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Cold acclimation and abscisic acid induced alterations in carbohydrate content in calli of wheat genotypes differing in frost tolerance
J. Plant Physiol. 161. 131 – 133 (2004) http://www.elsevier-deutschland.de/jplhp
Short Communication Cold acclimation and abscisic acid induced alterations in carbohydrate content in calli of wheat genotypes differing in frost tolerance Ildikó Kerepesi1 *, Éva Bányai-Stefanovits2, Gábor Galiba3 1
Department of Genetic and Molecular Biology, University of Pécs, 7624 Pécs, Ifjúság u. 6, Hungary
2
Department of Applied Chemistry, Szent István University, 1118 Budapest, Villányi u. 29 – 31, Hungary
3
Agricultural Research Institute of the Hungarian Academy of Sciences, 2462 Martonvásár P.O. Box 19, Hungary
Received February 27, 2002 · Accepted February 20, 2003
Summary The effect of cold and abscisic acid (ABA) treatment on soluble carbohydrate content was compared in callus cultures of wheat genotypes differing in frost tolerance. The effect of 5A chromosome substituted from the frost tolerant «Cheyenne» to the sensitive «Chinese Spring» on cold-induced carbohydrate accumulation was also determined. Following cold hardening, the increase in sucrose and fructan level in calli of tolerant varieties was significantly higher than those of the sensitive ones. In 5A substitution line higher sucrose and fructan content was detected than in recipient «Chinese Spring». Tendentiously, cold stress caused higher degree of changes in carbohydrate content than the exogenously applied ABA did. Comparing the accumulation pattern of the components of WSC measured in vitro to the previously published in vivo results it can be concluded that in the case of sucrose and fructans it was similar, while for the reducing sugars it was different. The regulatory role of chromosome 5A either in the development of freezing tolerance or carbohydrate accumulation was confirmed in dedifferentiated calli, as well. Key words: carbohydrates – cold hardening – freezing tolerance – tissue culture – wheat
Introduction Water-soluble carbohydrates accumulate (WSC), as compatible solutes within vegetative tissues of many plant species as a result of exposure to various abiotic stress conditions. Numerous authors have found a positive correlation between * E-mail corresponding author: [email protected]
sugar content and the degree of tolerance to abiotic stresses in cereals, suggesting that an higher sugar content increases hardiness (Suzuki 1989, Al Hakimi et al. 1995, Kerepesi et al. 1998). Apart from the cold treatment, exogenous application of plant hormone abscisic acid (ABA) also induces frost tolerance in both wheat seedlings (Veisz et al. 1996) and cultured cells (Galiba et al. 1993). The role of ABA is very important in 0176-1617/04/161/01-131 $ 30.00/0
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Ildikó Kerepesi, Éva Bányai-Stefanovits, Gábor Galiba
the cold signal transduction (Tëhtiharju and Palva 2001) and may affect sugar translocation by its specific interaction with potassium channel AKT3 (Lacombe et al. 2000). The analysis of chromosome substitution lines in wheat showed that major genes influencing frost tolerance (Fr1) and vernalization requirement (Vrn-A1) were localized on the long arm of 5A chromosome (Sutka 1981, Galiba and Sutka 1988). The 5A chromosome of winter wheat «Cheyenne» increased not only the freezing tolerance at the spring wheat «Chinese Spring» genetic background, but also increased the ABA and carbohydrate content during cold acclimation (Galiba et al. 1993, Vágújfalvi et al. 1999). It was shown that the gene regulating cold induced sucrose accumulation closely associated with the Vrn-A1 locus but separable from the Fr1 gene (Galiba et al. 1997, 2002). According to the proposed model of Fowler et al. (1999) the developmental genes act as master switches controlling the duration of the expression of low temperature-induced (LT) structural genes. Transition from the vegetative to the reproductive growth phase can be perceived as a critical step that initiates the down regulation of LT-induced genes (Fowler et al. 1996, Mahfoozi et al. 2001). As a result, full expression of cold hardiness genes only occurs in the vegetative phase and plants in the reproductive phase have a limited ability to cold acclimate. Accordingly, it was proposed that Vrn-A1 allele had a dominant pleiotropic effect for frost susceptibility (Fowler et al. 1999). Since the gene regulating cold-induced sucrose accumulation tightly linked to the Vrn-A1 it can be hypothesized that it is controlled by Vrn-A1 either directly or pleiotropically. The effect of developmental genes is suppressed by hormone treatment in vitro to keep the tissues in undifferentiated state. Our question was whether the changes in carbohydrate content in the heterotrophic callus tissue would follow the same pattern already described on the whole plant level. Accordingly, in this study the effect of ABA and cold hardening on the accumulation of soluble carbohydrates in wheat calli differing in frost tolerance was compared.
Chemical analyses Total water-soluble carbohydrate (WSC), glucose, fructose, sucrose, fructan and glucan content were determined on fresh callus materials (Kerepesi et al. 1996). The experimental design was a random complete block, with four replications. A replicate means one Petri dish containing 10 callus pieces. The data were analysed by the STATGRAPHICS (Statistical Graphics Corporation, Princeton USA) statistical package, using the t-test and ANOVA functions to assess significant differences between means.
Results and Discussion The percent survival of the two frost tolerant varieties «Mironovskaya 808» and «Cheyenne», proved to be significantly higher than the survival of «Chinese Spring» after the applied freezing temperatures. E. g.: After –11 ˚C treatment the survival rate of Mir, Ch, and CS was 62 % ± 5.3 (SD), 58 % ± 8.4 and 30 % ± 5.2, respectively. The substitution of chromosome 5A of CS with the same chromosome of Ch resulted in significantly increased frost tolerance (53 % ± 8.5) compared to CS. The results of this viability assay proved to be correspondent to plants acclimatized in controlled environment chambers (Sutka 1981). In vivo the cold treatment caused a considerable increase in WSC content (Öquist et al. 1993, Galiba et al. 1997). Conversely, in our in vitro system the WSC content increased only in the highly frost tolerant Mir and Ch genotypes (Table 1). More over glucose and fructose content remained constant in Mir, and Ch, but decreased considerably in CS and CS (Ch 5A) after the 6 week cold treatment (Table 1). In vivo a transient accumulation of reducing sugars was observed in the
Table 1. WSC, glucose and fructose content in calli of wheat genotypes exposure to cold acclimation and ABA treatment. Mean values ± SD are presented. Mir.
Ch
Cs/Ch5A
CS
mg/g fw
Materials and Methods Tissue cultures of Triticum aestivum L. cvs. were initiated from immature embryos of the highly frost tolerant winter wheat cvs. «Mironovskaya 808» (Mir) and «Cheyenne» (Ch), and of the frost sensitive «Chinese Spring» (CS), and of the frost tolerant CS/Ch 5A substitution line (Galiba and Sutka 1988, Galiba et al. 1993). To study the ABA effect on calli, filter-sterilised ABA was added to the medium in 40 mg/L and 100 mg/L concentrations before the agar started to solidify. The ABA treatment lasted one week in darkness at 26 ˚C. The 6 week long cold hardening, freezing at –11 and –13 ˚C and the triphenyltetrazolium viability assay were carried out as it was described by Galiba et al. (1993).
WSC Control Hardened ABS 40 ABS100
21.98 ± 3.01 36.74 ± 4.25 25.84 ± 3.47 28 ± 3.11
24.08 ± 3.76 35.19 ± 5.21 28.53 ± 3.44 28.43 ± 3.46
22.18 ± 3.34 27.84 ± 3.37 26.84 ± 3.24 26.11 ± 3.28
19.14 ± 3.16 24.11 ± 2.47 27.5 ± 03.13 26.84 ± 4.24
Glucose Control Hardened ABS 40 ABS 100
5.94 ± 0.76 6.86 ± 0.55 4.16 ± 0.61 5.85 ± 0.57
8.29 ± 0.77 7.71 ± 0.701 7.92 ± 0.66 6.35 ± 0.87
8.16 ± 0.91 2.88 ± 0.33 5.15 ± 0.64 6.72 ± 0.76
5.97 ± 0.67 1.86 ± 0.21 6.2 ± 0.97 4.48 ± 0.55
Fructose Control Hardeded ABS 40 ABS 100
6.67 ± 0.71 8.15 ± 0.76 6.48 ± 0.55 7.39 ± 0.84
9.46 ± 1.52 8.37 ± 0.97 9.14 ± 1.15 7 ± 0.67
9.28 ± 1.14 3.69 ± 0.47 6.81 ± 0.73 8.15 ± 0.91
7.67 ± 0.88 3.11 ± 0.47 7.92 ± 0.87 5.06 ± 0.64
Cold acclimation, abscisic acid and carbohydrates
Figure 1. Sucrose content in cold-hardened and ABA-treated wheat calli. The differences from the control were significant at the P < 0.05 (*); P < 0.01 (**) and P < 0.001 (***) levels.
133
the fructan concentration was observed following the ABA treatment. In this study we confirmed that the genes located on 5A chromosome play a central role in the regulation of both coldand ABA-induced fructan and sucrose accumulation. Comparing the carbohydrate accumulation pattern measured in vitro to in vivo systems we can conclude that the expression of the regulator gene situated in the chromosome 5A most likely is independent from the developmental phase. The effect of cold and ABA on soluble carbohydrate composition proved to be different: proportion of sugar content was more radically altered by cold treatment. This result is in agreement with the recent view about the role of ABA in the stress acclimation process, namely: ABA is only one of the factors which regulates stress adaptation (Shinozaki and Yamaguchi-Shinozaki 1997), therefore the regulation of some enzymes involved in the cold-induced carbohydrate metabolism is likely to be ABA independent. Acknowledgements. This work was supported by grants from the National Committee for Technological Development ‹Biotechnology 2000› (No. 02579/2000) and from the Hungarian National Research Found (No. T034277 and M28074).
Figure 2. Fructan content in cold-hardened and ABA-treated wheat calli. The differences from the control were significant at the P < 0.05 (*); P < 0.01 (**) and P < 0.001 (***) levels.
References
same genotypes reaching the maximum level between 19 and 35 day cold treatment. Afterwards the concentration decreased sharply back to the control level (Vágújfalvi et al. 1999). Characteristic, genotype-related increment in the components of soluble carbohydrates was detected in sucrose and fructan levels. Sucrose accumulation (Fig. 1) was significantly higher (P < 0.001) in the tolerant Mir Ch and Cs/Ch5A, than in the sensitive CS. Cold induced fructan accumulation (Fig. 2) was also the highest in Mir (8.98 mg/g fw) and the lowest in CS (5.57mg/g fw). Furthermore, the presence of the Ch 5A chromosome increased the sucrose and fructan concentrations at the CS genetic background to similar level like in Cheyenne. This accumulation pattern was very similar to the changes described in planta (Galiba et al. 1997, Vágújfalvi et al. 1999). The strict positive correlation between the fructan content and the freezing tolerance was also demonstrated in field studies (Olien and Clark 1993). The exogenously applied ABA increased the sucrose concentration in each genotype independently from their stress tolerance (Fig. 1). In this respect the 100 mg/L ABA concentration was more effective than the 40 mg/L. Opposite to this finding, ABA, only in the lower (40 mg/L) concentration, caused elevated fructan accumulation (Fig. 2). The 40 mg/L ABA induced fructan accumulation showed genotype-related degree: the highest concentration was detected in Mir followed by Ch and CS/Ch 5A. In the CS even some decrease in
Al Hakimi A, Monneveux P, Galiba G (1995) J Genet Breed 49: 237– 244 Galiba G, Kocsy G, Kerepesi I, Vágújfalvi A, Cattivelli L, Sutka J (2002) In: Li PH, Palva T (eds) Plant Cold Hardiness: Gene Regulation and Genetic Engeneer. Kluwer Academic/Plenum Publishers, New York pp 139–160 Galiba G, Kerepesi I, Snape JW, Sutka J (1997) Theor Appl Genet 95: 265 – 270 Galiba G, Sutka J (1988) Plant Breed 101: 132–136 Galiba G, Tuberosa R, Kocsy G, Sutka J (1993) Plant Breed 110: 237– 242 Fowler DB, Chauvin LP, Limin AE, Sarhan F (1996) Theor Appl Genet 93: 554 – 559 Fowler DB, Limin AE, Ritchie JT (1999) Crop Sci 39: 626 – 633 Kerepesi I, Tóth M, Boross L (1996) J Agric Food Chem 10: 3235 – 3239 Kerepesi I, Galiba G, Bányai É (1998) J Agric Food Chem 12: 5355 – 5361 Lacombe B, Pilot G, Michard E, Gaymard F, Sentenac H, Thibaud J-B (2000) Plant Cell 12: 837– 851 Mahfoozi S, Limin AE, Fowler DB (2001) Ann Bot 87: 751–757 Olien CR, Clark JL (1993) Crop Sci 85: 21– 29 Öquist G, Hurry VM, Huner NPA (1993) Plant Physiol 101: 245 – 250 Shinozaki K, Yamaguchi-Shinozaki K (1997) Plant Physiol 115: 327– 334 Sutka J (1981) Theor Appl Genet 59: 145–152 Suzuki M (1989) J Plant Physiol 134: 224 – 231 Tëhtiharju S, Palva T (2001) Plant J 26: 461– 470 Vágújfalvi A, Kerepesi I, Galiba G, Tischner T, Sutka J (1999) Plant Sci 144: 85 – 92 Veisz O, Galiba G, Sutka J (1996) J Plant Physiol 149: 439 – 443
Short Communication Cold acclimation and abscisic acid induced alterations in carbohydrate content in calli of wheat genotypes differing in frost tolerance Ildikó Kerepesi1 *, Éva Bányai-Stefanovits2, Gábor Galiba3 1
Department of Genetic and Molecular Biology, University of Pécs, 7624 Pécs, Ifjúság u. 6, Hungary
2
Department of Applied Chemistry, Szent István University, 1118 Budapest, Villányi u. 29 – 31, Hungary
3
Agricultural Research Institute of the Hungarian Academy of Sciences, 2462 Martonvásár P.O. Box 19, Hungary
Received February 27, 2002 · Accepted February 20, 2003
Summary The effect of cold and abscisic acid (ABA) treatment on soluble carbohydrate content was compared in callus cultures of wheat genotypes differing in frost tolerance. The effect of 5A chromosome substituted from the frost tolerant «Cheyenne» to the sensitive «Chinese Spring» on cold-induced carbohydrate accumulation was also determined. Following cold hardening, the increase in sucrose and fructan level in calli of tolerant varieties was significantly higher than those of the sensitive ones. In 5A substitution line higher sucrose and fructan content was detected than in recipient «Chinese Spring». Tendentiously, cold stress caused higher degree of changes in carbohydrate content than the exogenously applied ABA did. Comparing the accumulation pattern of the components of WSC measured in vitro to the previously published in vivo results it can be concluded that in the case of sucrose and fructans it was similar, while for the reducing sugars it was different. The regulatory role of chromosome 5A either in the development of freezing tolerance or carbohydrate accumulation was confirmed in dedifferentiated calli, as well. Key words: carbohydrates – cold hardening – freezing tolerance – tissue culture – wheat
Introduction Water-soluble carbohydrates accumulate (WSC), as compatible solutes within vegetative tissues of many plant species as a result of exposure to various abiotic stress conditions. Numerous authors have found a positive correlation between * E-mail corresponding author: [email protected]
sugar content and the degree of tolerance to abiotic stresses in cereals, suggesting that an higher sugar content increases hardiness (Suzuki 1989, Al Hakimi et al. 1995, Kerepesi et al. 1998). Apart from the cold treatment, exogenous application of plant hormone abscisic acid (ABA) also induces frost tolerance in both wheat seedlings (Veisz et al. 1996) and cultured cells (Galiba et al. 1993). The role of ABA is very important in 0176-1617/04/161/01-131 $ 30.00/0
132
Ildikó Kerepesi, Éva Bányai-Stefanovits, Gábor Galiba
the cold signal transduction (Tëhtiharju and Palva 2001) and may affect sugar translocation by its specific interaction with potassium channel AKT3 (Lacombe et al. 2000). The analysis of chromosome substitution lines in wheat showed that major genes influencing frost tolerance (Fr1) and vernalization requirement (Vrn-A1) were localized on the long arm of 5A chromosome (Sutka 1981, Galiba and Sutka 1988). The 5A chromosome of winter wheat «Cheyenne» increased not only the freezing tolerance at the spring wheat «Chinese Spring» genetic background, but also increased the ABA and carbohydrate content during cold acclimation (Galiba et al. 1993, Vágújfalvi et al. 1999). It was shown that the gene regulating cold induced sucrose accumulation closely associated with the Vrn-A1 locus but separable from the Fr1 gene (Galiba et al. 1997, 2002). According to the proposed model of Fowler et al. (1999) the developmental genes act as master switches controlling the duration of the expression of low temperature-induced (LT) structural genes. Transition from the vegetative to the reproductive growth phase can be perceived as a critical step that initiates the down regulation of LT-induced genes (Fowler et al. 1996, Mahfoozi et al. 2001). As a result, full expression of cold hardiness genes only occurs in the vegetative phase and plants in the reproductive phase have a limited ability to cold acclimate. Accordingly, it was proposed that Vrn-A1 allele had a dominant pleiotropic effect for frost susceptibility (Fowler et al. 1999). Since the gene regulating cold-induced sucrose accumulation tightly linked to the Vrn-A1 it can be hypothesized that it is controlled by Vrn-A1 either directly or pleiotropically. The effect of developmental genes is suppressed by hormone treatment in vitro to keep the tissues in undifferentiated state. Our question was whether the changes in carbohydrate content in the heterotrophic callus tissue would follow the same pattern already described on the whole plant level. Accordingly, in this study the effect of ABA and cold hardening on the accumulation of soluble carbohydrates in wheat calli differing in frost tolerance was compared.
Chemical analyses Total water-soluble carbohydrate (WSC), glucose, fructose, sucrose, fructan and glucan content were determined on fresh callus materials (Kerepesi et al. 1996). The experimental design was a random complete block, with four replications. A replicate means one Petri dish containing 10 callus pieces. The data were analysed by the STATGRAPHICS (Statistical Graphics Corporation, Princeton USA) statistical package, using the t-test and ANOVA functions to assess significant differences between means.
Results and Discussion The percent survival of the two frost tolerant varieties «Mironovskaya 808» and «Cheyenne», proved to be significantly higher than the survival of «Chinese Spring» after the applied freezing temperatures. E. g.: After –11 ˚C treatment the survival rate of Mir, Ch, and CS was 62 % ± 5.3 (SD), 58 % ± 8.4 and 30 % ± 5.2, respectively. The substitution of chromosome 5A of CS with the same chromosome of Ch resulted in significantly increased frost tolerance (53 % ± 8.5) compared to CS. The results of this viability assay proved to be correspondent to plants acclimatized in controlled environment chambers (Sutka 1981). In vivo the cold treatment caused a considerable increase in WSC content (Öquist et al. 1993, Galiba et al. 1997). Conversely, in our in vitro system the WSC content increased only in the highly frost tolerant Mir and Ch genotypes (Table 1). More over glucose and fructose content remained constant in Mir, and Ch, but decreased considerably in CS and CS (Ch 5A) after the 6 week cold treatment (Table 1). In vivo a transient accumulation of reducing sugars was observed in the
Table 1. WSC, glucose and fructose content in calli of wheat genotypes exposure to cold acclimation and ABA treatment. Mean values ± SD are presented. Mir.
Ch
Cs/Ch5A
CS
mg/g fw
Materials and Methods Tissue cultures of Triticum aestivum L. cvs. were initiated from immature embryos of the highly frost tolerant winter wheat cvs. «Mironovskaya 808» (Mir) and «Cheyenne» (Ch), and of the frost sensitive «Chinese Spring» (CS), and of the frost tolerant CS/Ch 5A substitution line (Galiba and Sutka 1988, Galiba et al. 1993). To study the ABA effect on calli, filter-sterilised ABA was added to the medium in 40 mg/L and 100 mg/L concentrations before the agar started to solidify. The ABA treatment lasted one week in darkness at 26 ˚C. The 6 week long cold hardening, freezing at –11 and –13 ˚C and the triphenyltetrazolium viability assay were carried out as it was described by Galiba et al. (1993).
WSC Control Hardened ABS 40 ABS100
21.98 ± 3.01 36.74 ± 4.25 25.84 ± 3.47 28 ± 3.11
24.08 ± 3.76 35.19 ± 5.21 28.53 ± 3.44 28.43 ± 3.46
22.18 ± 3.34 27.84 ± 3.37 26.84 ± 3.24 26.11 ± 3.28
19.14 ± 3.16 24.11 ± 2.47 27.5 ± 03.13 26.84 ± 4.24
Glucose Control Hardened ABS 40 ABS 100
5.94 ± 0.76 6.86 ± 0.55 4.16 ± 0.61 5.85 ± 0.57
8.29 ± 0.77 7.71 ± 0.701 7.92 ± 0.66 6.35 ± 0.87
8.16 ± 0.91 2.88 ± 0.33 5.15 ± 0.64 6.72 ± 0.76
5.97 ± 0.67 1.86 ± 0.21 6.2 ± 0.97 4.48 ± 0.55
Fructose Control Hardeded ABS 40 ABS 100
6.67 ± 0.71 8.15 ± 0.76 6.48 ± 0.55 7.39 ± 0.84
9.46 ± 1.52 8.37 ± 0.97 9.14 ± 1.15 7 ± 0.67
9.28 ± 1.14 3.69 ± 0.47 6.81 ± 0.73 8.15 ± 0.91
7.67 ± 0.88 3.11 ± 0.47 7.92 ± 0.87 5.06 ± 0.64
Cold acclimation, abscisic acid and carbohydrates
Figure 1. Sucrose content in cold-hardened and ABA-treated wheat calli. The differences from the control were significant at the P < 0.05 (*); P < 0.01 (**) and P < 0.001 (***) levels.
133
the fructan concentration was observed following the ABA treatment. In this study we confirmed that the genes located on 5A chromosome play a central role in the regulation of both coldand ABA-induced fructan and sucrose accumulation. Comparing the carbohydrate accumulation pattern measured in vitro to in vivo systems we can conclude that the expression of the regulator gene situated in the chromosome 5A most likely is independent from the developmental phase. The effect of cold and ABA on soluble carbohydrate composition proved to be different: proportion of sugar content was more radically altered by cold treatment. This result is in agreement with the recent view about the role of ABA in the stress acclimation process, namely: ABA is only one of the factors which regulates stress adaptation (Shinozaki and Yamaguchi-Shinozaki 1997), therefore the regulation of some enzymes involved in the cold-induced carbohydrate metabolism is likely to be ABA independent. Acknowledgements. This work was supported by grants from the National Committee for Technological Development ‹Biotechnology 2000› (No. 02579/2000) and from the Hungarian National Research Found (No. T034277 and M28074).
Figure 2. Fructan content in cold-hardened and ABA-treated wheat calli. The differences from the control were significant at the P < 0.05 (*); P < 0.01 (**) and P < 0.001 (***) levels.
References
same genotypes reaching the maximum level between 19 and 35 day cold treatment. Afterwards the concentration decreased sharply back to the control level (Vágújfalvi et al. 1999). Characteristic, genotype-related increment in the components of soluble carbohydrates was detected in sucrose and fructan levels. Sucrose accumulation (Fig. 1) was significantly higher (P < 0.001) in the tolerant Mir Ch and Cs/Ch5A, than in the sensitive CS. Cold induced fructan accumulation (Fig. 2) was also the highest in Mir (8.98 mg/g fw) and the lowest in CS (5.57mg/g fw). Furthermore, the presence of the Ch 5A chromosome increased the sucrose and fructan concentrations at the CS genetic background to similar level like in Cheyenne. This accumulation pattern was very similar to the changes described in planta (Galiba et al. 1997, Vágújfalvi et al. 1999). The strict positive correlation between the fructan content and the freezing tolerance was also demonstrated in field studies (Olien and Clark 1993). The exogenously applied ABA increased the sucrose concentration in each genotype independently from their stress tolerance (Fig. 1). In this respect the 100 mg/L ABA concentration was more effective than the 40 mg/L. Opposite to this finding, ABA, only in the lower (40 mg/L) concentration, caused elevated fructan accumulation (Fig. 2). The 40 mg/L ABA induced fructan accumulation showed genotype-related degree: the highest concentration was detected in Mir followed by Ch and CS/Ch 5A. In the CS even some decrease in
Al Hakimi A, Monneveux P, Galiba G (1995) J Genet Breed 49: 237– 244 Galiba G, Kocsy G, Kerepesi I, Vágújfalvi A, Cattivelli L, Sutka J (2002) In: Li PH, Palva T (eds) Plant Cold Hardiness: Gene Regulation and Genetic Engeneer. Kluwer Academic/Plenum Publishers, New York pp 139–160 Galiba G, Kerepesi I, Snape JW, Sutka J (1997) Theor Appl Genet 95: 265 – 270 Galiba G, Sutka J (1988) Plant Breed 101: 132–136 Galiba G, Tuberosa R, Kocsy G, Sutka J (1993) Plant Breed 110: 237– 242 Fowler DB, Chauvin LP, Limin AE, Sarhan F (1996) Theor Appl Genet 93: 554 – 559 Fowler DB, Limin AE, Ritchie JT (1999) Crop Sci 39: 626 – 633 Kerepesi I, Tóth M, Boross L (1996) J Agric Food Chem 10: 3235 – 3239 Kerepesi I, Galiba G, Bányai É (1998) J Agric Food Chem 12: 5355 – 5361 Lacombe B, Pilot G, Michard E, Gaymard F, Sentenac H, Thibaud J-B (2000) Plant Cell 12: 837– 851 Mahfoozi S, Limin AE, Fowler DB (2001) Ann Bot 87: 751–757 Olien CR, Clark JL (1993) Crop Sci 85: 21– 29 Öquist G, Hurry VM, Huner NPA (1993) Plant Physiol 101: 245 – 250 Shinozaki K, Yamaguchi-Shinozaki K (1997) Plant Physiol 115: 327– 334 Sutka J (1981) Theor Appl Genet 59: 145–152 Suzuki M (1989) J Plant Physiol 134: 224 – 231 Tëhtiharju S, Palva T (2001) Plant J 26: 461– 470 Vágújfalvi A, Kerepesi I, Galiba G, Tischner T, Sutka J (1999) Plant Sci 144: 85 – 92 Veisz O, Galiba G, Sutka J (1996) J Plant Physiol 149: 439 – 443