232 Physiology and Biochemistry of Endogenous Growth Regulators




March 25, 2009



Introduction What is Gibberellic Acid? Advantages and Disadvantages of GA Commercial Uses of GA a. Using GA Sprays on Navel Oranges b. Using GA Sprays on Lemon c. GA Application on Red Grapefruit and Tangerines d. GA Application on Mandarin


Physiology of Ripening in Citrus Regulation of Color Break in Citrus Citrus Chlorophyllase Dynamics at Ethylene-Induced Fruit Color-Break Conclusion References

INTRODUCTION Citrus is the most economically important fruit crop in the world, is grown in developed and developing countries and certainly constitutes one of the main sources of vitamin C. There is also an increasing demand of "high quality fresh citrus" driven by World Health Organization recommendations. Citrus contain the largest number of carotenoids found in any fruit and an extensive array of secondary compounds with pivotal nutritional properties such as vitamin E, pro-vitamin A, flavonoids, limonoids, polysaccharides, lignin, fiber, phenolic compounds, essential oils etc. These substances greatly contribute to the supply of anticancer agents and other nutraceutical compounds with anti-oxidant, inflammatory, cholesterol and allergic activities, all of them essential to prevent cardiovascular and degenerative diseases, thrombosis, cancer, atherosclerosis and obesity. Citrus fruits are also classified as hesperidiums, berries of very special organization characterized by a juicy pulp made of vesicles within segments. Thus, the combination of these characteristics suggests that the study of citrus fruit growth may reveal original regulation mechanisms based on specific molecular differences and/or even novel genes (Forment et al., 2005; Cercós et al., 2006; Terol et al., 2007 as cited by Iglesias, et al. 2007). Plant growth regulators have been used for many years to alter the behavior of fruit trees or fruit for the economic benefit of the fruit grower. Control of vegetative vigor, stimulation of flowering, regulation of crop load, reduction of fruit drop, and delay or stimulation of fruit maturity and ripening are important examples of processes in fruit trees and fruit that can be regulated with exogenous applications of plant growth regulators. Plant-growth regulators are a potential means of regulating various aspects of fruit maturity. The development of methods for bringing about early fruit maturity or delaying fruit maturation could prove of considerable advantage for numerous citrus varieties grown under a broad range of climatic conditions and subject to various marketing demands. The use of gibberellic acid (GA) is one of the most effective management tools available to citrus growers to improve rind quality and delay rind aging. The use of GA on citrus has been studied since the early 1960s and GA’s effect on increasing peel firmness and delaying peel senescence have been well documented. It has been demonstrated that Gibberellic Acid delays loss of green rind pigments of navel orange (Coggins and Hield, 1958; Eman, et al. 2007), Valencia orange (Coggins, Hield, and Garber, 1960), grapefruit (Coggins, Hield, and Burns, 1962; Ritenour, et al., 2005), tangerine (Soost and Burnett, 1961; Ritenour, et al., 2005), lemon (Coggins, Hield, and Boswell, 1960), lime (Burns et al., 1964), and satsuma mandarin (Nishiura and Iba, 1964). Regulation of chlorophyll breakdown at different physiological and developmental stages of the plant life cycle, particularly at fruit color-break, is still not well understood. This paper presents the effects of GA to delay ripening and prolong fruit storage of different citrus and describes the mechanistic basis of color break in citrus and the role of Chlase in chlorophyll breakdown.

What is Gibberellic Acid? Gibberellic Acid (GA) is a naturally-occurring plant growth hormone found in most plant tissues. It is a class of cyclic, diterpenoid hormones with an essential role in plant growth and development. It is involved in physiological processes such as stem elongation, fruit set, flowering, seed germination and fruit development, and is used in selected horticultural crops to manipulate flowering and fruit development. The highest concentration occurs in the immature seed and is what stimulates fruit to grow. GA stimulates both cell division and cell elongation. As discussed by Iglesias, et al. (2007), the endogenous GAs found in citrus fruits are mainly members of the 13-hydroxylation pathway [GA53, GA97, GA44, GA17, GA19, GA20, GA29, GA1, epi-GA1, and GA8 (Goto et al., 1989; Turnbull, 1989; Talon et al., 1990a, 1992)] leading to GA1, the bioactive GA (Zeevaart et al., 1993). This pathway also operates in vegetative tissues of citrus (Vidal et al., 2001, 2003) where is thought to control shoot growth and elongation (Fagoaga et al., 2007). Advantages of Using GA The primary benefits of GA treatment are a more desirable seasonal harvest pattern in relation to market demands, a larger percentage of fruit with a long storage life, and a decrease in the number of small yellow fruits. These effects permit more flexibility in harvesting and marketing. Since the sale of fresh fruit and peak production normally occurs when demand is low, these findings have important economic implications (Coggins et al., 1964). GA3 may reduce bloom and fruit set the spring following treatment. An increased fruit set during the following summer also may occur. Thus, the GA3 application can result in a delay in harvest of fruit. This allows for larger sized fruit. Disadvantages of Using GA The delay in loss of pigment can be a serious disadvantage, if conditions make it necessary to harvest while the fruits still retain chlorophyll, since consumers interpret this characteristic as a sign of immaturity. Treatment should be carried out after a marketable color has developed. Although delayed aging of grapefruit and Valencia orange rind tissues is a useful response, treatment of these fruits with GA results in two rind color problems. Gibberellic acid applied to green fruits delays loss of green pigments; applied to fruit of mature color, it enhances regreening.

COMMERCIAL USES OF GIBBERELLIC ACID Using GA Sprays on Navel Oranges GA spray was applied when the majority of the fruit are 30–50 mm in size in summer (January in southern Australian growing regions) to reduce the incidence of rind disorders such as albedo breakdown (creasing), water spot, and postharvest rind breakdown (Lindhout, K, 2008). It increased rind firmness, improves fruit quality and extends postharvest shelf-life by reducing fruit susceptibility to moulds. It improves the overall quality of fresh fruit, increasing packouts and market outturns. A GA spray applied before mid-February has minimal effect on rind colour. Research has shown that the summer GA spray can also enhance the effectiveness of the autumn GA spray on rind condition of late-harvested fruit. The positive effects that a summer GA spray has on rind quality can be seen in Figure 1-2. A 10 ppm GA spray applied in autumn delayed rind colour development and was used strategically to extend the harvest period (Fig. 3). However, the timing of the autumn application can significantly affect the length of time fruit take to reach full colour. Autumn GA sprays did not affect internal fruit maturity. As shown in Figure 4, Autumn GA sprays delay rind colour development. The earlier that GA sprays are applied during autumn, the longer fruit will take to reach full colour and the more likely that fruit will have uneven colouration.

Figure 1. A summer GA spray reduce the incidence of albedo break down and postharvest rind disorders (right) and increases rind firmness, resulting in improved fruit quality at market destinations.


Figure 2. Comparison of untreated (–GA) and GAtreated (+GA) navel orange fruit. The circles indicate cracks in the albedo tissue of untreated fruit, even though the fruit had no obvious visual symptoms of albedo breakdown.


Figure 3. The effect of an autumn GA spray on colour development in bellamy navel oranges, photographed in late may.

Figure 4. Rind colour development of Washington navel orange fruit sprayed with 10 ppm GA at different times. Arrows indicate when autumn GA sprays were applied. Fruit in the green section of the ‘suitable colour for export’ may require ethylene degreening.

Application of GA in water spray at 10–40 g a.i./acre can be used to reduce rind staining, water spot, and sticky rind (delayed aging and softening of rind). Preferred application time is 2 weeks before color break. If delayed coloring cannot be tolerated, apply after marketable color has developed. In both cases, there will be delayed rind aging but when it is applied after color has developed, considerable aging will have already occurred, resulting in less potential delay in aging. The effect of the later spray may be inadequate to provide the desired protection. Treatment with GA3 probably lowers intensity of puffy rind and fruit appears to be less susceptible to postharvest decay and mechanical injury. GA3 may result in a minor amount of leaf and fruit drop. Occasionally, leaf drop and fruit drop is excessive. When this happens, twig dieback can occur. Including 2,4-D in the GA3 spray may reduce these negative effects. There is little need for delaying fruit senescence on young trees. This plus the possibility of excessive leaf drop argue against the application of GA3 to young trees (UC IPM 2008). Separate and combination preharvest applications of GA3 and 2, 4-D are used widely in California to prolong the preharvest and postharvest life of navel orange fruit. 2, 4-D is used to

delay fruit abscission and GA3 to delay fruit maturation. Treatment of fruit with GA appears to substantially reduce water spot. When navel oranges age on the tree, the rind surfaces occasionally become sticky which cannot be removed by washing and packing operations but has been substantially reduced by preharvest treatments with GA (Coggins, 1973). GA3 delayed fruit coloration and rind softening for each cultivar. 2,4-D alone did not have any effect on fruit coloration, slightly delayed rind softening and significantly reduced fruit drop. Both compounds had long-lasting effects. GA3 slightly reduced the effectiveness of 2,4-D as a fruit-drop control agent. The combination of 2,4-D plus GA3 lengthened on-tree fruit storage and extended the harvest season (Mohamed, et al., 1990). Moreover, Eman, et al. (2007) also reported that treatments of GA on Washington Navel orange especially those included zinc sprays improved leaf N, K and Zn contents (Table 1-2). However data proved that GA sprays at 20 ppm were more effective than that at 10 ppm in term of fruit set, fruit retention and yield as fruit number or weight (Kg) per tree. Spraying 0.4% chelated zinc alone or with GA especially at 20 ppm significantly increased fruit set, fruit retention, decreased fruit drop and subsequently improved the yield as well as the physical and chemical fruit characteristics (Table 3-4). So, spraying 0.4% chelated zinc at mid February followed by spraying 0.4% chelated zinc + 20 ppm GA twice at (beginning of April and beginning of June) seems to be the promising treatment for increasing productivity of low yield Washington navel orange trees grown under sandy soil condition.

Using GA Sprays on Lemon High concentrations of GA applied to Lisbon lemon trees in the spring caused a significant delay in fruit maturity as well as a number of undesirable responses. Production during the treatment year was reduced, apparently as a result of increased fruit drop. Fruit drop and the percentage of harvested lemons without buttons were increased. The desirable feature of the treatment, a delay in yellowing, was marked: lemons from treated trees were greener as early as one month and for as long as seven months after treatment. Fruits treated with GA also appeared to develop a lemon-yellow color less rapidly in storage. Ultimate fruit size was not reduced by GA, but fruits of 3.6 to 4.7 cm in diameter at treatment time grew more slowly than untreated fruits. Growth rate of fruits that were larger or smaller at treatment time appeared to be normal. Delayed yellowing has been induced by GA applied to lemon trees at any time of the year. The response reflects an over-all delay in maturity rather than a simple delay in rind maturity as appears to be the case with navel orange. Very few undesirable responses, if any, occur when relatively low concentrations of GA are employed. In addition to the first-year effects on fruit color, fruit size, and seasonal harvest patterns, there is a change in harvest pattern in the second year. Probably this results from the influence of GA on flowering, which has been

observed in numerous trials. The volume of fruit produced early in the season is reduced, but treated trees produce more fruit during summer when the market demand is high. If GA is applied in two successive years, the seasonal harvest pattern is altered even more, since both direct and indirect effects are operative (Coggins et al., 1964 as cited by Iglesias, et al., 2007). Wright (2004) also reported that timely sprays of GA 3 when target crop is 0.5 to 0.75 full size and still green will delay lemon fruit maturity. When Gibberellic Acid (GA3) applied in storage wax to lemons, the result is delayed senescence, which maintains natural resistance to “Sour Rot” (Geotrichum candidum) and otherwise provides for a longer storage life. Some reports uses 10–20 g a.i./acre of GA spray during Oct.–Dec. in non desert areas to delay fruit maturity. Apply when target crop is 1/2 to 3/4 full size and still green. Reduces the number of small tree-ripe fruit and delays flowering, which shifts second year crop toward summer (UC IPM, 2008).

GA Application on ‘Fallglo’ Tangerines and ‘Red’ Grapefruit Gibberellic Acid (GA) has been reported to delay peel senescence of citrus fruits. Since physiological and pathological disorders of citrus tend to occur more frequently on senescent tissues, GA treatments are used in some citrus-growing areas to maintain quality through market channels (Ritenour, 2005). Preharvest sprays of GA at 30 g a.i. per acre with 0.05% Silwet (0.05%) increased peel puncture resistance, but also led to a slight reduction in total soluble solids (TSS). As expected, preharvest GA treatments delayed color development of 'Fallglo' tangerines at harvest and after degreening compared to the control containing only 0.05% Silwet. Extending ethylene exposure from 6 to 18 hours allowed GA-treated fruit to color better than non-GA treated fruit degreened for only 6 hours, but not better than control fruit degreened for 18 hours. Extending ethylene exposure to overcome GA-delayed color development enhance the development of anthracnose and total decay. Inhibition of color development in harvested 'Fallglo' tangerines dipped in 250 ppm GA + 0.05% Silwet was not significant immediately after degreening compared to the control (0.05% Silwet only), but was significant 18 days after degreening. GA treatments on 'Ruby' Red grapefruit resulted in phytotoxic injury when applied preharvest, but postharvest only when fruit were dipped in the GA solutions before degreening. The pre- nor postharvest application of gibberellic acid had beneficial effects on the maintenance of 'Fallglo' or 'Ruby' red grapefruit. Early application of GA + Silwet resulted in significantly greener fruit in ‘Fallglo’ and ‘Sunburst’ tangerines and ‘Minneola tangelo’ and ‘Marsh’ grapefruit. Early treatments of GA + Silwet also resulted in fruit with significantly greater peel puncture resistance than control fruit and reduced postharvest pitting of ‘Fallglo’ after one week of storage (Ritenour and Stover, 1999).

Table 5. Fruit quality of ‘Fallglo’ tangerines 1 d after harvest. Trees were sprayed with GA + Silwet or Silwet alone 17 d prior to harvest.

Table 6. Peel color of ‘Fallglo’ tangerines treated with GA + Silwet or Silwet alone 17 d prior to harvest. Fruit were degreened for either 6 or 18 h with 2 ppm ethylene at 85 °F (29 °C).

GA Application on Mandarin 20–40 g a.i./acre of GA is used to delay rind aging and softening and to reduce puffiness of rind. It is Applied about 2 weeks before color break, but only to groves where early harvest will not occur as this treatment delays coloring; satisfactory color was not expected until late January. Later GA3 applications produced undesirable results: applications made during coloring resulted in unacceptable variations in rind color and applications made after coloring caused preharvest rind staining (UC IPM, 2008). GA spray on Imperial Mandarins, reduced albedo breakdown, oleocellosis and watermark (Figure 5). The earlier the spray the less effect the GA have on delaying colour change. For instance, an early spray, fruit diameter 30 to 50 mm (early January), delayed colour change by 0 to 5 days instead of 2 to 3 weeks if sprayed in early February. The delay in colour change depends on timing, variety, the rate used, location and prevailing seasonal conditions. Also the earlier the spray the more effective the GA will be (George Morris).




Figure 5. (a) Albedo breakdown (sometimes known as creasing) is the separation of the mesocarp or albedo (the layer of white internal rind) from the exocarp or flavedo (the external rind) resulting in the rind developing creases. It is recognised by narrow sunken groves in the rind. In severe cases the groves intersect making the fruit appear lumpy and soft. It is a serious condition and may cause the fruit to split open under pressure when packed. (b) Watermark on Imperial Mandarins (known as ‘waterburn’ in the Eastern States) occurs during wet weather when the bottom of the fruit is wet for a prolonged period. (c) Oleocellosis (sometimes known as ‘oil spotting or burn’) occurs where there is mechanical injury to the fruit. It is caused by the rupturing of oil glands releasing toxic oil during harvest and/or transport from the field to the packinghouse. This oil kills the nearby cells of the flavedo. The oil from injured fruit can also cause oil spotting on the surface of adjacent fruit. It is more common when the fruit is turgid and the weather is cold and/or wet at harvest. The oil from the ruptured cells inhibits degreening and discolours the rind.

Physiology of Ripening in Citrus The physiological processes regulating fruit development and ripening have been extensively studied because of the importance of fruits as components for the human diet. In climacteric fruits and in particular in tomato, it has been demonstrated that ethylene controls ripening processes through the regulation of gene transcription and therefore, much information has been generated in the areas of ethylene biosynthesis and response (Giovannoni, 2004; as cited by Iglesias, et al., 2007). In contrast, the mechanism of ripening control in non-climacteric fruits, including citrus, is totally unknown. As the navel orange approaches legal maturity, the concentrations of chlorophyll pigments in the flavedo of the fruit decrease and carotenoid pigments increase. During the change in color from green to orange, the rind softens rapidly for a period and then continues softening at a slower rate throughout the harvest season (Lewis and Coggins, 1965; as cited by Iglesias, et al., 2007). Since color development commences prior to the attainment of legal maturity, softening actually begins before the start of the harvest season, continuing for as long as fruit remain on the tree. During the eight-month softening period, the capacity of rind tissue to utilize glucose decreases, sugars accumulate to approximately twice their initial concentration, and the ratio of potassium to calcium plus magnesium increases (Lewis et al., 1967 as cited by Iglesias, et al., 2007). These physiological changes suggest that mitochondrial and plasmalemma membrane systems become less functional during aging. The structure of citrus fruit and metabolic changes associated with the internal development is shown in Figure 6.

Figure 6. Structure of citrus fruit and metabolic changes associated with the internal development. (A) Internal structure of a ripe orange fruit. (B) Fruit growth is mostly due to cell divisions during phase I and to water accumulation and therefore cell enlargement, during phase II. At the beginning of phase III, growth is arrested and fruit starts a non-climacteric ripening process. (C) In contrast with peel flavedo, chlorophyll degradation and carotenoid biosynthesis in pulp proceed along phase II and therefore color break is reached earlier. This results in a progressive change in pulp color that contrasts with the rapid color break occurring in flavedo (D) Paralleling fruit growth, high amounts of soluble carbohydrates are translocated to the developing fruit. Thus, mature citrus fruit pulp accumulates high amounts of sucrose, glucose and fructose in a 2:1:1 ration. (E) Acid accumulation in fruit pulp takes place during phase I and the beginning of COMMERCIAL USES OF GA phase II the characteristic low acidity of ripe fruits.

Recent histological studies provide a structural basis for the soft condition that exists in senescent rind. In general, cells of the flavedo and albedo enlarge, become highly vacuolated, and change shape during development, maturation, and senescence. Many intercellular spaces develop, and cell walls appear to weaken and break, particularly in the albedo. Tissue of the senescent rind appears to be structurally weak, and the relatively low amount of cytoplasm suggests that the tissue may possess low metabolic activity. The events leading to senescence in navel orange rind appear to include pigment changes, softening of the tissue, increases in carbohydrates, some changes in carbohydrate metabolism, and changes in cation concentrations. All of these factors clearly are modified by GA, resulting in a delay in senescence. Rind disorders that appear to be associated with aging include rind staining, water spot, increased susceptibility to decay, puffy rinds, and accumulation of a rind exudate. Each of these disorders can be reduced by GA treatments. The reduced susceptibility of GA-treated fruits to rind staining (Eaks and Jones, 1959; Coggins et al., 1963; as cited by Iglesias, et al. 2007) is probably related to the retardation of the softening process. Rind staining is a physiological disorder associated with mechanical abrasion during harvesting, handling, and packing. Treatment with GA to reduce rind staining also delays loss of green pigments. Regulation of Color Break in Citrus Citrus are classified as non-climacteric fruit; however, a role for ethylene during ripening, and in particular in chlorophyll breakdown during fruit color-break, is well established (Purvis and Barmore, 1981; Goldschmidt et al., 1993; Porat et al., 1999; as cited by Shemer, et al., 2008). Furthermore, the application of exogenous ethylene dramatically enhances the speed of color-break of detached mature green citrus fruit, which is a slow process for fruit left untreated on trees (Amir-Shapira et al., 1987; Iglesias et al., 2001; as cited by Shemer, et al., 2008). As reported by Iglesias, et al. (2007), it has been known for a long time that regulatory mechanisms involving phytohormone control are deeply involved in chloroplast transformation (Bruinsma et al., 1975; McGlasson et al., 1978; Goldschmidt, 1988; Ben-Arie et al., 1995; Guis et al., 1997). Thus, ethylene is being used for the last half century to stimulate color change in citrus fruits during post-harvest storage. It has been suggested that ethylene is synthesized in citrus fruit through typical system I machinery although an additional system II-like process appears to operate in young fruitlets (Katz et al., 2004). Mature citrus fruits release very low amounts of ethylene although they respond to exogenous ethylene accelerating color break through both chlorophyll degradation and carotenoid deposition. Exogenous ethylene certainly accelerates chlorophyll disappearance and increases chlorophyllase activity (Trebitsh et al., 1993; Azuma et al., 1999; Jacob-Wilk et al., 1999; Fujii et al., 2007). In contrast, GA3 partly counteracts the ethylene-induced increase in chlorophyllase (Trebitsh et al., 1993) delaying de-greening (Cooper and Henry, 1968). This observation is consistent with many field observations of de-greening delay or even fruit re-greening associated with the GA treatment (Porat et al., 2001). It has also been suggested that color break of citrus fruit is controlled by nutrients. Based on data collected from in vitro studies on citrus epicarp,

Huff (1983, 1984) as cited by Iglesias, et al. (2007) suggested that citrus fruit might partially degreen in response to the accumulation of sugars. It has specifically been shown that the chloro- to chromoplast conversion in citrus fruit epicarps is stimulated by sucrose accumulation after an initial decrease in peel nitrogen content (Iglesias et al., 2001). In this work it is proposed that sugar regulation may operate via ethylene, whereas GA functions as a repressor of the ethylenesucrose stimulation. Therefore, GAs as well as nitrates are color break retardants since both delay the chloro- to chromoplast transition (Alós et al., 2006). While the mechanistic basis of color break in citrus has not yet been resolved, several other studies have suggested various factors that may contribute to pigment turnover. For example, it has been shown that chlorophyllase, a constitutively expressed gene, does not increase during natural fruit development (Jacob-Wilk et al., 1999). In addition, the expression patterns of carotenoid biosynthetic genes and an associated abundance of carotenoids during ripening have recently been reported (Kato et al., 2004; Rodrigo et al., 2004; Alós et al., 2006). It is noteworthy that citrus peel contains the greatest diversity of carotenoids of any fruit studied to date and their specific accumulation patterns are responsible for the broad range of colors exhibited by citrus fruits (Gross, 1987). Although the characteristic color of typical citrus varieties is mainly provided by the accumulation of 9-cis-violaxanthin and b-cryptoxanthin, there are also citrus-specific carotenoids such as b-citraurin and b-citraurinene that provide an attractive coloration and whose biosynthetic basis remains unknown (Oberholster et al., 2001). The regulation of color break in citrus fruits is summarized in Figure 7.

Figure 7. Regulation of color break in citrus fruits. External fruit ripening is depended upon the conversion of chloroto chromoplast and involves the progressive loss of chlorophylls and the gain of carotenoids, changing peel color from green to orange. The changes associated with external ripening are influenced by environmental conditions, nutrient availability and hormones. De-greening in subtropical areas generally takes place in mid-autumn when temperatures go down and day length diminishes. Depletion of nitrogen appears to be a pre-requisite for color break but carbohydrate accumulation is thought to stimulate the process. Similarly, ethylene promotes de-greening and gibberellins counteract this process. Interestingly, the chloro- to chromoplast conversion is a reversible process even from fully differentiated chromopalsts. During spring, temperature rises and day length increases and these new environmental conditions induce new re-growth that results in nitrogen uptake, carbohydrate utilization and gibberellin synthesis. These changes presumably drive re-greening of the fruit peel.

Citrus Chlorophyllase Dynamics at Ethylene-Induced Fruit Color-Break Fruit color-break is the visual manifestation of the developmentally regulated transition of chloroplasts to chromoplasts during fruit ripening and often involves biosynthesis of copious amounts of carotenoids concomitant with massive breakdown of chlorophyll. Regulation of chlorophyll breakdown at different physiological and developmental stages of the plant life cycle, particularly at fruit color-break, is still not well understood. The graphic model of the proteins involved in chlorophyll breakdown in higher plants is shown in Figure 8.

Figure 8. Graphic model of the proteins involved in chlorophyll breakdown in higher plants and chemical structures of chlorophyll and chlorophyll catabolites. The protein SGR, involved in stay-green mutations, is suggested to function upstream of the chlorophyll catabolic enzymes by destabilizing the LHC (Park et al., 2007). The enzyme chlorophyll b reductase is involved in maintaining the balance between chlorophylls by catalyzing the conversion of chlorophyll b to a. The catabolic steps of the chlorophyll breakdown pathway, which lead to loss of the typical green color, are outlined together with the structures of chlorophyll and the intermediate breakdown products as follows: (1) Chlase catalyzes the cleavage of the hydrophobic thylakoidanchoring phytol chain from the chlorophyll porphyrin ring, resulting in the product chlorophyllide; (2) removal of the Mg ion from chlorophyllide, yielding pheophorbide a, has not yet been shown to be an enzymatic step (labeled as Mgdechelatase); and (3) PaO catalyzes the cleavage of the porphyrin ring, resulting in the RCC, which is further metabolized by additional enzymes downstream, exported to the vacuole, nonenzymatically converted to nonfluorescent chlorophyll catabolites (NCCs), and stored indefinitely.

The dynamics of native chlorophyllase (Chlase) and chlorophyll breakdown in lemon (Citrus limon) fruit during ethylene-induced color-break is documented by Shemer, et al., (2008) using in situ immunofluorescence on ethylene-treated fruit peel (flavedo) tissue, that citrus Chlase is located in the plastid (Figure 9), in contrast to recent reports suggesting cytoplasmic localization of Arabidopsis (Arabidopsis thaliana) Chlases. At the intra-organellar level, Chlase signal was found to overlap mostly with chlorophyll fluorescence, suggesting association of most of the Chlase protein with the photosynthetic membranes. Confocal microscopy analysis showed that the kinetics of chlorophyll breakdown was not uniform in the flavedo cells. Chlorophyll quantity at the cellular level was negatively correlated with plastid Chlase accumulation; plastids with reduced chlorophyll content were found by in situ immunofluorescence to contain significant levels of Chlase, while plastids containing still-intact chlorophyll lacked any Chlase signal. Immunoblot and protein-mass spectrometry analyses were used to demonstrate that citrus Chlase initially accumulates as an approximately 35-kD precursor, which is subsequently Nterminally processed to approximately 33-kD mature forms by cleavage at either of three consecutive amino acid positions. Chlase plastid localization, expression kinetics, and the negative correlation with chlorophyll levels support the central role of the enzyme in chlorophyll breakdown during citrus fruit color-break.
Figure 9. Subcellular localization of Chlase in ethylene-treated citrus fruit peel by in situ immunofluorescence. Mature green lemon fruit were treated with ethylene (20 mL L21) at 25_C in the dark for 24 h in a 4-L sealed container. The container was ventilated once after 12 h, followed by injection of fresh ethylene, to maintain CO2 levels below 1%. The flavedo of the treated fruit was dissected and used for obtaining cross sections (70 mM thick) using a vibratome. Tissue sections were fixed and dressed with Alexa-488- conjugated goat antirabbit secondary antibody as a control (A) or affinity-purified anticitrus Chlase antibody (rabbit) followed by Alexa- 488-conjugated goat anti-rabbit secondary antibody (B and C). Fluorescence was visualized using a laser scanning confocal microscope. Images of representative cells (A and B) are presented as follows: 1, green fluorescence of the Alexa-488 secondary antibody corresponds to detection by anti-Chlase antibody; 2, red fluorescence corresponds to chlorophyll autofluorescence; 3, confocal image recorded simultaneously in transmitted and red fluorescence mode (i.e. chlorophyll fluorescence superimposed on the bright-field image); and 4, confocal image recorded simultaneously for red and green fluorescence (i.e. immunofluorescence resulting from detection by anti-Chlase superimposed on the chlorophyll autofluorescence). Chloroplasts demonstrating green fluorescence (i.e. detection by anti-Chlase antibody) were further analyzed by the confocal microscope colocalization program analysis tool. Images of representative chloroplasts (C) are presented as follows: 1, red fluorescence corresponds to chlorophyll autofluorescence; 2, green fluorescence of the Alexa-488 secondary antibody corresponds to detection by antiChlase antibody; and 3, sectors of yellow color indicate colocalization of chlorophyll autofluorescence and immunofluorescence resulting from detection by anti-Chlase antibody. Colocalized sectors in the fluorescing chloroplasts account for 76% of the total fluorescence observed. Scale bar 5 10 mm (A and B) and 2 mm (C).

CONCLUSION Plant Growth regulators are widely used in tree fruit production. They produce many beneficial effects on both trees and fruit. Many researches seek to expand the range of useful responses of fruit trees and fruits themselves to plant growth regulators by conducting trials of new products as well as new research directions already available for use in the tree fruit industry. The use of gibberellic acid (GA) is one of the most effective management tools available to citrus growers to improve rind quality and delay rind aging. Appropriate use of GA can result in better packouts and market outturns. Citrus are classified as non-climacteric fruit; however, a role for ethylene during ripening, and in particular in chlorophyll breakdown during fruit color-break, is well established. While ethylene is being used for the last half century to stimulate color change in citrus fruits during post-harvest storage, in contrast, GA3 partly counteracts the ethylene-induced increase in chlorophyllase, delaying de-greening. External fruit ripening is dependent upon the conversion of chloro- to chromoplast and involves the progressive loss of chlorophylls and the gain of carotenoids, changing peel color from green to orange. Chlase plastid localization, expression kinetics, and the negative correlation with chlorophyll levels support the central role of the enzyme in chlorophyll breakdown during citrus fruit color-break. While the mechanistic basis of color break in citrus has not yet been resolved, environmental conditions, nutrient availability and hormones influence the changes associated with external ripening. GA can be applied to citrus to improve rind firmness and quality at harvest, for increased packouts and market outturns, reduce albedo breakdown (creasing), reduce postharvest rind breakdown and extend postharvest shelf-life, and slow down rind colour development and delay harvesting. Physiological disorders such as creasing, splitting, puffing, peel pitting, etc. can be reduced in intensity or minimized using gibberellic acid, synthetic auxins or a mixture of both as reported by Agusti, et al. (2002). Fruit maturation (colouring) can be enhanced using ethepon or figaron and can be delayed using gibberellic acid. The effectiveness of some of these treatments can be significantly improved by adding mineral salts, mainly nitrogen compounds in the spray mix. Although delayed aging of grapefruit and Valencia orange rind tissues is a useful response, treatment of these fruits with GA results in two-rind color problems. Gibberellic acid applied to green fruits delays loss of green pigments; applied to fruit of mature color, it enhances regreening. Applying GA can be a costly exercise in terms of time and money, so it needs to be done right. The effects of GA are dependent on both the timing of application and the concentration of GA applied. The earlier the spray the less effect the GA has on delaying colour change. The delay in colour change depends on timing, variety, the rate used, location and prevailing seasonal conditions. Also the earlier the spray the more effective the GA will be. Manufactured GA is a product that has been available for many years and there are several products available. GA is highly effective when applied at the right time and at the right concentration. GA can be used to alter the growth pattern of plants considerably.

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