With a few exceptions, modern potato cultivars are all derived from one tuber bearing species of plants of the genus Solarium, i.e. Solatium tuberosum. Typically, the plant forms underground lateral shoots (stolons) with elongated internodes, hooked tips and characterised by diageotropic growth. Under specific environmental conditions e.g. short photoperiod, high light intensity, cool temperatures or low nitrogen levels, the stolons change their growth habit by dramatically reducing elongation and initiate radial growth (tuberisation, Figure 1).
The earliest changes associated with tuber formation are the cessation of cell divisions in the stolon apex and a marked increase in the mitotic index and starch and protein deposition in the sub-apical region of the stolon (3). During tuber expansion, cell division appears concentrated in the perimedullary region and shows a random orientation of the plane of cell division. At maturity, the starch-rich perimedullary region forms the major portion of the tuber.
1.1. The role of growth regulators
Tuberisation is stimulated by short days (long nights) although there is enormous genotyp-ic variation in the sensitivity to this requirement. The response pattern to tuberisation-induc-ing conditions is typical of a phytochrome response (4) and there is clear evidence that signals perceived in the leaves and transmitted to the stolons are involved in the control of tuberisation. Early work (5) showed that tuberisation could be induced in plants exposed to long pho-toperiod by grafting on leaves or stems from plants exposed to short days. More recently, Jackson et al. (6) produced transgenic plants of Solatium tuberosum spp. andigena (which naturally require day lengths of 12h or more to tuberise) with reduced levels of phytochrome B by antisense repression of the PHYB gene. The transgenic plants tuberise both under short and long days whilst wildtype plants only tuberise under long day conditions. Grafting experiments between the transgenic and wildtype plants indicated that phytochrome B-induced inhibition of tuberisation under short days is mediated through a transmissible stimulus (7). There is some evidence that the effects of phytochrome B on tuber formation are mediated by changes in the levels of, or sensitivity to, gibberellin (GA) in the stolon tip (discussed in 8). There is extensive literature on the dominant role played by GA in the tuberisation process. GA levels decline in leaves of potato plants exposed to short days (9 and references therein) and increase in various tissues under conditions that affect tuberisation such as low irradiance (10), high temperature (11) and continuous nitrate supply to plants grown in hydroponics (12). On the other hand, GA content markedly declines in the stolon tips at the very earliest stages of swelling (13). Indeed, it has been suggested that all the known effects of the environment and other hormones (e.g. abscisic acid [ABA]) on the tuberisation process modulate GA levels or antagonize the effects of GA, (8). Exogenous applications of GA inhibit tuberisation but promote stolon elongation (14). Exogenous GA also promotes shoot growth, thus mimicking the effect of long days (11), and reduces the allocation of assimilates to tubers (15).
The role of sucrose in the tuberisation process has been recently studied in detail in single-node cuttings of induced potato plants cultured in vitro (13). Under suitable conditions, (i.e. low nitrogen status, darkness), the axillary buds in the cuttings develop stolon-like shoots which eventually tuberise. A requirement for tuber formation is the inclusion of at least 2% sucrose in the medium. Above this concentration, tuberisation increases in a sucrose-concentration-dependent manner. Sucrose appears to influence specifically the GA concentration in the stolon tips, which dramatically declines upon tuberisation (ibid.). Inclusion of GA in the culture medium completely prevents tuberisation even at the highest sucrose concentrations used. Under specific conditions, tuberisation of the axillary buds in the in vitro system can be highly synchronous enabling a study of differential gene expression during potato tuber development (16). Tuberisation coincides with the up-regulation of the expression of several genes involved in carbohydrate metabolism including sucrose synthase (SuSy), ADPglucose pyrophosphorylase (AGPase), the major granule bound starch synthase (GBSSI) and branching enzyme (BE) (17). Sucrose is known to induce the expression of these genes and of others (i.e. soluble starch synthases (SSII and SSIII) involved in starch biosynthesis (18) as well as patatin (19) and this effect appears to be modulated by GA, at least in some cases (20). Exogenous GA inhibits starch synthesis (21) and the production of patatin (22) by potato tubers.
2. METABOLIC CHANGES DURING TUBER FORMATION 2.1. Carbohydrates
Under normal circumstances, massive accumulation of storage polymers, namely starch and protein (class I patatin) coincides with the cell proliferation and morphogenesis characteristic of tuber formation.
Storage product accumulation and morphogenesis are independently regulated processes. For example, transgenic plants with reduced AGPase activity produce tubers which accumulate sucrose instead of starch and have a drastically reduced storage protein content (23). On the other hand potato cuttings from which the axillary buds have been removed accumulate large quantities of starch and patatin in the petiole irrespectively of the photoperiod, which determines whether the bud develops into a stem or a tuber (20). There are reports of increased photosynthetic rates and translocation of assimilates at tuber initiation and it is conceivable that increases in sucrose levels in the stolon tip are conducive to up-regulation of genes involved in storage polymer biosynthesis. Gifford and Moorby (24) found that net photosynthetic assimilation rate doubled after initiation of tubers. Similarly, Dwelle et al. (25) reported an increase of 50% of gross photosynthesis after tuber initiation. Ewing (9) lists decreased stem growth, increased net photosynthetic rate and increased export of assimilate from the leaf as early changes associated with exposure of potato plants to short days. Burton (26) reported a 4.6-fold increase in sucrose levels in stolon tips of cv Record from 5-11 days prior to tuberisation to 1 day after tuber induction. Ross et al. (27) also reported higher (2-fold) sucrose levels in swelling stolon tips compared with non-swelling tips.
Figure 2. Soluble sugar content (a) and sucrose/hexose ratio (b) at different stages of tuber development (see fig 1). The mean FW of the stolon apex (stage 1) and developing tubers (2-6) were 0.012g, 0.21g, 0.5 lg, 1.5g, 3.17g and 5.09g (adapted from ref. 27)
The relative concentration of soluble sugars in stolon tips also markedly changes upon tuber initiation (Figure 2). A large increase in the sucrose/reducing sugars ratio during the early stages of tuber development was first noted by Appleman and Miller (28) and later reported by many others (27, 29, 30). In addition, Davies (30) and, later, Ross et al. (27) found a marked decline in the fructose pool and an increase in the glucose/fructose ratio in stolon tips following tuber initiation. A glucose:fructose ratio of around 2 was observed in the sub-apical region of non-tuberising stolon, increasing to around 10 during the early stages of tuberisation (ibid.). Glucose:fructose ratios as high as 100 have been recorded in tuberising stolon tips (31).
Davies and Oparka (32) attributed the rapid decline of fructose during the tuberisation of stolon to the induction of a hexose kinase specific for fructose (fructokinase). This was later confirmed by Ross et al. (27) who carried out a comparative analysis of enzymes involved in carbohydrate metabolism in non-tuberising or tuberising stolon tips. These authors also found that there was no difference in the activity of hexose kinase using glucose as substrate. In addition, tuberising stolons contained a 20-fold higher sucrose synthase (SuSy) activity compared with non-tuberising stolons whilst the opposite was observed with regard to acid and alkaline invertase activities. Broadly similar changes were observed by Appeldoorn et al. (33) in axillary buds of single node potato cutting cultured in vitro under tuberising or non-tuberising conditions. These authors also reported a sharp decline in cell wall-bound acid invertase during tuber formation. Overall, these data are consistent with a switch from an invertase-driven pathway of sucrose degradation in elongating stolons to a SuSy-mediated pathway in tuberising stolons.
Although SuSy activity increases during tuber formation, the enzyme has a lower affinity for sucrose (Km 130 mM; 34) than invertase (Km 16 mM; 35) and this could explain the accumulation of sucrose in tuberising tips. Sucrose accumulation would serve the purpose of up-regulating the expression of genes involved in storage metabolism. The marked decline in the fructose pool observed during tuberisation reflects the thermodynamical requirement for a sucrolytic flux mediated via SuSy given that this enzyme catalyses a reaction near equilibrium in vivo (36). This conclusion is supported by the finding that fructokinase (FK) activity correlates very well with that of SuSy during tuber development (27, 31).
In plants, the bulk of assimilates transport occurs primarily as mass flow driven by hydrostatic pressure differential between the phloem loading sites in the leaves and the ends of the phloem conduits in the sink regions. Transfer of assimilates from the sieve elements companion cell (SECC) complexes to recipient cells can occur via the symplast (i.e. through plas-modesmata connections) or via unloading into the apoplast followed by active uptake of individual components by the recipient cells. Sucrose may be taken up directly from the apoplast or following hydrolysis to hexoses by invertases present in the cell walls. The pathway of sucrose unloading in elongating stolons is not known. One interesting possibility is that the changes observed during the stolon-tuber transition may be attributable to a switch from apoplastic unloading route of assimilates to a symplastic one. This may be brought about by increases in plasmodesmatal frequency or functioning in the subapical region of the stolon at tuber initiation. The increased symplastic delivery of sucrose at these sites would result in increased sucrose concentrations in selective regions of the stolon. Circumstantial evidence in favour of this hypothesis is available. For example, stolons contain cell wall-bound invertase (33) which is absent from tubers (37). Moreover, there is no evidence of symplastic unloading of phloem tracers (e.g. carboxyfluoroscein) in the subapical region in elongating stolons (R. Viola, A.G. Roberts, S. Haupt and K.J. Oparka unpublished observations). On the other hand, abundant plasmodesmata connections exist between the sieve element-companion cell complexes (SECC) and the surrounding parenchyma in developing tubers (38). There is also strong, albeit indirect, evidence that, in common with many other storage sinks, the bulk of assimilates in the potato tuber is unloaded through the symplast (39).
Figure 3. Schematic representation of the pathway of sucrose unloading in storage parenchyma cells of developing potato tubers. Note that unloading in the cell occurs via plasmodesmata and the turgor-sensitive sucrose transport system (reference to the circle on the plasma membrane) on the plasma membrane functions as a retrieval mechanism for sucrose leaking into the apoplast (adapted from ref. 40).
Storage parenchyma cells of developing tubers also possess the capacity to take up exogenous sugars. The sucrose uptake mechanism is sensitive to p-chloromercuribenzene sulphonic acid (PCMBS), an inhibitor specific for sucrose transporters (40), and to the turgor pressure of the storage cells (41). These observations are rather puzzling given that sucrose is supposed to be moving symplastically between the cells. One possibility is that the plasma membrane transport system functions not to take up sucrose which is moving apoplastically, but rather to retrieve sucrose escaping from the symplast (Figure 3).
A similar mechanism of solute retrieval from the apoplast is commonly found in leaves (42) and may also be a general feature of parenchyma cells in storage sinks (43). The sucrose retrieval mechanism of the tubers storage parenchyma appears to be activated under conditions of low turgor which also decreases passive leakage of sucrose into the apoplast. This explains the apparent turgor sensitivity of sucrose uptake given also that leakage of sucrose and other solutes is enhanced under high turgor (44). The retrieval function for the sucrose transporter in growing tubers is supported by studies at the molecular level. A sucrose transporter gene encoding a highly hydrophobic protein of about 47 kDa was cloned from potato (SUTl) and RNA in situ hybridization studies localized its expression in the phloem in leaves (45). The expression profile of SUTl follows the sink-to-source transition in leaves and very low expression is found in developing potato tubers. Constitutive antisense repression of SUTl in transgenic plants induces a dramatic reduction in root development and tuber yield, consistent with the involvement of SUTl in apoplastic phloem loading of sucrose in potato leaves (46). On the other hand, tuber-specific antisense repression of SUTl has very little impact on tuber yield in the absence of a leaf phenotype (1) confirming that this carrier does not play a major role in sucrose import into the tuber cells. The main function of this carrier is likely to be the retrieval of sucrose along the translocation pathway (ibid.). The proportion of the tuber sucrose located in the apoplast appears significant. Targeted expression of yeast invertase to the cell walls of potato tubers results in a 90% decrease in the tuber sucrose content and up to 18-fold increase in glucose levels compared with wildtype tubers (47). The transgenic plants produce fewer but larger tubers compared with the wildtype and show an 18-30% increase in tuber yield (fresh weight), but no change in the total yield of tuber dry matter. These results clearly indicate that the bulk of sucrose unloading in potato tubers does not involve an apoplastic step.
There is no simple explanation as to why the transgenic plants should produce fewer and larger tubers. One possibility relates to the functionality of plasmodesmata which link the parenchyma cells to the SECC. It is now accepted that plasmodesmata are not static pores in the walls but controllable "valves" opening and closing in response to a number of internal stimuli (39), including pressure differential between cells (48). It is feasible that the water potential in the apoplast of transgenic tubers is decreased as a result of hexose accumulation in this compartment leading to a decreased turgor of sink cells. This would decrease the pressure differential between these cells and the SECC in the transgenic tubers and increase assimilate unloading. This hypothesis has been used by Sonnewald et al. (47) to explain the opposite effect on sink strength induced by the targeting of yeast invertase to the cell wall or to the cell cytosol of potato tubers. Cytoplasmic expression of yeast invertase induces accumulation of hexoses within the tuber cells leading to increased cell turgor and decreased water potential of the symplast. Glucose accumulation in the apoplast of transgenic tubers may act as a signal for cell division, leading to the production of larger tubers. There is evidence that changes in glucose levels affect the cell cycle in yeast cells (49, 50) and gene expression, metabolic activity and development in plants (51). For example, seed size differences between genotypes of Viciafaba have been attributed to different mitotic indexes which well correlate with the duration of apoplastic invertase activity and the hexose content (52). Appeldoorn et al. (33) proposed that metabolic signaling of glucose influx from the apoplast is responsible for the regulation of hexose kinase (glucose substrate) and invertase activity in elongating potato stolons. Tauberger et al. (53) observed high levels of glucose in swelling stolon tips and proposed that a defined level of glucose may be required for the initiation of tuber formation. In this context it is relevant to note that glucose can repress both GA synthesis and GA signaling in barley embryos (54) and GA is a known inhibitor of tuber formation and growth (3, 8, 9).
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