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Plant Cell Physiol. 46(8): 13661376 (2005) doi:10.1093/pcp/pci148, available online at www.pcp.oupjournals.

org JSPP 2005

Sucrose Synthase Controls Both Intracellular ADP Glucose Levels and Transitory Starch Biosynthesis in Source Leaves
Francisco Jos Muoz 1, 3, Edurne Baroja-Fernndez 1, 3, Mara Teresa Morn-Zorzano 1, Alejandro Miguel Viale 1, Ed Etxeberria 2, Nora Alonso-Casajs 1 and Javier Pozueta-Romero 1, *
Agrobioteknologiako Instituta, Nafarroako Unibertsitate Publikoa and Consejo Superior de Investigaciones Cientficas, Mutiloako etorbidea zenbaki gabe, 31192 Mutiloabeti, Nafarroa, Spain 2 University of Florida, IFAS, Department of Horticultural Sciences, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA ;
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The prevailing model on transitory starch biosynthesis in source leaves assumes that the plastidial ADPglucose (ADPG) pyrophosphorylase (AGP) is the sole enzyme catalyzing the synthesis of the starch precursor molecule, ADPG. However, recent investigations have shown that ADPG linked to starch biosynthesis accumulates outside the chloroplast, presumably in the cytosol. This finding is consistent with the occurrence of an alternative gluconeogenic pathway wherein sucrose synthase (SuSy) is involved in the production of ADPG in the cytosol, whereas both plastidial phosphoglucomutase (pPGM) and AGP play a prime role in the scavenging of starch breakdown products. To test this hypothesis, we have compared the ADPG content in both Arabidopsis and potato wild-type (WT) leaves with those of the starch-deficient mutants with reduced pPGM and AGP. These analyses provided evidence against the classical model of starch biosynthesis, since ADPG levels in all the starch-deficient lines were normal compared with WT plants. Whether or not SuSy is involved in the synthesis of ADPG accumulating in leaves was tested by characterizing both SuSy-overexpressing and SuSy-antisensed transgenic leaves. Importantly, SuSy-overexpressing leaves exhibited a large increase of both ADPG and starch levels compared with WT leaves, whereas SuSyantisensed leaves accumulated low amounts of both ADPG and starch. These findings show that (i) ADPG produced by SuSy is linked to starch biosynthesis; (ii) SuSy exerts a strong control on the starch biosynthetic process; and (iii) SuSy, but not AGP, controls the production of ADPG accumulating in source leaves. Keywords: ADPglucose Arabidopsis thaliana Solanum tuberosum Starch Sucrose Sucrose synthase.
Abbreviations: ADPG, ADPglucose; AGP, ADPG pyrophosphorylase; ASPP, adenosine diphosphate sugar pyrophosphatase; E4P, erythrose-4-phosphate; G1P, glucose-1-phosphate; G6P, glucose6-phosphate; NOS, nopaline synthase; 3PGA, 3-phosphoglycerate; pPGI, plastid phosphoglucose isomerase; pPGM, plastid phosphoglucomutase; RBCS, ribulose 1,5-bisphosphate carboxylase small subunit; 35S, cauliflower mosaic virus 35S promoter; SPS, sucrose phosphate synthase; SuSy, sucrose synthase; UGP, UDPglucose pyrophosphorylase; WT, wild type.
3 *

Introduction
Plants accumulate starch in their leaves during the day and degrade it during the subsequent night. Due to the diurnal rise and fall cycle of its levels, starch found in photosynthetically competent cells is termed transitory starch. Starch biosynthesis in leaves has generally been considered to take place exclusively in the chloroplast, and segregated from the sucrose biosynthetic process that takes place in the cytosol (Stitt et al. 1984, Okita 1992, Sonnewald 1992, Baroja-Fernndez et al. 2004) (Supplementary Fig. 1). According to this classical view, starch is considered to be the end-product of a unidirectional and vectorial pathway linked to the Calvin cycle wherein ADPglucose (ADPG) pyrophosphorylase (AGP) exclusively catalyzes the synthesis of the starch precursor molecule, ADPG, and functions as the major regulatory step in the gluconeogenic process (Caspar et al. 1985, Lin et al. 1988, MllerRber et al. 1992, Okita 1992, Stark et al. 1992). However, recent evidence has exposed inconsistencies in the classical view of transitory starch biosynthesis and previews the occurrence of an alternative pathway wherein ADPG linked to starch biosynthesis is produced de novo in the cytosol by sucrose synthase (SuSy) (Baroja-Fernndez et al. 2001, Baroja-Fernndez et al. 2004, Baroja-Fernndez et al. 2005) (Supplementary Fig. 1). According to this alternative view, both sucrose and starch biosynthetic pathways are tightly interconnected by means of ADPG producing SuSy activity (Delmer 1972, Porchia et al. 1999, Baroja-Fernndez et al. 2003), and by the action of an ADPG translocator located at the chloroplast envelope membranes (Pozueta-Romero et al. 1991, Baroja-Fernndez et al. 2004). Pulsechase and starch pre-loading experiments using isolated chloroplasts (Stitt and Heldt 1981, Fox and Geiger 1984), intact leaves (Scott and Kruger 1995, Walters et al. 2004) and cultured photosynthetic cells (Lozovaya et al. 1996) have provided evidence indicating that chloroplasts can synthesize and mobilize starch simultaneously. This observation is in agreement with reports dealing with gluconeogenic processes in bacteria (Gaudet et al. 1992, Belanger and Hatfull 1999, Guedon et al. 2000), animals (David et al. 1990, Massillon et al. 1995),

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These authors contributed equally to this work Corresponding author: E-mail, javier.pozueta@unavarra.es; Fax, +34-948232191. 1366

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heterotrophic plant organs (Sweetlove et al. 1996, NguyenQuoc and Foyer 2001) and amyloplasts (Pozueta-Romero and Akazawa 1993, Neuhaus et al. 1995). Accordingly, the alternative view on transitory starch biosynthesis assumes that both AGP and plastid phosphoglucomutase (pPGM) play vital roles in the scavenging of glucose units derived from starch breakdown (Pozueta-Romero et al. 1991, Baroja-Fernndez et al. 2001, Pozueta-Romero et al. 2003, Baroja-Fernndez et al. 2004). Against the idea that synthesis and breakdown of starch occur simultaneously in leaves, pulsechase experiments on illuminated Arabidopsis leaves have recently shown that none of the 14C incorporated into starch during a short pulse of 14CO2 was released during a subsequent chase (Zeeman et al. 2002). Based on these results, and assuming that starch biosynthesis occurs following a primer non-reducing-end mechanism of polysaccharide chain elongation, Zeeman et al. (2002) concluded that starch synthesis is not accompanied by significant turnover. This conclusion, however, had an erroneous basis, since it has been shown recently that starch biosynthesis occurs following a non-primer reducing-end insertion mechanism (Mukerjea and Robyt 2004). Taking into account that (i) amylase controls transitory starch degradation (Scheidig et al. 2002, Niittyl et al. 2004, Lloyd et al. 2005) and (ii) this enzyme liberates maltose from the non-reducing end of starch chains, it is not surprising that illuminated leaves exposed to a short pulse of 14CO2 do not release any label during the subsequent chase. Under light conditions, sucrose transiently accumulates in the photosynthetic cell (Stitt et al. 1983, Stitt et al. 1984, Gerhardt et al. 1987, Draborg et al. 2001, Smith et al. 2004). According to the alternative model of transitory starch biosynthesis, SuSy will produce from sucrose the ADPG necessary for starch biosynthesis. Concurrently, both AGP and pPGM will scavenge starch breakdown products in the chloroplast. During the night, however, sucrose is depleted, resulting in considerable decline in ADPG production by SuSy. In addition, AGP activity will also decline because of both redox inactivation of the enzyme (Hendriks et al. 2003) and the low 3-phosphoglycerate (3PGA)/Pi ratio (Kleczkowski 1999, Kleczkowski 2000, Crevilln et al. 2003). The alternative view is highly compatible with most, if not all, starch mutant and transgenic plants described to date. For example, the alternative view predicts that in the low starch phenotype resulting from deficiencies in either pPGM or AGP (Caspar et al. 1985, Lin et al. 1988, Mller-Rber et al. 1992), the recovery towards starch biosynthesis of the glucose units derived from the starch breakdown will also be deficient, resulting in a parallel decline of starch accumulation (BarojaFernndez et al. 2001, Baroja-Fernndez et al. 2004, BarojaFernndez et al. 2005). In this respect, it is worth mentioning that both leaves and heterotrophic organs totally lacking pPGM accumulate readily detectable amounts of starch (Saether and

Iversen 1991, Harrison et al. 1998), which indicates that ADPG sources other than AGP occur in plants. Leaves with a high starch/sucrose balance resulting when (i) export of triose phosphates from the chloroplast is constrained (Riesmeier et al. 1993, Heineke et al. 1994); (ii) either cytosolic phosphoglucose isomerase or fructose bisphosphatase activities are low (Neuhaus et al. 1989, Zrenner et al. 1996, Strand et al. 2000); and (iii) fructose-2,6-bisphosphate levels are high (Scott and Kruger 1995), are characterized by a high 3PGA/Pi ratio as compared with wild-type (WT) leaves. A high 3PGA/Pi ratio activates AGP. Under those circumstances, the alternative view predicts that the recovery towards starch biosynthesis of the glucose units derived from the starch breakdown will also be enhanced, resulting in a parallel increase of the starch/sucrose balance. We must emphasize that a high 3PGA/Pi ratio has been shown to stimulate leaf starch synthesis in the dark (Scott and Kruger 1995). The alternative model of starch biosynthesis in leaves predicts that both sucrose and starch biosynthetic pathways are tightly connected by means of ADPG-producing SuSy. In this respect, the occurrence of UDP-glucose pyrophosphorylase (UGP)-, cytosolic PGM- and sucrose phosphate synthase (SPS)-deficient plants is consistent with the alternative view (but not with the classical one), since these plants are characterized by containing low levels of both sucrose and starch in their leaves compared with WT leaves (Strand et al. 2000, Fernie et al. 2002, Kleczkowski et al. 2004). It is worth noting that leaves from the latter two types of plants have normal 3PGA/Pi ratios compared with WT leaves. The alternative view postulates that the Calvin cycle is not directly connected to the starch biosynthetic pathway by means of plastid PGI (pPGI). In fact, Calvin cycle intermediates such as erythrose-4-phosphate (E4P) and 3PGA are potent inhibitors of pPGI (Kelly and Latzko 1980, Dietz 1985, Backhausen et al. 1997), the stromal concentrations of these compounds in the illuminated chloroplast being much higher than their Ki values for pPGI (Bassham and Krause 1969, Dietz 1985). Furthermore, the stromal glucose-6-phosphate (G6P)/ fructose-6-phosphate ratio is quite low and displaced from equilibrium in the illuminated leaf (Dietz 1985), indicating that pPGI is strongly inhibited under conditions of active CO2 fixation and starch production. The presumption that the Calvin cycle is not connected to starch biosynthesis by means of pPGI does not conflict with the occurrence of starch-deficient pPGI mutants (Jones et al. 1986, Kruckeberg et al. 1989, Yu et al. 2000) since leaves from these plants have a reduced photosynthetic capacity that, in addition to reducing starch biosynthesis, causes a reduced growth phenotype. Using transgenic plants expressing ADP sugar pyrophosphatase (ASPP) from Escherichia coli (Moreno-Bruna et al. 2001) in either the cytosol or chloroplast, we recently provided strong evidence indicating that most of ADPG linked to starch biosynthesis accumulates outside the chloroplast, presumably in the cytosol (Baroja-Fernndez et al. 2004). Further-

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more, we also demonstrated that extraplastidial ADPG is intimately linked to starch biosynthesis. Our results strongly indicated that ADPG cannot accumulate in the chloroplast, most probably because it is transferred rapidly to starch, and/or because it spontaneously hydrolyzes under pH conditions occurring in the illuminated chloroplast. The implications of these findings evidently raised the question as to which of the two enzymes known to synthesize ADPG, i.e. AGP and SuSy, is the predominant source of cytosolic ADPG accumulating in leaves. To address this question, in this work we measured the ADPG content in starch-deficient Arabidopsis thaliana and potato (Solanum tuberosum L.) plants with reduced pPGM, AGP or SuSy (Caspar et al. 1985, Lin et al. 1988, MllerRber et al. 1992, Zrenner et al. 1995). Furthermore, we characterized both SuSy-overexpressing and -antisensed leaves, but which contain normal pPGM and AGP activities. The rationale behind this approach was that if ADPG accumulating in the photosynthetically active cell is exclusively produced in the chloroplast, plants with reduced activities of either pPGM or AGP will not accumulate ADPG. In addition, ADPG and starch levels in both SuSy-overexpressing and -antisensed leaves will be normal compared with WT leaves. Conversely, if ADPG accumulating in the cytosol is produced by SuSy, ADPG levels in starch-deficient plants with reduced plastid gluconeogenic enzymes will be normal compared with WT leaves. Furthermore, both ADPG and starch levels in SuSy-overexpressing leaves will be higher than in WT leaves, whereas SuSy-antisensed leaves will accumulate low amounts of both ADPG and transitory starch. The results presented in this communication undoubtedly recognize the essential participation of SuSy in the production of ADPG destined for the transitory starch biosynthetic process in leaves.

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Results and Discussion


ADPG extraction and measurement ADPG was extracted and measured by HPLC as described in Materials and Methods. To ensure the effectiveness of the ADPG extraction method, we added known amounts of commercially available ADPG to leaves immediately before homogenization. The added amounts of ADPG were comparable with values previously reported for leaves (Smith et al. 1990, Baroja-Fernndez et al. 2004), and their recoveries were 98 and 95% for potato and Arabidopsis leaves, respectively. We subsequently checked the reliability of the two distinct chromatographic methods of ADPG detection and measurement employed in this work by adding known amounts of ADPG to leaf extracts previously digested with purified ASPP (Moreno-Bruna et al. 2001). ASPP is a nucleotide-sugar hydrolase that specifically cleaves ADP-sugars (Bessman et al. 1996, Moreno-Bruna et al. 2001). Fig. 1A and B demonstrates that

Fig. 1 ADPG measurement in 100 l potato leaf extracts (A) without and (BH) with an ASPP digestion step prior to analysis by HPLC with a Waters and Associates system fitted to a Partisil-10-SAX column. In (BH), once the ASPP reactions were stopped, the indicated amounts of commercially available ADPG were added to the extracts and then analyzed by HPLC. Nucleotide elution times are: AMP, 3.2 min; UMP, 8.9 min; ADPG, 15.5 min; ADPribose, 18.3 min; UDPglucose, 21.8 min; ADP, 31.8 min; ATP, 35 min; UTP, 36.1 min. AU, absorbance units of A260. An equivalent type of analysis was performed using HPLC with pulsed amperometric detection on a DX-500 system fitted to a CarboPac PA10 column (not shown).

ASPP digestion exclusively and totally removed a substance that eluted at the level of ADPG. This substance was collected and hydrolyzed with ASPP. The products of the enzymatic reaction were subjected to ion chromatography and proved to

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Fig. 2 ADPG calibration curve for potato leaf extracts. The curve was obtained from chromatographic analyses illustrated in Fig. 1 using 100 l of leaf extracts. An equivalent curve was also obtained for Arabidopsis leaves (not shown). AU, absorbance units of A260

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be AMP and glucose-1-phosphate (G1P) (not shown), the overall results confirming the correct identification of ADPG. As illustrated in Fig. 1BH, this approach allowed us to produce accurate calibration curves for ADPG content in both Arabidopsis and potato leaves (Fig. 2). Using these calibration curves, we observed that ADPG levels in both Arabidopsis and potato leaves ranged between 0.25 and 0.35 nmol g1 FW (0.350.40 nmol mg1 Chl) (Fig. 1A). These values were comparable with those previously reported for WT pea leaves (0.6 nmol mg1 Chl) (Smith et al. 1990). AGP- and pPGM-deficient leaves have low levels of starch but normal levels of ADPG The classical view of starch biosynthesis, according to which AGP is the sole enzyme producing ADPG linked to starch biosynthesis, predicts that ADPG levels in starch-deficient leaves with reduced activities of either AGP or pPGM will be low when compared with WT leaves. Conversely, the alternative starch biosynthetic model, according to which SuSy catalyzes the de novo production of ADPG and AGP plays a role in the scavenging of the glucose units derived from the starch breakdown, predicts that intracellular levels of ADPG in leaves with low activities of chloroplast gluconeogenic enzymes will be normal when compared with those of WT plants (Baroja-Fernndez et al. 2001, Pozueta-Romero et al. 2003, Baroja-Fernndez et al. 2004, Baroja-Fernndez et al. 2005). As a first step to distinguish which of these two metabolic models may entail a predominant role in starch biosynthesis in source leaves, we measured the ADPG content in leaves of TC7 and TL25 Arabidopsis plants lacking pPGM and AGP, respectively (Caspar et al. 1985, Lin et al. 1988). The starchdeficient phenotype of these plants has been used as a crucially important argument to support the classical starch biosynthetic model (Okita 1992). As illustrated in Fig. 3A, meas-

Fig. 3 Starch-deficient AGP and pPGM mutant Arabidopsis leaves contain normal ADPG levels. Starch (A) and ADPG (B) levels in fully expanded source leaves of WT, TL25 (Lin et al. 1988) and TC7 (Caspar et al. 1985) plants. Analyses were conducted using fully expanded leaves after 9 h illumination (300 mol photons s1 m2). ADPG levels were measured by the two chromatographic methods described in Materials and Methods.

urements of starch content in Arabidopsis plants confirmed previous studies showing that both TL25 and TC7 plants contain low starch levels when compared with WT plants (Caspar et al. 1985, Lin et al. 1988). Most importantly however, this reduction in starch content was not accompanied by any measurable reduction in intracellular ADPG levels, which ranged between 0.25 and 0.35 nmol g1 FW (Fig. 3B). Normal levels of ADPG in starch-deficient plants with low AGP activity were not limited to Arabidopsis plants. As illustrated in Fig. 4A, and in conformity with the results of Mller-Rber et al. (1992), leaves of transgenic potato plants having low AGP activity accumulate less starch than leaves of WT plants. In clear contrast, however, ADPG levels in the transgenic lines were shown to be nearly identical to those found in WT plants (Fig. 4B). Collectively, these results strongly indicate that AGP is not the predominant source of ADPG accumulating in photosynthetic cells. Changes in SuSy expression are accompanied by changes in both ADPG content and rate of transitory starch biosynthesis Compared with reserve starch-storing organs, SuSy activity and starch content are considerably lower in source leaves. This enzyme is highly regulated (Huber et al. 1996, Nakai et al.

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Fig. 5 ADPG-producing SuSy activities in WT and both SuSy-antisensed and -overexpressing source leaves (both 35S-SuSy-NOS and RBCS-SuSy-NOS). The results are the mean SE of 10 independent plants per line.
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Fig. 4 Starch-deficient AGP-antisensed potato leaves contain normal ADPG levels. Starch (A) and ADPG (B) levels in fully expanded (fifth) source leaves from 5-week-old WT plants and plants with reduced AGP activity (AGP62 and AGP85, Mller-Rber et al. 1992). Analyses were conducted using fully expanded leaves after 9 h illumination (300 mol photons s1 m2). ADPG levels were measured by the two chromatographic methods described in Materials and Methods.

1998, Asano et al. 2002) and is involved in the sucrosestarch conversion process in sink organs (Zrenner et al. 1995, BarojaFernndez et al. 2003). SuSy is expressed in the mesophyll cells (Fu et al. 1995, Wang et al. 1999), though its physiological role still remains unknown. The alternative model of starch biosynthesis assumes that SuSy catalyzes the production of a sizable pool of ADPG linked to starch biosynthesis. Whether or not SuSy is responsible for the ADPG accumulating in leaves was tested by characterizing both SuSy-overexpressing and -antisensed leaves. The rationale behind this approach was that if this sucrolytic enzyme catalyzes the production of cytosolic ADPG linked to transitory starch biosynthesis in mesophyll cells, varying SuSy activities will be accompanied by changes in both ADPG and starch levels. We therefore proceeded to generate and characterize genetically modified potato plants expressing SuSy either constitutively or in the illuminated leaves [caulifower mosaic virus 35S promoter (35S)-SuSy-nopaline synthase (NOS) and ribulose 1,5-bisphosphate carboxylase small subunit (RBCS)-SuSy-NOS, respectively]. In addition, we characterized the leaves of S-112 and S-129 SuSy-antisensed potato plants (Zrenner et al. 1995). As illustrated in Fig. 5, ADPGproducing SuSy activities in the WT leaves were approxi-

Fig. 6 ADPG levels in fully expanded (fifth) source leaves from 6week-old WT and both SuSy-antisensed and -overexpressing plants (both 35S-SuSy-NOS and RBCS-SuSy-NOS). Leaf samples were taken and quenched in liquid nitrogen 9 h after the beginning of the light period. The results are the mean SE of 10 independent plants per line. ADPG levels were measured by the two chromatographic methods described in Materials and Methods.

mately 15 mU g1 FW, whereas SuSy activities in the leaves of the SuSy-overexpressing and -antisensed transgenic lines were 3590 and 810 mU g1 FW, respectively. Leaves of both SuSy-overexpressing and -antisensed plants were analyzed further for their ADPG and starch contents. As it is our experience that biochemical analyses are subject to considerable variation, we analyzed 10 plants per line to ensure the attainment of reliable data. Importantly, analyses of the ADPG content in leaves of both WT and transgenic plants (Fig. 6, Supplementary Fig. 2) revealed a pattern similar to that of ADPG-producing SuSy activities shown in Fig. 5. Most important was the fact that starch content patterns in both WT and transgenic leaves paralleled that of ADPG-producing SuSy activities shown in Fig. 5 (Fig. 7). During the day, sucrose accumulates in the source leaf (Stitt et al. 1983, Stitt et al. 1984, Gerhardt et al. 1987,

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Fig. 7 Starch levels in fully expanded (fifth) source leaves from 6week-old WT and both SuSy-antisensed and -overexpressing plants (both 35S-SuSy-NOS and RBCS-SuSy-NOS). Leaf samples were taken and quenched in liquid nitrogen 9 h after the beginning of the light period. The results are the mean SE of 10 independent plants per line.

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Draborg et al. 2001, Smith et al. 2004) followed by a decline during the night. If SuSy plays an important role in the starch biosynthetic process by channeling ADPG from sucrose to starch, it can be presumed that both ADPG and starch accumulation patterns will be determined by both sucrose availability and SuSy activity. We therefore compared the ADPG and starch accumulation patterns between WT, 35S-SuSy-NOS (line 5) and RBCS-SuSy-NOS (line 4) leaves throughout the photoperiod. As illustrated in Fig. 8A, these analyses revealed that the highest rate of transient starch accumulation in WT plants (7 nmol of glucose transferred to starch g1 FW min) can be well accounted for by the ADPG-producing SuSy activity measured in vitro (15 mU g1 FW, cf. Fig. 5). Furthermore, these analyses revealed that, essentially similar to the case of starch and sucrose, ADPG transiently accumulates in the photosynthetic cell during the light period (Fig. 8B) followed by a decline during the subsequent night. Most important, however, was the fact that the rates of both ADPG and transitory starch accumulation in both 35S-SuSy-NOS and RBCS-SuSy-NOS leaves were considerably higher than that of WT leaves. The increase in the rates of both ADPG and transitory starch accumulation in SuSy-enhanced lines during the day was mirrored by a similar increase in the rates of both ADPG and starch depletion during the night. Collectively, these results are a strong indication that SuSy plays a dominant role in the transitory starch biosynthetic process in source leaves. Furthermore, these results also indicate that (i) sucrose availability is a limiting step in the transitory starch biosynthetic process and (ii) SuSy catalyzes the production of cytosolic ADPG accumulating in the leaf.

Fig. 8 Time course analysis of (A) starch and (B) ADPG content in leaves of 5-week-old WT (circle), 35S-SuSy (line 5, square) and RBCSSuSy-NOS (line 4, triangle) plants grown in chambers under an 8 h/ 16 h light (300 mol photons s1 m2, 20C)/dark (20C) regime. At each time point, samples were taken from mature source leaves and the data represent the means SE of measurements on 10 plants per line.

SuSy overexpression is not accompanied by pleiotropic changes in enzymes directly connected to starch and sucrose metabolism In order to verify that increases in ADPG and starch contents in SuSy-overexpressing leaves were not due to changes in the activities of enzymes other than SuSy, we measured the maximum catalytic activities of a range of enzymes closely connected to starch and sucrose metabolism. These analyses revealed no significant changes in AGP, alkaline pyrophosphatase, UGP, SPS, acid invertase, total starch synthase, starch phosphorylase and total amylolytic activity (Table 1, Supplementary Table 1). SuSy overexpression does not affect the photosynthetic capacity of the plant Given that both ADPG and starch levels are higher in the leaves of SuSy-overexpressing plants than in the WT plants, we proceeded to investigate whether transgenic plants dis-

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Table 1

Enzyme activities (mU g1 FW) in WT and 35S-SuSy-NOS potato leaves WT 6 5 35S-SuSy-NOS 12 3 4 7

AGP 144 6 149 5 140 3 148 7 124 6 122 7 137 7 UGP 107 3 113 5 108 5 116 4 125 5 103 4 116 8 Acid invertase 118 9 137 11 104 8 155 9 125 7 120 9 146 12 SPS 2,855 220 3,279 245 3,235 362 3,211 264 2,875 303 2,542 231 3,611 316 Alkaline 756 61 879 80 880 70 717 65 786 72 762 74 762 75 pyrophosphatase Amylolytic activity 114 9 135 10 119 5 124 6 125 6 143 8 133 10 Total starch synthase 6.1 1 6.6 0.5 5.9 0.9 7.0 1 6.7 0.7 5.9 0.8 7.9 0.9 Starch phosphorylase 39 6 39 7 33 6 49 12 35 7 43 9 44 8
The activities were determined in samples from source leaves of 6-week-old plants grown in chambers at ambient CO2 conditions, 20C and at an irradiance of 300 mol photons s1 m2. Leaf samples were taken and quenched in liquid nitrogen 7 h after the beginning of the light period. The results are the mean SE of extracts from 10 independent plants per line.
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Table 2 Photosynthetic parameters of whole leaves attached to 6-week-old WT and SuSy-expressing plants grown in the same conditions as in Table 1 WT 6 5 35S-SuSy-NOS 12 3 4 7

O2 evolution 9.1 1.1 8.5 1.2 9.1 1.1 9.0 1.0 9.6 0.5 8.9 1.3 9.0 1.1 (mol m2 s1) Transpiration 1.8 0.4 2.3 0.3 2.0 0.3 1.9 0.5 2.2 0.4 1.8 0.2 1.8 0.3 (mmol m2 s1) Stomatal conductance of 170 41 200 25 180 20 190 35 180 30 130 20 150 40 CO2 (mmol m2 s1) Substomatal CO2 (ppm) 372 53 374 19 379 18 362 39 355 39 342 21 366 30 Chlorophyll (mg/g FW) 0.80 0.06 0.88 0.07 0.80 0.06 0.77 0.01 0.82 0.01 0.75 0.08 0.80 0.02
The results are the mean SE of extracts from 10 independent plants per line.

played increased photosynthetic activity. Towards this end, photosynthetic parameters of whole attached leaves in SuSyexpressing and control plants were compared at light intensities in which the plants were grown (300 mol photon m2 s1) and at ambient CO2 (350 ppm). Our results revealed no significant changes in the rates of O2 production, transpiration, CO2 stomatal conductance and substomatal CO2 concentration (Table 2, Supplementary Table 2). Furthermore, there were no significant differences between chlorophyll contents of the SuSy lines and WT plants. Therefore, increase of both ADPG and starch contents in the leaves of SuSy-overexpressing plants cannot be ascribed to increased photosynthetic capacities. Metabolic profile of SuSy-overexpressing leaves Leaves from both 35S-SuSy-NOS and RBCS-SuSy-NOS plants were shown to accumulate normal levels of glucose, fructose, 3PGA and Pi when compared with control plants (Table 3, Supplementary Table 3). In contrast, G1P levels in the SuSy-overexpressing leaves decreased up to 2-fold when compared with control plants. This observation is highly significant given previous observations that reducing the levels of

cytosolic ADPG is accompanied by an increase of total G1P levels (Baroja-Fernndez et al. 2004), strongly indicating that G1P and cytosolic ADPG pools are closely connected. Levels of G6P, an allosteric effector of SPS (Doehlert and Huber 1983, Huber and Huber 1996), were 3040% higher in the SuSyoverexpressing leaves than in the WT leaves. As a possible consequence of the stimulatory effect of high G6P levels on SPS, SuSy-overexpressing leaves were shown to accumulate 3040% more sucrose than WT leaves. Additional remarks In source leaves, SuSy plays a pivotal role in basic aspects of plant survival such as supplying energy for the sucrose loading and unloading process (Martin et al. 1993, Nolte and Koch 1993). Taking into account all the limitations that are inherent in basing conclusions on genetically engineered and mutant plants, results from the work presented in this communication show that SuSy exerts a strong control on the starch biosynthetic process in source leaves. Furthermore, evidence is provided that SuSy, but not AGP, catalyzes the production of ADPG accumulating in source leaves (Fig. 3, 4).

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Table 3

Metabolite levels (given in nmol g1 FW) in control (WT) and 35S-SuSy-NOS source leaves Control WT 6 5 35S-SuSy-NOS 12 3 4 7

Glucose 848 31 922 29 860 30 933 29 881 56 895 32 871 60 Fructose 996 43 1,035 67 1,094 17 1,022 10 1,067 58 1,078 63 817 41 Sucrose 1,012 27 1,529 48 1,402 68 1,642 58 1,307 35 1,317 35 1,391 70 G6P 244 28 329 15 320 25 291 27 355 23 298 12 315 9.8 G1P 22.7 1.9 15.5 2.1 10.3 1.1 9.9 1.2 9.5 1.5 15.2 1.9 11.4 1.8 3PGA 324 42 354 40 332 21 362 12 386 27 311 47 359 27 Pi 40.6 2.8 36.9 1.2 39.9 2.1 43.7 3.1 39.8 0.5 41.6 1.7 42.3 0.9 3PGA/Pi ratio 8.1 1.0 9.5 1.3 8.3 1.0 8.2 1.0 9.6 0.9 7.5 1.1 8.3 0.7
Leaf samples were taken from 6-week-old plants grown in chambers at ambient CO2 conditions, 20C and at an irradiance of 300 mol photons s1 m2. Leaves were taken and quenched in liquid nitrogen 7 h after the beginning of the light period. The results are the mean SE of extracts from 10 independent plants per line. Values that are significantly different from the control plants are marked in bold.
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SuSy has the capacity of producing ADPG from sucrose (Delmer 1972, Porchia et al. 1999, Baroja-Fernndez et al. 2003). It is therefore highly conceivable that the high levels of ADPG occurring in SuSy-overexpressing leaves (Fig. 6) are due to the direct conversion of sucrose into ADPG by means of SuSy. An alternative explanation for our results is that, essentially identical to the classical view on sucrosestarch conversion in heterotrophic tissues (Neuhaus and Emes 2000), SuSy converts sucrose into UDPG, which is then stepwise transformed to G1P and ADPG by the coupled reactions of UGP and AGP. This possibility, however, is highly unlikely because (i) UGP in leaves functions primarily in the production of UDPG rather than in its degradation and (ii) G1P levels in SuSy-overexpressing leaves are lower than in WT leaves (Table 3, Supplementary Table 3). Furthermore, this presumption indirectly implies the operation of a G1P transport machinery which, as shown by Quick et al. (1995), does not occur in the envelope membranes of chloroplasts. Also, and contrary to our recent findings showing the extraplastidial localization of ADPG (Baroja-Fernndez et al. 2004), this possibility would imply that ADPG in the SuSy-overexpressing leaves would accumulate in the chloroplast. A second alternative explanation for the occurrence of high levels of both ADPG and starch in the SuSy-overexpressing leaves is that high SuSy activity alters the exchange of Pi and triose phosphates between the cytosol and the chloroplast. This situation will lead to a high 3PGA to Pi ratio in the chloroplast, thus activating AGP. This possibility, however, is highly unlikely since the 3PGA to Pi ratio in SuSy-overexpressing leaves is normal when compared with WT leaves (Table 3, Supplementary Table 3). The overall data thus strongly indicate that the starch biosynthetic process occurring in the chloroplast is tightly linked to the cytosolic sucrose metabolism by means of ADPG-producing SuSy activity.

Materials and Methods


Plants, bacterial strains and media This work was carried out using plants regenerated from WT potato plants (S. tuberosum L. cv Desire), and plants transformed with either anti-AGP-1 (Mller-Rber et al. 1992, lines 62 and 85), anti-SuSy (Zrenner et al. 1995, lines S-112 and S-129), 35S-SuSyNOS terminator or RBCS-SuSy-NOS (see below). Columbia WT, TL25 and TC7 Arabidopsis plants were obtained from the Nottingham Arabidopsis Stock Centre. Unless otherwise indicated, plants were grown at ambient CO2 (350 ppm) for 46 weeks in growth chambers with a 16 h light (300 mol photons s1 m2) photoperiod and at a constant temperature of 20C. For biochemical analyses, fully expanded source leaves were harvested at the indicated times of illumination, immediately quenched in liquid nitrogen and stored at 80C before use. Agrobacterium tumefaciens cells, strain C58C1:GV2260, transformed with either pBIN35S-SuSy-NOS or pBINRBCS-SuSy-NOS (see below) were grown in yeast extract broth medium, with the appropriate selection using standard techniques. Production of SuSy-overexpressing potato plants For the production of 35S-SuSy-NOS plants, a complete Sus4 cDNA (EMBL accession no. AJ537575) was amplified by PCR using the primers 5-GTCTCGATAAGCTTCTGCCATGGCTGAACGTGTTTTGACTCG-3 (forward) and 5-CCGGTGGATCCCCCCTTCATTCACTCAGCAGCCAATGGAAC-3 (reverse) and a potato cDNA library. The approximately 2,400 bp PCR product obtained was cloned into pBluescript SK- to produce pSK-SuSy (Supplementary Fig. 3). The NcoIBamHI fragment of pSK-SuSy was ligated with the corresponding restriction sites of p35S-NOS (Baroja-Fernndez et al. 2004) to produce p35S-SuSy-NOS. This construct was digested successively with HindIII, T4 DNA polymerase and NotI. The fragment thus released was cloned into the pBIN20 plant expression vector (Hennegan and Danna 1998) previously digested successively with the enzymes HindIII, T4 DNA polymerase and EcoRI to produce pBIN35S-SuSy-NOS. For production of SuSy-overexpressing leaves, approximately 1,000 bp of the 5-upstream region of the tobacco RBCS gene (EMBL accession no. X02353) was amplified by PCR that confers expression to illuminated chloroplast-containing cells (Barnes et al. 1994). Towards this end, we used tobacco genomic DNA and the following primers: (forward) 5-AAGCTTGTGGGAACGAGATAAGGGC-3; (reverse) 5-CCATGGTTAATTACACTTAGACAGAAAGATTTCTC-

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SuSy controls starch production in leaves Production and purification of recombinant ASPP pET-ASPP cells (Moreno-Bruna et al. 2001) were grown in 100 ml of liquid LB medium to an absorbance at 600 nm of about 0.5 and then 0.3 mM isopropyl--D-thiogalactopyranoside (IPTG) was added. After 3 h, cells were centrifuged at 6,000g for 10 min. The pelleted bacteria were resuspended in 4 ml of His-bind binding buffer (Novagen), sonicated and centrifuged at 10,000g for 10 min. The supernatant thus obtained was subjected to His-bind chromatography (Novagen). The eluted His-tagged ASPP was then rapidly desalted by ultrafiltration on Centricon YM-10 (Amicon, Beford, MA, USA). SDSPAGE of the purified His-tagged ASPP preparation and subsequent stain revealed a single approximately 30 kDa band (not shown). Analytical procedures Starch in desalted plant extracts obtained by precipitation with 70% ethanol was measured by using an amyloglucosidase-based test kit (Sigma-Aldrich Chemical Co, St Louis, MO, USA). Chlorophyll was quantified according to the method of Wintermans and de Mots (1965). Photosynthetic parameters of the first fully expanded (fifth) leaves attached to the potato plants were determined under growth conditions by means of an Lci portable photosynthesis system (ADC BioScientfic Ltd., Hoddesdon, Herts, UK) in the same chamber where the leaves were grown. Supplementary material Supplementary material mentioned in the article is available to online subscribers at the journal website www.pcp.oupjournals.org.

TTTCTCTCTTTTCCTTTAACTTCC-3. The fragment thus obtained was cloned into pGEMT-easy vector (Promega) to produce pGEMTRBCSprom (Supplementary Fig. 4). This construct was digested with HindIII and NcoI and the fragment released was ligated with the corresponding restriction sites of p35S-SuSy-NOS to produce pRBCS-SuSyNOS. This final construct was digested successively with HindIII, T4 DNA polymerase and NotI. The resulting fragment was cloned into pBIN20 previously digested successively with the enzymes HindIII, T4 DNA polymerase and EcoRI to produce pBINRBCS-SuSy-NOS. Transfer of constructs into A. tumefaciens was carried out by electroporation. Subsequent transformation of potato plants was conducted as described by Baroja-Fernndez et al. (2004). Transgenic plants were selected on kanamycin-containing medium. The presence of the recombinant Sus4 was confirmed by Southern blot analyses using radiolabeled Sus4 cDNA as probe. A second screening of the transgenic lines was performed by SuSy activity. Six independent lines per construct were selected and used for all subsequent analyses described in this report. Enzyme assays All enzymatic reactions were carried out at 37C. Harvested leaves were immediately freeze-clamped and ground to a fine powder in liquid nitrogen with a pestle and mortar. To assay enzyme activity, 1 g of the frozen powder was resuspended at 4C in 5 ml of 100 mM HEPES (pH 7.5), 2 mM EDTA and 5 mM dithiothreitol. The suspension was desalted and assayed for enzymatic activities. We checked that this procedure did not result in loss of enzymatic activity as evidenced by comparing the activity in extracts prepared from the frozen powder and extracts prepared by homogenizing fresh tissue in extraction medium. AGP, acid invertase, UGP, alkaline pyrophosphatase, total starch synthase, starch phosphorylase, total amylolytic and SPS activities were assayed as described by Baroja-Fernndez et al. (2004). Measurements of SuSy were performed in the direction of ADPG synthesis. The assay mixture contained 50 mM HEPES (pH 7.0), 1 mM MgCl2, 15 mM KCl, 250 mM sucrose and 2 mM ADP. After 5 min at 37C, reactions were stopped by boiling the assay mixture for 1 min. ADPG was measured by HPLC on a Waters Associates system fitted with a Partisil-10-SAX column as described by Sweetlove et al. (1996). We define 1 U of enzyme activity as the amount of enzyme that catalyzes the production of 1 mol of product min1. Determination of soluble sugars Fully expanded leaves were harvested and immediately ground to a fine powder in liquid nitrogen with a pestle and mortar. A 0.5 g aliquot of the frozen powdered tissue was resuspended in 0.4 ml of 1.4 M HClO4, left at 4C for 2 h and centrifuged at 10,000g for 5 min. The supernatant was neutralized with K2CO3 and centrifuged at 10,000g. ADPG content in the supernatant was determined by using either one of the following methods: (i) by HPLC on a system obtained from P.E. Waters and Associates fitted with a Partisil-10-SAX column as described by Sweetlove et al. (1996); or (ii) by HPLC with pulsed amperometric detection on a DX-500 system (Dionex) fitted to a CarboPac PA10 column. To confirm further that measurements of ADPG were correct, ADPG eluted from either the Partisil-10-SAX or CarboPac PA10 columns was enzymatically hydrolyzed with purified E. coli ASPP (see below) and assayed for conversion into AMP and G1P. Glucose, sucrose, fructose, G1P and G6P were determined by HPLC with pulsed amperometric detection on a DX-500 system as described by Baroja-Fernndez et al. (2003). 3PGA and Pi were determined as described by Lytovchenko et al. (2002)

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Acknowledgments
This research was partially supported by the grants BIO200 1080 and BIO200401922 from the Comisin Interministerial de Ciencia y Tecnologa and Fondo Europeo de Desarrollo Regional (Spain). M.T.M.-Z. acknowledges the Spanish Ministry of Culture and Education for a pre-doctoral felloship. A.M.V. expresses his gratitude to the Spanish Ministry of Culture and Education for financial support. E.E. expresses his gratitude to the Spanish Ministry of Culture and Education, to the Consejo Superior de Investigaciones Cientficas and to the Public University of Nafarroa for their support. We thank Vanessa Rubio, Maria Jos Villafranca and Sorospen Barn Lacunza (I.A.R.N., Spain) for expert technical support. We are indebted to Dr. R. N. Trethewey (Metanomics GmbH, Berlin) who kindly made us a gift of the anti-AGP1 and anti-SuSy plants. We are also indebted to Axel Tiessen (Max Planck Institut, Golm, Germany), Eckehard Neuhaus (University of Kaiserslautern, Germany) and Kay Denyer (John Innes Centre, Norwich, UK) for critical discussions and readings of the manuscript.

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(Received April 27, 2005; Accepted June 3, 2005)