Starch in the Arabidopsis plant - Wiley Online Library

Starch/Sta¨rke 2012, 64, 421–434

DOI 10.1002/star.201100163

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REVIEW

Starch in the Arabidopsis plant Alison M. Smith Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Norwich, United Kingdom

The genetic and genomic resources available for the model plant Arabidopsis have allowed rapid progress in understanding the pathways of leaf starch synthesis and degradation. The pathway of starch synthesis is generally similar to that in other plants, but Arabidopsis research has permitted new insights into the mechanism of granule initiation. The pathway of starch degradation is very different from the ‘textbook’ version, largely derived from research on germinating cereal grains. The starch granule is attacked by b- rather than a-amylases, and this process is strongly dependent on a cycle of phosphorylation and dephosphorylation of the granule surface. The major product of granule degradation is maltose, which is exported to the cytosol where it is metabolised to hexose phosphate and then to sucrose. Cytosolic maltose metabolism requires a glucanotransferase and a glucosyl acceptor which is believed to be a complex heteroglycan. Plant productivity and yield are highly dependent on leaf starch turnover, and starch metabolism is coordinated with other factors and processes that determine growth rate. Research in Arabidopsis provides an important knowledge base for the study of starch metabolism in other, less tractable species.

Received: October 21, 2011 Revised: December 14, 2011 Accepted: December 14, 2011

Keywords: Arabidopsis / Leaf starch / Starch degradation / Starch granules / Starch synthesis

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Introduction

Abbreviations: DPE, glucanotransferase (disproportionating enzyme); GWD, glucan water dikinase; ISA, isoamylase (a debranching enzyme); LDA, limit dextrinase (a debranching enzyme); LSF, Like SEX FOUR, proteins similar to SEX4 glucan phosphate phosphatase; MEX1, MALTOSE EXCESS 1, a chloroplastic maltose transporter; NTRC, NADP-thioredoxin reductase C; PHS, glucan phosphorylase; PWD, phosphoglucan water dikinase; SBE, starch branching enzyme; SEX4, STARCH EXCESS 4, a glucan phosphate phosphatase; SS, starch synthase

plant growth, and permitted the identification of some of the proteins necessary for starch synthesis and degradation [1–8]. Arabidopsis is a particularly good species for this sort of forward genetic approach to the elucidation of pathways of starch turnover: it is a diploid, inbreeding species with a short life cycle and a very pronounced daily change in starch content in most of the leaves of its rosettes. Publication of the Arabidopsis genome sequence [9] greatly enhanced speed and ease of gene identification in mutants defective in starch synthesis and degradation. It also permitted reverse genetic approaches to identify the roles of the many Arabidopsis genes that putatively encode enzymes of starch metabolism (e.g. [10, 11]). Together, the forward and reverse genetic approaches have given rise to a very detailed understanding of the pathways of starch turnover in Arabidopsis leaves. From this base, current research is starting to discover how starch turnover is controlled in relation to the assimilation of carbon in photosynthesis and demand for carbon from growth and maintenance processes in the plant. Starch research traditionally recognises two sorts of starch. Transitory starch accumulates in leaves during

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There is a long history of research on starch in the Arabidopsis plant. In pioneering work prior to availability of the genome sequence of this model plant, mutants with altered leaf starch contents were isolated using iodine staining as a screening method (Fig. 1). These mutants provided information about the importance of starch for

Correspondence: Professor Alison M. Smith, Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom E-mail: [email protected] Fax: þ44-1603-450045

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Figure 1. Starch in the Arabidopsis plant. Top: rosettes stained with iodine at the end of the (left), and the end of the night (right). Rosettes were treated with hot ethanol to remove chlorophyll prior to staining with Lugol’s solution and washing in water. Plants stain darkly at the end of the day because of their high starch content. Starch reserves are almost depleted by the end of the night. Bottom: scanning electron micrographs of starch granules purified from leaves harvested at the end of the day (left), and after 9 h of darkness (right). The scale bars represent 5 mm.

the day and is degraded at night. Storage starch accumulates over longer periods, often in specialised storage organs, and is degraded to provide carbon and energy for re-growth or germination after periods of dormancy. Almost all of the research in Arabidopsis has been on leaf starch. The plant lacks organs that store starch in the sense that potato tubers, cereal grains or pea seeds store starch. However, starch is present in many nonphotosynthetic cell types throughout the plant. It is not yet clear whether these starch reserves are metabolised in essentially the same way as leaf starch, or via different pathways that may be more akin to those in storage organs of other plants. This review describes the nature and metabolism of leaf starch, and the control of its turnover in relation to plant growth. It then summarises the relatively sparse information on starch metabolism in other parts of the plant. It also considers the extent to which research on Arabidopsis has provided information of value in understanding starch turnover in other species, and in exploiting starch in commercially important plants. ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Leaf starch

Starch in the chloroplasts of Arabidopsis leaves occurs as flattened disc-like granules that are approximately 2 mm in diameter at the end of the day and 0.75 mm in diameter at the end of the night [12] (Fig. 1). In these respects it is typical of leaf starches in general. Unlike storage starches, which differ profoundly in granule size and morphology between species and types of organs, leaf starch granules are almost always small and disc-like [13–16]. Organisation of glucose polymers within the Arabidopsis leaf starch granule is in several respects similar to that of storage starches: granules are birefringent, suggesting a radial organisation, and small-angle X-ray scatter analysis is consistent with a semi-crystalline structure with a repeat of 9 nm [12]. Both the size and the transitory nature of leaf starch granules probably preclude the formation of the growth rings seen in storage starch granules. Growth rings typically occur with a periodicity of a few hundred nm, and in at least some storage organs are laid down on a daily basis. Leaf starch granules are typically only about 800 nm thick. They grow only during the light period each day and are substantially degraded during the night. Solubilised polymers from leaf starch granules can be separated into large, branched (amylopectin) and smaller, relatively unbranched (amylose) components [12, 17]. The amylopectin component has a polymodal distribution of branch lengths, with maxima in the distribution profile at chain lengths of about 12 and 22 glucose units (e.g. [18–20]). At the end of the day, leaf starch contains about 5% amylose [12]. Leaf starches in general have much lower amylose contents than the 20–30% found in storage starches (e.g. [21, 22]). Again this difference may reflect the transitory nature of leaf starch. Amylose is thought to be synthesised with the starch granule matrix. Given the short period over which leaf starch granules grow, there may simply be insufficient time for the amylose content to build up before the granule is subjected to degradation. The idea that differences between leaf and storage starches largely reflect the different lengths of time over which they are laid down is supported by observations on starch in Arabidopsis mutants in which deficiencies in starch degradation lead to the accumulation of starch, with relatively little night-time degradation, over periods of many days. The sex1 and sex4 mutants, deficient in enzymes necessary for normal granule degradation (see below) have leaf starch contents that are three to six times higher than those of normal (wild-type) plants at the end of the day [2, 23]. In mature leaves of sex1 mutants, starch content remains at these high levels throughout the night. Although starch content decreases in sex4 mutants at night, levels at the end of the night are many times higher than in wild-type leaves. The starch of www.starch-journal.com

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both of these mutants has a much higher amylose content than that of wild-type plants, and growth rings occur in sex4 starch granules [12, 23]. Thus accumulation of starch over a longer period in leaves gives rise to features more akin to storage starches.

3

Starch synthesis

The substrate for starch synthesis in leaves, ADPglucose, is derived from the Calvin–Benson cycle intermediate fructose 6-phosphate via the enzymes phosphoglucose isomerase (PGI), phosphoglucomutase (PGM) and ADPglucose pyrophosphorylase (Fig. 2). Loss of any one of these enzyme activities reduces starch synthesis in Arabidopsis leaves to less than 2% of wild-type levels [24, 25]. The residual starch synthesis in pgi and pgm mutants may utilise glucose 1-phosphate imported into the chloroplast from the cytosol [26]. It has also been suggested that ADPglucose may be synthesised via an unidentified enzyme in the cytosol, then imported into the chloroplast [25]: this route is likely to provide only a small fraction of the substrate for starch synthesis. The pathway of starch synthesis in plants is highly conserved. The substrate ADPglucose is used by starch synthases belonging to five classes [starch synthases (SS) I, II, III, IV and granule bound starch synthase (GBSS)] for elongation of glucose chains which are then branched by branching enzymes that largely belong to two classes [starch branching enzymes (SBE) I and II]. Synthesis of starch granules also requires two classes of debranching enzyme, isoamylases (ISA) 1 and 2. All five classes of starch synthase are active in Arabidopsis leaves. As in all other species and organs so far examined, GBSS is exclusively responsible for the synthesis of amylose. gbss mutants lack the amylose component of the starch granule but exhibit no other obvious phenotypes (Matilda Crumpton-Taylor, John Innes Centre, unpublished data). Mutant analyses show that each of the other four starch synthases plays a distinct role in the synthesis of amylopectin chains [19, 20, 27–29]. The analyses also reveal that isoforms of starch synthase and of starch branching enzyme do not act independently in the synthesis of starch polymers. The structures and quantities of starch polymers are determined by synergistic interactions between these activities. Precise definition of the contribution of each isoform to starch synthesis in a wild-type plant is further complicated by the fact that when one isoform is eliminated by a mutation, there may be changes in activities of other isoforms [19]. Starch branching enzymes are encoded by three genes in Arabidopsis. Two genes encode enzymes that are both of the SBEII class (BE2 and BE3) and are necessary for normal starch synthesis. Loss of BE2 (responsible for ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 2. Pathways of starch synthesis and degradation in the Arabidopsis leaf. Top: pathway of starch synthesis during the day. Calvin–Benson cycle intermediates are used for the synthesis of sucrose and starch. Starch is synthesised from fructose 6-phosphate (fructose 6P) inside the chloroplast. Sucrose is synthesised in the cytosol from triose phosphates, exported from the chloroplast via the triose phosphate transporter. SS: starch synthase. SBE: starch branching enzyme. ISA: isoamylase. Bottom: pathway of starch degradation at night. The granule surface is made accessible to the degradative enzymes by phosphorylation by glucan water dikinase (GWD, also called GWD1) and phosphoglucan water dikinase (PWD, also called GWD3), followed by dephosphorylation by the SEX4 glucan phosphate phosphatase. Starch polymers are hydrolysed by b-amylases and isoamylase 3 to yield maltose, and some longer malto-oligosaccharides which are further metabolised via the glucanotransferase DPE1. Maltose is exported from the chloroplast via the maltose transporter MEX1. In the cytosol it is converted to hexose phosphates via the glucanotransferase DPE2 and reactions putatively involving a heteroglycan as a glucosyl accepter and the glucan phosphorylase PHS2.


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almost all of the activity in the leaf) or BE3 individually slightly affects the chain-length profile of amylopectin in leaf starch. Intriguingly, loss of both isoforms prevents the accumulation of starch in the leaf. The accumulation of high levels of maltose in leaves of such mutants (be2 be3 mutants) indicates that in the absence of branching the linear polymers formed by starch synthases are immediately susceptible to attack by degradative enzymes [30]. Loss of the third BE, BE1, has no effect on starch polymer structure or content [30]. This isoform is only distantly related to the SBEI family defined from other species of plant: together with putative SBEs encoded in the poplar and rice genomes it represents a separate class of SBEs that has been designated as SBE III [31]. The effects of loss of SBE activities in the Arabidopsis leaf are different from those reported for maize and for pea. As in Arabidopsis, branching enzyme activity in leaves of both species is very largely accounted for by an isoform of the SBEII class (about 85% of the activity in both cases). Loss of this isoform in maize (the sbe2a mutant) results in the accumulation of large amounts of highly abnormal insoluble glucans and premature leaf senescence [16]. Loss of the isoform in pea (the r mutant) also strongly affects amylopectin structure, but reduces leaf starch content [21]. These different effects of loss of class II SBEs do not necessarily reflect species-specific roles for the enzymes. The three species may differ quantitatively in their complements of other starch metabolising enzymes, hence the outcomes of loss of SBEII may be different in each case. Synthesis of normal starch granules in Arabidopsis leaves requires the presence of both ISA1 and ISA2 [18, 32]. These two proteins form a heterotetramer capable of debranching amylopectin. In the absence of this activity, leaves accumulate reduced amounts of starch but substantial amounts of a soluble glucan called phytoglycogen. Phytoglycogen is more highly branched than amylopectin. Its branching frequency and pattern do not permit its organisation to form insoluble granules. The accumulation of phytoglycogen in isa1, isa2 and isa1 isa2 mutants led to the proposal that ISA removes some of the branches introduced into a ‘pre-amylopectin’ molecule by SBEs, achieving a branching pattern appropriate for organisation of the polymer into a semi-crystalline matrix [33]. This remains an attractive idea, but there is little definitive information about the mechanism of action of ISA during starch synthesis in wild-type plants in vivo. Interpretation of the phenotypes of mutant plants lacking debranching activities is complex [34], and 14C distribution in Arabidopsis leaves supplied with short pulses of 14 CO2 could be explained by direct incorporation of label from monomers into amylopectin chains at the granule surface rather than into a pre-amylopectin molecule [35]. ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The pathway described above explains how existing starch granules are elaborated, but not how new granules are initiated. In mature Arabidopsis leaves, chloroplasts contain an average of about six starch granules throughout the light–dark cycle [36] It is highly likely that the same starch granules persist over periods of many days, being substantially degraded during each night then growing during the light period via the pathway described above. However, the situation must be different in expanding leaves. Chloroplasts in immature leaves immediately following the cessation of cell division contain approximately twelve starch granules [36]. There are at least three rounds of chloroplast division during cell expansion, so there must be frequent initiation of new starch granules. Very little is known about starch granule initiation in these – or indeed any other – plant cells, but recent studies of the starch synthase 4 (ss4) mutant may shed light on this problem [29, 37]. ss4 mutants have only one starch granule per chloroplast. This phenotype is unique to ss4. Loss of any of the other isoforms appears not to affect granule number, even though some of the mutants have altered starch contents and granule morphology [20, 29]. Thus SS4 may play an essential role in the initiation of starch granules, perhaps by forming a protein complex capable of synthesising a specific glucan that nucleates granule formation [37].

4

Starch degradation

Whereas the pathway of starch synthesis in Arabidopsis leaves is broadly similar to ‘textbook’ pathways previously established for storage organs, this is not the case for the pathway of starch degradation. The ‘textbook’ pathway of degradation was largely derived from studies of germinating cereal grains. The major product of starch degradation in the endosperm – a dead tissue at the time of germination – is glucose, which is taken up into the embryo as the substrate for its growth. In contrast, starch degradation in leaves takes place in the chloroplasts of living cells and the major products are hexose phosphates and sucrose located in the cytosol. As might be anticipated, the pathway of starch degradation in leaves has proved to be very different from that in the endosperm. Elucidation of mutants unable to degrade starch at night has been central to discovery of the pathway. As for starch synthesis, a number of mutants were isolated early on [2]. However, establishing the nature of the lesions was more difficult than for starch synthesis mutants. Whereas all of the starch synthesis mutants thus far examined have mutations affecting proteins that were already known to be involved in starch synthesis, ‘candidate gene’ approaches were ineffective for starch degradation www.starch-journal.com

Starch/Sta¨rke 2012, 64, 421–434 mutants. Several of the mutations affecting starch degradation proved to be in genes encoding proteins not previously known to be involved in this process. Starch granules in Arabidopsis leaves are degraded by the actions of b-amylases and the debranching enzyme isoamylase 3 (ISA3), producing mainly maltose and a smaller amount of maltotriose and longer malto-oligosaccharides (Fig. 2). At least four of the nine b-amylases encoded in the Arabidopsis genome are chloroplastic. Analysis of mutant plants shows that BAM3 (b-amylase 3) is probably the major activity responsible for starch degradation: bam3 mutant plants have higher levels of starch than wild-type plants and lower levels of maltose at night [10]. Some other b-amylase isoforms may also contribute directly to starch degradation but at present their roles are incompletely understood [10, 38–40]. Mutants lacking ISA3 have slower rates of starch degradation than wild-type plants [32, 41] and an abundance of very short glucose chains at the granule surface [41], consistent with a major role for ISA3 in cleaving a1,6 linkages at the base of chains degraded by b-amylases. Although b-amylases and ISA3 are the major enzymes of granule degradation in wild-type plants, chloroplasts also contain a-amylase (AMY3), the debranching enzyme limit dextrinase (LDA; also called pullulanase, PU1), and glucan phosphorylase (PHS1). Mutant analysis shows that these enzymes are not individually required for normal patterns of starch turnover [11, 33, 34, 42]. However, analysis of mutants in which more than one enzyme of starch degradation is missing reveals that AMY3 and LDA can contribute to starch degradation. For example, although the lda mutant has normal starch turnover, the double mutant lda isa3 contains much more starch and has a slower rate of starch degradation than the isa3 mutant [41]. Thus in the absence of ISA3, LDA is important for starch debranching during degradation. Early studies of isolated chloroplasts suggested that the predominant attack on transitory starch granules was phosphorolytic – via glucan phosphorylase – rather than amylolytic. Chloroplasts supplied with phosphate exported products of starch degradation as triose phosphates, consistent with conversion of starch polymers to hexose phosphates and then triose phosphates (e.g. [43]). However, analysis of Arabidopsis mutants lacking PHS1 suggests that this pathway provides carbon for specific functions inside the chloroplast rather for export to the cytosol [42, 44]. Maltose produced by the actions of b-amylases on the starch granule is exported from the chloroplast to the cytosol [45–47] (Fig. 2). A chloroplastic maltose transporter necessary for this process, MEX1, was identified by screening a collection of mutants with altered starch degradation for lines with elevated levels of maltose [48]. The minor maltotriose product of starch degradation is metabolised by a glucanotransferase (a disproportionating ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

425 enzyme), DPE1 [49]. The main role of this enzyme is to catalyse the conversion of two maltotriose units to a maltopentaose and a glucose: in dpe1 mutants, maltotriose levels at night are elevated and starch turnover is reduced. The maltopentaose product of DPE1 can be further metabolised by b-amylases; the glucose product is presumed to be exported to the cytosol via the glucose transporter GlcT. Consistent with this idea, mutants lacking both MEX1 and GlcT have perturbations of carbohydrate metabolism and growth that are much more severe than those of either the mex1 or the glct mutants [50]. Analysis of mutants with reduced rates of starch degradation in their leaves at night led to the surprising discovery that degradation of the starch granule requires not only enzymes able to catalyse hydrolysis of starch polymers but also enzymes that catalyse phosphorylation and dephosphorylation of these polymers. Most starches contain some phosphate, covalently linked to glucose residues in amylopectin polymers. In Arabidopsis leaf starch at the end of the day, about one in 2000 glucose residues is phosphorylated. Phosphorylation was originally proposed to be important for starch degradation from research in potato, which showed that transgenic plants deficient in a protein associated with starch granules (R1) had reduced turnover of leaf starch and greatly reduced levels of starch phosphate [51]. This proposal was supported by the demonstration that the sex1 mutant of Arabidopsis, which has exceptionally high levels of leaf starch, lacks the R1 protein and is deficient in starch phosphate [52]. The R1 protein was subsequently shown to be a glucan water dikinase (GWD or GWD1), which catalyses the transfer of the b-phosphate of ATP onto glucose residues of a 1,4-linked glucans [53]. The importance of dephosphorylation for starch degradation was established through studies of another high-starch mutant, sex4. The sex4 mutation was found to lie in a gene initially believed to encode a protein phosphate phosphatase [54] but later recognised as a glucan phosphate phosphatase capable of removing the phosphate groups added by GWD [55]. Identification and reverse-genetic analysis of Arabidopsis genes encoding proteins related to GWD and SEX4 has now established the following picture (Fig. 2). Two phosphorylating enzymes, GWD and PWD (phosphoglucan water dikinase, also called GWD3 [56, 57]) are required for normal rates of starch degradation, respectively catalysing addition of phosphate groups to the C6- and C3-positions of glucose residues in amylopectin [58]. Two dephosphorylating enzymes, SEX4 and LSF2 (Like SEX4 2 [59]), are required for starch degradation. In vitro, SEX4 can remove phosphate groups from both the C3 and C6 positions [60], whereas LSF2 acts exclusively at the C3 position [59]. Why are phosphorylation and dephosphorylation required for normal starch degradation? In vitro experiwww.starch-journal.com

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ments have established that degradation of isolated starch granules by recombinant BAM and ISA3 is greatly enhanced by concomitant phosphorylation (achieved by addition of GWD and ATP) [61]. Degradation of pre-phosphorylated granules by BAM and ISA3 is enhanced by addition of SEX4 [55]. These results have led to the proposal that phosphorylation disrupts the regular organisation of polymers at the granule surface, facilitating access by the degrading enzymes. This proposal is supported by analyses and modelling of starch structure [61, 62] and by the demonstration that synthetic dextrin crystallites can be solubilised by addition of GWD plus ATP [63, 64]. Dephosphorylation is thought to be required for complete degradation of the glucan chains by BAM because action of this enzyme is blocked by the presence of phosphate residues on glucan chains [65, 66]. Many aspects of this process remain to be understood. The level of phosphate in purified starch is low, but the rate at which phosphate is turned over at the granule surface during the night is unknown. The amount and source of the ATP required for this process, and the spatial relationships between the phosphorylating and dephosphorylating enzymes and the starch degrading enzymes, likewise remain to be discovered. One interesting possibility is that at least some of these enzymes act together in complexes bound to the granule surface. First GWD, PWD (GWD3) and SEX4 can bind to starch both in vitro and in vivo, via established starch-binding domains [67–70]. Second, normal rates of starch degradation in Arabidopsis leaves require two apparently catalytically inactive, granuleassociated proteins, BAM4 and LSF1. These are members of the BAM and SEX4 classes, respectively, but lack amino acid residues thought to be essential for enzyme activity and have no activity when expressed as recombinant proteins. It has been proposed that these proteins may mediate complex formation by the catalytically competent enzymes necessary for granule degradation [10, 71]. Maltose exported from the chloroplast is converted to hexose phosphate in the cytosol via a pathway that is still not fully understood. The initial step is catalysed by a glucanotransferase, DPE2 (Fig. 2). Mutants lacking DPE2 resemble those lacking the maltose transporter MEX1 in having altered starch degradation and very high levels of maltose [72, 73]. DPE2 catalyses the cleavage of the a1,4 linkage of b-maltose with the release of one glucose and the transfer of the other glucose residue to an acceptor. The best acceptor in vitro is glycogen, the animal and bacterial storage glucan, but the nature of the acceptor in vivo is not known with certainty [72, 74, 75]. However, there is excellent circumstantial evidence that it is a complex arabinogalactan-like polysaccharide known as soluble heteroglycan (SHG). A specific subfraction of this heterogeneous pool of molecules (SHGL1) is located in the cytosol, has a relatively high content of glucose resiß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Starch/Sta¨rke 2012, 64, 421–434 dues and can bind to and act as an acceptor for recombinant DPE2 in in vitro assays. Importantly, the glucose residues added by DPE2 can be removed by recombinant PHS2, a ubiquitous cytosolic glucan phosphorylase that converts these residues to glucose 1-phosphate [75–79]. Thus in a test tube it is possible to convert maltose to hexose phosphate using recombinant DPE2 and PHS2 plus purified SHGL1. If this proves to be the major or sole route of utilisation of maltose derived from starch degradation at night, it will be important to discover more about the biosynthesis and evolutionary origins of the cytosolic SHG. The pathway of starch degradation in Arabidopsis leaves is more complex than the pathway in cereal endosperm at almost every step (compare Fig. 2 and Fig. 3). In germinating cereal grains the degradation of the starch granule is catalysed primarily by the endoamylase a-amylase rather than BAM and ISA3, and there is no direct evidence of a requirement for phosphorylation (although GWD influences grain filling and the growth of vegetative parts of wheat plants: [80] and references therein). Neither DPE1 nor ISA3 are of importance in the cereal endosperm; instead, the heterogeneous population of branched and linear glucans produced by a-amylolytic attack on the cereal starch granule is acted on by a-amylase, b-amylase

Figure 3. Pathway of starch degradation in a cereal endosperm. Starch granules are attacked by a-amylase to release branched and linear glucans, which are converted to maltose and glucose via limit dextrinase (a debranching enzyme), a-amylase, and b-amylase. Maltose is converted to glucose via a-glucosidase. Glucose is taken up by the embryo as its source of carbon for growth. www.starch-journal.com

Starch/Sta¨rke 2012, 64, 421–434 and the debranching enzyme limit dextrinase, to produce glucose and maltose [81]. The maltose is hydrolysed via an a-glucosidase to glucose that is taken up by the embryo [82]: there is no role for DPE2, SHG or PHS2 in cereal starch degradation. The greater complexity of the Arabidopsis pathway may well reflect the very different metabolic circumstances in which degradation occurs in leaves and endosperms. There is no subcellular compartmentation in the endosperm, and little or no requirement for fine control of starch degradation. In the leaf cell, the rate of starch degradation must be finely controlled to meet the metabolic needs of the plant during the night, and to allow integration with a host of other essential metabolic processes occurring in the same cell at the same time. The complexity of the Arabidopsis pathway allows for sensitive and responsive control of flux. For example, the combination in Arabidopsis of the exoamylolytic attack via b-amylase and the requirement for a cycle of phosphorylation and dephosphorylation potentially enables tight, multilevel control over the rate of release of a limited spectrum of carbohydrates from the granule surface. This is not possible in endosperm, in which an attack by a single enzyme produces a wide spectrum of products. The requirement for and nature of control of flux into and out of starch in the Arabidopsis leaf are discussed in Section 5. To what extent does pathway in Arabidopsis resemble that in other leaves? Key components including GWD, PWD (GWD3), ISA3, SEX4, MEX1 and DPE2 are conserved among plants and generally expressed in leaves. Proteomics studies show that these proteins are present, for example, in maize leaves [83]. The cytosolic heteroglycan proposed to be the acceptor for DPE2 has been found in leaves of several different species [84] and the apple gene homologous to MEX1 is able to complement the Arabidopsis mex1 mutant [85]. Loss or downregulation of GWD causes starch-excess phenotypes in leaves of tomato, maize, potato and Lotus japonicus [51, 86, 87], suggesting that a requirement for granule phosphorylation during starch degradation is very widespread. There are also indications that production of maltose via b-amylase followed by metabolism via DPE2 in the cytosol may be widespread. Down-regulation of a chloroplastic b-amylase or a cytosolic DPE2 causes a starch-excess phenotype in potato leaves [88–90]. The precise contributions and importance of enzymes involved in leaf starch degradation may differ between species. The impact of loss of GWD on plant growth is far more severe in Lotus japonicus than in Arabidopsis grown under the same conditions [86]. The contributions of the two debranching enzymes ISA3 and LDA to leaf starch degradation may also be species dependent. Loss of ISA3 strongly reduces starch degradation in rice as it does in Arabidopsis [91]. However, whereas loss of LDA has no ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

427 effect on starch turnover in Arabidopsis, the equivalent mutation in maize, pul1, reduces leaf starch turnover [92]. The distribution of proteins involved in starch degradation in maize leaves is unexpected [83]. Almost all of the starch turnover occurs in the bundle-sheath cells in this C4 species [93], and these cells are enriched relative to mesophyll cells in GWD, PWD (GWD3) and ISA3. However, the bundle sheath cells have less SEX4 (DSP4) protein than mesophyll cells [83]. A further complication is that whereas starch degradation occurs in bundle sheath cells, the enzymes of sucrose synthesis are located in the mesophyll cells [93]. Further work is required to elucidate the pathway of starch degradation in maize and other C4 leaves.

5 Control of starch synthesis and degradation Starch synthesis and degradation in leaves are tightly controlled. Starch synthesis during the day fulfils two functions. It acts as a buffer for temporary imbalances between the rates of CO2 assimilation and the rate of synthesis and utilisation of the major product, sucrose [94] (Fig. 2). Equally importantly, its accumulation provides a carbon reserve that is degraded to generate sucrose for the growth and maintenance of the plant during the following night [95, 96]. This section deals first with control of starch synthesis at the level of the pathway itself. It then deals with the broader issue of how starch turnover is integrated with changes in day length and the demand for carbon for plant growth. Recent research on the control of partitioning of carbon into starch during the day has focussed largely on the redox properties of the enzyme ADPglucose pyrophosphorylase. This enzyme is important in controlling flux of carbon from the Calvin–Benson cycle into starch synthesis. It is strongly activated by reduction of disulphide bridges that link its subunits, and plants expressing a redox-insensitive (constitutively active) form of the enzyme have altered patterns of starch turnover [97–99]. Both light levels and sugars influence the redox state of the enzyme in vivo [98, 100]. Thioredoxin, the signalling metabolite trehalose 6-phosphate and a chloroplastic NADP-thioredoxin reductase C (NTRC) have all been implicated in mediating the reduction of the enzyme in response to these signals [100–102]. At present, however, it is not clear whether and how control of ADPglucose pyrophosphorylase activity can permit both the short-term adjustment of the rate of starch synthesis in response to changes in CO2 assimilation and sucrose utilisation, and longer-term maintenance of a rate sufficient to supply an appropriate carbon reserve for growth during the following night. The extent of partitioning of carbon from the Calvin– Benson cycle into starch during the day and the rate of www.starch-journal.com

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starch degradation at night are both determined by day length. The shorter the light period in which the plant is grown, the higher the proportion of newly-assimilated carbon that is partitioned into starch (Fig. 4) [103–105]. Thus a plant in short days stores a higher proportion of its assimilated carbon for use at night than a plant in long days. The rate of utilisation of stored carbon at night is finely adjusted to the length of the night. Over a wide range of night lengths, from about 4 h up to 16 h, the rate of degradation is linear and is adjusted so that starch reserves are exhausted approximately at dawn (Fig. 4) [103, 104, 106]. The control of degradation thus allows for a rather constant supply of carbohydrate from starch throughout the night, and efficient utilisation of the reserves accumulated during the previous day [95]. Very little is known about how fluxes of carbon into and out of starch are adjusted according to day length. The regulatory networks involved may share features with those responsible for control of other, better-understood day-length dependent processes, such as the initiation of flowering [107]. Important clues about the control of starch degradation in relation to the length of the night have been obtained

Figure 4. Response of daily patterns of starch turnover to different day lengths. Grey/black bars and shading indicate periods of darkness. Top: starch accumulation and loss over a day–night cycle in plants grown in 12 h light, 12 h dark (left), 8 h light, 16 h dark (middle) and 16 h light, 8 h dark (right). Note that the rate of starch synthesis (and the proportion of assimilated carbon partitioned into starch) decreases as the length of the light period increases, and that in all three cases the rate of starch degradation is such that reserves are exhausted at dawn. Bottom: response of starch degradation to an unexpectedly early night. When plants grown in 12 h light, 12h dark are subjected to darkness after only 8 h of light (arrow), the rate of starch degradation during the subsequent night is adjusted so that the starch reserves last for the entire 16-h night; in other words the rate is only half that in plants given 12 h light. ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Starch/Sta¨rke 2012, 64, 421–434 from experiments in which plants are subjected to abnormal day–night regimes [103, 106]. In plants grown with a 12-h light period then subjected to an unexpectedly early night after only 8 h of light, the rate of starch degradation from the onset of darkness is half that on previous nights so that the reserves of starch last for the entire 16-h night (Fig. 4). This observation implies that, at the onset of darkness, information about the starch content of the leaf is integrated with information about the anticipated time until dawn to set an appropriate rate of starch degradation. There is good evidence that information about time until dawn is provided at least in part by the circadian clock [106]. The circadian clock may also prove to be important in the adjustment of starch synthesis and degradation to growth in different day lengths, discussed above. However, most aspects of the response to an early night, including the ‘sensing’ of starch content and the way in which the circadian clock actually influences starch degradation in the chloroplast, remain to be elucidated. One way in which flux through starch degradation might be controlled is at the level of expression of genes encoding the chloroplastic enzymes involved. This is superficially an attractive possibility, because transcripts encoding many of these enzymes show strong, coordinated patterns of change in abundance over the day that are driven by both the circadian clock and the light–dark cycle. They peak in the latter part of the light period, and are least abundant at the end of the night (e.g. [103, 108–110]). However, it is highly unlikely that these transcriptional changes influence the capacity for starch degradation during a single night. With the exception of GBSS [109], all of the proteins of starch degradation thus far examined show little or no change in abundance over the day–night cycle [11, 72, 103, 109]. It is probable that both the activation of the pathway in response to darkness [12] and the short-term control of flux are brought about by post-translational modulation of activities of one or more starch degrading enzymes. Several of these enzymes have properties that may be of importance, for example different degrees of binding to the starch granule during the day– night cycle, sensitivity to redox potential, and sumoylation [69, 70, 111–113]. Another intriguing possibility is that changes in flux are brought about at the level of formation of enzyme complexes, rather than modulation of individual enzymes. The daily turnover of leaf starch is essential for the normal growth of the Arabidopsis plant. Degradation of starch accumulated during the previous day provides most of the carbon required for the maintenance and growth of the plant during the night. Growth at night is specifically and strongly compromised in mutant plants that cannot accumulate starch, or have low rates of starch degradation. Unexpected extension of the night, beyond the normal dawn, also leads to rapid cessation of growth www.starch-journal.com

Starch/Sta¨rke 2012, 64, 421–434 [95, 114, 115]. It is increasingly clear that starch metabolism is coordinated with other processes that determine the overall growth rate of the plant in response to carbon availability and environmental conditions [95, 96, 116]. Thus the control of starch turnover cannot be understood in isolation – it must be studied in the context of whole-plant carbon allocation and growth. This is a rapidly developing field, making use of systems and modelling approaches and exploitation of natural variation for growth rate (e.g. [117, 118]).

6

Starch in other plant organs

In addition to the leaf mesophyll cells, many other cells in the Arabidopsis plant accumulate starch at some point during their development. These include the columella cells of the root cap, anther cells, cells in the outer integument of the seed coat and cells in specific zones within the embryo, guard cells and cells in the vascular bundles of the leaf [1, 119, 120]. Although some of these cell types contain chlorophyll (for example guard, vascular bundle and embryo cells), it is likely that their starch is synthesised from imported sucrose rather than from Calvin–Benson cycle intermediates. Imported sucrose is metabolised in the cytosol to glucose 6-phosphate, which enters the plastids via a glucose 6-phophate transporter. Evidence for this route of starch synthesis in cells other than mesophyll cells comes from studies of pgi1 mutants, which are unable to synthesise starch from Calvin–Benson cycle intermediates but can synthesise it from glucose 6-phosphate (see Fig. 2). These plants lack starch in mesophyll cells but retain at least some starch in other cell types [121]. It seems likely that the pathways of starch synthesis and degradation of starch are broadly similar in all starchaccumulating cells of the plant. Detailed analysis of expression in the root of genes encoding enzymes important for starch metabolism in the leaf – including two SBEs, four SSs, ISA1, ISA2, GWD, PWD (GWD3), SEX4, LSF2, ISA3, DPE1, MEX1 and DPE2 – showed that all were highly expressed in the starch-containing columella cells of the roots [121]. They were expressed only at low levels in 16 other cell types that do not contain significant amounts of starch. Mutant plants lacking plastidial PGM or subunits of ADPglucose pyrophosphorylase are almost starchless in all organs, and mutant plants lacking GWD accumulate large amounts of starch in many cell types [1, 2], including cells that contain almost no starch in wild-type plants (for example petal cells and cells in the mature seed coat). The effects of loss of some other enzymes important for starch turnover in the leaf differ between plant organs. For example, mex1, bam3 and isa3 mutants have apparently normal starch turnover in embryo cells [120], and vascular bundle cells of isa1 and isa2 mutants accumulate starch ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

429 rather than the phytoglycogen found in the mesophyll cells [18]. Interpretation of these differences is complicated by the fact there may be simultaneous synthesis and degradation of starch in at least some non-photosynthetic cell types (for example in embryo cells [120]): a reduced flux in one direction may be masked by the flux in the other direction. In general, starch in non-photosynthetic cells probably acts as a reserve of carbon that can buffer transient imbalances between carbon import and carbon utilisation in the cell. In some cell types, however, starch has more specific functions. Guard cells are specialised cells in the leaf epidermis that undergo rapid volume and hence shape changes that alter the aperture of the stomatal pores through which CO2 and water vapour enter and leave the leaf. Starch degradation in guard cells provides a counterion in the form of malate, allowing rapid uptake of potassium ions and thus cell expansion and the opening of the stomatal pore [122]. Starch turnover in this cell type is controlled in a very different way from the process in mesophyll cells. Starch content is generally inversely related to stomatal aperture. Degradation is not triggered by darkness, and appears to occur in response to blue light [123, 124]. The process requires BAM1, a b-amylase isoform which is not required for normal starch turnover in mesophyll cells [10, 111]. Starch in the endodermal cells of the hypocotyl and flowering stem and the columella cells of the root cap plays an essential role in gravity sensing and hence the growth of the shoot away from, and the root towards, the pull of gravity [125–127]. Starch-containing plastids (statoliths) in these cell types tend to sediment to the lowest point of the cell, triggering signalling systems that bring about directed cell expansion. Impaired gravitational responses are seen in starchless mutants [128– 131]. In the sex1 mutant, in which the starch content of gravity-sensing cells is abnormally high, hypocotyls show abnormally sensitive gravitational responses [132].

7

Conclusions

Rapid progress has been made in understanding the nature and control of starch metabolism in Arabidopsis. Research in the last decade has provided radically new insights into starch degradation in particular, and into the control of fluxes into and out of starch. This large body of knowledge has been derived from leaves of plants grown in highly-controlled and invariant conditions. It is pertinent to ask whether it is relevant to the study of starch metabolism in the non-photosynthetic starch-storing organs of crop plants. It is certainly true that information from Arabidopsis cannot simply be extrapolated to the seeds, roots and tubers of crop plants. There are several major and obvious differences, such as the completely different www.starch-journal.com

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pathways of starch degradation in Arabidopsis leaves and the endosperm of germinating cereal grains. However, the depth of understanding now available for Arabidopsis can inform and guide research in crop plants. For example, starch degradation is poorly understood in commercially important situations such as the ripening of banana fruit and post-harvest deterioration of roots and tubers including cassava. Little is known about the control of accumulation and loss of starch in forage, silage and bioenergy crops [133]. In cases like these, knowledge from Arabidopsis can provide a foundation for new approaches and biotechnological advances. The author has declared no conflict of interest.

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