RESEARCH ARTICLE

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, UKd Corresponding Author: szeeman@ethz.ch

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

chloroplasts.

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 (www.plantcell.org) is: Samuel C.

Zeeman (szeeman@ethz.ch).

ABSTRACT

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

INTRODUCTION

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

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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 (http://www.cazy.org/), 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

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(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 to work with SS4 in the granule initiation

process. PTST2 is proposed to interact with and provide SS4 with appropriate oligosaccharide

primers (Seung et al. 2017). The loss of PTST2 leads to a reduction in starch granule numbers

per chloroplast, a phenotype exacerbated by the additional loss of its homologue, PTST3, with

which it also interacts. PTST2 also interacts with MAR BINDING FILAMENT-LIKE

PROTEIN1 (MFP1) and MYOSIN-RESEMBLING CHLOROPLAST PROTEIN (MRC), also

called PROTEIN INVOLVED IN STARCH INITIATION1 (PII1)—two proteins containing

extensive predicted coiled-coil motifs. Both MFP1 and MRC influence the number of starch

granules formed per chloroplast, with mfp1 and mrc mutants having low numbers of granules

compared to wild-type plants (Seung et al. 2018, Vandromme et al. 2019). MRC further

directly interacts with SS4 (Vandromme et al. 2019). Presently, the mechanism(s) by which

this network of interacting proteins functions together to control granule initiation is not well

understood, nor is it known whether this protein network is complete. Here, we demonstrate

that the starch synthase-like protein, SS5, also influences the numbers of starch granules that

form in chloroplasts. SS5 is widely conserved across the plant kingdom and most closely

related to SS4. Yet, unlike the other starch synthases, SS5 lacks the C-terminal GT1 subdomain

that has been proposed to bind the donor substrate and is unlikely to function as a canonical

starch synthase. We show that SS5 interacts with MRC and propose that it serves to regulate

other components of the starch granule initiation network.

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RESULTS

Arabidopsis SS5 is a Conserved Noncanonical Starch Synthase with Unique Features

The canonical starch synthases SS1-SS4 are highly conserved in plants (Pfister and

Zeeman 2016). The presence of SS5 has also been reported in several plant species, and,

although bioinformatic analyses have indicated intriguing features (Liu et al. 2015, Helle et al.

2018, Qu et al. 2018), its function is unclear. To clarify this, we first used the protein sequences

of the soluble Arabidopsis starch synthases (SS1-SS5) as queries to isolate possible

orthologous sequences and create a phylogenetic tree (Supplemental Figure 1). In accordance

with previous observations (Liu et al. 2015, Helle et al. 2018), a number of the retrieved protein

sequences clustered together with At-SS5 (ABJ17089.1) into a separate SS5 clade (including

the rice (Oryza sativa) SS5 protein, Os-SS5; XP 015626202.1) that was most closely related to

the group of SS4 proteins, confirming that SS5 proteins are evolutionarily conserved. Despite

the generally broad phylogenetic representation of SS5 proteins, we noticed the apparent

absence of SS5 in Brachypodium distachyon. We further failed to identify SS5 orthologues in

the genomes of wheat and barley, suggesting a relatively recent gene loss within the Pooideae.

We next explored the features of the isolated SS5 proteins using bioinformatics and in

vivo and in vitro assays. Consistent with a putative role in starch metabolism, the algorithm

TargetP (Emanuelsson et al. 2000) predicted a chloroplast transit peptide at the N-terminus of

the At-SS5 protein (Figure 1A). To verify its plastidial localization in vivo, we cloned and

stably expressed in wild-type Arabidopsis plants an mCitrine-tagged version of the At-SS5

protein. We used a construct based on the genomic sequence of AT5G65685, including ~1.7

kb of upstream sequence containing the putative endogenous SS5 promoter (At-SS5pro), the 5’

UTR, and the complete intron-exon structure. Confocal laser scanning microscopy of those

lines confirmed that At-SS5-mCitrine localized to the chloroplast (Figure 1B), a feature that is

likely common to SS5 proteins because most have a predicted transit peptide. Interestingly, we

observed that At-SS5-mCitrine accumulated mostly in specific subchloroplastic locations, the

nature of which we could not conclusively determine.

The Arabidopsis SS5 gene has been reported to be truncated relative to its orthologues

(Pfister and Zeeman 2016, Helle et al. 2018). Our analysis confirmed that this is due to a

deletion of the sequence corresponding to the C-terminal GT1 subdomain of the canonical

starch synthases, a feature also observed in close Brassicaceae relatives of Arabidopsis thaliana

(Figure 1A, Supplemental Figure 2A). While other SS5 proteins also displayed distinct

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truncations (e.g. from Glycine max and Amborella trichopoda—together with At-SS5 hereafter

referred to as truncated SS5 proteins), most orthologues featured a C-terminal region that is

likely derived from, but not predicted to be a GT1 subdomain (Supplemental Figure 2A,

Supplemental Dataset 1). In accordance with previous observations, we will refer to this region

as GT1-like subdomain (Liu et al. 2015), and to SS5 proteins carrying a GT1-like subdomain

as nontruncated SS5 proteins. Whether truncated or not, all SS5 proteins that we isolated were

consistently predicted to feature a GT5 subdomain (Supplemental Dataset 1).

We next used the recently published crystal structure of the GT domain of At-SS4

(Nielsen et al. 2018) to explore structural features of SS5. The catalytic domain of At-SS4

adopts the classical GT-B type fold, with the active site in the cleft between the two Rossmann-

like subdomains involving residues from both subdomains (Nielsen et al. 2018). We used this

structure as a template to model structures of both At-SS5 and Os-SS5 as representatives of

truncated and nontruncated SS5 proteins, respectively. The modelled sequence of At-SS5

(corresponding to 257 amino acids covering the entire predicted At-SS5 GT5 subdomain)

overlapped well with the N-terminal Rossmann-like subdomain of At-SS4, as expected. The

Os-SS5 model covered the entire catalytic domain of At-SS4 (Supplemental Figure 2B). In

order to identify protein regions that might be relevant to the respective starch synthase

isoforms’ function, we mapped residues that were conserved or conservatively substituted onto

the protein structures—either within all analyzed SS4 orthologues (for At-SS4), all SS5

orthologues (for At-SS5) or the nontruncated SS5 orthologues (for Os-SS5). This approach

showed strong conservation of the GT1 subdomain of SS4 orthologues. However, the

equivalent sequence in nontruncated SS5 orthologues was poorly conserved (Figure 2A). As

expected, the catalytic cleft between the N- and C-terminal subdomains was strongly conserved

in SS4. Again, this was not the case for SS5, irrespective of whether the protein was C-

terminally truncated or not (Figure 2A).

These findings all suggest that SS5 lacks a functional catalytic domain, and is unlikely

to be an active glycosyltransferase. This is supported by the analysis of the conservation of key

amino acids and amino acid motifs. Both bacterial glycogen synthases and starch synthases

share a KXGGL motif in their GT5 subdomains (Figure 1A). Although the exact role of this

motif is not entirely clear (Furukawa et al. 1993, Gao et al. 2004), it is stringently conserved in

all canonical starch synthases, indicating a vital influence on enzymatic function. This motif is

not conserved in SS5 isoforms (Supplemental Figure 3A). Similarly, two conserved residues

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located in the GT1 subdomain in canonical starch and glycogen synthases (K854 and E929 in

At-SS4, corresponding to K305 and E377 in E. coli GS, respectively), suggested to be essential

for catalysis (Sheng et al. 2009b), are not conserved in SS5 isoforms, being either absent (i.e.

in At-SS5) or substituted (in nontruncated orthologues) (Supplemental Figure 3A). In SS4,

these residues interact with ADP and acarbose (Nielsen et al. 2018).

To validate experimentally that SS5 proteins are not functional glycosyltransferases,

recombinant At-SS5 and Os-SS5 proteins were expressed in and purified from E. coli

(Supplemental Figure 3B). We then performed in vitro starch synthase assays, providing these

purified proteins with 14C-labeled ADP-Glc and waxy maize starch as donor and acceptor

substrates, respectively. Neither SS5 protein was capable of incorporating any 14C into starch

under these conditions, whereas recombinant At-GBSS did (Supplemental Figure 3C). The

enzymatic inactivity of SS5 in this in vitro activity assay may be caused by a multitude of

experimental details, such as the use of the wrong substrate. We thus expressed At-SS5 in our

previously developed yeast system for the functional analysis of starch biosynthetic enzymes

and the heterologous production of starch-like polymers (Pfister et al. 2016). In this system, all

canonical Arabidopsis soluble SSs have been shown to produce glucans, presumably using the

endogenous substrates in the yeast cells. When expressing At-SS5, we saw no glucan staining

and thus no evidence of catalytic activity in two independent yeast strains, unlike strains

expressing At-SS1, At-SS2, At-SS3, or At-SS4 (Figure 3A and B) (Pfister et al. 2016). We also

attempted to express the Os-SS5 protein in this yeast system, but did not achieve reproducible

expression. Nonetheless, the combination of bioinformatics and activity assays strongly

suggests that SS5 proteins are not catalytically active as starch synthases.

The crystal structure of At-SS4 revealed a putative surface glucan binding site,

characterized by an aliphatic patch on the surface of the GT5 subdomain (Nielsen et al. 2018).

Interestingly, this site is strongly conserved not only in SS4 orthologues but also in SS5

homologues (Figure 2A; visible after a 90° turn, Supplemental Figure 3D). Potato (Solanum

tuberosum) SS5 has been found associated with potato starch granules in proteomic

experiments (Helle et al. 2018), suggesting that the protein binds to starch. To test glucan

binding for At-SS5, we incubated recombinant At-SS5 with waxy maize starch granules. This

assay indicated that recombinant At-SS5 could indeed bind to starch, albeit weakly (Figure

2B). Residues suggested to interact with glucans at the active site (such as D678 in At-SS4)

(Sheng et al. 2009b, Nielsen et al. 2018) that are strongly conserved in canonical starch

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synthases are not consistently conserved in SS5 (Figure 1A, Supplemental Figure 3D). This

suggests that glucan binding in At-SS5 may be mediated by the surface binding site. The At-

SS5 GT5 subdomain is preceded by an N-terminal extension containing predicted (Lupas et al.

1991) coiled-coil motifs (Figure 1A). Regions with coiled-coil motifs are also present in the

N-termini of SS3 and SS4 (Pfister and Zeeman 2016). The SS5 coiled-coil motif is conserved

in orthologues (Supplemental Figure 4).

SS5 is Weakly Expressed in Leaves

To study the role of SS5 in planta, we obtained three homozygous Arabidopsis lines

with T-DNA insertions in different regions of the At-SS5 gene, AT5G65685 (Figure 4A). No

phenotypic effects of the insertions were evident from an initial inspection of plant growth and

morphologies compared with the corresponding Columbia-0 (Col-0) wild type (Figure 4A).

We confirmed disruption of the SS5 transcript by endpoint RT-PCR using primer pairs

spanning the respective T-DNA insertion sites. As expected, no transcript fragments were

detected in the insertional regions of the respective lines (Figure 4B). Because transcript

fragments up- and downstream of the respective insertions were readily amplified for all three

lines, we further attempted to verify absence of At-SS5 on the protein level. Proteome

information from the publicly available database pep2pro (Baerenfaller et al. 2011) indicated

low protein abundance for At-SS5. Furthermore, the predicted molecular weight (~52 and ~48

kD, for full-length and after cTP cleavage, respectively) is similar to the large subunit of

RuBisCO (~53 kD), rendering its detection by SDS-PAGE and immunoblotting potentially

problematic. Polyclonal antibodies recognizing the recombinant SS5 protein (Figure 2B)

detected a faint band of the expected molecular weight in immunoblots of total protein extracts

from wild-type, but not ss5 leaves (Figure 4B). Its detection, however, was difficult due to

nonspecific cross reactions with other, presumably more abundant proteins and to the low SS5

protein levels. To determine native SS5 expression pattern, we analyzed wild type and ss5-1

plants transformed with the construct based on the genomic sequence of AT5G65685 that was

used to confirm plastidial localization (Figure 1B). Expression of chimeric At-SS5-mCitrine,

though low, was detectable when immunoblotting leaf protein extracts using anti-SS5

antibodies (Figure 5A). Again, endogenous SS5 could only be very faintly detected.

Fluorescence microscopy of intact young rosettes expressing At-SS5-mCitrine confirmed very

weak expression that was most visible in juvenile leaves (Figure 5B).

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SS5 Influences Both the Number and Size of Starch Granules in Chloroplasts

We investigated whether the loss of SS5 had any effect on amylopectin fine structure

by analyzing the chain length distribution (CLD) of ss5 starch. Consistent with the suspected

catalytic inactivity of SS5, the CLD of starch extracted from ss5 was indistinguishable from

that of the wild type (Supplemental Figure 5). Measurements of the starch content in whole

rosettes harvested at both the end of the day and the end of the night revealed a minor, but

statistically significant increase in starch content at the end of the night (Figure 6A). Similar

trends were observed when starch content was monitored over a complete day-night (diel)

cycle (Supplemental Figure 6A). We also tested whether the loss of SS5 affected the plant’s

ability to regulate starch degradation by subjecting plants to unexpected changes in the length

of the photoperiod. In wild-type plants, an unexpectedly long day elicits an increased rate of

degradation during the subsequent short night, while an unexpectedly short day elicits a

decreased rate during the subsequent long night (Scialdone et al. 2013). These patterns were

also observed in ss5 mutants, although in each case there was a slight increase in starch

throughout the measurements compared to the wild type (Supplemental Figure 6B).

Next, we used light and electron microscopy to examine the chloroplasts and starch

granules of ss5 mutants. Leaf samples were harvested at the end of the day, chemically fixed

and embedded in epoxy resin. Interestingly, quantitative analysis of the light micrographs

revealed that the ss5 mutant lines produced significantly fewer starch granules per chloroplast

than wild-type plants (Fig 6B). Sections from ss5 and the wild type had a grand mean ±

composite standard error of 1.85 ± 0.03 and 2.89 ± 0.08 starch granules per chloroplast section,

respectively. Note that these numbers are derived from 500 nm thick sections only, and that

the actual number of granules in the entire chloroplast volume will be higher in both cases as

only a fraction of the chloroplast is being sectioned. The shift towards lower granule number

per chloroplast in ss5 was comparable in all three T-DNA insertion lines (Figure 6B), and

milder than in mutants recently identified and analyzed by our laboratory and others (e.g. ss4,

ptst2, and mrc; (Roldán et al. 2007, Seung et al. 2017, Seung et al. 2018, Vandromme et al.

2019)). We purified and analyzed the starch granules of ss5 by scanning electron microscopy

(SEM), and, consistent with the impression from light micrographs, purified ss5 starch granules

appeared as flattened discoids but were much larger in diameter than wild-type granules

(Figure 6C). Furthermore, they appeared irregular in shape, as has been observed for other

abnormally large starch granules (Mahlow et al. 2014, Seung et al. 2017, Seung et al. 2018).

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These observations suggest that SS5, although probably not catalyzing a classic

glucosyltransferase reaction, does have a role in the regulation of starch granule initiation.

Surprisingly, the reduced granule number phenotype of ss5 was only partially

complemented by the expression of At-SS5-mCitrine. Furthermore, we observed a slight

dominant-negative effect when expressing this chimeric protein in the wild-type background

(Supplemental Figure 7). This suggests that the mCitrine-tagged protein version is only

partially functional and, when present, may interfere with the function of endogenous SS5.

Given that the very C-terminal amino acids in SS5 proteins are conserved (Supplemental

Figure 2A), we reasoned that the C-terminally placed tag may be the reason for dysfunction.

Therefore, we modified our construct, introducing a stop codon before the tag. Indeed, At-SS5

expression from this construct fully complemented the starch granule number phenotype of ss5

mutants already in the T1 population (Figure 7).

SS5 Interacts With Itself and MRC

Because SS5 has an N-terminal conserved coiled-coil region (Figure 1A, Supplemental

Figure 4), we reasoned that it may exert its influence on granule initiation by interacting with

other proteins. Therefore, we used both At-SS5-mCitrine expressed from the genomic

construct as well as At-SS5-YFP expressed under the UBIQUITIN10 promoter (UBQ10pro) as

baits in immunoprecipitation experiments. Like At-SS5-mCitrine, At-SS5-YFP had a slight

dominant-negative effect when expressed in the wild-type background and did not fully rescue

the mutant ss5 phenotype (Supplemental Figure 8). We used magnetic beads to capture and

enrich the baits via their mCitrine and YFP epitopes, respectively. When confirming bait

enrichment using anti-At-SS5 antibodies, we noticed that the immunoprecipitate of plants

expressing At-SS5-YFP in a wild-type background also contained a band corresponding to the

endogenous SS5 protein. This band was absent when the At-SS5-YFP protein was

immunoprecipitated from the ss5-1 background (Figure 8A, middle panel). This suggested that

SS5 was able to multimerize in vivo. We made use of this feature as an indicator of endogenous

At-SS5 migration in SDS-PAGE (Figure 4B). We also used it to test whether any of our ss5

mutant lines still expressed At-SS5 protein (e.g. a truncated form). As expected, this approach

failed to retrieve the endogenous SS5 protein in different ss5 T-DNA insertion lines

(Supplemental Figure 9).

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Next, we analyzed the At-SS5-mCitrine immunoprecipitate for other putative

interaction partners using mass spectrometry. In addition to stably expressed At-SS5-mCitrine,

we also used N-terminally polyhistidine-tagged At-SS5 recombinant protein purified from E.

coli as a bait in crude extracts from wild-type plants. With both approaches, we consistently

identified peptides of the MRC protein coprecipitating with At-SS5 baits, even when using

very stringent exclusion criteria (Supplemental Table 1). Two other proteins (At-AMT1 and

At-CHY4) were found in both immunoprecipitate types with our stringent exclusion criteria.

However, both are likely false positives, as both proteins are not predicted to localize to the

chloroplast (Loqué et al. 2006, Gipson et al. 2017).

We confirmed the presence of MRC in the At-SS5-YFP immunoprecipitate by

immunoblotting with antibodies raised against MRC (Figure 8A, bottom panel). MRC was

enriched by the SS5-YFP bait, regardless of whether it was expressed in wild-type or ss5 plants.

Next, we used plants expressing At-MRC-mCitrine (Seung et al. 2018) for a reverse

immunoprecipitation. Using this bait, we could detect coprecipitating endogenous At-SS5 in

the immunoprecipitate (Figure 8B), confirming the interaction between the two proteins in

vivo.

At-MRC was recently found to adopt a punctate subchloroplastic distribution (Seung

et al. 2018), similar to what we observe for At-SS5-mCitrine (Figure 1B). To determine

whether the two proteins localize to the same region in chloroplasts, we transiently expressed

At-SS5-YFP and At-MRC-CFP in tobacco leaves under the strong constitutive cauliflower

mosaic virus 35S promoter (CaMV35Spro). Consistent with the results from stable Arabidopsis

transformants, both At-MRC-CFP and At-SS5-YFP localized to discrete puncta when

expressed individually (Figure 8C). A significant fraction of At-SS5-YFP additionally

displayed a diffuse, likely stromal, localization. Interestingly, when coexpressing both proteins,

At-SS5-YFP and At-MRC-CFP colocalized to the same puncta, consistent with their physical

interaction.

To test our initial hypothesis that the N-terminal coiled-coil motif of At-SS5 mediates

the observed protein-protein interactions, we expressed an At-SS5 protein version lacking this

region (At-SS5Δcc-YFP) in Arabidopsis. Expression of the At-SS5Δcc-YFP protein version

did not elicit the dominant-negative phenotypic effects seen for expression of At-SS5-YFP in

the wild-type background, indicating that this deletion protein is not functional. Consistently,

expression of At-SS5Δcc-YFP in the ss5 mutant background did not strongly affect the starch

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granule phenotype (Supplemental Figure 10). To test whether this may be related to protein-

protein interactions, we used protein extracts of plants expressing At-SS5Δcc-YFP for

immunoprecipitations. We detected neither the endogenous At-SS5 nor At-MRC in the

immunoprecipitate when using this truncated protein as a bait (Figure 8D), suggesting both

that the conserved coiled-coil is directly or indirectly mediating homomeric and heteromeric

interactions and that these interactions are crucial to the protein’s function.

We further investigated if the homo- and heteromeric interactions that we observed for

At-SS5 are evolutionarily conserved by cloning and expressing the rice Os-SS5-YFP protein

in wild-type Arabidopsis under control of CaMV35Spro. As with fluorescently tagged At-SS5,

we observed a dominant-negative effect on starch granule numbers when expressing this

protein in the wild-type background (Supplemental Figure 10). When subjecting these plants

to an anti-YFP immunoprecipitation, we detected the Arabidopsis MRC protein both by

immunoblotting (Figure 8D, bottom panel) and by mass spectrometry (Supplemental Table 1),

indicating that the rice SS5 orthologue can also interact with At-MRC. However, we could not

detect the endogenous At-SS5 in the immunoprecipitate by immunoblotting (Figure 8D,

middle panel). Using mass spectrometry, two peptides matching At-SS5 were detected in one

of the replicates only (Supplemental Table 1). This experiment could not determine whether or

not Os-SS5 undergoes homomultimerization as does At-SS5.

SS5 Modifies Several Starch Granule Initiation Phenotypes

Our observations strongly suggest a physical interaction between MRC and SS5. This

is interesting since their mutant phenotypes are similar despite that of mrc being more severe;

the vast majority of chloroplast sections in mrc mutant plants contain one single, large starch

granule (Seung et al. 2018). We thus crossed the ss5-1 line with mrc and selected a homozygous

double mutant line to determine whether the additional loss of SS5 in the mrc background

would enhance the mrc phenotype. However, we could not discriminate between the

phenotypes of mrc and the mrc ss5 double mutant (Figure 9A). This suggests that SS5

influences starch granule numbers via the MRC protein.

Given that SS5 is most closely related to SS4 and that SS4 has a pivotal role in starch

granule initiation, we also created a ss4 ss5 double mutant. We found no significant difference

in starch contents of ss4 ss5 compared with the ss4 single mutant parent (Figure 9B). Iodine

stained rosettes harvested at the end of the light phase showed a characteristic ss4-like

12

heterogenous starch accumulation in leaves of different ages in the ss4 ss5 double mutant

(Figure 9C), whereas starch was homogenously distributed in ss5-1 rosettes, as in the wild type.

Quantification of starch granules in sections of ss4 ss5, however, revealed an even higher

fraction of chloroplasts failing to initiate any granules than in ss4 (Figure 9A). Consistent with

this, we measured a significant increase in ADP-Glc in ss4 ss5 compared to ss4 (Figure 9D).

Because the ss4 and ss5 phenotypes are additive rather than epistatic, we conclude that SS5

does not exert its function through SS4.

We also crossed ss5-1 to the mutants lacking PTST2 or PTST3, two homologous

proteins proposed to provide SS4 with malto-oligosaccharide substrates for granule initiation

(Seung et al. 2017). In both cases, we observed a shift towards lower starch granule numbers

(Figure 9A). This effect was relatively mild in the ptst2 ss5 double mutant compared to ptst2,

but particularly strong in the ptst3 ss5 double mutant compared to ptst3. Both ss5 and ptst3

single mutant phenotypes are mild (Seung et al. 2017) but the combination of both mutations

resulted in a strong phenotype that was similar to that of mrc. Last, we crossed ss5 to a mutant

of MFP1, a plastidial coiled-coil protein that was suggested to influence the sub-chloroplastic

localization of PTST2 (Seung et al. 2018). Here, we observed a granule number distribution

that was, surprisingly, shifted slightly towards more granules compared to the mfp1 parent

(Figure 9A). Because the difference was small, further investigation will be helpful to support

this observation. Together, these data suggest that SS5, acting via MRC, operates in a

previously unrecognized dimension of the starch granule initiation process.

DISCUSSION

Arabidopsis SS5 Is a Noncanonical Starch Synthase that Influences Starch Granule

Abundance and Morphology

We have shown that Arabidopsis SS5 is a noncanonical member of the plastidial starch

synthase family that, although lacking the glucosyl transferase activity associated with the

canonical members (Figure 3, Supplemental Figure 3C), influences the number and

morphology of starch granules that form in chloroplasts. The number of starch granules is

significantly reduced in three independent ss5 mutant lines compared to the wild type, and the

ss5 granules, though typically discoid, are abnormally large, with irregular margins (Figure 6B

and C). Total starch content in ss5 is largely comparable to wild-type levels, except for slightly

increased levels at the end of the dark phases, and this may be attributable to the alterations in

13

the granule numbers and sizes having a direct effect on the rate of degradation, as previously

suggested for SS4 (Roldán et al. 2007). However, amylopectin structure in the ss5 mutant lines

is unaffected (Supplemental Figure 5), suggesting SS5 plays a role in processes that directly

initiate or otherwise control the number of starch granules in chloroplasts, rather than in

amylopectin biosynthesis.

Starch granule initiation in Arabidopsis has received much attention recently, with a

multitude of novel proteins being discovered. SS4, the closest homologue of SS5, was the first

protein to be identified as both a mediator of starch granule initiation (Roldán et al. 2007) and

a determinant of starch granule morphology. The ss4 mutant phenotype is severe, with many

chloroplasts failing to produce any granules and with those that are produced displaying

morphological anomalies, i.e. they are more spherical than the normal discoid form (Roldán et

al. 2007). The subsequent discovery of PTST2 and PTST3 (Seung et al. 2017), and MFP1 and

MRC (Seung et al. 2018, Vandromme et al. 2019), all of which exert varying levels of influence

on granule numbers, suggests that granule initiation is a far from simple process. Interactions

among these proteins and with glucan substrates has fueled speculation about the molecular

mechanisms underpinning granule initiation. Present models place PTST2 and SS4 at the heart

of the process, with PTST2 binding long malto-oligosaccharides (> DP10) and presenting them

as substrates to SS4 for elongation into granule initials (Seung et al. 2017). The association of

PTST2 with thylakoid-associated MFP1 is suggested to determine where within the chloroplast

these initiation events take place. The importance attributed to SS4 and PTST2 in the model is

partly due to the strong phenotypic consequences of the loss of these proteins (Roldán et al.

2007, Crumpton‐Taylor et al. 2013, Ragel et al. 2013, Seung et al. 2017). In comparison, the

phenotypes of mfp1 and mrc are less severe (Seung et al. 2018), and that of ptst3 is even milder

(Seung et al. 2017) under standard experimental conditions.

We showed that ss5 plants produce lower numbers of starch granules. The ss5 mutant

phenotype is comparable to that of ptst3 (Figure 6B, Figure 9A). Together with the protein’s

relationship to SS4 (Figure 2A, Supplemental Figure 1), its capacity for carbohydrate binding

(Figure 2B), and its interaction with MRC (Figure 8A and B, Supplemental Table 1), this

strongly implicates SS5 as part of the protein network that establishes the correct number of

starch granules per chloroplast. The phylogenetically broad representation of SS5 proteins

(Supplemental Figure 1) indicates that this function may be well conserved.

14

SS5 Is Part of the Starch Granule Initiation Protein Network

Although our data show that SS5 is almost certainly not an active glucosyl transferase,

there are conspicuous similarities with SS4. We have shown in vitro that At-SS5 is capable of

binding glucans (Figure 2B) and that a putative surface carbohydrate binding site recently

identified in At-SS4 (Nielsen et al. 2018) is strongly conserved in SS5 proteins. Although the

significance of this site for SS4 function is not yet known, it is plausible that it plays a role in

substrate coordination or the association of the protein with the starch granule surface. Because

the glucosyltransferase active site is not conserved in SS5 (Figure 2A), we propose that the

observed binding to glucans is mediated by this site.

We also observed that SS5 interacts with itself and with MRC (Figure 8A and B), and

these are properties also reported for SS4 (Raynaud et al. 2016, Vandromme et al. 2019).

Interestingly, the ability to interact with At-MRC was conserved in the full-length SS5 from

rice (Figure 8D). It is uncertain whether the sites of interaction are common to SS4 and SS5

proteins. The N-terminal parts of both proteins feature predicted coiled-coil motifs, parts of

which show amino acid sequence similarity. Deletion of the coiled-coil from At-SS5 prevented

both multimerization and interaction with MRC (Figure 8D). However, the part of the SS4

protein implicated in dimerization by Raynaud et al. (2016) is not conserved in SS5, and the

part(s) mediating its interaction with MRC are unknown. Nevertheless, the N-terminal part of

SS4 was shown to be critical both for its function in terms of localizing the protein within the

chloroplast (Raynaud et al. 2016) and for controlling the morphology of the developing starch

granules (Lu et al. 2018). In contrast to ss4, granules from ss5 display an overall discoid

morphology. The same is true for mrc granules, suggesting that neither interaction of SS4 or

SS5 with MRC is involved in the regulation of granule morphology.

Our data suggest that SS5 may exert its function through its interaction with MRC, as

the MRC mutation was epistatic over that of SS5 (Figure 9A). The MRC protein is rich in

predicted coiled-coil motifs and likely serves some sort of scaffolding function, potentially

bringing SS4 and/or SS5 into contact with other protein factors. Unfortunately, besides SS4

and SS5 binding, the precise role of MRC itself in granule initiation is unclear. Based on the

analysis of fluorescent fusion proteins, its subchloroplastic localization appears to be

concentrated in discrete puncta (Seung et al. 2018, Vandromme et al. 2019). Interestingly, we

saw that SS5 localized to similar puncta when expressed as a fluorescent fusion protein in

Arabidopsis (Figure 1B), and that the localization pattern of MRC and SS5 overlapped when

15

the two proteins were coexpressed in tobacco (Figure 8C). Unlike MFP1, MRC behaves as a

soluble stromal protein in extracts, and the nature of the puncta formed by fluorescently tagged

MRC and SS5 thus remains unclear at this point. Furthermore, we again note that the SS5

fusion proteins used in this study were only partially functional. Even though they enabled us

to detect the interaction with MRC, which could be validated by reciprocal IP experiments, the

in-vivo localization studies should be treated as preliminary. The use of new lines and advanced

microscopy techniques capable of correlating fluorescent signals with the chloroplast

ultrastructure will provide the necessary plastid context to fully evaluate such localizations in

future.

The analysis of double mutants between ss5 and lines lacking other granule initiation

factors revealed intriguing genetic interactions. Whereas the observed epistasis of mrc over ss5

(Figure 9A) was clear and consistent with the observation of the two proteins interacting,

observations made for other double mutants were more difficult to interpret. In the absence of

SS4, SS5 still appeared to promote granule formation because the phenotype of the ss4 ss5

double mutant was more severe than that of the ss4 single mutant. Similarly, ptst2 ss5 had a

stronger phenotype than ptst2, which is consistent with the current hypothesis of PTST2 and

SS4 acting together (Seung et al. 2017). However, the phenotype of the ss5 mfp1 double mutant

was no more severe than that of mfp1; if anything, a slight alleviation of the phenotype was

observed. Strikingly, the ss5 ptst3 double mutant had a strongly enhanced, mrc-like phenotype

compared to its parental mutant lines (Figure 9A). Importantly, these data illustrate that granule

initiation can be greatly perturbed by the loss of factors that individually have small influences,

emphasizing that the roles of such factors should not be underestimated based on their single

mutant phenotypes. However, it also illustrates that proteins acting in the process of starch

granule initiation may not act in linear pathways, and that the analysis of mutant phenotypes

consequently may not follow strict epistatic principles.

Future studies are needed to address potential confounding factors—including the high

multiplicity and redundancy of the involved proteins—that may effectively mask aspects of

how starch granules are being formed. These future studies may include a detailed analysis of

higher-order mutants and the thorough exploration of the extent to which the involved proteins

colocalize and functionally interact in vivo. Such data will help us to understand how the

numbers of initiated starch granules are determined, the way they develop, and where within

the chloroplast this is most likely to happen. It will also help us to establish whether granules

16

can be initiated via different mechanisms and which protein complex configurations can

successfully do so, as discussed below. Parallel analyses will be required to clarify to which

extent the roles and interactions of proteins are conserved in species other than Arabidopsis

and in storage tissues. This is especially important since recent research has indicated

significant species- and tissue-specific consequences of the loss of individual proteins involved

in this aspect of starch metabolism (Peng et al. 2014, Saito et al. 2018, Wang et al. 2019, Zhong

et al. 2019, Chia et al. 2020).

SS5 Is Relevant in Starch Granule Initiation: How and Why?

The similarities between SS4 and SS5 indicate that their functions likely have common

mechanistic origins, yet functional divergence has occurred with the degeneration of catalytic

glycosyltransferase abilities in SS5. The mild phenotype of ss5 mutants and the observation

that SS5 acts in the absence of SS4 indicates that its role is related to aspects of granule

initiation that do not directly or exclusively require SS4. Yet both SS4 and SS5 interact with

MRC. These findings may be interpreted in several ways. On one hand, SS4, SS5, and MRC

(and possibly other factors) may constitute one large protein complex that is compromised to

a certain extent upon the absence of either constituent subunit. However, we did not detect SS4

among the potential interaction partners of SS5 in our immunoprecipitation experiments

(Supplemental Table 1). This could be due to difficulties in detecting weak or indirect

interactions. However, it is also possible that MRC may interact with either SS4 or SS5, but

not with both at the same time.

Thus an alternative explanation is that SS4 and SS5 act in separate protein complexes

that both—via similar or different mechanisms—promote the initiation of starch granules.

Given the enzymatic inactivity of SS5, a granule-initiating complex containing SS5 may

require incorporation of another canonical starch synthase. In the absence of SS4, SS3 can

initiate some granules (Szydlowski et al. 2009, Seung et al. 2016), so SS3 may be a good

candidate for this role. In this context, the fact that ss4 ss5 plants still produce some starch

would suggest that SS5 assists, but is not essential for SS3 to initiate granules. However, we

also did not detect a physical interaction between SS5 and SS3. As for SS4, it is difficult to

rule out the possibility of a weak, transient, or indirect interaction. Again we note that the

fluorescent tag, which influenced the function of SS5 somewhat, may have reduced or

prevented some functionally important interactions. The overall question remains as to why a

17

noncatalytic starch synthase would promote starch granule initiation by any of the canonical

starch synthases.

An intrinsic aspect of transitory starch metabolism is the recurrent alternation between

biosynthesis and degradation over the diel cycle, which intuitively implies the need for

reinitiation. However, the need for starch granule initiation may differ greatly depending on

the developmental stage of the leaf and on varying environmental conditions that may occur

over multiple diel cycles. For instance, it has been suggested that starch granule initiation is

closely coordinated with chloroplast division and maturation, which happens mostly as leaves

develop and expand (Crumpton-Taylor et al. 2012, Crumpton‐Taylor et al. 2013). Because

chloroplasts divide by fission, it would presumably be advantageous for each daughter

chloroplast to be equally endowed with starch and the biosynthetic apparatus for producing

it—something that is easiest to achieve by having multiple discrete granules. It is also possible

that the presence of just a few, or only one, large starch granule may impede the chloroplast

division process. These ideas are consistent with the observation that starch granule numbers

per chloroplast are highest in immature leaves and lower in mature leaves (Crumpton-Taylor

et al. 2012), even though the overall starch content is similar (Zeeman and Rees 1999).

Furthermore, young, actively growing leaves have high metabolic demands and might

fully deplete their starch reserves at night to fuel growth, and this could necessitate de novo

initiations each dawn. In this context, SS4 appears to be especially important, as young ss4

mutant leaves are essentially starchless (Crumpton‐Taylor et al. 2013). In contrast, cycles of

granule growth and shrinkage may predominate in mature source leaves (Crumpton-Taylor et

al. 2012), with remnant granules from the previous diurnal cycle serving as substrates for the

starch biosynthetic machinery at dawn. In such a case, de-novo initiations will be required less

frequently, or may even be suppressed. Consistent with this, inducible knock-down of SS4

expression only seemed to impact upon starch granule numbers in newly emerging tissue, but

not in mature leaves (Crumpton‐Taylor et al. 2013).

Even if the need for granule initiation is high in young tissues, this does not mean that

they are unnecessary in mature tissue; it is likely that individual chloroplasts undergo stochastic

fluctuations in granule numbers, and that low frequencies of new initiations may still be

necessary to adjust those fluctuating numbers. Compared to the initiations presumably

mediated by SS4 in developing/dividing chloroplasts, initiations in mature leaves would take

place under quite different conditions and alongside existing starch granules, the growth of

18

which may sequester active components of the biosynthetic machinery. In such a scenario, the

unique features of SS5 may promote initiation of new starch granules by binding glucan

substrates and localizing them via interaction with MRC. Alternatively, SS5 may shield nascent

granule initials from amylolytic degradation (Seung et al. 2016), thereby increasing their

lifetime and availability to the biosynthetic machinery. Further biochemical and molecular

genetic analyses, such as the characterization of the putative oligosaccharide surface binding

site and the expression of altered protein versions will help to resolve the precise function of

SS5 in the network of proteins enabling starch granule initiation.

METHODS

Phylogenetic and Sequence Analysis

Arabidopsis SS1, SS2, SS3, SS4, and SS5 sequences were retrieved from Phytozome and

orthologues from NCBI via BLASTp, searching the refseq_protein database. The three closest

prokaryotic GS sequences of At-SS3, At-SS4, and At-SS5 (WP 048672006.1, WP

059061331.1, and WP 026046064.1, respectively), as determined by searching the

Procaryotae, were included as outgroup. A multiple sequence alignment was created using

Multiple Sequence Comparison by Log-Expectation (MUSCLE) (Edgar 2004) and a

phylogenetic tree was constructed in MEGA (Tamura et al. 2013), using a maximum likelihood

method based on the Jones-Taylor-Thornton (JTT) matrix-based model (Jones et al. 1992) and

1000 bootstrap replications. Positions with less than 95% site coverage were eliminated. The

alignment used for tree construction in MEGA format is provided in Supplemental Dataset 4A,

and the corresponding tree file is provided in Supplemental Dataset 4B.

For analyses of specific motifs and residues mentioned in text and shown in

Supplemental Figures 3A and D, regions of interest were located in the multiple sequence

alignment created above based on previously published sequence information (Sheng et al.

2009b, Nielsen et al. 2018) and analyzed in respect to SS isoforms. Weblogos were constructed

using Weblogo3 (Crooks et al. 2004).

For analysis of the C-terminal deletion in SS5 isoforms, sequences belonging to the

SS5 family were complemented by the orthologous sequences of the close A. thaliana relatives

A. lyrata, Camelina sativa and Capsella rubella. An alignment was generated using MUSCLE

(Edgar 2004) in MEGA (Tamura et al. 2013).

19

Batch GT5 and GT1 predictions for starch synthase sequences were performed using

the HMMER web server (hmmer.org). Corresponding data can be found in Supplemental

Dataset 1. Domain predictions for the Arabidopsis starch synthase isoforms and Os-SS5

depicted in Figure 1A were predicted using TargetP (Emanuelsson et al. 2000), ChloroP

(Emanuelsson et al. 1999), SMART (Schultz et al. 1998), Coils (Lupas et al. 1991), and Pfam

(Sonnhammer et al. 1997). Shown in Figure 1A are AED93283.1 (At-SS1), AEE73621.1 (At-

SS2), AEE28775.1 (At-SS3), AEE84015.1 (At-SS4), ABJ17089.1 (At-SS5) and

XP_015626202.1 (Os-SS5).

Protein Structure Prediction and Surface Conservation

For prediction of the three-dimensional structure of At-SS5 and Os-SS5, the respective

primary sequences were used for homology modelling using SWISSMODEL (Waterhouse et

al. 2018) and the previously published structure of the At-SS4 catalytic domain (amino acids

533-1040 of the product of AT4G18240.1; SWISS-MODEL Template Library ID 6gne.1) as

template (Nielsen et al. 2018). The resulting models were visualized in Pymol

(http://pymol.org). For surface residue conservation of SS4, amino acid sequences belonging

to the SS4 family were aligned using Clustal Omega (Sievers et al. 2011). From this alignment,

conserved and conservatively substituted residues were identified and mapped to the Pymol

coordinates. This was similarly done for surface residue conservation of At-SS5 and Os-SS5,

whilst for the latter, C-terminally truncated SS5 sequences (from A. thaliana, G. max, and A.

trichopoda) were excluded prior to alignment. The pdb models for At-SS5 and Os-SS5 can be

found in Supplemental Datasets 5 and 6.

Plant Materials and Growth Conditions

Arabidopsis plants were grown under controlled conditions in CLF Plant Climatics,

Percival AR-95L fitted with fluorescent tubes (Philips 368290 F25T8/TL841 ALTO) and

supplemented with red LED panels or Kalte 3000 climate chambers fitted with fluorescent

tubes under a 12-h light and 12-h dark cycle unless otherwise specified. Light intensity was

fixed at 150 μmol photons m-2 s-1, the temperature was 20°C, and the relative humidity was

65%. Seeds were sown on soil (Klasmann TKS 2) or on 0.8% [w/v] agar plates containing ½-

strength Murashige and Skoog (MS) medium including vitamins (Duchefa Biochemie) at pH

5.7, and stratified at 4°C before germination.

20

T-DNA insertion lines for At5g65685 were obtained from the Salk Institute Genomic

Analysis Laboratory (Alonso et al. 2003) and from GABI-Kat (Kleinboelting et al. 2012). All

mutants used are in the Columbia-0 (Col-0) ecotype background: ss5-1: SALK_148945; ss5-

2: GABI_760E03; ss5-4: SALK_040191. Homozygous lines were isolated by PCR-based

genotyping. Primers matching the corresponding transformation vector were used to amplify

the region containing the border between T-DNA and genomic sequence, and the insertion sites

confirmed by Sanger sequencing. All crosses with ss5 in this study were done using the ss5-1

line, to the previously described ss4-1 (Roldán et al. 2007), ptst3-5 (Seung et al. 2017), mfp1-

1, mrc-3, and ptst2-7 (Seung et al. 2018) lines. Homozygous double insertional mutants were

selected as described above.

Cloning for Expression of Recombinant Protein, Purification of Polyhistidine-Tagged

Protein and Production of Anti-SS5 Antibody

Primers used for genotyping (unless described elsewhere) and molecular cloning are

listed in Supplemental Dataset 2. For the expression of polyhistidine-tagged At-SS5 in E. coli,

the At-SS5 coding sequence was amplified excluding the first 117 bp encoding the putative

chloroplast transit peptide. For the expression of polyhistidine-tagged Os-SS5, an E. coli codon

optimized version of starch synthase V precursor ACC78131.1 was synthesized (Invitrogen)

and amplified excluding the first 156 bp encoding the putative chloroplast transit peptide. At-

SS5 and Os-SS5 fragments were then cloned into the E. coli expression vector pProEX HTb

(Invitrogen) using BamHI/XhoI and NotI/XhoI, respectively, in frame with an N-terminal

poly(6x-)histidine-tag and transformed into BL21 CodonPlus ΔglgAP cells (Morán-Zorzano et

al. 2007, Szydlowski et al. 2009). Note that ACC78131.1 differs from XP_015626202.1 (used

for phylogeny and modelling) by one nonconserved amino acid C-terminal to the predicted

GT5 subdomain.

For recombinant protein purification, cells were grown in LB medium to an optical

density of 0.6. Recombinant protein expression was induced by addition of 1 mM isopropyl-β-

D-thiogalactopyranoside, followed by a 16-h incubation at 18.5°C. Cells were lysed and

polyhistidine-tagged proteins were purified using Ni2+-nitrilotriacetic acid (Ni2+-NTA)-agarose

affinity chromatography. Briefly, cells were pelleted at 3,000 rcf at 4°C for 15 min,

resuspended in lysis medium (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 40 mM imidazole, 1x

Protease inhibitor cocktail (PiC, Roche), 2 mM Dithiothreitol (DTT), 1 mg mL-1 lysozyme),

21

and incubated with slight shaking for 15 min at 4°C. Cells were broken by three cycles in a

microfluidizer (Microfluidics M-110P, Microfluidics Corp., USA), and insoluble material was

pelleted by centrifugation (10 min, 20,000 rcf, 4°C). The pellet containing inclusion bodies

was stored at -20°C for the purification of denatured protein for rabbit immunization (see

below). The lysate supernatant containing soluble proteins was incubated with Ni2+-NTA resin

(MN Protino) for 2 h at 4°C with on a spinning wheel. The resin was collected by centrifugation

(200 rcf, 1 min), resuspended in lysis medium, and washed five times with 50 mM Tris-HCl

pH 7.5, 300 mM NaCl, 40 mM imidazole, 2 mM DTT, 0.5% [v/v] Triton X-100 and five times

with 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 40 mM imidazole, 2 mM DTT. Proteins were

eluted at 4°C in 50 mM Tris-HCl pH 7.5, 300 mM NaCl, and increasing imidazole

concentrations (100 mM, 250 mM, 500 mM). Protein in each elution fraction was measured

using a Bradford Assay reagent (Bio-Rad). Protein-enriched fractions were pooled and

concentrated using Amicon Ultra Centrifugal Filter Units 3 kD MWCO, then exchanged into

50 mM Tris-HCl pH 7.5, 10% [v/v] glycerol, 2 mM DTT using NAP-5 (GE Healthcare)

columns. Final protein content was quantified using the Bradford reagent. Samples collected

during the expression and purification process were monitored by SDS-PAGE and Coomassie

staining.

For the purification of denatured recombinant polyhistidine-tagged At-SS5 protein

from inclusion bodies, the insoluble material obtained by cell lysis (see above) was

resuspended in 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10 mM imidazole, 8 M urea and stirred

at 4°C for 30 min. The suspension was clarified by centrifugation (10 min, 20,000 rcf, 20°C)

and the resulting supernatant incubated with Ni2+-NTA resin (MN Protino) for 90 min on a

spinning wheel (~30 rpm). The resin was then sedimented by centrifugation (500 rcf, 5 min,

4°C), washed 5 times with 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 20 mM imidazole, 8 M

urea and five times with 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 20 mM imidazole, 6 M urea.

Proteins were eluted in 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 250 mM imidazole, 5 M urea,

concentrated, exchanged into 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 4 M urea, and quantified

as above.

A polyclonal antibody raised against At-SS5 was ordered at Eurogentec (Seraing,

Belgium). Denatured recombinant At-SS5 purified from inclusion bodies (see above) served

as antigen for the immunization of rabbits. For the purification of anti-SS5 antibodies from

immune sera, recombinant polyhistidine-tagged At-SS5 protein was covalently coupled to

22

NHS-activated Sepharose. In brief, 1 mg At-SS5 protein in 0.2 M NaHCO3, 0.5 M NaCl, pH

8.3 was injected into a HiTrap NHS-activated HP column (GE Healthcare) for covalent

coupling. The column was washed and excess active groups were deactivated according to the

manufacturer’s instructions. Dilute rabbit immune serum was circulated over the ligand

coupled column for 1 h at 20°C. The column was then washed with phosphate buffered saline

(137 mM NaCl, 2.7 mM KCl, 8.7 mM Na2HPO4, 1.8 mM KH2PO4 pH 7.4) supplemented with

0.1% [v/v] Tween-20, and then with three alternating washes using 0.1 M NaHCO3, 0.5 M

NaCl, pH 9.5 and 0.1 M sodium acetate, 0.5 M NaCl, pH 4. Bound antibodies were eluted from

the column using a pH shift (0.1 M glycine-HCl, 0.5 M NaCl, pH 2.3) and immediately

neutralized with HEPES-KOH pH 8. The protein content in elution fractions was measured

using the Bradford assay reagent. Protein-enriched fractions were pooled and concentrated

using Amicon Ultra 3 kD MWCO spin filters. They were then exchanged into phosphate

buffered saline using a NAP10 column and 0.1% [w/v] bovine serum albumin (BSA) was

added before storage at -80°C.

Cloning of Expression Vectors, Expression in A. thaliana, and Imaging

The AT5G65685.1 cDNA was obtained from the RIKEN Bioresource Centre (clone

pda11967). The CDS was amplified from the cDNA and cloned into pCR8 using the

pCR8/GW/TOPO TA cloning kit (Thermo Scientific). For expression of At-SS5-YFP driven

by CaMV35Spro, At-SS5:pCR8 was recombined with pB7YWG2 (Karimi et al. 2002) between

CaMV35Spro and the in-frame C-terminal eYFP tag via Gateway LR (Invitrogen). For

expression driven by UBQ10pro, At-SS5:pCR8 was recombined with pUBC-YFP (Grefen et al.

2010) between UBQ10pro and the in-frame C-terminal eYFP tag (for simplicity, we will refer

to this tag as YFP) via Gateway LR clonase (Invitrogen). For expression of the Δcc protein

version, At-SS5:pCR8 was used in QuikChange (Agilent Technologies) site-directed

mutagenesis, with primers designed to delete the base pairs encoding the amino acids between

W68 and D120, representing the predicted coiled-coil of At-SS5. The resulting At-

SS5Δcc:pCR8 vector was then recombined with pUBC-YFP as described above.

For expression of At-SS5-mCitrine under control of the native Arabidopsis SS5pro, the

full genomic construct with 1704 bp (including the 5’UTR) upstream sequence containing the

At-SS5 promoter and the complete intron-exon structure (lacking the stop codon) was

amplified from genomic DNA with Gateway recombination sites and ligated into pJET1.2

23

using the CloneJET PCR Cloning Kit (ThermoFisher Scientific). Subsequent sequencing

revealed a point mutation, which was corrected using QuikChange site-directed mutagenesis

before recombination into pDONR221 via BP clonase (Invitrogen). At-SS5:pDONR221 was

then recombined as previously described (Seung et al. 2018) into pH7m34GW,0, resulting in

an in-frame fusion of the genomic fragment with a C-terminal mCitrine tag.

For the expression of untagged At-SS5 driven by SS5pro, the above-mentioned At-

SS5:pDONR221 was modified by site-directed mutagenesis to carry a stop codon at the 3’ end

of the genomic fragment. The resulting At-SS5*:pDONR221 was then recombined as described

above, in this case using mCherry:pDONR P2RP3 and the destination vector pB7m34GW,0.

For expression of Os-SS5-YFP driven by CaMV35Spro, the E. coli codon optimized

gene synthesis product (Invitrogen) of ACC78131.1 was cloned into pDONR221. The coding

sequence was then recombined into pB7YWG2,0 (Karimi et al. 2002) between CaMV35Spro

and the in-frame C-terminal eYFP (for simplicity, we will refer to this tag as YFP) tag.

For expression of At-MRC-CFP driven by CaMV35Spro, the AT4G32190.1 cDNA was

obtained from the RIKEN Bioresource Centre (clone pda01994). This clone contained an A to

G polymorphism that led to an amino acid change (D98→G) relative to the TAIR reference

sequence. The coding sequence was amplified with Gateway recombination sites and

recombined into pDONR221 via BP clonase. The resulting vector was used in a Quikchange

mutagenesis reaction to correct the polymorphism to match the TAIR reference sequence. The

corrected coding sequence was subsequently recombined into pH7CWG2 in-frame with a C-

terminal CFP tag.

For expression in plants, constructs were transformed into Agrobacterium tumefaciens

strains AGLØ (At-SS5 in pB7YWG2 only) or GV3101 by electroporation. For stable

expression in A. thaliana, wild type (ecotype Col-0) and ss5 plants were transformed using a

floral dip method (Zhang et al. 2006). Transformants were selected using their respective

resistance markers either on soil (Basta spraying) or on 0.8% [w/v] agar plates containing ½-

strength MS salts (with 25 mg mL-1 hygromycin B selection) and further analysis for transgene

expression by immunoblotting and/or RT-PCR. For At-SS5-YFP and At-SS5-mCitrine lines,

genomic T-DNA integration sites were determined by Thermal asymmetric interlaced (TAIL)

PCR (http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=3615) and

homozygous offspring were selected by PCR-based genotyping. Homozygous lines were used

for all experiments except those involving At-SS5Δcc-YFP and Os-SS5-YFP expressing

24

plants, which were performed on segregating but Basta-resistant T2 individuals and those

involving untagged At-SS5 expressing plants as well as At-SS5-YFP expressing ss5-2 and ss5-

4 plants, which were performed on Basta-resistant T1 individuals.

To determine the subcellular localization of SS5, leaf tissue from 35-d-old Col-0 plants

expressing At-SS5-mCitrine was imaged with a Zeiss LSM780 Confocal Imaging System. The

specimens were sequentially excited with either 514-nm (mCitrine) argon or 633-nm

(chlorophyll) helium-neon lasers. Images were acquired using filters ranging from 519 to 600

nm (mCitrine) and 647–721 nm (chlorophyll). At least 20 regions of interest (ROIs) from two

independent transgenic lines were imaged.

For fluorescence detection in whole Arabidopsis rosettes, 2-week-old seedlings grown

on 0.8% [w/v] agar plates containing ½-strength MS salts were imaged using a fluorescence

stereo microscope (Leica M205 FCA) and the filter set ET-YFP. Images were adjusted using

Fiji (Schindelin et al. 2012). Backgrounds were subtracted using a sliding paraboloid with a

rolling ball radius of 300 pixels, and a specific look-up table (LUT) was applied with the Min

and Max set to 9 to 199 for the overview images and 12 to 255 for the closeup images.

Transient expression in Nicotiana benthamiana was performed as described in Sharma

et al. (2018) except that the N. benthamiana plants were grown and incubated after infiltration

under a 12-h light/12-h dark cycle. The specimens were analyzed using a Zeiss LSM780

confocal imaging system using either 514-nm (YFP) and 458-nm (CFP) argon or 633-nm

(chlorophyll) helium-neon lasers. Image acquisition was done sequentially using filters ranging

from 526 to 624 nm (YFP), 463 to 509 (CFP) and 647 to 721 nm (chlorophyll). For protein

colocalizations, at least 20 ROIs were imaged from two independent biological replicates.

Two-Step Endpoint RT-PCR

For cDNA preparation, snap-frozen leaf tissue was homogenized for 1 min (Retsch

mixer mill at a vibration intensity of 30 s-1 using glass beads) and immediately mixed with

TRIzol reagent (Invitrogen). After addition of ice-cold chloroform and mixing, samples were

incubated at 20°C for 3 min, then centrifuged (15 min, 4°C, 12,000 rcf). The aqueous phase

was mixed with ice-cold isopropanol and incubated at 20°C for 10 min. RNA was pelleted by

centrifugation (10 min, 4°C, 12,000 rcf), washed twice with cold 75% [v/v] ethanol (with

centrifugation in between, 3 min, 4°C, 12,000 rcf), dried, and dissolved in water. Equal

amounts of total RNA were digested using DNAse I (Roche) and subsequently used for first

25

strand cDNA synthesis from poly(A)-tailed mRNA using the RevertAid Reverse Transcription

Kit (Thermo Scientific) and oligo(dT)18 primers. cDNA was used as a template for endpoint

PCR with 35 cycles. RT-PCR primers were designed such that at least one of the two primers

in each reaction spanned two exon borders.

In vitro Activity Assay

Reactions of 1 μg recombinant protein and 1.5 mg hydrated waxy maize starch in 100

mM Bicine-KOH pH 8, 25 mM KCH3CO2, 2 mM MgCl2, 5 mM DTT, 0.05% [w/v] BSA and

125 nM ADP-Glc [D-glucose-14C(U)] were incubated on a rotating wheel at 20°C. Reactions

were stopped by addition of SDS to a final concentration of 3.3% [w/v]. Starch was collected

by centrifugation (3 min at 20,817 rcf), washed once with water, and subsequently resuspended

in water. The amount of radioactivity incorporated into the starch by recombinant protein

activity was measured by scintillation counting. Three experimental replicates (N) were

performed for each protein/control reaction.

In vitro Starch Binding Assay

Starch binding was assessed as described previously (Seung et al. 2015), with minor

modifications. Recombinant polyhistidine-tagged At-SS5 protein was used at a final

concentration of 300 nM and was incubated with 30 mg hydrated waxy maize starch in 50 mM

HEPES-NaOH pH 7.5, 2 mM MgCl2, 1 mM DTT, 0.05% [w/v] BSA, 0.005% [v/v] Triton X-

100 for 45 min at 20°C. Starch was pelleted by centrifugation (30 s, 5,000 rcf) and an aliquot

of the supernatant collected as the soluble protein fraction (S). Substrates were then washed

and bound proteins eluted as described (Seung et al. 2015). Supernatant from the last wash and

the elution step were collected as the final wash (FW) and insoluble (I) fractions. S, FW, and I

fractions were mixed with SDS-PAGE loading buffer (final concentrations 100 mM DTT, 3%

[v/v] glycerol, 2% [w/v] SDS, 50 mM Tris-HCl pH 6.8, 0.005% [w/v] bromophenol blue) and

analyzed by immunoblotting (see below). Sephadex G-10 resin (GE-Healthcare) was used as

a substitute for starch in control samples. The protein portion found in the insoluble fraction

was estimated using Fiji (Schindelin et al. 2012) in relation to the respective soluble fraction

as described earlier (Kesten et al. 2016).

26

Heterologous Expression in Yeast

A summary of the yeast genotypes used in this study is found in Supplemental Table 2.

For the expression of At-SS5 in S. cerevisiae, the At-SS5 (At5g65685.1) CDS excluding the

first 117 bp encoding the putative chloroplast transit peptide and the S. cerevisiae 450-bp

GAL1pro were amplified by PCR. A Gly and Ser codon were inserted after the ATG start codon

of At-SS5 to improve the Kozak sequence. GAL1pro and At-SS5 CDS were then integrated into

the yeast integrative vector pGSY2 (Pfister et al. 2016) by USER fusion (Geu-Flores et al.

2007) to produce At-SS5:pGSY2.

For yeast transformations, haploid CEN.PK113-11C strains “L” and “4” were selected

for loss of the URA3 marker on SC plates supplemented with 0.1% [w/v] 5-fluoroorotic acid

(ThermoFisher) as described (Pfister et al. 2016). Purified ura3- strains were transformed with

At-SS5:pGSY2 and selected by prototrophy for uracil on SC-Ura plates. Yeast strains were

grown and harvested in complex medium as described (Pfister et al. 2016). Briefly, cells were

grown overnight in YPD and then inoculated into YPGal medium containing 2% [w/v]

galactose to induce protein expression.

For protein extracts, cells were harvested after 3 h of growth under inducing conditions,

collected by centrifugation (3 min, 3,000 rcf), washed twice in 50 mM Tris-HCl pH 8, 150 mM

NaCl, 1% [v/v] Triton X-100, 1 mM DTT, and directly homogenized in 50 mM Tris-HCl pH

8, 150 mM NaCl, 1% [v/v] Triton X-100, 1 mM DTT, 1X PiC by vortexing for 10 min in total

at 4°C with cooling in between with glass beads (acid-washed, 425 to 600 μm diameter). The

resulting lysate was cleared by centrifugation (5 min, 14,000 rcf, 4°C), the supernatant

containing soluble proteins mixed with SDS-PAGE loading buffer, and used for

immunoblotting. 15 μg total protein was loaded per sample. Light microscopy of iodine-stained

cells was done as described (Pfister et al. 2016).

Measurement of Starch Content

Entire nonflowering Arabidopsis rosettes (N = individual rosettes) were weighed,

snap frozen in liquid nitrogen, and extracted in cold 0.7 M HClO4 using either a glass

homogenizer or a Retsch mixer mill. The insoluble material was pelleted by centrifugation

for 5 min at 5,000 rcf and 4°C, washed in 80% [v/v] ethanol with in-between spins of 3 min

at 3,000 rcf and 4°C, and resuspended in water as previously described (Seung et al. 2017).

Starch was quantified using an enzyme-based spectrophotometric assay as described

previously (Smith and Zeeman

27

2006). Statistical differences between genotypes were assessed using unpaired, two-tailed t-

tests assuming heteroscedasticity, following Shapiro-Wilk testing for normality. Raw

measurements including statistical analyses and test statistics are provided in Supplemental

Dataset 3.

Iodine Staining of Arabidopsis Rosettes

Arabidopsis rosettes were harvested at the end of the light cycle, de-colorized in 80%

[v/v] ethanol, and subsequently stained in an iodine and potassium iodide solution (Lugol

solution; Sigma). Excess iodine was removed by washing in water before imaging.

Measurements of Chain Length Distribution

Starch from perchloric-acid extracts was debranched and applied to Dowex columns as

described previously (Hostettler et al. 2011, Pfister et al. 2016). Chains were separated on a

HPAEC-PAD system (Dionex ICS-5000, ThermoFisher Scientific) using a CarboPac PA-200

column, a flow rate of 0.4 mL min-1 and the following program: 0 to 12.5 min, a linear gradient

from 95% eluent A (100 mM NaOH) and 5% B (150 mM NaOH, 0.5 M sodium acetate) to

60% A and 40% B; 12.5 to 50 min, a linear gradient to 15% A and 85% B; 50 to 50.1 min, a

linear gradient to 95% A and 5% B; 50.1 – 70 min, column equilibration at 95% A and 5% B

(all eluent percentages are given as volume per volume). Peak areas were determined with

Chromeleon 7.2 software and summed within a degree of polymerization (DP) of 3 and 73.

Biological replicates (N) correspond to individually extracted Arabidopsis rosettes.

Purification of Arabidopsis Leaf Starch Granules and Scanning Electron Microscopy

Entire nonflowering Arabidopsis rosettes were harvested at the end of the day and snap

frozen in liquid nitrogen. Frozen leaf tissue was ground in liquid nitrogen using a mortar and

pestle and subsequently homogenized with 50 mM Tris-HCl pH 8, 0.2 mM EDTA, 0.5% [v/v]

Triton X-100 in a Waring Blender. The suspension was filtered through a 100 μm nylon net,

centrifuged (15 min, 3,000 rcf), and the resulting pellet was resuspended in 50 mM Tris-HCl

pH 8, 0.2 mM EDTA, 0.5% [v/v] Triton X-100. The suspension was sequentially filtered

through a 31 μm and a 15 μm nylon net and the starch granules separated from the filtrate at

2,500 rcf for 15 min over a Percoll cushion (95% [v/v] Percoll (Sigma-Aldrich), 5% [v/v] 0.5

M Tris-HCl, pH 8). The starch pellet was then washed in 0.5% [w/v] SDS, and excess SDS

28

removed by washing in water. Images were acquired by scanning electron microscopy as

described previously (Pfister et al. 2016).

Epoxy Resin Embedding of Leaf Tissue, Light Microscopy, and Quantification of Starch

Granule Number

Leaf samples were harvested at the end of the light period, chemically fixed, stained,

and embedded into Spurr epoxy resin (Sigma) using a Pelco BioWave Pro+ (Ted Pella) as

described in the Supplemental Method. Semi-thin sections of the resulting epoxy resin

embedded leaf pieces were stained with toluidine blue O before light microscopy. Microscopy

was performed using a Zeiss AxioImager Z2 microscope with an AxioCam monochrome

camera.

For analyses of granule numbers per chloroplast section, samples of replicate plants

(duplicates or triplicates, as specified; N) were chemically fixed and embedded as described

above. Semi-thin sections were imaged, ensuring that no overlap between images existed.

Starch granule sections per chloroplast section were counted and listed. Each experimental

batch with transgenic lines included the complete set of all transformed backgrounds as

controls, resulting in slight differences between histograms of Col-0 and ss5-1 depending on

the experimental batches. For each experiment, the minimum chloroplast count of all analyzed

replicates was determined and a randomized sample containing this count was selected from

each replicate. Those equilibrated samples were then merged for each genotype (giving rise to

n = total number of chloroplast sections accounted for in the analysis).

Extraction of Total Leaf Protein and Immunoprecipitation

For total leaf protein extracts, 7-mm leaf discs were snap frozen, ground in a Retsch

mixer mill (1 min at a frequency of 30 s-1 using glass beads) and subsequently extracted in

SDS-PAGE loading buffer or Biorad Laemmli Sample buffer supplemented with 50 mM DTT.

Immunoprecipitations using plants expressing mCitrine- and YFP-tagged proteins were

carried out as previously described (Seung et al. 2017). For mass spectrometry, bound proteins

were eluted with 50 mM Tris-HCl pH 8, 2% [w/v] SDS. Two independent transgenic lines

were used for each construct and two wild-type (Col-0) replicates lacking tagged bait were

used as controls in this case.

For immunoprecipitations using polyhistidine-tagged recombinant At-SS5 protein

expressed in E. coli as a bait, a wild-type (Col-0) extract was prepared similarly as above.

29

Recombinant protein was added to each aliquot of extract (n = 2; Rep1 and Rep2) and the

reactions were incubated for 1 h at 4°C on a rotating wheel. Magnetic μMACS beads (Miltenyi

Biotec) binding to the polyhistidine tag of the recombinant protein were then added and the

reactions incubated for another h at 4°C on a rotating wheel. Bead capture, washing and eluting

were performed as described above, except that the last wash step was done with wash buffer

II provided in the kit. Extract aliquots (n = 2) without the addition of recombinant protein were

used as a control in this case.

For mass spectrometry, proteins were precipitated with 10% [w/v] TCA, pelleted, and

washed twice with cold acetone. The protein pellet was dried, dissolved in 10 mM Tris-HCl

pH 8.2, 2 mM CaCl2 and digested with trypsin for 30 min at 60°C. Peptides were then dried,

dissolved in 0.1% [v/v] formic acid and analyzed by LC-MS/MS on a nanoAcquity UPLC

(Waters) coupled to a Digital PicoView source for electrospray ionization (New Objective) and

a Q Exactive mass spectrometer (Thermo Fisher Scientific). Peptides were trapped on a

Symmetry C18 trap column (5 μm, 180 μm × 20 mm; Waters) and separated on a BEH300 C18

column (1.7 μm, 75 μm × 150 m; Waters) at a flow rate of 250 nL/min using a gradient from

1% solvent B (0.1% [v/v] formic acid in acetonitrile)/99% solvent A (0.1% [v/v] formic acid

in water) to 40% solvent B/60% solvent A over a 90-min period. Mass spectrometry settings

were as follows: precursor scan range, 350 to 1500 m/z; resolution, 70,000; maximum injection

time, 100 ms; threshold, 3e6; fragment ion scan range, 200 to 2000 m/z; resolution, 35,000;

maximum injection time, 120 ms; and threshold, 1e5. Peptides were searched against the

TAIR10 Arabidopsis proteome database with the Mascot search engine (Matrix Science,

version 2.5.1), with fragment ion mass tolerance of 0.03 D, parent ion tolerance of 10.0 ppm,

and oxidation of methionine as a variable modification. Scaffold (Proteome Software) was used

to validate MS/MS-based peptide and protein identifications. Peptide identifications were

accepted if they achieved a false discovery rate (FDR) <0.1%, and protein identifications if

they achieved an FDR of <1% and a minimum of two identified peptides.

Proteins for which peptides were found in any control sample from both types of

immunoprecipitations were removed from the list of potential interaction partners, as were

proteins for which peptides could not be identified in all At-SS5-mCitrine and His-At-SS5

immunoprecipitation samples, leaving only three potential interactors apart from the bait.

Those potential interaction partners were then searched in the immunoprecipitated of Os-SS5-

30

YFP. At-SS5-mCitrine and Os-SS5-mCitrine immunoprecipitations originate from the same

experimental batch and thus share common control samples.

Immunoblotting and Coomassie Staining

For immunoblotting, proteins were separated by SDS-PAGE and subsequently

transferred to low fluorescent polyvinylidene difluoride (LF PVDF) membranes using the

Transblot-Turbo system (Biorad). Membranes were rinsed in Tris Buffered Saline (TBS; 20

mM Tris-HCl, pH 7.4, 150 mM NaCl) and air-dried. Dry membranes were sequentially rinsed

in methanol and TBS and subsequently incubated in blocking solution (TBS supplemented

with 5% [w/v] milk powder). Primary antibodies were applied in blocking solution overnight

at 4°C (at antibody-specific concentrations: anti-YFP/GFP (polyclonal/rabbit, Torrey Pines

Biolabs, catalog: TP401): 1:5,000, anti-actin (plant) (monoclonal/mouse, Sigma clone 10-B3

(MAbGPa)): 1:10,000, anti-SS5 (polyclonal, affinity purified, this study): 1:250, anti-MRC

(Seung et al. 2018): 1:200. In cases where actin was used as a loading control, the actin primary

antibody was applied simultaneously with the target antigen antibody. Proteins were observed

by near-infrared (NIR) detection, using IRDye secondary antibodies (Li-Cor) and an Odyssey

CLx detection system.

For Coomassie staining, polyacrylamide gels were transferred into Coomassie staining

solution (10% [w/v] ammonium sulfate, 2% [v/v] ortho-phosphoric acid, 10% [v/v] methanol,

0.1% [w/v] Coomassie blue G-250) overnight following SDS-PAGE. Gels were destained for

several hours in water before imaging.

Measurement of ADP-Glc Content

Entire Arabidopsis rosettes (N) were harvested at the end of the light period and

immediately snap frozen in liquid nitrogen. Frozen tissue was homogenized in liquid nitrogen

using mortar and pestle, and metabolites were extracted using a chloroform-methanol method

(Arrivault et al. 2009). Extracted metabolites were dried, dissolved in water, and analyzed as

described previously (Seung et al. 2016). Statistical differences between genotypes were

assessed using unpaired, two-tailed t-tests assuming heteroscedasticity, following Shapiro-

Wilk testing for normality (Supplemental Data Set 3).

31

Accession Numbers

Sequence data of the Arabidopsis genes studied in this article can be found in TAIR

(www.arabidopsis.org) under the following accession numbers: SS5 (AT5G65685), SS4

(AT4G18240), MRC (AT4G32190), PTST2 (AT1G27070), PTST3 (AT5G03420), and MFP1

(AT3G16000). Accession numbers for starch synthase orthologues that were used in this study

are indicated in Supplemental Figure 1.

Supplemental Data

Supplemental Figure 1. Maximum Likelihood Phylogenetic Tree of the Starch Synthases.

Supplemental Figure 2. SS5 Orthologues Feature a Conserved GT5-, but not a GT1

Subdomain.

Supplemental Figure 3. Key Residue Conservation in SS5 and in vitro Activity Test.

Supplemental Figure 4. SS5 Orthologues Feature a Conserved N-Terminal Coiled-Coil.

Supplemental Figure 5. Loss of At-SS5 Does Not Alter the Amylopectin Fine Structure.

Supplemental Figure 6. Diel Starch Turnover is Largely Unaffected in ss5.

Supplemental Figure 7. At-SS5-mCitrine Expression Driven by the Endogenous Promoter

From a Genomic Fragment Has a Dominant-Negative Effect in the Wild-Type Background

and Does Not Fully Rescue the ss5 Phenotype.

Supplemental Figure 8. At-SS5-YFP Expression Driven by UBQ10pro Has a Dominant-

Negative Effect in the Wild-Type Background and Does Not Fully Rescue the ss5 Phenotype.

Supplemental Figure 9. ss5 Mutants All Lack the Full-Length At-SS5 Protein.

Supplemental Figure 10. At-SS5Δcc-YFP Expression Does Not Influence Starch Granule

Numbers, Whereas Os-SS5-YFP Expression Has a Dominant-Negative Effect in the Wild-

Type (Col-0) Background Similar to At-SS5-mCitrine and At-SS5-YFP Expression.

Supplemental Table 1. Proteins Copurifying With SS5-Baits in Immunoprecipitation

Experiments.

32

Supplemental Table 2. Genotypes of Saccharomyces cerevisiae CEN.PK113-11C Strains

Used in the Present Study.

Supplemental Method. Epoxy Resin Embedding of Arabidopsis Leaf Sections for Light

Microscopy.

Supplemental Dataset 1. Output of HMMER Search.

Supplemental Dataset 2. Oligonucleotide Primers Used in This Study.

Supplemental Dataset 3. Measurements, Statistical Analyses, Test Statistics, and Transgenic

Lines.

Supplemental Dataset 4A. Alignment Used for the Phylogenetic Tree in Supplemental Figure

1.

Supplemental Dataset 4B. Phylogenetic Tree in Machine-Readable Format.

Supplemental Dataset 5. SWISS-MODEL Prediction of the Three-Dimensional Structure of

At-SS5.

Supplemental Dataset 6. SWISS-MODEL Prediction of the Three-Dimensional Structure of

Os-SS5.

Supplemental Dataset Legends. Descriptions of Data in Supplemental Datasets 1-6.

ACKNOWLEDGEMENTS

This work was funded by the Swiss National Science Foundation (grants CR32I3_166487 and

31003A_182570 to S.Z.). We thank the Functional Genomics Center Zurich and ScopeM (ETH

Zurich) for providing proteomic and microscopy facilities, respectively, and for their advice in

data interpretation.

AUTHOR CONTRIBUTIONS

M.R.A., D.S., and S.C.Z. designed the research. M.R.A., D.S., B.P., M.S., S.E., L.B., and I.N.

performed the research and analyzed data. M.R.A. and S.C.Z. wrote the article with input from

all authors.

33

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39

Figure 1. SS5 Proteins Differ Substantially from the Canonical Starch Synthases, but Localize to the Chloroplast.(A) Protein domain organization of the Arabidopsis soluble starch synthases SS1 to SS5. Note that the transit peptide (cTP) of SS3 is not predicted for the splice form resulting from the longest transcript. The rice SS5 orthologue (Os-SS5) is included because the At-SS5 structure is not representative of the majority of SS5 proteins. The dashed line in At-SS5 indicates the C-terminal deletion relative to the rice orthologue. Locations of amino acid residues and motifs mentioned in the text and Supplemental Figures 3A and 3D are highlighted in red (conserved in all canonical starch synthases) and blue (SS4-specific). Residue identities in the respective starch synthase isoform are specified. (B) Fluorescence images of representative leaf epidermal cells of a transgenic Arabidopsis thaliana plant expressing mCit-rine tagged At-SS5 under the control of the endogenous promoter. The images are orthogonal projections of several single images acquired in the Z plane. At-SS5-mCitrine adopts a punctate localization pattern (highlighted with white arrowheads). Bars = 10 μm.

B ChlorophyllmCitrine merge

At-S

S5-

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itrin

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A cTP GT5 GT1CBM53 coiled-coil

100 aa

At-SS1A T K EKTGGL

DWH

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NWE

At-SS5W YSVGPL

NWQ

At-SS2K A K EKTGGL

DWH

At-SS3K F K EKVGGL

DWS

At-SS4W689 F754 K854 E929K556VGGL

D678WQ

40

A B

Figure 2. SS5 Shares a Putative Surface Carbohydrate Binding Site With SS4.(A) Surface amino acid conservation in SS4 and SS5. The resolved crystal structure of the catalytic domain of At-SS4 (dark blue; Nielsen et al., 2018) was used to model corresponding three-dimensional structures of At-SS5 (cyan) and Os-SS5 (green). Amino acid conservation deduced from multiple sequence alignments of orthologs for each respective isoform was superimposed on the resulting models. The modelled structures are shown with ADP and acarbose (a glucosidase inhibitor used as an acceptor mimic; Nielsen et al., 2018) as ligands and maltose as a generic glucan at the surface binding site (carbon, oxygen, nitrogen, and phos-phate atoms in yellow, red, blue, and orange, respectively) shown in the same position for Os-SS5 and At-SS5 as for At-SS4. (B) Carbohydrate binding of recombinant polyhistidine-tagged At-SS5. S, soluble; FW, final wash, I, insoluble; Rep 1 to Rep 3, repli-cates. The immunoblot shows that some At-SS5 protein (black arrowheads) is detectable in the insoluble fraction when using maize starch as a binding reagent (variable levels, between 3-9% of the respective soluble portion). Note that some protein was detected in the insoluble fraction also when using crosslinked dextran beads (Sephadex G-10) as a nonstarch polymer. Further note the presence of a prominent double band, which is likely due to partial loss of the polyhistidine tag, in the soluble fraction.

fully conservedconservatively substituted

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41

Figure 3. Arabidopsis SS5 Does Not Produce Glucans in Saccharomyces cerevisiae.(A) At-SS1, At-SS2, At-SS3, At-SS4, and At-SS5 without their respective predicted chloroplast transit peptides were expressed in yeast cells purged of their endogenous glycogen-metabolic genes except for the glycogenin genes GLG1 and GLG2 as indicated (Pfister et al., 2016). Arabidopsis branching enzyme(s) and the bacterial ADP-glucose pyrophosphorylase (glgC; for the provision of ADP-Glc) were coexpressed as indicated. Six h after induction of heterologous protein expression, cells were stained with iodine to visualize glucan accumulation. Shown are representative light micrographs from two independent experiments that gave identical results. Bar = 10 μm. (B) Representative immu-noblot showing that At-SS5 was readily detected in soluble extracts of yeast at the expected size (white arrowhead) with the antibody raised against At-SS5.

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42

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3’UTRss5-4 (SALK_040191)

Figure 4. Arabidopsis SS5 Insertional Mutants Lack SS5.(A) Schematic representation of the At-SS5 genomic sequence (AT5G65685.1). Exons are represented as black boxes, introns as thin lines, untranslated regions (UTRs) as grey boxes. Triangles show sites of T-DNA insertions of lines used in this study. Photographs of 25-d-old Arabidopsis rosettes of the respective lines are shown below the gene model. Bar = 2 cm. (B) Confirmation of gene disruption in the T-DNA insertional mutants shown in (A). Upper panels, endpoint RT-PCR on cDNA preparations from wild-type (Col-0) plants and ss5 T-DNA lines. Colors correspond to the amplification products indicated below the gene model in (A). Note the absence of bands spanning the respective insertion sites. Lower panels, immunoblot of total leaf protein extracts probed with the antibody raised against At-SS5, showing a weak band corresponding to At-SS5 that is absent in the three T-DNA insertional mutant lines. An At-SS5-YFP anti-YFP immunoprecipitation sample (bait indicated by a black arrowhead) containing endogenous At-SS5 (indicated by a white arrowhead) as a prey was included as an indicator of native At-SS5 migration (ind); this lane was run on the same gel. Actin levels analyzed simultaneously on the blot (in red) served as a loading control.

Col-0 ss5-1 ss5-2 ss5-4

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43

Figure 5. Arabidopsis SS5 is Expressed to Low Levels In Planta.(A) Expression of At-SS5-mCitrine from a genomic fragment. Immunoblot of total leaf protein extracts. Two independent transgenic lines are shown per background. mCitrine-tagged and endogenous At-SS5 are indicated by black and white arrowheads, respectively. (B) Whole-ro-sette fluorescent imaging of two-week-old transgenic plants expressing At-SS5-mCitrine driven by the At-SS5 promoter from a genomic fragment. The same rosette was imaged in both overview (upper two rows) and close-up magnifications (lower two rows). Bars = 500 μm.

Brightfield

mCitrine

mCitrine

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Starch granule counts per chloroplast section

n = 552, N = 3n = 552, N = 3n = 552, N = 3n = 552, N = 3

Per

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Figure 6. Arabidopsis SS5 Insertional Mutants Produce Fewer, But Larger Starch Granules Per Chloroplast.(A) Starch content in whole Arabidopsis rosettes. Plants were harvested after 12 h light (end of the day; ED) and after 12 h dark (end of the night; EN). Error bars represent the standard error of the mean (SE). *** P < 0.001, based on t-tests (N = 4-6 biological replicates (rosettes); individual measurements are shown by red points). (B) Starch granule quantifications in sections of embedded ss5 leaf tissue, represented as histograms of granules per chloroplast section in red with the wild-type (Col-0) distribution underlaid in transparent gray. Individual chloroplast counts (n) are scattered into bins below histograms with hues differentiating the biological replicates (individually sampled rosettes; N). Representative light micrographs of the respective lines are shown above each histogram. Bar = 10 μm. (C) Scanning electron micrographs of purified wild-type (Col-0) and ss5 starch granules at a magnification of 15,000 X. Bar = 4 μm.

ss5-2

ss5-1Col-0

ss5-4

45

Figure 7. Complementation of the ss5 Phenotype by SS5 Expression From a Genomic Fragment.Overlay histograms represent the distribution of granule numbers per chloroplast section in each three replicate plants (Col-0, ss5-1) or three independent T1 transformants per background. Different hues are shown to account for variations between different transgene insertions in this experiment; black outlines represent the mean across all lines or replicates. Individual counts (n) are scattered into bins below histograms with hues corresponding to the respective transformant/replicate histogram. Representative micrographs of each transformant or control are shown above each histogram. Bar = 10 μm.

Col-0

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n = 1629Starch granule counts per chloroplast section

n = 1629 n = 1629

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47

Figure 8. Arabidopsis SS5 and MRC Interact.(A) At-SS5-YFP was expressed in different genetic backgrounds and immunoprecipitated via the YFP tag (black arrowheads). Shown are immunoblots using antibodies recognizing GFP/YFP, SS5, or MRC. Endogenous At-SS5 (white arrowhead) is enriched in the immuno-precipitate (IP) when the bait is expressed in a wild-type (Col-0) background, whereas At-MRC is enriched when it is expressed in either the wild-type or ss5 mutant background. Two independent transgenic lines are shown per genetic background. Actin, analyzed simultane-ously (in red), served as a loading control in anti-GFP and anti-SS5 immunoblots. (B) IP using At-MRC-mCitrine (yellow arrowhead) as bait. Endogenous At-SS5 (white arrowhead) is enriched in the immunoprecipitate. Note that endogenous MRC runs slightly higher in this blot than in (A), likely due to the polyacrylamide gradient gel used for SDS-PAGE in this case. (C) At-SS5-YFP and At-MRC-CFP were transiently expressed in tobacco leaves. When expressed together, the proteins colocalize to the same puncta (examples indicated by white arrowheads) in the chloroplast. (D) Deletion of the At-SS5 coiled-coil in the At-SS5Δcc-YFP bait abolishes both dimerization and interaction with At-MRC. The Os-SS5-YFP immunoprecipitate contains At-MRC, but not endogenous At-SS5. Protein bands are indicated with arrowheads as following: At-SS5-YFP: black; endogenous At-SS5: white; At-MRC: yellow; At-SS5Δcc-YFP: pink; Os-SS5-YFP: blue.

48

Figure 9. SS5 Exerts its Function Through MRC and Acts in the Absence of SS4.(A) Quantification of starch granule content, represented as histograms of counts per chloroplast section, in crosses of ss5-1 and mutantsof other proteins involved in starch granule initiation. Shown is the distribution upon loss of SS5 (red) in the wild-type (Col-0) or mutantbackground (transparent blue). Individual chloroplast counts (n) upon loss of SS5 are scattered into bins below the histogram with differ-ent hues representing biological replicates (N). Representative micrographs of the genetic background as well as the respective doublemutants are shown above each histogram. Note that plants used for this quantification come from the same experimental batch (andtherefore share the same Col-0 and ss5-1 quantifications) as the ones used for Figure 6B. Bar = 10 μm. (B) Whole-rosette measurementsof starch content in the ss4 ss5 double mutant (N = 4-6 rosettes). Plants were harvested after 12 h light (end of the day; ED) and after 12h dark (end of the night; EN). Individual data points are shown in red. (C) Iodine-stained rosettes harvested at ED. Wild types and ss5-1stain similarly. As in ss4, immature leaves of ss4 ss5 double mutants show very weak staining. Shown are two biological replicates foreach genotype. (D) ADP-glucose content measurements of the ss4 ss5 double mutant (N = 3-4 rosettes). Entire rosettes were harvestedat ED and metabolites extracted and quantified as described previously (Seung et al., 2016). Individual datapoints are shown in red. Errorbars in (B) and (D) represent the SE. Asterisks show statistical significance: *P < 0.05, **P < 0.01, and ***P < 0.001, based on t-tests.

******

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60

70

Starch granule counts per chloroplast sectionn = 368, N = 2n = 552, N = 3n = 552, N = 3n = 552, N = 3 n = 552, N = 3n = 368, N = 2

Per

cent

age

of c

hlor

opla

sts

ss4

D

2 cm

Col-0

ss5

ss4

ss4 ss5

**

***

**

****

0

1

2

3

4

5

6

7

8

9

49

DOI 10.1105/tpc.19.00946; originally published online May 29, 2020;Plant Cell

and Samuel C ZeemanMelanie Abt, Barbara Pfister, Mayank Sharma, Simona Eicke, Léo Bürgy, Isabel Neale, David Seung

Granule Initiation in ArabidopsisSTARCH SYNTHASE 5, a Noncanonical Starch Synthase-Like Protein, Promotes Starch

 This information is current as of October 4, 2020

 

Supplemental Data /content/suppl/2020/06/09/tpc.19.00946.DC2.html /content/suppl/2020/06/09/tpc.19.00946.DC1.html

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