Low Temperature and Breaking of Dormancy Effects on Respiration Rate, Sugars, Phenolics and Peroxidase Activity Changes in Inner Buds of Onion Allium

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1 Low Temperature and Breaking of Dormancy Effects on Respiration Rate, Sugars, Phenolics and Peroxidase Activity Changes in Inner Buds of Onion Allium cepa L. Benkeblia, N. (Department of Food Science, Rakuno Gakuen University, Ebetsu, Hokkaido , Japan). Low temperature and breaking of dormancy effects on respiration rate, sugars, phenolics and peroxidase activity changes in inner buds of onion (Allium cepa L.). Accepted 25 February Acta Agric. Scand., Sect. B, Soil and Plant Sci. 53: 16/20, # 2003 Taylor & Francis. Respiration rate (CO 2 production), soluble sugars, total phenolics and peroxidase activity (POD) were measured in inner bud tissues during the break of dormancy of onion bulbs treated for one week at 08C and stored in the dark at 208C. Results showed that after eight weeks, the RR CO2 of sprouted control onions was 52% higher than that of unsprouted control onions. High concentrations of soluble sugars (glucose, fructose and sucrose) were observed for cold treated bulbs (from 9 to 20 mg g 1 FW) after two weeks following cooling, but in control bulbs high concentrations were only observed after eight weeks. In cold-treated onions, a slight increase in phenolics (from 0.17 to 0.2 mg g 1 FW) was observed after one week, and then there was a decrease to 0.12 mg g 1 FW after five weeks. In inner buds of control bulbs, an increase from 0.17 to 0.2 mg g 1 FW was observed during the first five weeks and then a decrease to 0.15 mg occurred during the three last weeks when the bulbs began to sprout. Peroxidase activity followed the same pattern as phenolics, with a decrease (38%) in cold-treated bulb after four weeks. In control samples, a decrease of 25% was noted after eight weeks. This decrease in POD activity coincided with the decrease in phenolics and the onset of sprouting. With cold treatment, total sprouting was observed after eight weeks. In comparison, only 20% of the control bulbs started sprouting after the same period. N. Benkeblia Department of Food Science, Rakuno Gakuen University, Ebetsu, Hokkaido , Japan Key words: Allium cepa, CO 2 production, oligosaccharides, phenols, POD, sprouting. Introduction Onion bulbs (Allium cepa L.) are the most widely cultivated vegetables in the world and are widely used for culinary purposes. During their storage, bulbs are exposed to changes in environmental conditions such as atmospheric composition and temperatures which can affect their respiration rate (O 2 consumption and CO 2 production). During this period, high catabolism is considered to be the main cause of changes in quality. These changes, occurring during post-harvest 16 DOI: /

2 Low temperature and breaking of dormancy effects storage, are principally heat production, transpiration, and sprouting and rotting. Dormancy of onion bulbs, which is defined as a temporary suspension of visible growth of the meristematic tissues of the sprouts, has a major impact on the storage of bulbs. One of the most popular approaches in the study of dormancy has been to study the physiological basis of dormancy in sprouts during exposure to rest-breaking treatments at low temperature (Dennis, 1987; Benkeblia & Selselet-Attou 1999). The rest effect disappeared with time at all storage temperatures, but did so more quickly at low temperatures (Abdallah & Mann, 1963; Benkeblia & Selselet-Attou, 1999). Gradual changes in the biochemistry of the bulbs throughout the dormancy period were observed, and low temperatures caused compositional changes and physiological disorders in tissues (Benkeblia & Selselet-Attou, 1999; Benkeblia et al., 2000, 2002). However, the role of phenolics and peroxidase in the dormancy process and in the sprouting of onions was not a subject of any known investigation, and remains unclear despite the large amount of literature available on biochemistry and physiology of phenolics (Shahidi & Naczk, 1995; Mann et al., 1994) and peroxidase (Greppin et al., 1986; Rodriguez, 1990) in other vegetables, particularly fruits. The present investigation was carried out to determine the effects of low temperature (08C) on breaking of dormancy of onion bulbs and the physiological and biochemical changes in the inner buds during sprouting. Respiration rate assessment Respiration rate (RR) was determined by the glass jar technique on whole onion bulbs without any treatment (Benkeblia et al., 2000). At specific time intervals (1, 2, 3, 4 and 5 hours), gas samples (50 ml) were taken through a silicon septum and analyzed by gas chromatograph (MTI, model M200, Fremont, USA). RR was calculated by linear regression from the CO 2 depletion curve and expressed as mmole kg 1 h 1. The mean value of respiration rate was determined from triplicate measurements. Soluble sugars analysis Glucose, fructose and sucrose content were determined by HPLC as described by Doyon et al. (1991). Samples (5 g) of freeze-dried inner bud tissues were homogenized in 50 ml of water using a Sorvall blender. The homogenate was heated for 30 minutes in a boiling water bath. After cooling, the homogenate was centrifuged for 15 minutes at /g and the supernatant was filtered on a 0.25 mm Millipore filter. The sugars were separated by HPLC using a Varian 5000 model fitted with a Polyspher CH-CA column (300/7.8 mm. Merck) set at 808C and a differential refractometer detector. The mobile phase was DDI water at a flow rate of 0.5 ml min 1. Sugars were identified and quantified by comparison with authentic samples (Sigma Co, St Louis, USA) and each determination was run in triplicate. Materials and methods Onions Onions (organic product, free of any pre-harvest chemical treatments) c.v. Rouge Amposta, which had been freshly harvested and dried in the field for two weeks were obtained from the local market, sorted for uniformity and absence of defects, packed in commercial plastic (PVC) trays each of 12 kg and placed at 188C prior to treatment. Cold treatment and storage Immediately after cold treatment (08C-1 week) which was given 18 days after harvesting, onions were stored in the dark at 208C and 65% relative humidity. Sprouting was counted from the first week of cold treatment. Thirty bulbs were cut longitudinally twice a week to check for sprouting (appearance of first green internal leaves) and soluble sugars; total phenolics and peroxidase activity were assessed simultaneously on nine bulbs. Total phenolics determination Total phenolics were extracted as described by Brenes et al. (1992), and determined according to AOAC method (1984). Samples (10 g) were mixed with 80 ml aqueous EtOH (80%) and 20 ml of metabisulphite (25 mm), homogenized for 30 s and left for 15 min at 48C. The homogenate was filtered and filtrate used for phenolics determination. Total phenolics were quantified colorimetrically at 730 nm, using chlorogenic acid (Sigma Co, St Louis, USA) as a standard. Peroxidase extraction and assay POD was extracted and assayed as described by Gunes & Bayindirh (1993). Fresh tissues (10 g) were mixed with 50 ml of phosphate buffer (ph 7.0) and blended for 5 min in the blender at minimal speed. The homogenate was centrifuged for 15 min at /g and the supernatant was removed for POD assay. One ml of extract was mixed with 1 ml of guaiacol (45 mm), 1 ml of H 2 O 2 (0.2 M) and 18 ml of phosphate buffer (ph 6.5). After 10 min incubation at room temperature, absorbance was measured at 430 nm. 17

3 N. Benkeblia One unit of POD activity was defined as change in absorbance of min 1. Statistical analysis All determinations were carried out in triplicate. The data from the different samples were compared by Student test (t) to determine the effect of temperature and breaking of dormancy on respiration rate, sugars, phenolics and peroxidase activity using Statistica 7.0 software. Results and discussion The effect of sprouting on respiration rate of control onion bulbs is reported in Fig. 1. After eight weeks at 208C, RR of sprouted onion increased from 0.17 to 0.29 mmole CO 2 kg 1 h 1. The increase in respiration rate is the consequence of physiological changes during this period corresponding to the breaking of dormancy and the onset of sprouting. This result is consistent with the findings of Loogheed & Franklin (1975) who reported a value of 0.24 mmole CO 2 kg 1 h 1 of sprouted onions stored at 218C for two months. In cold treated bulbs, soluble sugars decreased over the first week (during cold treatment) from 15 to 9 mg g 1 FW, but increased sharply to 20 mg g 1 FW between two and three weeks (Fig. 2). There was then a decrease to 5 mg after four weeks from the start of the cold treatment. During the last four weeks, soluble sugars increased slightly from 5 to 8 mg 100 g 1 FW. The concentration of soluble sugars in the control samples peaked after six weeks, in both cases the peaks coinciding with the onset of sprouting. Similar peaks of glucose and soluble sugars were observed in onion bulbs treated at 0 and 98C during weeks two and three (Benkeblia & Selselet-Attou, 1999). Similar results were reported by Rutherford (1981) but a high glucose Fig. 2. Changes in soluble sugars (glucose, fructose and sucrose) in inner buds of onion bulbs during breaking of dormancy (k control, j chilled). concentration was noted by Pak et al. (1995) during the onset of bulbs sprouting. During the first week, the concentration of total phenolics in the inner buds of cold treated bulbs increased from 0.17 to 0.2 mg g 1 FW, and then it decreased and reached a value of 0.12 mg g 1 FW after five weeks, finally increasing to 0.18 mg during the last three weeks (Fig. 3). In control samples, concentration of total phenolics increased slightly from 0.17 to 0.2 g g 1 FW during the first five weeks, and then decreased progressively to 0.15 mg g 1 FW. Despite their unclear role in bulb dormancy, phenolics could have indirect involvement in the sprouting process and low temperature triggers a signal to reduce these compounds, promoting sprouting. Benkeblia & Selselet-Attou (1999) reported that total phenolics content decreased between two and six weeks in onion bulbs treated at 0 and 98C during the second and third weeks. Close, but barely comparable with onion involvement of phenolic acids, in sprouting of potato buds was reported by Cvirkova et al. (1994), the lack of Fig. 1. Effect of breaking of dormancy on respiration rate of control onion bulbs after eight weeks at 208C. Fig. 3. Changes in phenolics in inner buds of onion bulbs during breaking of dormancy (k control, j chilled). 18

4 Low temperature and breaking of dormancy effects comparability being due to the biological and physiological differences between the two species. Peroxidase activity showed a similar pattern in both cold treated and control bulbs, although at different timescales (Fig. 4). In cold treated bulbs, POD activity decreased from 1.55 to 1.1 U g 1 FW during the first four weeks and remained stable during the last three weeks. On the other hand in control samples, this decrease to 1.1 U g 1 FW was reached only after eight weeks. The role of POD in sprouting of onion has not yet been established; however the role of peroxidases, particularly in their degrading activity on IAA in other vegetables has been widely investigated, as IAA is considered an effective promoter of sprouting (with cytokinins) (Thomas, 1969). On the other hand, Benkeblia & Selselet-Attou (1999) noted that POD decreased more rapidly between two and four weeks in onion bulbs treated at 98C during weeks 2 and 3, than in those cooled at 08C. The total breaking of dormancy of cold treated onion bulbs was observed after eight weeks (50% was noted within the 6 th week) (Fig. 5). In comparison, only 20% of the control bulbs started sprouting after this period. Effect of low temperature on breaking of dormancy of onion bulbs and other bulbous vegetables was reported previously. However, some flower bulbs are sensitive to chilling while others need higher temperatures for sprouting (Le Nard, 1982; Lang, 1996). Statistical analysis of data showed a highly significant effect (at P B/0.05) of chilling on changes in sugars and peroxidase activity. A highly significant effect of sprouting on respiration rate was also observed. In contrast, no significant effect of temperature on changes in phenolics was detected. This nonsignificant effect could be due to the different levels of phenolics observed during eight weeks of storage. Fig. 4. Changes in POD activity in inner buds of onion bulbs during breaking of dormancy (k control, j chilled). Fig. 5. Effect of chilling (08C/1week) on sprouting of onion bulbs (j control, I chilled). Conclusion Results of this investigation indicate that cold treatment of 08C could induce break of dormancy. Low temperatures caused a decrease in phenolics and peroxidase activity, which could be involved directly or indirectly in extending the dormancy period of onion bulbs. Consistent with previous results, it is likely that cold treatment of rest organs induces a signal triggering biochemical modifications and breaking of dormancy. But the question remains: what is the exact nature of this signal? Our current work using molecular biology tools will possibly give us some clarification on the nature of this signal. References Abdallah, A. A. & Mann, L. K Bulb development in the onion (Allium cepa L.) and the effect of storage temperature and bulb rest. Hilgardia 35, 85/112. A.O.A.C (Association of Official Analytical Chemists) Official methods of analysis. 14 th edition, Washington. Benkeblia, N., Varoquaux, P., Shiomi, N. & Sakai, H Effect of irradiation, MH and CIP treatments on respiration rate and oligosaccharides variations in onion bulbs in store. Int. J. Food Sci. Technol. 37, 169/176. Benkeblia, N., Varoquaux, P., Gouble, B. & Selselet-Attou, G Respiratory parameters of onion bulbs. Effects of irradiation and temperature. J. Sci. Food Agric. 80, 1772/1778. Benkeblia, N. & Selselet-Attou, G Changes in oligosaccharides, phenolics and peroxidase activity in inner bud of onion bulbs during break of dormancy by low temperatures. Acta Agric. Scand. 49, 98/102. Brenes, M., Garcia, P., Duran, M. C. & Garrido, A Concentration of phenolic compounds changes in storage brines of ripe olives. J. Food Sci. 58, 347/350. Cvirkova, M., Sukhova, L., Eder, J. & Korableva, N Possible involvement of abscissic acid, ethylene and phenolic acids in potato tuber dormancy. Plant Physiol. Biochem. 32, 685/691. Dennis, F. J Two methods of studying rest: temperature alternation and genetic analysis. HortScience 22, 820/824. Doyon, G., Gaudreau, G., St-Gelais, D., Beaulieu, Y. & Randal, C. J Simultaneous HPLC determination of organic acids, sugars and alcohols. Can. Inst. Food Sci. Technol. 24, 87/94. 19

5 N. Benkeblia Greppin, C., Penel, C. & Gaspar, T Molecular and physiological aspects of plant peroxidases. Geneva University Press, Geneva, 478 pp. Gunes, B. & Bayindirh, A Peroxidase and lipoxygenase inactivation during blanching of green beans, green peas and carrots. Lebens. Wiss. Technol. 22, 406/410. Lang, G. A Plant dormancy: physiology, biochemistry and molecular biology. CAB International, Wallingford, Oxon, 386 pp. Le Nard, M Physiology and storage of bulbs: concepts and nature of dormancy. In: Lieberman, M (ed.), Post-harvest physiology and crop preservation. Plenum Press, New York, 191/235 pp. Loogheed, E. C. & Franklin, R. C Air flow influence on CO 2 production of apple fruits, potato tubers and onion bulbs. HortScience 10, 388/390. Mann, J., Davidson, R. S. & Harborne, J. B Natural products: their chemistry and biological significance. Longman Scientific & Technical, London, 376 pp. Pak, C., Van der Plas, L. H. & De Boer, A. D Importance of dormancy and sink strength in sprouting of onion Allium cepa L. during storage. Physiol. Plant. 94, 277/283. Rodriguez, R Plant ageing: basic and applied approaches. Plenum Press, New York, 480 pp. Rutherford, P. P Some biochemical changes in vegetables during storage. Ann. Appl. Biol. 98, 538/541. Shahidi, F. & Naczk, M Food phenolics: sources, chemistry, effects and applications. Technomic Publishing, Lancaster, 331 pp. Thomas, T. H The role of growth substances in the regulation of onion bulb dormancy. J. Exp. Bot. 20, 124/

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