992 and 1993, respectively) (Tables 1 and 2). Na+ and Mg2 + ions were also present, but their levels were low and remained unaffected by stress conditions, whereas non-reducing sugars showed a marked reduction under water deficit (about 36 and 32% on average in 1992 and 1993, respectively). For all species, Ca2 + and K+ were the most important of the cations (138.3 and 97.8 mM in 1992, and 167.8 and 162.4 mM in 1993 on average, respectively), whereas Cl− was present in only small amounts (9.05 mM as the mean), representing only 3% of the total content of ions in both years (Tables 1 and 2). Furthermore, reducing sugars represented the major organic solutes (62.0 and 72.4 mM in the first and second year, respectively), representing about 64% of the analyzed organic compounds in both years; while the levels of non-reducing sugars were two- to eightfold lower than those of reducing sugars. The accumulation of most solutes among species was statistically significant in both years (Tables 1 and

69.7 83.8 196.4 176.8 138.6 138.1

5.9 6.6 9.5 11.9 8.2 8.9 ns ** ns

164.8 175.1

9.9 9.5

ns ** ns

123.3 116.5

7.6 7.5

Ca2+ (mM)

** ** **

75.9 119.6

77.2de 161.1a

47.8f 83.7cde

108.3bc 131.3b

70.4ef 102.5cd

K+ (mM)

ns ns ns

14.8 17.0

12.5 20.8

11.2 14.3

20.6 15.8

15.0 17.1

Mg2+ (mM)

ns ** ns

8.8 8.9

11.9 15.8

7.1 4.9

11.4 10.6

4.7 4.2

Na+ (mM)

** ** **

56.8 67.1

87.6b 112.8a

36.8e 41.6e

43.7e 42.2e

59.2d 71.9c

RSa (mM)

** ** ns

22.9 14.7

27.5 24.6

17.9 9.5

19.3 9.0

27.0 15.6

NSa (mM)

ns ** **

79.8 81.8

115.2b 137.4a

54.7d 51.0d

63.0d 51.2d

86.2c 87.5c

TWSSa (mM)

** ** **

10.5 25.0

12.6cd 34.0a

8.1d 17.7bc

8.7d 19.3b

12.6cd 29.1a

Proline (mM)

** ** ns

−0.66 −1.41

−0.99 −1.80

−0.43 −1.13

−0.55 −1.13

−0.68 −1.59

cw (MPa)

** ** ns

−1.11 −1.45

−1.22 −1.66

−0.99 −1.23

−1.09 −1.42

−1.15 −1.48

c 100 (MPa) p

ns, *, ** represent not significant and significant at the 0.05 and 0.01 probability levels, respectively. Means (n=4) within a column not followed by the same letter are significantly different at PB0.05. a RS, reducing sugars; NS, non-reducing sugars; TWSS, total water soluble sugars.

Berseem Well-watered Water-stressed Crimson Well-watered Water-stressed Persian Well-watered Water-stressed Squarrosum Well-watered Water-stressed Mean Well-watered Water-stressed F 6alues Treatment Species Interaction

Cl− (mM)

– ns –

0.33

0.44

0.23

0.33

0.34

OA (MPa)

Table 1 Organic and inorganic solute content, leaf water potential (cw), leaf osmotic potential at full turgor (c 100 p ) and osmotic adjustment (OA) of four annual clovers under well-watered and water-stressed conditions measured at late flowering (216 days from sowing) in 1992

116 A. Iannucci et al. / Europ. J. Agronomy 16 (2002) 111–122

193.2 186.2 156.3 158.3 125.0 136.9 175.6 210.5 162.5 173.0 ns ** ns

12.9 10.1 9.9 7.2 7.7 7.3 11.1 10.8 10.4 8.9 ns ** ns

Ca2+ (mM)

* ** ns

155.4 169.4

186.7 220.6

143.3 138.5

144.9 142.3

146.7 176.2

K+ (mM)

ns * ns

28.6 30.5

31.6 36.1

22.1 23.3

26.1 27.5

34.5 35.1

Mg2+ (mM)

ns ns ns

19.3 19.7

22.2 25.1

17.7 16.5

20.9 17.0

16.2 20.3

Na+ (mM)

** ** ns

62.8 82.1

70.6 106.6

54.8 69.4

56.2 69.8

69.6 82.5

RSa (mM)

** ** **

16.0 10.9

15.7ab 14.8b

13.6bc 8.8d

16.1ab 9.2d

18.7a 11.0cd

NSa (mM)

** ** ns

78.8 93.0

86.3bc 121.3a

68.4d 78.2bcd

72.3cd 78.9bcd

88.3bc 93.5b

TWSSa (mM)

** ** **

16.6 38.9

14.5d 41.8ab

14.2d 30.2c

19.7d 35.4bc

18.1d 48.1a

Proline (mM)

** ** ns

−0.86 −2.15

−1.09 −2.55

−0.80 −1.86

−0.75 −2.02

−0.80 −2.16

cw (MPa)

** ** ns

−1.29 −1.62

−1.40 −1.92

−1.21 −1.36

−1.17 −1.41

−1.40 −1.81

c 100 (MPa) p

ns, *, ** represent not significant and significant at the 0.05 and 0.01 probability levels, respectively. Means (n= 4) within a column not followed by the same letter are significantly different at PB0.05. a RS, reducing sugars; NS, non-reducing sugars; TWSS, total water soluble sugars.

Berseem Well-watered Water-stressed Crimson Well-watered Water-stressed Persian Well-watered Water-stressed Squarrosum Well-watered Water-stressed Mean Well-watered Water-stressed F 6alues Treatment Species Interaction

Cl− (mM)

– ns –

0.34

0.52

0.20

0.24

0.41

OA (MPa)

Table 2 Organic and inorganic solute content, leaf water potential (cw), leaf osmotic potential at full turgor (c p100) and osmotic adjustment (OA) of four annual clovers under well-watered and water-stressed conditions measured at late flowering (220 days from sowing) in 1993

A. Iannucci et al. / Europ. J. Agronomy 16 (2002) 111–122 117

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118

2). Squarrosum and Persian clovers showed the highest and lowest ion and organic solutes concentrations, respectively. Proline content was significantly different among species in both years; berseem showed the highest values. To examine the contribution of the various osmotically active organic and inorganic compounds identified in the clover leaves, the c 100 p measured was compared to the solute potential evaluated according to the Boyle– Van’t Hoff relation. Generally, about 80% of cellular osmotic potential measured was attributable to the sum of the osmotically active solutes (85% in 1993 and 70% in 1992 as the mean, respectively) (Tables 3 and 4). Because the organic solute percentage was unchanged (19%) in contribution to c 100 p 1992 and 1993, the difference registered between years for the total contribution to c 100 was asp cribed to ion concentrations (K+, Mg2 + and Na+, principally). In fact, quantitatively, in both treatments of all species, the inorganic solute contribution to c 100 was several times higher than p

that of organic solutes, representing about 51 and 66% as the mean in 1992 and 1993, respectively. Although there were not statistically significant differences between treatments in both years, all solutes examined accounted for a higher percentage of c 100 under well-watered than under waterp stressed conditions (73 and 66% in 1992 and 89 and 73% in 1993, respectively); this was probably due to an increase in ion contribution in well-watered plants. The organic solute contribution to c 100 was similar in both treatments and years p (about 19%), because a reduction (about 1.6%) in non-reducing sugar and an increase (about 2.2%) in proline contributions were observed under water-stressed treatment. Clear differences among the clover species for the importance of the assayed substances with respect to the c 100 were found in 1992. In fact, p crimson and squarrosum clovers showed the highest total percentage contribution (79 and 85%, respectively), the Ca2 + and K+ concentrations accounting for this difference. Persian clover

Table 3 Relative contribution of inorganic and organic solutes to leaf osmotic potential at full turgor in four annual clovers under well-watered and water-stressed conditions measured at late flowering (216 days after sowing) in 1992

Berseem Well-watered Water-stressed Crimson Well-watered Water-stressed Persian Well-watered Water-stressed Squarrosum Well-watered Water-stressed Mean Well-watered Water-stressed F 6alues Treatment Species Interaction

Sum of solutes (mM)

c 100 p (%)

Sum inorganic ions (mM)

c 100 p (%)

Sum organic compounds (mM)

c 100 (%) p

320d 364cd

68 60

221 248

47 41

99c 117b

21 19

387bc 413bc

87 71

315 342

70 59

72d 71d

16 12

205f 262e

50 52

142 193

35 38

63d 69d

16 14

435b 558a

87 82

308 386

62 57

128b 171a

26 25

337 399

73 66

246 292

53 48

90 107

19 17

** ** *

ns ** ns

** ** ns

ns ** ns

* ** **

ns ** ns

ns, *, ** represent not significant and significant at the 0.05 and 0.01 probability levels, respectively. Values within a column not followed by the same letter are significantly different at PB0.05.

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Table 4 Relative contribution of inorganic and organic solutes to leaf osmotic potential at full turgor in four annual clovers under well-watered and water-stressed conditions measured at late flowering (220 days after sowing) in 1993

Berseem Well-watered Water-stressed Crimson Well-watered Water-stressed Persian Well-watered Water-stressed Squarrosum Well-watered Water-stressed Mean Well-watered Water-stressed F 6alues Treatment Species Interaction

Sum of solutes (mM)

c 100 p (%)

Sum inorganic ions (mM)

c 100 p (%)

Sum organic compounds (mM)

c 100 (%) p

510 570

89 77

404 428

70 58

106cd 142b

18 19

450 467

94 81

358 352

75 61

92de 114c

19 20

399 431

80 77

316 323

64 58

83e 108cd

17 19

528 666

92 85

427 503

74 64

101cde 163a

18 21

471 533

88 79

376 401

70 59

95 132

18 20

* ** ns

ns ns ns

ns ** ns

* ns ns

** ** *

ns ns ns

ns, *, ** represent not significant and significant at the 0.05 and 0.01 probability levels, respectively. Values within a column not followed by the same letter are significantly different at PB0.05.

showed the lowest values for both inorganic and organic compounds. cw was linearly related to RWC, and the relationship was identical for all species as evidenced by the slopes of the four linear regressions that were not significantly different (P 50.05) from the pooled linear equation (Fig. 3a). Leaf turgor values for each species were derived from plots of cp and cw obtained by fitting the quadratic equation to data recorded during the experiment (Fig. 3b). According to the relationship cw =cp +cp, cp is taken to be the difference between the response line and the line of equality at any value of cw. Leaf osmotic potential decreased with decreases in cw and the four clover species showing the same relationship (the slopes were not significantly different from the pooled equation for P 5 0.05), evidenced a similar turgor maintenance ability. A turgor potential of 0 MPa was reached at a cw mean value of − 2.4 MPa.

4. Discussion The fact that berseem and squarrosum clovers had more negative cw values throughout the experimental period (data not shown) also under well-watered conditions, could be due to differences in root system related to the higher resistance of plant to water flow. Bittman and Simpson (1989) reported that low cw may be the consequence of low water uptake and hydraulic flow rates within the plants, or high water loss rates. The relationship between RWC and cw has often been used to quantify the dehydration tolerance of tissues: tissues which maintain a high RWC as cw decreases are more tolerant to dehydration. In our experiment the clover species showed the same degree of tolerance to dehydration. Furthermore, the existence of a significant relationship between cw and cp values observed in all species suggests the importance of solute

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accumulation process in maintaining low water potential, which could involve selective increase of particular solutes. However, all species showing the same cp values, and, as consequence, similar cp values for a wide range of cw, evidenced the same capacity to osmoregulate. Increased concentrations of inorganic ions as a consequence of water deficits have not been commonly found in the tissue of higher plants (Hsiao, 1973). However, Graham and Ulrich (1972) and Greenway and Klepper (1969) noted that the permeability of plant cells and roots to mineral nutrients and their accumulation in various organs is sensitive to water status. Calcium is the major inorganic cation found in clover leaves, but its osmotic implication is not yet well-known. Recent investigations indicated that Ca2 + ions control water-use efficiency by causing stomatal closure (Ruiz and Mansfield, 1994). Potassium ions also,

Fig. 3. Relationship between RWC and leaf water potential (a), and between osmotic potential and leaf water potential (b) for four clover species. The data set for each species consisted of 16 observations.

appear to be in sufficient concentration in clover leaves such that they might be important in the osmotic adjustment phenomenon. K+ ions are known to be quite soluble and to play a key osmoregulatory role in the guard cells and similarly in turgor maintenance (Talbott and Zeiger, 1996). The concentrations of a variety of organic compounds are known to increase in some plant tissues subjected to water stress (Hsiao, 1973). Leaves of all clovers studied subjected to water stress synthesized more reducing sugars and proline than wellwatered plants. Carbohydrates have been shown to make a major contribution to osmotic adjustment in the growing regions of leaves (Munns and Weir, 1981), stems (Meyer and Boyer, 1981) and roots (Sharp and Davies, 1979). Furthermore, the decrease in non-reducing sugar concentrations under drought has been reported also for other legumes and ascribed to the inhibition of photosynthesis due to turgor loss (Yan et al., 1994). Differences in proline accumulation under water-stressed treatment were found among clover species. However, a relationship existed between the degree of stress (MPa) and the accumulation of organic solutes (mM), as evidenced by the positive correlation between cw and reducing sugar and proline fitted to linear (r= 0.63**) and quadratic (r= 0.85**) models, respectively. The significant relationships indicated, in agreement with the results of Navari-Izzo et al. (1990) that the metabolic differences among species may reflect differences in water status achieved, rather than metabolic differences at a given water status. The contribution of various solutes to osmotic adjustment may depend on several factors such as plant species, growth stage and light level under which plants are grown (Jones et al., 1981). Although Ca2 + and K+ ions as well as reducing sugars constitute the major fractions of the solute pool in the youngest fully expanded clover leaves, their percent contribution to c 100 was not inp creased under water-stressed treatment. Furthermore, the total contribution of the quantified solutes to c 100 was lower under stress conditions. p As a consequence, it seems that osmotic adjustment is a highly regulated process, involving metabolites besides those analyzed in this experiment. In fact, other organic solutes have been

A. Iannucci et al. / Europ. J. Agronomy 16 (2002) 111–122

reported as osmotically active substances; particularly McManus et al. (2000) reported that pinitol is the major solute present in mature leaves of white clover (Trifolium repens L.) when subjected to a significant water deficit. According to the results obtained by Handa et al. (1983) in cultured cells of tomato (Lycopersicon esculentum Mill) adapted to water stress, the maximum contribution to osmotic potential was made by reducing sugars and by K+ ions; but only proline levels appears to change markedly in terms of relative contribution to osmotic adjustment. Thus, synthesis of proline may represent the most specifically regulated metabolic event during water-stress development. However, Iannucci et al. (2000) found in berseem leaves that osmotic adjustment started before proline levels began to increase. Furthermore, Kameli and Lo¨ sel (1993) indicated reducing sugars as more sensitive indicators of the degree of stress and of potential tolerance, than proline which increased later and to the same extent in tolerant or non-tolerant durum wheat (Triticum durum Desf.) varieties. The constancy of osmotic adjustment values in spite of different stress intensities between years, indicated that under our experimental conditions, clover plants showed a limit to the accumulation of osmotic solutes. Furthermore, in agreement with the reports of Martin et al. (1997) in winter wheat (Triticum aesti6um L.), although the solutes detected could be important in the adaptation of each species to water-stressed environments, their synthesis and that of other metabolites was apparently insufficient to significantly influence osmotic adjustment, as indicated by the lack of differences among clover species for this trait. Because genotypic differences were evident in leaf water parameters, but not for the degree of tolerance to dehydration, turgor maintenance and osmotic adjustment capacity, we conclude that clover species had a different sensitivity to soil water depletion and other physiological and/or morphological traits (such as leaf conductance or root length and rooting depth) must be investigated to explain the performance of each species under water stress. However, according to Lewitt (1972), the tendency of crimson and Persian clovers to maintain high leaf water potentials

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would be an example of a stress-avoidance mechanism. Furthermore, these species are characterized by a shorter growth cycle than other clovers. Therefore, their ability to yield under water-stress conditions may be ascribed to their capacity to avoid dehydration by means of ontogenic characteristics as well as ecological strategies.

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