全 文 ：miR156 靶基因 SPL在植物中的系统分布和进化模式分析*
凌立贞1,2, 张书东1**
(1 中国科学院昆明植物研究所中国西南野生生物种质资源库, 云南 昆明摇 650201;
2 中国科学院研究生院, 北京摇 100049)
摘要: Squamosa promoter鄄binding protein鄄like genes (SPLs) 在植物发育过程中具有重要作用。 很多 SPLs 被
miR156调节, 然而, 对于它们在植物中的系统分布和进化模式还知之甚少。 本文对 9 个测序物种 (藻类,
苔藓, 石松, 单子叶和双子叶植物) 的 183 个 SPLs 进行了生物信息学分析。 结果表明 miR156 应答元件
(MREs) 仅在陆生植物 SPLs中发现, 藻类中不存在。 系统进化分析显示陆生植物 SPLs 分为两大分支:
group I和 group II。 MiR156 靶基因仅分布于 group II, 表明它们有着共同的祖先。 Group II进一步分为 7 个
亚支 (IIa鄄IIg), miR156 靶基因分布在除 IId外的其余 6 个亚支的特定 SPLs。 系统分类与基因结构的相关
性反映了 SPL靶基因结构上的变化。 在进化过程中, 它们可能发生外显子的丢失且伴随 MRE的丢失。 另
外, 基因重复对 SPL靶基因的丰度变化影响很大, 尤其是被子植物与低等植物分歧后它们数量明显增加。
以拟南芥为模式植物分析发现串联重复和片段重复是 SPL靶基因扩张的主要机制。
关键词: 系统分析; 基因重复; 基因结构; microRNA; 转录因子
中图分类号: Q 78摇 摇 摇 摇 摇 摇 摇 文献标识码: A摇 摇 摇 摇 摇 摇 摇 文章编号: 2095-0845(2012)01-033-14
Unraveling the Distribution and Evolution of miR156鄄targeted
SPLs in Plants by Phylogenetic Analysis
LING Li鄄Zhen1,2, ZHANG Shu鄄Dong1**
(1 Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China;
2 Graduate University of Chinese Academy of Sciences, Beijing 100049, China)
Abstract: Squamosa promoter鄄binding protein鄄like genes (SPLs) are critical during plant development and mostly
regulated by miR156. However, little is known about phylogenetic distribution and evolutionary patterns of miR156鄄
targeted SPLs. In this study, 183 SPLs from nine genome鄄sequenced species representing algae, bryophytes, lyco鄄
phyte, monocots, and eudicots were computationally analyzed. Our results showed that miR156 responsive elements
(MREs) on SPLs were present in land plants but absent from unicellular green algae. Phylogenetic analysis revealed
that miR156鄄targeted SPLs only distributed in group II not group I of land plants, suggesting they originated from a
common ancestor. In addition, group II were further divided into seven subgroups ( IIa鄄IIg) and miR156鄄targeted
SPLs distributed in some specific members of SPLs from six subgroups except subgroup IId. Such distribution pattern
was well elucidated by gene structure evolution of miR156鄄targeted SPLs based on the correlation of phylogenetic
classification and gene structure. They could suffer from the exon loss events combined with MREs loss during evolu鄄
tion. Moreover, gene duplication contributed to the abundance of miR156鄄targeted SPLs, which had significantly in鄄
creased after angiosperms and lower plants split. With Arabidopsis as the model species, we found segmental and
tandem gene duplications predominated during miR156鄄targeted SPLs expansion. Taken together, these results pro鄄
植 物 分 类 与 资 源 学 报摇 2012, 34 (1): 33 ~ 46
Plant Diversity and Resources摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 DOI: 10. 3724 / SP. J. 1143. 2012. 11117
*
**
Fundation items: The Chinese Academy of Sciences Large鄄Scale Scientific Facility (2009鄄LSF鄄GBOWS鄄01)
Author for correspondence; E鄄mail: sdchang@ mail. kib. ac. cn; Tel +86-871-5223137
Received date: 2011-08-16, Accepted date: 2011-11-07
作者简介: 凌立贞 (1979-) 女, 博士, 主要从事植物进化研究。 E鄄mail: linglizhen@ mail. kib. ac. cn
vide better insights in understanding the function diversity and evolution of miR156鄄targeted SPLs in plants.
Key words: Phylogenetic analysis; Gene duplication; Gene structure; MicroRNA; Transcription factor
Abbreviations: CDS, coding sequence; CR, Chlamydomonas reinhardtii; DBD, DNA鄄binding domain; miRNAs,
microRNAs; MITEs, miniature inverted repeat transposable elements; ML, maximum鄄likelihood; MREs, miR156
responsive elements; NJ, Neighbor鄄Joining; NLS, nuclear localization signal; SBP, Squamosa promoter鄄Binding
Protein; SPL, Squamosa promoter鄄binding protein鄄like gene
摇 Squamosa promoter鄄binding protein鄄like genes
( SPLs) encode plant鄄specific transcription factors
(TFs) that share a highly conserved Squamosa pro鄄
moter Binding Protein (SBP) domain and recognize
similar target DNA sequences. This SBP鄄domain
spans 79 amino acids residues and features a se鄄
quence鄄specific DNA鄄binding domain (DBD). The
DBD contains two zinc鄄binding sites assembled as
Cys鄄Cys鄄His鄄Cys (Cys2HisCys) and Cys鄄Cys鄄Cys鄄His
(Cys3His) (Yamasaki et al., 2004) and a highly
conserved bipartite nuclear localization signal (NLS)
in C鄄terminal (Birkenbihl et al., 2005). It has been
proved that the SBP鄄domain specifically binds to se鄄
quences containing a palindromic GTAC core motif
(Birkenbihl et al., 2005; Cardon et al., 1997).
Elevated studies have described the functions of
members of SBP鄄box genes in different plant orga鄄
nisms through analysis of either their loss鄄of鄄function
or gain鄄of鄄function mutants. It is known that SPLs
are critical in diverse biological processes, including
seed germination and seedling development (Martin
et al., 2010), leaf development ( Moreno et al.,
1997), phase transition (Gandikota et al., 2007;
Wang et al., 2009; Wu and Poethig, 2006), fruit
ripening (Manning et al., 2006), copper homeosta鄄
sis (Kropat et al., 2005; Yamasaki et al., 2009)
and grain yield ( Jiao et al., 2010; Miura et al.,
2010). In fact, it is difficult to point out the exact
functions of SPL transcription factors in development
because of their extreme genetic redundancy and the
regulatory complexity. Recently, the variety of ele鄄
gant approaches elucidated the regulatory mode of
SPLs and miR156 at different stages of plant deve鄄
lopment. Their interplay provides the paradigms for
how these SPLs exert their functions in development.
For example, the low鄄level expression of SPLs in
miR156鄄overexpress mutant prolonged the juvenile
phase in both maize (Chuck et al., 2007) and Ara鄄
bidopsis (Wu and Poethig, 2006). Another case is
the validation of miR156鄄miR172 gene regulation
cascades regulated by SPL9 from Arabidopsis juve鄄
nile to adult phase transition. In this case, evidence
has been obtained for the direct regulation of miR172b
by SPL9, a miR156 target (Wu et al., 2009). Over
the past few years, many researchers have been wor鄄
king to reveal the functions of the miR156鄄regulated
developmental programs through analyzing the spatio鄄
temporal expression patterns of miR156 and its targets,
as well as characterizing the mutations in Arabidopsis
and maize. The regulatory functions of SPL transcrip鄄
tion factors in relation to miR156 were documented in
several reviews (Chen et al., 2010; Fornara and Coup鄄
land, 2009; Nonogaki, 2010; Poethig, 2010).
A large number of work has shown that miR156
families and their targeted SPLs are conserved through鄄
out land plants. With respect to miR156 families, the
evolutionary study has benefited from large鄄scale small
RNA sequencing / cloning project from many species,
in particular the most ancient land plants ( e. g.
moss). The comparision of the mature miR156 se鄄
quences showed that miR156 family was conserved be鄄
tween core eudicots and mosses, elaborating their pres鄄
ence in the earliest common ancestor of land plants. In
addition, the targeted SPLs for miR156 families are al鄄
so conserved in plants. For example, a conserved SBP
protein target of miR156 has been cloned from the moss
Physcomitrella patens and shown to be cleaved within
the predicted target site (Arazi et al., 2005). More
recently, Guo and his colleagues have demonstrated
that there is nearly a perfect conservation of the
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miR156 target site in SPLs for all land plants ana鄄
lyzed but not conserved in the unicellular green alga
Chlamydomonas reinhardtii (Guo et al., 2008).
Current evidences indicate that considerable di鄄
vergence of the functions of SPLs (including miR156鄄
targeted SPLs) exists in plants. For example, in
Arabidopsis SPL3, SPL4 and SPL5 appear to function
mostly in the control of flowering time and phase
change (Fornara and Coupland, 2009; Wu and Po鄄
ethig, 2006), whereas SPL9 and SPL15 have strong
effects on leaf initiation (Schwarz et al., 2008). In
addition, studies displayed SBP鄄box genes were di鄄
versified during evolution by analyzing gene struc鄄
tures, phylogeny, and motif elements (Guo et al.,
2008; Riese et al., 2007; Yang et al., 2008 ).
Take motif elements for example, some of them was
conserved between moss and seed plants ( Guo et
al., 2008; Riese et al., 2007), whereas others are
species鄄specific after the split of monocotyledon and
dicotyledon (Yang et al., 2008). Although the pre鄄
vious studies illustrated the diversity of SBP鄄box
gene family in plants during evolution, they did not
detail the evolutionary pathway of miR156鄄targeted
SPLs. For example, when such a large set of impor鄄
tant MREs has been established in the SPLs? Why
some of SBP鄄box genes are targeted by miR156,
while others were not. What are the differences be鄄
tween miR156鄄targeted SPLs and non鄄targeted SPLs
during evolution? These aforementioned questions in鄄
trigue us to glean about the evolutionary information
of targeted SPLs over long evolutionary timescales.
The survey of miR156鄄targeted SPLs occurrence in
plants and mapping this information onto the com鄄
prehensive plant phylogeny will update our know鄄
ledge on their origin and facilitate interpretation of
evolutionary pathway and function divergence among
distantly related plant species. In addition, the com鄄
plete sequencing of numerous plant species genome
(in particular, the green algae and moss) promotes
the comprehensive collection of information on the
SBP鄄box genes. Currently, two integrative transcrip鄄
tion factor libraries exploited are available online,
which documented SBP鄄box TF family and other TF
families in lower and higher plants (He et al., 2010;
Perez鄄Rodriguez et al., 2009). These resources allow
us to perform extensive phylogenetic analyses for
miR156鄄targeted SPLs and explore evolutionary histo鄄
ry based on their phylogenetic distribution.
Materials and methods
SPL sequences collection
The protein, domain and mRNA sequences of
SBP鄄box genes were downloaded from PlnTFDB v3. 0
( Riano鄄Pachon et al., 2007 ). The collected se鄄
quences included nine genome鄄sequenced species:
one alga ( Chlamydomonas reinhardtii), one moss
(Physcomitrella patens), one lycophyte (Selaginella
moellendorffii), three eudicots ( Arabidopsis thali鄄
ana, Populus trichocarpa and Vitis vinifera ) and
three monocots (Oryza sativa subsp. japonica, Zea
mays and Sorghum bicolor) ( Table 1 and Supple鄄
mentary Table 1). Sequence data for gene and CDS
( Coding Sequence ) were downloaded from DOE
Joint Genome Institute ( JGI) ( http: / / www. jgi.
doe. gov / ) and several species genome annotation
databases: The Arabidopsis Information Resource
(TAIR) 10 genome release (http: / / www. arabidop鄄
sis. org / ), TIGR rice genome annotation database
release 6. 1 (http: / / rice. plantbiology. msu. edu / ),
and maize sequence genome database release 5b. 60
( http: / / www. maizesequence. org / index. html ).
The transcript sequences of grape SBP鄄box genes
were conducted blast analysis to obtain their corre鄄
sponding gene sequences and CDS in Phytogome v6. 0
(http: / / www. phytozome. net) . Some obsolete locus
identifiers and new added SPLs uniformly adopted
the locus identifiers from JGI or species genome an鄄
notation database. A total of 183 SBP鄄box genes
were obtained and the complete catalog of them is a鄄
vailable in Supplementary Table 1, including the ob鄄
solete or new added genes.
Prediction of miR156 responsive elements within
SPL genes
Mature miR156 sequences of eight species (not
531 期摇 摇 摇 LING Li鄄Zhen et al. : Unraveling the Distribution and Evolution of miR156鄄targeted SPLs in Plants by …摇 摇 摇
found in green algae) were downloaded from the
miRBase database (Release 17. 0) (Kozomara and
Griffiths鄄Jones, 2011). They were used to predict
SPL targets by using miRU with default settings
(Zhang, 2005). To further increase the stringency
of predicted miR156 targets, we used empirical pa鄄
rameters as a second filter (Schwab et al., 2005).
These algorithms were designed to reflect molecular
target recognition mechanisms that are assumed to
apply to miRNA target recognition. The empirical
parameters used in this study were as follows: no
mismatch at positions 10 and 11; no more than one
mismatch at positions 2鄄12; no more than two con鄄
secutive mismatches downstream of position 13; the
total number of mismatch no more than 3 with minor
modification. By applying the above rules, our anal鄄
ysis led to the prediction of 61 SPL genes as the pu鄄
tative targets for miR156 family ( Table 1 and Ap鄄
pendix 1).
Sequence alignment and phylogenetic analysis
Protein and domain sequences from the above
nine species were initially aligned using CLUSTALX
(Thompson et al., 1997) and manually adjusted in
Se鄄Al software v2. 0 ( http: / / evolve. zoo. ac. uk)
whenever necessary. Only the SBP鄄box domains
were used for the phylogenetic analysis, because the
protein sequences showed no consensus sequences
when SBP鄄box domains were masked. We used
PHYLIP (v3. 6) (http: / / www. bioinformatics. uth鄄
scsa. edu / www / phylip / ) to construct the neighbor鄄
joining ( NJ) and maximum鄄likelihood (ML) tree
following Guo爷s method (Guo et al., 2008). Sup鄄
port values were assessed using 1000 replicate boot鄄
strap tests, only the clades with bootstrap value
higher than 50 were shown.
Intron / exon structure and sequence logo analysis
The CDS and genomic sequences of SPL genes
were used to derive intron / exon structure with Gene
Structure Display Server (GSDS, http: / / gsds. cbi.
pku. edu. cn / ) . The sequence logos were performed
using the WebLogo at the URL: http: / / weblogo.
berkeley. edu / logo. cgi.
Chromosomal distribution and duplication analysis
The location of SBP鄄box genes on chromosomes in
Arabidopsis was mapped by the Chromosome Map Tool
at TAIR ( http:/ / arabidopsis. org / jsp / Chromosome鄄
Map / tool. jsp webcite). Gene duplications and their
presence on duplicated chromosomal segments were in鄄
vestigated using “Paralogous in Arabidopsis thaliana冶
with the default parameters set (Blanc et al., 2003;
Vision et al., 2000; Wang et al., 2008). Only the
blocks containing SBP鄄box genes were retained, and
then genes detected were mapped on the chromo鄄
somes and linked to each other by lines manually.
Results and discussion
MREs are specific to SPLs of land plant lineages
A total of 183 SPL genes were obtained from
nine species, which represented the main lineages of
the green plants: green alga (C. reinhardtii), moss
(P. patens), lycophyte ( S. moellendorffii), mono鄄
cots (rice, sorghum and maize) and eudicots (Ara鄄
bidopsis, grape and poplar) (Table 1 and Appendix
1). These genomes have been fully sequenced and
all the putative members of the SBP鄄box gene family
have been identified according to their domain struc鄄
ture (Perez鄄Rodriguez et al., 2009). For example,
the SPLs from green algae had reached to 23, which
was 3 times more than those of the previous report
(Guo et al., 2008) when the genome sequence has
not been released (Table 1). Therefore, these spe鄄
cies open the possibility for a comprehensive analysis
of MREs within SPL genes. High鄄confidence prediction
Table 1摇 The number of SBP鄄box genes in nine representative plants
Lineage Organism SPLs Targeted SPLs
Alga Chlamydomonas reinhardtii 23 0
Moss Physcomitrella patens 14 5
Lycophyte Selaginella moellendorffii 11 0
Oryza sativa 19 12
Monocots Sorghum bicolor 19 9
Zea mays 33 13
Arabidopsis thaliana 17 11
Eudicots Populus trichocarpa 29 6
Vitis vinifera 18 5
Total 183 61
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of miR156 targets were performed by miRU based on
sequence complementarity and evolution conservation
(Zhang, 2005). To further increase the stringency of
predicted miR156 targets, we used empirical parame鄄
ters as a second filter (Schwab et al., 2005). These
parameters considered more algorithm features in鄄
stead only sequence complementarity and conserva鄄
tion (see Materials and Methods in detail) . Finally,
61 out of 183 SPL genes were the putative target
genes for miR156 family with high probability (Ta鄄
ble 1 and Appendix 1). We roughly estimated the
accuracy of putative targets by seeking confirmations
for experimental data. For example, all the putative
targets in Arabidopsis and rice have been experimen鄄
tally validated by several independent laboratories
(Li et al., 2010; Xie et al., 2006; Xing et al.,
2010), indicating that our prediction of conserved
miR156 targets was highly accurate. The predicted
results showed that MREs were not found in green
algae and Selaginella moellendorffii, while they were
observed in other seven land plants. With respect to
green algae, we noticed that no miR156 homologous
have been identified after publishing its genome se鄄
quence (Worden et al., 2009). To further affirm
our prediction, we used the members of miR156
from all other land plants to predict MREs within
SPLs of green algae. The result indicated that there
were still no MREs found in SPLs of green algae.
Meanwhile, previous studies indicated that no uni鄄
versal miRNA regulatory pathways ( including
miR156鄄regulatory pathway ) were existed among
land plants and green algae (Guo et al., 2008; Mol鄄
nar et al., 2007). Therefore, we can conclude that
the miR156 targets were indeed not appeared in uni鄄
cellular green algae. On the contrary, the miR156
homologous and SPL genes were indentified in Sela鄄
ginella moellendorffii, but the MREs were not pre鄄
dicted in our analysis. One possible explanation is
the interactive sites miR156 and SPLs had more than
four mismatches and did not serve as MREs by using
prediction criteria in this study ( data not shown).
All together, these above analyses concluded that
miR156鄄regulatory pathway had arisen after the di鄄
vergence multicellular land plants and unicellular
green algae.
Phylogenetic distribution of miR156 targeted鄄SPLs
in land plants
To understand the evolution history of these tar鄄
geted SPLs, we constructed an unrooted neighbor鄄
joining ( NJ) tree for all the SPLs of land plants
(Fig. 1). In addition, we obtained another tree with
similar topology using maximum鄄likelihood ( ML)
method (data not shown). As shown in Fig. 1, all
SBP鄄box gene sequences of land plants were resolved
into two major clades (group I and group II) . Ten
SPL genes with the SBP鄄domain of four Cys residues
from moss, lycophyte and several flowering plants
formed group I ( Fig. 1 and Fig. 2: A). A large
number of SPLs from each land plant lineage were
clustered into group II, where they were further di鄄
vided into seven subgroups ( IIa鄄IIg). The group II
had the SBP鄄domain with a Cys3 His motif, which
was different from group I but same to CR group
(Fig. 2: B, C). Based on the phylogenetic data,
we speculated that the last common ancestor of land
plants had at least two classes of SBP鄄box genes. In鄄
spection of miR156 targets displayed distribution in
group II but not in group I (Fig. 1), indicating that
they originated from a common ancestor and had
arisen after the divergence of group I and group II.
Similarly, an uneven distribution of the miR156
targets on different subgroups of group II was also
apparent ( Fig. 1 and Table 2). At first, with the
exception of subgroup IId, targeted SPLs were wide鄄
ly existed in remaining six subgroups. It was obvious
that these targeted SPLs restrictedly distributed in
some members of six branches (Fig. 1). More strik鄄
ingly, we found a lineage鄄specific distribution pat鄄
tern of targeted SPLs. For example, two targeted
SPLs in subgroup IIe were both from moss, whereas
subgroup IIa, IIb, IIf only contained angiosperm tar鄄
geted SPLs (Fig. 1 and Table 2). A similar distri鄄
bution pattern of miR172 binding sites, a down鄄
stream regulatory factor of miR156 targets, that were
731 期摇 摇 摇 LING Li鄄Zhen et al. : Unraveling the Distribution and Evolution of miR156鄄targeted SPLs in Plants by …摇 摇 摇
only restricted to some member sequences of the eu鄄
AP2 group of the AP2鄄like family was also reported
(Kim et al., 2006). The restricted distribution of
miR156 targets suggested their involvement in targe鄄
ting of selected SBP鄄box genes in distinct lineages
and therefore in the regulation of particular functions
of lineage鄄specific characters. Secondly, the abun鄄
dance of miR156鄄targeted SPLs in angiosperm linea鄄
ges was largely different on evolutionary timescale. If
angiosperm SPLs clustered with lower plant SPLs (e.
g. moss SPLs), suggesting that these angiosperm
SPLs were early evolved and vice verse. Among seven
subgroups, angiosperm SPLs and lower plants SPLs
shared four subgroups (IIc, IId, IIe and IIf), where
20 out of 70 SPLs were targeted by miR156. Howev鄄
er, the remaining three subgroups (IIa, IIb and IIg)
only possessed angiosperm SPL genes, where 35 tar鄄
geted SPLs were detected among 55 SPLs (Table 2).
By comparing the abundance of targeted SPLs in angi鄄
osperms across subgroups over different evolutionary
timescales, we concluded that targeted SPLs of angio鄄
sperms mainly increased after angiosperms and lower
plants split. More importantly, we found a majority
of targeted SPLs enriched in gene pairs among diffe鄄
rent angiosperm lineages of group II. Therefore, it
implied that gene duplication lead to the increasing
of miR156 targets in angiosperms after the diver鄄
gence of angiosperms from lower plants.
Fig. 1摇 Phylogenetic tree of SBP鄄box genes across different species. SBP鄄box domain sequences of nine plant species were analyzed;
an unrooted tree was constructed using Neighbour鄄Joining ( NJ) method, bootstrap 1000 replicates. : green algae, :
moss, : lycophyte, : monocots, : eudicots. Note: The digits inside the branches indicate the support values and
those outside the branches indicate the number of miR156鄄targeted SPLs (see Appendix 1 in detail)
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Fig. 2摇 Sequence logos of the SBP鄄box domain. The sequence logos of SBP鄄box domain of group I (A), group II (B),
CR group (C). The overall height of letter at each position represents the degree conservation. The two
conserved zinc finger structures and NLS are indicated in three different groups
Table 2摇 The distribution of miR156鄄targeted SPLs
on each subgroup of group II
Subgroup No. of SPLs Lower plantsa Higher plantsb
Subgroup IIa 22 0 (0) 22 (15)
Subgroup IIb 11 0 (0) 11 (11)
Subgroup IIc 16 7 (3) 9 (2)
Subgroup IId 27 4 (0) 23 (0)
Subgroup IIe 24 10 (2) 14 (0)
Subgroup IIf 25 1 (0) 24 (18)
Subgroup IIg 25 0 (0) 25 (10)
Total 150 23 (5) 128 (56)
a and b The digit in bracket indicates the number of miR156鄄targeted
SPLs in lower plants and higher plants, respectively
Gene structure analysis of miR156鄄targeted SPLs
The phylogenetic distribution patterns of miR156
targets could shed light on the evolutionary pathway
that shaped their history. To investigate this possi鄄
bility, we analyzed and compared the gene structure
between the targeted SPLs and non鄄targeted SPLs be鄄
cause gene structure is an important indicator to
classify the different genes. As such, we reconstruc鄄
ted an NJ tree based on 36 SBP鄄box proteins from
eudicots (Arabidopsis) and monocots (Oryza sativa)
and carried out intron / exon structure analysis (Fig.
3: A). It is notable that the intron / exon structure
correlated with the classification of SPL genes based
on the phylogenetic analysis. For example, SPL
genes in subgroup IId and group I had 11 and 10
exons respectively, while all SPL genes of subgroup
IIa and IIf had four and three exons respectively
(Fig. 3: B). The apparent correlation between in鄄
tron / exon structures and the classes of SPL genes
was probably due to the expansion of SPLs in each
clade by ancient and recent duplication events. The
alternative possibility was that the SPL genes intron /
exon structures could have certain level of stability at
the late stages of evolution of angiosperms. There鄄
fore, this good correlation between phylogenetic rela鄄
tionship and gene structure was contributed to under鄄
standing the evolution of gene structure of targeted
SPLs and interpreting their distribution patterns.
931 期摇 摇 摇 LING Li鄄Zhen et al. : Unraveling the Distribution and Evolution of miR156鄄targeted SPLs in Plants by …摇 摇 摇
Fig. 3摇 Intron / exon structure in conjunction with phylogenetic tree for the Arabidopsis and rice SBP鄄box proteins and the structure of targeted
SPLs in moss. (A) Schematic diagram of phylogenetic tree reconstructed from a complete alignment of 17 Arabidopsis and 19 rice SBP鄄box pro鄄
teins. (B) Intron / exon structures of SBP鄄box genes of Arabidopsis and rice. (C) Intron / exon structures of targeted SPLs in moss. The genes
marked the star in the phylogenetic tree were regulated by miR156. As shown in the legend, blank boxes stand for CDS, horizontal lines stand
for the introns, black boxes are UTR regions, and vertical bars indicate the position of MREs. The phylogenetic relationships of groups and sub鄄
groups were presented in Figure 1. Four SBP鄄box proteins not assigned the clades corresponding to the domain tree were bold and italic
04摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 植 物 分 类 与 资 源 学 报摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 第 34 卷
摇 摇 As shown in Fig. 3B, the coding sequences of
SBP鄄box genes were interrupted by a variable num鄄
ber of exons ranging from two to eleven. The targe鄄
ted SPL genes from different subgroups had similar
structure features with two to four exons. In addi鄄
tion, all the targets belonged to a monophyletic
clade. By contrast, non鄄targeted SPLs were the most
divergent in gene structures and could be classified
into two classes according to the phylogenetic posi鄄
tion. One class of non鄄targeted SPLs lied outside of
the monophyletic clade that the targeted SPLs be鄄
longed to, such as the genes from subgroup IId.
This class of non鄄targeted SPLs contained more ex鄄
ons (at least ten exons) than the targeted SPLs but
was similar to SPL genes of group I. Therefore, we
speculate that the targeted SPLs might suffer from
exon loss events during evolution. Furthermore,
moss, an early鄄branching species of land plants,
could provide a window into the early evolution of
targeted SPLs in land plants. Fig. 3C shows that a
portion of targeted SPLs in moss possessed the
ancient gene structure with exons ranging from 6 to
13, such as targeted SPLs 69445, 93998 and 168927.
These exons of miR156鄄targeted SPLs might be lost
at different dimensions. At first, previous studies
suggested that SBP鄄domains lied in the first two ex鄄
ons and possessed the conserved intron position
(Guo et al., 2008; Xie et al., 2006). The authors
found that part of moss SBP鄄box genes had some ex鄄
ons at the upstream of SBP鄄domain, providing the
evidence of exons loss from 5爷鄄end flanking of SBP鄄
domain. In our study, we found the MREs within
the above three targeted SPLs in moss lied in exon
regions excluding the last ones. By contrast, most
MREs were located in the last exon and some of
them began to reside in 3爷 UTR regions. These re鄄
sults indicated the exon of targeted SPLs might also
be lost from the 3爷鄄end regions. A mechanistic ex鄄
planation for these scenarios suggested that the exons
might be lost from the 3爷鄄portion of SPLs because of
homologous recombination of their cDNAs ( Derr,
1998; Mourier and Jeffares, 2003).
The second class of non鄄targeted SPLs ( e. g.
the genes from subgroup IIe) had the similar gene
structure to targeted SPLs and also possessed no
more than four exons ( Fig. 3: A, B). However,
they embedded within the same monophyletic clade
as targeted SPLs. One impossible explanation was
that these non鄄targeted SPLs might be originally tar鄄
geted by miR156 followed by the loss of miR156
binding sites. To test this hypothesis, we further an鄄
alyzed the phylogenetic relationship across targeted
SPLs because the paraphyly of miRNA targets on the
phylogenetic tree may account for MRE loss. In鄄
deed, five targeted SPL genes (e. g. LOC_Os08g39890
and LOC_Os09g31438 from subgroup IIb) and one
target gene (LOC_Os04g46580) from subgroup IIg
formed paraphyletic branch (Fig. 3: A). However,
LOC_Os04g46580 and non鄄targeted SPL genes (e.
g. LOC _Os02g08070 and LOC _ Os04g56170 ) in
subgroup IIe clustered each other in a branch. Such
distribution pattern of MREs suggested a loss of
miR156 targeting or alternatively a gain of miR156
targeting in closely related genes. This could be evi鄄
dence of loss of a MRE after duplication event, be鄄
cause the latter scenario was less likely unless
recombinational events or gene conversion events
were involved. Overall, these analyses revealed tar鄄
geted SPLs mainly experienced the exon loss events
following by some MREs loss during evolution.
Gene duplication of miR156鄄targeted SPLs in
Arabidopsis
Apart from the relatedness of gene structure,
gene duplication was also an important factor to in鄄
fluence the distribution pattern of targeted SPLs. As
shown in Fig. 3A, more than an half of SBP鄄box
genes constituted gene pairs, such as 12 paralogous
gene pairs and 2 orthologous gene pairs indentified
based on protein analysis. We observed the paralo鄄
gous gene pairs in each lineage were mainly regula鄄
ted by miR156. For example, 5 out of 7 paralogous
gene pairs were miR156 targets in Arabidopsis. This
result suggested that the duplication events in re鄄
spective lineage were the main resource of targeted
141 期摇 摇 摇 LING Li鄄Zhen et al. : Unraveling the Distribution and Evolution of miR156鄄targeted SPLs in Plants by …摇 摇 摇
SPLs and influenced the abundance of them on phy鄄
logenetic tree. Therefore, it is important to study the
duplication mechanisms to interpret the distribution
pattern. In our study, we focused on Arabidopsis.
This species genome has undergone at least two
large鄄scale segmental duplication events, which had
great impact on amplification of members of a gene
family ( including targeted SPLs) in the genome.
One was the recent polyploidy duplication, which
occurred before Arabidopsis and Brassica rapa split
about 24-40 Mya. The other was an older duplica鄄
tion between chromosomal blocks after the diver鄄
gence of monocot鄄eudicot around 120 Mya (Blanc et
al., 2003; Bowers et al., 2003; Vision et al., 2000).
Considering these factors, we investigated SBP鄄box
family gene duplication and distribution on all five
Arabidopsis chromosomes. The recent segmental
polyploidy duplicated blocks were explored by the
“Paralogons in Arabidopsis thaliana冶 search engine
(Wang et al., 2008).
Fig. 4 showed that there were three pairs of re鄄
cent duplicated blocks containing SBP鄄box genes.
Both regions on chromosome 1 containing AT1G20980
and AT1G76580 were duplicated segmental block
pairs. The region containing AT1G53160 on chro鄄
mosome 1 and the region containing AT3G15270 on
chromosome 3 were duplicated segmental block
pairs. The regions on chromosome 2 and on chromo鄄
some 3 comprised two duplicated segmental block
pairs, such as AT2G42200 and AT3G57920, AT2G
47070 and AT3G60030. Among four segmental
pairs, there were two duplicated gene pairs targeted
by miR156. All of these segmentally duplicated
genes were also found to be paralogous in the phylo鄄
genetic analysis as shown in Fig. 1. The results indi鄄
cated that segmental duplication was a major way for
SBP鄄box gene birth ( in particular the targeted
SPLs) for Arabidopsis.
Fig. 4摇 Chromosomal distribution and duplication events for Arabidopsis SBP鄄box genes. Diagram of five chromosomes
of Arabidopsis was depicted, 17 SBP鄄box family genes were distributed on these chromosomes. Only the duplicated
regions containing SBP鄄box genes are shown. Black lines connect corresponding sister gene pairs in duplicated blocks
(Blank boxes) . AT1G27360 and AT1G27370, AT5G50570 and AT1G50670 are clustered as tandem repeats
24摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 植 物 分 类 与 资 源 学 报摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 第 34 卷
摇 Besides, two tandem duplication events were
also found on chromosome 1 and 5, respectively.
AT1G27360 and AT1G27370 were two genes with
high similarity of DNA sequence and only 1 kb dis鄄
tance on the chromosome 1. The other two genes,
AT5G50570 and AT1G50670 had almost consensus
similarity, although they depart from about 31 kb
distance. The two gene pairs were targeted by miR156.
All together, large鄄scale segmental duplication and
tandem duplication events in Arabidopsis increased
the abundance of targeted SPLs and appeared to
have exclusively contributed to the current comple鄄
xes of the targeted SPLs and their gene family.
Acknowledgements: The authors kindly thank Dr. Yuxiao
Zhang ( Kunming Institute of Botany, Chinese Academy of
Sciences) for revising the manuscript. The authors also thank
Professor Aizhong Liu ( Xishuangbanna Tropical Botanical
Garden, Chinese Academy of Sciences) for his constructive
advices.
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Appendix 1摇 The catalog of 183 SBP鄄box genes in nine species
No. Species Locus ID Target siteposition No. Species Locus ID
Target site
position
1 Arabidopsis thaliana AT2G33810 3爷 UTR 14 AT2G47070
2 (Arabidopsis) AT3G15270 3爷 UTR 15 AT3G60030
3 AT1G27360 CDS 16 AT5G18830
4 AT1G27370 CDS 17 AT1G76580
5 AT1G53160 CDS 18 Chlaymydomonas reinhardtii 93505
6 AT1G69170 CDS 19 (green algae) 96716
7 AT2G42200 CDS 20 101247
8 AT3G57920 CDS 21 101657
9 AT5G43270 CDS 22 105679
10 AT5G50570 CDS 23 106739
11 AT5G50670 CDS 24 108149
12 AT1G02065 25 108444
13 AT1G20980 26 115124
44摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 植 物 分 类 与 资 源 学 报摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 第 34 卷
Continued
No. Species Locus ID Target siteposition No. Species Locus ID
Target site
position
27 115254 72 90199
28 118761 73 97909
29 120852 74 Populus trichocarpa 743829 3爷 UTR
30 121606 75 (Poplar) 570289 CDS
31 121939 76 576281 CDS
32 170753 77 733659 CDS
33 171833 78 755123 CDS
34 186869 79 769914 CDS
35 195928 80 179090
36 288620 81 179183
37 290479 82 197948
38 291579 83 216243
39 405089 84 226094
40 414856 85 235814
41 Oryza sativa subsp. japonica LOC_Os04g46580 3爷UTR 86 245406
42 (rice) LOC_Os01g69830 CDS 87 263406
43 LOC_Os02g04680 CDS 88 267542
44 LOC_Os02g07780 CDS 89 274234
45 LOC_Os06g45310 CDS 90 286316
46 LOC_Os06g49010 CDS 91 286321
47 LOC_Os07g32170 CDS 92 298307
48 LOC_Os08g39890 CDS 93 409154
49 LOC_Os08g41940 CDS 94 412443
50 LOC_Os09g31438 CDS 95 415293
51 LOC_Os09g32944 CDS 96 560022
52 LOC_Os11g30370 CDS 97 647067
53 LOC_Os01g18850 98 656549
54 LOC_Os02g08070 99 656553
55 LOC_Os03g61760 100 798319
56 LOC_Os04g56170 101 832886
57 LOC_Os05g33810 102 833398
58 LOC_Os06g44860 103 Sorghum bicolor 4160487 CDS
59 LOC_Os08g40260 104 (sorghum) 4160700 CDS
60 Physcomitrella patens 69445 CDS 105 4748489 CDS
61 (moss) 74968 CDS 106 5003160 CDS
62 93998 CDS 107 5003651 CDS
63 168927 CDS 108 5042307 CDS
64 168928 CDS 109 5054656 CDS
65 8925 110 5059026 CDS
66 19787 111 5062217 CDS
67 19788 112 4112095
68 29422 113 4163165
69 29851 114 4785561
70 74970 115 4814910
71 83876 116 4862557
541 期摇 摇 摇 LING Li鄄Zhen et al. : Unraveling the Distribution and Evolution of miR156鄄targeted SPLs in Plants by …摇 摇 摇
Continued
No. Species Locus ID Target siteposition No. Species Locus ID
Target site
position
117 4974192 151 Zea mays GRMZM2G163813 3爷 UTR
118 4985600 152 (Maize) GRMZM2G040785 CDS
119 5047795 153 GRMZM2G061734 CDS
120 5059084 154 GRMZM2G065451 CDS
121 5060486 155 GRMZM2G097275 CDS
122 Selaginella moellendorffii 17777 156 GRMZM2G101511 CDS
123 (Lycophyte) 28598 157 GRMZM2G126018 CDS
124 28626 158 GRMZM2G148467 CDS
125 28629 159 GRMZM2G307588 CDS
126 28630 160 GRMZM2G390470 CDS
127 28635 161 GRMZM2G414805 CDS
128 49859 162 GRMZM2G450128 CDS
129 59543 163 GRMZM2G460544 CDS
130 59991 164 GRMZM2G024760
131 79699 165 GRMZM2G036297
132 437670 166 GRMZM2G058588
133 Vitis vinifera GSVIVT00002776001 CDS 167 GRMZM2G067624
134 (grape) GSVIVT00017032001 CDS 168 GRMZM2G080065
135 GSVIVT00017953001 CDS 169 GRMZM2G081127
136 GSVIVT00019157001 CDS 170 GRMZM2G098557
137 GSVIVT00025360001 CDS 171 GRMZM2G101499
138 GSVIVT00002800001 172 GRMZM2G102758
139 GSVIVT00002959001 173 GRMZM2G106798
140 GSVIVT00003071001 174 GRMZM2G109354
141 GSVIVT00004625001 175 GRMZM2G113779
142 GSVIVT00008511001 176 GRMZM2G126827
143 GSVIVT00018616001 177 GRMZM2G133279
144 GSVIVT00019158001 178 GRMZM2G133646
145 GSVIVT00019711001 179 GRMZM2G138421
146 GSVIVT00019851001 180 GRMZM2G156621
147 GSVIVT00027720001 181 GRMZM2G156756
148 GSVIVT00028195001 182 GRMZM2G168229
149 GSVIVT00030009001 183 GRMZM2G169270
150 GSVIVT00037879001
Data resource: Green alge, Moss, Lycophyte, Poplar and Sorghum (http: / / www. jgi. doe. gov / genome鄄projects / , the release version is 4. 0, 1. 1,
1. 0 and 1. 0 for the first four species, respectively); Grape (http: / / www. phytozome. net, v6. 0); Arabidopsis (http: / / www. arabidopsis. org / ,
release 10); Rice (http: / / rice. plantbiology. msu. edu / , v6. 1); Maize ( http: / / www. maizesequence. org / index. html, release 5b. 60) . The
gene sequences and CDS of SBP鄄box genes of each species were downloaded from the above databases. All the transcripts, protein and domain se鄄
quences were downloaded from PlnTFDB (v3. 0) (http: / / plntfdb. bio. uni鄄potsdam. de / v3. 0 / ) .
Noting: the 10 obsolete locus identifiers of SPL genes: GRMZM2G006850, GRMZM2G015007, GRMZM2G020881, GRMZM2G075639,
GRMZM2G090058, GRMZM2G108162, GRMZM2G114243, GRMZM2G145615, GRMZM2G154844 and GRMZM2G160932. Another 6 new
added SPL genes: GRMZM2G307588, GRMZM2G390470, GRMZM2G414805, GRMZM2G450128, GRMZM2G460544 and AT1G76580. All the
altered genes were from maize except for AT1G76580 (from Arabidopsis) .
64摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 植 物 分 类 与 资 源 学 报摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 摇 第 34 卷