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Previous experiments have shown that carbohydrate partitioning in leaves of potato (Solanum tuberosum L.) plants can be modified by antisense repression of ...
Plant Physiol. (1997) 115: 471-475

Antisense Repression of Both ADP-Glucose Pyrophosphorylase and Triose Phosphate Translocator Modifies Carbohydrate Partitioning in Potato leaves' Andrea Hattenbach, Bernd Müller-Rober, Cabriele Nast, and Dieter Heineke* lnstitut für Biochemie der Pflanze, Untere Karspüle 2, 0-37073 Gottingen, Germany (A.H., D.H.); Max-Plancklnstitut für Molekulare Pflanzenphysiologie, Karl-Liebknecht-Strasse 25, Haus 20, D-I 4476 Golm, Germany (B.M.-R.); a n d lnstitut für Genbiologische Forschung Berlin G m b H , lhnestrasse 63, D-14195 Berlin, Germany (6.M.-R., G.N.) The ratio of light-to-dark export of carbohydrate can be modified by using transgenic plants with altered starch accumulation capacity. In one set of transgenic potato (Solanum tuberosum L. cv Désirée) plants the TPT protein, catalyzing the export of TP from the chloroplast stroma in exchange for Pi (Fliege et al., 1978), was reduced by about 30%by using the antisense technique (Riesmeier et al., 1993). The reduction of the export capacity for TPs led to increased starch synthesis during the light period, allowing Pi recycling in the chloroplast. There was, however, no effect on the rate of CO, assimilation or on tuber yield, at least with the growth conditions that were used. The surplus starch, accumulated during the light period, was degraded during the night. Since starch degradation in chloroplasts results partly in the release of Glc (Stitt and Heldt, 1981), which is exported from the chloroplasts via the hexose translocator (Schafer et al., 1977), only a part of the starch-degradation products requires the TPT for export from the chloroplast during the night. This explains why in these transformants only a minor proportion of the photoassimilates was exported from the leaves during the day and the major part was exported during the night (Heineke et al., 1994). This acclimation was accompanied by a higher activity of Rubisco and AGPase. In addition, the chloroplastic 3-PGA level was increased, whch is known to stimulate starch synthesis (Heineke et al., 1994). In a second set of potato plants, the capacity of starch synthesis was reduced by a leaf-specific antisense inhibition of the AGPase plants (Leidreiter et al., 1995).A reduction in the amount of starch was only achieved when the enzyme activity was reduced by about 90%. As with TPT plants, there was no reduction in the rate of CO, assimilation or tuber yield observed. From these observations it was concluded that Suc synthesis in these transformants was increased during the day and was accompanied by an increase in assimilate export during the day period.

Previous experiments have shown that carbohydrate partitioning in leaves of potato (Solanum tuberosum L.) plants can be modified by antisense repression of the triose phosphate translocator (TPT), favoring starch accumulation during the light period, or by leafspecific antisense repression of ADP-glucose pyrophosphorylase (ACPase), reducing leaf starch content. These experiments showed that starch and sucrose synthesis can partially replace each other. To determine how leaf metabolism acclimates to an inhibition of both pathways, transgenic potato (S. tuberosum 1. cv Désirée) plants, with a 30% reduction of the TPT achieved by antisense repression, were transformed with an antisense cDNA of the small subunit of ACPase, driven by the leaf-specific ST-LS1 promoter. These double-transformed plants were analyzed with respect to their carbohydrate metabolism, and starch accumulation was reduced in all lines of these plants. In one line with a 50% reduction of AGPase activity, the rate of CO, assimilation was unaltered. In these plants the stromal level of triose phosphate was increased, enabling a high rate of triose phosphate export in spite of the reduction of the TPT protein by antisense repression. In a second line with a 9 5 % reduction of ACPase activity, the amount of chlorophyll was significantly reduced as a consequence of the lowered triose phosphate utilization capacity.

Suc and starch are the prominent products of CO, assimilation in many plants. Whereas most of the SUCis exported from the source leaves during the light period, starch is accumulated in the chloroplast stroma and degraded during the night. The amount of CO, assimilates that are transiently partitioned into starch differs among plant species (Chatterton and Sylvius, 1979, 1980; Gordon et al., 1980; Sicher et al., 1984; Gerhardt et al., 1987; Li et al., 1992).The temporary storage of photoassimilates as starch during the day allows the export of photoassimilates via the phloem to continue during the night at 60 to 15%of the corresponding rate during the light period (Fondy and Geiger, 1982; Hendrix and Huber, 1986; Kalt-Torres et al., 1987; Heineke et al., 1994; Riens et al., 1994).

Abbreviations: AGPase, ADP-Glc pyrophosphorylase; AT, double transformants inhibited for both triose phosphate translocator and ADP-Glc pyrophosphorylase; bisP, bisphosphate; DHAP, dihydroxyacetone phosphate; FBPase, Fru-1,6-bisphosphatase; 3PGA, 3-phosphoglycerate; TI', triose phosphate; TPT, triose phosphate translocator.

This work was supported by the Deutsche Forschungsgemeinschaft (grant no. He 565/17-4). * Corresponding author; e-mail [email protected]; fax 49-551395749. 471

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These previous experiments showed that carbohydrate partitioning between Sue and starch is sufficiently flexible to compensate for decreased activities of starch biosynthesis or of TP transport without a loss of plant productivity. The question arises, how do plants respond when both TP export and TP utilization for synthesis of starch are decreased? To answer this question, transformants were generated in which both AGPase and TPT activities were reduced by an antisense technique, and the effect of this transformation on metabolism was analyzed. MATERIALS AND METHODS

A chimeric gene with a leaf-specific ST-LS1 promoter (Stockhaus et al., 1989), designed for antisense repression of the small subunit of AGPase, was transformed into transgenic potato (Solarium tuberosum L. cv Desiree) plants with repressed chloroplastic TPT (Riesmeier et al., 1993). The binary vector carrying the chimeric gene was constructed as follows: the EcoRI fragment of plasmid B22-1 (harboring the potato tuber AGPase small subunit cDNA; Miiller-Rober et al., 1990) was isolated and, after a fill-in reaction with T4 DNA polymerase, cloned into the BamHI site (blunt-ended) of a plant expression cassette containing the ST-LS1 promoter and the polyadenylation signal of the T-DNA octopine synthase gene in a pUC18-based plasmid (von Schaewen, 1989). A plasmid that contained the AGPase cDNA in the antisense orientation with respect to the promoter was selected by restriction analysis. An EcoRI/Sail fragment (promoter/cDN A/terminator) was transferred to binary vector pBIB-HYG, allowing plant transformation with hygromycin selection (Becker, 1990). Plants inhibited for TPT expression (Riesmeier et al., 1993) were transformed via Agrobacterium tumefaciens, according to the method of Dietze et al. (1995). Transgenic plants were screened for reduced amounts of AGPase protein in the leaves by immunoblot analysis, as described previously (Miiller-Rober et al., 1992; Leidreiter et al., 1995). Plants were propagated from tissue cultures and grown in pots (16 cm in diameter) for 10 weeks in a climatized growth chamber in a 12-h light/ 12-h dark cycle with a PPFD of 300 /rniol m~ 2 s"1. Photosynthesis rates of leaves attached to the plants were determined under growth conditions by a portable IR gasexchange system (LCA 3, ADC, Hoddeston, UK). Each leaf was monitored for 24 h, and the rate of CO2 assimilation was calculated from the values taken during the light period. Maximum photosynthesis rates were determined in a leaf disc electrode (Hansatech Instruments, King's Lynn, Norfolk, UK) in saturating light (2000 /xrnol m~ 2 s"1) and a high-atmospheric CO2 concentration as described by Delieu and Walker (1981). Samples for the determination of metabolite levels and enzyme activities were harvested from plants at the end of the light period. Samples for the assay of enzymes were extracted in 50 mM Hepes-KOH, pH 8.0, 5 mM MgCl2,1 mM EGTA, 1 mM EDTA, 5 mM DTT, 0.5 mM PMSF, and 10% glycerol. AGPase activity was determined spectrophotometrically in 80 mM Hepes-KOH, pH 8.0, 10 mM MgCl2, 10 mM 3-PGA, 1 mM ADP-Glc, 0.6 mM NADP, 10 JUM Glc-1,6bisP, 3 mM DTT, 17 nkat of phosphoglucomutase, and 42

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nkat of Glc-6-P dehydrogenase. The absorption change was fairly constant during the measuring period (Leidreiter et al., 1995). Rubisco activity was quantified by incorporation of 14CO2 into acid-stable products after Rubisco was fully activated by incubation with 100 mM MgCl2 and 150 mM NaHCO3 (Heineke et al., 1994). The contents of phosphorylated intermediates were determined spectrophotometrically after extraction with 10% perchloric acid, with a recovery of >95% for Glc-6-P, Fru-6-P, and 3-PGA, and >85% for Fru-l,6-bisP and DHAP (Heineke et al., 1994). Sugar levels were analyzed spectrophotometrically after extraction with chloroform / methanol (Leidreiter et al., 1995). For the analysis of the subcellular distribution of Fru-l,6-bisP and Fru-6-P potato leaves were lyophilized and fractionated in nonaqueous medium, as described in detail by Heineke et al. (1994). Significance was tested by comparing the results of transformant leaves with those from the wild type by using Student's t test with P = 0.05. RESULTS AGPase Activity, Phenotype, and Photosynthesis

To examine the effect of an antisense repression of AGPase on the metabolism of transformants with a 30% reduced TPT protein, it was necessary to compare the AT plants with both wild-type and TPT antisense plants. Two lines of transformants (AT 50 and 104), differing in their degree of antisense repression, were analyzed (Fig. 1). In one line (AT 50) AGPase activity was reduced by about 50%, and in the other (AT 104) AGPase activity was reduced by about 95% (Table I). No visible difference was found between the phenotypes of wild-type, TPT, and AT 50 plants, but AT 104 plants had reduced leaf chlorophyll contents. The rates of CO2 assimilation were determined in two conditions. To measure the rates of CO2 assimilation at ambient conditions in the growth chamber, a portable IR gas analyzer was used. Maximum rates of CO2 assimilation were determined by a leaf disc electrode in saturating light and at a saturating CO2 concentration. In growth conditions the rates of CO2 assimilation of wild-type and transformant lines were identical; only that of transformant AT 104 was reduced, concurring with a lower chlorophyll content. No such difference was observed when using chlorophyll as the basis for the calculation (Table I). At high

1 2 3 4

Figure 1. Western-blot analysis of AGPase protein using antiAGPase antibody as described by Muller-Rober et al. (1992) and Leidreiter et al. (1995). Lane 1, AT 50; lane 2, AT 104; lane 3, TPT; and lane 4, wild type.

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Table 1. Activity of AGPase and Rubisco, chlorophyll content, and rate of CO, assimilation in leaves from wild-type, TPT antisense, and AT potato plants

Data are mean values

-C_

SE.

Wild TvDe

Activitv

TPT

AT 50

AT 104

2.7 i 0.44 =100

1.2” 5 0.44 44

0.13= 2 0.04 ( n = 6) 5

~~

AGPase pmol m-2 s-’ % Chlorophyll pg cm-’ Rubisco pmol mg-’ chlorophyll s-’ Rate of CO, assimilation Ambient pmol m-* s-’ pmol mg-’ chlorophyll h-’ Maximum pmol m-2 s-’ pmol mg-’ chlorophyll h-’ a

2.2 t 0.68 81 40 +- 5

39

2.1 2 0.3

2.9

*5 -C_

1.2

41 t 7

27“

rC_

3 (n = 5)

3.2“ L 0.4 ( n = 7)

3.3” t 0.9

8.2 71.9

7.9 71.4

7.6 63.8

5.9 ( n = 3) 77.0

23.0 202

13.3 120

11.7 98

6.3 ( n = 4) 82

Difference from wild type is significant using Student’s t test with P = 0.05.

light and a high CO, concentration, however, each line responded differently. The highest rate of CO, assimilation was found in wild-type leaves. The reduction in the rate of CO, assimilation, which was already observed in TPT leaves, was even more pronounced in AT plants and was independent of the chlorophyll content. It should be noted that in AT 104 the rate of CO, assimilation at ambient and at maximum conditions was nearly identical, indicating that in this line the rate of CO, assimilation was saturated even at the relatively low light intensity used during measurement in ambient conditions. Activity of Rubisco and Content of Phosphorylated lntermediates

It was shown earlier that in TPT transformants the Rubisco capacity was increased (Heineke et al., 1994). This was also true for the AT transformants. In a11 lines the

maximum Rubisco activity per unit of chlorophyll was 1.5 times higher than in wild-type leaves (Table I). The levels of some of the phosphorylated intermediates of the Calvin cycle and the starch and SUCsynthesis pathways responded similarly to the transformation in TPT and AT transformants, whereas others responded differently. In a11 transformants the 3-PGA content was higher than in wild-type leaves. DHAP and Fru-1,6-bisP, which were slightly higher in TPT plants, were significantly increased in the AT transformants; Glc-6-P and Fru-6-P were nearly identical. These changes led to decreased 3-PGA-to-DHAP and increased Fru-1,6-bisP-to-Fru-6-Pratios (Table 11).In two other sets of plants the subcellular localization of Fru-1,6-bisP and Fru6-I‘ in leaves of the TPT and the AT 104 transformant was determined by nonaqueous fractionation (Table 111).In these sets the Fru-1,6-bisP-to-Fru-6-P ratio in AT 104 plants was slightly lower than in those leaves shown in Table 11, but the tendencies were identical. Whereas no differences were

Table 11.

Contents of phosphorylated intermediates of Suc and starch synthesis pathways and of carbohydrates and amino acids in leaves from wild-type, TPT antisense potato plants, and AT at the end of the light period

Data are mean values t- SE ( n = 5 for phosphorylated intermediates and n = 10 for others). Metabolite 3-PCA DHAP CIC-6-P Fru-6-P Fru-I ,6-bisP Ratio 3- PG N D HA P Fru-I ,6-bisP/Fru-6-P Glc Fru SUC

Starch

Sum of amino acids a

Wild Tvpe 7.7 0.4 7.6 2.8 0.9

t 3.0 i 0.1 t 1.9 2 0.6 t 0.4

18.6 0.32 42 t 49 74 2 65 124 t 20 1100 t 320 233 2 516

TPT

AT 50

nmol cm-’ 13.3a 2 4.9 11.6 t 3.8 0.6 t 0.2 0.9” 2 0.3 7.0 i 2.2 6.0 i 1.3 3.0 2 0.8 2.9 t 0.5 2.0” t 1.1 2.9” 1.9

*

21.1 0.67 8 t 16 19= i 22 105a t 18 4600a ? 1400 262 t 5660

13.4 0.98 10 2 15 25 i 23 105 t 29 2900” -C_ 1200 264 t 458

Difference from wild type is significant using Student’s t test with P = 0.05.

AT 104

11.3 1.O” 6.5 2.4 3.0”

t 3.4 t 0.3 5 1.5

2 0.5 2 1.3

11.1 1.27 2” t 3 14” 5 10 69“ rC_ 14 440a t 150 230 t 457

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Table 111. Subcellular distribution of Fru- 7,6-bisP and Fru-6-P in TPT antisense and AT 104 potato plants at the end of the light period Results are mean values ? SE from six gradients from two sets of plants. Plant and Sugar

T PT Fru-l,6-bisP (nmol mg-’ chlorophyll) Fru-6-P (nmol mg-’ chlorophyll) Fru-l,6-bisP/Fru-6-P (ratio) AT 104 Fru-l,6-bisP (nmol mg-’ chlorophyll) Fru-6-P (nmol mg-‘ chlorophyll) Fru-I,6-bisP/Fru-6-P (ratio)

Stroma

Cytosol

3528 2424 1.5

18?9 41 2 13 0.4

46213 21 2 13 2.2

1225 36?8 0.3

found in the distribution of Fru-6-P in either transgenic line, in AT 104 the stromal content of Fru-1,6-bisP was increased and that in the cytosol decreased compared with the TPT plant, resulting in an increased Fru-1,6-bisP-to-Fru-6-Pratio in the chloroplast stroma. Effect of Transformation on the Carbohydrate and Amino Acid Content of the Leaves

Antisense repression of TPT led to an increase in starch accumulation during the light period (Heineke et al., 1994). In the transformant AT 50 the inhibition of AGPase by 50% reduced the starch content at the end of the light period by about 40%, and in the transformant AT 104 an inhibition of AGPase by 95% resulted in about 90% reduction of the starch content (Table 11).For a closer inspection of the role of AGPase activity in starch synthesis, not only the steadystate levels but the synthesis rates are needed. Net starch accumulation during the light period can be followed by measuring starch content at the beginning and at the end of the light period. Plotting the amount of starch accumulated during the light period against the extractable AGPase activity showed that starch accumulation in these plants was directly correlated with the amount of extractable enzyme (Fig. 2). For TPT and AT plants, but not for the wild type, there was a correlation between both parameters. In a11 transformants the contents of Glc and Fru were reduced, whereas that of Suc was less affected. No changes were found in the amino acid content, and the amino acid pattern was only slightly altered (data not shown).

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1995). The reduction of the capacities of both pathways, depending on the degree of AGPase activity, influenced leaf metabolism differently. In AT 50 plants the amount of carbohydrate transiently stored in starch was reduced by about 50% of that in TPT plants, with an unaltered rate of COp assimilation. This observation implies that in spite of the reduced translocator activity the TP export from the chloroplast was nearly as high as in wild-type plants. A further reduction in AGPase activity, however, does not seem to be compensated for by an increased TP export. In AT 104 plants an altered phenotype was found with lower chlorophyll content in the leaves. This observation can be interpreted as an acclimation of the CO, assimilation to the decreased capacity for utilizing TP. Activation State of AGPase and TP Export

The correlation between starch accumulation and AGPase activity in TPT and AT plants indicates that AGPase cannot be further activated by allosteric effectors (Fig. 2). Plants obviously lose their flexibility in partitioning carbohydrate to starch, and surplus carbohydrate can then be exported only by the TPT. In spite of the reduced translocator activity some flexibility is left at this step. The TPT catalyzes a reversible counterexchange of Pi, DHAP, and 3-PGA, and the relative transport rates of these compounds depend on their relative concentrations (Fliege et al., 1978). An increased stromal concentration of DHAP would increase its export into the cytosol. The subcellular distribution of DHAP is difficult to determine because of its low concentration, but the whole-leaf content of DHAP was significantly higher in AT plants than in wild-type and TPT plants (Table 11). An accumulation of DHAP in the stroma would explain its higher export rate, which seemed to be sufficient to compensate for the reduced AGPase activity in AT 50 plants. Regulation of the Stromal FBPase

A comparison of the contents of Calvin cycle intermediates in TPT and AT plants showed a specific increase in the

DlSCUSSlON Carbohydrate Partitioning in TPT, AGPase, and AT Transformants

The aim of this research was to determine in which way carbohydrate partitioning can be influenced by the antisense repression of key enzymes. Reduction of TPT activity by about 30% reduced TP export and more carbohydrate was directed into starch. During the dark period starch was degraded and probably exported by the hexose translocator (Heineke et al., 1994).Antisense repression of the leaf AGPase reduced the starch content and enhanced the photoassimilate export during the light period (Leidreiter et al.,

O0

05

10

15

20

25

30

itarch awmulation (pmal m ’ d ‘ )

Figure 2. Starch accumulation and activity of AGPase of leaves from wild-type, TPT, and AT potato plants. ACPase activities (?SE; n = 6) were taken from Table I, and starch accumulation was calculated from the starch content (in Glc units) at the end of the light period and at the end of the dark period (for details, see Table 11).

Reduction of ADP-Clc Pyrophosphorylase and Triose Phosphate Translocator

DHAP and Fru-1,6-bisP contents (Table 11), with the increase of Fru-1,6-bisP restricted t o t h e stroma (Table 111). The higher stromal Fru-1,6-bisP-to-Fru-6-P ratio is a good indication that the stromal FBPase i s involved i n t h e regulation of carbohydrate partitioning. Stromal FBPase is known t o be activated by the thioredoxin system, and the activity of t h e activated enzyme is controlled by severa1 Calvin cycle intermediates (Buchanan et al., 1971; Gardemann e t al., 1986). The analysis of t h e transgenic plants shows that fine t u n i n g of stromal FBPase restricts t h e form a t i o n of sugar monophosphates to the decreased demands of ribulose-1,5-bisP regeneration and starch synthesis. Surplus TP accumulates i n the stroma t o enhance its export. Apparently, a regulation of stromal FBPase is responsible for compensating for t h e reduction of t h e starch synthesis capacity in AT plants. The Capacity for Utilizing TP Can Limit the Rate of CO, Assimilation in Vivo Sharkey (1985) showed that under some conditions the utilization of TPs for SUCa n d starch synthesis could limit the rate of CO, assimilation. This limitation was observed only in transients and w a s characterized by an increase in t h e stromal 3-PGA concentration a n d a decrease of t h e ATP-to-ADP ratio (Sharkey e t al., 1986). The question remained whether such a limitation really occurred under i n vivo conditions. AT 104 plants show how plants acclimate to a long-term reduction i n t h e capacity t o utilize TP. In these plants a n increase i n light intensity and CO, supply d i d n o t influence the rate of CO, assimilation i n leaf discs (Table I). Unlike t h e short-term experiments of Sharkey (1985) and Sharkey e t al. (1986), our d a t a indicate that in AT 104 plants 3-PGA accumulation w a ç moderate a n d there w a s no indication of a decrease in the ATP-to-ADP ratio, which would have been recognizable from an increased 3-PGA-to-DHAP ratio (Heber e t al., 1986). The long-term acclimation is therefore mainly characterized by a reduction of chlorophyll (Table I). ACKNOWLEDGMENTS

We would like to thank L. Willmitzer and his co-workers for providing the TPT antisense plants and J. Dietze for transformation of potato plants. Received February 10, 1997; accepted June 19, 1997. Copyright Clearance Center: 0032-0889/97/ 115/0471/OS. LITERATURE ClTED

Becker D (1990) Binary vectors which allow the exchange of plant selectable markers and reporter genes. Nucleic Acids Res 18: 203 Buchanan BB, Schiirmann P, Kalberer PP (1971) Ferredoxinactivated fructose diphosphatase of spinach chloroplasts. J Biol Chem 246 5952-5959 Chatterton NJ, Sylvius JE (1979) Photosynthate partitioning into starch in soybean leaves. I. Effects of photoperiod versus photosynthetic period duration. Plant Physiol 6 4 749-753 Chatterton NJ, Sylvius JE (1980) Photosynthate partitioning into leaf starch as affected by daily photosynthetic period duration in six species. Physiol Plant 49: 141-144 Delieu T, Walker DA (1981) Polarographic measurement of photosynthetic oxygen evolution by leaf disks. New Phytol89: 165-178 Dietze J, Blau A, Willmitzer L (1995) Agrobacterium-mediated transformation of potato (Solanum tuberosum). In I Potrykus, G

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Spangenberg, eds, Gene Transfer to Plants, Springer Laboratory Manual. Springer, Berlin, pp 24-29 Fliege R, Flügge U-I, Werdan K, Heldt HW (1978) Specific transport of inorganic phosphate, 3-phosphoglycerate and triose phosphates across the inner membrane of the envelope in spinach chloroplasts. Biochim Biophys Acta 502 232-247 Fondy BR, Geiger DR (1982) Diurnal pattern of translocation and carbohydrate metabolism in source leaves of Beta vulgavis L. Plant Physiol 70: 671-676 Gardemann A, Schimkat D, Heldt HW (1986) Control of CO, fixation. Regulation of stromal fructose-l,6-bisphosphatasein spinach by pH and Mg2+ concentration. Planta 168: 536-545 Gerhardt R, Stitt M, Heldt HW (1987) Subcellular metabolite levels in spinach leaves. Regulation of sucrose synthesis during diurnal alterations in photosynthetic partitioning. Plant Physiol 83: 399407 Gordon AJ, Ryle GJA, Webb G (1980) The relationship between sucrose and starch during 'dark export from leaves of uniculm barley. J Exp Bot 31: 845-850 Heber U, Neimanis S, Dietz KJ, Viil J (1986) Assimilatory power as a driving force in photosynthesis. Biochim Biophys Acta 852: 144155 Heineke D, Kruse A, Flügge U-I, Frommer WB, Riesmeier JW, Willmitzer L, Heldt HW (1994) Effect of antisense repression of the chloroplast triose-phosphate translocator on photosynthetic metabolism in transgenic potato plants. Planta 193: 174-180 Hendrix DL, Huber SC (1986) Diurnal fluctuations in cotton leaf carbon export, carbohydrate content, and sucrose synthesizing enzymes. Plant Physiol 81: 584-586 Kalt-Torres W, Kerr PS, Usuda H, Huber SC (1987)Diumal changes in maize leaf photosynthesis. Carbon exchange rate, assimilate export rate, and enzyme activities. Plant Physiol83: 283-288 Leidreiter K, Heineke D, Heldt HW, Miiller-Rober B, Sonnewald U, Willmitzer L (1995) Leaf-specific antisense inhibition of starch biosynthesis in transgenic potato plants leads to an increase in photoassimilate export from source leaves during the light period. Plant Cell Physiol 36: 615-624 Li B, Geiger DR, Shieh W-J (1992) Evidence for circadian regulation of starch and sucrose synthesis in sugar beet leaves. Plant Physiol 99: 1393-1399 Müller-Rober B, Sonnewald U, Willmitzer L (1992) Inhibition of the ADP-glucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes. EMBO J 11:1229-1238 Miiller-Rober BT, Kossmann J, Hannah LC, Willmitzer L, Sonnewald U (1990) One of two different ADP-glucose pyrophosphorylase genes from potato responds strongly to elevated levels of sucrose. Mo1 Gen Genet 224: 136-146 Riem B, Lohaus G, Winter H, Heldt HW (1994) Production and diurnal accumulation of assimilates in leaves of spinach (Spinacia olerucea L.) and barley (Hordeum vulgaue L.). Planta 192 497-501 Riesmeier JW, Flügge U-I, Schulz B, Heineke D, Heldt H-W, Willmitzer L, Frommer WB (1993) Antisense repression of the triose phosphate translocator affects carbon partitioning in transgenic potato plants. Proc Natl Acad Sci USA 9 0 6160-6164 Scháfer G, Heber U, Heldt HW (1977) Glucose transport into spinach chloroplasts. Plant Physiol 60: 286-289 Sharkey TD (1985) O,-insensitive photosynthesis in C, plants. Its occurrence and its possible explanation. Plant Physiol 78: 71-75 Shatkey TD, Stitt M, Heineke D, Gerhardt R, Raschke K, Heldt HW (1986) Limitation of photosynthesis by carbon metabolism. 11. O,-insensitive CO, uptake results from limitation of triose phosphate utilization. Plant Physiol 81: 1123-1129 Sicher RC, Kremer DF, Harries WG (1984) Diurnal carbohydrate metabolism of barley primary leaves. Plant Physiol 7 6 165-169 Stitt M,Heldt HW (1981) Physiological rates of starch breakdown in isolated intact spinach chloroplasts. Plant Physiol68: 755-761 Stockhaus J, Schell J, Willmitzer L (1989) Correlation of the expression of the nuclear photosynthetic gene ST-LS1 with the presence of chloroplasts. EMBO J 8 2445-2451 von Schaewen A (1989) Untersuchungen zur ER-vermittelten, subzellularen Kompartimentierung fremder Proteine in hoheren Pflanzen. PhD thesis. Freie Universitat, Berlin