Extensive and uniform new flushing can be induced in various citrus cultivars by simple removal of leaves; this is much more pronounced in Rio Red grapefruit and Owari Satsuma than others (Figures 1 and 2). Similar stimulatory effect of leaf removal and chemicals to induce bud break has been noted in other plant species such as peach, apple and grape plants [32
]. In addition, it has been previously documented that removal of apical meristems induces lateral bud sprouting [33
]. Thus, tip pruning has been used to induce lateral bud sprouting in macadamia but buds did not sprout uniformly [16
]. Our results show that simple defoliation in citrus can be an effective method to induce flushing at will for research related to citrus greening disease and to investigate metabolic regulation
of bud break. The ability to manipulate and predict bud break would be invaluable for efficient farm management practices such as proper scheduling of spray applications of insecticides to control ACP. The fact that even the presence of a petiole was as effective in suppressing lateral bud growth, just as the presence of the whole leaf inhibited bud break, it indicates that nutritional factors such as carbohydrates supply from leaves play little role in the bud break of citrus axillary buds.
Our findings are supported by reports that photosynthetic activity within the growing bud tissue could supply sugars needed for bud break in Valencia oranges [34
]. In other species, however, supply of carbohydrates from leaves was considered important for bud break [35
]. The dramatic difference between intact and removed petiole on bud break would relate to possible hormonal stimulus as result of wounding effect; and of course, production of hormonal stimuli in response to wounding have been reported in the literature [36
]. Since the development of leaves after bud break occurs quite quickly (Figure 2), the plant soon becomes capable producing photosynthates
for the plant needs even after complete defoliation; although complete defoliation of entire plant may not be necessary and only few branches may be defoliated to produce new flush.
While simple defoliation could effectively cause bud burst in citrus, chilling treatment of defoliated trees for 8 weeks further significantly (p<0.05) increased the percentage of bud breaks (Figure 4) and the observation is consistent with results obtained through winter defoliation in apples [39
]. In addition, it is not surprising that 8 weeks of chilling resulted in the production of flowers from the bud burst since induction of flowering with various levels of chilling is well known phenomenon in many plant species including citrus [40
The fact that chilling of defoliated trees shifted bud break from vegetative to reproductive bud growth indicates that metabolic changes within bud tissue from external stimuli are primarily responsible for flower induction in Rio Red grapefruit and this observation is similar to what we observed for flower induction in olives; i.e., developmental change in auxiliary buds could occur independent of leaf metabolism [43
Levels of several polyphenols significantly changed when defoliation induced vegetative bud break; however, the changes were quite dramatic when chilling treatment was given to defoliated plants that resulted in reproductive bud growth (Figures 4-6). For example, there was no significant change in levels of hesperidin, Apigenin-7-glucoside, and only 20% increase of Naringenin during the sprouting of vegetative buds compared to non-sprouting control buds, while their levels increased by 246%, 150%, and 190% respectively in buds sprouting to produce flowers compared to buds sprouting to become vegetative shoot (Figures 5 and 6). Naringin also increased by 62% in buds sprouting to produce flowers, but in buds sprouting to become vegetative shoot (i.e., defoliation but no chilling) its levels actually decreased by 21%. Increases in poly phenols during reproductive bud break and development have been reported for different plant species [17
]. The only polyphenols that actually increased significantly during the sprouting of vegetative buds were chlorogenic acid and naringenin, and out of these two only naringin continued to remain higher in flowering buds compared to vegetative buds; the chlorogenic acid levels remained unchanged in buds that turned into reproductive structures (Figures 5 and 6). Thus, increases in the levels of chlorogenic acid were distinctly related to bud break in Rio Red grapefruits that may be a good biological marker of vegetative growth if there was no accompanying dramatic increase in hesperidin, apigenin-7-glucoside, and naringenin. However, past studies have given contrasting result regarding correlations between chlorogenic acid levels and developmental changes such as vegetative versus reproductive growth. For example, higher levels of chlorogenic acid
were found in leaves of flowering olive trees versus nonflowering tree but exogenous application of chlorogenic acid had no effect on floral differentiation [45
]. High levels of chlorogenic acid were found in Lonicera japonica
flowers, but in Asparagus officinalis,
flowering was closely related to decreases in chlorogenic acid levels. In Chrysanthemum morifolium
light quality that increased chlorogenic acid had no effect on induction of flowering, and application of chlorogenic acid had no response on dormant buds of peaches [49
]. Exogenous applications of chlorogenic acid on peaches had no effect on bud break, but in Impatiens balsamina
the application of chlorogenic acid alone and in combination with gibberellic acid promoted the initiation of floral buds [50
]. These reports indicate that the role of chlorogenic acid could be different in different plant species. Our findings therefore, provide a valuable information regarding rise in the levels of chlorogenic alone (or in combination with naringenin) in dormant grapefruit buds as indicator of vegetative bud sprouting, and since it does not increase in flowering buds it is valuable pointer for flushing as opposed to flowering.
Phillips in 1962 [52
], reported that Naringenin inhibit the bud breaking effect of gibberellin, and therefore, concluded that naringenin acts as modulator of bud break in peaches; but, Luna et al. [53
] could not find any positive correlations between levels of naringenin and rest period in peach buds. However, Biggs [50
] reported that exogenous application of naringenin had positive effect on bud break in peaches. Both increase and decrease in the levels of naringenin related to bud growth have been reported in different experimental conditions [22
]. Apparently, studies with different plants under different experimental condition and sampling protocols are responsible for lack of consistency regarding correlations between naringenin levels and the bud break. Our experiments designed to capture changes in naringenin levels, along with other polyphenols, just at the time of bud break in grapefruit provide an accurate estimation of indicator molecules such as naringenin, hesperidin, and apigenin-7-glucoside that dramatically increase at the onset of flowering bud break. Thus, while rise in levels of chlorogenic acid, in combination with rise in Naringenin, indicate vegetative bud break in grape fruits, a dramatic rise in hesperidin, in combination with apigenin and naringenin indicate the onset of flowering bud break. This is valuable information that may be used to develop specific sensor for predicting bud break in grapefruit [55
In general, this study clearly demonstrates that the presence of leaves in different citrus cultivars inhibit axillary bud break which were released soon after defoliation. Since even the presence of small petiole alone was as effective in inhibiting bud growth, just as the whole leaf, it indicates that supply of sugars or photosynthates from leaves play little role in regulating bud break in grapefruit. On the other hand, it appears that physical injury through complete defoliation may trigger hormonal stimulation that in turn starts the metabolic process leading to bud break. Majority of bud break from defoliation tend to develop vegetative buds, but, as anticipated, chilling treatment results in majority of bud breaks into flowers. Since differentiation of buds into vegetative or reproductive structures occurred in the absence of leaves (i.e. strictly by chilling defoliated plants induced flowering) it indicates that in grapefruit metabolic process leading to flowering occur within the resting bud tissue. Changes in the levels of specific polyphenols occur when buds begin to sprout as vegetative shoot, marked by increase in chlorogenic acid and naringenin levels, or develop into reproductive structures that accompany dramatic rise in hesperidin, naringenin, and Apigenin-7-Glucoside. Thus, pattern with which changes in levels of different polyphenols
occur at the onset of bud break in grapefruit provide good indications for bud break into vegetative or reproductive structures.