MBE Advance Access published July 11, 2012

Divergent Evolutionary Pattern of Starch Biosynthetic Pathway

Genes in Grasses and Dicots
Chun Li,1,2 Qi-Gang Li,1 Jim M. Dunwell,3 and Yuan-Ming Zhang*,1
1

State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Agriculture, Department of Crop Genetics and
Breeding, Nanjing Agricultural University, Nanjing, Peoples Republic of China
2
Henan Sesame Research Center, Henan Academy of Agricultural Sciences, Zhengzhou, Peoples Republic of China
3
School of Biological Sciences, University of Reading, Whiteknights, Reading, United Kingdom
*Corresponding author: E-mail: soyzhang@njau.edu.cn.
Associate editor: Neelima Sinha

Starch is the most widespread and abundant storage carbohydrate in crops and its production is critical to both crop yield and
quality. In regard to the starch content in the seeds of crop plants, there is a distinct difference between grasses (Poaceae) and
dicots. However, few studies have described the evolutionary pattern of genes in the starch biosynthetic pathway in these two
groups of plants. In this study, therefore, an attempt was made to compare evolutionary rate, gene duplication, and selective
pattern of the key genes involved in this pathway between the two groups, using five grasses and five dicots as materials. The
results showed 1) distinct differences in patterns of gene duplication and loss between grasses and dicots; duplication in grasses
mainly occurred before the divergence of grasses, whereas duplication mostly occurred in individual species within the dicots;
there is less gene loss in grasses than in dicots, 2) a considerably higher evolutionary rate in grasses than in dicots in most gene
families analyzed, and 3) evidence of a different selective pattern between grasses and dicots; positive selection may have
occurred asymmetrically in grasses in some gene families, for example, ADP-glucose pyrophosphorylase small subunit.
Therefore, we deduced that gene duplication contributes to, and a higher evolutionary rate is associated with, the higher
starch content in grasses. In addition, two novel aspects of the evolution of the starch biosynthetic pathway were observed.
Key words: starch synthesis pathway, gene duplication, evolutionary rate, divergent selection, grasses, dicots.

Introduction
Monocots and dicots are two main classes of angiosperms.
Dicots include soybean, oilseed rape, and related species,
whereas monocots include wheat, corn, and rice. The distinct
differences in morphological and physiological features between monocots and dicots, such as the structure and chemical components of the seed, have been described by Raven
and Johnson (2002). The evolutionary divergence of the genetic factors underlying these features is of great interest.
Grasses (Poaceae), including the most important cereal
crops, are typical monocots. Monocot seeds are rich in
starch, whereas dicot seeds are generally rich in lipid or
protein. Therefore, it can be expected that there has been
evolutionary divergence in the genes controlling starch biosynthesis between these two groups of plants.
Starch is the most widespread and abundant storage carbohydrate in crops and its production is critical to both crop
yield and quality (Umemoto et al. 2002). To date, most studies relating to the genes of starch biosynthesis have focused
on two aspects, namely molecular genetics and evolution
(Whitt et al. 2002; Tetlow et al. 2004; Han et al. 2007; Wu
et al. 2008; Comparot-Moss and Denyer 2009; Zeeman et al.
2010). First, in the area of molecular genetics, the pathway of
starch biosynthesis and the genes encoding the various enzymes have been shown to be remarkably conserved in dicots

and monocots; and four enzymes, which play a major role in

starch biosynthesis, are suggested to be unique in the two
plant kingdoms. These enzymes are ADP-glucose pyrophosphorylase (AGPase), starch synthase (SS), starch branching
enzyme (SBE) and debranching enzyme (DBE). Each of them is
further divided into different subunits or subtypes, for example, two subunits (AGPase large subunit [AGPL] and AGPase
small subunit [AGPS]) for AGPase, five classes (granule-bound
starch synthase [GBSS and SSI-IV]) for SS, two main classes
(classes I and II) for SBE, and two types (isoamylase [ISA] and
pullulanase [PUL]) for DBE (Tetlow et al. 2004; ComparotMoss and Denyer 2009; Zeeman et al. 2010).
Second, various evolutionary and domestication studies of
the genes in the starch biosynthetic pathway have been presented previously. For example, Whitt et al. (2002) surveyed
nucleotide diversity of six genes, namely Amylose extender1
(Ae1), Brittle2 (Bt2), Shrunken1 (Sh1), Shrun-ken2 (Sh2),
Sugary (Su1), and Waxy (Wx), which play major roles in the
starch biosynthetic pathway in maize, Zea mays ssp.
parviglumis, and uncovered evidence of positive selection
on three of these genes (Ae1, Su1, and Bt2). Han et al.
(2007) analyzed the phylogeny of the SBEII gene and found
different duplication patterns between grasses and dicots. Wu
et al. (2008) investigated the fate of duplicated genes following the ancient whole-genome duplication (WGD) in rice and

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Mol. Biol. Evol.

doi:10.1093/molbev/mss131

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Research
article

Abstract

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Li et al. . doi:10.1093/molbev/mss131

showed that genes involved in starch synthesis are preferentially retained in rice compared with Arabidopsis. In the most
recent study, it was shown that the diversification of GBSS
genes is mainly associated with genome-wide duplication
throughout the evolutionary history of monocots and eudicots (Cheng et al. 2012).
All these evolutionary studies imply that the evolutionary
pattern of the starch pathway in grasses is different from that
in dicots. However, there are few comprehensive studies to
confirm this viewpoint. In this study, therefore, we compared
evolutionary rate, gene duplication, and selective pattern of
the key genes involved in the starch biosynthetic pathway
between grasses and dicots, using five grasses and five dicots
as materials. The results should shed light on how evolutionary divergence contributes to differences in morphological
and physiological features between these two groups of
plants.

similarity, and therefore it allowed the detection of orthologs

among both slowly and rapidly evolving genes.

Materials and Methods

The codon sequences were first aligned using the PRANK

codon model (Loytynoja and Goldman 2005) and then translated into amino acid sequences. When necessary, sequences
were further edited and aligned manually. Phylogenetic tree
reconstruction was performed with both Neighbor Joining
(NJ) and Bayesian approaches using the amino acid sequences. In the NJ method, the phylogenetic analyses were
conducted using the Molecular Evolutionary Genetics
Analysis 4.0 program (Tamura et al. 2007). The parameter
setups were as follows: model: p-distance; bootstrap: 1,000
replicates; and gap/missing data: pairwise deletion.
In the Bayesian method, the analyses were conducted
using MrBayes v3.1 (Ronquist and Huelsenbeck 2003) with
the Jones, Taylor, Thornton substitution model (Jones et al.
1992), four chains, one million generations, and two runs.
Trees were sampled every 100 generations, discarding a
burn-in of 250,000 generations.

Sequences were collected using the method described by

Tatusov et al. (2000) with a slight modification. Briefly, our
method includes the following steps:
1) Protein-coding transcripts of Brachypodium distachyon
(JGI v1.0, International Brachypodium Initiative), Oryza
sativa (MSU Release 6.0, Ouyang et al. 2007), Setaria
italica (version 1.1, DoE Joint Genome Institute),
Z. mays (5a release, Maize Genome Project), Sorghum
bicolor (v1.0 release, Paterson et al. 2009), Arabidopsis
thaliana (TAIR release 9, The Arabidopsis Genome
Initiative), Glycine max (Glyma1.0, Soybean Genome
Project, DoE Joint Genome Institute), Medicago truncatula (Mt3.0, M. truncatula genome sequencing project),
Populus trichocarpa (v2.0 release, The Joint Genome
Institute), and Ricinus communis (release v0.1, Chan et
al. 2010) were downloaded from JGI (http://www.phyto
zome.net/) or maizesequence (http://www.maize
sequence.org) and used to construct a local BLAST database using BLAST 2.2.24;
2) An all-against-all protein sequence comparison was
performed;
3) In all the comparisons carried out in step 2, the ones with
Arabidopsis AGPL1 as a query were identified, and the
obvious paralogs were collapsed;
4) All interspecies Best Hits (BeTs) of AtAGPL1 and its paralogs were detected;
5) Steps 3 and 4 were repeated with the resulting sequences
as secondary BLASTp queries until no new sequence was
found;
6) Clusters of Orthologous Groups (COGs) were formed
using the protocol of Tatusov et al. (2000); and
7) COGs with Arabidopsis AGPS1, GBSS, SSI, SSII, SSIII, SSIV,
BEI, BEII, ISAI, ISAII, ISAIII, and PUL as a query were formed
in the same way.
This approach was based on the consistency between
genome-specific best hits, rather than the absolute level of
2

Gene duplication and loss within each gene family were analyzed principally by checking each subfamily manually. Each
gene family is assumed to have at least two subclades (the
grass and dicot subfamily) and at least five members from
each species in each subfamily (supplementary fig. S3,
Supplementary Material online). If there are two or more
grass or dicot subclades, or if these subclades have two or
more members from the same species, it was concluded that
there is one or more duplications. If there is a member missing
from any species within the grass or dicot subclade, it was
concluded that this is a gene loss. Every gene loss was further
confirmed by searching GenBank.

Phylogenetic Analyses

Estimation of dN:dS Ratios

The codon sequences were aligned using the PRANK codon
model (Loytynoja and Goldman 2005), alignment gaps were
manually deleted, and then used for the following calculations. The ratio of nonsynonymous substitutions per
nonsynonymous site (dN) to synonymous substitutions per
synonymous site (dS) (! value) of homologous gene pairs was
computed with the maximum likelihood method in Codeml
from the PAML package (v4.4; Yang 2007). Saturation effects
were avoided by discarding the gene pairs for which dS > 2
(Ramsay et al. 2009).
To test for variation of the ! ratio among different
branches in gene trees, a branch-specific model was used
and conducted in Codeml. This type of branch-specific
model allows the ! ratio to vary among branches in the
phylogeny, and it can be used to test whether there are different ! values on particular lineages (Yang 1998); thus, this
model can be compared with the one-ratio model that assumes a constant ! value across all branches using the likelihood ratio test (LRT). In these calculations, truncated
sequences were removed and tree topologies were adjusted
manually.

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Retrieval of Sequences

Estimation of Gene Duplication and Loss

Starch Biosynthetic Pathway Genes in Grasses and Dicots . doi:10.1093/molbev/mss131

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Detection of Positively Selected Sites

The data sets used in dN:dS ratio estimation were further used
in the selection analysis conducted in the program Fitmodel
0.5.3 (Guindon et al. 2004). Fitmodel is similar to PAML (Yang
2007) and uses maximum likelihood for estimating parameters of sequence evolution. Moreover, it also estimates switching parameters, such that FitmodeL allows changes between
selection patterns to occur through time (i.e., switches between selection patterns in the phylogenetic tree at individual
sites) (Guindon et al. 2004). The models M3 and M3 + 1,
described in Yang and Nielsen (2002) and Guindon et al.
(2004), were employed in this analysis.

Results

grasses (or dicots) form distinct subfamily clades with high

support (mostly >90% bootstrap value) in all the phylogenetic trees (fig. 2 and supplementary fig. S2, Supplementary
Material online). In the trees of AGPL type 3, AGPS, GBSS, SSII,
SSIII, and SBEII, different gene classes also form distinct subfamily clades. For example, SBEIIa and SBEIIb form grass subfamilies 1 and 2, respectively (supplementary fig. S2,
Supplementary Material online). These results are supported
by previous results from different species and quantities of
sequence, for example, five clades in the AGPL tree in higher
plants (Georgelis et al. 2007). In each grass or dicot subfamily
clade, the gene tree is largely concordant with the species tree
(Soltis 1999) (supplementary fig. S3, Supplementary Material
online).

Data Collection and Phylogenetic Analyses

Fourteen COGs were obtained and are shown in figure 1 and
supplementary figure S1 (Supplementary Material online).
Homologs of AGPS, AGPL, GBSS, SSI, SSII, SSIII, and SSIV are
included in a single COG (fig. 1 and supplementary fig. S1,
Supplementary Material online); homologs of BE are separated into three COGs (supplementary fig. S1,
Supplementary Material online), corresponding to classes I
and II and a third class (Wang et al. 2010), respectively; and
homologs of DBE are separated into four COGS (supplementary fig. S1, Supplementary Material online), corresponding to
ISAI-III and PUL, respectively.
Phylogenetic tree reconstruction was performed by the
Bayesian and NJ methods for each of the COGs; both methods yielded identical topologies (data not shown). Genes from

Distinct Differences for Gene Duplication and

Patterns of Loss between Grasses and Dicots
Gene duplication can be distinguished into recent and old
duplications according to the time it occurred (Ohno 1970).
In this study, the grass- (duplication before the grasses divergence) and dicot-specific duplications (duplication before the
dicots divergence) were termed as old duplications, and the
species-specific (duplication within a species) ones were
termed as recent duplications. Among 14 gene families,
each one was separately analyzed in detail for the recent
and old duplications. The results are shown in table 1,
figure 2, and supplementary figure S2 (Supplementary
Material online). As for the old duplication, it occurred approximately seven times in six gene families (AGPL, AGPS,
3

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FIG. 1. The COG of the AGPS gene family. Solid lines show symmetrical BeTs (Best Hits) and broken lines show asymmetrical BeTs. Genes from the same
species are adjacent. Each gene ID is indicated, and the prefix Rc denotes IDs from Ricinus communis.

Li et al. . doi:10.1093/molbev/mss131

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GBSS, SSII, SSIII, and SBEII) in the grasses and once in the AGPS
gene family in the dicots. Considering the recent duplication,
it occurred approximately 3 times in 3 gene families (AGPS,
SSIII, and SSIV) in the grasses and more than 20 times in most
of the gene families in the dicots (except SBEIII, ISAIII, and
PUL). Note that in the dicots, most evidence of recent duplication was found in G. max.
Considering gene losses, those specific to the grass species
only occurred in the AGPL and SSII gene families and those
specific to the dicot species occurred in nearly all the families
analyzed and the majority of the missing members are from
the species M. truncatula (table 1, fig. 2, and supplementary
fig. S2, Supplementary Material online). Obviously, there are
fewer gene losses in the grasses.
As a result, there are distinct differences in patterns of gene
duplication and loss between grasses and dicots. These results
are consistent with the observations that there are more
copies of starch synthesis genes in Z. mays and O. sativa
than in A. thaliana (Yan et al. 2009), the genes involved in
synthesis and catabolism of saccharides are preferentially retained in rice (Wu et al. 2008), and the proposition that the
4

genes encoding the starch pathway originated from a WGD

event in an early ancestor of the grasses (Comparot-Moss and
Denyer 2009; Yan et al. 2009).

Starch Pathway Genes Evolve Rapidly in Grasses

To determine whether the starch pathway genes are under
different evolutionary constraints in grasses and dicots, the !
values for the above genes were calculated. We applied two
methods: the pairwise comparison approach and the
branch-specific model.
In the pairwise comparison approach, we calculated the
pairwise ! values within each grass or dicot subfamily and
compared their averages between two subfamilies. The result
showed that the average ! values of nine grass subfamilies
(subfamily of AGPL type 1, subfamily 2 of AGPL type 3, subfamilies 1 and 2 of AGPS, subfamilies 2 and 3 of SSII, subfamily
1 of SSIII and SSIV, and subfamily 2 of SBEII) are significantly
higher than that of the corresponding subfamilies composed
of the homologs from the dicots; and three grass subfamilies
(subfamily 1 of AGPL type 3 and 4 and subfamily 2 of GBSS)
show the opposite result (fig. 3).

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FIG. 2. Phylogenetic relationship of sequences within the AGPS gene family by NJ method with bootstrap support above 50% shown at the nodes.
Square boxes indicate duplication events, and the broken lines indicate absent genes, lost from either those species or not yet sequenced. Branch A
indicates the most recent common ancestor to grass subfamily 2. WGD indicates the ancient WGD event that occurred approximately 70 Ma in the
grasses. Gene names, based on previous references or BLAST results, are listed after the Gene IDs.

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Table 1. Gene Duplication and Loss in the Gene Families of the Starch Biosynthetic Pathway.
Types
Old
Grass
Dicot
Recent
Grass
Dicot

Gene Duplicationa

Gene Loss

specific
specific

AGPL type 3*; AGPS*; GBSS*; SSII**; SSIII*; SBEII*

AGPS*

species specific
species specific

AGPS*; SSIII*; SSIV*

AGPL type 1*, 2** and 3***; AGPS***; GBSS****; SSI*; SSII**; SSIII*;
SSIV**; SBEI*; SBEII**; ISAI*; ISAII*

AGPL type 3*
AGPL type 4; AGPS; GBSS; SSI; SSII;
SSIII; SSIV; SBEI; SBEII; SBEIII;
ISAII; PUL

The number of asterisks indicates the number of replications.

the selection is mainly located either in the grass or in the

dicot subclade of the gene trees. This result provides evidence
of divergent selection between grasses and dicots (table 3). In
addition, positive selection may also have driven evolution in
the AGPL group 4, GBSS, ISAII, and ISAIII gene families,
although no or slightly positive selection sign (!3  1.040)
was observed (table 3). The probable reason is due to the
low power derived from the small number of sequences.

Discussion
In this study, the major genes involved in starch biosynthesis,
that is AGPL, AGPS, GBSS, SSI-IV, SBEI-III, ISAI-III, and PUL, were
used as materials to compare the evolutionary pattern of
these genes between grasses and dicots. As a result, two distinct evolutionary features were observed: more old duplications and a higher evolutionary rate of the genes in the
grasses. Given the fact that the grass seeds are rich in
starch, while the dicot seeds are rich in protein or lipid, we
hypothesized that the old duplication contributes to, and the
higher evolutionary rate is associated with, the higher starch
content in the grasses.

Divergent Selection between Grasses and Dicots

Hypothesis 1, Gene Duplication Contributes to

Higher Starch Content in Grass Seed

To assess the potential for selection on gene families in the

starch synthesis pathway, we performed a selection analysis
using the Fitmodel of Guindon et al. (2004), and compared
the sites under positive selection between the grass and the
dicot subfamilies in each gene tree. The advantage of the
Fitmodel approach, rather than the PAML model, is that it
does not require a priori knowledge of the positions in the
tree that are potentially experiencing positive selection, and
thus, it is more appropriate for this analysis. For every data set
analyzed, the M3+1 model was favored over the M3 model.
The parameter estimates of the M3+1 model suggest that
most sites ( 87%, p1 + p2) are identified to be under purifying selection with !1  0.012 and !2  0.391 in all the
genes (table 3); and 1% to 9% (p3) of sites within the genes
in type 1 and 3 of AGPL, AGPS, SSI-IV, and SBEI-III and two DEB
(ISAI and PUL) gene families are identified to be under positive selection with an !3 value considerably larger than one.
Among these positive selection sites, the number of sites for
genes in AGPS, SSIV, SBEIII, and PUL gene families is significantly asymmetric across grasses and dicots, suggesting that

In this study, the grass subfamilies of AGPL type 3, AGPS, GBSS,

SSII, SSIII, and SBEII gene families were shown to have experienced one or two rounds of duplication before the grasses
divergence (table 1, fig. 2, and supplementary fig. S2,
Supplementary Material online). Of these duplications, the
ones that gave rise to the subfamilies 1 and 2 of AGPL type
3, AGPS, GBSS, SSIII, and SBEII and subfamilies 2 and 3 of SSII
(fig. 2 and supplementary fig. S2, Supplementary Material
online) were suggested to have been created through the
ancient WGD event that occurred approximately 70 Ma in
grasses (Paterson et al. 2004; Comparot-Moss and Denyer
2009; Yan et al. 2009; Pan et al. 2011). Following the WGD,
the duplicates underwent a process of subfunctionalization:
For the genes of AGPL type 3, the one from subfamily 1 is
assumed to encode plastidial AGPLs, whereas the one from
subfamily 2 is assumed to encode cytosolic AGPLs
(Comparot-Moss and Denyer 2009); for the AGPS genes,
those from subfamily 1 are known to encode plastidial subunits in leaf and cytosolic subunits in seed endosperm by the
use of the alternate first exon, whereas the genes from grass
5

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However, there is a shortcoming in the pairwise comparison approach: It does not take into account the evolutionary
rate of the most recent common ancestor of the subfamily
(e.g., branch A in fig. 2). To overcome the issue, we applied the
branch-specific model to the gene trees to check the changes
in the evolutionary constraints across different subfamilies. In
this approach, the model assumes two or more ! ratios,
which correspond to the number of subfamilies. As a result,
this model was favored over the one-ratio model by the LRT
(P < 0.05) in all the gene trees except for SSI and ISAI; and the
estimated ! values of the subfamilies confirm the result of the
pairwise comparison approach. More importantly, these results show that some of the grass subfamilies, which did not
exhibit a higher ! value than their dicot subfamilies in the
pairwise comparison approach, exhibit a higher ! value when
the most recent common ancestor is taken into account
(table 2).
Therefore, all the grass subfamilies examined, except for
subfamily 1 of AGPL type 3 and SBEII and subfamilies of AGPL
type 4, GBSS, and ISAI, have a considerably higher ! value
than their corresponding dicot equivalent (table 2). This
result suggests that these genes evolve rapidly in grasses.

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Table 2. LRT Statistic and Parameters from Branch Model of PAML.

Gene Family/Subfamily
AGPL
Type 1
Type 3
Type 4
AGPS
SS
GBSS
SSI
SSII
SSIII
SSIV
SBE
SBEI
SBEII
SBEIII
DBE
ISAI
ISAII
ISAIII
PUL

Null Hypothesis

Alternative Hypothesis

ln L

ln L

6,556.04
10,741.28
5,908.11
8,243.16

0.074
0.113
0.097
0.033

6,551.64
10,737.01
5,902.29
8,218.80

Grass = 0.095; Dicot = 0.061

Grass1a = 0.055; Grass 2a = 0.262; Dicot = 0.103
Grass = 0.073; Dicot = 0.125
Grass 1a = 0.045; Grass 2a = 0.064; Dicot = 0.019

13,938.81
7,779.76
12,886.63
23,127.17
15,032.69

0.111
0.100
0.114
0.169
0.158

13,927.50
7,779.71
12,856.93
23,076.27
15,027.01

Grass1a = 0.106 M; Grass2 = 0.077; Dicot = 0.133

Grass = 0.102; Dicot = 0.099
Grass 1 = 0.120; Grass 2a = 0.159; Grass 3 = 0.176; Dicot = 0.076
Grass 1a = 0.248; Grass 2 = 0.169; Dicot = 0.111
Grass = 0.181; Dicot = 0.134

9,291.88
13,201.58
11,671.75

0.011
0.070
0.120

9,280.23
13,191.18
11,668.39

Grass = 0.138; Dicot = 0.077

Grass 1 = 0.061; Grass 2a = 0.100; Dicot = 0.060
Grass = 0.140; Dicot = 0.106

11,634.27
10,935.65
10,181.83
14,383.27

0.111
0.162
0.115
0.158

11,634.01
10,932.39
10,177.50
14,379.68

Grassa = 0.116; Dicot = 0.107

Grass = 0.195; Dicot = 0.147
Grass = 0.142; Dicot = 0.102
Grassa = 0.184; Dicot = 0.143

LRT
Statistic

8.8
88.96
11.64
48.72

3.01e-3
<1.0e-12
6.46e-4
2.63e-11

22.62
0.1
59.4
101.8
11.36

1.23e-5
0.752
7.90e-13
<1.0e-12
7.50e-4

23.3
20.8
6.72

1.39e-6
3.04e-5
9.53e-3

0.52
6.52
8.66
7.18

0.471
1.07e-2
3.25e-3
7.37e-3

NOTE.P-values were calculated from Twice the log-likelihood difference between the two models compared to a distribution with df = the difference for the number of
parameters between the two models.
a
The genes from these subfamilies were suggested to be mainly expressed in the endosperm of the grasses.

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FIG. 3. Pairwise estimation of ! values in each grass or dicot subfamily of gene family: (A) AGPL type 1, 3, 4 and AGPS; (B) GBSS and SSI-IV; (C) SBEI-III;
and (D) ISAI-III and PUL. In each of the gene families, a significance test on the difference between grass subfamily and dicot subfamily was carried out,
and its result, listed in supplementary table S1 (Supplementary Material online), is indicated by n.s. (no significance). *0.01 < P  0.05 and **P  0.01.

11,437.12

10,758.39

9,947.50

14,124.90

ISAI

ISAII

ISAIII

PUL

11,554.07

SBEIII

p1 p2 p3
x1 x2 x3
0.48 0.35 0.17
0.003 0.070 0.375
0.55 0.43 0.02
0.027 0.220 0.853
0.31 0.32 0.07
0.014 0.205 0.682
0.84 0.15 0.01
0.007 0.182 1.282
0.51 0.35 0.14
0.010 0.175 0.559
0.65 0.34 0.01
0.015 0.294 8.334
0.61 0.31 0.08
0.016 0.244 0.923
0.43 0.44 0.13
0.017 0.244 0.842
0.44 0.48 0.08
0.179 0.265 0.842
0.69 0.29 0.02
0.019 0.333 2.252
0.66 0.29 0.05
0.007 0.179 0.668
0.53 0.42 0.05
0.017 0.247 1.427
0.62 0.33 0.05
0.013 0.276 1.035
0.45 0.42 0.13
0.019 0.223 0.852
0.52 0.35 0.13
<.001 0.187 0.811
0.46 0.38 0.16
0.013 0.218 0.864

M3 Modela

14,090.21**

9,939.22**

10,748.98**

11,409.15**

11,527.44**

12,947.2**

9,174.00**

14,826.22**

22,680.34**

12,599.16**

7,633.18**

13,735.55**

8,173.32**

5,857.56**

10,659.58**

6,510.01**

ln L

p1 p2 p3
x1 x2 x3
0.79 0.20 0.01
0.007 0.368 3.517
0.54 0.42 0.04
<.001 0.224 1.192
0.66 0.25 0.09
0.070 0.213 0.806
0.87 0.12 0.01
0.004 0.223 1.527
0.64 0.26 0.10
<.001 0.264 1.026
0.67 0.31 0.02
0.006 0.304 5.132
0.71 0.24 0.05
0.003 0.376 1.858
0.58 0.35 0.07
0.010 0.389 1.383
0.54 0.39 0.07
<.001 0.335 1.301
0.72 0.26 0.02
0.012 0.391 2.721
0.72 0.25 0.03
0.001 0.247 1.139
0.63 0.33 0.04
0.001 0.322 3.439
0.69 0.25 0.06
<.001 0.350 1.243
0.49 0.38 0.13
0.006 0.236 1.036
0.62 0.28 0.10
<.001 0.257 1.040
0.59 0.33 0.08
0.004 0.350 1.805

M3+1 Modela

188, 262, 419, 508, 743, 798

31, 100, 172, 515

19, 61, 62, 111, 136, 156, 160, 361, 369, 479,
599, 677
96, 119, 218, 274, 605, 612

266, 297, 654

61, 434, 663

52, 76, 133, 167, 213, 266, 273, 281, 288,

353, 368, 379, 435, 454
15, 16, 33, 47, 120, 262, 315, 327, 371, 384,
542, 549, 651, 687, 734, 738, 761, 841, 852
1, 49, 89, 140, 168, 169, 179, 182, 187, 197,
229, 278, 330, 422, 527
14, 132

124, 128, 166, 182, 186, 194, 254, 311, 351,

359, 365, 380, 387, 445, 460, 462
100, 301

222, 246, 281, 388, 428

96, 149, 172, 201, 368, 416, 440

Dicots Specific

34, 51, 75, 91, 100, 156, 157, 238, 258, 307,
316, 318, 321, 327, 332, 477, 494, 527,
604, 619, 650, 824, 829, 841, 863

2, 25, 28, 81, 127, 145, 148, 152, 233, 315,

339, 353, 354, 488, 498, 518, 541, 577, 628
80, 173, 602

68, 141, 191, 214, 238, 270, 284, 368, 403,

533, 588, 629, 661, 698, 709, 762, 835, 851
105, 125, 324, 661, 682, 705

8, 351, 584

134, 331, 359, 370, 373, 421, 460, 534, 571,

679
29, 43, 83, 91, 127, 272, 808

94, 98, 99, 272, 278, 409, 487

82, 179, 191, 202, 223, 249, 275, 286, 348,

368, 372, 450, 455, 471, 495
59, 109, 122

19, 75, 115, 151, 202, 204, 238, 290, 297,

316, 332, 375, 408, 431

148, 179, 196, 203, 208, 287, 364

Positive Sites (M3+1 Model)

4, 125, 211, 239, 240, 345, 371, 401, 405

66, 100

Grasses Specific

Downloaded from http://mbe.oxfordjournals.org/ at University of Reading on March 6, 2015

pi proportion of sites that fall into the !i site class, i = 1, 2, 3.

**P  0.01, and the probabilities are obtained by LRT (df = 2) of the models M3 and M3 + 1.

DBE

12,964.60

14,858.01

SSIV

SBEII

22,727.50

SSIII

9,179.54

12,662.74

SSII

SBEI

7,645.76

SSI

SBE

13,787.51

GBSS

SS

5,862.01

Type 4

8,185.64

10,679.19

Type 3

AGPS

6,519.57

Type 1

AGPL

ln L

Subfamily

Gene
Family

Table 3. Likelihood Ratio Test Statistic and Parameters Estimated from the M3 and M3+1 Models in Fitmodel.

Starch Biosynthetic Pathway Genes in Grasses and Dicots . doi:10.1093/molbev/mss131

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MBE

Li et al. . doi:10.1093/molbev/mss131

Hypothesis 2, the Higher Evolutionary Rate Is

Associated with Higher Starch Content in Grass Seed
The above results showed that most genes examined exhibit a
considerably higher evolutionary rate in grasses than in dicots
(fig. 3 and table 2). This conclusion is favored when we consider only the genes mainly expressed in the endosperm in
grasses. Based on the previous studies, the genes from grass
subfamily of 1 and 2 in AGPL type 3 and AGPS, 1 in GBSS and
SSIII, and 2 in SSII and SBEII, ISAI, and PUL, were suggested to
be mainly expressed in the endosperm (Beatty et al. 1999;
Mizuno et al. 2001; Hirose and Terao 2004; Dian et al. 2005;
Ohdan et al. 2005; Yan et al. 2009; Chen et al. 2011) (table 2).
All these subfamilies, except those in AGPL type 3 and GBSS,
exhibit a higher evolutionary rate in the grasses than the
corresponding subfamilies comprising their homologs from
the dicots.
The above results suggest that the higher evolutionary
rate is associated with the higher starch content of grass
seed. However, the reason for this association is not obvious.
The possible explanation is that strong positive selection on
a large numbers of residues leads to a rapid rate of evolution; and the supporting evidence is that we found a
8

significant positive selection signature in genes from the

grasses (table 3).

Possible Explanation for the Evolutionary Features of

the Starch Biosynthetic Genes in Grasses
Two basic questions of evolutionary biology are How does
it happen? and What is the consequence? The above two
hypotheses partly answered the latter. It is also tempting to
speculate how it happens. We provide a possible explanation
in terms of why the grass starch biosynthetic genes exhibit
two such distinct features.
Our speculation involves proteinprotein interactions.
More and more evidences support the concept that enzymes
involved in starch biosynthesis form a functional multiprotein
complex. For example, in rice, the amylose extender (ae) mutation lacking SBEIIb caused a dramatic reduction in the activity of soluble SSI (Nishi et al. 2001); in maize, the ae mutant
lines lacking SBEIIb also show loss of activity of SBEI and altered properties of an ISA-type DBE (Colleoni et al. 2003), and
mutant lines lacking both PUL and ISAI cause a loss in SBEIIa
activity, although the amount of SBEIIa protein is apparently
unchanged (James et al. 2003); and in wheat, it was confirmed
by mass spectrometry that SSI, SSIIa, and SBEII form as components of one or more protein complexes in amyloplasts
(Tetlow et al. 2008).
Provided that enzymes involved in starch biosynthesis
form functional multiprotein complexes, if a gene encoding
the enzymes had a sister through duplication and consequently conferred a selective advantage to the plant, the
sister would be maintained; and subsequently, the duplicates
of the other genes in the pathway could be created through
convergent evolution. Similarly, if a gene in the complex
evolves more rapidly, other genes were also expected to
show a similar pattern.
In conclusion, we deduced that there are two novel aspects
of the evolution of the starch biosynthesis pathway. First, the
results of the gene duplication analyses suggest that the
whole-starch biosynthetic pathway was duplicated and
then diverted in grasses. Second, the evolutionary rate of
the two grass subfamilies of the AGPS gene family at the
top of the pathway is greater than that of the dicot subfamily
(table 2), contrary to the previous finding that upstream
genes in a biosynthetic pathway evolve more slowly than
downstream genes (Rausher et al. 1999; Lu and Rausher
2003). The reason for the two novel findings needs to be
addressed in future studies.

Supplementary Material
Supplementary figures S1S3 and supplementary table S1 are
available at Molecular Biology and Evolution online (http://
www.mbe.oxfordjournals.org/).

Acknowledgments
We are grateful to Dr Stephane Guindon (Department of
Statistics, University of Auckland, New Zealand) for his helpful
discussion in the process of this study. This work was supported by National Key Basic Research Program of China

Downloaded from http://mbe.oxfordjournals.org/ at University of Reading on March 6, 2015

subfamily 2 are known to encode cytosolic subunits (Rosti et

al. 2006; Rosti and Denyer 2007; Comparot-Moss and Denyer
2009); for the GBSS, SSII, SSIII, and SBEII genes, those from one
of the subfamilies are suggested to be mainly expressed in
seed endosperm, whereas those from the other are suggested
to be mainly expressed in other tissues (Beatty et al. 1999;
Mizuno et al. 2001; Hirose and Terao 2004; Dian et al. 2005;
Ohdan et al. 2005; Yan et al. 2009; Chen et al. 2011).
There is some evidence to suggest that the emergence of
the duplicates promotes seed starch content: 1) The synthesis
of ADPglucose is an important step in starch biosynthesis,
and this occurs in the plastid in all plants, including grasses.
However, in cereal endosperm, ADPglucose is also synthesized
in the cytosol through AGPase, which is encoded by the genes
arising from the ancient WGD of grasses. In all the grasses
tested to date, 8595% of the AGPase activity in the endosperm is the cytosolic form (Comparot-Moss and Denyer
2009). This cytosolic activity is proposed to lead to a greater
amount of carbon committed to starch in the endosperm
(Beckles et al. 2001; James et al. 2003; Comparot-Moss and
Denyer 2009), 2) SSIIa that also arose from the ancient WGD
of grasses appears also to be key to starch biosynthesis. Loss of
this enzyme in maize, wheat, rice, and starchy legume pea
results in reduced starch content in the seed (Morell et al.
2003; Keeling and Myers 2010), and 3) There are two functional isoforms each for GBSSI and SSII in starchy seed
legumes (but this is not the case in soybean seed, which is
rich in protein), with one of the two isoforms mainly expressed in cotyledons and thus showing a pattern of convergent evolution with the GBSSII and SSII genes in grasses (Pan
et al. 2009). These three lines of evidence lend some support
to the hypothesis that gene duplication may contribute to
higher starch content in grass seeds.

Starch Biosynthetic Pathway Genes in Grasses and Dicots . doi:10.1093/molbev/mss131

(2011CB109300), National Natural Science Foundation of

China (30971848), Fundamental Research Funds for the
Central Universities (KYT201002), Specialized Research Fund
for the Doctoral Program of Higher Education (2010009711
0035), and a project funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions.

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