STARCH SYNTHASE 5, a Noncanonical Starch Synthase-Like 2020-05-29آ  RESEARCH ARTICLE STARCH SYNTHASE

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    STARCH SYNTHASE 5, a Noncanonical Starch Synthase-Like Protein,

    Promotes Starch Granule Initiation in Arabidopsis

    Melanie R. Abta, Barbara Pfistera, Mayank Sharmaa, Simona Eickea, Léo Bürgya, Isabel Nealea,b,

    David Seunga,c, Samuel C. Zeemana,d

    a Institute of Molecular Plant Biology, Swiss Federal Institute of Technology in Zurich (ETH Zurich),

    Universitätstrasse 2, 8092 Zurich, Switzerland b Present address: St John’s College, University of Cambridge, Cambridge CB2 1TP c Present address: John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK d Corresponding Author:

    Short title: Role of SS5 in Starch Granule Initiation

    One-sentence summary: A widely conserved but noncatalytic starch synthase-like protein interacts

    with the known granule-initiating factor MRC and regulates the number of starch granules formed in


    The author responsible for distribution of materials integral to the findings presented in this article in

    accordance with the policy described in the Instructions for Authors ( is: Samuel C.

    Zeeman (


    What determines the number of starch granules in plastids is an enigmatic aspect of starch metabolism.

    Several structurally and functionally diverse proteins have been implicated in the granule initiation

    process in Arabidopsis, with each protein exerting a varying degree of influence. Here, we show that a

    conserved starch synthase-like protein, STARCH SYNTHASE 5 (SS5), regulates the number of starch

    granules that form in Arabidopsis chloroplasts. Among the starch synthases, SS5 is most closely related

    to STARCH SYNTHASE 4 (SS4), a major determinant of granule initiation and morphology. However,

    unlike SS4 and the other starch synthases, SS5 is a noncanonical isoform that lacks catalytic

    glycosyltransferase activity. Nevertheless, loss of SS5 reduces starch granule numbers that form per

    chloroplast in Arabidopsis and ss5 mutant starch granules are larger than wild-type granules. Like SS4,

    SS5 has a conserved putative surface binding site for glucans and also interacts with MYOSIN-

    RESEMBLING CHLOROPLAST PROTEIN (MRC), a proposed structural protein influential in starch

    granule initiation. Phenotypic analysis of a suite of double mutants lacking both SS5 and other

    proteins implicated in starch granule initiation allows us to propose how SS5 may act in this process.

    Plant Cell Advance Publication. Published on May 29, 2020, doi:10.1105/tpc.19.00946

    ©2020 American Society of Plant Biologists. All Rights Reserved


    Green plants and algae produce transitory starch as a temporary storage compound that

    provides energy during phases of darkness that would otherwise result in deleterious energy

    starvation (Stitt and Zeeman 2012). As a dense, compact, and osmotically inert carbohydrate

    polymer, starch allows the efficient storage of photoassimilates directly within the chloroplast.

    Transitory starch takes the form of discrete, lenticular (discoid) granules that occur between

    the thylakoid membranes (Streb and Zeeman 2012). In Arabidopsis leaves, chloroplasts

    reportedly contain five to seven granules, a number that was shown to be correlated with

    chloroplast volume (i.e. larger chloroplasts have more starch granules) (Crumpton-Taylor et

    al. 2012). The situation is different in starch-containing storage organs where different types

    or populations of starch granules have been described. Some amyloplasts (e.g. in potato tubers)

    are reported to contain just one simple granule (Ohad et al. 1971), whereas other amyloplasts

    (e.g. in rice) initiate multiple granules that grow together to form compound granules

    (Matsushima et al. 2010, Toyosawa et al. 2016). In other cases, such as in wheat or barley,

    amyloplasts contain distinct populations of large and small granules that are initiated at

    different times (Tomlinson and Denyer 2003).

    Starch consists of glucose units that are condensed into two distinct polysaccharides—

    amylopectin and amylose—by ⍺-1,4- and ⍺-1,6-glycosidic linkages. The predominant

    component, amylopectin, has ⍺-1,4-linked glucan chains connected by ⍺-1,6-bonds to form a

    branched molecule with a tree-like (racemose) structure that contains clusters of unbranched

    chain segments. Neighboring linear chain segments within clusters form double helices that

    pack tightly into crystalline lamellae. These crystalline lamellae alternate with amorphous

    lamellae, which contain the branched chain segments connecting the clusters (Streb and

    Zeeman 2012). Amylose, a minor component of starch and a mostly linear glucan made from

    ⍺-1,4 linked glucose units, is thought to be synthesized within the amorphous lamellae (Denyer

    et al. 2001). Amylose is not strictly required for the formation of starch granules, but it may

    increase the efficiency of glucan storage by occupying residual space in the semicrystalline

    amylopectin matrix.

    Three enzyme classes are needed to make branched, crystallization-competent

    amylopectin. First, starch synthases (SSs) elongate glucan chains by catalyzing the formation

    of ⍺-1,4 glycosidic bonds using ADP-Glucose (ADP-Glc) as a glucosyl donor. Second,

    branching enzymes (BEs) introduce ⍺-1,6 glycosidic linkages by catalyzing glucanotransferase


  • reactions. Third, and less intuitively, debranching enzymes (DBEs) hydrolyze some of the ⍺-

    1,6 branch points introduced by BEs, and this is thought to promote crystallization by refining

    the branching pattern (Streb and Zeeman 2012). Plants possess genes encoding multiple

    isoforms of SSs, BEs, and DBEs, and these isoforms have distinct roles in amylopectin

    synthesis. For example, in Arabidopsis, six genes are described as encoding SSs. Four isoforms

    (SS1-SS4) have been implicated in amylopectin biogenesis (Delvallé et al. 2005, Zhang et al.

    2005, Roldán et al. 2007, Zhang et al. 2008) and are thought to be active at the granule surface.

    The fifth isoform, granule bound starch synthase (GBSS), is responsible for amylose

    production (Denyer et al. 2001). The last isoform, SS5, has not been assigned a specific

    function, which is likely due to its very unusual features (Liu et al. 2015, Helle et al. 2018, Qu

    et al. 2018).

    At the protein level, the canonical SSs (SS1-SS4 and GBSS) share a conserved

    catalytic domain. They are glycosyltransferases (GTs) with a GT-B fold (Carbohydrate Active

    Enzymes (CAZy) database; Lombard et al., 2014), meaning that their structure consists of two

    similar Rossmann-like subdomains that are connected via a hinge region. It is proposed that

    the N-terminal subdomain binds the acceptor substrate and the C-terminal subdomain binds the

    donor substrate; the active site is thus formed in between the two (Qasba et al. 2005, Sheng et

    al. 2009a). Based on amino acid sequence similarity, GTs have been further classified into 109

    GT families in the CAZy database. Both ADP-Glc-utilizing bacterial glycogen synthases and

    plant starch synthases are assigned to GT family 5 (, and their N-terminal

    and C-terminal subdomains are denoted as GT5 and GT1 subdomains, respectively.

    Despite the similarities in their catalytic domains, plant SS isoforms differ significantly

    at their N-termini, which are variable in length and contain either no conserved predicted

    domains (GBSS, SS1), predicted coiled-coil motifs (SS4 and some SS2 orthologues), or both

    coiled-coil motifs and carbohydrate binding modules (CBMs: in the case of SS3, three CBMs

    of the family 53). At the enzymatic level, SSs seem to differ mostly in their acceptor substrate

    preferences. The loss of individual SS1, SS2 or SS3 isoforms results in characteristic changes

    in the amylopectin fine structure (Pfister and Zeeman 2016). A special role has been assigned

    to SS4, however. In Arabidopsis, this isoform strongly influences both the numbers and

    morphology of starch granules produced. Rather than forming five to seven discoid starch

    granules, chloroplasts from Arabidopsis ss4 mutants contain far fewer granules which are

    nearly spherical rather than discoid, and many chloroplasts fail to produce any granules at all


  • (Roldán et al. 2007, Szydlowski et al. 2009). This starch granule phenotype is accompanied by

    a substantial accumulation of ADP-Glc and mild chlorosis, which probably results from a

    deleterious shortage of adenylates for photosynthesis (Crumpton‐Taylor et al. 2013, Ragel et

    al. 2013). These observations have led to the hypothesis that SS4 is a key factor in starch

    granule initiation. Consistent with this hypothesis, the partial loss-of-function of SS4 in wheat

    has similar effects on the numbers of granules formed in leaves (Guo et al. 2017).

    Recent research has identified additional proteins that influence starch granule

    initiation in Arabidopsis (Seung et al. 2017, Seung et al. 2018, Vandromme et al. 2019). First,

    PROTEIN TARGETING TO STARCH 2 (PTST2), a protein containing predicted coiled-coil

    motifs and a family 48 CBM, has been shown