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-Planck-
lnstitut für Molekulare Pflanzenphysiologie, Karl-Liebknecht-Strasse 25, Haus 20, D-I 4476 Golm, Germany (B.M.-R.); and lnstitut für Genbiologische Forschung Berlin GmbH, lhnestrasse 63,
D-14195 Berlin, Germany (6.M.-R., G.N.)
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 leaf- specific 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 re- duced 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 reduc- tion of the TPT protein by antisense repression. In a second line with a 95% reduction of ACPase activity, the amount of chlorophyll was significantly reduced as a consequence of the lowered triose phos- phate utilization capacity.
Suc and starch are the prominent products of CO, as- similation in many plants. Whereas most of the SUC is exported from the source leaves during the light period, starch is accumulated in the chloroplast stroma and de- graded 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).
This work was supported by the Deutsche Forschungsgemein-
The ratio of light-to-dark export of carbohydrate can be modified by using transgenic plants with altered starch ac- cumulation capacity. In one set of transgenic potato (Sola- num tuberosum L. cv Désirée) plants the TPT protein, cata- lyzing 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 recy- cling 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, accu- mulated 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 ex- ported 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 ex- ported 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 inhibi- tion of the AGPase plants (Leidreiter et al., 1995). A reduc- tion 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, assimila- tion 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.
Abbreviations: AGPase, ADP-Glc pyrophosphorylase; AT, dou- ble transformants inhibited for both triose phosphate translocator and ADP-Glc pyrophosphorylase; bisP, bisphosphate; DHAP, di- hydroxyacetone phosphate; FBPase, Fru-1,6-bisphosphatase; 3- PGA, 3-phosphoglycerate; TI', triose phosphate; TPT, triose phos- phate translocator.
472 Hattenbach et al. Plant Physiol. Vol. 115, 1997
These previous experiments showed that carbohydratepartitioning between Sue and starch is sufficiently flexibleto compensate for decreased activities of starch biosynthe-sis or of TP transport without a loss of plant productivity.The question arises, how do plants respond when both TPexport and TP utilization for synthesis of starch are de-creased? To answer this question, transformants were gen-erated in which both AGPase and TPT activities werereduced by an antisense technique, and the effect of thistransformation 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 ofthe small subunit of AGPase, was transformed into trans-genic potato (Solarium tuberosum L. cv Desiree) plants withrepressed chloroplastic TPT (Riesmeier et al., 1993). Thebinary vector carrying the chimeric gene was constructed asfollows: the EcoRI fragment of plasmid B22-1 (harboring thepotato tuber AGPase small subunit cDNA; Miiller-Rober etal., 1990) was isolated and, after a fill-in reaction with T4DNA polymerase, cloned into the BamHI site (blunt-ended)of a plant expression cassette containing the ST-LS1 pro-moter and the polyadenylation signal of the T-DNA octo-pine synthase gene in a pUC18-based plasmid (von Schae-wen, 1989). A plasmid that contained the AGPase cDNA inthe antisense orientation with respect to the promoter wasselected by restriction analysis. An EcoRI/Sail fragment(promoter/cDN A/terminator) was transferred to binaryvector pBIB-HYG, allowing plant transformation with hy-gromycin selection (Becker, 1990).
Plants inhibited for TPT expression (Riesmeier et al., 1993)were transformed via Agrobacterium tumefaciens, accordingto the method of Dietze et al. (1995). Transgenic plants werescreened for reduced amounts of AGPase protein in theleaves by immunoblot analysis, as described previously(Miiller-Rober et al., 1992; Leidreiter et al., 1995). Plants werepropagated from tissue cultures and grown in pots (16 cm indiameter) for 10 weeks in a climatized growth chamber in a12-h light/ 12-h dark cycle with a PPFD of 300 /rniol m~2 s"1.Photosynthesis rates of leaves attached to the plants weredetermined under growth conditions by a portable IR gas-exchange system (LCA 3, ADC, Hoddeston, UK). Each leafwas monitored for 24 h, and the rate of CO2 assimilation wascalculated from the values taken during the light period.Maximum photosynthesis rates were determined in a leafdisc electrode (Hansatech Instruments, King's Lynn, Nor-folk, UK) in saturating light (2000 /xrnol m~2 s"1) and ahigh-atmospheric CO2 concentration as described by Delieuand Walker (1981).
Samples for the determination of metabolite levels andenzyme activities were harvested from plants at the end ofthe light period. Samples for the assay of enzymes wereextracted in 50 mM Hepes-KOH, pH 8.0, 5 mM MgCl2,1 mMEGTA, 1 mM EDTA, 5 mM DTT, 0.5 mM PMSF, and 10%glycerol. AGPase activity was determined spectrophoto-metrically in 80 mM Hepes-KOH, pH 8.0, 10 mM MgCl2, 10mM 3-PGA, 1 mM ADP-Glc, 0.6 mM NADP, 10 JUM Glc-1,6-bisP, 3 mM DTT, 17 nkat of phosphoglucomutase, and 42
nkat of Glc-6-P dehydrogenase. The absorption change wasfairly constant during the measuring period (Leidreiter etal., 1995). Rubisco activity was quantified by incorporationof 14CO2 into acid-stable products after Rubisco was fullyactivated by incubation with 100 mM MgCl2 and 150 mMNaHCO3 (Heineke et al., 1994). The contents of phosphor-ylated intermediates were determined spectrophotometri-cally after extraction with 10% perchloric acid, with a re-covery 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 afterextraction with chloroform / methanol (Leidreiter et al.,1995). For the analysis of the subcellular distribution ofFru-l,6-bisP and Fru-6-P potato leaves were lyophilizedand fractionated in nonaqueous medium, as described indetail by Heineke et al. (1994). Significance was tested bycomparing the results of transformant leaves with thosefrom 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 ofAGPase on the metabolism of transformants with a 30%reduced TPT protein, it was necessary to compare the ATplants with both wild-type and TPT antisense plants. Twolines of transformants (AT 50 and 104), differing in theirdegree of antisense repression, were analyzed (Fig. 1). In oneline (AT 50) AGPase activity was reduced by about 50%, andin the other (AT 104) AGPase activity was reduced by about95% (Table I). No visible difference was found between thephenotypes of wild-type, TPT, and AT 50 plants, but AT 104plants had reduced leaf chlorophyll contents.
The rates of CO2 assimilation were determined in twoconditions. To measure the rates of CO2 assimilation atambient conditions in the growth chamber, a portable IRgas analyzer was used. Maximum rates of CO2 assimilationwere determined by a leaf disc electrode in saturating lightand at a saturating CO2 concentration. In growth condi-tions the rates of CO2 assimilation of wild-type and trans-formant lines were identical; only that of transformant AT104 was reduced, concurring with a lower chlorophyllcontent. No such difference was observed when using chlo-rophyll as the basis for the calculation (Table I). At high
1 2 3 4
Figure 1. Western-blot analysis of AGPase protein using anti-AGPase antibody as described by Muller-Rober et al. (1992) andLeidreiter et al. (1995). Lane 1, AT 50; lane 2, AT 104; lane 3, TPT;and lane 4, wild type. www.plantphysiol.orgon June 11, 2020 - Published by Downloaded from
Reduction of ADP-Clc Pyrophosphorylase and Triose Phosphate Translocator 473
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
a 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 mea- surement 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 SUC synthesis pathways responded similarly to the transformation in TPT and AT transfor- mants, whereas others responded differently. In a11 transfor- mants 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 trans- formants; 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-P ratios (Table 11). In two other sets of plants the subcellular localization of Fru-1,6-bisP and Fru- 6-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 car- bohydrates 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 Wild Tvpe TPT AT 50 AT 104
3-PCA 7.7 t 3.0 DHAP 0.4 i 0.1 CIC-6-P 7.6 t 1.9 Fru-6-P 2.8 2 0.6 Fru-I ,6-bisP 0.9 t 0.4 Ratio
3 - PG N D H A P Fru-I ,6-bisP/Fru-6-P 0.32
Glc 42 t 49 Fru 74 2 65 SUC 124 t 20 Starch 1100 t 320 Sum of amino acids 233 2 516
18.6
nmol cm-’ 13.3a 2 4.9
0.6 t 0.2 7.0 i 2.2 3.0 2 0.8
2.0” t 1.1
11.6 t 3.8 0.9” 2 0.3 6.0 i 1.3 2.9 t 0.5
2.9” * 1.9
21.1 13.4
8 t 16 19= i 22
105a t 18 4600a ? 1400
262 t 5660
0.67 0.98 10 2 15 25 i 23
105 t 29 2900” -C_ 1200
264 t 458
11.3 t 3.4 1 .O” t 0.3 6.5 5 1.5 2.4 2 0.5
3.0” 2 1.3
11.1 1.27
2” t 3 14” 5 10 69“ rC_ 14
440a t 150 230 t 457
a Difference from wild type is significant using Student’s t test with P = 0.05.
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-P ratio 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 steady- state 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 parame- ters. 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).
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 anti- sense 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 AG- Pase reduced the starch content and enhanced the photoas- similate export during the light period (Leidreiter et al.,
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 car- bohydrate 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 chloro- plast 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 AG- Pase 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 carbo- hydrate to starch, and surplus carbohydrate can then be exported only by the TPT. In spite of the reduced translo- cator 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 in- crease its export into the cytosol. The subcellular distribu- tion 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 intermedi- ates in TPT and AT plants showed a specific increase in the
O 0 0 5 1 0 1 5 20 2 5 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 475
DHAP and Fru-1,6-bisP contents (Table 11), with the in- crease of Fru-1,6-bisP restricted t o the stroma (Table 111). The higher stromal Fru-1,6-bisP-to-Fru-6-P ratio is a good indication that the stromal FBPase is involved i n the reg- ulation of carbohydrate partitioning. Stromal FBPase is known t o be activated by the thioredoxin system, and the activity of the activated enzyme is controlled by severa1 Calvin cycle intermediates (Buchanan et al., 1971; Garde- mann et al., 1986). The analysis of the transgenic plants shows that fine tuning of stromal FBPase restricts the for- mat ion of sugar monophosphates to the decreased de- mands of ribulose-1,5-bisP regeneration and starch synthe- sis. Surplus TP accumulates i n the stroma to enhance its export. Apparently, a regulation of stromal FBPase is re- sponsible for compensating for the reduction of the 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 SUC a 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 the stromal 3-PGA concentration a n d a decrease of the ATP-to-ADP ratio (Sharkey e t al., 1986). The question re- mained 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 the capacity to utilize TP. In these plants a n increase i n light intensity and CO, supply d id not influence the rate of CO, assimilation i n leaf discs (Table I). Unlike the short-term experiments of Sharkey (1985) and Sharkey e t al. (1986), our data indicate that in AT 104 plants 3-PGA accumulation waç moderate and there w a s no indication of a decrease in the ATP-to-ADP ratio, which would have been recognizable from an in- creased 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 transforma- tion 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
Chatterton NJ, Sylvius JE (1979) Photosynthate partitioning into starch in soybean leaves. I. Effects of photoperiod versus pho- tosynthetic 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 photo- synthetic 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
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 trans- port of inorganic phosphate, 3-phosphoglycerate and triose phosphates across the inner membrane of the envelope in spin- ach 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-bisphosphatase in 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
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 in- crease 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 regula- tion 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, Son- newald U (1990) One of two different ADP-glucose pyrophos- phorylase genes from potato responds strongly to elevated lev- els 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 76 165-169
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, sub- zellularen Kompartimentierung fremder Proteine in hoheren Pflanzen. PhD thesis. Freie Universitat, Berlin
