Starch Granule Initiation in Arabidopsis Thaliana Chloroplasts

The Plant Journal (2021) doi: 10.1111/tpj.15359

FOCUSED REVIEW Starch granule initiation in Arabidopsis thaliana chloroplasts

Angel Merida 1 and Joerg Fettke2,* 1Institute of Plant Biochemistry and Photosynthesis (IBVF), Consejo Superior de Investigaciones Cientıﬁcas (CSIC), Universidad de Sevilla (US), Avda Americo Vespucio, 49, Sevilla 41092, Spain, and 2Biopolymer Analytics, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, Building 20, Potsdam-Golm 14476, Germany

Received 1 April 2021; revised 14 May 2021; accepted 22 May 2021. *For correspondence (e-mail [email protected]).

SUMMARY The initiation of starch granule formation and the mechanism controlling the number of granules per plastid have been some of the most elusive aspects of starch metabolism. This review covers the advances made in the study of these processes. The analyses presented herein depict a scenario in which starch synthase isoform 4 (SS4) provides the elongating activity necessary for the initiation of starch granule formation. However, this protein does not act alone; other polypeptides are required for the initiation of an appropriate number of starch granules per chloroplast. The functions of this group of polypeptides include providing suitable substrates (maltooligosaccharides) to SS4, the localization of the starch initiation machinery to the thylakoid membranes, and facilitating the correct folding of SS4. The number of starch granules per chloroplast is tightly regulated and depends on the developmental stage of the leaves and their metabolic status. Plastidial phosphorylase (PHS1) and other enzymes play an essential role in this process since they are necessary for the synthesis of the substrates used by the initiation machinery. The mechanism of starch granule formation initiation in Arabidopsis seems to be generalizable to other plants and also to the synthesis of long-term storage starch. The latter, however, shows speciﬁc features due to the presence of more isoforms, the absence of constantly recurring starch synthesis and degradation, and the metabolic characteristics of the storage sink organs.

Keywords: starch granules, starch metabolism, starch granule initiation, starch granule number per chloroplast, starch morphology, Arabidopsis thaliana.

INITIATION OF THE STARCH GRANULE In plants, starch appears in the form of granules in the plas- After cellulose, starch is the most abundant polymer in the tids (mainly in chloroplasts of photosynthetic tissues or amy- biosphere. It plays an essential role in those organisms that loplasts of storage organs such as tubers, endosperm, or accumulate it (photosynthetic eukaryotes and some roots) (Ball and Morell, 2003). The size, number, and morphol- cyanobacteria) (Cenci et al., 2014). Starch is water-insoluble, ogy of these granules vary greatly depending on the species in contrast to glycogen, which is the storage carbohydrate of and even the organ analyzed (Tetlow and Bertoft, 2020). nearly all other organisms. Starch granules are composed of two different polymers: The harvested parts of our staple crop plants are starch- amylose and amylopectin. In both of these glucans, thou- storing organs. Starch is one of the main contributors to the sands of glucosyl residues possessing the a-configuration are human diet in terms of calories, and it is often used as animal linked together. In starch (and in glycogen as well), there are feed. Furthermore, there is also an increasing demand from essentially only two types of glycosidic linkages that intercon- non-food industries for starch as a renewable material (Zee- nect these glucosyl residues: linear chains of a-1,4-linked resi- man et al., 2010). The synthesis and degradation of starch has dues and a-1,6-linked branches. Amylopectin is the main therefore been the subject of much research efforts, all with component of starch, and has a large number of branches the final aim of obtaining more productive crops or modified ordered in clusters that confer a semi-crystalline structure to starches for industrial applications (Zeeman et al., 2010). the granule. In contrast, amylose, the minor component, has a

© 2021 The Authors. 1 The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 2 Angel Merida and Joerg Fettke small number of branches and no known structure (Tetlow et al., 2007). The amylopectin chain-length profiles, reflect- and Bertoft, 2020). The enzymatic activities involved in the for- ing alterations to the inner granule structure, of ss4 mation of starch are relatively simple: ADP-glucose pyrophos- mutants are similar to those of the WT. No changes in the phorylase (AGPase, EC 2.7.7.27) provides ADP-glucose, which activity of the main enzymes involved in the synthesis or is one substrate used by starch synthases (SSs, ADP-Glc:a- degradation of starch were observed, except for an 1,4-glucan a-4-glucosyl transferases, EC 2.4.1.21) to elongate increase in the cytosolic and plastidial phosphorylase activ- the glucan polymer chains. Starch-branching enzyme (SBE, a- ities (see below). It was proposed that SS4 is involved in 1,4-glucan branching enzyme, EC 2.4.1.18) establishes the the priming of starch granule formation (Roldan et al., branches, and starch-debranching enzyme (DBE or isoamy- 2007). lase, EC 3.2.1.68) eliminates excess branches, allowing the SS4 is a GT-B-fold glycosyltransferase classified within crystallization of clusters (Ball et al., 1996; Brust et al., 2013). the GT5 family in the Carbohydrate Active Enzyme (CAZy) This scenario becomes more complex as a consequence of database (Lombard et al., 2013). It has starch catalytic the presence of different isoenzymes for each activity and the (GT5) and glycosyltransferase 1 (GT1) domains in its C- discovery of new proteins involved in the synthesis of the terminal region. These domains are highly conserved granule. These isoenzymes show different substrate affinities, among the SS4 homologs of different species and among and they form different complexes with other enzymes the different isoforms of SSs (Leterrier et al., 2008; Liu involved in starch synthesis and degradation, modulating et al., 2015). In addition, SS4 has a long N-terminal region their activities (Ahmed et al., 2015; Ball et al., 2011; Kotting€ with some features exclusive to this isoform. This region is et al., 2010; Mehrpouyan et al., 2021). Moreover, for some less phylogenetically conserved, although all SS4 analyzed enzymes, for example, plastidial phosphorylase (PHS1, EC have long coiled-coil domains. A domain of approximately 2.4.4.1), their participation in starch synthesis, degradation, or 50 amino acids, highly conserved among all the SS4 both is still a matter of discussion. enzymes analyzed to date, separates the coiled-coil and Overall, the concerted action of all these polypeptides is glycosyltransferase regions (Raynaud et al., 2016). SS4 is necessary to form the starch granule. The initiation of not found soluble throughout the stroma. On the contrary, starch synthesis remains obscure, in contrast to that of localization experiments involving translational fusion with glycogen. For the latter, its synthesis initiation is known to green fluorescence protein (GFP) have indicated SS4 to be depend on a self-glycosylating protein – glycogenin (EC associated with specific areas of the thylakoid membranes 2.4.1.186; Lomako et al., 1988) – that generates short a- known as plastoglobules (Gamez-Arjona et al., 2014). The glucan chains, which are further elongated to form the N-terminal part of the protein is responsible for its localiza- entire glycogen molecule. No functional glycogenin or tion pattern (Raynaud et al., 2016), and the interaction of equivalent has been reported for higher plants. this region with the plastoglobule-associated proteins fib- However, together with advances in the understanding of rillins 1a and 1b has been shown, although the elimination the different steps necessary for the synthesis and degrada- of these fibrillins does not alter the initiation of starch tion of the starch polymers, relevant progress has been made granule formation (Gamez-Arjona et al., 2014). Using trans- in the research on how starch granule formation is initiated. lational fusions of the N-terminal part of SS4 with Agrobac- Most of the available data refer to transitory starch, and this terium glycogen synthase (GS), Lu et al. (2018) showed that focuses on that of the Arabidopsis thaliana leaf starch. There- this region is also somehow involved in altering the shape of fore, the present review will focus on this example. starch granules, which may establish a link between the localization of SS4 and the shape of granules. THE CENTRAL ROLE OF SS4-DEPENDENT INITIATION OF SS4 has an active catalytic site and shows SS activity STARCH GRANULE FORMATION IN ARABIDOPSIS using amylopectin, glycogen, or some maltooligosaccha- CHLOROPLASTS rides (MOS) as substrates when the polypeptide is The chloroplasts of Arabidopsis Columbia-0 usually contain expressed in E. coli (Brust et al., 2013; Szydlowski et al., four to seven starch granules of lenticular shape, although 2009). The active form of SS4 seems to be a homodimer, the granule number per chloroplast varies slightly between and the conserved region localized between the coiled-coil different accessions (Crumpton-Taylor et al., 2012; Malinova and glycosyltransferase regions is necessary for its forma- et al., 2014). In the wild type (WT), the number seems to be tion (Raynaud et al., 2016). correlated to chloroplast volume (Crumpton-Taylor et al., SS4 was the first element described to be involved in 2012). This restricted number seems to be linked to the coor- the initiation of starch granule formation, but this complex dinated initiation of starch granule synthesis and therefore process requires the participation of other players. Some to an existing regulation network. of these elements have been recently described. Seung Mutants lacking SS isoform 4 (SS4) have just one or two et al. (2017) showed that Protein Targeting To Starch 2 starch granules per chloroplast. These are considerably lar- (PTST2) interacts with SS4, and its elimination leads to a ger than those in WT and show a rounded shape (Roldan phenotype similar to that of ss4 mutants. PTST2 contains,

© 2021 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2021), doi: 10.1111/tpj.15359 Starch granule initiation in Arabidopsis 3 as does its homolog PTST1, a carbohydrate-binding the change is minor compared to that seen in ptst2 domain (Lohmeier-Vogel et al., 2008; Seung et al., 2017). It mutants. Unlike PTST2, there is no evidence that PTST3 has been proposed that PTST2 facilitates granule initiation interacts with SS4. Although PTST3 seems to be involved by delivering suitable substrates to SS4 (Seung et al., in the initiation of starch granules in Arabidopsis, its func- 2017). Moreover, PTST2 has been shown to interact with tion remains unclear. two other plastidial polypeptides: MAR-BINDING OTHER STARCH SYNTHASES INVOLVED IN INITIATION FILAMENT-LIKE PROTEIN1 (MFP1) and MYOSIN- OF STARCH GRANULE FORMATION RESEMBLING CHLOROPLAST PROTEIN (MRC) (Seung et al., 2018). MRC is also known as PROTEIN INVOLVED IN It has recently been described that the elimination of SS STARCH INITIATION (PII1) (Vandromme et al., 2019). The isoform 5 (SS5) in Arabidopsis reduces the number of elimination of MFP1 or PII1 reduces the number of starch starch granules synthesized per chloroplast, indicating that granules per chloroplast, but the granules, although bigger it may also be involved in the initiation of their formation than WT, maintain their lenticular shape. Unlike ss4 (Abt et al., 2020). This protein is found in all green plants mutants, in which the distribution of starch granules is not and is phylogenetically related to SS4, and it has been sug- homogeneous in the plant (indeed they are almost absent gested that both result from a gene duplication event that in young leaves), the distribution of starch granules in may have occurred before the appearance of algae (Liu mfp1 and pii1 mutants is homogeneous throughout the et al., 2015). Abt et al. (2020) showed that SS5 is a non- rosette leaves (Seung et al., 2018; Vandromme et al., 2019). canonical SS isoform that lacks catalytic glycosyltrans- MFP1 was identified in tomato (Solanum lycopersicum)by ferase activity. Mutants lacking SS5 have fewer starch its ability to bind to a matrix attachment region of DNA granules per chloroplast, although the phenotype is less (Meier et al., 1996). MFP1 is a large coiled-coil protein with severe than that of ss4 mutant plants. Like SS4, SS5 forms a C-terminal DNA-binding domain and a predicted N- a homodimer in vivo, interacts with PII1, and localizes to terminal transmembrane domain (Gindullis and Meier, specific points on the thylakoid membranes (Abt et al., 1999). More recently, it has been shown to be associated 2020). The pii1 mutation seems to be epistatic over that of with the thylakoid membranes, with its C-terminal DNA- SS5, suggesting that SS5 exerts its function through its binding domain oriented towards the stroma. It is associ- interaction with PII1. In contrast, the ss4 and ss5 pheno- ated in vivo with nucleoids, suggesting a function for types are additive rather than epistatic, suggesting that MFP1 at the interface between chloroplast nucleoids and SS5 does not exert its function through SS4 (Abt et al., the developing thylakoid membrane system (Jeong et al., 2020). However, the function of SS5 in the initiation of 2003). In addition to these functions, it has been suggested starch granule formation remains unclear. that MFP1 is responsible for the localization of PTST2 asso- Thus, SS4 remains the main protein with enzymatic ciated with specific points of the thylakoid membranes activity among the different elements that make up the (Seung et al., 2018). PII1 interacts with SS4 and shows the starch granule initiation machinery described to date, and same dot-like localization pattern, suggesting PII1 is also as such should have ultimate responsibility for the elonga- associated with specific points of the thylakoid membranes tion of the primer that gives rise to the starch granule. Nev- (Vandromme et al., 2019). However, although these two ertheless, Arabidopsis plants lacking SS4 still synthesize polypeptides seem to interact in vivo, analyses of the local- starch granules, indicating that another elongating activity ization of PII1 in ss4 mutants and SS4 in pii1 mutants indi- takes over the function of SS4. Analyses of double and tri- cate that their localization patterns do not depend on each ple SS mutants indicate that SS isoform 3 (SS3) may be other (Vandromme et al., 2019). The significance of the responsible for the synthesis of starch in the absence of interaction between SS4 and PII1 is unclear; unlike PTST2, SS4. Indeed, most of the chloroplasts in ss3 ss4 double PII1 does not contain a carbohydrate-binding module, mutants lack starch granules (Szydlowski et al., 2009). SS3 which suggests that it does not provide an adequate sub- has the GT5 and GT1 domains characteristic of the rest of strate to SS4. As commented above, the localization of the SSs. It also has a long N-terminal region containing SS4 associated with specific points of the thylakoid mem- three carbohydrate-binding domains necessary for its branes does not depend on PII1. It has been suggested that activity and the binding of the enzyme to glucan molecules PII1 might be required for the correct folding of SS4 or its (Busi et al., 2007; Wayllace et al., 2010). These features interaction with some other factor needed by the starch- enable this enzyme to bind and elongate MOS present in priming machinery (Vandromme et al., 2019). PTST2 has the chloroplast and to initiate starch granule formation another homolog in Arabidopsis, PTST3. This gene is not (see below). However, it is not clear whether SS3 carries phylogenetically well conserved, and several plant species, out this function by interacting with the rest of the initia- including grasses, appear to have lost it (Seung et al., tion machinery. No interaction of SS3 with other elements 2017). The elimination of PTST3 in Arabidopsis leads to a that interact with SS4, such as PII1 or PTST2, has been reduction in the number of starch granules in leaves, but described, and SS3 seems to be localized mainly

© 2021 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2021), doi: 10.1111/tpj.15359 4 Angel Merida and Joerg Fettke surrounding the surface of the starch granule and not associated with specific points on the thylakoid membranes, as most of the elements involved in the initiation of granule formation (Gamez-Arjona et al., 2014). These points suggest that the SS3-mediated initiation of starch granule formation is different from the canonical initiation described above. Notwithstanding, Arabidopsis is still able to synthesize starch even without SS4 and SS3, and some chloroplasts with large, spherical starch granules characteristic of SS4-lacking mutants are seen in the ss3 ss4 double mutant (D’Hulst and Merida, 2012; Figure 1). This indicates that yet other elongating activities can replace the function of SS4 and SS3, although they do so ineffi- ciently since just a few chloroplasts in such mutants contain starch (Figure 1). The elongating activities present in chloroplasts of ss3 ss4 mutants are provided by GBSS, SS1, SS2, and PHS1. None of these enzymes has a noncatalytic starch-binding domain, which may explain their very low efficiency in terms of elongating MOS and initiat- ing starch granule formation. OTHER PROTEINS INVOLVED IN GRANULE INITIATION In addition to SS4 (and partially SS3 and SS5) and its interacting partners, other proteins, which so far have been not directly linked with SS4, influence the starch granule number significantly. PHS1 is one of these. A lack of PHS1 has minor effects on starch metabolism, and causes no alteration in starch granule formation initiation (Malinova et al., 2014). However, the additional lack of either MEX1, a maltose exporter responsible for maltose release from the chloroplast at night during starch degradation (Niittyla€ et al., 2004), or disproportionating enzyme 2 (DPE2, EC 2.4.1.25), involved in maltose metabolism in the cytosol (Chia et al., 2004; Fettke et al., 2006; Lu and Sharkey, 2004), results in a massive reduction in starch granule number. dpe2 phs1 double mutants show the strongest reduction with one starch granule per chloroplast, whereas in mex1 phs1 double mutants mostly two or three granules per chloroplast are seen. In both double mutants, the amount Figure 1. The presence of starch granules in the Arabidopsis ss3 ss4 double of SS4 is not reduced. Interestingly, the reduction in starch mutant. (a) WT and ss3 ss4 plantlets were depigmented and subsequently stained with Lugol solution. (b) Magnification of a leaf of the ss3 ss4 mutant granule number is only observed when a dark phase stained with Lugol solution. The arrow indicates a starch granule. (c) Light exists. Under continuous light the starch granule number microscopy image of a thin section of an ss3 ss4 leaf stained by the periodic increases to WT values (Malinova et al., 2014a, 2014b, acid–Schiff reaction for carbohydrates. The arrow shows the localization of a starch granule. 2017, 2018). Since PHS1 is putatively involved in plastidial maltodextrin metabolism, a connection between MOS and the initiation of starch granule formation is plausible. acceptor-free or unprimed synthesis by the typical elongating enzymes (SSs and PHS1) has been reported. For Ara- THE ORIGIN OF MALTOOLIGOSACCHARIDES bidopsis SSs, maltose was shown to be the shortest MOS can be produced in various ways. However, two sce- acceptor; glucose and ADP-glucose alone cannot be used narios have to be envisaged: the generation of MOS (i) as a starting point for producing longer MOS (Brust et al., when starch synthesis and degradation are already under- 2013). Arabidopsis phosphorylases also require a glucan way and (ii) when starch synthesis is not yet underway. In acceptor (at least of degree of polymerization [DP] 4; Fettke the latter case, it is not clear how the de novo synthesis of et al., 2005, 2006). However, reports exist of rapid maltose MOS occurs; so far, no (or hardly any) evidence of production in the light (e.g., Szecowka et al., 2013). A

© 2021 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2021), doi: 10.1111/tpj.15359 Starch granule initiation in Arabidopsis 5 possible explanation for this is the export of glucose phos- number of granules per chloroplast in these mutants is phates out of the chloroplast during photosynthesis. Glu- highly flexible, since they reflect the current rather than the cose 6-phosphate as well as glucose 1-phosphate previous light regime (Malinova et al., 2017). Furthermore, transporters in the chloroplast have been described (Fettke the mutants show increased amounts of a wide range of and Fernie, 2015; Fettke et al., 2011; Kunz et al., 2010; Mali- MOS compared with WT (Malinova et al., 2014). nova et al., 2019). These phosphorylated monosaccharides It was recently shown that the elimination of the amy- are also connected via the existing phosphoglucomutase lolytic enzyme a-amylase 3 (AMY3) partially supresses the (EC 5.4.2.2) activity inside and outside of the chloroplast growth and morphological phenotypes of ss4 mutants and (Caspar et al., 1985; Egli et al., 2010; Malinova et al., 2014). ss3 ss4 double mutants (Seung et al., 2016). In the amy3 Furthermore, the interconversion of fructose 6-phosphate ss3 ss4 triple mutant, most of the chloroplasts have no (an intermediate of the Calvin–Benson cycle) via phospho- starch, but those that do contain many granules (Seung glucoisomerase (EC 5.3.1.9) to glucose 6-phosphate is pos- et al., 2016). It has been suggested that the amylolytic sible (Kunz et al., 2014; Yu et al., 2000). In the cytosol, the activity of AMY3 eliminates the MOS that could be used by phosphorylase PHS2 can transfer the glucosyl residue of elongating enzymes to synthesize a starch granule (Seung glucose 1-phosphate to cytosolic-soluble heteroglycans, et al., 2016). The involvement of amylolytic activities in the even if their origin is unclear (Fettke et al., 2005; Fettke and control of the starch granule number had previously been Fernie, 2015; Ruzanski et al., 2013). Thus, heteroglycans shown in other plant species. The reduction of isoamylase with longer glucan chains can be formed, which eventually activity in transgenic potato (Solanum tuberosum) tubers allow the release of maltose via the action of DPE2 (Fettke and mutant barley (Hordeum vulgare) seeds lead to the et al., 2006; Fettke and Fernie, 2015) or cytosolic b-amylase accumulation of large numbers of tiny starch granules (Monroe and Storm, 2018). This maltose can be trans- (Burton et al., 2002; Bustos et al., 2004). In this respect, it is ported into the chloroplast via MEX1 (this transport has worth noting that ss3 ss4 double mutant plants have more been shown to be bidirectional) (Lu et al., 2006), where it starch in the cotyledons (P. Ragel, 2012, PhD Thesis, Fig- functions as a primer for elongation to longer MOS via the ure 1). Thus, it would be interesting to see if the amylolytic various SS isoforms, especially SS4. Moreover, DPE1 in activity is lower in these organs so that 2021 the formation the chloroplast is also able to generate longer MOS using of a starch granule is more probable in the cotyledons of maltose, although it does so relatively slowly (EC 2.4.1.25; this mutant. Kartal et al., 2011). DEVELOPMENTAL AND METABOLIC IMPLICATIONS The generation of MOS by already existing starch turnover is much simpler. During both the synthesis and the The phenotype of ss4 plants depends on the developmen- degradation of starch, MOS are easily produced. During tal stage of the leaves. Thus, most of the chloroplasts in starch synthesis, MOS are produced by the trimming mature leaves contain one large starch granule, whereas in action of DBEs (isoamylase 1 [ISA1] and ISA2) (Delatte young leaves they are devoid of starch (Crumpton-Taylor et al., 2005; Myers et al., 2000; Wattebled et al., 2005). et al., 2013). dpe2 phs1 double mutants have different Starch degradation generates a huge amount of maltose numbers of starch granules in mature and young leaves, and short MOS by the action of a- and b-amylases, ISA3, but the effect is opposite to that seen in ss4 plants (Mali- and limited dextrinase (Fettke et al., 2012). MOS longer nova et al., 2014). In contrast, ss5 plants have starch in than DP 4 can be elongated by PHS1. This, plus evidence both young and mature leaves (Abt et al., 2020). Similar from dpe2 phs1 double mutants (see below), may indicate results have been reported for mfp1 and pii1 plants (Seung the involvement of PHS1 in starch initiation. et al., 2018; Vandromme et al., 2019). Furthermore, dpe2 In summary, several alternative pathways exist for MOS phs1 double mutant plants shows the abovementioned generation. They may well substitute for one another, alteration in starch granule formation initiation under dif- since phs1, dpe2, mex1, and dpe1 mutants and isa1 isa2 ferent light/dark regimes. It is likely that differences in double mutants show no obvious alteration in starch gran- starch granule number will be seen for other mutants that ule formation initiation (Chia et al., 2004; Critchley et al., lack the proteins involved in starch granule formation initi- 2001; Delatte et al., 2005; Malinova et al., 2014; Niittyla€ ation. These examples indicate that both the developmen- et al., 2004). Further evidence of this redundancy is pro- tal and the metabolic state of the leaf influence the vided by mex1 phs1 and dpe2 phs1 double mutants; in initiation of starch granule formation. both, the maltose production described above is blocked The starch degradation rate is adjusted to the amount of and therefore MOS generation is too. In principle, this starch accumulated at the beginning of the dark phase. allows the reduction in starch granule formation initiation This control avoids the complete degradation of starch to be explained. However, under continuous light, no during the night and prevents periods of starvation that reduction in starch granule number occurs. Repetitive will be deleterious for the growth of the plant (Graf et al., switching between different photoregimes shows that the 2010; Scialdone et al., 2013). This indicates a connection

© 2021 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2021), doi: 10.1111/tpj.15359 6 Angel Merida and Joerg Fettke between the degradation and the synthesis of starch, and 2020). Environmental conditions also affect the regulation hence with the initiation of starch granule formation. dpe2 of the number of starch granules. Maize (Zea mays) and phs1 double mutant plants illustrate this idea on starch potato plants grown at low temperature changed the num- granule formation initiation. In these mutants, the lack of a ber of starch granules, likely by alterations of plastidial dark phase (continuous light) results in a nearly normal phosphorylase activity (Orawetz et al., 2016; Satoh et al., number of starch granules per chloroplast, indicating that 2008). the dark phase per se or starch degradation in the dark IMPACT OF ALTERED STARCH GRANULE NUMBER ON may be responsible for the reduction of the starch granule OTHER STARCH PARAMETERS number. Blockage of starch degradation in the dpe2 phs1 background by an additional mutation in the gene coding Arabidopsis WT starch is typically thin and discoid, while for the a-glucan, water dikinase (GWD, EC 2.7.9.4; Ritte ss4 and dpe2 phs1 plants have bigger and more spherical et al., 2006), a key enzyme in the phosphorylation/dephos- granules (Malinova et al., 2017). ss5 mutant granules are phorylation cycle at the starch granule surface, which initi- typically discoid, but with irregular margins (Abt et al., ates starch degradation (Fettke et al., 2012), results in an 2020). Other mutants in starch metabolism, although not increase in starch granule number, indicating that starch involved in the initiation starch granule formation, such as breakdown is likely involved in its regulation (Malinova gwd, starch excess 4 (sex4), or like starch excess four 1 and Fettke, 2017). This idea is supported by the increase of (lsf1) (Comparot-Moss et al., 2010; Kotting€ et al., 2009; the number of starch granules per chloroplast in the gwd Mahlow et al., 2014), also show changes in granule mor- single mutant with respect to that found in WT (Mahlow phology, which point to an impact of metabolism on gran- et al., 2014). Moreover, it was reported that phosphoryla- ule morphology (Figure 2). The molecular basis of the tion of starch by GWD also occurs during starch synthesis relation between starch granule morphology and metabo- (Hejazi et al., 2014). lism is still completely unknown. dpe2 phs1 ss4 triple For metabolic implications, not only restrictions on sub- mutants and dpe2 ss4 double mutants have perfectly strates and products can be considered. In ss4, the sub- spherical, large or even very large granules, a distinct phe- strates for starch synthesis, ADP-glucose and MOS, are notype that may be the result of simultaneous alterations available for the remaining SSs (Malinova et al., 2017; in both starch synthesis and degradation. Reports of a Ragel et al., 2013). The high accumulation of ADP-glucose dominant function of SS4 in normal starch granule mor- (with an approximately 100-fold increase compared with phology, especially of its N-terminal domain (Lu et al., WT) was thought to reflect that SS4 acts upstream of all 2018), are unlikely, since dpe2 phs1, mex1 phs1, and dpe2 other SSs, which are limited in starch formation when SS4 mex1 double mutants express SS4 but show more spheri- is lacking. However, dpe2 phs1 double mutants show only cal starch granules (Malinova et al., 2017), suggesting that a very moderate accumulation of ADP-glucose, although starch granule morphology is a feature that is ultimately the number of starch granules is strongly reduced. MOS determined by the balance between different factors, such are also accumulated in this double mutant. Furthermore, as the localization of granule formation initiation and the ss4 mutants, dpe2 phs1 double mutants, and dpe2 phs1 metabolic state of the chloroplast. ss4 triple mutants show an increase in the number of The idea that metabolism is of central importance for starch granules per chloroplast when the light phase is starch granule parameters is further supported by the fact elongated, which is not accompanied by a reduction in the that several mutants that reveal alterations in size and/or intracellular levels of ADP-glucose. Overall, these data sug- morphology also show differences in the chain length dis- gest a precise regulation of the number of starch granules tribution pattern, and thus, at least in part, in the inner that is not strictly dependent on the availability of sub- structure of starch granules or in the starch granule surface strates and relevant enzymes. The regulatory mechanisms properties (Mahlow et al., 2014). are as yet largely unknown. The elucidation of these mech- Overall, a direct link between granule number per anisms is hindered by massive alterations in the metabo- chloroplast, granule size, and granule morphology is lism produced by changes in starch turnover in those demonstrated. Unfortunately, there is still a huge gap in mutants (Malinova et al., 2014). However, these observed our understanding of the various parameters, which in part comprehensive metabolic alterations reflect the central sig- is conditioned by the time and resolution limitations of the nificance of starch turnover for plant metabolism. In addi- available methods and techniques (see also Compart et al., tion, the regulation seems to be tissue- and organ-specific. 2021). Leaf guard cells show fewer starch granules per chloro- EVOLUTIONARY CONSERVATION OF THE STARCH plast than mesophyll cells, and are differently affected by GRANULE FORMATION INITIATION MECHANISM mutations in the abovementioned proteins (Santelia and Lunn, 2017). Similarly, mutations in these proteins differen- This review focuses on recent advances in our knowledge tially affect the columella cells in root tips (Seung et al., of the initiation of the formation of transitory starch

Figure 2. Starch granule parameters. Laser confo- Ws ss4-2 dpe2-1/ss4-2 phs1a/ss4-2 dpe2-1/phs1a/ss4-2 cal scanning microscopy and scanning electron microscopy of chloroplast and isolated starch granules, respectively, of wild-type plants and various mutants related to starch granule formation initiation and starch degradation.

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Col-0 bam3ptst2 pwd sex1-8

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Col-0 sex1-8

granules in Arabidopsis leaves, but it is not well known chloroplasts and amyloplasts, in which transitory and how generalizable this knowledge is to other starch- long-term starch is accumulated, respectively, is the accumulating organisms, especially crops that exhibit absence of thylakoid membranes in the latter. Many of the long-term storage of starch, which is the most economi- elements involved in the initiation of starch granule forma- cally important type of starch. The research performed to tion in chloroplasts are associated to the thylakoid mem- date indicates that many of the elements involved in the branes, likely providing specific points for the initiation of starch formation initiation machinery in Arabidopsis are granule formation. Further studies are necessary to under- conserved in other plants (other elements are missing in stand how these points are established in plastids lacking some groups, such as PTST3 in grasses; Seung et al., thylakoid membranes. 2017), where they carry out a similar function to those Most of the newly identified proteins of the starch initia- described in Arabidopsis. For example, SS4 is required for tion machinery are absent in green algae (Seung et al., normal starch granule formation initiation in wheat endo- 2017, 2018), suggesting the presence of a core set, which sperm amyloplasts (Hawkins et al., 2021). However, the includes SS4 and PHS1, that is shared by green algae and absence of daily turnover in long-term starch storage and land plants. The necessity and advantage of the potentially the different metabolic environment in storage organs newly established initiation mechanism is unclear, but it means that the impact of the different elements of the initi- could be related to the increase in granule number per ation machinery on the synthesis of starch is different to chloroplast frequently observed in land plants compared that described in Arabidopsis chloroplasts. For instance, to algae. Evolutionary studies of starch granule formation the elimination of SS3a and SS4b in rice (Oryza sativa) initiation in different groups of land plants and green algae leads to a change in granule morphology from polyhedral are needed to define the core elements necessary for the to spherical granules and to a reduction in the starch con- initiation of starch granule formation and to understand tent in grains (Toyosawa et al., 2016), although this pheno- the selective pressure that might explain the differences type is not as severe as that of the Arabidopsis ss3 ss4 between these organisms. double mutant, probably because of the presence of other Finally, red algae, glaucophytes, cryptophytes, dinoflag- isoforms of SS3 and SS4. Another difference between ellates, and Apicomplexa parasites store a similar type of

newly identiﬁed proteins involved in starch granule formation initiation in land plants are not found in algae (Seung et al., 2017, 2018). Therefore, SS4 and PHS1 may represent proteins of the original initiation mechanism that is still used in algae and which has been adapted to the needs of land plants.

ACKNOWLEDGMENTS The authors thank Julia Compart and Qingting Liu for help during preparation of the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (DFG‐FE 1030/2‐1, DFG‐FE 1030/5‐1, and DFG‐FE 1030/6‐1) and by grant PGC2018‐096851‐B‐ C22 from the Spanish Ministry of Science and Innovation (MICINN) and the European Fund for Regional Development. Open Access funding enabled and organized by Projekt DEAL. Figure 3. Starch granule synthesis. In starch granule formation initiation, the chloroplastidial maltodextrin pool is used by Protein Targeting To AUTHOR CONTRIBUTIONS Starch 2 (PTST2) and starch synthase isoform 4 (SS4). PTST2 interacts with PII1 and MPFP1. Protein–protein interactions between SS4 and PHS1 and AM and JF designed and wrote the manuscript. PII1 are shown. A lack of the related proteins strongly influences the number of starch granules. CONFLICT OF INTEREST The granule formation initiation complex is formed by starch synthases (SSs), branching enzymes (BEs), and debranching enzymes (DBEs), and a No conflict of interest is declared. starch granule is formed. The chloroplastidial maltodextrin pool is affected by starch synthesis in the light via DBEs and by starch degradation in the DATA AVAILABILITY STATEMENT dark (black lines). The de novo synthesis of maltodextrins is shown by gray lines and covers the transport processes over the plastidial membranes. All relevant data can be found within the manuscript and Dashed arrows represent multiple enzymatic reactions. its supporting materials. 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