Characterization of multiple SPS knockout mutants reveals redundant functions of the four Arabidopsis sucrose phosphate synthase isoforms in plant viability, and strongly indicates that enhanced respiration and accelerated starch turnover can alleviate the blockage of sucrose biosynthesis

1 Title: Characterization of multiple SPS knockout mutants reveals redundant functions of the four Arabidopsis sucrose phosphate synthase isoforms in plant viability, and strongly indicates that enhanced respiration and accelerated starch turnover can alleviate the blockage of sucrose biosynthesis Authors: Abdellatif Bahaji¶1, Edurne Baroja-Fernández¶1, Adriana Ricarte-Bermejo1, Ángela María Sánchez-López1, Francisco José Muñoz1, Jose M. Romero2, María Teresa Ruiz2, Marouane Baslam1, Goizeder Almagro1, María Teresa Sesma1 and Javier Pozueta- Romero*1 Affiliations: (1) Instituto de Agrobiotecnología (CSIC/UPNA/Gobierno de Navarra). Iruñako etorbidea 123, 31192 Mutiloabeti, Nafarroa, Spain. (2) Instituto de Bioquímica Vegetal y Fotosíntesis. Universidad de Sevilla-CSIC. Avenida Américo Vespucio 49. Isla de la Cartuja, 41092, Sevilla, Spain. *Corresponding author: Javier Pozueta Romero. Address: Instituto de Agrobiotecnología. Iruñako etorbidea 123, 31192 Mutiloabeti, Nafarroa, Spain. E-mail: javier.pozue[email protected]. Phone: (34) 948168009. FAX: (34) 948232191 ¶A.B. and E.B.-F. have equally contributed to this work. Abbreviations: An, net photosynthetic CO2 fixation rate; ADPG, ADP-glucose; AGP, ADPG pyrophosphorylase; Ci, intercellular CO2 concentrations; CL, continuous light; DAG, days after germination; F1,6P2, fructose-1,6-bisphosphate; F6P, fructose-6- phosphate; G1P, glucose-1-phosphate; G6P, glucose-6-phosphate; G6PDH, G6P dehydrogenase; gs, stomatal conductance; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HK, hexokinase; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; OPPP, oxidative pentose phosphate pathway; PEP, phospho-enol- pyruvate; 3-PGA, 3-phosphoglycerate; PGI, phosphoglucose isomerase; 6PGDH, 6- phosphogluconate dehydrogenase; PGM, phosphoglucomutase; Pi, inorganic orthophosphate; PK, pyruvate kinase; PPase, alkaline pyrophosphatase; S6P, sucrose-6- phosphate; SDH, succinate dehydrogenase; SPP, sucrose-phosphate phosphatase; SPS, 2 sucrose-phosphate synthase; SS, starch synthase; SuSy, sucrose synthase; TCA, tricarboxylic acid cycle; TPT, triose-phosphate/phosphate translocator; U, unit of enzyme activity; UDPG, UDP-glucose; UGP, UDPG pyrophosphorylase; WT, wild type 3 ABSTRACT We characterized multiple knock-out mutants of the four Arabidopsis sucrose phosphate synthase (SPSA1, SPSA2, SPSB and SPSC) isoforms. Despite their reduced SPS activity, spsa1/spsa2, spsa1/spsb, spsa2/spsb, spsa2/spsc, spsb/spsc, spsa1/spsa2/spsb and spsa2/spsb/spsc mutants displayed wild type (WT) vegetative and reproductive morphology, and showed WT photosynthetic capacity and respiration. In contrast, growth of rosettes, flowers and siliques of the spsa1/spsc and spsa1/spsa2/spsc mutants was reduced compared with WT plants. Furthermore, these plants displayed a high dark respiration phenotype. spsa1/spsb/spsc and spsa1/spsa2/spsb/spsc seeds poorly germinated and produced aberrant and sterile plants. Leaves of all viable sps mutants, except spsa1/spsc and spsa1/spsa2/spsc, accumulated WT levels of nonstructural carbohydrates. spsa1/spsc leaves possessed high levels of metabolic intermediates and activities of enzymes of the glycolytic and tricarboxylic acid cycle pathways, and accumulated high levels of metabolic intermediates of the nocturnal starch-to-sucrose conversion process, even under continuous light conditions. Results presented in this work show that SPS is essential for plant viability, reveal redundant functions of the four SPS isoforms in processes that are important for plant growth and nonstructural carbohydrate metabolism, and strongly indicate that accelerated starch turnover and enhanced respiration can alleviate the blockage of sucrose biosynthesis in spsa1/spsc leaves. Keywords: Carbohydrate metabolism, development, functional interaction, genetic redundancy, growth, sucrose 4 1. Introduction In the majority of higher plants sucrose is the main end-product of photosynthesis. It serves as the mobile form of photoassimilate that is transported from leaves to sink organs. This disaccharide plays a major role in growth, and also acts as a signaling molecule in the control of the expression of genes involved in multiple processes such as central carbon and nitrogen metabolisms [1], storage of proteins [2], cell cycle and differentiation [3], flowering [4] and seed development [5]. During the day, photosynthetically fixed carbon is either retained within the chloroplast of leaf mesophyll cells to fuel the synthesis of transitory starch, or exported to the cytosol as triose phosphates by means of the triose-phosphate/phosphate translocator (TPT) to be converted into activated forms of hexoses and sucrose (Fig. S1A). Sucrose is synthesized by the action of two enzymes: sucrose-phosphate synthase (SPS, which catalyzes the conversion of fructose-6-phosphate (F6P) and UDP-glucose (UDPG) into sucrose-6- phosphate (S6P)), and sucrose-phosphate phosphatase (SPP, which hydrolyzes S6P to produce sucrose) (Fig. S1A). During the night, starch is mobilized to produce maltose that is transported to the cytosol by means of the MEX1 translocator (Fig. S1B). Once in the cytosol maltose is converted into heteroglycans, glucose-1-P (G1P), UDPG, S6P and sucrose by the stepwise reactions of the cytosolic disproportionating enzyme DPE2, glucan phosphorylase PHS2, UDPG pyrophosphorylase (UGP), SPS and SPP [6-8] (Fig. S1B). Glucose, another starch breakdown product, can be transported to the cytosol by means of the pGlcT transporter [9]. Once in the cytosol, glucose can be converted into glucose-6-P (G6P), G1P, UDPG, S6P and sucrose by the stepwise reactions of hexokinase (HK), phosphoglucomutase (PGM), UGP, SPS and SPP [9] (Fig. S1B). SPS is a key control point of carbon flux into sucrose that is regulated by a hierarchy of mechanisms including posttranslational modification via protein phosphorylation, activation by G6P and inhibition by inorganic orthophosphate (Pi) [10,11], and transcriptional regulation of SPS gene expression [12,13]. SPS isoforms in the many plant species examined to date are encoded by a small SPS multigene family. Studies of the predicted amino acid sequences and gene structure have shown that the Arabidopsis SPS family consists of four SPS genes, referred to as AtSPSA1 (At5g20280), AtSPSA2 (At5g11110), AtSPSB (At1g04920) and AtSPSC (At4g10120) [14,15]. Genome- 5 wide expression analyses (https://www.geneinvestigator.ethz.ch) and comparative studies of SPS gene expression in Arabidopsis [14,15] provided evidence for distinct, but partially overlapping spatial and temporal expression patterns for the four SPS genes. Metabolic studies of an spsa1/spsc double knockout Arabidopsis mutant revealed effects on growth and leaf nonstructural carbohydrate metabolism in this mutant [15]. Thus spsa1/spsc plants cultured under 8 h light/16 h dark photoregime displayed a dwarf phenotype. Also, these plants accumulated low levels of sucrose and moderately high levels of both starch and maltose when compared with wild type (WT) plants, strongly indicating that SPSA1 and SPSC have overlapping functions in aspects related with growth and leaf nonstructural carbohydrate metabolism. According to Volkert et al. [15], the increase in starch was probably not due to an increased partitioning of carbon into starch, but was rather caused by an impaired starch mobilization during the night due to impairment in downstream metabolization of maltose. Mutants impaired in TPT and cytosolic fructose 1,6-bisphosphatase display a nearly WT growth phenotype [16-19], strongly indicating the operation in these mutants of mechanism(s) of diurnal sucrose biosynthesis additional/alternative to that illustrated in Fig. S1A, and showing that TPT and cytosolic fructose 1,6-bisphosphatase are not essential for plant viability. While SPS catalyzes an undoubtedly crucial step in sucrose biosynthesis, the challenge still remains to determine if SPS is an essential function for plant viability and if, in addition to the functional overlapping occurring between SPSA1 and SPSC in planta, there are other functional interactions between the four SPS isoforms. Towards the end of exploring possible interactions between the four SPS isoforms in planta, and between sucrose biosynthesis and other metabolic pathways when SPS-mediated sucrose production is limited, in this work we conducted a comprehensive study of different multiple SPS knock-out mutants. Results presented in this work show that, in Arabidopsis, (a) SPS is essential for plant viability, implying that sucrose is mainly synthesized through the SPS-SPP pathway and (b) the four SPS isoforms are functionally redundant in processes that are important for plant growth, vegetative and reproductive development, and nonstructural carbohydrate metabolism. Furthermore, the results provide strong evidence supporting the occurrence in illuminated spsa1/spsc leaves of mechanisms alleviating the blockage of the starch-to-S6P conversion process 6 such as accelerated starch turnover, and channeling of starch breakdown products towards the glycolytic, oxidative pentose phosphate (OPP) and tricarboxylic acid cycle (TCA) pathways. 2. Materials and methods 2.1. Plants, growth conditions and sampling Unless otherwise indicated WT Arabidopsis thaliana L. (Heynh) ecotype Columbia and T-DNA insertion lines in this background were cultured in soil in growth chambers under the indicated photoperiod conditions (light intensity of 90 µmol photons s–1 m-2) (22oC during the light period and 18oC during the dark period). Harvested source leaves were immediately freeze-clamped and ground to a fine powder in liquid nitrogen with a pestle and mortar. 2.2. Production of multiple T-DNA knock-out lines The T-DNA insertion mutants spsa1 (SALK_119162), spsa2 (SALK_064922), spsb (GABI_368F01) and spsc (SAIL_31_H05) were obtained from the European Arabidopsis Stock Center (NASC) (Fig. S2). The T-DNA insertion in spsa1 mutant is in the third intron, whereas the T-DNA insertions in spsa2, spsb and spsc are in the fifth, ninth and fifth exon of SPSA2, SPSB and SPSC, respectively (Fig. S2). By crossing these mutants, self-pollinating the resulting heterozygous mutants, and PCR screening for homozygous progeny using the oligonucleotide primers listed in Table S2 we produced the spsa1/spsa2, spsa1/spsb, spsa1/spsc, spsa2/spsb, spsa2/spsc, spsb/spsc, spsa1/spsb/spsc, spsa1/spsa2/spsb, spsa1/spsa2/spsc, spsa2/spsb/spsc, and spsa1/spsa2/spsb/spsc mutants (Table S1, Fig. S3). The knock-out status of the T-DNA mutants was confirmed by RT-PCR for SPS transcripts using specific primers that spanned the T-DNA insert site of each gene (Table S3). To this end, total RNA was extracted from leaves using the trizol method according to the manufacturer´s procedure (Invitrogen). RNA was treated with RNAase free DNAase (Takara). RT-PCR was conducted with SuperScript III one-step RT-PCR with Platinum Taq DNA polymerase kit (12574-018; Invitrogen) using 100 ng of RNA and the SPSA1, SPSA2, SPSB and SPSC specific primers listed in Table S3. 18S RNA was used 7 as the positive control. PCR products were separated on 1% (w/v) agarose gels containing ethidium bromide and visualized by ultraviolet light. SPSA1, SPSA2, SPSB and SPSC PCR products were detected in WT plants, but were undetectable in spsa1, spsa2, spsb and spsc mutants, respectively (Fig. S4). 2.3. Enzyme assays One g of the frozen powder (see above) was resuspended at 4oC in 3 mL of 100 mM HEPES (pH 7.5), 2 mM EDTA, 2 mM dithiothreitol, 1 mM PMSF and 10 mL/L protease inhibitor cocktail (Sigma P9599), and centrifuged at 14,000 x g for 20 min. The supernatant was desalted by ultrafiltration on Vivaspin 500 centrifugal concentrator (Sartorius, Ref. VS0102) and the protein extract thus obtained was assayed for enzymatic activities. ADP-glucose (ADPG) pyrophosphorylase (AGP) and UGP activities were measured following the two-step assay method described in [20]. Phosphoglucose isomerase (PGI) and sucrose synthase (SuSy) activities were measured as described in [21] and [22], respectively. PGM and amylolytic activities were assayed as described in [23] and [24], respectively. Acid and alkaline invertases were measured as described in [25]. HK activity was assayed as described in [26]. Alkaline pyrophosphatase (PPase) and SPS were measured as described in [27]. Starch synthase (SS) activity was measured as described in [28]. Fructose-1,6-bisphosphate (F1,6P2) aldolase, glyceraldehyde-3- phosphate dehydrogenase (G3PDH), 3-phosphoglycerate (3PGA) kinase and pyruvate kinase (PK) were measured as described in [29]. G6P dehydrogenase (G6PDH), 6- phosphogluconate dehydrogenase (6PGDH) and isocitrate dehydrogenase (IDH) were measured as described in [30]. Malate dehydrogenase (MDH) and succinate dehydrogenase (SDH) were measured as described in [31] and [32], respectively. One unit (U) is defined as the amount of enzyme that catalyzes the production of 1 µmol of product per min. 2.4. Non-reducing western blot analyses of AGP For non-reducing western blots of AGP, 50 mg of the homogenized frozen material (see above) was extracted in cold 16% (w/v) trichloroacetic acid in diethyl ether, mixed, and stored at –20oC for at least 2 h as described in [20]. The pellet was collected by 8 centrifugation at 10,000 x g for 5 min at 4oC, washed 3 times with ice-cold acetone, dried briefly under vacuum, and resuspended in 1x Laemmli sample buffer containing no reductant. Protein samples were separated on 10% SDS-PAGE, transferred to nitrocellulose filters, and immunodecorated by using antisera raised against maize AGP as primary antibody [20], and a goat anti-rabbit IgG alkaline phosphatase conjugate (Sigma) as secondary antibody. 2.5. Native gel assay for PGM activity PGM zymograms were performed essentially as described in [23]. Protein extracts (see above) of both WT and spsa1/spsc leaves were loaded onto a 7.5% (w/v) polyacrylamide gel. After electrophoresis gels were stained by incubating in darkness at room temperature with 0.1 M Tris-HCl (pH 8.0), 5 mM G1P, 1 mM NAD+, 4 mM MgCl2, 0.2 mM methylthiazolyldiphenyl-tetrazolium bromide (Sigma M5655) and 0.25 mM phenazine methosulfate (Sigma P9625) and 1 U/mL of G6PDH from Leuconostoc mesenteroides (Sigma G8404). 2.6. Analytical procedures ADPG content was measured by HPLC-MS/MS as described in [33]. 3PGA was determined as described in [34]. For measurement of sucrose, glucose, maltose and fructose, a 0.1 g aliquot of the frozen leaf powder (see above) was resuspended in 1 mL of 90% ethanol, left at 70oC for 90 min and centrifuged at 13,000 x g for 10 min. For measurement of G6P, F6P, F1,6P2, S6P, UDPG and G1P 0.1 g aliquot of the frozen leaf powder was resuspended in 1 mL of 1 M HClO4, left at 4oC for 2 h and centrifuged at 10,000 x g for 5 min. The supernatant was neutralized with K2CO3 and centrifuged at 10,000 x g. Sucrose, glucose, fructose, maltose, F6P, F1,6P2, G6P, S6P and G1P from the above supernatants were determined by HPLC with pulsed amperometric detection on a DX-500 Dionex system by gradient separation with a CarboPac 10 column according to the application method suggested by the supplier (100 mM NaOH/100 mM sodium acetate to 100 mM NaOH/500 mM sodium acetate in 40 min). UDPG was measured as described in [35] by HPLC on a system obtained from Waters Associates fitted with a Partisil-10-SAX column. Starch was measured by using an amyloglucosydase–based test 9 kit (Boehringer Mannheim, Germany). Oxaloacetate, pyruvate, phospho-enol-pyruvate (PEP) and isocitrate were determined as described in [36]. Recovery experiments were carried out by the addition of known amounts of metabolites standards to the frozen tissue slurry immediately after addition of extraction solutions. The difference in the measured amounts between the samples with and without added standards was used as an estimate of the percentage of recovery. All data were corrected for loss during extraction. 2.7. Iodine staining and microscopic localization of starch granules Leaves harvested at the end of the light period were fixed by immersion into 3.7% formaldehyde in phosphate buffer. Leaf pigments were then removed in 96% ethanol. Rehydrated samples were stained in iodine solution (KI 2% (w/v) I2 1% (w/v)) for 30 min, rinsed briefly in deionized water and photographed. Samples for sectioning were immersed in cryoprotective medium OCT (Tissue-Tec, USA) and frozen at -50ºC. Cryosections of 10 µm thick were obtained in AS620 Cryotome (Shandon, England). After thawing, sections were stained in iodine solution for 2 min at room temperature, mounted to microscope slides and observed using a stereomicroscope Olympus MVX10 (Japan). Microphotographs were captured with video camera DP72 (Olympus, Japan) and Cell D software (Olympus, Japan). 2.8. Gas exchange determinations Fully expanded apical leaves were enclosed in a LCipro portable photosynthesis system (ADC BioScientfic Ltd., Hoddesdon, Herts). Gas exchange parameters including net photosynthetic CO2 fixation rate (An), stomatal conductance (gs) and intercellular CO2 concentrations (Ci) were measured at 25ºC with photosynthetic photon flux densities of 90 and 350 µmol m-2 s-1 and CO2 concentration of 450 µmol mol-1. An was calculated using equations developed in [37]. gs values were determined as described in [38]. The rate of mitochondrial respiration in the dark was determined by measuring the rate of CO2 evolution in the dark. 2.9. Statistical analysis The data presented are the means of three independent experiments, with 3-5 replicates 16 levels of maltose and of intermediates in the maltose-to-S6P conversion process in leaves of spsa1/spsc plants cultured under CL conditions should be high when compared with WT leaves. Furthermore, due to feedback inhibition of amylolytic breakdown of starch by excess maltose, levels of starch in spsa1/spsc leaves should be high when cultured under CL conditions. As shown in Fig. 6A, spsa1/spsc plants cultured under CL conditions displayed a dwarf phenotype. Furthermore, their leaves accumulated higher levels of G6P, G1P and UDPG than WT leaves, and exceedingly higher levels of starch and maltose than WT leaves (Fig. 6B). Expectedly, sucrose and S6P contents in spsa1/spsc were lower than in WT leaves (Fig. 6B). The overall data thus provided strong evidence that (a) continuously illuminated spsa1/spsc leaves degrade starch, and (b) the accumulation of high levels of maltose, G6P, G1P and UDPG, and low levels of sucrose and S6P in leaves of spsa1/spsc plants cultured under CL conditions is the consequence of impaired conversion of maltose into S6P. We also measured maltose and starch contents at the end of the light and dark periods in leaves of WT and spsa1/spsc plants cultured under three different photoregimes (12 h light/12 h dark, 16 h light/8 h dark and 20 h light/4 h dark), and compared them with those of leaves of plants cultured under CL conditions. As shown in Fig. 7, irrespective of the photoregime, WT and spsa1/spsc leaves accumulated lower levels of starch and higher levels of maltose at the end of the dark period than at the end of the light period, indicating the occurrence of amylolytic starch breakdown during the night. Furthermore, irrespective of the photoperiod condition and day moment, starch and maltose contents in spsa1/spsc leaves were exceedingly higher than those of WT leaves. Such differences in maltose and starch contents between leaves of WT and spsa1/spsc plants cultured under 12 h light/12 h dark, 16 h light/8 h dark and 20 h light/4 h dark photoregimes were much more pronounced than those occurring between leaves of WT and spsa1/spsc plants cultured under 8 h light/16 h dark photoperiod conditions ([15] and data not shown). Moreover, maltose and starch contents at the end of the light period in leaves of spsa1/spsc plants cultured under 16 h light/8 h dark and 20 h light/4 h dark photoregimes were similar to those of leaves of spsa1/spsc plants cultured under CL conditions (Fig. 7). The overall data thus strongly indicate that amylolytic production of maltose occurs not only during the night, but also during the day in spsa1/spsc leaves. 17 4. Discussion Results presented in this work describing the phenotypic and metabolic characteristics of different multiple SPS knockout mutants (summarized in Table S1) provide strong evidence that (a) the four SPS isoforms overlap in functions that are essential for normal seed development and germination and subsequent development of the plant, (b) the four SPS isoforms play redundant functions in process that are important for nonstructural carbohydrate metabolism (c) SPSA2 expression does not compensate the detrimental effects caused by the complete loss of SPSA1+SPSB+SPSC expression, (d) SPSB expression in the total absence of SPSA1+SPSA2+SPSC expression is enough to guarantee plant viability, and (e) SPSA1 expression in the total absence of SPSA2+SPSB+SPSC expression, and SPSC expression in the total absence of SPSA1+SPSA2+SPSB expression guarantee normal development, growth and nonstructural carbohydrate metabolism of the plant. That (a) the spsa1/spsc mutant has a reduced vegetative and reproductive growth phenotype that is exacerbated in the spsa1/spsa2/spsc mutant, and (b) the spsa1/spsb/spsc and spsa1/spsa2/spsb/spsc mutants (both totally lacking SPSA1 and SPSC expression) are not viable (Fig. 1) strongly indicates that SPSA1 and SPSC (and to a lesser extent SPSA2 and SPSB) play predominant roles in processes that are important for fertility, development and growth. This is further strengthen by the observations that the spsa1/spsa2/spsb and spsa2/spsb/spsc mutants display a WT phenotype and produce fertile seeds (Fig. 1 and data not shown). In vitro, SuSy catalizes the reversible conversion of sucrose and NDP into the corresponding NDPG and fructose [48]. It primarily works as a sucrose-degrading enzyme in planta, playing important roles in the regulation of carbon partitioning into various sink tissues or organs and in phloem loading and unloading. Although SuSy can potentially synthesize sucrose from UDPG and fructose, results presented in this work showing that (a) spsa1/spsa2/spsb/spsc mutants are not viable (Fig. 1), and (b) spsa1/spsc and spsa1/spsa2/spsc leaves accumulate reduced levels of sucrose (Fig. 3), provide strong evidence that (a) SuSy plays a minor role, if any, in the sucrose biosynthetic process, and (b) sucrose is mainly synthesized through the SPS-SPP pathway in Arabidopsis. 18 Comparative studies of SPS gene expression in Arabidopsis using quantitative RT-PCR and promoter-reporter gene expression techniques showed that SPSA1 is expressed in all tissues except roots and, together with SPSC, constitutes the major SPS gene expressed in leaves [14,15]. SPSA2 is mainly expressed in roots, whereas SPSB is predominantly expressed in seeds and reproductive organs [14,15]. In addition, genomewide expression analyses (https://www.geneinvestigator.ethz.ch, [54]) showed strong expression of SPSA1 in most organs and tissues, medium/moderate expression levels of SPSA2, SPSB and SPSC in most organs and tissues, and high expression of SPSB in seeds. It is thus conceivable that the poor germination and aberrant growth of spsa1/spsb/spsc is due to impairments in processes mediated by SPSA1, SPSB and SPSC that are essential for fertilization and/or seed development and subsequent development and growth of the plant. Recently, Brauner et al. [55] reported that growth reduction in near-starchless pgm1 mutants impaired in pPGM is caused by exaggerated root respiration. Taking into account that respiration in spsa1/spsc and spsa1/spsa2/spsc plants is exceedingly higher than in WT plants (Fig. 2) it is highly conceivable that restricted growth of these mutants (Figs. 1 and 6) is ascribed not only to impairments in leaf sucrose synthesis and subsequent provision of photosynthate to the rest of the plant, but also to exaggerated respiration. The stunted growth phenotype and high maltose content of the spsa1/spsc and spsa1/spsa2/spsc mutants (Fig. 1) is reminiscent of that observed in the mex1 and dpe2 mutants whose phenotypes have been ascribed to impairment in the maltose to sucrose conversion process during the night [6-8]. Results presented in this work showing that (a) the levels of all metabolic intermediates in the nocturnal maltose to S6P conversion pathway in spsa1/spsc leaves are higher than in WT leaves when plants are cultured under CL conditions (Fig. 6), (b) maltose contents at the end of the light period in leaves of spsa1/spsc plants cultured under different photoregimes are similar to those of leaves of spsa1/spsc plants cultured under CL conditions (Fig. 7B), and (c) the levels of glycolytic, OPPP and TCA metabolic intermediates and enzymatic activities in spsa1/spsc leaves are higher than in WT leaves (Fig. 5) point to the occurrence in spsa1/spsc leaves of mechanism(s) involving simultaneous synthesis and mobilization of starch during the day similar to that previously reported for mutants impaired in TPT [16- 19 18], and channeling of starch breakdown products towards the glycolytic, OPPP and TCA as schematically illustrated in Fig. S11. Previous studies have shown that enhancement of plastidic PGM stimulates photosynthetic carbon flow into starch [56]. It is thus conceivable that elevated plastidic PGM occurring in spsa1/spsc and spsa1/spsa2/spsc plants (Fig. 4 and Fig. S9) will favor the scavenging of starch breakdown products, thus making up a substrate (starch) cycle. Both activation of starch cycling and enhanced respiration can be considered as alleviation mechanisms that would contribute to compensate the detrimental effects caused by the blockage of sucrose biosynthesis in spsa1/spsc and spsa1/spsa2/spsc leaves. ACKNOWLEDGEMENTS This work was partially supported by the Comisión Interministerial de Ciencia y Tecnología and Fondo Europeo de Desarrollo Regional (Spain) [grant numbers BIO2010- 18239, BIO2013-49125-C2-1-P, BIO2008-02292 and BIO2011-28847-C02-02]. A.M.S- L. acknowledges a predoctoral fellowship from the Spanish Ministry of Science and Innovation. M.B. acknowledges a post-doctoral fellowship from the Public University of Navarra. 20 REFERENCES 1. I. Ciereszko, H. Johansson, L.A. Kleczkowski, Sucrose and light regulation of a cold-inducible UDP-glucose pyrophosphorylase gene via a hexokinase-independent and abscisic acid-insensitive pathway in Arabidopsis, Biochem. J. 354 (2001) 67- 82. 2. M. Zourelidou, M. de Torres-Zabala, C. Smith, M.W. Bevan, Storekeeper defines a new class of plant-specific DNA-binding proteins and is a putative regulator of patatin expression, Plant J. 30 (2002) 489-497. 3. M.A. Blázquez, R. Green, O. Nilsson, M.R. Sussman, D. Weigel, Gibberelins promote flowering of Arabidopsis by activating the LEAFY promoter, Plant Cell 10 (1998) 791-800. 4. R.W. King, Y. Ben-Tal, A florigenic effect of sucrose in Fuchsia hydrida is blocked by gibberellin-induced assimilate competition, Plant Physiol. 125 (2001) 488-496. 5. D. Iraqi, F.M. Tremblay, Analysis of carbohydrate metabolism enzymes and cellular contents of sugars and proteins during spruce somatic embryogenesis suggests a regulatory role of exogenous sucrose in embryo development, J. Exp. Bot. 52 (2001) 2301-2311. 6. T. Chia, D. Thorneycroft, A. Chapple, G. Messerli, J. Chen, et al., A cytosolic glucosyltransferase is required for conversion of starch to sucrose in Arabidopsis leaves at night, Plant J. 37 (2004) 853-863. 7. T. Niittylä, G. Messerli, M. Trevisan, J. Chen, C. Smith, et al., A previously unknown maltose transporter essential for starch degradation in leaves, Science 303 (2004) 87-89. 8. S.E. Weise, K.S. Kim, R.P. Stewart, T.D. Sharkey, β-maltose is the metabolically active anomer of maltose during transitory starch degradation, Plant Physiol. 137 (2005) 756-761. 9. M.H. Cho, H. Lim, D.H. Shin, J.S. Jeonv, S.H. Bhoo, et al., Role of the plastidic glucose translocator in the export of starch degradation products from the chloroplasts in Arabidopsis thaliana, New Phytol. 109 (2011) 101-112. 21 10. S.C. Huber, J.L. Huber, Role and regulation of sucrose-phosphate synthase in higher plants, Ann. Rev. Plant Physiol. Plant Mol. Biol. 47 (1996) 431-444. 11. H. Winter, S.C. Huber, Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes, Crit. Rev. Plant Sci. 19 (2000) 31-67. 12. Y. Gibon, O.E. Bläsing, N. Palacios-Rojas, D. Pankovic, J.H.M. Hendriks, et al., Adjustment of diurnal starch turnover to short days: inhibition of carbohydrate utilization, accumulation of sugars and post-translational activation of ADP-glucose pyrophosphorylase in the following light period, Plant J. 39 (2004) 847-862. 13. M. Okamura, N. Aoki, T. Hirose, M. Yonekura, C. Ohto, et al., Tissue specific and diurnal change in gene expression of the sucrose phosphate synthase gene family in rice, Plant Sci. 181 (2011) 159-166. 14. J. Sun, J. Zhang, C. Larue, S. Huber, Decrease in leaf sucrose synthesis leads to increased leaf starch turnover and decreased RuBP regeneration-limited photosynthesis but not Rubisco-limited photosynthesis in Arabidopsis null mutants of SPSA1, Plant Cell Environ. 34 (2011) 592-604. 15. K. Volkert, S. Debast, L.M. Voll, H. Voll, I. Schießl, et al., Loss of the two major leaf isoforms of sucrose-phosphate synthase in Arabidopsis thaliana limits sucrose synthesis and nocturnal starch degradation but does not alter carbon partitioning during photosynthesis, J. Exp. Bot. 65 (2014) 5217-5229. 16. R.E. Häusler, N.H. Schlieben, B. Schulz, U.-I. Flügge, Compensation of decreased triose phosphate/phosphate translocator activity by accelerated starch turnover and glucose transport in transgenic tobacco, Planta 204 (1998) 366-376. 17. A. Schneider, R.E. Häusler, Ü. Kolukisaoglu, R. Kunze, E. van der Graaff, et al., An Arabidopsis thaliana knock-out mutant of the chloroplast triose phosphate/phosphate translocator is severely compromised only when starch synthesis, but not starch mobilization is abolished, Plant J. 32 (2002) 685-699. 18. R.G. Walters, D.G. Ibrahim, P. Horton, N.J. Kruger, A mutant of Arabidopsis lacking the triose-phosphate/phosphate translocator reveals metabolic regulation of starch breakdown in the light, Plant Physiol. 135 (2004) 891-906. 22 19. J.A. Rojas-González, M. Soto-Suárez, A. García-Díaz, M.C. Romero-Puertas, L.M. Sandalio, et al., Disruption of both chloroplastic and cytosolic FBPase genes results in a dwarf phenotype and important starch and metabolite changes in Arabidopsis thaliana, J. Exp. Bot. 66 (2015) 2673-2689. 20. J. Li, G. Almagro, F.J. Muñoz, E. Baroja-Fernández, A. Bahaji, et al., Posttranslational redox modification of ADP-glucose pyrophosphorylase in response to light is not a major determinant of fine regulation of leaf starch accumulation in Arabidopsis, Plant Cell Physiol. 53 (2012) 433-444. 21. T.W.A. Jones, L.D. Gottlieb, E. Pichersky, Reduced enzyme activity and starch level in an induced mutant of chloroplast phosphoglucose isomerase, Plant Physiol. 81 (1986) 367-371. 22. E. Baroja-Fernández, F.J. Muñoz, J. Li, A. Bahaji, G. Almagro, et al., Sucrose synthase activity in the sus1/sus2/sus3/sus4 Arabidopsis mutant is sufficient to support normal cellulose and starch production, Proc. Natl. Acad. Sci. U.S.A. 109 (2012) 321-326. 23. T. Caspar, S.C. Huber, C. Somerville, Alterations in growth, photosynthesis, and respiration in a starchless mutant of Arabidopsis thaliana (L.) deficient in chloroplast phosphoglucomutase activity, Plant Physiol. 79 (1985) 11-17. 24. X. Liu, S. Zhang, X. Shan, Y.G. Zhu, Toxicity of arsenate and arsenite on germination, seedling growth and amylolitic activity of wheat, Chemosphere 61 (2005) 293-301. 25. E. Baroja-Fernández, F.J. Muñoz, M. Montero, E. Etxeberria, M.T. Sesma, et al., Enhancing sucrose synthase activity in transgenic potato (Solanum tuberosum L.) tubers results in increased levels of starch, ADPglucose and UDPglucose and total yield, Plant Cell Physiol. 50 (2009) 1651-1662. 26. A. Renz, L. Merlo, M. Stitt, Partial purification from potato tubers of three fructokinases and three hexokinases which show differing organ and developmental specificity, Planta 190 (1993) 156-165. 27. E. Baroja-Fernández, F.J. Muñoz, A. Zandueta-Criado, M.T. Morán-Zorzano, A.M. Viale, et al., Most of ADP-glucose linked to starch biosynthesis occurs outside the 23 chloroplast in source leaves, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 13080- 13085. 28. J. Li, E. Baroja-Fernández, A. Bahaji, F.J. Muñoz, M. Ovecka, et al., Enhancing sucrose synthase activity results in increased levels of starch and ADP-glucose in maize (Zea mays L.) seed endosperms, Plant Cell Physiol. 54 (2013) 282-294. 29. M.M. Burrell, P.J. Mooney, M. Blundy, D. Carter, F. Wilson, et al., Genetic manipulation of 6-phosphofructokinase in potato tubers, Planta 194 (1994) 95-101. 30. M. Leterrier, J.B. Barroso, R. Valderrama, J.M. Palma, F.J. Corpas, NADP- dependent isocitrate dehydrogenase from Arabidopsis roots contributes in the mechanism of defence against the nitro-oxidative stress induced by salinity, ScientificWorldJournal (2012) 694740. doi: 10.1100/2012/694740. 31. T. Tomaz, M. Bargard, I. Pracharoenwattana, P. Lindén, C.P. Lee, et al., Mitochondrial malate dehydrogenase lowers leaf respiration and alters photorespiration in plant growth in Arabidopsis, Plant Physiol. 154 (2010) 1143- 1157. 32. S. Huang, N.L. Taylor, E. Ströher, R. Fenske, A.H. Millar, Succinate dehydrogenase assembly factor 2 is need for assembly and activity of mitochondrial complex II and for normal root elongation in Arabidopsis, Plant J. 73 (2013) 429-441. 33. A. Bahaji, E. Baroja-Fernández, A.M. Sánchez-López, F.J. Muñoz, J. Li, et al., HPLC-MS/MS analyses show that the near-starchless aps1 and pgm leaves accumulate wild type levels of ADPglucose: further evidence for the occurrence of important ADPglucose biosynthetic pathway(s) alternative to the pPGI-pPGM- AGP pathway, PLOS ONE 18;9(8) (2014) e104997. 34. A. Lytovchenko, K Bieberich, L. Willmitzer, A.R. Fernie, Carbon assimilation and metabolism in potato leaves deficient in plastidial phosphoglucomutase, Planta 215 (2002) 802-811. 35. L.J. Sweetlove, M.M. Burrell, T. ap Rees, Starch metabolism in tubers of transgenic potato (Solanum tuberosum) with increased ADPglucose pyrophosphorylase, Biochem. J. 320 (1996) 493-498. 24 36. L.-S. Chen, Q. Lin, A. Nose, A comparative study on diurnal changes in metabolite levels in the leaves of three ceassulacean acid metabolism (CAM) species, Ananas comosus, Kalanchoë daigremontiana and K. pinnata, J. Exp. Bot. 53 (2002) 341- 350. 37. S. von Caemmerer, G.D. Farquhar, Some relationships between the biochemistry and photosynthesis and the gas exchange of leaves, Planta 153 (1981) 376-387. 38. P.C. Harley, F. Loreto, G.D. Marco, T.D. Sharkey, Theoretical considerations when estimating the mesophyll conductance to CO2 flux by analysis of the response of photosynthesis to CO2, Plant Physiol. 98 (1992) 1429-1436. 39. B. Egli, K. Kölling, C. Köhler, S.C. Zeeman, S. Streb, Loss of cytosolic phosphoglucomutase compromises gametophyte development in Arabidopsis, Plant Physiol. 154 (2010) 1659-1671. 40. J.H.M. Hendriks, A. Kolbe, Y. Gibon, M. Stitt, P. Geigenberger, ADP-glucose pyrophosphorylase is activated by posttranslational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species, Plant Physiol. 133 (2003) 838-849. 41. A. Cornish-Bowden, J.H.S. Hofmeyr, M.L. Cárdenas, Strategies for manipulating metabolic fluxes in biotechnology, Bioorg. Chem. 23 (1995) 439-449. 42. S. Thomas, P.J.F. Mooney, M.M. Burrell, D.A. Fell, Finite changes analysis of glycolytic intermediates in tuber tissue of lines of transgenic potato (Solanum tuberosum) overexpressing phosphofructokinase, Biochem. J. 322 (1997) 111-117. 43. S.C. Zeeman, A. Tiessen, E. Pilling, K.L. Kato, A.M. Donald, et al., Starch synthesis in Arabidopsis. Granule synthesis, composition, and structure, Plant Physiol. 129 (2002) 516-529. 44. M. Stitt, S.C. Zeeman, Starch turnover: pathways, regulation and role in growth, Curr. Opin. Plant Biol. 15 (2012) 1-11. 45. M. Stitt, H.W. Heldt, Simultaneous synthesis and degradation of starch in spinach chloroplasts in the light, Biochim. Biophys. Acta 638 (1981) 1-11. 46. T.C. Fox, D.R. Geiger, Effects of decreased net carbon exchange on carbohydrate metabolism in sugar beet source leaves, Plant Physiol. 76 (1984) 763-768. 25 47. V.V. Lozovaya, O.A. Zabotina, J.M. Widholm, Synthesis and turnover of cell-wall polysaccharides and starch in photosynthetic soybean suspension cultures, Plant Physiol. 111 (1996) 921-929. 48. A. Bahaji, J. Li, A.M. Sánchez-López, E. Baroja-Fernández, F.J. Muñoz, et al., Starch biosynthesis, its regulation and biotechnological approaches to improve crop yields. Biotechnol. Adv. 32 (2014) 87-106. 49. M. Szecowka, R. Heise, T. Tohge, A. Nunes-Nesi, D. Vosloh, et al., Metabolic fluxes in an illuminated Arabidopsis rosette, Plant Cell 25 (2013) 694-714. 50. R. Mikkelsen, K.E. Mutenda, A. Mant, P. Schürmann, A. Blennow, α-glucan, water dikinase (GWD): a plastidic enzyme with redox-regulated and coordinated catalytic activity and binding affinity, Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 1785-1790. 51. F. Sparla, A. Costa, F. Lo Schiavo, P. Pupillo, P. Trost, Redox regulation of a novel plastid-targeted β-amylase of Arabidopsis, Plant Physiol. 141 (2006) 840-850. 52. C. Valerio, A. Costa, L. Marri, E. Issakidis-Bourguet, P. Pupillo, et al., Thioredoxin-regulated β-amylase (BAM1) triggers diurnal starch degradation in guard cells, and in mesophyll cells under osmotic stress, J. Exp. Bot. 62 (2010) 545-555. 53. M.A. Glaring, K. Skryhan, O. Kötting, S.C. Zeeman, A. Blennow, Comprehensive survey of redox sensitive starch metabolizing enzymes in Arabidopsis thaliana, Plant Physiol. Biochem. 58 (2012) 89-97. 54. P. Zimmermann, M. Hirsch-Hoffmann, L. Hennig, W. Gruissem, GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox, Plant Physiol. 136 (2004) 2621-2632. 55. K. Brauner, I. Hörmiller, T. Nägele, A.G. Heyer, Exaggerated root respiration accounts for growth retardation in a starchless mutant of Arabidopsis thaliana, Plant J. 79 (2014) 82-91. 56. K. Uematsu, N. Suzuki, T. Iwamae, M. Inui, H. Yukawa, Expression of Arabidopsis plastidial phosphoglucomutase in tobacco stimulates photosynthetic carbon flow into starch synthesis, J. Plant Physiol. 169 (2012) 1454-1462. Figure 2 0 2 1 4 3 5 7 6 Dark respiration (µmol CO2.m-2.s-1) WT spsa1/spsa2 spsa1/spsb spsa1/spsc spsa2/spsb spsa2/spsc spsb/spsc spsa1/spsa2/spsb spsa1/spsa2/spsc spsa2/spsb/spsc * * Figure 3 Glucose (µmol/g FW) 0 0.4 0.2 0.6 1.0 0.8 1.2 0 0.4 0.2 0.6 1.0 0.8 1.2 Fructose (µmol/g FW) 0 0.4 0.2 0.6 1.0 0.8 1.2 Starch (µmol glucose/g FW) 0 100 50 150 250 200 300 350 400 450 UDPG (nmol/g FW) 0 20 10 30 50 40 60 70 G6P (nmol/g FW) 0 200 100 300 500 400 600 700 G1P (nmol/g FW) 0 20 10 30 50 40 60 70 80 90 ADPG (nmol/g FW) 0 0.2 0.1 0.3 0.5 0.4 S6P (nmol/g FW) 0 2 1 3 5 6 7 4 1.4 1.6 1.8 2.0 Sucrose (µmol/g FW) ** * * * * ** * * ** * * WT spsa1/spsa2 spsa1/spsb spsa1/spsc spsa2/spsb spsa2/spsc spsb/spsc spsa1/spsa2/spsb spsa1/spsa2/spsc spsa2/spsb/spsc spsa1/spsa2/spsb spsa1/spsa2/spsc spsa2/spsb/spsc WT spsa1/spsa2 spsa1/spsb spsa1/spsc spsa2/spsb spsa2/spsc spsb/spsc Maltose (µmol/g FW) 0 2 1 3 5 4 6 7 8 * * Figure 4 0 100 300 400 500 600 200 SPS (mU/g FW) 0 1.0 0.5 2.0 2.5 3.0 3.5 1.5 PGM (U/g FW) * * * *** * * * * **** * spsa1/spsa2 WT WT spsa1/spsb spsa1/spsc spsa2/spsb spsa2/spsc spsb/spsc spsa1/spsa2/spsb spsa1/spsa2/spsc * * spsa2/spsb/spsc spsa1/spsa2/spsb/spsc spsa1/spsa2 spsa1/spsb spsa1/spsc spsa2/spsb spsa2/spsc spsb/spsc spsa1/spsa2/spsb spsa1/spsa2/spsc * * spsa2/spsb/spsc Figure 5 0 50 100 150 200 250 WT spsa1/spsc WT spsa1/spscWT spsa1/spsc WT spsa1/spsc WT spsa1/spsc WT spsa1/spsc F6P (nmol/g FW) 0 100 200 300 400 500 600 WT spsa1/spsc Oxalacetate (nmol/g FW) 0 100 200 300 400 500 600 3PGA (nmol/g FW) 0 100 200 300 400 500 600 F1,6P2 (nmol/g FW) WT spsa1/spsc WT spsa1/spsc 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Piruvate (µmol/g FW) 0 100 200 300 400 500 PEP (nmol/g FW) 0 0.5 1.0 1.5 2.0 2.5 PGI (U/g FW) 0 0.2 0.4 0.6 0.8 1.0 1.2 G6PDH (U/g FW) 0 0.2 0.4 0.6 0.8 1.0 1.2 6PGDH (U/g FW) WT spsa1/spsc 0 0.5 1.0 1.5 2.0 2.5 3.0 MDH (U/g FW) WT spsa1/spsc 0 100 200 300 400 500 600 IDH (mU/g FW) WT spsa1/spsc WT spsa1/spsc WT spsa1/spsc 0 0.5 1.0 1.5 2.0 2.5 0 0.5 1.0 1.5 2.0 2.5 PK (U/g FW) 3PGA kinase (U/g FW) 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 G3PDH (U/g FW) 0 20 40 60 80 100 120 140 160 WT spsa1/spsc Aldolase (mU/g FW) 0 100 200 300 400 500 600 WT spsa1/sps c SDH (mU/g FW) A B 0 100 200 300 400 500 600 WT spsa1/spsc Isocitrate (nmol/g FW) Figure 6 0 50 100 150 200 250 300 350 400 450 Starch (µmol glucose/g FW) 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 0 100 200 300 400 500 600 Sucrose (µmol/g FW) G6P (nmol/g FW) 0 1 2 3 4 5 6 7 8 Maltose (µmol/g FW) WT spsa1/spsc WT spsa1/spsc WT spsa1/spsc WT spsa1/spsc WT spsa1/spsc WT spsa1/spsc WT spsa1/sps c 0 10 20 30 40 50 60 G1P (nmol/g FW) 0 10 20 30 40 50 60 UDPG (nmol/g FW) 0 1 2 3 4 5 S6P (nmol/g FW) B A WT spsa1/spsc Figure 7 Starch (µmol glucose/g FW) 0 End of night 12 h light/12 h dark WT spsa1/spsc WT spsa1/spsc 100 50 150 250 200 300 350 400 450 500 A B Maltose (µmol/g FW) Maltose (nmol/g FW) End of light End of night 16 h light/8 h dark WT spsa1/spsc WT spsa1/spsc End of light End of night 20 h light/4 h dark WT spsa1/spsc WT spsa1/spsc End of light CL WT spsa1/spsc 0 End of night 12 h light/12 h dark WT spsa1/spsc WT spsa1/spsc 2 1 3 5 4 6 7 8 9 10 11 12 13 14 12 h light/ 12 h dark 16 h light/ 8 h dark 20 h light/ 4 h dark CL End of light End of night 16 h light/8 h dark WT spsa1/spsc WT spsa1/spsc End of light End of night 20 h light/4 h dark WT spsa1/spsc WT spsa1/spsc End of light CL WT spsa1/spsc 0 50 150 200 250 300 100 Table S1: sps mutants used in this work and their phenotypes Designation Description Source Rosette Growth Flower and silique growth, and seed production Nonstructural carbohydrate content spsa1/spsa2 spsa1 and spsa2 double mutant This work Like WT Like WT Like WT spsa1/spsb spsa1 and spsb double mutant This work Like WT Like WT Like WT spsa1/spsc spsa1 and spsc double mutant This work Reduced Reduced -High levels of UDPG, G1P, glucose and G6P, and very high levels of maltose and starch in plants cultured under different photoperiod conditions. -Low levels of sucrose and S6P. -High levels of OPPP, glycolytic and TCA intermediates and enzymatic activities. spsa2/spsb spsa2 and spsb double mutant This work Like WT Like WT Like WT spsa2/spsc spsa2 and spsc double mutant This work Like WT Like WT Like WT spsb/spsc spsb and spsc double mutant This work Like WT Like WT Like WT spsa1/spsb/spsc spsa1, spsb and spsc triple mutant This work Aberrant Sterile _ spsa1/spsa2/spsb spsa1, spsa2 and spsb triple mutant This work Like WT Like WT Like WT spsa1/spsa2/spsc spsa1, spsa2 and spsc triple mutant This work Reduced Reduced Comparable to spsa1/spsc spsa2/spsb/spsc spsa2, spsb and spsc triple mutant This work Like WT Like WT Like WT spsa1/spsa2/spsb/spsc spsa1, spsa2, spsb and spsc quadruple mutant This work Aberrant Sterile _ Table S2. Primers used for PCR screening of sps mutants. For further details about annealing positions of RP, LP and T-DNA specific primers and PCR analyses of sps mutants, see Fig. S2 and Fig. S3. Mutant Primer Sequence spsa1 (SALK_ 119162) RP (SPSA1) 5’-aggaaaaagaagcacagaggc-3’ LP (SPSA1) 5’-accagcatcagcatagtgtcc-3’ LBb1 (T-DNA) 5’-gcgtggaccgcttgctgcaact-3’ spsa2 (SALK_ 064922) RP (SPSA2) 5’-ccagctactctgaaccgtctg-3’ LP (SPSA2) 5’-tgcaagacttacaaggttcgc-3’ LBb1 (T-DNA) 5’-gcgtggaccgcttgctgcaact-3’ spsb (GABI_368F01) RP (SPSB) 5’-ttgctatgacaatgagggagc-3’ LP (SPSB) 5’-ttaaccggtgaatcaaccttg-3’ Gabi (T-DNA) 5’-cccatttggacgtgaatgtagacac-3’ spsc (SAIL_31_H05) RP (SPSC) 5’-ttttcaatatgctcgtgggac-3’ LP (SPSC) 5’-gcgggaaaggacttataccac-3’ LB3 (T-DNA) 5’-tagcatctgaatttcataaccaatctcgatacac-3’ Table S3. Primers used in RT-PCR analyses Gene Direction Sequence 18S RNA Forward 5´-gggcattcgtatttcatagtcagag-3´ At3g41768 Reverse 5´-cggttcttgattaatgaaaacatcct-3´ SPSA1 Forward 5´-aggaaaaagaagcacagaggc-3´ At5g20280 Reverse 5´-accagcatcagcatagtgtcc-3´ SPSA2 Forward 5´-ccagctactctgaaccgtctg-3´ At5g11110 Reverse 5´-caatgtaaggttcgcaagctc-3´ SPSB Forward 5´-ttgctatgacaatgagggagc-3´ At1g04920 Reverse 5´-ttaaccggtgaatcaaccttg-3´ SPSC Forward 5´-gatgataaatcaagtcgaaacc -3´ At4g10120 Reverse 5´-cattcgtggcatgtatctacc-3´ Triose-P Chloroplast Cytosol 1 2 4 5 5 678 678 3 CO2 Tri ose-P FBP F6P F6P G6P G1P A B Calvin-Benson cycle Supplemental Figure 1 Chloroplast Cytosol Glucose Maltose Glucose Heteroglycan S6P Sucrose G6P G1P UDPG S6P Sucrose UDPG Starch 12 13 14 5’ G6P G1P ATP ADPG 9 10 4’ FBP 1’ F6P 2’ F6P Maltose 11 12’ Starch Supplemental Figure 7 WT spsa1/spsc 0 0 0.4 0.2 0.8 1.0 1.2 1.4 0.6 PPase (U/g FW) 0 100 50 200 250 300 150 AGP (mU/g FW) 0 10 5 20 25 30 35 15 SS (mU/g FW) 0 1.0 0.5 2.0 2.5 3.0 3.5 1.5 α-amylase (U/g FW) 40 20 80 100 120 60 SuSy (mU/g FW) 0 50 150 200 250 300 100 UGP (mU/g FW) 0 1 3 4 5 6 2 HK (mU/g FW) 0 2 1 4 5 6 3 β-amylase (U/g FW) 0 400 200 800 1000 600 Acid invertase (mU/g FW) 0 400 200 800 600 Alkaline invertase (mU/g FW) WT spsa1/spsa2 spsa1/spsb spsa1/spsc spsa2/spsb spsa2/spsc spsb/spsc spsa1/spsa2/spsb spsa1/spsa2/spsc spsa2/spsb/spsc spsa1/spsa2/spsb spsa1/spsa2/spsc spsa2/spsb/spsc WT spsa1/spsa2 spsa1/spsb spsa1/spsc spsa2/spsb spsa2/spsc spsb/spsc Supplemental Figure 8 WT PGM1 spsa1/spsc Supplemental Figure 9 PGM2 PGM3 100 kDa 50 WT spsa1/spsc Supplemental Figure 10 S6P Sucrose 78 UDPG 6 Triose-P Chloroplast Cytosol Maltose Glucose Maltose Glucose Heteroglycan CO2 CO2 Triose-P FBP F6P F6P G6PG6PG1P ATP ADPG G1P Glycolysis TCA OPPP Calvin-Benson cycle CO2 TCA Starch 5 4 15 16 14’ 4’ 5’ 9 10 11 1212’ 13 14 Supplemental Figure 11 S6P Sucrose 78 UDPG 6 Chloroplast Cytosol Glucose Maltose Glucose Heteroglycan Triose-P FBP F6P F6P G6P G1P Glycolysis OPPP 5 4 15 16 12 13 14 A B Maltose 11 12’ Starch