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Changes in SH-containing compounds and catalase activity in apricot flower bud during the winter season
Scientia Horticulturae 73 Ž1998. 1–9
Changes in SH-containing compounds and catalase activity in apricot flower bud during the winter season R. Viti
a,)
, S. Bartolini
b
a
Dipartimento di ColtiÕazione e Difesa delle Specie Legnose, Sezione di ColtiÕazioni Arboree, UniÕersita’ degli Studi di Pisa, Via del Borghetto 80, 56123 Pisa, Italy b Scuola Superiore di Studi UniÕersitari e di Perfezionamento ‘S. Anna’, Via G. Carducci 40, 56124 Pisa, Italy Accepted 14 September 1997
Abstract Accumulation and variations of sulfhydryl compounds ŽSH., reduced glutathione ŽGSH. and catalase activity during the flower bud development cycle and their relationship were studied in two apricot cultivars, ‘S. Castrese’ and ‘Portici’, which differ in production behaviour in different years and environments. The changes in catalase activity, SH and GSH showed three distinct phases from November to February. In the first two phases, a gradual increase in catalase activity and reduced glutathione content was observed, paralleled by a decrease in total SH concentration. During phase 3 Žearly February., at the same time with an increase in total SH and GSH, catalase activity showed a tendency to decrease. The two cultivars showed a difference in flower bud response, which was detected as early as the first observations in autumn. In particular, ‘S. Castrese’ was found to present constant and elevated catalase activity and a greater GSH content. q 1998 Elsevier Science B.V. Keywords: Armeniaca Õulgaris; Sulfhydryl compounds; Glutathione; Catalase activity
1. Introduction Sulfhydryl groups ŽSH. occur in plant protoplasm mainly as components of proteins, essential amino acids Žcysteine wCSHx. and peptide Žglutathione wGSHx. ŽLevitt, 1980.. )
Corresponding author.
0304-4238r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 2 3 8 Ž 9 7 . 0 0 1 2 7 - 1
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R. Viti, S. Bartolinir Scientia Horticulturae 73 (1998) 1–9
Glutathione is the predominant low-molecular-weight sulfhydryl compound present in most living cells, maintaining protein thiol groups in the reduced state ŽFahey et al., 1975.. In plants, glutathione is involved in many metabolic detoxification processes ŽLevitt et al., 1961; Levitt, 1980.. In addition, it represents the main reserve and long-distance transport form of reduced sulfhydryls, which are indispensable for protein synthesis ŽRennenberg, 1982.. Both in photosynthesizing and non-photosynthesizing tissue, glutathione is found in two forms: the oxidated form ŽGSSG., which is converted into the reduced form ŽGSH. by glutathione reductase ŽGR. ŽFoyer and Halliwell, 1976.. The balance between GSH and GSSG maintains the SH of intracellular proteins in oxidated state ŽHalliwell and Foyer, 1978.. GSH may protect membranes from free radical damage by trapping oxygen radicals in the aqueous phase ŽBarclay, 1988.. It has been hypothesized ŽAmberger, 1984. that an increase in glutathione could stimulate the breaking of rest. Catalase activity could play a role in this process. It has been shown that in peach and grapevine, catalase activity decreases during exposure to low temperatures and dormancy breaking ŽKaminsky and Rom, 1974; Nir et al., 1986.. The decrease in catalase activity is thought to cause an increase in peroxide content and favour a shift from the Embden–Meyerhoff Parnas system ŽEMP. to the pentose phosphate ŽPP. pathway, leading to an increase in reduced nucleotide production essential for intensified metabolism ŽNir et al., 1984.. The role of glutathione in stone fruit flower buds has not been investigated. The objective of this research was to study the changes in sulfhydryl compounds and catalase activity during apricot flower bud growth. Two apricot cultivars, ‘S. Castrese’ and ‘Portici’ were compared. ‘S. Castrese’ consistently yields well in all environments, whereas ‘Portici’ can present scanty and inconstant yield. This irregular productivity is prevalently due to the appearance of elevated percentages Žabove 35%. of flower bud anomalies during the dormant period ŽGuerriero et al., 1991; Viti and Monteleone, 1991a..
2. Materials and methods Trials were carried out on container-grown Ž30 l. 5-year-old apricot trees of cultivars ‘S. Castrese’ and ‘Portici’, maintained in the open at sea level experimental station in Pisa ŽTuscan.. Trees were grafted to Myrabolan B, irrigated daily with tap water and complex nutrient mixture was added at periodic intervals. Periodically, from November to February, 600–700 flower buds were randomly collected from 1-year-old mixed twigs of 10 plants per cultivar. The following parameters were measured: Ža. flower bud growth: determined by increase in fresh weight of 100 buds; Žb. catalase actiÕity: determined in 1 g sample of flower buds using a floating disc procedure based on liberation of O 2 due to catalase activity and measured indirectly by recording the time Žs. required for an enzyme saturated paper disc to rise to the surface of a 3% hydrogen peroxide solution ŽGagnon et al., 1959.. The lowest values correspond to the highest activities. This indirect method has been utilized for its rapid and easy-to-perform assay and for the observed relationship between enzyme activity to enzyme concentration ŽKaminsky and Rom, 1974.; Žc. total water extractable sulfhydryl
R. Viti, S. Bartolinir Scientia Horticulturae 73 (1998) 1–9
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compounds (protein SH q non-protein SH): determined in 1 g samples of homogenized flower buds, using the 5,5’-dithio-bis-Ž2-nitrobenzoic acid. ŽDTNB. according to the method of Ellman Ž1959.. Both 1 cm3 0.2 M potassium phosphate ŽpH 8.0. and 0.1 cm3 10 mM DTNB in 0.2 mM potassium phosphate ŽpH 7.0. were added to 0.5 cm3 of supernatant. The absorbance was measured at 412 nm ŽGrill et al., 1979., and corrections were made for the absorbance of the sample and that of DTNB ŽGrill et al., 1979.; Žd. reduced glutathione content: determined in the deproteinized supernatant, as the difference between the DTNB-reactive compound content of a non-incubated sample and that of a sample incubated with glyoxalase I together with methylglyoxal, according to De Kok et al. Ž1981.. From November 1 Žat 50% of leaf drop. minimum and maximum daily temperatures were used to calculate Chill Units ŽC.U.., according to Richardson et al. Ž1974.. Sampling dates were also converted in DLD: Days after 50% of Leaf Drop ŽDLD 1 s November 1..
3. Results and discussion 3.1. Catalase actiÕity and flower bud growth The changes in catalase activity showed three distinct phases ŽFig. 1.. From November to January 9, the activity was initially low and rose gradually. During this first phase, the catalase activity levels in ‘S. Castrese’ were almost double than in ‘Portici’.
Fig. 1. Evolution of catalase activity Žs. from November to February detected in flower buds. The sample dates reported in x-axis are also converted in Days after Leaf Drop ŽDLD.. The correspondent Chill Units cumulated ŽC.U.. are indicated. Bar indicate LSD P F 0.05.
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R. Viti, S. Bartolinir Scientia Horticulturae 73 (1998) 1–9
During the second phase, no change in catalase activity was observed. In this phase, the activity was highest and similar in the two cultivars. A third phase was noted afterwards, in which catalase activity decreased gradually to the initial values. In ‘Portici’, decrease was rapid and started in late January. In ‘S. Castrese’, no decrease in catalase activity was recorded until February 3. During this phase, the activity was significantly higher in ‘S. Castrese’ than in ‘Portici’. Flower bud fresh weight ŽFig. 2. did not change during the first and the second phase Žcorresponding to accumulation of roughly 1100 and 1300 C.U., respectively.. During phase 3, bud fresh weight increased. Weight increase was faster for ‘S. Castrese’ than for ‘Portici’. The changes in catalase activity observed are in agreement with earlier reports for peach ŽKaminsky and Rom, 1974. and apricot ŽScalabrelli et al., 1991.. The decrease in catalase activity coincided with the resumption of active bud growth. It has been suggested that the decrease in catalase activity leads to an increase in peroxide content and to the activation of the pentose phosphate pathway ŽSimmonds and Simpson, 1972; Nir et al., 1984.. Throughout the study, catalase activity was higher in ‘S. Castrese’ than in ‘Portici’, even during the bud growth resumption. This trend could explain the different appearance of flower cup anomalies of these cultivars over several years of observations: in ‘S. Castrese’, very low percentage Ž15%. was recorded, while ‘Portici’ was characterized by high incidence Ž) 35%. ŽViti and Monteleone, 1991b.. It has been suggested that a high catalase activity could perform a detoxifying function, providing protection against cell damage caused by oxidative events ŽFridovich, 1978; Patterson et al., 1984..
Fig. 2. Fresh weight Žg. of 100 flower buds detected from November to February. The sample dates reported in x-axis are also converted in DLD. The correspondent C.U. cumulated are indicated. Bars indicate standard errors of means.
R. Viti, S. Bartolinir Scientia Horticulturae 73 (1998) 1–9
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Fig. 3. Changes in the levels of total water extractable sulfhydryl compounds ŽSH. Ž m molrg fresh weight. in the flower buds from November to February. The sample dates reported in x-axis are also converted in DLD. The correspondent C.U. cumulated are indicated. Bars indicate standard errors of means.
3.2. Total water extractable sulfhydryl compounds (protein SH q non-protein SH) and reduced glutathione content The overall trend of the changes for total SH compounds was similar for both cultivars ŽFig. 3.. An elevated SH content was found during the first period and ‘S. Castrese’ had greater SH concentration than ‘Portici’, when no increase in flower bud
Fig. 4. Changes in the levels of reduced glutathione Žnmolrg fresh weight. in the flower buds from November to February. The sample dates reported in x-axis are also converted in DLD. The correspondent C.U. cumulated are indicated. Bars indicate standard errors of means.
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R. Viti, S. Bartolinir Scientia Horticulturae 73 (1998) 1–9
Fig. 5. Relationship between reduced glutathione ŽGSH. and flower bud fresh weight in cultivar ‘Portici’ Ža. and ‘S. Castrese’ Žb.. Confidence interval Ž P F 0.05..
R. Viti, S. Bartolinir Scientia Horticulturae 73 (1998) 1–9
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fresh weight was observed ŽFig. 2.. In the second period, a sudden SH decrease was recorded, to reach the lowest levels at the beginning of February. The SH content increased again during the resumption of bud growth. SH levels observed during the bud development period were in overall agreement with findings on leaves of Picea abies ŽEsterbauer and Grill, 1978. and other several species ŽLevitt et al., 1961.. The greater SH content observed during the winter period could be the necessary condition for the flower bud cold hardiness. Several authors also suggested that the elevated SH compound concentration increases the stress resistance ŽGrill et al., 1979; De Kok et al., 1981; Soldatini et al., 1992.. The changes in GSH content showed the lowest concentration in November, followed by a gradual increase ŽFig. 4.. GSH content was higher in ‘S. Castrese’ than in ‘Portici’ and the most substantial difference was recorded in November. During dormancy, a low GSH concentration would appear to correspond to decrease in bud metabolic activity. The high concentrations of oxidated form ŽGSSG. effectively inhibit initiation of protein synthesis and lead to conversion of polysomes to monosomes ŽKosower et al., 1972.. During the first phase, the elevated GSH levels observed in ‘S. Castrese’ could favour a better bud protection, preventing oxidation of protein sulfhydryl groups and thereby facilitating repair of stress-induced damage of the membrane ŽJoselyn, 1972.. The maximum GSH concentration was observed during the period of rapid bud growth and a curvilinear correlation between GSH and flower bud fresh weight was noted ŽFig. 5a,b.. In this period, GSH has been involved in metabolic activation of protein synthesis after the breaking of rest ŽEsterbauer and Grill, 1978; Guy and Carter, 1982.. Moreover, in apple buds high GSH content has been associated with rapid growth ŽWang et al., 1991..
4. Conclusions The two cultivars showed a difference in flower bud response, which was detected as early as the first observations in autumn. In particular, ‘S. Castrese’ was found to present constant and elevated catalase activity and a greater GSH content. These results appear to be in agreement with the observations on production behaviour of the two cultivars ŽGuerriero et al., 1991..‘S. Castrese’ always shows elevated production while ‘Portici’ can present scanty and inconstant yield, prevalently due to a dramatic drop of flower buds before flowering. These flower buds were damaged with browning and necrosis on the different floral organs ŽViti and Monteleone, 1991a.. Moreover, the vascular continuity between flower bud axis and ovary were stabilized considerably later in ‘Portici’ than in ‘S. Castrese’ ŽBartolini and Giorgelli, 1994.. It is probably that the better nutritional preparation associated to the elevated GSH content and catalase activity of ‘S. Castrese’ flower buds from early development stages onwards, allowing buds to develop more successfully during the rapid growth stage. However, further in-depth researches will be required in order to obtain confirmation of these preliminary results and the explanatory hypotheses put forward.
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R. Viti, S. Bartolinir Scientia Horticulturae 73 (1998) 1–9
Acknowledgements This research was supported through the contribution of the Research National Council ŽC.N.R... References Amberger, A., 1984. Uptake and metabolism of hydrogen cyanamide in plants. In: Weaver, R.J. ŽEd.., Proceeding of Bud Dormancy of Grapevines. Univ. of California, Davis, pp. 5–10. Barclay, L.R.C., 1988. The cooperative antioxidant role of glutathione with a lipid-soluble and water-soluble antioxidant during peroxidation of liposomes initiated in the aqueous phase and in the lipid phase. J. Biol. Chem. 263, 16138–16142. Bartolini, S., Giorgelli, F., 1994. Observations on development of vascular connections in two apricot cultivars. Adv. Hort. Sci. 8, 97–100. De Kok, L.J., De Kan, P.J.L., Tanczos, O.G., Kuiper, P.J.C., 1981. Sulphate induced accumulation of glutathione and frost-tolerance of spinach leaf tissue. Physiol. Plant 53, 435–438. Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77. Esterbauer, H., Grill, D., 1978. Seasonal variation of glutathione and glutathione reductase in needles of Picea abies. Plant Physiol. 61, 119–121. Fahey, R.C., Brody, S., Mikolajczyk, S.D., 1975. Changes in the glutathione thio-disulfide status of Neurospora crassa conidia during germination and aging. J. Bacteriol. 121, 144–151. Foyer, C.H., Halliwell, B., 1976. The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133, 21–25. Fridovich, I., 1978. The biology of oxygen radicals. Science 201, 875–880. Gagnon, M., Hunting, W.M., Esselen, W.B., 1959. New method for catalase determination. Anal. Chem. 31 Ž1., 144–146. Grill, D., Esterbauer, H., Klosch, U., 1979. Effect of sulphur dioxide on glutathione in leaves of plants. Environ. Pollut. 19, 187–194. Guerriero, R., Massai, R., Monteleone, P., 1991. Osservazioni comparative sulla biologia fiorale e di fruttificazione di 15 cultivar di albicocco nel litorale toscano. Agricoltura e Ricerca 122, 39–50. Guy, C.L., Carter, J.V., 1982. Effect of low temperature on the glutathione status of plant cells, In: Li, P.H., Sakai, A. ŽEds.., Plant Cold Hardiness and Freezing Stress, Vol. 2. Academic Press, New York, pp. 169–179. Halliwell, B., Foyer, C.H., 1978. Properties and physiological function of a glutathione reductase purified from spinach leaves by affinity chromatography. Planta 139, 7–17. Joselyn, P.C., 1972. Biochemistry of the Thiol Group. Academic Press, New York. Kaminsky, W., Rom, R., 1974. A possible role of catalase in the rest of peach, Prunus persica Sieb. and Zucc., flower buds. J. Am. Soc. Hort. Sci. 99 Ž1., 84–86. Kosower, N.S., Vanerhoff, G.A., Kosower, E.M., 1972. Glutathione: VIII. The effects of glutathione disulphide on initiation of protein synthesis. Biochim. Biophys. Acta 772, 623–637. Levitt, J., 1980. Responses of Plants to Environmental Stresses, Vol. I. Academic Press. Levitt, J., Sullivan, C.J., Johansson, N., Pettit, R.M., 1961. Sulfhydryls—a new factor in frost resistance. Plant Physiol., pp. 611–616. Nir, G., Shulman, Y., Fabersteich, L., Lavee, S., 1984. The involvement of catalase in the dormancy of grapevine buds. In: Weaver, R.J. ŽEd.., Proceedings of Bud Dormancy of Grapevines. Potential and Practical Uses of Hydrogen Cyanamide on Grapevines. Univ. of California, Davis, pp. 40–43. Nir, G., Shulman, Y., Fabersteich, L., Lavee, S., 1986. Changes in the activity of catalase ŽEC 1.11.1.6.. in relation to the dormancy of grapevine Ž Vitis Õinifera L.. buds. Plant Physiol. 81, 1140–1142. Patterson, B.D., Payne, L.A., Chen, Y., Graham, D., 1984. An inhibitor of catalase induced by cold in chilling-sensitive plants. Plant Physiol. 76, 1014–1018. Rennenberg, H., 1982. Glutathione metabolism and possible biological roles in higher plants. Phytochemistry 21 Ž12., 2771–2781.
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Richardson, E.A., Seeley, S.D., Walker, D.R., 1974. A model for estimating the completion of rest for ‘Redhaven’ and ‘Elberta’ peach trees. Hort. Sci. 9, 331–332. Scalabrelli, G., Viti, R., Cinelli, F., 1991. Changes in catalase activity and dormancy of apricot buds in response to chilling. Acta Hort. 293, 267–274. Simmonds, J.A., Simpson, G.M., 1972. Regulation of Krebs cycle and pentose phosphate pathway activities in the control of dormancy of AÕena fatua. Can. J. Bot. 50, 1041–1048. Soldatini, G.F., Ranieri, A., Lencioni, L., Lorenzini, G., 1992. Effects of continuous SO 2 fumigation on SH-containing compounds in two wheat cultivars of different sensitivities. J. Exp. Bot. 43 Ž251., 797–801. Viti, R., Monteleone, P., 1991a. Observations on flower bud growth in some low yield varieties of apricot. Acta Hort. 293, 327–330. Viti, R., Monteleone, P., 1991b. Etude et characterisation des anomalies de developement des bourgeons a fleur de l’abricotier. Agriculture, pp. 31–43. Wang, S.Y., Jiao, H.J., Faust, M., 1991. Changes in ascorbate, glutathione, and related enzyme activities during thidiazuron-induced bud break of apple. Physiol. Plant 82, 231–236.
Changes in SH-containing compounds and catalase activity in apricot flower bud during the winter season R. Viti
a,)
, S. Bartolini
b
a
Dipartimento di ColtiÕazione e Difesa delle Specie Legnose, Sezione di ColtiÕazioni Arboree, UniÕersita’ degli Studi di Pisa, Via del Borghetto 80, 56123 Pisa, Italy b Scuola Superiore di Studi UniÕersitari e di Perfezionamento ‘S. Anna’, Via G. Carducci 40, 56124 Pisa, Italy Accepted 14 September 1997
Abstract Accumulation and variations of sulfhydryl compounds ŽSH., reduced glutathione ŽGSH. and catalase activity during the flower bud development cycle and their relationship were studied in two apricot cultivars, ‘S. Castrese’ and ‘Portici’, which differ in production behaviour in different years and environments. The changes in catalase activity, SH and GSH showed three distinct phases from November to February. In the first two phases, a gradual increase in catalase activity and reduced glutathione content was observed, paralleled by a decrease in total SH concentration. During phase 3 Žearly February., at the same time with an increase in total SH and GSH, catalase activity showed a tendency to decrease. The two cultivars showed a difference in flower bud response, which was detected as early as the first observations in autumn. In particular, ‘S. Castrese’ was found to present constant and elevated catalase activity and a greater GSH content. q 1998 Elsevier Science B.V. Keywords: Armeniaca Õulgaris; Sulfhydryl compounds; Glutathione; Catalase activity
1. Introduction Sulfhydryl groups ŽSH. occur in plant protoplasm mainly as components of proteins, essential amino acids Žcysteine wCSHx. and peptide Žglutathione wGSHx. ŽLevitt, 1980.. )
Corresponding author.
0304-4238r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 3 0 4 - 4 2 3 8 Ž 9 7 . 0 0 1 2 7 - 1
2
R. Viti, S. Bartolinir Scientia Horticulturae 73 (1998) 1–9
Glutathione is the predominant low-molecular-weight sulfhydryl compound present in most living cells, maintaining protein thiol groups in the reduced state ŽFahey et al., 1975.. In plants, glutathione is involved in many metabolic detoxification processes ŽLevitt et al., 1961; Levitt, 1980.. In addition, it represents the main reserve and long-distance transport form of reduced sulfhydryls, which are indispensable for protein synthesis ŽRennenberg, 1982.. Both in photosynthesizing and non-photosynthesizing tissue, glutathione is found in two forms: the oxidated form ŽGSSG., which is converted into the reduced form ŽGSH. by glutathione reductase ŽGR. ŽFoyer and Halliwell, 1976.. The balance between GSH and GSSG maintains the SH of intracellular proteins in oxidated state ŽHalliwell and Foyer, 1978.. GSH may protect membranes from free radical damage by trapping oxygen radicals in the aqueous phase ŽBarclay, 1988.. It has been hypothesized ŽAmberger, 1984. that an increase in glutathione could stimulate the breaking of rest. Catalase activity could play a role in this process. It has been shown that in peach and grapevine, catalase activity decreases during exposure to low temperatures and dormancy breaking ŽKaminsky and Rom, 1974; Nir et al., 1986.. The decrease in catalase activity is thought to cause an increase in peroxide content and favour a shift from the Embden–Meyerhoff Parnas system ŽEMP. to the pentose phosphate ŽPP. pathway, leading to an increase in reduced nucleotide production essential for intensified metabolism ŽNir et al., 1984.. The role of glutathione in stone fruit flower buds has not been investigated. The objective of this research was to study the changes in sulfhydryl compounds and catalase activity during apricot flower bud growth. Two apricot cultivars, ‘S. Castrese’ and ‘Portici’ were compared. ‘S. Castrese’ consistently yields well in all environments, whereas ‘Portici’ can present scanty and inconstant yield. This irregular productivity is prevalently due to the appearance of elevated percentages Žabove 35%. of flower bud anomalies during the dormant period ŽGuerriero et al., 1991; Viti and Monteleone, 1991a..
2. Materials and methods Trials were carried out on container-grown Ž30 l. 5-year-old apricot trees of cultivars ‘S. Castrese’ and ‘Portici’, maintained in the open at sea level experimental station in Pisa ŽTuscan.. Trees were grafted to Myrabolan B, irrigated daily with tap water and complex nutrient mixture was added at periodic intervals. Periodically, from November to February, 600–700 flower buds were randomly collected from 1-year-old mixed twigs of 10 plants per cultivar. The following parameters were measured: Ža. flower bud growth: determined by increase in fresh weight of 100 buds; Žb. catalase actiÕity: determined in 1 g sample of flower buds using a floating disc procedure based on liberation of O 2 due to catalase activity and measured indirectly by recording the time Žs. required for an enzyme saturated paper disc to rise to the surface of a 3% hydrogen peroxide solution ŽGagnon et al., 1959.. The lowest values correspond to the highest activities. This indirect method has been utilized for its rapid and easy-to-perform assay and for the observed relationship between enzyme activity to enzyme concentration ŽKaminsky and Rom, 1974.; Žc. total water extractable sulfhydryl
R. Viti, S. Bartolinir Scientia Horticulturae 73 (1998) 1–9
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compounds (protein SH q non-protein SH): determined in 1 g samples of homogenized flower buds, using the 5,5’-dithio-bis-Ž2-nitrobenzoic acid. ŽDTNB. according to the method of Ellman Ž1959.. Both 1 cm3 0.2 M potassium phosphate ŽpH 8.0. and 0.1 cm3 10 mM DTNB in 0.2 mM potassium phosphate ŽpH 7.0. were added to 0.5 cm3 of supernatant. The absorbance was measured at 412 nm ŽGrill et al., 1979., and corrections were made for the absorbance of the sample and that of DTNB ŽGrill et al., 1979.; Žd. reduced glutathione content: determined in the deproteinized supernatant, as the difference between the DTNB-reactive compound content of a non-incubated sample and that of a sample incubated with glyoxalase I together with methylglyoxal, according to De Kok et al. Ž1981.. From November 1 Žat 50% of leaf drop. minimum and maximum daily temperatures were used to calculate Chill Units ŽC.U.., according to Richardson et al. Ž1974.. Sampling dates were also converted in DLD: Days after 50% of Leaf Drop ŽDLD 1 s November 1..
3. Results and discussion 3.1. Catalase actiÕity and flower bud growth The changes in catalase activity showed three distinct phases ŽFig. 1.. From November to January 9, the activity was initially low and rose gradually. During this first phase, the catalase activity levels in ‘S. Castrese’ were almost double than in ‘Portici’.
Fig. 1. Evolution of catalase activity Žs. from November to February detected in flower buds. The sample dates reported in x-axis are also converted in Days after Leaf Drop ŽDLD.. The correspondent Chill Units cumulated ŽC.U.. are indicated. Bar indicate LSD P F 0.05.
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R. Viti, S. Bartolinir Scientia Horticulturae 73 (1998) 1–9
During the second phase, no change in catalase activity was observed. In this phase, the activity was highest and similar in the two cultivars. A third phase was noted afterwards, in which catalase activity decreased gradually to the initial values. In ‘Portici’, decrease was rapid and started in late January. In ‘S. Castrese’, no decrease in catalase activity was recorded until February 3. During this phase, the activity was significantly higher in ‘S. Castrese’ than in ‘Portici’. Flower bud fresh weight ŽFig. 2. did not change during the first and the second phase Žcorresponding to accumulation of roughly 1100 and 1300 C.U., respectively.. During phase 3, bud fresh weight increased. Weight increase was faster for ‘S. Castrese’ than for ‘Portici’. The changes in catalase activity observed are in agreement with earlier reports for peach ŽKaminsky and Rom, 1974. and apricot ŽScalabrelli et al., 1991.. The decrease in catalase activity coincided with the resumption of active bud growth. It has been suggested that the decrease in catalase activity leads to an increase in peroxide content and to the activation of the pentose phosphate pathway ŽSimmonds and Simpson, 1972; Nir et al., 1984.. Throughout the study, catalase activity was higher in ‘S. Castrese’ than in ‘Portici’, even during the bud growth resumption. This trend could explain the different appearance of flower cup anomalies of these cultivars over several years of observations: in ‘S. Castrese’, very low percentage Ž15%. was recorded, while ‘Portici’ was characterized by high incidence Ž) 35%. ŽViti and Monteleone, 1991b.. It has been suggested that a high catalase activity could perform a detoxifying function, providing protection against cell damage caused by oxidative events ŽFridovich, 1978; Patterson et al., 1984..
Fig. 2. Fresh weight Žg. of 100 flower buds detected from November to February. The sample dates reported in x-axis are also converted in DLD. The correspondent C.U. cumulated are indicated. Bars indicate standard errors of means.
R. Viti, S. Bartolinir Scientia Horticulturae 73 (1998) 1–9
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Fig. 3. Changes in the levels of total water extractable sulfhydryl compounds ŽSH. Ž m molrg fresh weight. in the flower buds from November to February. The sample dates reported in x-axis are also converted in DLD. The correspondent C.U. cumulated are indicated. Bars indicate standard errors of means.
3.2. Total water extractable sulfhydryl compounds (protein SH q non-protein SH) and reduced glutathione content The overall trend of the changes for total SH compounds was similar for both cultivars ŽFig. 3.. An elevated SH content was found during the first period and ‘S. Castrese’ had greater SH concentration than ‘Portici’, when no increase in flower bud
Fig. 4. Changes in the levels of reduced glutathione Žnmolrg fresh weight. in the flower buds from November to February. The sample dates reported in x-axis are also converted in DLD. The correspondent C.U. cumulated are indicated. Bars indicate standard errors of means.
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R. Viti, S. Bartolinir Scientia Horticulturae 73 (1998) 1–9
Fig. 5. Relationship between reduced glutathione ŽGSH. and flower bud fresh weight in cultivar ‘Portici’ Ža. and ‘S. Castrese’ Žb.. Confidence interval Ž P F 0.05..
R. Viti, S. Bartolinir Scientia Horticulturae 73 (1998) 1–9
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fresh weight was observed ŽFig. 2.. In the second period, a sudden SH decrease was recorded, to reach the lowest levels at the beginning of February. The SH content increased again during the resumption of bud growth. SH levels observed during the bud development period were in overall agreement with findings on leaves of Picea abies ŽEsterbauer and Grill, 1978. and other several species ŽLevitt et al., 1961.. The greater SH content observed during the winter period could be the necessary condition for the flower bud cold hardiness. Several authors also suggested that the elevated SH compound concentration increases the stress resistance ŽGrill et al., 1979; De Kok et al., 1981; Soldatini et al., 1992.. The changes in GSH content showed the lowest concentration in November, followed by a gradual increase ŽFig. 4.. GSH content was higher in ‘S. Castrese’ than in ‘Portici’ and the most substantial difference was recorded in November. During dormancy, a low GSH concentration would appear to correspond to decrease in bud metabolic activity. The high concentrations of oxidated form ŽGSSG. effectively inhibit initiation of protein synthesis and lead to conversion of polysomes to monosomes ŽKosower et al., 1972.. During the first phase, the elevated GSH levels observed in ‘S. Castrese’ could favour a better bud protection, preventing oxidation of protein sulfhydryl groups and thereby facilitating repair of stress-induced damage of the membrane ŽJoselyn, 1972.. The maximum GSH concentration was observed during the period of rapid bud growth and a curvilinear correlation between GSH and flower bud fresh weight was noted ŽFig. 5a,b.. In this period, GSH has been involved in metabolic activation of protein synthesis after the breaking of rest ŽEsterbauer and Grill, 1978; Guy and Carter, 1982.. Moreover, in apple buds high GSH content has been associated with rapid growth ŽWang et al., 1991..
4. Conclusions The two cultivars showed a difference in flower bud response, which was detected as early as the first observations in autumn. In particular, ‘S. Castrese’ was found to present constant and elevated catalase activity and a greater GSH content. These results appear to be in agreement with the observations on production behaviour of the two cultivars ŽGuerriero et al., 1991..‘S. Castrese’ always shows elevated production while ‘Portici’ can present scanty and inconstant yield, prevalently due to a dramatic drop of flower buds before flowering. These flower buds were damaged with browning and necrosis on the different floral organs ŽViti and Monteleone, 1991a.. Moreover, the vascular continuity between flower bud axis and ovary were stabilized considerably later in ‘Portici’ than in ‘S. Castrese’ ŽBartolini and Giorgelli, 1994.. It is probably that the better nutritional preparation associated to the elevated GSH content and catalase activity of ‘S. Castrese’ flower buds from early development stages onwards, allowing buds to develop more successfully during the rapid growth stage. However, further in-depth researches will be required in order to obtain confirmation of these preliminary results and the explanatory hypotheses put forward.
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