BROTOMAX : A POSSIBLE MODULATOR OF FLAVANOID EXPRESSION IN FRUIT.CITRUS

J.M. Botía, M.D. Fuster, A. Ortuño, F. Sabater, I. Porras, A. García-Lidón and J.A. Del Río. Dpto Biología Vegetal (Fisiología Vegetal). Facultad de Biología. Universidad de Murcia. 30100 Murcia. Dpto Citricultura. Centro de Investigación y Desarrollo Agroalimentario (CIDA). La Alberca. 30150 Murcia.

ABSTRACT.

We describe the stimulatory action of Brotomax on the synthesis and/or accumulation of the flavanone hesperidin in the fruit and leaves of tangelo Nova. The results are of great interest given the importance of this flavanone in the pharmaceutical industry and the possible physiological role that hesperidin may play in the growth processes and defence mechanisms of plants.

Fifty six days after the application of Brotomax at 0.3 % concentration, the level of hesperidin increased by around 23 % in both fruit and plants. Higher concentrations (e. g. 0.5 %) or a double treatment at 0.3 % produced no further increases.

The results obtained reaffirm the suggestion we made in previous studies (Fuster et al., 1994 a) that the action of Brotomax is involved in modulation of the processes of hesperidin synthesis via the shikimate pathways, although it might also act in the mobilisation of this flavanone from the leaves to the fruit.

INTRODUCTION.

In previous studies (Fuster et al., 1994 a) we showed that Brotomax might stimulate the synthesis of flavonoids in tangelo Nova fruits when the product is applied during the first stages of fruit development.

We also confirmed that flavanone synthesis in citrus occurs in both leaves and fruits and that a transport mechanism exists from the former to the latter (Castillo et al., 1995).

Given the above, we proposed to study whether the effect of Brotomax on the synthesis and/or accumulation of the most abundant flavanone in tangelo Nova, hesperidin (Del Río et al., 1992, 1995), also occurs in leaves, and to ascertain the optimum concentration of Brotomax in the stimulation of such processes in this hybrid.

Due to the interest of the pharmacological industry for this flavanone because of its effect on vascular permeability (Gabor, 1988), studies on its synthesis and modulation in citrics would have a double interest, both from the applied point of view and for basic research since new information would be added to our scant knowledge on the biosynthetic pathway and the possible physiological role of this flavanone in sp. and concerning the possible regulation of secondary metabolism in plants.

We used eight year old tangelo Nova trees, a hybrid of Clementine mandarine (

B) x Tangelo Olrlando ( x Macf.) grown in a commercial plantation

in Alhama, Murcia (S.E. Spain).

Brotomax was provided by Agrométodos S.A. (Madrid, Spain).

Sixteen trees were distributed in four alternate rows, four trees per treatment and the corresponding control (untreated trees). Brotomax was applied recently after fruit set (90 % of the flower petals fallen), five litres per tree being applied at 0.3 or 0.5 % (single applications) and 0.3 % for the double applications (7 days apart).

. At different times after treatment (11, 28, 42 and 56 days) twenty fruits or leaves (5 per tree) were taken for each of the assays and their corresponding controls in order to measure the equatorial diameter and the fresh and dry weight (in fruit) or the fresh and dry weight (leaves). The mean values of these determinations at the different times were used to express the evolution in growth of both organs studied.

For each of the above mentioned treatments and times, four whole fruit and four leaves from near the apex were dried (50 ºC in a forced air oven), with three repetitions per assay. Once dried, the flavanone, hesperidin, was extracted and quantified by high performance liquid chromatography, as previously described (Del Río et al., 1992, 1995).

Citrus

C. reticulata C. reticulata C. paradisi

MATERIALS AND METHODS.

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Plant material

Reagent

Treatments

Measurement of growth

Isolation and Measurement of Hesperidin

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RESULTS AND DISCUSSION

No significant differences were observed between the growth curves obtained for the control fruit and leaves and those treated with a single application of 0.3 % or 0.5 % Brotomax and a double application of 0.3 %.

The variations in the diameter and fresh and fry weight of the fruit are not shown, while Figure 1 illustrates variations in the fresh and dry weight of the leaves at the different times assayed. At the concentrations used in this experiment, then, it is clear that Brotomax has no effect on the processes of growth of tangelo Nova fruit or leaves, which agrees with the findings of previous studies (Fuster et al., 1994 a).The levels of hesperidin observed in the control fruit and fruit treated are shown in Table 1. It can be seen that 56 days after treatment with 0.3 % Brotomax the fruit showed an approximate 23 % increase in hesperidin compared with the untreated fruit at the same age. No significant differences in these levels were obtained with the increased concentration (0.5 %) or with the double application of 0.3 % Brotomax.

Figure 1.- Growth curve of tangelo Nova leaves. The mean values of the fresh weight ( A) and fry weight (g B) of 20 leaves are represented control( ) , treated with single application of Brotomax 0.3 % ( ) or 0.5 % ( ). Similar results were obtained with a doble application of 0.3 % Brotomax (data not shown). Error bars indicate SE.

g,,

As in the case of the fruit, the 0.3 % Brotomax concentration also induced the greatest increase in hesperidin levels in leaves (Table 2), the increase in this case being above 23 % at 56 days. The higher concentration (0.5 %) and double application of 0.3 % Brotomax did not significantly modify these results.

Moreover, the fact that the stimulating effect of 0.3 % Brotomax on the synthesis of hesperidin in leaves is noted earlier and in a more pronounced way (56 % increase after 42 days, see Table 2) than in fruit (23 % increase after 56 days, see Table 1) suggests a possible effect of Brotomax on hesperidin synthesis and/or accumulation in leaves and on its subsequent transport towards the fruit. This does not rule out a possible effect on its synthesis and/or accumulation in fruit.

Finally, the results obtained suggest that a 0.3 % concentration of Brotomax is the optimum for stimulating hesperidin synthesis/accumulation in tangelo Nova.The results also confirm our previous suggestion (Fuster et al., 1994 a) that the action of Brotomax must be linked to the modulation of the processes of hesperidin synthesis via the sikimate pathway, although it may also effect the mobilization of this flavanone from the leaves to the fruit.

LITERATURE CITED

Castillo J, Benavente 0, Sabater F, Del Río JA (1995). Flavonoid translocation in Citrus. Plant Physiol. (enviado).

Del Río JA, Ortuño A, Cebrián F, García-Lidón A, Porras I, Sabater F (1992). Efecto de 6-benzilamino-purina sobre los niveles de hesperidina presentes en frutos de tangelo Nova. En: Metabolismo y Modo de Acción de Fitohormonas. 111 Simposium. Univ. de Murcia p. 347-360.

Del Río JA, Fuster MD, Sabater F, Porras I, García Lidón A, Ortuño A (1995). Effect of benzylaminopurine on the flavanones hesperidin, hesperetin- 7-0-glucoside and prunin in tangelo Nova fruits. Journal of Agricultural and Food Chemistry (en prensa).

Fuster MD, Porras 1, García Lidón A, Sabater F, Ortuño A, Del Río JA (1994a). Efecto del Brotomax sobre el crecimiento y producción de flavonoides en frutos de tangelo Nova. Levante Agrícola 33: 60-64.

Fuster MD, Benavente García 0, Castillo J, Ortuño A, Sabater , F, Porras I, García Lidón A, Del Rio JA (1994b). Hesperidina: un posible modulador del catabolismo auxínico en tangelo Nova. En: Metabolismo y Modo de Acción de Fitohormonas. IV Simposium. Univ. de Valencia. 24-29.

Gábor M (1 988). Szent-Gybrgyi and the Bioflavonoids: New Results and Perspectives of Pharmacological Research Into Benzo-Pyrone Derivatives. In: Cody V, Middleton E Jr, Harborne JB, Beretz A (eds) Plant Flavonoids in Biology and Medicine II: Biochemical, Cellular, and Medicinal Properties. Alan R Liss, Inc, New York. PP. 1-15.