Starch Biosynthesis, Its Regulation and Biotechnological Approaches to Improve Crop Yields

JBA-06696; No of Pages 20 Biotechnology Advances xxx (2013) xxx–xxx

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Biotechnology Advances

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Starch biosynthesis, its regulation and biotechnological approaches to improve crop yields

Abdellatif Bahaji a,1, Jun Li a,1, Ángela María Sánchez-López a, Edurne Baroja-Fernández a, Francisco José Muñoz a, Miroslav Ovecka a,b, Goizeder Almagro a, Manuel Montero a, Ignacio Ezquer a, Ed Etxeberria c, Javier Pozueta-Romero a,⁎ a Instituto de Agrobiotecnología (CSIC/UPNA/Gobierno de Navarra), Mutiloako etorbidea z/g, 31192 Mutiloabeti, Nafarroa, Spain b Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Cell Biology, Faculty of Science, Palacky University, Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republic c University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850-2299, USA article info abstract

Available online xxxx Structurally composed of the glucose homopolymers amylose and amylopectin, starch is the main storage carbohydrate in vascular plants, and is synthesized in the plastids of both photosynthetic and non-photosynthetic cells. Keywords: Its abundance as a naturally occurring organic compound is surpassed only by cellulose, and represents both a ADPglucose cornerstone for human and animal nutrition and a feedstock for many non-food industrial applications including – Calvin Benson cycle production of adhesives, biodegradable materials, and ﬁrst-generation bioethanol. This review provides an up- Carbohydrate metabolism date on the different proposed pathways of starch biosynthesis occurring in both autotrophic and heterotrophic Genetic engineering organs, and provides emerging information about the networks regulating them and their interactions with the Microbial volatiles ﬁ MIVOISAP environment. Special emphasis is given to recent ndings showing that volatile compounds emitted by microor- Plant–microbe interaction ganisms promote both growth and the accumulation of exceptionally high levels of starch in mono- and dicoty- Endocytosis ledonous plants. We also review how plant biotechnologists have attempted to use basic knowledge on starch Starch futile cycling metabolism for the rational design of genetic engineering traits aimed at increasing starch in annual crop species. Sucrose synthase Finally we present some potential biotechnological strategies for enhancing starch content. © 2013 Elsevier Inc. All rights reserved.

Contents

1. Introduction...... 0 2. Proposedstarchbiosyntheticpathways...... 0 2.1. Starchbiosynthesisinleaves...... 0 2.1.1. A classic model of starch biosynthesis according to which the starch biosynthetic pathway is linked to the Calvin–Benson cycle by means of plastidialphosphoglucoseisomerase...... 0 2.1.2. Models of starch biosynthesis according to which the starch biosynthetic pathway is not linked to the Calvin–Benson cycle by meansofplastidialphosphoglucoseisomerase...... 0 2.2. Starchbiosynthesisinheterotrophicorgans...... 0 2.2.1. Mechanismsofsucroseunloadinginheterotrophicorgans...... 0 2.2.2. Proposed mechanisms of sucrose–starchconversioninheterotrophiccells...... 0

Abbreviations: ADPG, ADPglucose; AGP, ADPG pyrophosphorylase; AGPP, ADPG pyrophosphatase; A/N-inv, alkaline/neutral invertase; BT1, Brittle 1; F6P, fructose-6-phosphate; G1P, glucose-1-phosphate; G6P, glucose-6-phosphate; GBSS, granule bound starch synthase; GPT, glucose-6-phosphate translocator; GWD, glucan, water dikinase; HvBT1, Hordeum vulgare BT1; MCF, mitochondrial carrier family; MVs, microbial volatiles; NPP, nucleotide pyrophosphatase/phosphodiesterase; MIVOISAP, MIcrobial VOlatiles Induced Starch Accumulation Pro- cess; NTRC, NADP-thioredoxin reductase C; OPPP, oxidative pentose phosphate pathway; Pi, orthophosphate; 3PGA, 3-phosphoglycerate; pHK, plastidial hexokinase; pPGI, plastidial phosphoglucoseisomerase; pPGM, plastidial phosphoglucomutase; PPi, inorganic pyrophosphate; pSP, plastidial starch phosphorylase; RSR1, Rice Starch Regulator1; SuSy, sucrose synthase; SS, starch synthase; T6P, trehalose-6-phosphate; Trx, thioredoxin; UDPG, UDPglucose; UGP, UDPG pyrophosphorylase; WT, wild type; ZmBT1, Zea mays BT1. ⁎ Corresponding author. Tel.: +34 948168009; fax: +34 948232191. E-mail address: [email protected] (J. Pozueta-Romero). 1 These authors contributed equally to this work.

3. Microbialvolatilespromotebothgrowthandaccumulationofexceptionallyhighlevelsofstarchinmono-anddicotyledonousplants...... 0 4. Biotechnologicalapproachesforincreasingstarchcontentinheterotrophicorgans...... 0 4.1. EnhancementofAGPactivity...... 0 4.2. Increasingsupplyofstarchprecursorstotheamyloplast...... 0 4.3. Increasingsupplyofsucrosetoheterotrophiccells...... 0 4.4. Enhancementofsucrosebreakdownwithintheheterotrophiccell...... 0 4.5. EnhancementofexpressionofstarchsynthaseclassIV...... 0 4.6. Blockingstarchbreakdown...... 0 4.7. Alteringtheexpressionofglobalregulators...... 0 4.8. Enhancingtrehalose-6-phosphatecontent...... 0 4.9. Potentialstrategiesforincreasingstarchcontentinheterotrophicorgans...... 0 4.9.1. Over-expressionofBT1...... 0 4.9.2. BlockingtheactivityofADPGbreakdownenzymes...... 0 4.9.3. EctopicexpressionofSuSyintheplastid...... 0 4.9.4. Blockingthesynthesisofstorageproteins...... 0 5. Conclusionsandmainchallenges...... 0 Acknowledgments...... 0 References...... 0

1. Introduction industrial needs and social demands. However, while much is known about starch metabolism, there are still major gaps in our knowledge Starch is the main storage carbohydrate in vascular plants. Its abun- that prevent breeders and biotechnologists from advancing in their at- dance as a naturally occurring compound of living terrestrial biomass is tempts to increase starch content in a predictable way (Chen et al., surpassed only by cellulose, and represents the primary source of calo- 2012). ries in the human diet. Because starch is the principal constituent of In this paper, we first review recent advances in the biochemistry of the harvestable organ of many agronomic plants, its synthesis and accu- starch biosynthesis and its regulation, and provide an update on different mulation also influences crop yields. proposed pathways of starch biosynthesis occurring in both autotrophic Synthesized in the plastids of both photosynthetic and non- and heterotrophic organs. Special emphasis is given to recent findings photosynthetic cells, starch is an insoluble polyglucan produced by showing that volatile compounds emitted by microorganisms promote starch synthase (SS) using ADP-glucose (ADPG) as the sugar donor mol- both growth and the accumulation of exceptionally high levels of starch. ecule. Starch has two major components, amylopectin and amylose, both Finally, we assess the progress in molecular strategies for increasing of which are polymers of α-D-glucose units. Amylose is a linear polymer starch in annual crop species by genetic engineering approaches. of up to several thousand glucose residues, whereas amylopectin is a larger polymer regularly branched with α-1,6-branch points. These 2. Proposed starch biosynthetic pathways two molecules are assembled together to form a semi-crystalline starch granule. The exact proportions of these molecules and the size and Starch is found in the plastids of photosynthetic and non- shape of the granule vary between species and between organs of the photosynthetic tissues. Mature chloroplasts occurring in photosynthet- same plant. The diversity of both composition and physical parameters ically active cells possess the capacity of providing energy (ATP) and of starches from different botanical sources gives rise to their diverse fixed carbon for the synthesis of starch during illumination. By contrast, processing properties and applications in both non-food sectors such as production of long-term storage of starch taking place in amyloplasts of sizing agents in textile and paper industry, adhesive gums, and biode- reserve organs such as tubers, roots and seed endosperms depends gradable materials, and in food industries, particularly in bakery, thick- upon the incoming supply of carbon precursors and energy from the ening, confectionary and emulsification (Burrell, 2003; Mooney, 2009; cytosol. This difference between the metabolic capacities of chloroplasts Slattery et al., 2000). Starch is also used as a feedstock for first generation and amyloplasts has led to the generally accepted view that the path- bioethanol production (Goldemberg, 2007). From an industrial perspec- way(s) involved in starch production are different in photosynthetic tive, the utilization of starch as a cheap and renewable polymer and clean and non-photosynthetic cells. energy source is becoming increasingly attractive as a consequence of social concerns about industrial wastes generated from fossil fuels. In general, the harvested parts of our staple crop plants are hetero- 2.1. Starch biosynthesis in leaves trophic starch-storing organs. Worldwide annual starch production of cereal seeds (rice, maize, wheat, barley, etc.) is approximately In leaves, a portion of the photosynthetically fixed carbon is retained 2 billion tons, whereas worldwide annual starch production of roots within the chloroplasts during the day to synthesize starch, which is (e.g. cassava and taro) and tubers (e.g. potato and sweet potato) ex- then remobilized during the subsequent night to support non- ceeds 700 million tons (Food and Agriculture Organization of the Unit- photosynthetic metabolism and growth by continued export of carbon ed Nations, http://faostat.fao.org), much of which is destined to non- to the rest of the plant. Due to the diurnal rise and fall cycle of its levels, food purposes. In 2012 for instance, 75 million tons of starch were foliar starch is termed “transitory starch”. extracted and used in many industrial applications (http://www. Transitory starch is a major determinant of plant growth (Sulpice starch.dk/). The increasing demand of starch from non-food industries, et al., 2009), and its metabolism is regulated to avoid a shortfall of car- the continuing rapid increase in the world's population, predicted to bon at the end of the dark period. It is typically degraded in a near-linear reach 9 billion by 2050, and the growing loss of arable land to urbaniza- manner during darkness, with only a small amount remaining at the tion have spurred on efforts to synthesize plants with higher starch end of the night. When changes in the day length occur that leads to a content. Therefore, a thorough understanding of the mechanisms in- temporary period of carbon starvation, the carbon budget is rebalanced volved in starch metabolism and regulation is critically important for by increasing the rate of starch synthesis, decreasing the rate of starch the rational design of experimental traits aimed at improving yields in breakdown and, consequently, decreasing the rate of growth during agriculture, and producing more and better polymers that fitboth darkness. The importance of starch turnover in plant growth and

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Triose-P Triose-P 1’ 1 Calvin-Benson cycle FBP FBP 2’ 3 2 F6P 4’ F6P 4 G6P 5’ G6P 5 Sucrose G1P Starch 9 G1P 10 6 8 ADPG 7 UDPG Sucrose-P

Chloroplast Cytosol

Fig. 1. Classic interpretation of the mechanisms of starch and sucrose syntheses in leaves. The enzymes are numbered as follows: 1, 1′, fructose-1,6-bisphosphate aldolase; 2, 2′,fructose

1,6-bisphosphatase; 3, PPi: fructose-6-P 1-phosphotransferase; 4, 4′,PGI;5,5′, PGM; 6, UGP; 7, sucrose-phosphate-synthase; 8, sucrose phosphate phosphatase; 9, AGP; and 10, SS. According to this view, the starch biosynthetic process resides exclusively in the chloroplast and is segregated from the sucrose biosynthetic pathway that takes place in the cytosol.

AGP is allosterically activated by 3PGA, and inhibited by Pi APS1 and pPGM antisensed potato plants accumulate low levels of starch (Kleczkowski, 1999). This property makes the production of ADPG high- when compared with WT leaves (Lytovchenko et al., 2002; Müller- ly sensitive to changes in photoassimilate (e.g. 3PGA) availability in the Röber et al., 1992; Muñoz et al., 2005). Furthermore, Arabidopsis pgm chloroplast, and helps to coordinate photosynthetic CO2 fixation with null mutants (Caspar et al., 1985; Kofler et al., 2000), adg1-1 mutants starch synthesis in leaves. AGP activity is also subjected to APS redox with less than 3% of the WT AGP activity (Lin et al., 1988; Wang regulation (Hendriks et al., 2003; Kolbe et al., 2005; Li et al., 2012; et al., 1998), and APS1 T-DNA null mutants completely lacking AGP Michalska et al., 2009). Under oxidizing conditions the two APS subunits activity (Bahaji et al., 2011a; Ventriglia et al., 2008) accumulate are covalently linked via an intermolecular disulfide bridge, thus less than 2% of the WT starch. Further evidence supporting that forming a stable dimer within the heterotetrameric AGP enzyme (Fu AGP plays a crucial role in starch biosynthesis in leaves comes from et al., 1998; Hendriks et al., 2003). Conversely, under reductive condi- alkaline pyrophosphatase-silenced plants of Nicotiana benthamiana, tions APS monomerization is accompanied by activation of AGP activity. since their leaves accumulate ca. 60% of the WT starch (George et al., APS dimerization markedly decreases the activity of AGP and alters its 2010). kinetic properties, making it less sensitive to activation by 3PGA and increasing its Km for ATP (Hendriks et al., 2003). In turn, APS 2.1.2. Models of starch biosynthesis according to which the starch monomerization activates AGP, which is then sensitive to fine control biosynthetic pathway is not linked to the Calvin–Benson cycle by means through allosteric activation by 3PGA. of plastidial phosphoglucose isomerase By examining the correlation between APS redox status, AGP activity A vast amount of biochemical and genetic data appear to support the and starch levels in leaves, different works have provided evidence that starch biosynthetic pathway illustrated in Fig. 1 according to which (a) transitory starch accumulation is finely regulated by APS post- the whole starch biosynthetic process resides exclusively in the chloro- translational redox modification in response to environmental inputs plast, (b) pPGI links the Calvin–Benson cycle with starch biosynthesis, such as light, sugars and biotic stress (Gibon et al., 2004; Hendriks and (c) AGP is the sole source of ADPG linked to starch biosynthesis. et al., 2003; Kolbe et al., 2005; J. Li et al., 2011). However, using aps1 However, in recent years an increasing volume of evidence has been Arabidopsis plants ectopically expressing a redox-insensitive, mutated provided that supports the occurrence of additional/alternative starch APS1 form, Li et al. (2012) and Hädrich et al. (2012) have independently biosynthetic pathways involving the cytosolic and plastidial compart- shown that the rates of transitory starch accumulation in these plants ments wherein the supply of ADPG is not directly linked to the are comparable to that of WT leaves. In addition, Li et al. (2012) report- Calvin–Benson cycle by means of pPGI. Thus, Fettke et al. (2011) ed WT rates of starch accumulation in aps1 plants expressing in the found that the envelope membranes of chloroplasts in mesophyll cells chloroplast a redox-insensitive AGP from Escherichia coli, and concluded possess a yet to be identified G1P transport machinery enabling the in- that post-translational redox modification of APS1 in response to light is corporation of cytosolic G1P into the stroma, which is subsequently not a major determinant of fine regulation of transitory starch accumu- converted into starch by the stepwise AGP and SS reactions. According lation in Arabidopsis leaves. to these authors, such mechanism would only allow the accumulation Plastidial NADP-thioredoxin reductase C (NTRC) plays an im- of 1% of the WT starch, and would explain the occurrence of some portant role in protecting plants against oxidative stress (Pulido trace amounts of starch in the pPGM mutants (Caspar et al., 1985; et al., 2010; Serrato et al., 2004). Serrato et al. (2004) and Michalska Muñoz et al., 2005; Streb et al., 2009). et al. (2009) reported that Arabidopsis ntrc mutants grown at Kunz et al. (2010) found that leaves of the pgi1-2 null mutant im- 180 μmol photons s−1 m−2 are small and their leaves accumulate paired in pPGI activity accumulate 10% of the WT starch content, low levels of starch when compared with WT leaves. Furthermore, which is restricted to bundle sheath cells adjacent to the mesophyll Michalska et al. (2009) reported that ntrc leaves exhibit a decrease in and stomatal guard cells. This mutant exhibits high G6P transport activ- the extent of APS1 activation (reduction) by light. These authors ity as a consequence of the elevated expression of GPT2 (Kunz et al., thus concluded that NTRC is a major determinant of both light- 2010), a gene that codes for a G6P/Pi translocator (GPT) mainly operat- dependent APS1 redox status and transitory starch accumulation. ing in heterotrophic tissues where the imported G6P can be used for the However, Li et al. (2012) have recently found that the size of synthesis of starch and fatty acids or to drive the oxidative pentose Arabidopsis ntrc mutants is similar to that of WT plants when phosphate pathway (Bowsher et al., 2007; Kammerer et al., 1998; cultured at 90 μmol photons s−1 m−2. Under these conditions, APS1 Kang and Rawsthorne, 1996; L. Zhang et al., 2008). According to Kunz redox status and starch accumulation rates in leaves of ntrc mutants et al. (2010) the unexpected high levels of starch occurring in leaves are similar to those of WT leaves, strongly indicating that NTRC plays of pPGI mutants can be ascribed to high GPT2-mediated incorporation a minor role, if any, in the control of both light-dependent APS1 redox of G6P into the chloroplast of bundle sheath cells adjacent to the meso- status and transitory starch accumulation in Arabidopsis cultured under phyll and stomatal guard cells, where this hexose-phosphate can be non-stressing conditions. then converted to starch by means of pPGM, AGP and SS as schematical- In vitro assays using heterologously expressed potato and pea AGP ly illustrated in Fig. 2. This interpretation, however, is hardly reconcil- have shown that APS can be activated by plastidial thioredoxins (Trxs) f able with previous studies carried out by the same group showing and m (Ballicora et al., 2000; Geigenberger et al., 2005), indicating that that the starch-deficient phenotype of pPGI mutants can be totally these proteins could act as major determinants of APS redox status and reverted to WT starch phenotype by the ectopic expression of GPT2 of fine regulation of transitory starch accumulation. However, Li et al. (Niewiadomski et al., 2005), and with the fact that leaves of the pgi1- (2012) found no differences in the rate of starch accumulation between 2/gpt2 double mutant accumulate as much as 60% of the starch occur- WT leaves and leaves of homozygous trxf1, trxf2, trxm2andtrxm3mu- ring in pgi1-2 leaves (Kunz et al., 2010). Furthermore, considering that tants. Furthermore, Thormählen et al. (2013) reported that starch levels (a) most of leaf starch accumulates in the mesophyll cells, (b) micro- in trxf1 leaves were only slightly reduced when compared with WT scopic analyses revealed that stomatal guard cells and bundle sheath leaves. In addition, Sanz-Barrio et al. (2013) have shown that APS redox cells adjacent to the mesophyll of pPGI mutants accumulate nearly status in high-starch tobacco leaves overexpressing Trxf and Trxm genes WT starch content (Kunz et al., 2010; Tsai et al., 2009), and (c) guard from the plastid genome is similar to that of WT leaves. The overall cells of WT leaves possess a G6P transport machinery (Overlach et al., data thus indicate that, individually, plastidial Trxs are not major determi- 1993), it is difficult to understand how guard cells and bundle sheath nants of APS1 redox status-controlled transitory starch content. cells adjacent to the mesophyll of pPGI mutants can accumulate as Genetic evidence showing that transitory starch biosynthesis occurs much as 10% of the WT starch as a consequence of high GPT2 activity. solely by the AGP pathway has been obtained from the characterization Needless to say, further investigations will be necessary to understand of leaves with reduced pPGM and AGP activities. For example, leaves of how and where pgi mutants accumulate ca. 10% of the WT starch.

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Calvin- Triose-P Triose-P Benson cycle 1 FBP 3 2 F6P 4 G6P G6P GPT 5’ G1P Starch 9 10 ADPG

Chloroplast Cytosol

Fig. 2. Suggested model of starch biosynthesis in bundle sheath cells adjacent to the mesophyll and stomatal guard cells of pgi-2Arabidopsismutants (from Kunz et al., 2010). Numbering of enzyme activities is the same as in Fig. 1.

Gerrits et al. (2001) have provided evidence that a sizable pool of analyses (Baroja-Fernández et al., 2013). Third, HPLC:MS analyses of sucrose in the plant cell has a plastidial localization. Consistent with the triple ss3/ss4/aps1 mutant impaired in AGP and SS class III and IV re- the occurrence of a plastidial pool of sucrose, Murayama and Handa vealed that leaves from this mutant accumulate ca. 40-fold more ADPG (2007) and Vargas et al. (2008) reported the presence of a functional than WT leaves (Ragel, 2012). alkaline/neutral invertase (A/N-inv) inside plastids. Noteworthy, SuSy catalyzes the reversible conversion of sucrose and NDP into Vargas et al. (2008) reported that leaves of Arabidopsis mutants impaired fructose and the corresponding NDP-glucose, where N stands for uridine, in A/N-inv accumulate ca. 70% of the WT starch content. The same au- adenosine, guanosine, cytidine, thymidine or inosine. Although UDP is thors hypothesized that chloroplastic sucrose plays a sensing role in a generally considered to be the preferred nucleoside diphosphate for mechanism of control of carbon partitioning and sucrose/starch ratio SuSy, ADP also serves as an effective substrate to produce ADPG wherein glucose and fructose generated by A/N-inv are sensed through (Baroja-Fernández et al., 2003, 2012; Cumino et al., 2007; Delmer, plastidial hexokinase (pHK) (Giese et al., 2005; Olsson et al., 2003). How- 1972; Nakai et al., 1998; Porchia et al., 1999; Silvius and Snyder, 1979; ever, a possibility cannot be ruled out that, as schematically illustrated in Zervosen et al., 1998). SuSy is highly regulated at both transcriptional Fig. 3, plastidial sucrose acts as precursor for starch biosynthesis, its and post-transcriptional levels (Asano et al., 2002; Ciereszko and A/N-inv breakdown products being converted into starch by means Klezkowski, 2002; Déjardin et al., 1999; Fu and Park, 1995; Hardin of pHK, pPGM and AGP. et al., 2004; Koch et al., 1992; Pontis et al., 1981; Purcell et al., 1998;for Sufficient evidence exists to support the view that a sizable pool of a review see Kleczkowski et al., 2010). Although this sucrolytic enzyme ADPG in leaves accumulates in the cytosol of photosynthetically compe- is very active in reserve organs, it also expresses in the mesophyll cells tent cells. This hypothesis is compatible with the occurrence of cytosolic of source leaves (Fu et al., 1995; Wang et al., 1999), its activity in potato ADPG metabolizing enzymes such as ADPG phosphorylase (McCoy et al., and Arabidopsis leaves greatly exceeding the minimum needed to sup- 2006), glucan synthases (Tacke et al., 1991), sucrose synthase (SuSy) and port normal rate of starch accumulation during illumination (Baroja- ADPG hydrolases (Olejnik and Kraszewska, 2005; Rodríguez-López et al., Fernández et al., 2012; Muñoz et al., 2005). Accordingly, a metabolic 2000), and with the occurrence in the chloroplast envelope membranes model of transitory starch biosynthesis has been proposed wherein (a) of yet to be molecularly identified ADPG transport machineries whose both sucrose and starch metabolic pathways are tightly interconnected activities can account for rates of starch accumulation occurring in leaves by means of cytosolic ADPG producing enzymes such as SuSy (acting (Pozueta-Romero et al., 1991a). Genetic evidence supporting the occur- when cytosolic sucrose transiently accumulates during illumina- rence of important ADPG source(s), other than AGP, has been obtained tion), and by the action of an ADPG translocator located at the chlo- from different sources. First, leaves of transgenic potato and Arabidopsis roplast envelope membranes, and (b) both AGP and pPGM play an plants ectopically expressing in the cytosol a bacterial ADPG hydrolase important role in the scavenging of glucose units derived from starch (Moreno-Bruna et al., 2001) accumulate lower ADPG levels than WT breakdown occurring during starch biosynthesis and during the bio- leaves, as determined by HPLC analyses (Bahaji et al., 2011a; Baroja- genesis of the starch granule (Bahaji et al., 2011a; Baroja-Fernández Fernández et al., 2004). Second, ADPG content in the leaves of AGP and et al., 2005; Muñoz et al., 2005, 2006)(Fig. 4). Thus, the net rate of pPGM mutants is comparable to that of WT leaves, as confirmed by starch accumulation is determined by the balance between the both HPLC (Bahaji et al., 2011a; Muñoz et al., 2005)andHPLC:MS rates of SuSy-mediated ADPG synthesis in the cytosol, import of

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Calvin- Triose-P Triose-P Benson cycle 1-8

Sucrose Sucrose 11 Fructose Glucose 12 12 4’ F6P G6P

5’ Starch 10 9 ADPG G1P

Chloroplast Cytosol

Fig. 3. Suggested model of starch biosynthesis where sucrose entering the plastid is broken down by A/N-inv to produce fructose and glucose, the latter being converted to G6P by means of pHK. G6P is subsequently metabolized to starch by the stepwise reactions of pPGM, AGP and SS. Enzyme activities 1–10 and 4′ and 5′ are the same as in legend of Fig. 1.Otherenzymes involved are numbered as follows: 11, A/N-inv; 12, pHK.

cytosolic ADPG to the chloroplast, starch synthesis, starch break- warrant starch production and rapid connection of starch metabolism down and by the efficiency with which starch breakdown products with other metabolic pathways. can be recycled back to starch via the coupled reactions of pPGM The postulated starch biosynthetic pathways involving the plastidial and AGP. Accordingly, this view predicts that the recovery toward and cytosolic compartments are consistent with still enigmatic results starch biosynthesis of the glucose units derived from the starch obtained from experiments carried out more than 50 years ago showing breakdown will be deficient in pPGM and AGP mutants, resulting in that 14C in the glucose moiety of sucrose, starch, UDP-glucose and a parallel decline of starch accumulation. hexose-Ps is asymmetrically distributed in green leaves exposed to 14 The occurrence of a substrate or “futile” cycle as a result of the simul- CO2 for a short period of time (Gibbs and Kandler, 1957; Havir and taneous synthesis and breakdown of starch in leaves is not surprising Gibbs, 1963; Kandler and Gibbs, 1956). Unlike the “classic” view on tran- since pulse-chase and starch-preloading experiments using isolated sitory starch biosynthesis (Fig. 1) predicting that green leaves exposed to 14 14 chloroplasts (Fox and Geiger, 1984; Stitt and Heldt, 1981), intact leaves CO2 for a short period of time should synthesize starch with Csym- (Scott and Kruger, 1995; Walters et al., 2004), or cultured photosyn- metrically distributed in the glucose moiety, the proposed mechanisms thetic cells (Lozovaya et al., 1996) have shown that the illuminated of starch synthesis illustrated in Figs. 2–4 provideapossibleexplanation chloroplasts can synthesize and mobilize starch simultaneously. Fur- for solving the above enigma. As shown in Fig. 4, triose-Ps produced in thermore, enzymes involved in starch breakdown such as SEX1 and the Calvin–Benson cycle are exported to the cytosol where they can be BAM1 are redox-activated during the light under different environmen- channeled into the oxidative pentose-P pathway (OPPP), thereby leading tal conditions (Mikkelsen et al., 2005; Sparla et al., 2006; Valerio et al., to a randomization of the carbons giving rise to the asymmetric 14Cdis- 2010). Futile cycles lead to a net consumption of ATP, which in some in- tribution observed in hexose-Ps, nucleotide-sugars and sucrose upon ex- 14 stances can be estimated at about 60% of the total ATP produced by the posure to CO2 for a short period of time. Cytosolic G1P, G6P, sucrose cell (Alonso et al., 2005; Hill and ap Rees, 1994). Although these cycles and/or ADPG will be then transported into the chloroplast and utilized appear to waste energy without any apparent physiological reason, as precursors for the synthesis of starch molecules possessing glucose they form part of the organization of the plant central metabolism, molecules with asymmetric 14C distribution. and contribute to its flexibility (Rontein et al., 2002). As presented Genetic evidence consistent with the occurrence of a link between su- above, starch acts as a major integrator in the plant growth that accu- crose and transitory starch metabolism can be obtained from sucrose- mulates to cope with temporary starvation imposed by the environ- phosphate-synthase and cytosolic PGM deficient plants (Fernie et al., ment (Sulpice et al., 2009). It is thus conceivable that highly regulated 2002; Strand et al., 2000), since their leaves contain low levels of both su- starch futile cycling may entail advantages such as sensitive regulation crose and starch as compared with WT leaves. Further genetic evidence and rapid metabolic channeling toward various pathways in response can be obtained from sedoheptulose-1,7-bisphosphate overexpressing to physiological and biochemical needs of the plant. Therefore, due to plants (Lefebvre et al., 2005; Miyagawa et al., 2001), since their leaves the importance of starch in the overall plant metabolism and growth, are characterized by having high levels of both sucrose and starch when it is tempting to speculate that both redundancy of ADPG sources and compared with WT leaves. Finally, genetic evidence indicating that leaf starch futile cycling have been selected during plant evolution to sucrose and starch metabolic pathways are linked by SuSy has been

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Calvin- Triose-P Triose-P OPPP Benson cycle Sulfolipids Fatty acids, Hexose-Ps OPPP, etc. 1’- 4’ 1-8 UDPG GPT 13 Sucrose G6P 5’ G1P 12 ADP Glucose 15 16 9 14 Fructose Starch 10 ADPG ADPG ?

Chloroplast Cytosol

Fig. 4. Suggested interpretation of the mechanism of starch biosynthesis in leaves according to which (a) ADPG is produced in the cytosol and transported to the chloroplast by the action of a yet to be identified ADPG translocator, (b) AGP and pPGM play an important role in the scavenging of glucose units derived from starch breakdown, and (c) starch metabolism is connected with other metabolic pathways through the metabolites generated by the starch futile cycle. Enzyme activities 1–10 and 1′–5′ are the same as in legend of Fig. 1. Other enzymes involved are numbered as follows: 12, pHK; 13, plastidial UGP (Okazaki et al., 2009); 14, plastidial AGPP; 15, pSP (Zeeman et al., 2004); and 16, SuSy. Starch to glucose conversion would involve the coordinated actions of amylases, isoamylases and disproportionating enzymes (Asatsuma et al., 2005; Fulton et al., 2008; Kaplan and Guy, 2005). Note that starch futile cycling (indicated with red arrows) may entail advantages such as rapid metabolic channeling toward various pathways (such as fatty acid (Periappuram et al., 2000), OPPP under stress conditions (Zeeman et al., 2004) and sulfolipid biosynthesis (Okazaki et al., 2009)) in response to physiological and biochemical needs. This view predicts that the recovery toward starch biosynthesis of the glucose units derived from the starch breakdown will be deficient in pPGM and AGP mutants, resulting in a parallel decline of starch accumulation and enhancement of soluble sugars content since starch breakdown derived products (especially glucose and G6P) will leak out the chloroplast through very active glucose translocator (Cho et al., 2011)and GPT2 (which is highly expressed in pgi, pgm and agp mutants, Kunz et al., 2010). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) obtained from SuSy-overexpressing potato plants whose leaves accumu- uptake of extracellular sucrose and subsequent transport to the vacuole late higher ADPG and starch contents than WT leaves (Muñoz et al., is not in conflict with transport through membrane-bound carriers 2005). Further endeavors based on the characterization of mutants totally given that cell homeostasis can be better maintained if both mechanisms lacking SuSy will be necessary to confirm (or refute) the involvement of operate in parallel (Etxeberria et al., 2005a). Consistent with this view, SuSy in starch biosynthesis in leaves. Baroja-Fernández et al. (2006) reported that carrier-mediated sucrose import in starved cells of sycamore (Acer pseudoplatanus L.) plays an im- – 2.2. Starch biosynthesis in heterotrophic organs portant role in the overall sucrose starch conversion process during the initial period of sucrose unloading, whereas endocytosis may play an im- Sucrose produced in leaves is imported by the heterotrophic organs portant role in transporting the bulk of sucrose to the vacuole and its and used as carbon source for energy production and starch synthesis in subsequent conversion into starch after prolonged period of sucrose the amyloplast. As in the case of leaves, various mechanisms have been unloading. proposed to describe starch biosynthesis in the heterotrophic organs, which together with the mechanisms of sucrose unloading, will be 2.2.2. Proposed mechanisms of sucrose–starch conversion in heterotrophic presented and discussed below. cells Sucrose entering the heterotrophic cell by any of the mechanisms 2.2.1. Mechanisms of sucrose unloading in heterotrophic organs described above must be metabolized to molecules that can be Unloading of sucrose arriving from aerial parts of the plant into sink transported to the amyloplast for subsequent conversion into starch. organs occurs either symplastically (moving via plasmodesmata) or Because both chloroplasts and amyloplasts are ontogenically related, apoplastically. The route depends on the species, organ or tissue. In and because chloroplasts possess a very active triose-P translocator the apoplastic delivery of sucrose to rapidly growing sinks, apoplastic connecting the plastidial and cytosolic compartments, it was originally sucrose that has been released into the extracellular space can be split assumed that cytosolic triose-Ps entering the amyloplast by means of by cell wall-bound invertases into glucose and fructose for subsequent a triose-P translocator act as precursors for starch biosynthesis (Boyer, uptake into the cell by hexose transporters. Alternatively, sucrose can 1985). However, NMR spectroscopy experiments of starch biogenesis be taken up by plasma membrane-bound sink specificsucrosetrans- in wheat and maize grains using 13CNMR(Hatzfeld and Stitt, 1990; porters of non-dividing storage sinks (Sauer, 2007). Sucrose is then Keeling et al., 1988) demonstrated that a C-6 molecule, not a triose-P transported via yet-to-be-identified tonoplast sucrose transporters to (C-3), is the precursor of starch biosynthesis in amyloplasts. Further in- the vacuole, which serves as temporary reservoir. Apoplastic sucrose vestigations of the enzyme capacities of amyloplasts (Entwistle and ap can also be taken up by a sucrose-induced endocytic process, and Rees, 1988; Frehner et al., 1990) supported the view that C-6 molecules transported to the central vacuole of heterotrophic cells (Etxeberria entering amyloplasts are the precursors of starch biosynthesis. et al., 2005a, 2005b;forareviewseeEtxeberria et al., 2012). Endocytic Depending on the nature of the C-6 molecule entering the amyloplast

(G6P or ADPG), and depending on the enzymes involved in ADPG regulation (Gómez-Casati and Iglesias, 2002; Hylton and Smith, 1992; synthesis, various mechanisms have been proposed to explain the Kleczkowski et al., 1993; Weber et al., 1995). sucrose–starch conversion process in heterotrophic cells. In heterotrophic organs of dicotyledonous plants, sucrose entering the cytosolic compartment of the heterotrophic cell is broken down by SuSy 2.2.2.1. Models of sucrose–starch conversion in heterotrophic cells to produce fructose and UDPglucose (UDPG), the latter being converted according to which AGP is the sole source of ADPG linked to starch to G1P and PPi by UDPG pyrophosphorylase (UGP). G1P is subsequently biosynthesis. It is widely assumed that AGP is the sole source of ADPG metabolized to G6P by means of the cytosolic phosphoglucomutase. in heterotrophic organs of mono- and dicotyledonous plants. Maximal Cytosolic G6P then enters the amyloplast, where it is converted to starch in vitro AGP activity greatly exceeds the minimum required to support by the sequential activities of pPGM, AGP and SS (Fig. 5A). Unlike chloro- the normal rate of starch accumulation in starch storing organs plasts, amyloplasts are unable to photosynthetically generate ATP and (Denyer et al., 1995; Li et al., 2013; Weber et al., 2000). This would indi- therefore, cytosolic ATP must enter the amyloplast to produce ADPG by cate that AGP is not a rate-limiting step in the sucrose–starch conver- means of AGP. The presence of an ATP/ADP translocator in the envelope sion process in many heterotrophic organs. In fact, the flux control membranes of amyloplasts has been firmly established by different labo- coefficient of AGP has been estimated to be as low as 0.08 in some het- ratories (Pozueta-Romero et al., 1991b; Tjaden et al., 1998), and evidence erotrophic organs (Denyer et al., 1995; Rolletschek et al., 2002; Weber about its relevance in the starch biosynthetic process has been provided et al., 2000). As presented above, AGP is a highly regulated enzyme. by Tjaden et al. (1998) and Geigenberger et al. (2001) who reported However, whereas evidence has been provided that starch syn- that potato tubers with decreased plastidic ATP/ADP transporter activities thesis is regulated by post-translational redox modification of AGP exhibit reduced starch content. and 3PGA/Pi balance in potato tubers (Tiessen et al., 2003), AGP in devel- Genetic evidence demonstrating the importance of SuSy in the oping barley, wheat, pea and bean seeds is insensitive to 3PGA and Pi sucrose–starch conversion process in heterotrophic organs of

10 Starch ADPG

PPi Carriers/Endocytosis 9 Sucrose Sucrose ATP UDP 16 ADP ADP G1P Fructose ATP ATP 5 56 G6P G6P G1P UDPG GPT UTP PPi Amyloplast

Cytosol

Carriers/Endocytosis Starch Sucrose Sucrose UDP 16 Fructose 10 9 6 ADPG ADPG G1P UDPG PPi ATP UTP PPi

Amyloplast

Cytosol

Fig. 5. Classic interpretation of the mechanisms of sucrose–starch conversion in (A) heterotrophic cells of dicotyledonous plants and (B) cereal endosperm cells according to which AGP is the sole source of ADPG linked to starch biosynthesis. Note that in heterotrophic cells of dicotyledonous plants (a) AGP is exclusively localized in the plastid, (b) the cytosolic carbon substrate compound entering the amyloplast for subsequent conversion into starch is G6P, and (c) cytosolic UGP is involved in the sucrose–G6P conversion process. Plastidial and cytosolic G1P pools are likely connected by means of a yet to be identiﬁed G1P translocator occurring in amyloplasts (Fettke et al., 2010). Note also that in cereal endosperms cells (a) most of AGP has a cytosolic localization, and (b) the cytosolic carbon substrate compound entering the amyloplast for subsequent conversion into starch is ADPG. Numbering of enzyme activities is the same as in legends of Figs. 1–4.

Please cite this article as: Bahaji A, et al, Starch biosynthesis, its regulation and biotechnological approaches to improve crop yields, Biotechnol Adv (2013), http://dx.doi.org/10.1016/j.biotechadv.2013.06.006 A. Bahaji et al. / Biotechnology Advances xxx (2013) xxx–xxx 9 dicotyledonous plants comes from the reduced levels of starch in potato sources, other than AGP, of ADPG linked to starch biosynthesis. This hy- tubers and carrot roots exhibiting low SuSy activity (Tang and Sturm, pothesis is compatible with the occurrence in the amyloplast envelope 1999; Zrenner et al., 1995). In addition, genetic evidence showing that membranes of ADPG transporters (Naeem et al., 1997; Pozueta- starch biosynthesis in heterotrophic organs involves the incorporation Romero et al., 1991b; Shannon et al., 1998), and with the occurrence of G6P and its subsequent conversion into ADPG by means of pPGM in heterotrophic organs of cytosolic ADPG metabolizing enzymes such and AGP has been obtained from the characterization of Vicia as ADPG phosphorylase (Dankert et al., 1964; McCoy et al., 2006; narbonensis embryos expressing antisense GPT (Rolletschek et al., Murata, 1977)andSuSy.AspresentedinSection 2.1.2, SuSy is a highly 2007) (these embryos, however, expressed lower levels of starch- regulated enzyme that can produce ADPG from sucrose and ADP. It related genes such as AGP and SuSy encoding genes), genetically plays a predominant role in determining both sink strength and engineered pea seeds and potato tubers with reduced pPGM phloem loading, and in the entry of carbon into metabolism in (Harrison et al., 1998; Tauberger et al., 2000)andfromV. narbonensis nonphotosynthetic cells. Maximum in vitro ADPG producing SuSy activ- seeds and potato tubers with reduced AGP activity (Müller-Röber ity is particularly high in starch storing organs, being comparable to that et al., 1992; Weber et al., 2000). These organs all exhibit reduced starch ofAGPinpotatotubers(Baroja-Fernández et al., 2009)and3–4fold content when compared with their corresponding WT organs. higher than that of AGP in developing barley and maize endosperms Using disks from WT and pPGM antisensed potato tubers incubated (Baroja-Fernández et al., 2003; Li et al., 2013). Furthermore, maximal with radiolabeled G1P, Fettke et al. (2010) reported that G1P can be ef- ADPG producing SuSy activity in heterotrophic organs such as develop- ficiently taken up by potato tuber parenchyma cells and converted to ing maize endosperm greatly exceeds the minimum required to support starch. Moreover, these authors reported that exogenously added the normal rate of starch accumulation in this organ (Li et al., 2013), G1P-dependent starch synthesis was diminished in tuber disks of pota- which would indicate that ADPG producing SuSy activity is not a rate to plants with low plastidial starch phosphorylase (pSP) activity. Fettke limiting step in the sucrose–starch conversion process in heterotrophic et al. (2010) thus proposed that G1P can be taken up by amyloplast by a organs. yet to be identified transporter to be subsequently used as substrate for Essentially in line with investigations describing the occurrence of pSP- and/or AGP-mediated starch biosynthesis. However, although this simultaneous synthesis and breakdown of glycogen in heterotrophic hypothesis can explain the results obtained using potato tuber disks, it bacteria and animals (Bollen et al., 1998; David et al., 1990; Gaudet conflicts with previous reports using pSP antisensed potato tubers et al., 1992; Guedon et al., 2000; Massillon et al., 1995; Montero et al., showing that, in planta, the lack of pSP did not exert any effect on starch 2011), pulse chase as well as starch pre-loading experiments using iso- accumulation (Sonnewald et al., 1995). Furthermore, this hypothesis is lated amyloplasts and heterotrophic organs have provided evidence hardly reconcilable with the strong reduction of starch content in tubers about the occurrence of simultaneous synthesis and breakdown of of pPGM antisensed potato tubers, indicating that plastidial G6P is starch in non-photosynthetic cells of plants under conditions of active linked to starch biosynthesis (Tauberger et al., 2000). starch accumulation (Neuhaus et al., 1995; Pozueta-Romero and Unlike dicotyledonous plants where AGP is exclusively localized in Akazawa, 1993; Sweetlove et al., 1996). The occurrence of starch futile the plastidial compartment, most of AGP in cereal endosperm cells has cycling in heterotrophic organs has been further fortified by recent a cytosolic localization (Beckles et al., 2001; Denyer et al., 1996; studies showing that global regulators of storage substance accumula- Johnson et al., 2003; Kleczkowski, 1996; ThornbjØrnsen et al., 1996a, tion such as FLOURY ENDOSPERM2 (FLO2) positively co-regulate the 1996b; Villand and Kleczkowski, 1994). Consistently, most of ADPG has expression of starch synthesis and breakdown genes during starch a cytosolic localization in cereal endosperm cells (Liu and Shannon, accumulation in developing rice seeds (She et al., 2010)(see 1981; Tiessen et al., 2012). Import studies using amyloplasts isolated Section 4.7), and by the fact that down-regulation of starch breakdown from maize and barley endosperms of WT plants and starch-deficient/ functions in genetically engineered wheat and rice plants results in ADPG-excess maize Zmbt1 and barley Hvbt1 mutants provided genetic increased grain yield (Hakata et al., 2012; Ral et al., 2012)(see evidence that Zea mays brittle1 (BT1) (ZmBT1) and Hordeum vulgare Section 4.6). Furthermore, starch breakdown enzymatic activities are BT1 (HvBT1) are membrane proteins that can incorporate cytosolic high in heterotrophic organs (Li et al., 2013). Consistently, “alterna- ADPG into the amyloplast (Liu et al., 1992; Möhlmann et al., 1997; tive/additional” models for the sucrose–starch conversion process in Patron et al., 2004; Shannon et al., 1996, 1998). Furthermore, cell frac- heterotrophic organs of dicotyledonous and monocotyledonous plants tionation studies revealed that most of ADPG accumulating in “high- have been proposed according to which (a) cytosolic enzymes such as ADPG” developing endosperms of the Riso 13 Hvbt1 mutant has a cyto- SuSy catalyze directly the de novo production from sucrose of ADPG, solic localization (Tiessen et al., 2012). The overall data thus indicate which is subsequently imported into the amyloplast by the action of that, in cereal endosperm cells, BT1 proteins facilitate the transfer of an ADPG translocator, and (b) pPGM and AGP play an important role cytosolic ADPG into the amyloplast for starch biosynthesis and are in the scavenging of the glucose units derived from the starch break- rate-limiting steps in this process. Based on this evidence, a starch bio- down (Fig. 6). Accordingly, these models preview that the net rate of synthesis model has been proposed for cereal endosperms wherein the starch accumulation in heterotrophic cells is determined by the balance stepwise reactions of SuSy, UGP and AGP take place in the cytosol to con- between the rates of ADPG synthesis in the cytosol, import of cytosolic vert sucrose into ADPG, which enters the amyloplast by means of BT1 to ADPG to the amyloplast, starch synthesis and starch breakdown, and be utilized as glucosyl donor for starch biosynthesis (Fig. 5B) (Denyer by the efficiency with which starch breakdown products can be recycled et al., 1996; Villand and Kleczkowski, 1994). Genetic evidence demon- back to starch via the coupled reactions of pPGM and AGP. Also, these strating the importance of SuSy in the sucrose–starch conversion process models predict that the recovery toward starch biosynthesis of starch in cereal endosperms comes from QTL analyses in maize endosperms breakdown products will be deficient in pPGM and AGP mutants, (Thévenot et al., 2005) and from the reduced levels of starch in maize resulting in a parallel decline of starch accumulation. seeds exhibiting low SuSy activity (Chourey and Nelson, 1976). Evidence The occurrence of the sucrose–starch conversion pathway illustrat- showing the importance of AGP in starch biosynthesis in cereal endo- ed in Fig. 6A implies that neither UDPG produced by SuSy, nor cytosolic sperms comes from AGP mutants exhibiting reduced starch content hexose-Ps derived from the action of UGP on UDPG, are involved in (Johnson et al., 2003; Tsai and Nelson, 1966). starch biosynthesis in heterotrophic organs of dicotyledonous plants. This view is consistent with the unexpected WT starch content pheno- 2.2.2.2. Models of sucrose–starch conversion in heterotrophic cells type of UGP antisensed potato tubers (Zrenner et al., 1993), and with according to which both SuSy and AGP are involved in the synthesis of the WT G6P and G1P contents of starch-deficient SuSy antisensed pota- ADPG linked to starch biosynthesis. Sufficient evidence exists to support to tubers (Baroja-Fernández et al., 2003). Also, this view is coherent the view that heterotrophic organs possess in the cytosol important with the unexpected starch-deficient phenotype of transgenic potato

Starch 10 16 Carriers/Endocytosis ADPG ADPG Sucrose Sucrose Fructose ADP

9 15 Glucose 12 GPT 5 G6P G1P Amyloplast

Cytosol

Fructose ADP Carriers/Endocytosis Starch 10 ADPG ADPG Sucrose Sucrose 16

PPi PPi UDP 9 9 16 ATP ATP Fructose Glucose 15 12 GPT 5 6 G6P G1P G1P UDPG Amyloplast UTP PPi

Cytosol

Fig. 6. Suggested interpretation of the mechanisms of sucrose–starch conversion in (A) heterotrophic cells of dicotyledonous plants and (B) cereal endosperms according to which (a) both SuSy and AGP are involved in the synthesis of ADPG linked to starch biosynthesis, (b) the cytosolic carbon substrate compound entering the amyloplast forsubsequentconversioninto starch is ADPG, (c) pPGM and AGP are involved in the scavenging of starch breakdown products, and (d) the rate of starch accumulation would be the result of the net balance between starch synthesis and breakdown. Note that in heterotrophic cells of dicotyledonous plants neither cytosolic hexose-phosphates, nor cytosolic UGP is involved in the sucrose–starch conversion process. Note also that in cereal endosperm cells SuSy catalyzes the direct conversion of sucrose into both ADPG and UDPG, the latter being converted to ADPG in the cytosol by the stepwise reactions of UGP and AGP. This view predicts that in pPGM and AGP mutants the recovery toward starch biosynthesis of the glucose units derived from the starch breakdown will be deﬁcient, resulting in a parallel decline of starch accumulation since starch breakdown products (especially glucose and G6P) will leak out the amyloplast. Numbering of enzyme activities is the same as in legends of Figs. 1–4.

tubers heterologously expressing in the cytosol either yeast invertase spp., and from some fungal species inhibit growth in Arabidopsis plants plus glucokinase (Trethewey et al., 1998) or bacterial sucrose phos- (Splivallo et al., 2007; Tarkka and Piechulla, 2007; Vespermann et al., phorylase (Trethewey et al., 2001), since the high sucrolytic activity 2007). Given the lack of knowledge on how microbial volatiles (MVs) af- occurring in these tubers leads to drastic reduction of cytosolic sucrose fect reprogramming of carbohydrate metabolism in plants, Ezquer et al. levels, thus limiting ADPG-producing SuSy activity. (2010a) explored the effect on starch metabolism of volatiles released Genetic evidences demonstrating the signiﬁcance of ADPG- from different microbial species ranging from Gram-negative and producing SuSy in the sucrose–starch conversion process in heterotro- Gram-positive bacteria to different fungi. Toward this end, the authors phic cells of both mono- and dicotyledonous plants are the same as measured the starch and soluble sugar contents in leaves of plants those presented in Section 2.2.2.1. cultured in the presence or in the absence of adjacent microbial cultures. Noteworthy, Ezquer et al. (2010a) reported that, in the absence of phys- 3. Microbial volatiles promote both growth and accumulation of ical contact between plant and microbe, all microbial species tested exceptionally high levels of starch in mono- and (including plant pathogens and microbes that normally do not interact dicotyledonous plants with plants) emitted volatiles that promoted enhancement of the intracellular ADPG levels and a rapid accumulation of exceptionally high Microbes synthesize and emit many volatile compounds. Volatile levels of starch in leaves of both mono- and dicotyledonous plants. Levels emissions from rhizobacterial isolates of Bacillus spp. promote growth of starch reached by MV-treated leaves were comparable to those occur- in Arabidopsis plants by facilitating nutrient uptake, photosynthesis and ring in heterotrophic organs such as tubers and cereal seeds. This phe- defense responses, and by decreasing glucose sensing and abscisic acid nomenon, designated as MIVOISAP (for MIcrobial VOlatiles Induced levels (Ryu et al., 2003, 2004; H. Zhang et al., 2008, 2009). In contrast, Starch Accumulation Process), was also accompanied by strong promo- volatiles from Pseudomonas spp., Serratia spp. and Stenotrophomonas tion of growth. Therefore, MIVOISAP cannot be ascribed to utilization

Please cite this article as: Bahaji A, et al, Starch biosynthesis, its regulation and biotechnological approaches to improve crop yields, Biotechnol Adv (2013), http://dx.doi.org/10.1016/j.biotechadv.2013.06.006 A. Bahaji et al. / Biotechnology Advances xxx (2013) xxx–xxx 11 of surplus photosynthates and/or ATP for starch biosynthesis that would unsuccessful, for improving starch accumulation in heterotrophic organs occur under growth arrest conditions. of plants of agronomic interest. Finally we will present some potential Transcriptome, metabolite content and enzyme activity analyses of strategies for enhancing starch content. potato leaves exposed to volatiles emitted by the plant pathogen Alternaria alternata revealed that starch over-accumulation was accom- 4.1. Enhancement of AGP activity panied by enhanced 3PGA/Pi ratio and up-regulation of SuSy, acid invertase inhibitors, SSIII and SSIV, starch branching enzyme, GPT2, and Most attempts to increase starch accumulation have focused on en- synthesis of phosphatidylinositol-phosphates implicated in processes hancing AGP activity. The focus on AGP arose originally from the belief such as endocytosis and vesicle traffic(Ezquer et al., 2010a). that this enzymatic reaction catalyzes a “rate-limiting step” in the pro- MIVOISAP was also accompanied by down-regulation of acid invertase, cess of starch synthesis and thus, increases in its activity would concom- plastidial Trxs, plastidial β-amylase and pSP, and proteins involved in itantly result in enhanced starch accumulation. the conversion of plastidial triose-phosphates into cytosolic G6P. No One strategy for enhancing AGP activity in plants consisted of heter- changes were found neither in the expression levels of AGP encoding ologously expressing glgC16 (a mutant variant of the E. coli glgC gene) genes nor in the redox status of APS1. Furthermore AGP-antisensed po- that encodes an allosterically insensitive AGP form. Recent studies, tato leaves accumulated exceptionally high levels of starch in the pres- however, have shown that the allosterically sensitive glgC equally ence of MVs. The overall data thus indicated that potato MIVOISAP is the serves the same purpose, since ectopic glgC expression restores WT consequence of transcriptionally and post-transcriptionally regulated starch content in aps1 Arabidopsis mutants (Li et al., 2012). First at- metabolic reprogramming involving (a) up-regulation of ADPG- tempts to increase AGP activity in plants were made in potatoes producing SuSy, SSIII and SSIV, and proteins involved in the endocytic through expression of glgC16 under the control of the constitutive 35S uptake and traffic of sucrose, and (b) down-regulation of acid invertase promoter. Using this approach, Stark et al. (1992) reported that when and starch breakdown enzymes. During potato MIVOISAP there also oc- the product of glgC16 was targeted to plastids, tubers from the trans- curs a down-regulation of ATP-consuming functions involved in inter- genic lines accumulated more starch than control tubers, although nal amino acid provision such as proteases and enzymes involved in there was no correlation between starch levels and AGP activity in dif- de novo synthesis of amino acids. Ezquer et al. (2010a, 2010b) thus hy- ferent lines. Subsequent attempts to reproduce these results on a differ- pothesized that, under conditions of limited ATP-consuming protein ent cultivar of potato expressing glgC16 under the control of a tuber- breakdown occurring during exposure to MVs, the surplus ATP and car- specific promoter did not render tubers with increased levels of starch bon are diverted from protein metabolism to starch biosynthesis. and ADPG, although they showed increased levels of AGP activity Time-course analyses of starch and maltose contents in illuminated (Sweetlove et al., 1996). Ectopic expression of allosterically insensitive Arabidopsis leaves of plants exposed to volatiles emitted by A. alternata E. coli AGP has also been used in other crops such as rice (Nagai et al., revealed that both starch synthesis and β-amylase-dependent starch 2009; Sakulsingharoj et al., 2004), maize (Wang et al., 2007) or cassava degradation were enhanced upon MV treatment, although the final (Ihemere et al., 2006). In these cases enhanced AGP activity in seeds balance was positive for synthesis over breakdown (J. Li et al., 2011). resulted in enhanced biomass, but there was no increase in the starch The increase of starch content in illuminated leaves of MV-treated hy1/ content on a fresh weight basis. cry1, hy1/cry2 and hy1/cry1/cry2 Arabidopsis mutants was many-fold Other attempts at achieving increased starch content in cereal endo- lower than that of WT leaves, indicating that Arabidopsis MIVOISAP is sperms have used modified variants of the AGP large subunit, which subjected to photoreceptor-mediated control. Transcriptomic analyses contributes little to the catalytic activity of the enzyme but is important of Arabidopsis leaves exposed to A. alternata volatiles revealed changes in determining its regulatory properties. Giroux et al. (1996) obtained in the expression of genes involved in multiple processes. However, un- maize lines expressing Sh2r6hs (a mutated variant of the AGP large like potato, no changes could be observed in the expression of starch re- subunit gene), which encodes an enzyme that is allosterically insensi- lated genes (J. Li et al., 2011). Also, unlike potato plants, Arabidopsis tive. The starch content of these lines was higher than in control lines; MIVOISAP was accompanied by 2-3-fold increase of the levels of reduced however there was no increase in starch as a percentage of seed weight (active) form of APS1. Using different Arabidopsis knockout mutants J. Li and, instead, these lines had significantly heavier seeds (Giroux et al., et al. (2011) observed that the magnitude of the MV-induced starch ac- 1996). Ectopic expression of Sh2r6hs has been conducted in maize, cumulation was low in mutants impaired in SSIII, SSIV and NTRC. Unlike rice and wheat (Meyer et al., 2004; Smidansky et al., 2002, 2003). In WT leaves, no changes in the redox status of AGP occurred in ntrc these cases enhanced AGP activity in seeds resulted in increased indi- mutants, providing evidence that MV-promoted monomerization vidual seed weight and seed yield per plant. (activation) of AGP is mediated by NTRC. The overall data thus strongly Most recently N. Li et al. (2011) have shown that ectopic expression indicated that Arabidopsis MIVOISAP involves a photocontrolled, tran- of either the BT2 gene coding for the small subunit maize AGP, or SH2 scriptionally and post-transcriptionally regulated network wherein pho- gene coding for the maize AGP large subunit, results in enhanced seed toreceptors and post-translational changes (e.g. changes in the redox weight and starch content. status) of plastidial enzyme(s) such as AGP, SSIII and SSIV play important roles. Consistent with the possible involvement of changes in redox sta- 4.2. Increasing supply of starch precursors to the amyloplast tus of SSs in MIVOISAP, Glaring et al. (2012) have recently reported that SSI and SSIII are reductively activated enzymes. As indicated above, synthesis of storage starch in the amyloplast requires the incorporation of ATP and carbon skeletons from the cytosol. 4. Biotechnological approaches for increasing starch content in Tjaden et al. (1998) and Geigenberger et al. (2001) provided evidence heterotrophic organs that increasing the supply of ATP to the plastid in potato tubers could be a strategy to enhance tuber starch content, since transgenic potato Despite the monumental progress made in understanding the genetic tubers ectopically expressing a plastidic ATP/ADP transporter from and biochemical mechanisms of starch biosynthesis in plants, up to the Arabidopsis under the control of the cauliflower mosaic virus 35S pro- late 2000s relatively little had been achieved in terms of increasing starch moter accumulated 2-fold more ADPG and 16%–36% more starch per content and yield using biotechnological approaches, particularly in het- gram fresh weight than control tubers. Although these results would erotrophic organs of cereal crops (Smith, 2008). However, within the last suggest that starch content is very sensitive to changes in the transport 5 years, a series of major developments has produced significant results machinery that supplies ATP, more recent studies using transgenic po- that indicate the likelihood of enhancing starch production. We next tato plants ectopically expressing the Arabidopsis ATP/ADP transporter assess the progress made in molecular strategies, both successful and under the control of a tuber specific promoter did not result in increased

Please cite this article as: Bahaji A, et al, Starch biosynthesis, its regulation and biotechnological approaches to improve crop yields, Biotechnol Adv (2013), http://dx.doi.org/10.1016/j.biotechadv.2013.06.006 12 A. Bahaji et al. / Biotechnology Advances xxx (2013) xxx–xxx levels of starch (L. Zhang et al., 2008). Furthermore, these studies bacterial sucrose phosphorylase or fructokinase, that facilitate the con- showed that increasing G6P import to the amyloplast by ectopic expres- version of sucrose or sucrose breakdown products (i.e. glucose and fruc- sion of a pea GPT had no effect on tuber starch content or tuber yield. By tose) into cytosolic hexose-Ps. However, contrary to expectations, contrast, double transformants simultaneously expressing the pea GPT transgenic tubers expressing in the cytosol bacterial sucrose phosphor- and the Arabidopsis ATP/ADP transporter exhibited an enhanced tuber ylase, fructokinase or yeast invertase plus bacterial glucokinase all yield and starch content, the overall data indicating that both ATP/ADP exhibited reduced starch content (Davies et al., 2005; Trethewey et al., and GPT share control of the starch content and tuber yield, most prob- 1998, 2001). Despite extensive research, the mechanisms leading to ably by co-limiting substrate (ATP and hexose-P) supply to AGP. these unexpected results remain obscure (see Section 2.2.2.2). Indirect evidence that increasing ATP supply to the amyloplast is a More successful attempts to promote sucrose–starch conversion strategy for increasing starch content comes from the studies carried have centered on enhancing SuSy activity. The focus on SuSy arose out by Regierer et al. (2002) who reported that constitutive from the belief that this sucrolytic enzyme is a major determinant of downregulation of expression of plastidial adenylate kinase, an enzyme sink strength and starch accumulation in both mono- and dicotyledon- that catalyzes the inter-conversion of ATP with AMP and ADP, increased ous plants. First attempts to increase SuSy activity were conducted in the ADPG and starch contents of potato tubers (see also patent WO 02/ potatoes through expression of StSUS4 under the control of the constitu- 097101 A1). These changes were accompanied by increases in the total tive 35S promoter. Baroja-Fernández et al. (2009) reported that UDPG, adenylate pools (including ATP), although not by changes in the adenyl- ADPG and starch contents in SuSy overexpressing tubers were higher ate energy charge or by changes in developmental phenotype. Regierer than in WT tubers. Furthermore, starch yield per plant was shown to et al. (2002) thus hypothesized that adenylate kinase acts in the ATP- be higher in SuSy-overexpressing plants than in WT plants, under consuming direction, competing for the same ATP pool with AGP. The both greenhouse and open field conditions (Baroja-Fernández et al., increase in starch content (2 fold) is the largest so far reported for any 2009). “High starch” SuSy-overexpressing tubers exhibited a trend- targeted manipulation of starch synthesis. wise increase of AGP activity. Furthermore, they exhibited reduced acid invertase activity, which together with previous studies showing 4.3. Increasing supply of sucrose to heterotrophic cells that down-regulation of SuSy is accompanied by a concomitant reduction of starch levels and enhancement of acid invertase activity The occurrence of high levels of sucrose transporters in heterotrophic (Zrenner et al., 1995) indicates that (a) SuSy, but not acid invertase, is organs where apoplastic sucrose unloading occurs (e.g. developing rice, involved in the sucrose–starch conversion process, (b) SuSy and acid in- maize, barley, faba bean, and wheat seeds) has led to the conclusion vertase compete for the same substrate (sucrose), and (c) the balance that sucrose transporters may play a role in sucrose uptake into endo- between SuSy- and acid invertase-mediated sucrolytic pathways is a sperm cells for the synthesis of storage compounds during grain filling major determinant of starch accumulation in potato tubers. Interesting- (Aoki et al., 1999, 2002, 2003; Harrington et al., 1997; Hirose et al., ly enough SuSy overexpressing tubers exhibited other characteristics of 1997; Sivitz et al., 2005; Weschke et al., 2000). Supporting this hypothe- agronomic interest such as substantial increase in antioxidant activity sis, Scofield et al. (2002) reported that antisense inhibition of the rice su- and resistance to enzymatic browning when compared to control tu- crose transporter OsSUT1 leads to impaired grain filling, the final grain bers. Protection against browning was ascribed to the involvement of weight being reduced. Toward the aim of evaluating the potential of im- SuSy in the production of UDPG necessary for glycosylation and stabili- proved sucrose uptake capacity on wheat grain quality and yield zation of polyphenols (patent WO 2010/055186 A1). Weichert et al. (2010) produced transgenic wheat plants ectopically ex- Most recently Li et al. (2013) produced transgenic maize plants ectop- pressing the barley sucrose transporter HvSUT1 under the control of an ically expressing StSUS4 under the control of the constitutive UBI promot- endosperm-specific promoter. The authors reported that, although pro- er. The five independent transgenic lines characterized produced seeds tein content in transgenic grains was higher than in WT grains, starch exhibiting higher SuSy activity, higher ADPG content and ca. 10% more concentration was unchanged. starch than WT seeds at the mature stage. Furthermore, StSUS4 express- In very early stages of potato tuber development, i.e. during the elon- ing seeds contained a higher amylose/amylopectin balance that WT gation phase of stolon growth, apoplasmic sucrose unloading predomi- seeds. SuSy (and AGP) activity in WT seeds greatly exceeded the nates over symplastic unloading. Subsequent tuberization process minimum required to support normal rate of starch accumulation in de- involves a switch from apoplastic to symplastic phloem unloading veloping maize endosperms (Li et al., 2013). Consequently, SuSy over- (Viola et al., 2001). Although this would apparently imply that sucrose expression should not lead to enhancement of starch content. To explain transporters play a minor role in sucrose unloading in developing tubers the observed positive effect of SuSy over-expression on starch content, and subsequent uptake of sucrose in parenchymatic cells, Kühn et al. the authors argued that the rate of starch accumulation is the result of (2003) reported that reduction of StSUT1 expression in antisensed potato the balance between SuSy–AGP–ADPG transporter-SS-mediated starch plants leads to reduced yield during early stages of tuber development, synthesis, and amylase- and/or SP-mediated starch breakdown. There- indicating that in this phase StSUT1 plays an important role for sugar fore, up-regulation of SuSy results in a concomitant increase of the transport. To evaluate whether it is possible to improve potato plant per- balance between starch synthesis and breakdown, thereby leading to en- formance by overexpressing a sucrose transporter Leggewie et al. (2003) hancement of starch accumulation. The enhancement of ADPG content in produced transgenic potato plants heterologously expressing a spinach SuSy over-expressing lines (also occurring in AGP over-expressing rice sucrose transporter under the control of the 35S promoter. These plants endosperms, Nagai et al., 2009) would indicate that one or more reactions produced tubers with WT levels of starch content and yield. Thus, the constrain carbon flux from cytosolic ADPG into starch. Li et al. (2013) thus overall data obtained using both mono- and dicotyledonous crops hypothesized that the transport of ADPG is perhaps the more important would indicate that up-regulation of sucrose transport is not a satisfacto- limiting factor in starch synthesis in SuSy over-expressing maize ry strategy for increasing starch content in heterotrophic organs. endosperms. The overall data thus indicate that enhancement of SuSy activity 4.4. Enhancement of sucrose breakdown within the heterotrophic cell represents a useful strategy for increasing starch accumulation and yield in heterotrophic organs of both mono- and dicotyledonous crops. Inspired on the widely accepted view that cytosolic hexose-Ps are precursors for starch biosynthesis in heterotrophic organs of dicotyle- 4.5. Enhancement of expression of starch synthase class IV donous plants (see Fig. 5A), attempts to direct more carbon into starch production in potato tubers consisted of heterologously expressing in Five distinct classes of SSs are known in plants: granule-bound SS the cytosol enzymes such as yeast invertase plus bacterial glucokinase, (GBSS), which is responsible for the synthesis of amylose, and soluble

SS classes I, II, III, and IV (SSI, SSII, SSIII, and SSIV, respectively), which are dikinases (GWD) involved in starch phosphorylation required for amy- responsible for the synthesis of amylopectin. Using developing wheat lase mediated starch breakdown are expressed in starch storing organs. endosperms exposed to SS inactivating temperatures, Keeling et al. Supporting the above hypothesis that reducing starch breakdown (1993) reported that SSs have a starch biosynthesis flux control coeffi- would be a viable means of increasing starch content in storage organs cient approaching to unity. This and the fact that maximum catalytic ac- (see above), Ral et al. (2012) recently reported that RNAi-mediated tivity of SS is close to metabolic flux through starch biosynthesis in down-regulation of GWD in wheat endosperm under the control of an several starch storing organs would indicate that SS is a major site of endosperm-specific promoter resulted in an increase in grain yield deregulation of starch synthesis, and therefore, a good target for the termined by increases in seed weight, tiller number, spikelets per production of genetically engineered “high-starch” plants. However, head and seed number per spike (see also patent WO 2009/067751 ectopic expression of SS genes has hardly ever been used to increase A1). These changes were not accompanied by changes in the percentage the accumulation of starch. This is mainly due to the presence of differ- of starch per total dried weight, which is a phenomenon comparable to ent classes of SSs in plants interacting with each other in a protein com- that occurring in AGP overexpressing plants (see above). The work of plex and displaying a specific role in the synthesis of the final Ral et al. (2012) provided a promising mechanism for enhancing bio- architecture of the starch granule (Hennen-Bierwagen et al., 2008). mass and grain yield in wheat, with potential application to other Consequently, changes in the expression of a single SS may lead to pro- crops. Further evidence supporting that down-regulation of starch deg- found and unpredictable effects not only on starch structure, but also on radation may contribute to enhance starch content in developing seeds starch content, revealing that SS isoforms have specificbutinter- has been provided by Hakata et al. (2012), who reported that suppres- dependent roles in starch polymer synthesis. This problem is well sion of α-amylase genes improves the quality of rice grain when plants exemplified by the results obtained using transgenic potato plants ex- are cultured under conditions of high temperature. pressing the E. coli glycogen synthase (an enzyme that catalyzes the same reaction as SS) whose tubers accumulated less starch than WT tu- 4.7. Altering the expression of global regulators bers. Furthermore, the starch from the transgenic tubers had altered structural properties (Shewmaker et al., 1994). SnRK1 (for Sucrose non-fermenting-1-related kinase-1) is a serine/ Roldán et al. (2007) reported that SSIV T-DNA mutants accumulate threonine protein kinase that forms a complex with sucrose non- only one large starch granule in Arabidopsis chloroplasts. In addition, fermenting (SNF1) and AMP-activated protein kinase (AMPK). It acts Szydlowski et al. (2009) reported that Arabidopsis SSIII/SSIV double T- as a central integrator of transcription networks in plant stress and en- DNA mutants accumulate very reduced starch levels, the overall data ergy signaling, and as such, plays a role in regulating metabolism in re- indicating that SSIV plays a key role both in the starch granule initiation sponse to carbon availability (Baena-González and Sheen, 2008; Baena- process and in starch accumulation, being mandatory to render the regu- González et al., 2007). lar number of starch granule found in WT plants. That elimination of SSIV Upon sensing the energy deficit associated with stress or nutrient results in both reduction of number of starch granules and starch content deprivation, SnRK1 triggers extensive transcriptional changes that con- in Arabidopsis leaves suggested that the number of starch granules could tribute to restoring homeostasis. In general, SnRK1 activates genes in- represent a regulatory step in the control of starch accumulation volved in nutrient remobilization and represses those involved in (D'Hulst and Mérida, 2010). Confirming this hypothesis Gámez-Arjona biosynthetic processes and storage, thus providing alternative sources et al. (2011) reported that SSIV overexpression results in enhanced levels of energy through catabolism of amino acids and starch during nutrient of transitory starch in Arabidopsis leaves. Noteworthy, high levels of starch starvation. Thus, SnRK1 positively regulates the expression of starch accumulated in the end of the day were completely mobilized during the breakdown genes, since antisense expression of SnRK1 reduces α- night in SSIV overexpressing plants, allowing them to grow at a higher amylase gene expression in cultured wheat and rice embryos during rate than WT plants. Gámez-Arjona et al. (2011) also reported that ectop- sugar starvation (Laurie et al., 2003; Lu et al., 2007). Intriguingly, al- ic expression of SSIV results in increased levels of reserve starch and yield though the attributed role of SnRK1 is to repress functions involved in in tubers of transgenic potato plants cultured both under greenhouse and biosynthetic processes and storage, SnRK1 has been shown to be neces- open field conditions. Interestingly enough, SSIV overexpression was sary for starch synthesis, since (a) it is required for SuSy gene expression accompanied by pleiotropic increase in AGP and SuSy activities, reduction in potato tubers (Debast et al., 2011; Purcell et al., 1998), and (b) of acid invertase activity and enhancement of ADPG content. Gámez- McKibbin et al. (2006) reported that tubers of SnRK1 overexpressing po- Arjona et al. (2011) thus hypothesized that enhanced starch accumula- tato plants accumulate up to 30% more starch than control tubers. As tion in SSIV overexpressing tubers would be the result of enhanced discussed by McKibbin et al. (2006), starch content enhancement SSIV, AGP and SuSy activities and reduced acid invertase. Further investi- resulting from SnRK1 overexpression can be ascribed to up-regulation gations will be necessary to determine whether ectopic expression of SSIV of SuSy and AGP activities occurring in the SnRK1 overexpressing represents a useful strategy for increasing starch content and yield in lines. Although more research using different crop species is necessary other crops of agronomic interest such as cereal crops. to investigate the role of SnRK1 in the control of starch metabolism, the work of McKibbin et al. (2006) provided evidence that SnRK1 4.6. Blocking starch breakdown overexpression represents a useful strategy for increasing starch accumulation and yield in heterotrophic organs. As discussed above (see Section 2.2.2.2), substantial evidence has Genome-wide co-expression analyses conducted to identify candi- been provided showing the occurrence of simultaneous synthesis and date regulators for starch biosynthesis in rice revealed that Rice Starch breakdown of starch in some starch accumulating heterotrophic cells. Regulator1 (RSR1), an APETALA2/ethylene-responsive element binding It is thus highly conceivable that starch accumulated by heterotrophic protein family transcription factor, negatively co-regulates the expres- organs with active starch turnover is the result of the net balance be- sion of many starch synthesis genes including those coding for AGP, tween starch synthesis and breakdown (Baroja-Fernández et al., 2003, SSs, GBSS, branching enzymes and starch debranching enzymes (Fu 2009; Li et al., 2013; Muñoz et al., 2006). Consequently, down- and Xue, 2010). RSR1 highly expresses in rice organs and tissues with regulation of starch breakdown functions should be a viable means of reduced starch content such as roots, seedlings and leaves, and ex- increasing starch content in heterotrophic organs. presses to much lesser extent in developing seed endosperms. Although It is generally accepted that α-amylase plays a central role in endo- starch content and amylose/amylopectin balance in RSR1 over- sperm starch degradation. This activity is high in developing seed endo- expressing seeds were shown to be comparable to those of WT seeds, sperms during the process of starch accumulation (Hakata et al., 2012; seeds of rsr1 mutants displayed increased amylose content as a conse- Li et al., 2013). On the other hand, genes encoding glucan, water- quence of up-regulation of GBSS expression (Fu and Xue, 2010), had

WT starch content and were larger than WT seeds, a typical phenotype capacities of amyloplasts isolated from Zmbt1 and Hvbt1 seeds indicated of AGP over-expression (see above). It thus appears that down- that, in cereal endosperms, BT1 proteins facilitate the transfer of regulation of RSR1 is a good strategy for increasing starch yield in rice. cytosolic ADPG into the amyloplast for starch biosynthesis and are She et al. (2010) identified FLO2 as a gene highly expressing in devel- rate-limiting steps in this process. This has led to the proposal that up- oping rice seeds coincident with starch accumulation whose mutation regulation of BT1 function would be a strategy for increasing starch con- results in both reduced rice grain size and reduced amylose/amylopectin tent in cereal seeds (Chen et al., 2012; Slattery et al., 2000). balance. The same authors reported that FLO2 positively co-regulates the BT1 proteins belong to the mitochondrial carrier family (MCF) of expression of genes coding for many starch synthesis enzymes such as proteins that are involved in the transport of metabolites across the mi- AGP (AGPL1–4, AGPS1, AGPS2a and AGPS2b), SuSy (SuSy1 and SuSy2), tochondrial membrane (Haferkamp, 2007; Millar and Heazlewood, SSs (SSIIb, SSIIIb, SSIVa), GBSS and branching enzymes (BE1 and BEIIb), 2003). Although MCF proteins are all presumed to be targeted to the mi- and enzymes involved in starch breakdown such as α-amylase tochondrial inner membrane (Millar and Heazlewood, 2003), some of (Amy3C, Amy3D and Amy3E), isoamylases (ISA1 and ISA2) and them occur in other subcellular compartments. Kirchberger et al. pullulanase. Importantly, FLO2 overexpressing rice lines produce larger (2007, 2008) for instance provided evidence that BT1 is exclusively and heavier grains compared with WT plants, providing evidence that localized to the inner plastidial envelope membranes of plastids. How- FLO2 overexpression is a good strategy for increasing starch content ever, more recent studies have shown that BT1 proteins are dually and yield in rice (She et al., 2010). Noteworthy, FLO2 belongs to a family targeted to mitochondria and plastids (Bahaji et al., 2011c). of rice genes whose orthologs widely exist in plants. Provided the func- Kirchberger et al. (2008) reported that homozygous Atbt1 tion of FLO2 orthologs encoded proteins is similar to that of FLO2, it is Arabidopsis mutants displayed an aberrant growth and sterility pheno- likely that up-regulation of FLO2 orthologs in other plant species may re- type, which was ascribed to impairments in export of newly synthe- sult in enhanced starch content and yield. Further research will be neces- sized adenylates from plastids to the cytosol. However, Bahaji et al. sary to confirm or refute this hypothesis. (2011b) have recently shown that the aberrant growth and sterility phenotype of Atbt1 mutants could be reverted to WT phenotype by spe- 4.8. Enhancing trehalose-6-phosphate content cific delivery of AtBT1 to mitochondria, indicating that AtBT1 may play crucial roles, other than transport of newly synthesized adenylates Trehalose-6-phosphate (T6P) is a sugar signal of emerging signifi- from plastids to the cytosol, that are connected to mitochondrial metab- cance that has been suggested to regulate starch biosynthesis via Trx- olism. Taking into account that (a) similar to AtBT1, ZmBT1 is dually mediated posttranslational redox activation of AGP in response to sucrose targeted to plastids and mitochondria, (b) similar to homozygous in leaves, reporting on metabolite status between cytosol and chloroplast Atbt1 Arabidopsis mutants, homozygous Zmbt1 mutant seeds germinate (Kolbe et al., 2005; Lunn et al., 2006; Schluepmann et al., 2003). poorly and produce plants with low vigor (Mangelsdorf, 1926), (c) mi- Leaves of transgenic plants with enhanced T6P have increased redox tochondria do not synthesize starch, and (d) there are many “low activation of AGP and increased starch levels, whereas leaves of plants starch” maize mutant seeds that germinate and produce plants that with reduced T6P show the opposite effect (Kolbe et al., 2005). The oc- grow normally, Bahaji et al. (2011a) hypothesized that (a) the aberrant currence of a positive correlation between intracellular T6P levels, growth and sterility phenotype of homozygous Zmbt1 mutants would redox AGP activation and rates of starch synthesis in Arabidopsis (Kolbe not be ascribed to starch deficiency and/or to reduced ADPG transport et al., 2005; Lunn et al., 2006), and the promotion of SnRK1-dependent activity in endosperm amyloplasts, and (b) similar to AtBT1, ZmBT1 redox activation of AGP by trehaloseorsucrosefeedingtopotatotuber would be involved in yet to be identified processes occurring in mito- disks (Tiessen et al., 2003) led to the hypothesis that T6P is a signal of su- chondria, other than ADPG transport, that are important for normal crose status that promotes starch accumulation in a SnRK1-mediated growth and fertility that indirectly influence starch accumulation. Need- process involving redox AGP activation (Geigenberger et al., 2005; less to say further studies are necessary to confirm or refute the latter Tiessen et al., 2003). However, this proposal conflicts with recent find- hypothesis and to know whether up-regulation of BT1 constitutes a ings showing that T6P is a strong inhibitor of SnRK1 (Martínez-Barajas valuable strategy for increasing starch content and yields. et al., 2011; Y. Zhang et al., 2009) and with the fact that genes normally induced by SnRK1 are repressed by T6P and vice-versa (Y. Zhang et al., 4.9.2. Blocking the activity of ADPG breakdown enzymes 2009). To investigate the role of T6P signaling in starch biosynthesis in An alternative approach to enhancing starch content in crops would heterotrophic organs Debast et al. (2011) generated transgenic potato be to reduce the rate of ADPG degradation. Plants possess a widely dis- plants with high levels of T6P as a consequence of the heterologous ex- tributed enzymatic activity, designated as ADPG pyrophosphatase pression of E. coli OtsA (which encodes a T6P synthase). They also charac- (AGPP), that catalyzes the hydrolytic breakdown of ADPG to G1P and terized transgenic potato plants with low levels of T6P as a consequence AMP (Rodríguez-López et al., 2000). Two different protein entities are of the ectopic expression of E. coli OtsB (which encodes a T6P phospha- responsible for this activity: “Nudix” hydrolases and nucleotide tase). These authors reported that tubers from transgenic lines with ele- pyrophosphatase/phosphodiesterases (NPP). Nudix hydrolases have vated T6P levels displayed reduced starch content. On the other hand, been suggested to act as “housecleaning” enzymes that prevent accu- tubers from lines with reduced T6P accumulated WT starch content, mulation of reactive nucleoside diphosphate derivates, cell signaling although they showed a strongly reduced yield as a consequence of molecules or metabolic intermediates by diverting them to metabolic down-regulation of cell proliferation and growth. Transcriptional profil- pathways in response to biochemical and physiological needs ing of “low T6P” tubers revealed that SnRK1 target genes are strongly up- (McLennan, 2006). AGPPs belonging to this family have been found regulated, which confirms the inhibitory effect of T6P on SnRK1 activity. both in the plastidial and cytosolic compartments (Muñoz et al., 2008; Although Debast et al. (2011) concluded that T6P plays an important role Olejnik and Kraszewska, 2005). Up-regulation of the plastidial AGPP for potato tuber growth, their results indicated that altering T6P intracel- Nudix hydrolase in transgenic potato plants results in reduced levels lular levels is not a good strategy for increasing starch content in hetero- of starch content in both leaves and tubers, indicating that this enzyme trophic organs of plants. has access to the pool of ADPG linked to the biosynthesis of both transitory and reserve starch in leaves and tubers, respectively (Muñoz et al., 4.9. Potential strategies for increasing starch content in heterotrophic organs 2008). The control of the intracellular levels of ADPG linked to starch production likely represents an important, though still poorly investi- 4.9.1. Over-expression of BT1 gated point of control of starch metabolism. Continued molecular ge- As pointed out in Section 2.2.2.1, data on ADPG content in seeds of netic analysis will be needed to determine whether AGPPs play a Zmbt1 maize and Hvbt1 barley mutants, and on ADPG transport crucial role in preventing metabolic flux toward starch in heterotrophic

Please cite this article as: Bahaji A, et al, Starch biosynthesis, its regulation and biotechnological approaches to improve crop yields, Biotechnol Adv (2013), http://dx.doi.org/10.1016/j.biotechadv.2013.06.006 A. Bahaji et al. / Biotechnology Advances xxx (2013) xxx–xxx 15 organs of plants, and whether down-regulation of the AGPP function that autophagy contributes to starch degradation in N. benthamiana constitutes a good strategy for enhancing starch content and yield in leaves, cytosolic starch granule-like structures being sequestered major crops. by autophagic bodies and delivered to vacuoles for their subsequent breakdown. Needless to say, further studies will be necessary to 4.9.3. Ectopic expression of SuSy in the plastid evaluate the relevance of such links in the overall starch metabolism. As presented in Section 2.1.2., a sizable pool of sucrose has a plastid- Although the study of endocytosis in higher plants remained dor- ial localization (Gerrits et al., 2001;seeSection 2.1.2). SuSys from mant because of the notion that this mechanism would not work cyanobacteria are highly specific for ADP and have high affinity for against the high turgor pressure in plant cells, mounting evidence this nucleotide (Curatti et al., 2000; Figueroa et al., 2013; Porchia et al., has been compiled during the last years showing that endocytosis 1999). Thus, a more radical possibility for increasing the starch content is essential for many processes in plants (Müller et al., 2007; Samaj of crop plants would be to express SuSy (especially from cyanobacterial et al., 2005), including carbohydrate metabolism. We now know origin) in the plastid to produce ADPG. that in heterotrophic organs where apoplastic sucrose unloading occurs endocytosis may play an important role in transporting the bulk 4.9.4. Blocking the synthesis of storage proteins of sucrose from the apoplast to the vacuole and its subsequent con- During development of some heterotrophic organs such as tubers version into starch (see Section 2.2.1). Thus, further investigations and seed endosperms, cell division is followed by cell elongation, differ- will be necessary to understand the molecular and signaling mecha- entiation and storage. When cell division ceases, a sizable pool of carbon nisms involved in this process. and ATP is mainly utilized to produce massive amount of storage starch Starch biosynthesis is a highly regulated process, both at transcrip- and proteins. We must emphasize that a similar phenomenon has been tional and post-transcriptional level that is interconnected with a wide observed in bacterial cultures entering the stationary phase, where ATP variety of cellular processes and metabolic pathways. Its regulation in- and carbon are diverted from processes required for cell division and volves a complex and an as yet not well defined assemblage of factors growth to glycogen production (Montero et al., 2009; Rahimpour that are adjusted to the physiological status of the cell. However, to et al., 2013). date most studies on genetic regulation of starch metabolism have fo- Storage protein production is expensive in terms of carbon and ATP cused on the effects of single genes on starch biosynthesis, and analyses costs and it is highly likely that both storage protein and starch biosyn- of the effect of global regulatory factors on starch metabolism, as well as thetic processes compete for the same ATP and carbon pools. Therefore, systematic studies of the regulatory mechanisms of starch biosynthesis blocking storage protein synthesis during the latter phase of develop- are still scanty. Evidence has been provided that, similar to the bacterial ment could be a potential strategy for increasing starch content in het- system where glycogen synthesis and breakdown genes are co- erotrophic organs. One way to do it would consist in altering the expressed and co-regulated with genes involved in diverse biological expression of factors globally controlling the production of storage pro- processes (Montero et al., 2011), starch metabolism genes are co- teins. This, however, is difficult since it has been reported that the ex- expressed and coordinated with other functions such as amino acid pression of genes involved in storage starch and protein synthesis is and fatty acid biosynthesis (Fu and Xue, 2010; Giroux et al., 1994; She highly coordinated by the same factors (Giroux et al., 1994; She et al., et al., 2010; Tsai et al., 2009). Systematic analyses-based characteriza- 2010). One alternative would consist in moderately down-regulating tion of the interactome of starch metabolism-related genes and proteins the expression of genes directly or indirectly involved in the synthesis with genes and proteins involved in other biological processes, and the of amino acids by using promoters that are specifically expressed during identification of global factors controlling the expression of core starch the latter part of the heterotrophic organ development. In this respect metabolism functions will be crucially important to rationally design we must emphasize that accumulation of exceptionally high levels of molecular strategies aimed at enhancing storage starch content in het- starch in leaves of plants exposed to MVs was accompanied by reduc- erotrophic organs of main crops. In this respect, we must emphasize tion of the levels of amino acids as a consequence of down-regulation that genome-wide co-expression analyses, which are based on the as- of genes directly or indirectly involved in amino acid biosynthesis (see sumption that genes with similar expression patterns are more likely Section 3, Ezquer et al., 2010a). to be functionally related, have proven to be a powerful tool to identify regulatory factors in transcriptional networks regulating starch metab- 5. Conclusions and main challenges olism in crop species such as rice and potato (Ferreira et al., 2010; Fu and Xue, 2010). Similar type of comparative transcriptome and prote- The discrepancies between the different proposed models of ome analyses between different plant organs of WT plants and mutants starch biosynthesis in leaves (illustrated in Figs. 1–4) and heterotro- with altered starch content should be conducted using a wide range of phic organs (Figs. 5 and 6), and the unexpected failures of some mo- species of agronomic interest to identify major determinants and global lecular strategies aimed at improving storage starch content in regulators of starch biosynthesis. plants denotes that our knowledge of the starch biosynthetic process MV-promoted accumulation of exceptionally high levels of starch is is still at a rudimentary level. Better understanding of the regulation a recently discovered mechanism for the elicitation of plant carbohy- of starch synthesis will require additional research, either at the bio- drate metabolism by microbes that still needs to be investigated in chemical, cell biological and molecular levels, which in turn will more detail before it can be fully understood and used as a source of allow plant breeders and biotechnologists to advance in their ideas and tools for strategies to complement breeding and biotechnolo- attempts to increase starch in a predictable way. Additional knowl- gy programs aimed at improving crop yields. At short run, further efforts edge on resource allocation within the plant, sucrose uptake in het- and research will be necessary to identify the volatiles and sensing erotrophic cells, sources of ADPG, mechanisms of ADPG transport mechanisms involved in MIVOISAP. in plastids, interaction of the plant with both biotic and abiotic environmental factors, and metabolic interaction of the plastidial com- Acknowledgments partment with other cellular compartments will be crucial. In this last respect we must emphasize that recent studies have demon- This research was partially supported by the grants BIO2010-18239 strated the occurrence of strong, but unexplored metabolic links be- from the Comisión Interministerial de Ciencia y Tecnología and Fondo tween plastids and endomembrane organelles. Thus, Nanjo et al. Europeo de Desarrollo Regional (Spain) and IIM010491.RI1 from the (2006) and Kitajima et al. (2009) have shown that α-amylase and Government of Navarra, and by Iden Biotechnology. M.O. was partly NPPs traffic between endoplasmic reticulum-Golgi and the plastidial supported by grant N. ED0007/01/01 Centre of the Region Haná for Bio- compartment. Furthermore, Wang et al. (2013) have recently shown technological and Agricultural Research.

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