The major organic acid associated with pulp total acidity is citrate, which begins to accumulate during stage II of fruit development, when the fruit and its juice vesicle cells enlarge rapidly . The accumulation continues for a few weeks, reaching a peak when the fruit volume is about 50% of its final value, then the acid declines gradually as the fruit matures. In most varieties, there is a slight increase in sugar content early in fruit development, but the major increase occurs during stage III, when the acid content declines . In citrus, the major translocated sugar is sucrose and in many varieties, it accumulates to double the level of glucose or fructose . Maturation index, which determines the fruit’s internal quality, is the ratio between total soluble solids and total acidity. As the acid content declines toward harvest, sugars account for most of the TSS. As already noted, climate plays a major role in fruit development and maturation. Most of the commercial citrus cultivars were selected or bred in the subtropical regions of the world, and they are therefore adapted to regions where maturation occurs during the cool season . In hot climates, such as in the tropics, fruit maturation is accelerated and the major factor affected by temperature is the fruit acid level, with a linear relationship between the accumulation of heat hours and acid decline . Therefore, in hot climates, the fruit reaches its maturation index faster than in colder climates, black plastic planting pots and tends to be too sweet. However, this is only part of the problem. Citrate catabolism is associated with an increase in alcohols, aldehydes and other secondary metabolites associated with reduced flavor and fruit decay .
Therefore, in hot climates, the time during which the fruit is harvestable and marketable is considerably shortened, and fruit decay occurs faster than in the colder regions . Hot climate has the opposite effect on color break, which requires the correct number of cold night-time to develop . Therefore, not only the fruit decay faster in hot climates, but their color does not fully develop, and in extreme cases may even remain green. One of the expected outcomes of climate change is warmer winter temperatures with shorter cold-night times . As most citrus cultivars are harvested during this season, the effect of global warming is expected to be negative on both internal and external citrus fruit quality.Sink strength is determined by the sink’s size and activity . In crop plants, it is defined in practice by yield parameters , and quality parameters, such as carbohydrate and protein levels. Fruit size is genetically controlled, but physiological parameters, such as sink position in relation to other sinks and source tissues, and the time it takes to develop, also affect sink size and therefore, its strength . In tomato, there are over 30 loci that define fruit size, with many genes acting to control cell division at various developmental stages . Practically, sink activity is defined as the rate of photo assimilate translocation and their contribution to growth and developmental processes relative to their accumulation. To simplify the discussion, we will refer here only to sugars, as the major photo assimilates in fruit in general, and in citrus fruit in particular. As with many other plant species, in citrus, sucrose is the major sugar translocated from the leaves to the fruit .
During fruit maturation, it is the major accumulated sugar, with a sucrose:glucose:fructose ratio of 2:1:1 in many cultivars and references therein. In many cases, sucrose accumulation is detected early in fruit development, indicating a higher translocation than utilization rate . As discussed further on, sucrose catabolism into hexoses within the fruit provides the central mechanism controlling sink activity, and therefore sink strength. Sucrose is hydrolyzed either to fructose and UDPglucose by sucrose synthase , a bidirectional enzyme, or to glucose and fructose by invertase, a unidirectional enzyme . Following hydrolysis, glucose and fructose are phosphorylated to glucose-6-phosphate and fructose-6-phosphate by hexose kinase and fructokinase, respectively, while UDP-glucose is phosphorylated to glucose- 1-phosphate by UDP-glucose phosphorylase. While SuSy is cytosolic, sucrose hydrolysis by invertase is performed in the apoplasm by cell-wall invertases, in the cytosol by neutral/ alkaline invertases, and in the vacuole by acidic invertases. The enzyme is modulated post-translationally by invertase inhibitor, which might act in vivo, but not necessarily in vitro . In a few plant systems, it has been shown that alteration of the activities of SuSy and various forms of invertase results in altered yield and/or carbohydrate levels, and thus altered sink strength. For example, one amino acid change in the tomato cell-wall invertase LIN5 enhanced specific activity of the enzyme, the rate of sucrose uptake and, overall, BRIX . Increased expression of cucumber SuSy induced sucrose and starch accumulation and increased fruit size .
Transgenic down regulation of tomato SuSy resulted in reduced sucrose uptake early in fruit development, reduced fruit set, reduced fruit number and reduced fruit size . Similarly, reduced expression of acid invertase and SuSy in muskmelon and cucumber, respectively, reduced fruit size and sucrose level . Phenotypes associated with reduced sink strength were also demonstrated in carrot roots by down regulating vacuolar and cell-wall invertases as well as SuSy . Taken together, these studies demonstrated the importance of invertases and SuSy for sink strength and supported the notion that sink strength is controlled, at least in part, within the sink cells and/or at translocation points, i.e., zones of phloem unloading.Sugar transport from the leaf to the collecting phloem is defined as sugar or phloem loading, and its release from the transport system, the releasing phloem, into the sink cell is defined as sugar or phloem unloading . Movement of photo assimilates from the leaves to the sink through the stem via the transport phloem is a complex process. Although a major driving force is the concentration gradient between source and sink according to the pressure flow hypothesis , long-distance movement requires in and out movement of solutes from the transport system to the surrounding tissue, temporal accumulation, and energy investment . The mechanism of sugar unloading has been investigated in a number of fruit and other sink organs, such as tomato, grapeberry, cucumber, apple, walnut and potato tuber, by microscopy, fluorescent dyes, immunolocalization of sugar transporters, and use of transporter inhibitors . Unloading is operated by two major mechanisms, symplasmic, in which sugar transport occurs through the plasmodesmata connecting the transport cells and the sink cells, and apoplasmic, where sink cells are not connected symplasmically to the transport cells, and transport must therefore cross membranes through the apoplasm, usually using facilitated transport mechanisms . In some fruit, such as cucumber, apple, and kiwifruit, unloading is apoplasmic throughout fruit development . In other cases, such as tomato fruit and grape berry, there is a shift between symplasmic and apoplasmic unloading , whereas in the potato tuber, the shift is from apoplasmic to symplasmic . In jujube, two shifts occur during fruit development; apoplasmic unloading early in fruit development shifts to symplasmic unloading, which then shifts back to apoplasmic unloading close to ripening . The two types of unloading occur simultaneously during walnut fruit and seed development, symplasmic in the seed and apoplasmic in the fruit . Overall, symplasmic unloading is considered faster than apoplasmic unloading . Therefore, the mechanism is dependent on the rates of sink development and photo assimilate utilization versus the rate and form of their accumulation, in order to avoid an increase in osmoactive molecules in the cytosol. For instance, drainage pot in tomato fruit and grape berry, fruit development and sugar utilization are rapid during the first half of their development, requiring symplasmic unloading. Later, when the fruit shifts from a utilizing to accumulating sink, the unloading rate is reduced by shifting it to apoplasmic. During the first half of potato tuber development, it accumulates soluble, osmoactive sugars, and therefore apoplasmic unloading is required, but during later stages, starch is accumulated, allowing a faster unloading. Jujube fruit is characterized by rapid growth during the middle stages of its development, and therefore apoplasmic unloading is interrupted by symplasmic unloading during this stage.As phloem unloading has never been investigated directly in citrus fruit, the mechanisms in the various fruit tissues are unclear . Nevertheless, potential mechanisms can be discussed based on the following: the kinetics of sugar transport into the various fruit tissues, vascular bundles, segment epidermis, stalk of the juice sac, and juice sac, as determined using photo assimilate distribution by 14CO2-feeding of leaves, through either continuous labeling or pulse-chase experiments , the steady state distribution of sucrose and hexoses, as well as the activities of sugar-metabolizing enzymes and their protein levels in the various fruit tissues, especially during intensive sugar uptake . While the activities of sugar-metabolizing enzymes have been well-studied and characterized, understanding their physiological role during the various stages of fruit development is more challenging.
In tomato, for instance, about 20 days postanthesis, SuSy activity decreased and the activity of an apoplasmic invertase, eventually identified as LIN5, was induced . This shift was associated with the well-studied shift from symplasmic to apoplasmic unloading and with the conversion of the fruit from utilizing to accumulating sink . It might be concluded, therefore, that in tomato fruit, SuSy activity is required to maintain a high rate of sucrose utilization, whereas invertase activity is associated with hexose accumulation. Clearly, the equivalent information is still missing in citrus fruit. In the following, photo assimilate movement and distribution, as well as the activities of sugar metabolizing enzymes and their protein levels are described for the various fruit tissues . In most of the studies, the activities of the various forms of invertases are defined by their pH optima and solubility. Herein, alkaline/neutral-soluble invertase is referred to as cytosolic invertase, acid-soluble invertase as vacuolar invertase, and acid-insoluble invertase as cell-wall invertase .The vascular bundle terminates near the segment epidermis. However, albedo cells are present between the bundle and the segment epidermis, and therefore phloem unloading is expected to occur primarily into albedo cells before sugar reaches the pulptissue . In young fruitlets and fruit, the albedo is the major tissue; cell division terminates within 4–5 weeks post-anthesis, and fruit growth during stage II is achieved by pulp expansion . Therefore, it might be assumed that most of the sugar in the albedo is transported, while only a minor part of it is required for albedo cell metabolism and development. Pulse-chase experiments demonstrated that most of the radiolabel remains in the post-phloem compartment, i.e., albedo cells, for about 24 h before reaching the pulp tissues, juice sac and segment epidermis . This slowing of sugar movement could indicate that most of the transport is via the apoplasmic path. The presence of insoluble acid invertase activity could provide an indication for this type of unloading; such activity has been detected in albedo cells, although at a lower level than in other tissues—the vascular bundle, segment epidermis, and juice sacs . In addition, the albedo contained considerable activities of SuSy, vacuolar invertase and cytosolic invertase ; in fact, the activity of vacuolar invertase was strongest in the albedo compared to other fruit tissues, indicating active storage of sucrose/hexoses in the albedo after unloading. Obviously, the presence of plasmodesmata and a symplasmic pathway between the vascular bundle and albedo cells cannot be ruled out at this stage.As already noted, the segment epidermis provides a continuous layer with the juice sac epidermis . It is considered part of the transport tissues, and therefore enzymatic activities are sometimes reported for the vascular bundle and segment epidermis together , although in other cases they are separated . A considerable percentage, about 30%, of the total radiolabel could be recovered in the epidermis, with maximal accumulation between 24 and 48 h after feeding in a pulse-chase experiment using grapefruit . With continuous labeling, about 50% of the total radiolabel was recovered in the segment epidermis within 24 h. However, when radiolabeled sugars were quantified in Satsuma mandarin after 48 h of feeding with 14CO2, the segment epidermis displayed the lowest amount per fresh weight or per fruit . These discrepancies could be due to different experimental designs or reflect cultivar differences. Regardless, the segment epidermis provides a strong sink, and movement of photo assimilates from this sink to the juice sac cells cannot be ruled out. Sucrose hydrolysis in the segment epidermis was mediated by relatively high activities of SuSy and soluble invertase .