Anthocyanin accumulation in apple and peach shoots
during cold acclimation
P. Leng
a, H. Itamura
b, H. Yamamura
b, X.M. Deng
a,*a
Department of Horticulture, China Agricultural University, Beijing 100094, China b
Faculty of Agriculture, Shimane University, Nishikawatsu 1060, Matsue 690, Japan
Accepted 9 April 1999
Abstract
Experiments were conducted to investigate the effects of low temperatures on anthocyanin accumulation in apple and peach shoots. The anthocyanin concentration in both the apple and peach shoots increased rapidly during cold acclimation reaching the peak value in early December. The anthocyanin might be accumulated in shoots as a result of leucoanthocyanidin conversion. When apple and peach shoots collected in mid-January were subjected to various subzero temperature treatments, their anthocyanin concentration tended to increase and reached the peak value near the killing temperature, whereas the leucoanthocyanidin concentration was reduced as the temperature decreased.#2000 Elsevier Science B.V. All rights reserved.
Keywords: Anthocyanin; Freezing tolerance; Cold acclimation; Malus domesticaBorkh.;Prunus persicaBatch
1. Introduction
Anthocyanin is one of the flavonoid compounds that are synthesised in plants through shikimic acid pathway using phenylalanine as the substrate. Plant shoots accumulate anthocyanin in vacuoles of epidermal and sub-epidermal cells, turning red when they are subjected to low temperatures. According to Leonchenko (1988), anthocyanin content in shoot cortexes shows a canonical correlation with the freezing tolerance of apple trees. The synthesis of
* Corresponding author. Tel.: +86-10-62892479; fax: +86-10-62891638.
anthocyanin and other phenolic compounds were also found to be related to cold acclimation in other tree species (Parker, 1962; Chalker-Scott, 1989; Leng et al., 1993, 1995).
The objectives of this experiment were to investigate seasonal changes in the concentrations of anthocyanin and leucoanthocyanidin along with assessing the effects of subzero temperature treatments on the concentrations of anthocyanin and leuceanthocyanidin in shoot cortical tissues of a selected variety of cultivated apple and peach trees.
2. Materials and methods
2.1. Plant materials
The experiment was carried out at Shimane University in Matsue, Japan. Samples of one-year-old shoots were collected from mature peach (Prunus persicaBatch. cv. Okubo) and apple trees (Malus domesticaBorkh. cv. Fuji) on the orchard of Shimane University Farm every month from September 1991 to March 1992.
2.2. Subzero temperature treatments
In late January, the shoots were collected and divided into seven groups. They were put into a vinyl plastic bag and subjected to various subzero temperature treatments. The temperature dropped from 58C toÿ58C,ÿ108C,ÿ158C,ÿ208C, ÿ258C, ÿ308C within 5 h in each grouping. The temperature set of each group
was constantly maintained for 5 h. Each grouping was returned to 58C in 5 h, respectively. Each temperature treatment had three replicates.
2.3. Determination of anthocyanin and leucoanthocyanidin concentrations
Upper parts of the one-year-old shoots periodically collected from the mature trees on the orchard and of the shoots subjected to subzero temperature treatments, were both used to extract anthocyanin and leucoanthocyanidin. The bark of the shoots, 1 g in weight, were ground, put into a taper bottle with 100 ml methanol containing 0.1% HCl, and incubated at 238C for 3 h. After filtration, the solution was divided into two equal portions of 50 ml each. One of the portions was used to determine anthocyanin concentration with a spectrophotometer at 540 nm (Bate-Smith, 1954; Segel, 1976).
bath at 1008C for 10 min. Leucoanthocyanidin was converted into anthocyanin (Bate-Smith, 1954). After filtration, the liquid sample was diluted 10 times with a result volume of 500 ml. From this resulting volume, 10 ml was used to determine the total concentration of anthocyanin with a spectrophotometer at 540 nm. The concentration of leucoanthocyanidin was calculated as the difference between the concentration levels of the anthocyanin measured in these two portions.
2.4. Determination of freezing tolerance
Shoots, whether they were periodically collected from the orchard or subjected to subzero temperature treatments in January as previously described, were washed with tap water three times, dried indoors at room temperature and cut into pieces of 1 cm in length. The sliced 5 g samples were soaked in 40 ml of water at 208C for 10 h. The first conductance (C1) was measured with a conductivity
gauge (C-173 Horiba, Tokyo, Japan). The second conductance (C2) was measured
only after the sample was killed in boiling water for 30 min and soaked in 40 ml of water at room temperature for 12 h. Electrolyte leakage (E) was calculated as follows (Sun et al., 1987):
EC1=C2100% (1)
The freezing tolerance of fruit trees was defined as the temperature at which E
was equal to 50% (Sukumaran and Weiser, 1972).
In addition, shoots subjected to the subzero temperature treatments were also placed in a growth chamber with artificial light of 12 h and a temperature of 208C. The surviving percentage of shoots was determined 20 days later.
3. Results
3.1. Changes in the freezing tolerance of apple and peach shoots and concentrations of anthocyanin and leucoanthocyanidin
The freezing tolerance of apple and peach shoots increased as the air temperature dropped from autumn to winter reaching maximum tolerance in late January (Fig. 1(a) and (b)). Apple shoots showed a relatively strong tolerance to freezing temperatures until late March. Peach shoots rapidly lost their freezing tolerance in early February.
level during January. In early and middle February, the anthocyanin concentration increased again as air temperature dropped after heavy snow fall. In late February, it decreased rapidly as air temperature increased (Fig. 1(a) and (c)).
The leucoanthocyanidin concentration was higher in early autumn (Fig. 1(d)). The concentration decreased from October to December followed by an increase until early February. The variation in the total concentration of anthocyanin and leucoanthocyanidin between Septmber and April was not significant, apparently independent of air temperature (Fig. 1(a) and (d)).
3.2. Effects of subzero temperature treatments on the concentrations of anthocyanin and leucoanthocyanidin in apple and peach shoots
When apple and peach shoots were subjected to subzero temperature treatments, their anthocyanin concentration increased rapidly with a decrease in temperature. The concentration reached its maximum level atÿ258C (Fig. 2(a)).
In contrast, the concentration of leucoanthocyanidin in apple and peach shoots reduced with the decrease in temperature (Fig. 2(b)).
As shown in Fig. 2(c), there was no injury to apple and peach shoots above
ÿ158C. When the peach and apple shoots were subjected to temperatures below ÿ158 and ÿ208C, respectively, their electrolyte leakage significantly increased,
probably suggesting that the cell membrane was injured. The injured apple shoots were able to recover later, whereas the injury to the peach shoots was irreversible. The lethal temperature was ÿ25.38 and ÿ28.58C for peach and apple shoots,
respectively.
The freezing tolerance of the apple and peach shoots was greater in comparison to the buds of those same trees (Fig. 2(d)). The percentage of apple bud survival approached zero when the temperature dropped to ÿ258C, but the lethal
temperature for the shoots wasÿ28.58C. The survival percentage of peach buds
and shoots began to decrease at ÿ108 andÿ158C, respectively, and dropped to
zero for both atÿ258C.
4. Discussion
et al., 1993). The concentrations of anthocyanin in apple and peach shoots were higher than that in persimmons. The patterns of their changes were consistent in all plants tested. The anthocyanin may be accumulated in shoots as a result of leucoanthocyanidin conversion. However, after the anthocyanin reached a peak level in December, its concentration decreased with the continuous decline in the air temperature. At this time, it is unclear whether anthocyanin was converted into other compounds or if its synthesis was inhibited by severe temperatures.
In early February, local air temperature dropped suddenly after a heavy snow. The increase in the concentration of anthocyanin may be linked to this severe weather condition. The data collected from the in vitro experiment supported this conclusion. When shoots containing a low level of anthocyanin in mid-January were subjected to various subzero temperature treatments in the laboratory, the concentration of anthocyanin increased as the temperature decreased and reached peak volume near the lethal temperature (Fig. 2(a)).
The injury caused due to freezing temperatures is mainly attributed to the destruction of plant cell membrane systems by active oxygen and other free radicals (Jiang and Jing, 1993). It has been reported that phenols in plants can remove active oxygen and other free radicals (Nakayama, 1993; Shen et al., 1993, 1995; Luo and Wang, 1994). Anthocyanin accumulated in vacuoles may protect the vacuole membrane from low temperature injury. However, the role of anthocyanin played in membrane protection remains to be elucidated in the future.
Acknowledgements
The authors thank Eric M. Mapes and Dena A. Kram for reviewing the English of the text.
References
Bate-Smith, E.C., 1954. Leucoanthocyanins 1. Detection and identification of anthocyanins formed from leucoanthocyanins in plant tissues. Biochem. J. 58, 122±125.
Chalker-Scott, L., 1989. Spectrophotometric measurement of leached phenolic compounds as an indicator of freeze damage. J. Amer. Soc. Hort. Sci. 114, 315±319.
Jiang, M.Y., Jing, J.H., 1993. Generation of hydroxyl radical in plants and its relation to the initiation of lipid peroxidation. (in Chinese, with English abstract). Plant Physiology Communications 29, 300±305.
Leng, P., Itamura, H., Yamamura, H., 1993. Freezing tolerance of severalDiospyrosspecies and kaki cultivars as related to anthocyanin formation. (in Japanese, with English abstract). J. Japan Soc. Hort. Sci. 61, 795±804.
Leonchenko, V.G., 1988. Relationship between anthocyanin accumulation and freezing resistance in apple shoots. (in Russian). Fruit Growing and Viticulture 2, 26±27.
Luo, G.H., Wang, A.G., 1994. The scavenging effect of plant polyphenolics on superoxide radicals. (in Chinese, with English abstract). J. Tropical and Subtropical Bot. 2, 95±99.
Nakayama, T., 1993. Assays of antioxidants with mammalian cultured cells. (in Japanese, with English abstract). Nippon Shokuhin Kogyo Gakkaishi 40, pp. 218±223.
Parker, J., 1962. Relationships among cold hardiness, water-soluble protein, anthocyanins and free sugars inHedera helixL. Plant Physiol. 37, 809±813.
Segel, I.H., 1976. Biochemical Calculations, Second edn. John Wiley and Sons, New York, pp. 441. Shen, S.R., Yang, X.Q., Yang, F.J., 1993. Co-ordinated synergetic effect of catechins during their
anti-oxidation. (in Chinese, with English abstract). J. Tea Sci. 13, 141±146.
Shen, S.R., Zhao, Y.F., Yang, X.Q., 1995. Mechanism of tea polylphenols on protective actions of bio-macromolecule against free radicals. (in Chinese, with English abstract). J. Zhejiang Agric. Univ. 21, 361±365.
Sukumaran, N.P., Weiser, C.J., 1972. Anexcised leaflet test for evaluating potato frost tolerance. HortScience 7, 467±468.
