11.4 - Carbon accumulation

Fig 11.09.png

Figure 11.9. Carbohydrate economy in developing fruit is derived by import and recycling of photoassimilates between different metabolic compartments. Sucrose or less commonly other forms of translocated carbon (Table 11.1) arrive via phloem conduits and are loaded into cytoplasmic compartments. As shown schematically, carbohydrate can then be partitioned to vacuolar storage or converted to other sugars and, in the form of hexose phosphate, transferred to plastids where it is used to synthesise starch. Each step is reversible, and as happens during ripening, starch in plastids is transformed back to sugars that subsequently accumulate in vacuoles as indicated. Other specialised organelles (oleosomes) store lipids while mitochondria draw upon imported and locally fixed carbon for ATP generation. (Original diagram courtesy R.L. Bieleski)

During development, photoassimilate is stored in fruit (and other sink organs such as root vegetables, seeds and flowers) in either a soluble or insoluble form (Figure 11.9). Fruit that store carbon in a soluble form (e.g. berry fruit, peach, persimmon, melon, grape, citrus) need to remain on the plant until nearly ripe if they are to survive postharvest storage and meet customer expectations. In most cases the major and rapid increase in soluble sugar content does not occur until late in development, signalling the beginning of ripening. Because the sugar source is the parent plant, harvesting such fruit too early reduces their final sugar content to unacceptable levels. In contrast, there are other fruit that store their carbon in insoluble forms, particularly starch. This allows greater efficiency in accumulating carbon, as the storage product is more compact, osmotically inactive and better segregated from metabolic processes. Examples are avocado, which stores carbon as both starch and lipid, and kiwifruit, apple, pear, mango, papaya and banana, all of which store carbon as starch.

In fruit that store carbon as sugars and organic acids during development, colour changes followed by the initiation of softening signal that fruit are becoming mature and ready to harvest. The challenge facing postharvest physiologists is to assess when such fruit are still sufficiently firm to allow easy handling and storage, but have enough sugar for a true ripe flavour to develop. A developing crop is typically monitored with a simple refractometer test (soluble solids concentration based on refractive index of expressed juice). In grape, a rapid rise in sugar content (beginning at ‘veraison’) may need to reach SSC values of around 20% (with, say, 17% sugar and 2% acid as the main soluble components) before a successful harvest can be assured.

There is more to good flavour than high sugar content, with sugar to acid ratio being particularly important, so that a combination of SSC to acid ratio with a minimum SSC level may be required. For example, New Zealand mandarins may not be exported to Japan if the SSC to acid ratio is below 10 (when the proportion of acid is too high). In other crops such as melon, the acid component is unimportant, and sugar content primarily dictates fruit quality.

In fruit that store carbon as starch, time of picking is less dependent on sugar content, since a doubling or more of sugar concentration by starch hydrolysis can still take place after the fruit are picked. Once starch utilisation has started, fruit can be picked without much detriment to final eating quality. SSC measurements alone are then insufficient, and measurements of total solids, dry matter, oil content and starch concentration are used as well. For example, in kiwifruit sequential measurements of SSC are combined with dry matter measurements; in apple and pear, the pattern of starch distribution within the fruit is recorded along with SSC and ethylene measurements; in mango, total solids (and fruit shape, flesh colour and firmness) are recorded; in papaya, colour changes and sequential SSC measurements are recorded; in avocado, dry matter, sometimes in combination with oil content, is used. In banana, shape or ‘fullness’ of fruit is an important criterion rather than starch level because bananas can be harvested over a remarkably wide range of maturities and still ripen satisfactorily.

Thus, when fruit store carbon in insoluble forms, they can often be harvested while still hard, lending greater flexibility to postharvest handling and ensuring a longer storage life.

11.4.1 - Sugar storage

Fig 11.10.png

Figure 11.10. Grape undergoes an abrupt change in physiology midway through development. For about 8 weeks after flowering, berry volume increases steadily but fruit are hard (low deformability) and sugar content low. At 'veraison' invertase activity rises abruptly and reducing sugar content increases rapidly, reaching about 20% of fresh weight when ripe. Berries attain full size by 10–12 weeks, and approach an asymptote in sugar content 2–3 weeks later. (Based on Davies and Robinson (1996) Plant Physiol. 111, 275-283)

In sugar-storing fruit a major shift in metabolism generally takes place when fruit expansion is almost complete, heralding a rapid increase in sugar content. Unloading of sugars from the phloem usually occurs by a symplastic route, but in some species is interrupted by an apoplastic step. Control points for sugar entry and accumulation by fruit include:

  1. Rate of sugar production by leaves and delivery to transport pathways;
  2. Reallocation of sugar from supporting vegetative growth towards fruit growth;
  3. Enhanced unloading of sugar from transport streams into fruit;
  4. Enhanced transfer of sugar across plasma membranes into cells or through plasmodesmatal connections between cells;
  5. Onward metabolism of sugar in the cytoplasm, or transfer to storage in vacuoles;
  6. Increased respiratory utilisation of sugar to provide energy for metabolic processes.

As with any biological system, multiple controls operate concurrently to drive a given pattern of maturation. Such events lead us to more robust indicators of ripeness, and improved ways of manipulating maturation to yield higher sugar content and better handling properties.

In tomato, there are different genotypes that accumulate either hexoses or sucrose. Most cultivars are hexose accumulators, in which acid invertase is active during growth and ripening. In transgenic tomatoes in which acid invertase activity was suppressed by expression of an antisense invertase transgene, sucrose accumulation occurred in a normally hexose-accumulating cultivar (Klann et al. 1996). Conventional breeding studies using crosses between sucrose-accumulating and hexose-accumulating types of tomato showed that an acid invertase gene is not transcribed during ripening of the sucrose accumulators, and that sucrose accumulators therefore lack acid invertase (Harada et al. 1995).

In melon, where sucrose is the main sugar to increase, there is a corresponding decrease in acid invertase and an increase in sucrose phosphate synthase (SPS) activity (this synthesises sucrose from hexose phosphate and adenylated precursors). A complex metabolic transition to allow sucrose accumulation occurs at the end of fruit growth and onset of ripening, of which the loss of soluble acid invertase activity is only one component (Dai et al. 2011).

However, in grape berries where hexoses begin to accumulate at veraison and reach very high levels (Figure 11.10), SPS, sucrose synthase and hexokinase activities all increase, but acid invertase mRNA abundance and activity both peak just prior to or at veraison and then decline. This suggests that in grape factors other than invertase activity regulate hexose accumulation. Two sucrose transporters were up-regulated at veraison, and it is possible that these regulate ripening-associated sugar accumulation from the apoplast into the parenchyma cells (Davies et al. 1999). Consistent with this suggestion, studies using tracers have shown that at veraison phloem unloading switches from a symplastic to an apoplastic route (Zhang et al. 2006).

11.4.2 - Starch storage

Fruit that store starch switch from starch synthesis during development to starch hydrolysis during ripening. Starch–sugar interconversion involves a larger number of enzymes and a greater complexity of control than is required for sugar storage alone. No transgenic plants have yet been reported in which a starch-storing fruit or lipid-storing fruit has been altered to store only sugars, or vice versa, but results with potato suggest it could be possible. In potato, control of both insoluble carbon storage and sugar to starch conversion has been attained using sense and antisense constructs to alter the expression of specific carbohydrate enzymes (Stitt and Sonnewald 1995).

Fig 11.11.png

Figure 11.11. Kiwifruit show some dramatic changes in physiological status during development. For 24 weeks after flowering, fruit are hard and sugar content is low. Meanwhile starch content (not shown) rises to about 12% of fresh mass. Ripening in fruit harvested around 24 weeks can be triggered by exposure to an external source of ethylene even though the fruit are not yet producing ethylene by themselves, and are incapable of an autocatalytic response. Within a week of such treatment starch becomes hydrolysed and sugar concentration rises from 7% to 15%. Fruit then soften rapidly and several cell wall-modifying enzymes increase in activity. Fruit do eventually show a peak in ethylene production, but not until ripening is well under way. (Figure based on data from J. Win)

Kiwifruit provide an example of biochemical changes in a starch-storing fruit (Figure 11.11). They are normally harvested with starch contents ranging from 4 to 10% (dry matter concentrations between 14 and 20%; SSC between 6.2 and 12%). At harvest, the main sugars are sucrose, glucose and fructose. As fruit ripen after harvest, sucrose content increases only slightly, while fructose and glucose increase in parallel to become the predominant sugars in ripe fruit. Labelling with radioactive precursors indicates that all three sugars are actively synthesised during ripening, and there are increases in the activities of a number of sucrose-metabolising enzymes, particularly SPS and invertase. The starch-degrading enzyme α-amylase increases two-fold.

The organic acid content of kiwifruit also changes after harvest. At room temperature malate decreases and citrate increases while quinate remains unchanged. However, during cool storage (0–4°C), malate increases. Such changes are enough to alter the flavour balance in the ripe fruit. Where fruit store carbon in an insoluble form, there are several potential control points for sugar metabolism (Figure 11.9) including:

  1. Hydrolysis of starch to glucose;
  2. Transfer of sugar precursors from starch-containing plastids (amyloplasts);
  3. Synthesis or degradation of sucrose;
  4. Synthesis of hexoses;
  5. Transfer of sugar to vacuoles or export from cells;
  6. Carbon flow between sucrose and malate or citrate;
  7. Production of CO2 from sugar or acid precursors;
  8. Transfer of malate or citrate across the vacuolar or mitochondrial membranes.

Interference in any of these processes should affect ripening and/or flavour development after harvest. Such interference may be physical (as in storage temperature), chemical (as in atmosphere composition) or genetic (by modifying activities of specific proteins which control flow between particular metabolites).