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Short-term Solute Changes in Leaves and Roots of Cultivated and Wild Tomato Seedlings Under Salinity
]. Plant Physiol. Vol. 147. pp. 463-468 (1995)
Short-term Solute Changes in Leaves and Roots of Cultivated and Wild Tomato Seedlings Under Salinity M.
C. BOLARlN, A. SANTA-CRUZ,
E.
CAYUELA,
and F.
PEREZ-ALFOCEA
Centro de Edafologia y Biologia Aplicada del Segura, CSIC, Apdo 4195, E-30080, Murcia, Spain Received May 7, 1995 . Accepted August 17, 1995
Summary
The daily variations of inorganic and organic solutes with salinity (140 mM NaCI) were determined in seedlings of cultivated tomato (Lycopersicon esculentum Mill.) and its wild salt tolerant relative L. pennellii (Correll) D'Arcy for 8 days, with the objective to identify physiological traits in either leaves or roots contributing to the salt tolerance. A greater accumulation of leaf Cl- and, especially, Na+ was found in L. pennellii than in L. esculentum, while root Na+ concentrations increased similarly in both tomato species over the salinization period. The changes induced by salinity on the NaiK and NaiCa ratios were different in both tomato species; in leaves, the increases of both ratios with time of salinization were higher in L. pennellii than in L. esculentum, while in roots both ratios, mainly the NaiCa ratio, increased more in L. esculentum than in L. pennellii in the time course. The accumulation patterns of total soluble sugars were also different in both tomato species: although a higher sugar accumulation was found in L. esculentum than in L. pennellii at the end of the salinization period, an earlier sugar accumulation occurred in leaves and roots of L. pennellii. The proline accumulation was greater in the cultivated than in the wild tomato species, and earlier in roots than in leaves, but in no case did it occur in the first days of salinization. -y-aminobutyric acid (GABA) was the only amino acid that increased significantly from the beginning of the salt stress in leaves and roots of L. esculentum, which did not occur in L. pennellii.
Key words: NaCl salinity, tomato (Lycopersicon), ion contents, sugars, amino acids. Abbreviations: SS = total soluble sugars; Pro = proline; GABA = -y-aminobutyric acid. Introduction
High salt tolerance has been reported for various wild relatives of the cultivated tomato (Rush and Epstein, 1981; Bolarin et al., 1991). However, despite several attempts to enhance the salt tolerance of the cultivated tomato using the tolerance of related wild species of Lycopersicon, no commercial success has been achieved. An alternative approach is to attempt to accumulate physiological traits that contribute to tolerance within a single genotype (Cuartero et al., 1992). Therefore, a better understanding of the physiological processes and the identification of the specific physiological traits conferring salinity tolerance could playa major role in the development of breeding strategies. The fact that unclear results have been obtained until now, despite that plant response to salinity is one of the most widely researched sub© 1995 by Gustav Fischer Verlag, Stuttgart
jects in plant physiology, may be due to various factors, such as: a) the long-term salt effects are very different from those of short-term (Thiel et al., 1988), b) the majority of the data in the studies of salt tolerance are for harvests made at a single time-point, whereas there is very little information on changes that occur with time (Schachtman and Munns, 1992), and c) few studies have compared responses between leaf and root tissues despite that the primary effect of salinity is on the roots (Sharp, 1990). Munns (1993) indicated that one approach towards understanding the mechanisms of salt tolerance is to follow the series of events that exposure to salinity initiates. In this work, the daily variations of inorganic and organic solutes with the salinity were determined in leaves and roots of L. esculentum and its wild salt-tolerant relative L. pennellii for 8 days, with the objective to identify early traits of salt tolerance in order to develop breeding
464
M. C. BOURlN, A. SANTA-CIlUZ, E. CAYUELA, and F. PEllEz-AuoCEA in a Rank Hilger amino acid autoanalyzer using the ninhydrin post-column reaction. Solute contents are expressed on the basis of tissue water. The changes in different solutes with NaCl salinization were tested by one-way analysis of variance (ANOVA) using Standard Methods SYSTAT. Means were compared between treatments by LSD at the 0.05 confidence levels using Student's t-test.
strategies for transferring the higher level of salinity tolerance from wild relatives to cultivated tomato.
Materials and Methods
Plant material and culture conditions Seeds of L. esculentum Mill. cv. P-73 and L. pennellii (Correll) D'arcy accession PE-47 (collected in Peru by Cuartero et al., 1984) were used. Seeds were germinated and plants grown in a controlled culture chamber under the following conditions: a temperature and relative humidity of 28 ± 2°C and 90%, respectively, for germination; the controlled culture chamber was programmed to simulate the natural diurnal changes in temperature and light intensity for plant growth: 28/18 °C (lightldark) and 16 h light with a PPFD at the plant level of 24S/81I1molm- 2 s· 1 (400-700nm). The relative humidity was 60-70%. Plants were grown in trays (60x40x12) containing washed silica sand. Each tray was perforated at the bottom and then placed in another tray. Plants were irrigated twice a day with deionized water during the germination period. From the appearance of the first leaf until the end date, the plants were irrigated daily with Hoagland's solution; at first, a half strength Hoagland's solution was used, but was raised to a full-strength Hoagland's solution after 2 weeks until the final harvest. Nutrient solution (or deionized water during germination) was pumped into each tray from the bottom to the surface of the sand and then drained back to storage tanks, using a semi-automatic system. The nutrient solutions were completely replaced with fresh solutions twice weekly or more often. NaCl treatments (0 and 140 mM) were applied in a direct form when the plants had between 2 and 3 true leaves (15 days old).
Results
Plant growth and water content Root and shoot dry weights decreased significantly with salinity in L. esculentum, whereas they increased in L. pennelIii after 8 days of salt treatment (Table 1). In control medium, water contents were similar in roots of both species. In shoot, they were lower in L. esculentum than in L. pennel. Iii (Table 1). No great differences in the water content decreases with salinity were found in both species.
Ion concentration Leaf Na+ and CI- concentrations increased with time of salinization in the NaCl-treated plants of both tomato species, but the accumulation of both ions was much greater and quicker in L. pennellii than in L. esculentum (Fig. 1). At the end of the salinization period, the leaves of L. pennellii accumulated more Na+ than CI-, whereas no great differences were found in L. esculentum. Only the Na+ concentrations were determined in roots, with the two species showing similar Na+ accumulations at the end of the salinization period. There were no significant differences between the leaf K+ concentrations of control and treated-plants of L. esculentum throughout the period of the experiment (Table 2); these contents even increased with salinity in roots (between 2nd and 6th day). In L. pennellii a similar trend to that shown by L. esculentum was found in roots; in contrast, leaf K + concentrations decreased significantly from the second day of salt treatment. Similar Ca2 + concentrations were found in the control and NaCl-treated leaves of L. esculentum, and in roots this nutrient only decreased significantly with salinity at the end of the salinization period (Table 2). L. pennellii showed a similar response to L. esculentum in leaves, whereas the Ca2 + concentrations in roots showed either no decrease or an increase with salinity. The increases of leaf Na+ IK+ and Na+ ICa2+ ratios with time of salinization were higher in L. pennellii than in L. es-
Plant harvest and analyses Three replications of eighteen plants for each species and treatment were harvested daily from day 0 Gust prior to the time that salt treatment was applied) until day 8 after salinization. Plants were harvested usually 4 h after the onset of the light period. Leaves and roots were separated, rinsed in deionized water, blotted carefully with tissue paper and weighed. A part of this material was dried at 70°C for 72 h. At the end of the experiment, fresh and dry weights of the roots and shoots were determined. Tissue water content was obtained from the (FW-DW)/DW ratio. Dried ground plant material (500 mg) was digested in nitric and perchloric acids (2: 1) for cation analyses. The Na+, K+ and Ca2 + contents were determined by atomic absorption spectrophotometry (LaCh was previously added). Separate 100 mg samples were extracted for 30 min with 40mL of deionized water, and Cl- content was determined in the extract by potentiometric titration with AgN0 3• Analyses of SS and free amino acids were made on fresh plant material, according to Alarcon et al. (1993). Sugars were determined by the method of Jermyn (1975). Free amino acids were determined
Table 1: Dry weights (mg plant-I) and water content (mgmg- I DW) in root and shoot of control and NaCl-treated plants of L. esculentum and L. pennellii at the end of the experiment (8 days). Dry weight L. esculentum
NaClmM 0
140
Water content L. pennellii
L. esculentum
L. pennellii
Root
Shoot
Root
Shoot
Root
Shoot
Root
Shoot
353a* 234b
2959 a 1868b
41 b 50a
749b 1036a
12.8a 10.3 b
14.0a 11.5b
12.5 a 10.2b
15.0a 13.1 b
* Means within a column followed by different letters are significantly different at the 0.05 probability level.
Short-term solute changes in tomato seedlings under salinity
course. Thus, by day 8, the Na+ /Ca2 + ratio values in the roots of salt-treated plants were 3.05 and 1.14 in the cultivated and wild species, respectively.
culentum, especially the Na+ /K+ ratio after day 4 (Fig. 2). 2
However, both ratios, mainly the Na+ /Ca + ratio, increased more in roots of L. esculentum than of L. pennellii in the time L. esculentum
125
r
L. pennellii
Leaf
100 75 50
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2.0 125
100
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75
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25
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0.5 0
"0 125
+ Z
1.5
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25
465
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100 75
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"" +--Z"" U
50 25
1
~
2.0
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1.0
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246802468
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0
0 2 4
Time (days)
6 8
0 2 4 6 8
Time (days)
Fig. 1: Time course of leaf and root N a+ concentrations (0) and leaf CI- concentrations (D) in L. esculentum and L. pennellii plants grown under control medium (open symbols) and 140mM NaCI (closed symbols). Values are means of 3 replicates. Vertical bars on data points are ± SE of the mean (not shown when smaller than the symbol).
Fig. 2: Time course of Na+/K+ and Na+/Ca2 + ratios in leaves and roots of L. esculentum (0) and L. pennellii (D) plants grown under control medium (open symbols) and 140mM NaCl (closed symbols). Values are means of 3 replicates. Vertical bars on data points are ± SE of the mean (not shown when smaller than the symbol).
Table 2: Potassium and calcium concentrations (mol m -3 in tissue water) of leaves and roots of L. esculentum and L. pennellii plants grown under control and saline conditions. NaCI mM L. esculentum Leaf Root L. pennellii Leaf Root L. esculentum Leaf Root L. pennellii Leaf Root
Time from salinization (days) 2
0
0 140 0 140
112.7
0 140 0 140
78,4
89,4
101.5
0 140 0 140
115.6
0 140 0 140
144.8
37.2
43.3
3
4
5
6
7
8
127.7 a 133.8 a 97,4b 131.5 a
128.1 a 136.7 a 96.9b 117.9 a
119.8 a 116.2 a 103.5 a 103,4 a
119.3 a 103.6a 86.7 a 74.2 a
119.3 a* 108.0a 91.2 a 89.2a
120.8 a 116.9 a 106.6 b 130.7 a
130.8 a 126.2 a 77.6b 97.7 a
Potassium 136.3 a 148.2 a 80.6b 117.2 a
94.6a 95.8a 104.1 a 97.9a
106.5 a 83.1 b 116.0 a 90.0b
111.9 a 97.1 a 89.6a 94.2 a
113.0a 93.9b 96.0a 106.1 a
108.2a 85.ob 102.2 b 129.2 a
103.6a 82,4b 126.1 a 146.2 a
103.5 a 81.4b 122.2 b 153.5 a
101.5 a 68.1 b 122.9a 126.7 a
121.8 a 122.0a 32.2a 27,4 a
107.1 a 112.8 a 42.3 a 48.7 a
111.5 a 108.7 a 29.7a 32.3a
Calcium 115,4 a 115.2 a 33.9a 32.3 a
112.0 a 115.5 a 27.0a 31.1 a
104.1 a 116.2 a 26.2a 27.3 a
127.8 a 127.5 a 37.1 a 25.6b
113.9a 96.0a 35.8a 21.1 b
147.8a 148,4 a 38,4 a 38.6a
121.6 a 135.0 a 45,4 a 39.2a
132.3 a 121.6 a 38,4 b 47.6a
135.0 a 128.9 a 40.7b 49.9 a
119.0 a 124.8 a 40.9b 51.9a
112.7 a 97,4 a 52.8a 49.6 a
108.7 a 104.0 a 50.9b 61.3 a
110.8 a 102.9 a 59.5 a 60.0 a
* For each plant part, values within each column followed by different letters are significantly different at the 0.05 probability level.
M. C. BOLAR1N, A. SANTA-CRUZ, E. CAYUELA, and F. PWZ-ALFOCEA
466
Organic solutes
L. esculentum leaves accumulated three times more 55 under salt stress than those of L. pennellii at the end of salinization period (Fig. 3). However, in the wild species they increased significantly in the first two days of treatment, which did not occur in the cultivated species. In roots of L. esculentum, an important increase (twice that of the control plants) was only registered at day 4, followed by a decrease up to the end of the salinization period. L. pennellii showed a similar response in root to that found in leaf.
L. esculentum
L. pennellii
60
Leaf
50
.r 8
40 30
'0
20
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10
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Discussion
0
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0246802468
Time (days) Fig. 3: Time course of total soluble sugars in leaves and roots o.f L. esculentum and L. pennellii plants grown under control medIUm (open circles) and 140 mM N aCI (closed circles). Values are means of 3 replicates. Vertical bars on data points are ± SE of the mean (not shown when smaller than the symbol).
6
The most significant changes in the amino acid concentrations with salinity were found in Pro and GABA (Fig. 4). The other amino acids either remained unaltered or did not show a clear tendency (data not shown). In leaves of L. escu· lentum, the highest increase of Pro with salinity was found between days 7 and 8. The Pro concentrations in roots of L. esculentum tended to increase until day 6, but these increases were small at first, with the greatest increase taking place between days Sand 6. After day 6, an opposite tendency was found. In L. pennellii the Pro increases were smaller than in L. esculentum, especially in roots. The GABA contents increased in leaves and roots of NaCl-treated plants of L. esculentum during the first days of treatment, but they decreased by day 6 (Fig. 4). In this period, the highest increases found in root corresponded to the lowest in leaf. In L. pennelli~ there were no notable differences between the GABA concentrations of the control and NaCI-treated plants in leaves and roots over the salinization period, except for a tendency to increase in leaves from dayS.
L. esculentum
L. pennellii
Leaf
4
4
L. esculentum
A higher salt tolerance has been reported in L. pennellii than in L. esculentum (Dehan and Tal, 1978), which is in accordance to the results obtained in this experiment: salinity reduced plant growth of L. esculentum when the salt stress was applied for 8 days, while in L. pennellii it was increased. The higher salt tolerance of L. pennellii can be due to its inclusion mechanism, since the accumulation of leaf CI- and, especially, Na+ is much greater in L. pennellii than in L. escu· lentum. However, it did not occur in roots, which indicates that only the saline ion accumulation in leaf could be used to discriminate the salinity tolerance in tomato when the salt stress is applied for a short term. The maintenance of K + concentration in leaves of L. escu· lentum with salinity suggests a typical glycophytic mechanism of the cultivated species with a high K +-selectivity, L. pennellii
3 2
,-.,2
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8
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8 '-'
::======; Root
8 4
p..
2
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o2
4 6 8 024 6 8 Time (days)
o
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Time (days)
Fig. 4: Time course of Pro and GABA concentrations in leaves and roots of L. esculen· tum and L. pennellii plants grown under control medium (open circles) and 140mM NaCI (closed circles). Values are means of 3 replicates. Vertical bars on data points are ± SE of the mean (not shown when smaller than the symbol).
Short-term solute changes in tomato seedlings under salinity
while the K+ decrease in leaves of L. pennellii could be due to the ability of the wild species to substitute Na+ for K+ (Marschner, 1986). However, the root K+ concentrations did not decrease with salinity in L. pennellii over the salinization period. Taking into account that L. pennellii shows a high transport rate of Na+ and CI- to the leaf, the contributions of these ions to the osmotic adjustment of the root could not be sufficient and, consequently, the role of K + would be to contribute to osmotic adjustment (Perez-Alfocea et al., 1993). The maintenance, or even increase, of Ca2 + concentration in roots of L. pennellii could be inducing the absence of decrease in K +, since the presence of Ca2 + seems to be necessary for K+IN a+ selectivity and for the maintenance of an appropriate K+ concentration in plant cells (Subbarao et al., 1990). An interesting result is that the K +IN a+ and, especially, the Na+ ICa2 + ratio values are lower in roots of L. pennellii than in L. esculentum, which could be related, at least partially, to the higher salt-tolerance of the wild species. Moreover, these ratios in the rooting medium appear as better indicators of salt stress than the concentration of Na+ alone, such as was indicated by Rengel (1992). The lower salt tolerance of cultivated tomato may be related to the greater energetic cost of leaf osmotic adjustment with sugars as opposed to Na+ or Cl- (Yeo, 1981), according to the low accumulation of saline ions and great sugar accumulation at the end of treatment in leaves of L. esculentum, whereas the opposite response was shown by L. pennellii. This may also be due to the fact that plant growth reduction causes a sugar accumulation that occurred in the cultivated but not in the wild species. Therefore, the fast sugar increases found in L. pennellii (during the first two days) seem not to be due to the same events as the increases found in L. esculentum. These solutes could contribute to the osmotic adjustment while the accumulation of inorganic solutes from substrate is not sufficient. The most important fact does not seem to be the sugar accumulation in itself but the earlier sugar accumulation, which indicates that L. pennellii responds more quickly to salinity than L. esculentum. It would also be interesting to determine whether each species accumulates either different sugars or different levels of each one under salinity. Thus, Sacher and Staples (1985) indicated that the levels of myo-inositol in leaves and roots of tomato genotypes were positively correlated with their salt tolerances. Proline and GABA were the two amino acids that showed the most different responses between the two tomato species as a consequence of salt stress. In relation to the Pro accumulation with salinity, our results agree with those obtained by Tal et al. (1979), since the Pro increase was smaller in the wild species than in the cultivated one. Sudhakar et al. (1993) indicated that the accumulation of Pro in salt stressed greengram seedlings was mainly due to the fact that the activity of the oxidizing enzymes was significantly inhibited in both shoots and, mainly, roots, which would also explain the higher levels of Pro in L. esculentum, the tomato species more affected by salinity. Our results also show that Pro accumulation starts only when a high sugar accumulation occurred, as reported previously in wheat leaves under water stress (Kameli and Losel, 1995). It can be concluded that Pro accumulation is a salt-sensitive trait in tomato, with the differences between the cultivated and wild tomato species
467
being greater and earlier in roots than in leaves. Perez-Alfocea et al. (1994), in a study carried out in leaves and calli of cultivated and wild tomato species, indicated that not only Pro but also GABA accumulation could be used as salt-sensitive sensors at both organization levels when the salt treatments were applied for a long term. However, the only free amino acid that increased significantly from the beginning of salt treatment was GABA, this occurring only in leaves and roots of L. esculentum, which suggests that an increase of GABA is an earlier trait than the increase of Pro. Acknowledgements
We wish to thank Mr. E. Hasler for reviewing the manuscript in the English language and Mrs. M. R. Rojo for her technical assistance. This work has been supported by Project AIR 3 CT94-1508.
References ALARCON, J. J., M. J. SANCHEZ-BLANCO, M. C. BOLA!UN, and A. TORRECILLAS: Water relations and osmotic adjustment in Lyeopersicon eseulentum and L. pennellii during short term salt exposure and recovery. Physiol. Plant. 89, 441-447 (1993). BOLA!UN, M. c., F. G. FERNANDEZ, V. CRUZ, and J. CUARTERO: Salinity tolerance in four wild tomato species using vegetative yieldsalinity response curves. J. Amer. Soc. Hort. Sci. 116, 286-290 (1991). CUARTERO, J., F. NUEZ, and A. DIAz: Catalog of collections of Lyeopersieon and L. pennellii from Northwest of Peru. TGC Report 34,43 -46 (1984). CUARTERO, J., A. R. YEO, and T. J. FLOWERS: Selection of donors for salt-tolerance in tomato using physiological traits. New Phytol. 121,63 -69 (1992). DEHAN, K. and M. TAL: Salt tolerance in the wild relatives of the cultivated tomato: Responses of Solanum pennellii to high salinity. lrrig. Sci. 1, 71-76 (1978). JERMYN, M. A.: Increasing the sensitivity of the anthrone method for carbohydrate. Anal. Biochem. 68, 332-335 (1975). KAMELI, A. and D. M. UiSEL: Contribution of carbohydrates and solutes to osmotic adjustment in wheat leaves under water stress. J. Plant Physiol. 145, 363-366 (1995). MARSCHNER, H.: Mineral nutrition in higher plants. Academic Press Inc., London, 1986. MUNNS, R.: Physiological process limiting plant growth in saline soils: some dogmas and hypotheses. Plant Cell Environ. 16, 1524 (1993). PEREZ-ALFOCEA, F., M. T. ESTAN, M. CARO, and G. GUERRIER: Osmotic adjustment in Lycopersieon eseulentum and L. pennellii under NaCI and polyethylene glycol 6000 iso-osmotic stresses. Physiol. Plant. 87, 493-498 (1993). PEREZ-ALFOCEA, F., A. SANTA-CRUZ, G. GUERRIER, and M. C. BOLA!UN: NaCl stress induced solute changes on leaves and calli from leaf of Lycopersieon eseulentum, L. pennellii and their interspecific hybrid. J. Plant Physiol. 143, 106-111 (1994). RENGEL, Z.: The role of calcium in salt toxicity. Plant Cell Environ. 15,625-632 (1992). RUSH, D. W. and E. EpSTEIN: Breeding and selection for salt tolerance by the incorporation of wild germplasm into a domestic tomato. J. Amer. Soc. Hort. Sci. 106, 699-704 (1981). SACHER, R. F. and R. C. STAPLES: Inositol and sugars in adaptation of tomato to salt. 77, 206-210 (1985).
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M. C. BOLARIN, A. SANTA-CRUZ, E. CAYUELA, and F. PEREZ-ALFOCEA
SCHACHTMAN, D. P. and R. MUNNS: Sodium accumulation in leaves of Triticum species that differ in salt tolerance. Aust. J. Plant Physiol. 19, 311-340 (1992). SHARP, R. E.: Comparative sensitivity of root and shoot growth and physiology to low water potentials. In: Importance of Root to Shoot Communication in the Responses to Environmental Stress. pp. 29-44. British Society for Plant Growth Regulation. Monograph 21 (1990). SUBBARAO, G. V., C. JOHANSEN, M. K. JANA, and J. V. KUMAR KAo: Effect of the sodium/calcium ratio in modifying salinity responses of pigeonpea (Pajanus cajan). J. Plant Physiol. 136, 439-443 (1990). SUDHAKAR, c., P. S. REDDY, and K. VEERANJANEYULU: Effect of salt stress on the enzymes of proline synthesis and oxidation in
J. Plant Physiol. 141,621-623 (1993). TAL, M., A. KATz, H. HEiKIN, and K. DEHAN: Salt tolerance in the wild relatives of the cultivated tomato: Proline accumulation in Lycopersicon esculentum Mill., L. peruvianum Mill. and Solanum pennellii Cor. treated with NaCl and polyethylene glycol. New Phytol. 82, 349-355 (1979). THIEL, G., J. LYNCH, and A. LXUCHU: Short-term effects of salinity stress on the turgor and elongation of growing barley leaves. J. Plant Physiol. 132, 38-44 (1988). YEO, A. R.: Salt tolerance in the halophyte Suaeda maritima L. Dum.: intracellular comparmentation of ions. J. Exp. Bot. 32, 487 -497 (1981). Greengram (phaseolus aureus Roxb.) seedlings.
Short-term Solute Changes in Leaves and Roots of Cultivated and Wild Tomato Seedlings Under Salinity M.
C. BOLARlN, A. SANTA-CRUZ,
E.
CAYUELA,
and F.
PEREZ-ALFOCEA
Centro de Edafologia y Biologia Aplicada del Segura, CSIC, Apdo 4195, E-30080, Murcia, Spain Received May 7, 1995 . Accepted August 17, 1995
Summary
The daily variations of inorganic and organic solutes with salinity (140 mM NaCI) were determined in seedlings of cultivated tomato (Lycopersicon esculentum Mill.) and its wild salt tolerant relative L. pennellii (Correll) D'Arcy for 8 days, with the objective to identify physiological traits in either leaves or roots contributing to the salt tolerance. A greater accumulation of leaf Cl- and, especially, Na+ was found in L. pennellii than in L. esculentum, while root Na+ concentrations increased similarly in both tomato species over the salinization period. The changes induced by salinity on the NaiK and NaiCa ratios were different in both tomato species; in leaves, the increases of both ratios with time of salinization were higher in L. pennellii than in L. esculentum, while in roots both ratios, mainly the NaiCa ratio, increased more in L. esculentum than in L. pennellii in the time course. The accumulation patterns of total soluble sugars were also different in both tomato species: although a higher sugar accumulation was found in L. esculentum than in L. pennellii at the end of the salinization period, an earlier sugar accumulation occurred in leaves and roots of L. pennellii. The proline accumulation was greater in the cultivated than in the wild tomato species, and earlier in roots than in leaves, but in no case did it occur in the first days of salinization. -y-aminobutyric acid (GABA) was the only amino acid that increased significantly from the beginning of the salt stress in leaves and roots of L. esculentum, which did not occur in L. pennellii.
Key words: NaCl salinity, tomato (Lycopersicon), ion contents, sugars, amino acids. Abbreviations: SS = total soluble sugars; Pro = proline; GABA = -y-aminobutyric acid. Introduction
High salt tolerance has been reported for various wild relatives of the cultivated tomato (Rush and Epstein, 1981; Bolarin et al., 1991). However, despite several attempts to enhance the salt tolerance of the cultivated tomato using the tolerance of related wild species of Lycopersicon, no commercial success has been achieved. An alternative approach is to attempt to accumulate physiological traits that contribute to tolerance within a single genotype (Cuartero et al., 1992). Therefore, a better understanding of the physiological processes and the identification of the specific physiological traits conferring salinity tolerance could playa major role in the development of breeding strategies. The fact that unclear results have been obtained until now, despite that plant response to salinity is one of the most widely researched sub© 1995 by Gustav Fischer Verlag, Stuttgart
jects in plant physiology, may be due to various factors, such as: a) the long-term salt effects are very different from those of short-term (Thiel et al., 1988), b) the majority of the data in the studies of salt tolerance are for harvests made at a single time-point, whereas there is very little information on changes that occur with time (Schachtman and Munns, 1992), and c) few studies have compared responses between leaf and root tissues despite that the primary effect of salinity is on the roots (Sharp, 1990). Munns (1993) indicated that one approach towards understanding the mechanisms of salt tolerance is to follow the series of events that exposure to salinity initiates. In this work, the daily variations of inorganic and organic solutes with the salinity were determined in leaves and roots of L. esculentum and its wild salt-tolerant relative L. pennellii for 8 days, with the objective to identify early traits of salt tolerance in order to develop breeding
464
M. C. BOURlN, A. SANTA-CIlUZ, E. CAYUELA, and F. PEllEz-AuoCEA in a Rank Hilger amino acid autoanalyzer using the ninhydrin post-column reaction. Solute contents are expressed on the basis of tissue water. The changes in different solutes with NaCl salinization were tested by one-way analysis of variance (ANOVA) using Standard Methods SYSTAT. Means were compared between treatments by LSD at the 0.05 confidence levels using Student's t-test.
strategies for transferring the higher level of salinity tolerance from wild relatives to cultivated tomato.
Materials and Methods
Plant material and culture conditions Seeds of L. esculentum Mill. cv. P-73 and L. pennellii (Correll) D'arcy accession PE-47 (collected in Peru by Cuartero et al., 1984) were used. Seeds were germinated and plants grown in a controlled culture chamber under the following conditions: a temperature and relative humidity of 28 ± 2°C and 90%, respectively, for germination; the controlled culture chamber was programmed to simulate the natural diurnal changes in temperature and light intensity for plant growth: 28/18 °C (lightldark) and 16 h light with a PPFD at the plant level of 24S/81I1molm- 2 s· 1 (400-700nm). The relative humidity was 60-70%. Plants were grown in trays (60x40x12) containing washed silica sand. Each tray was perforated at the bottom and then placed in another tray. Plants were irrigated twice a day with deionized water during the germination period. From the appearance of the first leaf until the end date, the plants were irrigated daily with Hoagland's solution; at first, a half strength Hoagland's solution was used, but was raised to a full-strength Hoagland's solution after 2 weeks until the final harvest. Nutrient solution (or deionized water during germination) was pumped into each tray from the bottom to the surface of the sand and then drained back to storage tanks, using a semi-automatic system. The nutrient solutions were completely replaced with fresh solutions twice weekly or more often. NaCl treatments (0 and 140 mM) were applied in a direct form when the plants had between 2 and 3 true leaves (15 days old).
Results
Plant growth and water content Root and shoot dry weights decreased significantly with salinity in L. esculentum, whereas they increased in L. pennelIii after 8 days of salt treatment (Table 1). In control medium, water contents were similar in roots of both species. In shoot, they were lower in L. esculentum than in L. pennel. Iii (Table 1). No great differences in the water content decreases with salinity were found in both species.
Ion concentration Leaf Na+ and CI- concentrations increased with time of salinization in the NaCl-treated plants of both tomato species, but the accumulation of both ions was much greater and quicker in L. pennellii than in L. esculentum (Fig. 1). At the end of the salinization period, the leaves of L. pennellii accumulated more Na+ than CI-, whereas no great differences were found in L. esculentum. Only the Na+ concentrations were determined in roots, with the two species showing similar Na+ accumulations at the end of the salinization period. There were no significant differences between the leaf K+ concentrations of control and treated-plants of L. esculentum throughout the period of the experiment (Table 2); these contents even increased with salinity in roots (between 2nd and 6th day). In L. pennellii a similar trend to that shown by L. esculentum was found in roots; in contrast, leaf K + concentrations decreased significantly from the second day of salt treatment. Similar Ca2 + concentrations were found in the control and NaCl-treated leaves of L. esculentum, and in roots this nutrient only decreased significantly with salinity at the end of the salinization period (Table 2). L. pennellii showed a similar response to L. esculentum in leaves, whereas the Ca2 + concentrations in roots showed either no decrease or an increase with salinity. The increases of leaf Na+ IK+ and Na+ ICa2+ ratios with time of salinization were higher in L. pennellii than in L. es-
Plant harvest and analyses Three replications of eighteen plants for each species and treatment were harvested daily from day 0 Gust prior to the time that salt treatment was applied) until day 8 after salinization. Plants were harvested usually 4 h after the onset of the light period. Leaves and roots were separated, rinsed in deionized water, blotted carefully with tissue paper and weighed. A part of this material was dried at 70°C for 72 h. At the end of the experiment, fresh and dry weights of the roots and shoots were determined. Tissue water content was obtained from the (FW-DW)/DW ratio. Dried ground plant material (500 mg) was digested in nitric and perchloric acids (2: 1) for cation analyses. The Na+, K+ and Ca2 + contents were determined by atomic absorption spectrophotometry (LaCh was previously added). Separate 100 mg samples were extracted for 30 min with 40mL of deionized water, and Cl- content was determined in the extract by potentiometric titration with AgN0 3• Analyses of SS and free amino acids were made on fresh plant material, according to Alarcon et al. (1993). Sugars were determined by the method of Jermyn (1975). Free amino acids were determined
Table 1: Dry weights (mg plant-I) and water content (mgmg- I DW) in root and shoot of control and NaCl-treated plants of L. esculentum and L. pennellii at the end of the experiment (8 days). Dry weight L. esculentum
NaClmM 0
140
Water content L. pennellii
L. esculentum
L. pennellii
Root
Shoot
Root
Shoot
Root
Shoot
Root
Shoot
353a* 234b
2959 a 1868b
41 b 50a
749b 1036a
12.8a 10.3 b
14.0a 11.5b
12.5 a 10.2b
15.0a 13.1 b
* Means within a column followed by different letters are significantly different at the 0.05 probability level.
Short-term solute changes in tomato seedlings under salinity
course. Thus, by day 8, the Na+ /Ca2 + ratio values in the roots of salt-treated plants were 3.05 and 1.14 in the cultivated and wild species, respectively.
culentum, especially the Na+ /K+ ratio after day 4 (Fig. 2). 2
However, both ratios, mainly the Na+ /Ca + ratio, increased more in roots of L. esculentum than of L. pennellii in the time L. esculentum
125
r
L. pennellii
Leaf
100 75 50
.rS
2.0 125
100
q
75
~
0
...
.~
25
0
0
:::.:: 1.0
--Z""
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f
L"'i
+
'-'
I
Root
0.5 0
"0 125
+ Z
1.5
+
S £ 50 Sw
25
465
..jf
3.0 .9 .....
100 75
;'<
"" +--Z"" U
50 25
1
~
2.0
;H
1.0
~
246802468
J.--/
0
0 2 4
Time (days)
6 8
0 2 4 6 8
Time (days)
Fig. 1: Time course of leaf and root N a+ concentrations (0) and leaf CI- concentrations (D) in L. esculentum and L. pennellii plants grown under control medium (open symbols) and 140mM NaCI (closed symbols). Values are means of 3 replicates. Vertical bars on data points are ± SE of the mean (not shown when smaller than the symbol).
Fig. 2: Time course of Na+/K+ and Na+/Ca2 + ratios in leaves and roots of L. esculentum (0) and L. pennellii (D) plants grown under control medium (open symbols) and 140mM NaCl (closed symbols). Values are means of 3 replicates. Vertical bars on data points are ± SE of the mean (not shown when smaller than the symbol).
Table 2: Potassium and calcium concentrations (mol m -3 in tissue water) of leaves and roots of L. esculentum and L. pennellii plants grown under control and saline conditions. NaCI mM L. esculentum Leaf Root L. pennellii Leaf Root L. esculentum Leaf Root L. pennellii Leaf Root
Time from salinization (days) 2
0
0 140 0 140
112.7
0 140 0 140
78,4
89,4
101.5
0 140 0 140
115.6
0 140 0 140
144.8
37.2
43.3
3
4
5
6
7
8
127.7 a 133.8 a 97,4b 131.5 a
128.1 a 136.7 a 96.9b 117.9 a
119.8 a 116.2 a 103.5 a 103,4 a
119.3 a 103.6a 86.7 a 74.2 a
119.3 a* 108.0a 91.2 a 89.2a
120.8 a 116.9 a 106.6 b 130.7 a
130.8 a 126.2 a 77.6b 97.7 a
Potassium 136.3 a 148.2 a 80.6b 117.2 a
94.6a 95.8a 104.1 a 97.9a
106.5 a 83.1 b 116.0 a 90.0b
111.9 a 97.1 a 89.6a 94.2 a
113.0a 93.9b 96.0a 106.1 a
108.2a 85.ob 102.2 b 129.2 a
103.6a 82,4b 126.1 a 146.2 a
103.5 a 81.4b 122.2 b 153.5 a
101.5 a 68.1 b 122.9a 126.7 a
121.8 a 122.0a 32.2a 27,4 a
107.1 a 112.8 a 42.3 a 48.7 a
111.5 a 108.7 a 29.7a 32.3a
Calcium 115,4 a 115.2 a 33.9a 32.3 a
112.0 a 115.5 a 27.0a 31.1 a
104.1 a 116.2 a 26.2a 27.3 a
127.8 a 127.5 a 37.1 a 25.6b
113.9a 96.0a 35.8a 21.1 b
147.8a 148,4 a 38,4 a 38.6a
121.6 a 135.0 a 45,4 a 39.2a
132.3 a 121.6 a 38,4 b 47.6a
135.0 a 128.9 a 40.7b 49.9 a
119.0 a 124.8 a 40.9b 51.9a
112.7 a 97,4 a 52.8a 49.6 a
108.7 a 104.0 a 50.9b 61.3 a
110.8 a 102.9 a 59.5 a 60.0 a
* For each plant part, values within each column followed by different letters are significantly different at the 0.05 probability level.
M. C. BOLAR1N, A. SANTA-CRUZ, E. CAYUELA, and F. PWZ-ALFOCEA
466
Organic solutes
L. esculentum leaves accumulated three times more 55 under salt stress than those of L. pennellii at the end of salinization period (Fig. 3). However, in the wild species they increased significantly in the first two days of treatment, which did not occur in the cultivated species. In roots of L. esculentum, an important increase (twice that of the control plants) was only registered at day 4, followed by a decrease up to the end of the salinization period. L. pennellii showed a similar response in root to that found in leaf.
L. esculentum
L. pennellii
60
Leaf
50
.r 8
40 30
'0
20
'-'
10
8
a til
bJl
::l
Discussion
0
til
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Root
50
'0 til
40
'3
30
~
20 10 OL.....~~~.......,
0246802468
Time (days) Fig. 3: Time course of total soluble sugars in leaves and roots o.f L. esculentum and L. pennellii plants grown under control medIUm (open circles) and 140 mM N aCI (closed circles). Values are means of 3 replicates. Vertical bars on data points are ± SE of the mean (not shown when smaller than the symbol).
6
The most significant changes in the amino acid concentrations with salinity were found in Pro and GABA (Fig. 4). The other amino acids either remained unaltered or did not show a clear tendency (data not shown). In leaves of L. escu· lentum, the highest increase of Pro with salinity was found between days 7 and 8. The Pro concentrations in roots of L. esculentum tended to increase until day 6, but these increases were small at first, with the greatest increase taking place between days Sand 6. After day 6, an opposite tendency was found. In L. pennellii the Pro increases were smaller than in L. esculentum, especially in roots. The GABA contents increased in leaves and roots of NaCl-treated plants of L. esculentum during the first days of treatment, but they decreased by day 6 (Fig. 4). In this period, the highest increases found in root corresponded to the lowest in leaf. In L. pennelli~ there were no notable differences between the GABA concentrations of the control and NaCI-treated plants in leaves and roots over the salinization period, except for a tendency to increase in leaves from dayS.
L. esculentum
L. pennellii
Leaf
4
4
L. esculentum
A higher salt tolerance has been reported in L. pennellii than in L. esculentum (Dehan and Tal, 1978), which is in accordance to the results obtained in this experiment: salinity reduced plant growth of L. esculentum when the salt stress was applied for 8 days, while in L. pennellii it was increased. The higher salt tolerance of L. pennellii can be due to its inclusion mechanism, since the accumulation of leaf CI- and, especially, Na+ is much greater in L. pennellii than in L. escu· lentum. However, it did not occur in roots, which indicates that only the saline ion accumulation in leaf could be used to discriminate the salinity tolerance in tomato when the salt stress is applied for a short term. The maintenance of K + concentration in leaves of L. escu· lentum with salinity suggests a typical glycophytic mechanism of the cultivated species with a high K +-selectivity, L. pennellii
3 2
,-.,2
";'
8
'0 0
8 '-'
::======; Root
8 4
p..
2
1
o2
4 6 8 024 6 8 Time (days)
o
02468
024 6 8
Time (days)
Fig. 4: Time course of Pro and GABA concentrations in leaves and roots of L. esculen· tum and L. pennellii plants grown under control medium (open circles) and 140mM NaCI (closed circles). Values are means of 3 replicates. Vertical bars on data points are ± SE of the mean (not shown when smaller than the symbol).
Short-term solute changes in tomato seedlings under salinity
while the K+ decrease in leaves of L. pennellii could be due to the ability of the wild species to substitute Na+ for K+ (Marschner, 1986). However, the root K+ concentrations did not decrease with salinity in L. pennellii over the salinization period. Taking into account that L. pennellii shows a high transport rate of Na+ and CI- to the leaf, the contributions of these ions to the osmotic adjustment of the root could not be sufficient and, consequently, the role of K + would be to contribute to osmotic adjustment (Perez-Alfocea et al., 1993). The maintenance, or even increase, of Ca2 + concentration in roots of L. pennellii could be inducing the absence of decrease in K +, since the presence of Ca2 + seems to be necessary for K+IN a+ selectivity and for the maintenance of an appropriate K+ concentration in plant cells (Subbarao et al., 1990). An interesting result is that the K +IN a+ and, especially, the Na+ ICa2 + ratio values are lower in roots of L. pennellii than in L. esculentum, which could be related, at least partially, to the higher salt-tolerance of the wild species. Moreover, these ratios in the rooting medium appear as better indicators of salt stress than the concentration of Na+ alone, such as was indicated by Rengel (1992). The lower salt tolerance of cultivated tomato may be related to the greater energetic cost of leaf osmotic adjustment with sugars as opposed to Na+ or Cl- (Yeo, 1981), according to the low accumulation of saline ions and great sugar accumulation at the end of treatment in leaves of L. esculentum, whereas the opposite response was shown by L. pennellii. This may also be due to the fact that plant growth reduction causes a sugar accumulation that occurred in the cultivated but not in the wild species. Therefore, the fast sugar increases found in L. pennellii (during the first two days) seem not to be due to the same events as the increases found in L. esculentum. These solutes could contribute to the osmotic adjustment while the accumulation of inorganic solutes from substrate is not sufficient. The most important fact does not seem to be the sugar accumulation in itself but the earlier sugar accumulation, which indicates that L. pennellii responds more quickly to salinity than L. esculentum. It would also be interesting to determine whether each species accumulates either different sugars or different levels of each one under salinity. Thus, Sacher and Staples (1985) indicated that the levels of myo-inositol in leaves and roots of tomato genotypes were positively correlated with their salt tolerances. Proline and GABA were the two amino acids that showed the most different responses between the two tomato species as a consequence of salt stress. In relation to the Pro accumulation with salinity, our results agree with those obtained by Tal et al. (1979), since the Pro increase was smaller in the wild species than in the cultivated one. Sudhakar et al. (1993) indicated that the accumulation of Pro in salt stressed greengram seedlings was mainly due to the fact that the activity of the oxidizing enzymes was significantly inhibited in both shoots and, mainly, roots, which would also explain the higher levels of Pro in L. esculentum, the tomato species more affected by salinity. Our results also show that Pro accumulation starts only when a high sugar accumulation occurred, as reported previously in wheat leaves under water stress (Kameli and Losel, 1995). It can be concluded that Pro accumulation is a salt-sensitive trait in tomato, with the differences between the cultivated and wild tomato species
467
being greater and earlier in roots than in leaves. Perez-Alfocea et al. (1994), in a study carried out in leaves and calli of cultivated and wild tomato species, indicated that not only Pro but also GABA accumulation could be used as salt-sensitive sensors at both organization levels when the salt treatments were applied for a long term. However, the only free amino acid that increased significantly from the beginning of salt treatment was GABA, this occurring only in leaves and roots of L. esculentum, which suggests that an increase of GABA is an earlier trait than the increase of Pro. Acknowledgements
We wish to thank Mr. E. Hasler for reviewing the manuscript in the English language and Mrs. M. R. Rojo for her technical assistance. This work has been supported by Project AIR 3 CT94-1508.
References ALARCON, J. J., M. J. SANCHEZ-BLANCO, M. C. BOLA!UN, and A. TORRECILLAS: Water relations and osmotic adjustment in Lyeopersicon eseulentum and L. pennellii during short term salt exposure and recovery. Physiol. Plant. 89, 441-447 (1993). BOLA!UN, M. c., F. G. FERNANDEZ, V. CRUZ, and J. CUARTERO: Salinity tolerance in four wild tomato species using vegetative yieldsalinity response curves. J. Amer. Soc. Hort. Sci. 116, 286-290 (1991). CUARTERO, J., F. NUEZ, and A. DIAz: Catalog of collections of Lyeopersieon and L. pennellii from Northwest of Peru. TGC Report 34,43 -46 (1984). CUARTERO, J., A. R. YEO, and T. J. FLOWERS: Selection of donors for salt-tolerance in tomato using physiological traits. New Phytol. 121,63 -69 (1992). DEHAN, K. and M. TAL: Salt tolerance in the wild relatives of the cultivated tomato: Responses of Solanum pennellii to high salinity. lrrig. Sci. 1, 71-76 (1978). JERMYN, M. A.: Increasing the sensitivity of the anthrone method for carbohydrate. Anal. Biochem. 68, 332-335 (1975). KAMELI, A. and D. M. UiSEL: Contribution of carbohydrates and solutes to osmotic adjustment in wheat leaves under water stress. J. Plant Physiol. 145, 363-366 (1995). MARSCHNER, H.: Mineral nutrition in higher plants. Academic Press Inc., London, 1986. MUNNS, R.: Physiological process limiting plant growth in saline soils: some dogmas and hypotheses. Plant Cell Environ. 16, 1524 (1993). PEREZ-ALFOCEA, F., M. T. ESTAN, M. CARO, and G. GUERRIER: Osmotic adjustment in Lycopersieon eseulentum and L. pennellii under NaCI and polyethylene glycol 6000 iso-osmotic stresses. Physiol. Plant. 87, 493-498 (1993). PEREZ-ALFOCEA, F., A. SANTA-CRUZ, G. GUERRIER, and M. C. BOLA!UN: NaCl stress induced solute changes on leaves and calli from leaf of Lycopersieon eseulentum, L. pennellii and their interspecific hybrid. J. Plant Physiol. 143, 106-111 (1994). RENGEL, Z.: The role of calcium in salt toxicity. Plant Cell Environ. 15,625-632 (1992). RUSH, D. W. and E. EpSTEIN: Breeding and selection for salt tolerance by the incorporation of wild germplasm into a domestic tomato. J. Amer. Soc. Hort. Sci. 106, 699-704 (1981). SACHER, R. F. and R. C. STAPLES: Inositol and sugars in adaptation of tomato to salt. 77, 206-210 (1985).
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M. C. BOLARIN, A. SANTA-CRUZ, E. CAYUELA, and F. PEREZ-ALFOCEA
SCHACHTMAN, D. P. and R. MUNNS: Sodium accumulation in leaves of Triticum species that differ in salt tolerance. Aust. J. Plant Physiol. 19, 311-340 (1992). SHARP, R. E.: Comparative sensitivity of root and shoot growth and physiology to low water potentials. In: Importance of Root to Shoot Communication in the Responses to Environmental Stress. pp. 29-44. British Society for Plant Growth Regulation. Monograph 21 (1990). SUBBARAO, G. V., C. JOHANSEN, M. K. JANA, and J. V. KUMAR KAo: Effect of the sodium/calcium ratio in modifying salinity responses of pigeonpea (Pajanus cajan). J. Plant Physiol. 136, 439-443 (1990). SUDHAKAR, c., P. S. REDDY, and K. VEERANJANEYULU: Effect of salt stress on the enzymes of proline synthesis and oxidation in
J. Plant Physiol. 141,621-623 (1993). TAL, M., A. KATz, H. HEiKIN, and K. DEHAN: Salt tolerance in the wild relatives of the cultivated tomato: Proline accumulation in Lycopersicon esculentum Mill., L. peruvianum Mill. and Solanum pennellii Cor. treated with NaCl and polyethylene glycol. New Phytol. 82, 349-355 (1979). THIEL, G., J. LYNCH, and A. LXUCHU: Short-term effects of salinity stress on the turgor and elongation of growing barley leaves. J. Plant Physiol. 132, 38-44 (1988). YEO, A. R.: Salt tolerance in the halophyte Suaeda maritima L. Dum.: intracellular comparmentation of ions. J. Exp. Bot. 32, 487 -497 (1981). Greengram (phaseolus aureus Roxb.) seedlings.