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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Water stress stimulates sucrose synthesis and inhibits starch synthesis in wild-type tubers. Antisense and co-suppression potato transformants with decreased expression of sucrose–phosphate synthase (SPS) have been used to analyse the importance of SPS for the regulation of this water-stress induced change in partitioning. (i) In the absence of water stress, a 70–80% decrease in SPS activity led to a 30–50% inhibition of sucrose synthesis and a slight (10–20%) increase of starch synthesis in tuber discs in short-term labelling experiments with low concentrations of labelled glucose. Similar changes were seen in short-term labelling experiments with intact tubers attached to well-watered plants. Provided plants were grown with ample light and water, transformant tubers had a slightly lower water and sucrose content and a similar or even marginally higher starch content than wild-type tubers. (ii) When wild-type tuber slices were incubated with labelled glucose in the presence of mannitol to generate a moderate water deficit (between –0.12 and –0.72 MPa), there was a marked stimulation of sucrose synthesis and inhibition of starch synthesis. A similar stimulation was seen in labelling experiments with wild-type tubers that were attached to water-stressed wild-type plants. These changes were almost completely suppressed in transformants with a 70–80% reduction of SPS activity. (iii) Decreased irrigation led to an increase in the fraction of the dry-matter allocated to tubers in wild-type plants. This shift in allocation was prevented in transformants with reduced expression of SPS. (iv) The results show that operation of SPS and the sucrose cycle in growing potato tubers may lead to a marginal decrease in starch accumulation in non-stressed plants. However, SPS becomes a crucial factor in water-stressed plants because it is required for adaptive changes in tuber metabolism and whole plant allocation.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Sucrose–phosphate synthase (SPS) is proposed to be a key enzyme in the regulation of sucrose synthesis (Huber & Huber 1992a; Huber & Huber 1996; Huber et al. 1992; Stitt et al. 1987). In leaves, changes in SPS activity often correlate with changes in the rate of sucrose synthesis and export (Huber & Israel 1982; Rocher et al. 1989; Stitt et al. 1988), and SPS can be regulated by a hierarchy of several interacting mechanisms, including (i) regulation of gene expression (Huber & Huber 1996); (ii) covalent modification via reversible phosphorylation (Huber & Huber 1996); and (iii) allosteric regulation via metabolites (Doehlert & Huber 1984). Two phosphorylation sites are known for spinach-leaf SPS: phosphorylation of Ser164 is involved in light activation (Huber & Huber 1992b), whereas Ser424 in the water-stress activation of SPS (Toroser & Huber 1997). These properties allow SPS to regulate sucrose synthesis and allocation between sucrose and starch in response to light/dark signals and changes in the sucrose demand. The importance of SPS for the regulation of sucrose biosynthesis in leaves has been confirmed by studies with transgenic tomato plants overexpressing a maize SPS gene (Galtier et al. 1993; Worrell et al. 1991).

SPS is also present in growing tissues, including potato tubers (Geigenberger & Stitt 1993; Geigenberger et al. 1997), sugar cane stems (Zhu et al. 1997), heterotrophic Ricinus cotyledons (Geigenberger & Stitt 1991), kiwi fruits (MacRae et al. 1992) and ripening bananas (Hubbard et al. 1990). Two possible roles for SPS in tissues that import and degrade sucrose have been proposed. (i) SPS could allow re-synthesis of sucrose after import via apoplastic cleavage. However, potato tubers contain high SPS activity, even though sucrose is imported sym-plastically (Oparka et al. 1992). (ii) SPS could be involved in a regulatory cycle in which sucrose is simultaneously degraded and re-synthesised. This cycle has been shown to operate in potato tubers (Geigenberger & Stitt 1991; Geigenberger & Stitt 1993; Geigenberger et al. 1997) and other plant tissues (Hubbard et al. 1990; MacRae et al. 1992; Zhu et al. 1997), and could facilitate sensitive regulation of sucrose mobilisation in response to changes in the supply of, and the demand for, sucrose (Geigenberger et al. 1995).

Geigenberger et al. (1997) proposed that this sucrose cycle also modulates starch–sucrose interconversions during water stress. It is well established that water stress leads to decreased starch synthesis and increased sucrose synthesis in leaves (Fox & Geiger 1985; Moorby et al. 1975; Morgan 1984; Munns & Weir 1981; Quick et al. 1989; Steward 1971; Zrenner & Stitt 1991). Conversion of sucrose to starch represents a potentially important site at which sinks too could regulate their internal osmotic potential. During symplasmic import, sucrose will be entering the parenchyma cells of potato tubers at a local concentration of about 500 mm (Schurr 1991; Weiner et al. 1991). About half is converted to starch (Burton 1986; Geigenberger & Stitt 1993; Oparka & Wright 1988a; Oparka & Wright 1988b). Water stress has been shown to inhibit starch synthesis (Hnilo & Okita 1989; Oparka & Wright 1988a; Oparka & Wright 1988b; Oparka et al. 1990) and stimulate re-synthesis of sucrose (Geigenberger et al. 1997) in tissue slices from growing tubers. Based on investigations of the effect of water stress on SPS activity, metabolite levels and fluxes in tuber slices, Geigenberger et al. (1997) proposed that potato tuber SPS is activated by water stress, leading to an increased rate of sucrose synthesis. The resulting decrease of hexose phosphates and especially glycerate-3-phosphate (3PGA) then inhibits ADP-glucose pyrophosphorylase, leading to a decrease of ADPglucose and an inhibition of starch synthesis.

The present study tests these ideas about the role of SPS in growing potato tubers, using potato transformants with decreased expression of SPS. We investigate whether the decreased expression of SPS (i) has a large impact on partitioning between sucrose and starch in non-stressed tubers; (ii) prevents the water-stress induced stimulation of sucrose synthesis in growing tubers; and (iii) affects water-stress induced changes in carbon partitioning at the whole-plant level.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Generation of plants with decreased expression of SPS

Line-5 transformants were produced using a construct in which the entire open reading frame of SPS-P4 was linked in antisense configuration to the 35S CaMV promoter, and line 1 transformants were produced by linking a 2.0 kb fragment of SPS-P1 (including a 957 bp 5′-non translated region and 45% of the coding sequence) in sense configuration to the 35S CaMV promoter. SPS transcript level and SPS activity were decreased in the antisense and co-suppression lines (Krause et al. 1998). Potato plants contain several forms of SPS differing in their apparent molecular weight (Reimholz et al. 1997). The transformants contained smaller amounts of two closely related forms with apparent molecular weights of 125 (SPS-1a) and 127 kDa (SPS-1b), whereas the minor SPS form with a molecular weight of 135 kDa (SPS-2) was not reduced (Reimholz et al. 1997). Similar changes in SPS transcript levels, SPS protein and SPS activity were seen in the leaves of the plants used in the present study (data not shown).

SPS expression in growing tubers

The growing tubers of transformants contained decreased amounts of SPS transcript (Fig. 1a) and decreased SPS protein (Fig. 1b). To allow identification of the SPS forms, a sample from cold-stored tubers was included. Growing wild-type tubers contained mainly SPS-1a, with a small amount of SPS-2, whereas SPS-1b was not detectable. The band corresponding to SPS-1a was strongly decreased in the antisense lines 5–15 and 5–7, and very low in lines 1–74 and 5–59. As in leaves (see above), SPS-2 was less strongly affected. To assess the effect on overall SPS activity, an assay was used that contained saturating concentrations of hexose phosphates (Fig. 1c). Compared to wild-type tubers, overall SPS activity is decreased to 44%, 38%, 20% and 14% of the wild-type activity in the transformant lines 5–15, 5–7, 1–74 and 5–59, respectively. ADP glucose pyrophosphorylase (AGPase) and sucrose synthase activities were similar in wild-type and transformant tubers (data not shown).

image

Figure 1. SPS expression in tubers.

(a) Steady-state Sps transcript. Twenty micrograms of total RNA were loaded per lane.

(b) SPS protein detected by immunoblotting. Top arrow indicates SPS-2; second arrow, SPS-1b; third arrow, SPS-1a. Bottom arrow on left side, molecular weight marker, 116 kDa.

(c) Activity under optimal assay conditions: Vmax assay (12 mm Fru6P, 36 mm Glc6P and 6 mm UDPGlc).

(d) Activity under limiting assay conditions: Vsel assay (2 mm Fru6P, 6 mm Glc6P, 6 mm UDPGlc and 5 mm Pi).

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SPS activity was also measured in an assay that contained limiting hexose phosphate concentrations and phosphate (Vsel; Fig. 1d). In these assay conditions, the reduction of SPS activity in the transformants was much smaller. A similar effect was found in the leaves (data not shown). This weak inhibition of SPS activity in the transformants in the Vsel assay could be explained by post-translational activation of the remaining enzyme leading to changes in the kinetic properties. Alternatively, it could be explained if SPS-2, which represents a large portion of the residual SPS protein in the transformants (Fig. 1b), has a higher affinity for hexose phosphates or a lower sensitivity to inhibition by phosphate. To distinguish between these possibilities, tuber slices from line 5–59 were incubated with the protein phosphatase inhibitors 5 nmol g–1 FW calyculin or 19 nmol g–1 FW okadaic acid for 3 h, and then assayed for SPS activity in the presence of saturating hexose phosphates (Vmax assay) or the presence of limiting hexose phosphates plus phosphate (Vsel assay). The inhibitor concentrations were high enough to reverse the activation of potato tuber SPS by mannose or high mannitol in wild-type tubers (Reimholz et al. 1994; Reimholz et al. 1997). The ratio of Vsel activity to Vmax activity was higher in 5–59 than in the wild-type tubers in discs prepared directly from the tubers (0.20 ± 0.034 compared to 0.10 ± 0.004, mean ± SE, n = 3, original data not shown) and remained high at the end of a 3 h incubation, irrespective of whether the discs were incubated in the absence of protein phosphatase inhibitors (0.28 ± 0.03) or with okadaic acid (0.28 ± 0.04) or calyculin (0.24 ± 0.03). These results indicate that the high ratio found in the transformant is not primarily due to protein phosphorylation.

Metabolism in water-stressed tuber discs

Discs from wild-type tubers and four transformant lines (5–15, 5–7, 1–74 and 5–59) were incubated for 2 h with 2 mm14C glucose and 10, 100 or 300 mm mannitol, corresponding to an external water deficit of –0.02, –0.24 and –0.72 MPa, respectively. SPS activity responded to water stress in wild-type material as described previously (Geigenberger et al. 1997). Maximum SPS activity (measured in the assay that contained saturating hexose phosphates; Vmax) remained unaltered (Fig. 2a). SPS activity (measured in the assay that contained limiting hexose phosphates and phosphate, Vsel) (Fig. 2b) and the ratio of Vsel activity/Vmax activity (not shown) increased twofold as the mannitol concentration increased. The increase of Vsel in water-stressed wild-type tubers is prevented by protein phosphatase inhibitors (Geigenberger et al. 1997).

image

Figure 2. Metabolism of 2 mm[14C]glucose in potato slices of plants with reduced SPS incubated at 10, 100 and 300 mm mannitol.

After 90 min pre-incubation, [14C]glucose was added for 2 h until slices were harvested to determine radioactivity in sucrose and starch.

(a,b) SPS activity measured in the slices after 3 h incubation, (a) Vmax activity, (b) Vsel activity. (c) Uptake of [14C]glucose. (d,e) Distribution of label in sucrose (d) and starch (e) as a percentage of uptake. (f) Relation of label between sucrose and starch. Data are means ± SE, n = 3–4.

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Maximum SPS activity was inhibited by 35–40%, 40–55%, 63–72% and 67–78% in 5–15, 5–7, 1–74 and 5–59, respectively. The magnitude of the inhibition was not modified by the mannitol treatment (Fig. 2a). Increasing mannitol led to an increase of the Vsel activity in 5–15 and 5–7, but not in 1–74 and 5–59 (Fig. 2b). In the latter two lines, the Vsel/Vmax ratio was already between 0.3 and 0.4 in 10 mm mannitol (data not shown) and did not increase further at high mannitol.

Glucose uptake was stimulated at high mannitol (Fig. 2c) (see also Geigenberger et al. 1997). Discs from wild-type tubers and the transformants showed similar rates of [14C]glucose uptake (Fig. 2c). In the following figure panels, the labelling of the different fractions is given as a percentage of the absorbed label. In wild-type material, label incorporation into sucrose increased (Fig. 2d) and label incorporation into starch decreased (Fig. 2e) as the mannitol concentration was increased (see also Geigenberger et al. 1997). This response was modified in the discs from transformants. When discs in 10 mm mannitol are compared, decreased expression of SPS inhibited label incorporation into sucrose (Fig. 2d). However, only a small portion of the label was converted to sucrose in non-stressed discs, and the inhibition of sucrose synthesis was not accompanied by a significant increase of label incorporation into starch (Fig. 2e). When discs in 100 mm or 300 mm mannitol are compared, the transformants contained less label in sucrose (Fig. 2d), and more label in starch (Fig. 2e) than wild-type discs. This change was seen in all four transformant lines at 100 mm mannitol, but only in transformant lines 1–74 and 5–59 with the largest decrease in expression of SPS at 300 mm mannitol. The increase in partitioning of label to sucrose that is typical for water-stressed wild-type material (see Geigenberger et al. 1997) was partially inhibited in line 1–74 and almost totally prevented in line 5–59 (Fig. 2d). The accompanying decrease in partitioning of label to starch was almost totally suppressed in 1–74 and 5–59 (Fig. 2e). The water stress-induced increase of the sucrose-to-starch labelling ratio was partly or completely suppressed in 1–74 and 5–59 (Fig. 2f).

Metabolism was investigated at a wider range of mannitol concentrations in discs from wild-type tubers and the antisense line 5–59 (Fig. 3). SPS activity was decreased by 75% in this batch of 5–59 tubers, compared to wild-type tubers (data not shown). Tuber slices were incubated in 2 mm[14C]glucose with 0, 50, 150, 250 or 500 mm mannitol, corresponding to an external water deficit of 0, –0.12, –0.36, –0.60 and –1,2 MPa, respectively. Discs incubated on buffer gained marginally in fresh weight (see also Geigenberger et al. 1997), discs incubated on 150–250 mm mannitol lost 6–10% of their fresh weight, and discs incubated on 500 mm mannitol were under severe stress and lost 34% of their initial fresh weight (Fig. 3a). Similar changes in disc weight were found for wild-type and transformant material.

image

Figure 3. Metabolism of 2 mm[14C]glucose in potato slices of plants with reduced SPS incubated at 0, 50, 150, 250 and 500 mm mannitol as in Fig. 2 

(a) Fresh weight per disc. (b) Uptake of [14C]glucose. (c) Label remaining in glucose as percentage of uptake. (d,e) Incorporation of label into (d) sucrose and (e) starch. (f) Relation of label between sucrose and starch. Data are means ± SE, n = 3–4.

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Discs from wild-type and 5–59 tubers had similar rates of glucose uptake and metabolism (Fig. 3b,c). As the mannitol concentration was increased, wild-type discs incorporated more label into sucrose and less label into starch (Fig. 3d,e). This response was altered in 5–59. At mannitol concentrations up to 250 mm (corresponding to –0.6 MPa), the stimulation of sucrose synthesis was almost completely suppressed in 5–59 (Fig. 3d), and the inhibition of starch synthesis (Fig. 3e) was much weaker than in wild-type discs. Whereas the sucrose-to-starch labelling ratio increased dramatically in the wild-type, it remained at a low value in the transformant (Fig. 3f). More severe water deficits (500 mm mannitol, corresponding to –1.2 MPa) led to a stimulation of sucrose synthesis and inhibition of starch synthesis, although the changes were less marked than in wild-type discs.

Metabolism in intact non-stressed growing tubers attached to the plant

Growing tubers of well-watered plants were harvested at the start of the photoperiod and analysed for metabolite levels (Table 1). A similar sucrose level was found in tubers from wild-type plants and the transformants. Decreased expression of SPS was accompanied by a marked and significant increase of UDPGlc, a slight increase of Glc6P and Fru6P, and a slight and non-significant increase of 3PGA especially in lines 1–74 and 5–59 where SPS expression was most strongly inhibited.

Table 1.  Metabolite levels in intact growing tubers of potato plants with reduced expression of SPS, harvested at the start of the photoperiod
MetaboliteWild-type5–151–745–59
  1. Results are means ± SE, n = 5–6 separate tubers from different plants. Data are given as nmol gFW–1, except sucrose which is given as μmol gFW–1.

Sucrose13.8 ± 4.815.3 ± 0.810.7 ± 2.914.1 ± 4.0
UDPGlc35.8 ± 2.597.0 ± 17.459.0 ± 18.883.1 ± 20
Glc-1-P6.0 ± 1.45.6 ± 1.16.7 ± 1.38.0 ± 1
Glc-6-P90.3 ± 7.388.1 ± 27.8107.6 ± 9.4114.7 ± 13.1
Fru-6-P19.0 ± 2.217.5 ± 2.620.7 ± 0.723.0 ± 2.4
3-PGA51.6 ± 2.651.3 ± 5.757.1 ± 2.365.5 ± 2.0

Fluxes were investigated by injecting carrier-free [14C]glucose into intact tubers that were attached to the plant. This experiment was carried out with line 5–59. SPS activity was decreased by 70% in 5–59 (445 ± 5 nmol gFW–1 min–1 in wild-type tubers, 135 ± 10 nmol gFW –1 min–1 in transformant tubers, original data not shown). The experiment commenced 4 h into the photoperiod. Wild-type and 5–59 tubers metabolised the 14C glucose at the same rate (data not shown). Tubers from 5 to 59 showed a small significant decrease in the labelling of sucrose and a slight but non-significant increase in the labelling of starch (original data not shown). The ratio of label in sucrose to label in starch showed a small but significant decrease from 0.19 ± 0.005 in the wild-type tubers to 0.12 ± 0.021 in the transformant tubers (significant, P < 0.05).

Starch content was investigated in five harvests from independently grown batches of well-watered plants (Table 2). No significant differences in the starch content were observed between the genotypes when the plants were grown in the winter season in a greenhouse in Heidelberg under low light conditions (Table 2). When grown under higher irradiance in a growth chamber, there was a trend to a slightly higher tuber starch content in the transformants compared to wild-type tubers, but the increase was only significant in one experiment with 5–59.

Table 2.  Starch content in developing tubers from 8- to 10-week-old plants with reduced expression of SPS
Growth conditionsReplicatesWild-type5–155–71–745–59
  • Results are shown for five different trials and are the mean ± SE (n is indicated in the table). Data are expressed as μmol glucose-units gFW–1.

  • *

    Not analysed.

Greenhouse, Januaryn = 10–14346 ± 18375 ± 25– *288 ± 10326 ± 16
Greenhouse, Novembern = 10–12387 ± 44322 ± 29367 ± 31326 ± 31387 ± 12
Growth chamber, 12 h light, 250μEn = 6–8463 ± 32497 ± 43567 ± 29519 ± 18521 ± 29
Growth chamber, 14 h light, 250μEn = 8–10476 ± 34592 ± 41– 553 ± 37552 ± 34
Growth chamber, 14 h light, 350 μEn = 15618 ± 20– – 673 ± 65824 ± 58

Metabolism in intact water-stressed growing tubers on the plant

Wild-type potato plants and two transformants with a strong reduction of SPS (1–74 and 5–59) were grown in a growth cabinet under 350 μmol photons m–2 s–1 irradiance with daily watering for 6 weeks, by which time tubers had been initiated and then divided into three groups. One group was watered daily until harvest, 17 days later. A second group was not watered during the first 12 days, by which time the relative water content of the soil had decreased to 14% and the leaves were visibly wilted. For the next 5 days, the pots were weighed and the daily water loss substituted by rewatering, to keep the water content of the soil at a low but constant level. During this period, the leaves remained wilted. Water stress will decrease the rate of photosynthesis and therefore, as a control, a third group of wild-type plants were transferred to low light (20 μmol photons m–2 s–1) for the last 17 days.

Water stress led to a 38% increase of Vmax SPS activity in wild-type tubers (Fig. 4a). There was an even larger increase of SPS activity when it was assayed with limiting hexose phosphates and phosphate (Fig. 4b), and the ratio of Vsel to Vmax activity increased (not shown) as in water-stressed discs (see above). This increase of SPS activity and of the Vsel to Vmax ratio did not occur in low light. The transformants showed a strong reduction of SPS activity that was retained under water stress (Fig. 4a).

image

Figure 4. Metabolism of high specific activity [14C]glucose injected into intact tubers attached to non-stressed or water-stressed potato plants.

Wild-type potato plants and two lines of transformants were grown in a growth cabinet under 350 μE m–2 s–1 irradiance with daily watering for 6 weeks, at which point tubers had already been initiated. The plants were then divided into three groups. One group was watered daily through to harvest 17 days later. A second group was not watered during the first 12 days. After this time interval leaves were visibly wilted and the relative water content of the soil was decreased down to 14%. For the last 5 days, pots were weighted and the daily water loss substituted by rewatering to keep the water content of the soil at a low but constant level. During this time, leaves remained in a wilted condition. A third group of wild-type plants were transferred to low light (20 μE m–2 s–1) and harvested 17 days later.

(a,b) SPS activity in the tubers, (a) Vmax activity, (b) Vsel activity. (c–f) Metabolism of carrier-free [14C]glucose injected into intact tubers attached to the plants: (c) label metabolised as percentage of total injected, (d–f) label incorporation into (d) sucrose, (e) starch, (f) glycolytic intermediates (anions+cations), as percentage of label metabolised. Data are the mean ± SE, n = 4 separate tubers from four different plants.

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To investigate fluxes, high specific activity [14C]glucose was injected into intact tubers attached to the plants (Fig. 4c–f). In daily watered plants, the transformants showed a decrease in labelling of sucrose (Fig. 4d), a slight increase in labelling of starch (Fig. 4e), and the ratio of label in sucrose:label in starch decreased from 0.08 ± 0.01 in the wild-type to 0.042 ± 0.007 and 0.034 ± 0.011 in 1–74 and 5–59, respectively (mean ± SE, n = 4). Water stress led to a threefold stimulation of sucrose synthesis in wild-type plants, but did not stimulate sucrose synthesis in the transformants. Water stress led to a 33% inhibition of label incorporation into starch in wild-type tubers, but had a much weaker inhibitory effect in 1–74 and 5–59 (20% and 10% inhibition of starch synthesis, respectively). During water stress, the sucrose:starch labelling ratio increased approximately fivefold to 0.456 ± 0.228 in wild-type tubers, whereas in 1–74 and 5–59 it remained very low (0.043 ± 0.029 and 0.039 ± 0.006, respectively). During water stress, a larger proportion of the label was directed into glycolysis leading to increased labelling of ionic components (mainly phosphate esters, organic acids and amino acids; see Fig. 4f).

In tubers of wild-type plants transferred to low light, label in starch decreased and labelling of glycolytic products and intermediates markedly increased but, in contrast to water-stressed wild-type tubers, no significant increase in the labelling of sucrose was observed and the sucrose-to-starch labelling ratio remained low (0.148 ± 0.025, n = 4).

Effect of water stress on biomass allocation in whole potato plants

Sucrose levels remained high in tubers growing on water-stressed wild-type plants, whereas they decreased by 75% in low light (Fig. 5a). Tubers of the transformants contained marginally less sucrose than wild-type tubers, and this trend became more marked in water-stressed transformants (Fig. 5a). Tubers of the transformants had a significantly lower water content compared to the wild-type, and this trend was also sustained under water stress (Fig. 5b). Water stress led to a slight but non-significant increase of the starch content on a dry-weight basis in wild-type and transformants tubers (Fig. 5f).

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Figure 5. Effect of water-stress on biomass allocation in whole potato plants.

Plants used in the same experiment as in Fig. 4 were harvested to determine: (a) the sucrose content of the tubers, (b) the water content of the tubers, (c) shoot dry-weight, (d) tuber dry-weight, (e) the ratio between shoot and tuber dry-weights, and (f) the starch content of the tubers on a dry-weight basis. Data for sucrose, starch and water content of the tubers are the mean ± SE, n = 12 separate tubers from four different plants. Data for biomass allocation are the mean ± SE, n = 4 different plants.

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Changes in whole plant allocation were also investigated. In wild-type plants, water stress led to a larger reduction of shoot dry weight (50%) (Fig. 5c) than of tuber dry weight (29%) (Fig. 5d). As a result, the ratio of tuber dry weight to shoot dry weight rose in wild-type plants under water stress (Fig. 5e). This increase of the tuber-to-shoot ratio was not seen in wild-type plants in low light (Fig. 5e), even though there was a similar overall decrease of plant size (Fig. 5c,d). Well-watered transformants had a slightly higher tuber to shoot ratio than wild-type plants (Fig. 5e). However, water stress led to a larger inhibition of tuber growth in the transformants (43–48%) than in wild-type plants (30%) (Fig. 5d). The tuber:shoot ratio of the transformants therefore remained unaltered or decreased slightly in response to water stress (Fig. 5e), rather than increasing as in wild-type plants.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Reduction of SPS has no major impact on metabolism in non-stressed tubers

Changes in SPS expression led to adjustments in individual pools and fluxes, but SPS does not represent a major site for the control of the net rate of starch accumulation in non-stressed tubers. In labelling experiments with tuber discs (Figs 2 and 3) and intact tubers (Fig. 4), a 70–80% decrease in SPS expression resulted in a 50% inhibition of sucrose synthesis, demonstrating that SPS is active in vivo and does contribute to the re-synthesis of sucrose. However, the resulting stimulation of starch synthesis was small because sucrose synthesis only accounts for a small proportion of the total carbon fluxes in non-stressed tubers. Analyses of harvested tubers from well-watered plants (Table 2 and Fig. 5a) yielded a similar picture. Although decreased expression of SPS led to a slight decrease of the sucrose (Table 1, Fig. 5a) and water (Fig. 5b) content of the tubers, and a slight increase of tuber dry-matter (Fig. 5d) and the starch content (Table 2), the changes were small and were not seen in low irradiance (Table 2). Total tuber yield was slightly increased because tuber weight accounted for a slightly larger proportion of the overall plant weight (Fig. 5).

Decreased expression of SPS impairs the water-stress induced changes in partitioning in growing tubers

Starch accumulation occurs simultaneously with tuber growth (Reeve et al. 1973; Schneiders et al. 1988) and must be co-ordinated with the synthesis of other cell components and the maintenance of a suitable turgor for cell growth and expansion (see Patrick 1990). Since growing tubers are attached to the rest of the plant via the xylem, their water content can fluctuate by up to 10–20% every day (Baker & Moorby 1969; Plodowska et el. 1989; Schneiders et al. 1988). Based on investigations of the effect of water stress on SPS activity, metabolite levels and fluxes in tuber slices, Geigenberger et al. (1997) proposed that moderate water stress (up to –0.72 MPa) activates SPS and stimulates sucrose synthesis, and that the resulting decline in metabolite levels in turn restricts ADP-glucose pyrophosphorylase activity. A direct effect on starch synthesis was not evident until more severe water stress (–1.2 MPa) was applied, when an increase of ADPGlc provided evidence for an inhibition of starch synthase or branching enzyme activities.

The present experiments demonstrate that a 70–80% decrease of overall SPS activity almost completely suppresses the water stress-induced stimulation of sucrose synthesis in discs exposed to water deficits of up to –0.72 MPa (Figs 2 and 3), and in intact tubers on water stressed plants (Fig. 4). Decreased expression of SPS also weakens or suppresses the accompanying water stress-induced inhibition of starch synthesis. These results demonstrate (i) that SPS plays an important role in the stimulation of sucrose synthesis in response to water stress; and (ii) that the inhibition of starch synthesis observed in wild-type tubers at these water deficits is a consequence of the higher rate of sucrose synthesis.

In the more severely water-stressed disc (–1.2 MPa), the impact of SPS on the change in partitioning was weaker (Fig. 3). This is consistent with the proposal (Geigenberger et al. 1997) that severe water stress leads to an additional and more direct inhibition of starch synthesis.

Decreased expression of SPS affects the changes in biomass allocation in water-stressed whole potato plants

Although water stress leads to reduced dry-matter accumulation in the whole plant (see Burton 1986), a larger percentage of the dry-matter is allocated to tuber growth (Fig. 5). This ability to increase dry-matter transfer to the tubers is impaired in transformants with reduced expression of SPS (Fig. 5). Two factors could contribute: (i) reduced expression of SPS in the tubers prevents the stimulation of sucrose synthesis (see above) which could impair the ability of the tuber to attract water and sustain growth during water deficits; and (ii) reduced expression of SPS in the leaves may restrict sucrose export to the tubers. To separate the contribution of these two factors, transgenic plants will have to be generated where SPS is reduced in a tissue-specific manner using tuber or leaf specific promoters.

In conclusion, our results show that SPS contributes to a cycle of sucrose degradation and resynthesis in non-stressed tubers. They also show that SPS becomes important for acclimation to water stress, and provides direct evidence that sucrose synthesis is important for the ability of the plant to cope with water deficits.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Generation of transgenic plants

cDNA clones SPS-P1 and SPS-P4 (EMBL accession number X73477) with 99% identity of amino acid sequence were used to produce line-5 transformants using a construct in which the entire open reading frame of SPS-P4 was linked in antisense configuration to the 35S CaMV promoter, and line 1 transformants by linking a 2.0 kb fragment of SPS-P1 (including a 957 bp 5-´non-translated region and 45% of the coding sequence) in sense configuration to the 35S CaMV promoter as in Krause et al. (1998).

Growth of plants

Transformants and wild-type potato (Solanum tuberosum L. cv. Desirée) plants (Saatzucht Fritz Lange, Bad Schwartau, Germany) were grown in a growth chamber (250 or 350 μmol photons m–2 s–1 irradiance, 20°C, 50% relative humidity) under a 14 h/10 h day/night regime, or in the greenhouse with supplementary light (80 μmol photons m–2 s–1), in 3 litre pots in soil supplemented with Hakaphos grün (100 g per 230 litre soil, BASF, Ludwigshafen, Germany). Growing tubers from 10- to 12-week-old plants were used that had high activities of sucrose synthase (around 1000 nmol g FW–1 min–1) which is taken as an indicator for rapidly growing tubers (Merlo et al. 1993).

Immunodetection of SPS protein and determination of steady state SPS mRNA levels

Western analysis was as described in Reimholz et al. (1997). Total RNA was purified as in Logeman et al. (1987), capillary blotted, the RNA was covalently fixed to the blotting membrane (Duralon-UV membrane, Stratagene) by UV cross-linking (Stratalinker 1800, Stratagene), and hybridisation and detection of the Sps-transcript carried out using a polymerase chain reaction (PCR) amplified digoxigenin-labelled probe (sp4) according to the Boehringer DIG-System, and detected on Hyperfilms-ACL (Amersham). The blotted membrane was always checked for equal loading by visual inspection of the rRNA under UV-light.

Analysis of enzyme activities

Sucrose–phosphate synthase (SPS) was extracted, spin-desalted and immediately assayed (Geigenberger et al. 1997). Aliquots were snap-frozen in liquid nitrogen and assayed for sucrose synthase and ADPGlc pyrophosphorylase (Merlo et al. 1993).

Labelling experiments with tubers slices

Labelling experiments were carried out with tuber slices from growing tubers attached to the mother plant (Geigenberger et al. 1997). Discs (diameter 8 mm, thickness 2 mm) were cut, washed three times in 20-fold excess medium, immediately incubated in 10 mm 2-(N-morpholino) ethane sulfonic acid (Mes) (pH 6.5; KOH), 2 mm glucose, and different mannitol concentrations up to 500 mm (see legends for details), and 90 min later [U-14C]glucose (Amersham-Buchler, Braunschweig, Germany; final specific activity 38 kBq μmol–1) was added, the discs were incubated for 2 h, harvested, washed three times in buffer, and frozen in liquid nitrogen.

Labelling experiments with intact tubers

Labelling experiments were carried out on intact tubers of 10-week-old plants in a growth chamber (Geigenberger et al. 1994). Tubers were excavated, taking care not to bend the stolons, a fine bore-hole (1–2 mm) was made through the middle using a metal hypodermic needle, filled with 70 μm[U-14C]glucose (specific activity 10.8 MBq μmol–1), incubated for 2 h, and a concentric ring of tissue (diameter 0.8 cm) was removed for analysis. During the whole experiment, tubers remained attached to their mother plants via their stolons.

Fractionation of 14C-labelled tissue extracts

Tuber material was extracted and analysed for ionic components and sugars as in Geigenberger et al. (1997). The insoluble material left after ethanol extraction was analysed for label in starch (Merlo et al. 1993).

Metabolite analysis

Tissue slices were frozen in liquid N2, taking care that the slices fell into liquid N2 immediately. After extraction of frozen material (approximately 0.5 gFW) with trichloroacetic acid (Jellito et al. 1992), hexose-phosphates, UDPGlc and 3PGA were measured (Merlo et al. 1993). The recovery of small, representative amounts of each metabolite through the extraction, storage and assay procedures has been documented (Hajirezaei & Stitt 1991; Jellito et al. 1992). Sucrose and starch were measured as in Geigenberger et al. (1998).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 199).

References

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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Footnotes
  1. EMBL accession number X73477.