Polyphenol oxidase activity in mango Original article Polyphenol oxidase activity in mango (Mangifera indica L. ) in relation to flowering behaviour and the malformation incidence Ram Roshan Sharmaa*, Chhavi Nath Singhb, Apurba Millan Goswamia a Division of Fruits Polyphenol oxidase activity in mango (Mangifera indica L.) and Horticultural Technology, in relation to flowering behaviour and the malformation incidence. Indian Agricultural Abstract — Introduction. Biennial bearing and malformation are the two present problems Research Institute, of mango which are threatening the very existence of a global mango industry in India. Many New Delhi-110012, India factors are closely associated with these problems but, presently, those really responsible are b not known exactly. Polyphenol oxidase (PPO) plays vital roles in higher plants and, hence, its Department of Horticulture, activity was measured in swollen buds at panicle initiation in regular and biennial bearing CCR College, mango cultivars to establish a relation between the PPO activity and the flowering behaviour Muzaffarnagar, U.P., India or the malformation incidence. Materials and methods. Six regular (Sensation, Eldon, Tommy Atkins, Amrapali, Neelum and Bangalora) and six biennial bearing cultivars (Langra, Chausa, Edward, St. Alexandrina, Extrema and Bombay Green) were studied. PPO activity was meas- ured in fully swollen buds by preparing a crude enzyme extract at 4 °C. Catecholase activity was measured by using 4-methyl catechol (4-MC) as the substrate, while 4-methyl phenol (p-cresol) was used for cresolase. Results and discussion. Both catecholase and cresolase activities were higher in the swollen buds in the year 2000 than in 1999. Regular bearers exhib- ited low PPO activity and showed the higher incidence of malformation as compared to the biennial bearing cultivars.Whatever their type of production, cultivars exhibited significant vari- ations in PPO activities and malformation incidence. Conclusion. A strong negative correla- tion between PPO activity and number of panicles, and PPO activity and malformation inci- dence, suggests that the PPO activity is inversely related to both flowering and malformation incidence in mango. India / Mangifera indica / production / enzymic activity / catechol oxidase / flowering / malformations / fruits Activité de la polyphénol oxydase chez la mangue (Mangifera indica L.) en relation avec la floraison et l’observation de malformation. Résumé — Introduction. La production bisannuelle et la malformation du fruit sont les deux problèmes actuels qui menacent l’existence même d’une industrie globale de la mangue en Inde. Beaucoup de facteurs ont été étroitement associés à ces problèmes mais, à ce jour, ceux qui en sont réellement responsables ne sont pas exactement connus. La polyphénol oxydase (PPO) joue un rôle essentiel chez les plantes supérieures et, par conséquent, son activité a été mesurée dans des bourgeons gonflés au stade de l’initiation florale, chez des cultivars de man- guiers à productions régulières ou bisannuelles, afin d’établir une relation entre l’activité de la PPO et la floraison ou l’observation de malformations. Matériel et méthodes. Six cultivars à production régulière (Sensation, Eldon, Tommy Atkins, Amrapali, Neelum et Bangalora) et six autres à production bisannuelles (Langra, Chausa, Edward, St. Alexandrina, Extrema et Bom- bay Green) ont été étudiés. L’activité de la PPO a été mesurée dans les bourgeons entièrement * Correspondence and reprints gonflés en préparant un extrait brut d’enzymes à 4 °C. L’activité de la catécholase a été mesu- rée en employant le méthyle-4 catéchol (4-MC) comme substrat tandis que le méthyl-4 phénol (p-crésol) a été employé pour étudier la crésolase. Résultats et discussion. Les activités de la Received 16 October 2000 catécholase et de la crésolase ont été plus élevées en 2000 qu’en 1999. Les cultivars à produc- Accepted 2 January 2001 tion régulière ont montré une basse activité de la PPO et davantage de malformations que les cultivars produisant tous les deux ans. Quel que soit leur type de production, les cultivars ont montré des variations significatives d’activités de la PPO et de taux de malformation des fruits. Conclusion. Une forte corrélation négative entre l’activité de la PPO et le nombre d’inflores- Fruits, 2001, vol. 56, p. 219–224 cences, et entre cette même activité et le taux de malformations suggère que l’activité de la PPO © 2001 Cirad/EDP Sciences est inversement liée à la floraison et aux anomalies de formation du fruit chez le manguier. All rights reserved Inde / Mangifera indica / production / fréquence de récolte / activité enzymatique / RESUMEN ESPAÑOL, p. 224 catéchol oxydase / floraison / malformation / fruits Fruits, vol. 56 (4) 219 R.R. Sharma et al. 1. Introduction 2. Materials and methods Mango, the choicest fruit of India, occupies Five uniformly maintained full bearing a pre-eminent place amongst fruit crops plants (14–15 year old) were selected for the and is acknowledged as “the king of fruits” studies. Each of them were regular (‘Sensa- in this country. There are about 1600 culti- tion’, ‘Eldon’, ‘Tommy Atkins’, ‘Amrapali’, vars of mango in the world, of which about ‘Neelum’ and ‘Bangalora’) or biennial 1200 exist in India [1]. However, only a few bearing (‘Langra’, ‘Chausa’, ‘Edward’, cultivars have attained commercial status in ‘St. Alexandrina’, ‘Extrema’ and ‘Bombay the world mango industry. Green’), with a known history of regular- There are many inherent problems asso- ity / bienniality. In the years 1999 and 2000, ciated with the mango industry in the world, fully swollen buds at the beginning of pan- but the problem of biennial bearing is one icle initiation (March) were excised and of the major problems as it renders mango used for measuring polyphenol oxidase cultivation less remunerative to the growers (both catecholase and cresolase) activity. and is thus the major bottleneck in the Enzyme assay was done in 24 samples per expansion of the mango industry. Several plant per variety. The total number of pan- aspects like climatological factors [2], age icles healthy and malformed panicles in and size of the shoot [2–4], carbon:nitrogen each variety were counted during March ratio [5, 6], and hormonal imbalance [6–8] and averaged for calculating malformation may be closely associated with biennial incidence. The data on PPO activity of both bearing, but, at the present time, no defi- the years were averaged, pooled and nite knowledge could explain which factors analysed by using simple split plot design are responsible for biennial bearing in [17]. Correlations between PPO activity and mango [9]. Polyphenol oxidase (PPO), a number of panicles (flowering behaviour), widely distributed enzyme in plants, has PPO activity and malformation incidence been studied thoroughly to assess its role were calculated. in higher plants. Mayer and Harel [10] reported its association with in vivo syn- 2.1. Preparation of crude enzyme thesis and accumulation of phenolic com- extract pounds. Further, polyphenols have been reported to be associated with the forma- The crude enzyme extract was prepared at tion of IAA – an auxin [11] and auxins are 4 oC as per the procedure of Sharma et al. reported to be closely associated with veg- [16]. The swollen buds were chopped and etative growth and subsequent flowering in mixed before preparing enzyme extract. mango [6–8], although Briggs and Ray [12] One g of chopped material was homoge- and Tomaszewski and Thimman [13] have nised with 5 mL of 100 mM of phosphate shown their doubts about this role of PPO. buffer (pH 7.3) containing 10 mM of sodium Malformation, a disorder of international ascorbate in a blender for 15 s, filtered significance, affects with variable intensity, through four layers of gauze and cen- different mango cultivars (both regular and trifuged at 3,000 g for 30 min. The precipi- biennial bearers) in North India and in other tate was re-extracted for 15 min with 5 mL parts of the world [14–16] and its incidence of 1.5% Triton-X-100, prepared in 100 mM has been inversely related to PPO activity phosphate buffer (pH 7.3). The final vol- [16]. Thus, PPO activity (catecholase and ume of the extract was made up to 25 mL cresolase) may have some relation to flow- with phosphate buffer (pH 7.3). The filtrate ering, and studies were therefore conducted was then centrifuged at 15,000 g for 1 h. An to establish a relation between PPO activ- ammonium sulphate fractionation was car- ity and flowering behaviour, PPO activity ried out, and the fraction precipitating and malformation incidence, and flowering between 45% and 95% saturation was col- behaviour and malformation incidence in lected and re-dissolved. After dialysis, this mango. solution was used as an enzyme source. 220 Fruits, vol. 56 (4) Polyphenol oxidase activity in mango 2.2. Enzyme assay enzyme activity was represented as change in absorbance at 400 nm per g of tissue ∆ × –1 × –1 Both catecholase and cresolase activities weight per min ( A400 g min ). were measured spectrophotometrically at 400 nm as per the procedure of Sharma et al. [16]. 3. Results Catecholase activity was measured by using 30 mM of 4-methyl catechol (4MC) as substrate, made in 10 mM of sodium acetate 3.1. Polyphenol oxidase activity buffer (pH 4.5). To 1 mL crude enzyme Polyphenol oxidase (catecholase and creso- extract, 3 mL of 100 mM phosphate buffer lase) activity varied widely in different cul- (pH 7.3) was added. To this mixture, 1 mL tivars and both the seasons. Catecholase of substrate was added at zero time and the and cresolase activities were higher both in change in absorbance at 400 nm was regular and biennial bearers during the year recorded in a CL-1200 spectrophotometer. 2000 than in 1999. However, the rise in The cresolase activity was also measured activities was comparatively higher in bien- similarly, except that 4-methyl phenol nial bearers than in regular bearers (p-cresol) was used as substrate made in (table I ).