Download A Genome-Wide Survey of the NAC Transcription

A Genome-Wide Survey of the NAC
Transcription Factor Family in
Monocots and Eudicots
Mohammed Nuruzzaman
S National Institute of Agrobiological Sciences
Saitama University, Japan
Akhter Most Sharoni, Kouji Satoh, Hiroaki Kondoh, Aeni Hosaka, Shoshi Kikuchi
Plant Genome Research Unit, Agrogenomics Research Center
National Institute of Agrobiological Sciences (NIAS), Japan
1
Introduction
Transcription factors are proteins that modulate gene expression by binding to specific cis-acting promoter elements, thereby activating or repressing the transcription of target genes (Wray et al., 2003).
Gene specific transcription factors are DNA-binding regulatory proteins that activate or repress the basal
transcription apparatus at target gene promoters. Transcription factors are grouped into families based on
the sequence of their DNA-binding domains (DBD) (Luscombe & Thornton, 2002).
The NAC genes constitute one of the largest families of plant-specific transcription factors and are
present in a wide range of land plants. The NAC acronym was derived from the first three genes that
were discovered to contain NAC domains, namely NAM (No Apical Meristem) from petunia (Petunia
hybrida), ATAF1-2, and CUC2 (Cup-Shaped Cotyledon) from Arabidopsis. NAC proteins commonly
possess a conserved NAC domain at the N-terminus, which consists of approximately 150 to 160 amino
acid residues and is divided into five sub-domains (Ooka et al., 2003). The function of the NAC domain
has been associated with nuclear localization, DNA binding, and the formation of homodimers or heterodimers with other proteins that contain NAC domains (Olsen et al., 2005). The C-terminal regions of
NAC proteins are highly divergent (Ooka et al., 2003) and are responsible for the observed regulatory
differences between transcriptional activation activities of NAC proteins (Xie et al., 2000; Yamaguchi et
al., 2008; Jensen et al., 2010). The divergent C-terminal regions of these proteins generally operate as
functional domains and act as transcriptional activators or repressors (Tran et al., 2004; Hu et al., 2006;
Kim et al., 2007a). The C-terminal regions are large and have protein-binding activity. C-terminal NAC
domains bind calmodulin proteins in Arabidopsis (Kim et al., 2007a), which suggests that more complex
mechanisms exist for transcriptional regulation by NAC proteins. Most studies have found that NAC proteins function as transcriptional activators of related gene expression. AtNAM, ATAF1, AtNAC2, and
AtNAC3 were reported to be transcriptional activators in a yeast assay system (He et al., 2005).
ANAC019, ANAC055, and ANAC072 (RD26) were characterized as transcriptional activators in a plant
protoplast assay system (Tran et al., 2004). NAC1 showed a modest level of activation capacity in both
yeast and protoplasts (Xie et al., 2000).
Genes in the NAC family have been shown to regulate a wide range of developmental processes including seed development (Sperotto et al., 2009), embryo development (Duval et al., 2002), shoot apical
meristem (Kim et al., 2007b), fiber development (Ko et al., 2007), leaf senescence (Guo et al., 2005a),
and cell division (Kim et al., 2006). Additionally, AtNAC1 gene expression is induced by lateral root development, which in turn is regulated by the hormone auxin (Xie et al., 2000). Many proteins in the NAC
family were identified and found to be involved in many diverse functions, such as hormonal signal
transduction (Greve et al., 2003) and the developmental processes (Peng et al., 2009) of various plant
species.
Several NAC proteins have been identified because they interact with other proteins of biological
importance during defense and response to biotic stress. Rice with mutant rim1-1 is resistant to infection
by dwarf virus (Yoshii et al., 2009; Satoh et al., 2011). RIM1 is a negative regulator that suppresses the
signals induced by viral infection, which enhanced the defense system mediated by Jasmonic acid (Yoshii et al., 2010). The StNAC (Solanum tuberosum) gene is induced in response to Phytophthora infestans
infection (Collinge & Boller, 2001). Furthermore, numerous NAC proteins are involved in the response
of plants to abiotic stresses, such as drought, salinity, cold, and submergence. For example, AtNAC072
(RD29), AtNAC019, AtNAC055, and ANAC102 from Arabidopsis (Fujita et al., 2004; Tran et al., 2004;
Christianson et al., 2009), BnNAC from Brassica napus (Hegedus et al., 2003), and stress NAC (SNAC)
such as SNAC1, SNAC2/OsNAC6, OsNAC5, and OsNAC10 from rice (Hu et al., 2006, 2008; Nakashima
et al., 2007; Sperotto et al., 2009; Zheng et al., 2009; Jeong et al., 2010) are involved in the responses to
various environmental stresses.
In rice genome, there are 141 genes in the NAC family (Fang et al., 2008) and later Nuruzzaman et
al. (2010) reported 151 and 117 NAC genes in rice and Arabidopsis, respectively. To date, a few of these
genes have been characterized but most of the NAC family members have yet to be studied, despite the
likelihood that these genes play important roles in the physiology of plants. Substantial experimental
work will be required to determine the specific biological function of each NAC gene. Through phylogenetic analysis, it has become apparent that this large family of transcription factors consists of subgroups
that are closely related to each other (Kranz et al., 1998; Reyes et al., 2004; Tian et al., 2004). A functional analysis of each transcription factor in the NAC family should be performed, taking into account
functional redundancy. As part of this process, an assessment of the structural relationships between all
Arabidopsis NAC family proteins would serve as a guide for predicting the function of proteins that have
yet to be studied. Moreover, the current availability of the rice genome sequence also allows a comparative analysis of the NAC family between rice and Arabidopsis, which is useful for studying the functional
and evolutional diversity of the transcription factor in plants. Although Ooka et al. (2003) first described
a comprehensive analysis of the NAC family between rice and Arabidopsis, and they studied only 75 and
105 NAC proteins in rice and Arabidopsis, respectively.
In the present study, we provided genome-wide survey of the classification, conserved motif outside
of the NAC domain motif among the three species. We first characterized motifs outside of the NAC
domain in rice and then compared with those of Arabidopsis, which are widespread among the NAC proteins as well as motifs specific to only in a few groups. We compared potential gene birth-and-death
events between the rice and Arabidopsis NAC genes. We first indentified the NAC genes from rice, Arabidopsis, and citrus, and then conducted phylogenetic analyses to divide them into groups. Moreover,
Ka/Ks ratio was used to explore the mechanisms of the rice NAC genes and 3 evolutionary stages were
divergence after their duplication. For the synteny analysis, 352 NAC genes were used from monocots
and eudicots. The resulting classification, motifs, evolution and divergence patterns, and Ka/Ks and
synteny analysis will be useful for future studies of the biological functions of the NAC family and other
transcription factors.
2
Materials and Methods
2.1
Database Searches
Multiple database searches were performed to collect all members of the NAC family in rice, Arabidopsis, and citrus. First, the conserved NAM DNA-binding domains of known NAC proteins
(Os11g03300/OsNAC10, Jeong et al., 2010) were used to search (BLASTP program) against the predicted protein database of rice genome (Michigan State University, MSU; http://rice.plantbiology.msu.edu/),
in National Centre for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/), and in the ensembl plants (http://plants.ensembl.org/index.html). The protein sequences satisfied E≤1e-10 was selected as the candidate proteins, and amino acid sequences whose lengths <100 were excluded for the following analysis. Second, the HMM profile of the NAM domain in the Pfam database (http://pfam
.sanger.ac.uk/; Finn et al., 2010) was used to search the annotated rice protein database. The nonredundant sequences resulted from these two methods were then compared with the NAC family in the
rice transcription factor databases (RiceTFDB, http://ricetfdb.bio.uni-potsdam.de/v2.1/; Riano-Pachon et
al., 2007) and the previously reported annotation of this family (Ooka et al., 2003; Fang et al., 2008;
Nuruzzaman et al., 2010). All of the non-redundant putative NAC protein sequences were manually
checked for the NAM domain. The HMM profile of the NAM domain was reconstructed with the HMM
build program in the HMMER (Eddy, 1998) (HMMER version 2.1). Similarly, we used the BLAST programs (BLASTP) available in The Arabidopsis Information Resource (TAIR) Arabidopsis databases and
the NCBI Arabidopsis genome database search for members of the Arabidopsis NAC gene family.
Physcomitrella patens NAC genes and Selaginella NAC genes were surveyed based on homology with
protein sequences of the NAC domain at the NCBI web site.
2.2
Alignment, Phylogenetic Analysis, and Motif Detection
Multiple alignments of the NAC protein sequences were performed with CLUSTALW (Thompson et al.,
1994; http://www.genome.jp/tools/clustalw/). A phylogenetic tree was constructed with the aligned NAC
protein sequences using MEGA (version 4.0; http://www.megasoftware.net; Tamura et al., 2007) and the
Neibhbor-Joining (NJ) method with the following parameters: Poisson correction, pairwise deletion, and
bootstrap (1000 replicates). The amino acid variation rates were also obtained. Motif detection was performed using MEME (Bailey et al., 2006) (MEME version 4.8.1, http://meme.sdsc.edu/meme/
meme.html) with default settings, maximum number of motifs to find was set at 15, and motif length at
6-100.
2.3
Gene Locations on Chromosomes and Duplications
To determine the location of AtNAC genes on five chromosomes, the Chromosome Map Tool
(http://www.arabidopsis.org/jsp/ChromosomeMap/tool.jsp) at TAIR was used. Gene duplications and
their presence on duplicated chromosomal segments were investigated using ‘‘Paralogons in Arabidopsis’’ (http://wolfe.gen.tcd.ie/athal/dup) with the default parameters set to a minimum threshold for paired
proteins. OsNAC was located on rice chromosomes according to the positions specified in the
http://rice.plantbiology.msu.edu (Ouyang et al., 2007). We identified genome duplications of rice in the
http://rice.plantbiology.msu.edu with a maximum permitted distance between collinear gene pairs of 500kb (http://rice.plantbiology.msu.edu/semental_dup/index.shtml). We considered genes to be tandemly
duplicated if two OsNAC genes were separated by three or fewer gene loci according to the
http://rice.plantbiology.msu.edu. These genes were not included previously (Nuruzzaman et al., 2010).
2.4
Synteny Analysis and Ka/Ks Computing
Syntenic information from all examined genes was collected from the Plant Genome Duplication Database (PGDD, http://chibba.agtec.uga.edu/duplication) and using the Codeml procedure of the PAML
program (Suyama et al., 2006) the rate of non-synonymus substitution (Ka), the rate of synonymous substitutions (Ks) and Ka/Ks were determined. The dates of the duplication events were calculated by the
equation T=Ks/2λ, for rice, the λ= 6.5×10−9 (Yang et al., 2008).
3
Results
3.1
Phylogenetic Relationships between Members of the NAC Family in Rice and Arabidopsis
To confirm classifications and to analyse the phylogenetic relationships, multiple alignment analyses of
the amino acid sequences of the NAC domain in the 135 rice NAC proteins and 116 Arabidopsis NAC
proteins were performed (Supplementary Figure 1). Residues Leu19 was completely conserved among
all 251 proteins in both species (Supplementary Figure 1). In addition, >90% of the NAC family members contain Glu13 and Trp47 residues. Based on alignment, an NJ phylogenetic tree was generated with
bootstrap analysis (1000 replicates). As shown in Supplementary Figure 2, the phylogenetic tree divided
the NAC family proteins of rice and Arabidopsis into 18 groups, designated groups I to XVI (Supplementary Figure 3), in accordance with the classification described by Nuruzzaman et al. (2010). For example, some groups [e.g., I (ONAC2) and XVII] are species-specific in the present study. Comparative
analyses of the phylogenetic tree suggested that the classification of the rice NAC family was almost similar and applicable to the Arabidopsis NAC family.
3.2
Conserved motifs outside of the NAC domain in rice and Arabidopsis
First, the putative conserved motifs in NAC family proteins in rice (Supplementary Tables 1, 2) and both
rice and Arabidopsis were investigated using MEME version 4.8.1 (Supplementary Table 3). We select a
motif which is present at least in the three NAC genes. Most members in the same group shared one or
more motifs outside the NAC domain (Figure 1).
For example, the VIII (SND) group consisted of ten unigenes (e.g., Os04g59470 and Os08g01330)
and contained four conserved motifs (Figure 1). All of the unigenes in this group contain the CMVIII-1
motif in the C-terminal region; this was reported as a WVVCR conserved motif; the CMVIII-1 motif is
similar to the CMV-1 from group V (NAM/CUC3) (Figure 1, Supplementary Tables 1, 2). Some conserved motifs identified in the Arabidopsis NAC family were also examined in the deduced amino acid
sequences of NAC unigenes. For example, the WQ and LP motifs were found in members of group VIII
(SND) as the CMVIII-2 and CMVIII-3 motifs in both rice and Arabidopsis (Supplementary Table 3, Figs
1, 2). In addition, serine-rich (group V as the CMV-4 motif and in group VI as the CMVI-2 motif), glutamine-rich (in group VIII as the CMVIII-4 motif, in group X as the CMX-2 motif, and in group XV as
the CMXV-2 motif) amino acid sequences were detected in both rice and Arabidopsis (Figure 1, Supplementary Table 3). These previously were often designated as transcriptional activation domains (Liu
et al., 1999), but their functions were not rigorously demonstrated. A WVLCR motif, designated as
CMXI-1 was a characteristic feature of group XI (SNAC) in both species (Figure 1, Supplementary Table
3).
In addition to the conserved motifs between rice and Arabidopsis (Figure 2), there were also some
species-specific motifs in the NAC family (Supplementary Table 3). For example, the CMI-1 to CMI-3
motifs in group I (ONAC2), the CMII-1 and CMII-2 motifs in group II (ONAC3), the CMV-2 motif in
group V (NAM/CUC3), the CMVII-1 and CMVII-2 motifs in group VII (NAC22), and the CMXIV-1
and CMXIV-2 motifs in group XIV (ONAC1) occurred only in rice NAC proteins. Similarly, the
CMXVII-1 motif in group XVII and the CMXVIII-1 motif in group XVIII occurred only in Arabidopsis
(Supplementary Table 3). The functions of these motifs remain unknown.
Figure 1: Phylogenetic relationship among the rice NAC genes, (except IV, XII and XVI). Bootstrap values from 1000 replicates were used to assess the robustness of the trees. Each box represents the NAC domain. Protein structures of every group are shown. The same colors of motifs
were conserved in different groups. Conserved motifs are summarized in Supplementary Tables
1, 2. Classification by Nuruzzaman et al. (2010) is indicated in parentheses.
Figure 2: The WQ and LP motif-like sequences conserved in the C-terminal region of groups I
and IV NAC proteins (rice and Arabidopsis). A and B, An alignment of the sequences of the Cterminal regions of group VIII proteins. The conserved motifs are underlined. (*) Indicates identical and conserved amino acid residues of the aligned sequences. Consensus amino acid residues are given below the alignment. The ‘‘x’’ in the sequence indicates no conservation at this
position.
3.3
Comparative Analysis of the NAC Gene Family between Rice and Citrus
To determine the phylogenetic relationships of the NAC family genes in rice and citrus, a multiple sequence alignment was performed using amino acid sequences in the NAC domain. This analysis revealed
that those amino acid residues which might be involved in some form of physical contact with DNA are
also conserved among most of the rice NAC proteins and citrus NAC proteins (Supplementary Figure 4).
Residues, Glu13 and Leu19 were completely conserved among all 161 proteins in both species (Supplementary Figure 4). In addition, >91% and 98% of the NAC family members contain Leu14 and Trp47
residues, respectively (Supplementary Figure 4). The phylogenetic tree containing rice and citrus NAC
genes was constructed, and the phylogram were classed into 18 groups, namely groups I–XVII and a solo
group XVIII (Supplementary Figure 5), according to the report of Nuruzzaman et al. (2010). The comparative analysis of conserved motifs indicated that most of the motifs conserved in the rice and Arabidopsis NAC families also existed in the citrus NAC families (Supplementary Table 4). However, some
motifs, for example CMIII-1 in group III and CMIX-3 in group IX existing in both rice and Arabidopsis
NAC families Supplementary Table 3, were not found in the citrus NAC family (Supplementary Table
4). In contrast, the motifs CMIX-2 (EExWE/L) and CMXIII-1 (SxCRL/V) belonging to groups IX and
XIII, identified in both the rice and citrus NAC families, were not found in the Arabidopsis NAC family
(Supplementary Table 4). No rice NACs were assigned to groups XVII and XVIII (Supplementary Figs.
2, 5) that were specific to Arabidopsis and citrus.
3.4
Evolution and divergence of the NAC family genes in rice and Arabidopsis
Large segmental duplications of chromosomal regions during evolution, followed by gene loss, smallscale duplications and local rearrangement, have created the present complexities of plant genome (Bowers et al., 2003). The rice and Arabidopsis genome has undergone several rounds of genome-wide duplication events (Vision et al., 2000; Blanc et al., 2003; Tian et al., 2005; Wu et al., 2008). Duplication has
greatly impacted the amplification of this gene family in the genome. Therefore, in this study we investigate the relationship between the genetic divergence within the NAC family and the gene duplications in
rice and Arabidopsis. Gene duplication is the primary driving force in the development of new gene functions in the evolution of genetic systems and genomes (Moore & Purugganan, 2003). A total of 135 rice
NAC genes could be localized on the 12 chromosomes with an obviously uneven distribution (Figure 3A).
Rice NAC genes were present in all regions on a single chromosome (i.e., at the telomeric ends, near the
centromere, and in between). Comparatively high densities of OsNAC genes were observed in specific
chromosomal regions, including the bottom of chromosome 3, and the top of chromosome 11. To find
possible relationships between OsNAC genes and potential genome duplication events, we mapped 13
paralogous pairs of OsNAC genes. Nine of these pairs predicted chromosomal/segmental duplications by
Nuruzzaman et al. (2010) (Figure 3A, blue colored genes, linked with blue lines). Four additional pairs of
OsNAC genes are close paralogs (Figure 3A, green colored genes), and are new duplication events in rice
genome. Thus, 19% of the OsNAC family might have evolved from putative rice genome duplication
events. A total of 38 OsNACs showed tandem or local duplication. We indentified one duplication event
(Os08g02160 and Os08g02300) on chromosome 8 but 36 genes were found by Nuruzzaman et al. (2010).
Therefore 28% of OsNACs are organized in clusters and likely have evolved via local or tandem duplications.
To clarify the relationship between the genetic divergence within the NAC family and gene duplication events in Arabidopsis, we located the 112 AtNAC genes on the Arabidopsis chromosomes based on
the location information provided in the TAIR database (http://www.Arabidopsis.org). Of the 116 AtNAC
genes, 112 are distributed throughout the chromosomes, while only a few genes are found on the short
arms of chromosomes 2 and 4 (Figure 3B). We noted that 22 segmental duplication events in Arabidopsis
were observed (Figure 3B colored squares, Supplementary Table 5), which were originated from a polyploidy event occurred around 24 to 40 million years ago, probably close to the emergence of the crucifer
family (Blanc et al., 2003; Cannon et al., 2004). The putative protein sequences of those 22 pairs were
clustered with bootstrap value higher than 700 in the phylogenetic tree (Figure 2). Together, these genes
represent 40% of all AtNACs, indicating that large-scale segmental duplication events have contributed
largely to the current complexity of the NAC gene family in Arabidopsis. Paired genes such as
AT3G15170 and AT5G53950, AT3G61910 and AT2G46770, AT3G44290 and AT5G22290, and
AT3G15510 and AT1G52880 are the result of recently duplicated segmental chromosomes and were
assigned to groups V/NAM, VIII/SND, IX/TIP, and XI/SNAC, respectively. Interestingly, AT3G15510,
AT1G61110, and AT5G46590 have an additional segment on chromosomes 1 (AT1G52880) and 4
(AT4G27410 and AT4G17980); the phylogenetic relationships (Figure 3B) suggest that they are closely
related to each other. These genes were listed as duplicated genes in the segmental duplications dataset
maintained at “Paralogons in Arabidopsis” (http://www.wolfe.gen.tcd.ie/athal/dup). The most segmentally duplicated genes were found in groups IX/TIP and XI/SNAC (Supplementary Table 5). Only 6% of
AtNACs (seven pairs, black bars) are organized in clusters, and likely have evolved via local or tandem
duplications (Figure 3B, Supplementary Table 6).
Taken together, it appears that the NAC gene families expanded differently in rice and Arabidopsis.
Chromosomal segment duplications mainly contributed to the expansion of both OsNACs and AtNACs
giving rise to 19% and 39% of NAC genes, respectively. But tandem or local duplication occurred less
frequently in Arabidopsis (7 pairs, 6% of all NACs) than rice (15 clusters, 28%). However, the rice genome is three times the size of that of Arabidopsis. The reason for this could be the variable status of
whole genome duplications in rice and Arabidopsis (Yu et al., 2005).
During the course of gene evolution, NAC family genes in moss Physcomitrella patens (9 genes;
e.g., pp_gw1.5.134.1, pp_gw1.159.34.1, pp_gw1.44.97.1, pp_gw1.70.85.1, pp_gw1.117.35.1, and
pp_gw1.161.49.1) and in Selaginella (28 genes; e.g., sl_gw1.0.2553.1, sl_gw1.122.81.1, sl_gw1.79.346.1,
sl_gw1.14.891.1, sl_gw1.46.350.1, and sl_gw1.1.2023.1) were also created. A preliminary examination
using BLAST indicates that pp_gw1.5.134.1 and sl_gw1.0.2553.1, pp_gw1.159.34.1 and
sl_gw1.79.346.1, pp_gw1.70.85.1 and sl_gw1.14.891.1, pp_gw1.44.97.1 and sl_gw1.46.350.1, and
sl_gw1.1.2023.1 encode NAC proteins belonging to group V, VIII, IX, X, and XI respectively (Supplementary Figure 6), which indicates that the phylogenetic topology of the NAC family had already been
created before the divergence of vascular plants.
3.5
Duplication and Evolution Analysis of the Osnac Genes
The Ka/Ks ratio is a measure to explore the mechanism of gene divergence after duplication. Ka/Ks = 1
means neutral selection, Ka/Ks < 1 means purifying selection, and Ka/Ks > 1 means accelerated evolution with positive selection. We calculated 10 duplicated pairs in the OsNAC gene family (Table 1). The
Ka/Ks ratio of 9 pairs (e.g., Os02g06950/Os06g46270 and Os02g34970/Os04g35660) were less than 1,
suggesting purifying selection on these 9 duplicated pairs; however, the Ka/Ks ratio of
Os02g18460/Os02g18470 was more than 1, suggesting positive selection on the one duplicated pair.
Figure 3: Chromosome numbers are indicated at the top of each species. (A) Straight lines connect the OsNAC genes presented on duplicated chromosomal segments are marked by blue line
(green color genes are new identified), and tandemly duplicated gene clusters are marked by
black and green bars, (green bar is new identified). (B) The locations of the NAC family genes
on the Arabidopsis chromosomes. The colored squares after the genes presented on duplicated
chromosomal segment and thick black rectangle joined tandem repeated genes. AT1G61110,
AT3G15510, and AT5G46590 showed double duplicated chromosomal segments. There were
two colored squares beside these genes.
Duplication events for 5 pairs (Os02g06950/Os06g46270, Os02g34970/Os04g35660,
Os02g36880/Os04g38720, Os03g03540/Os10g38834 and, Os03g21060/Os07g48450) occurred within
last 70 to 50 million years, after origin of grasses and before divergence of rice and maize, according to
the first whole-genome duplication events of grass genomes (Gaut, 2002). Duplication events for 3 pairs
(Os01g01430/Os01g01470, Os08g02160/Os08g02300, and Os11g03300/Os12g03040) occurred within
last 50 to 20 million years, after divergence of rice and maize, but before Zizaniinae and Oryzinae were
separated from each other (Guo et al., 2005b). Duplication events for the other two pairs
(Os03g56580/Os07g04560 and Os02g18460/Os02g18470) occurred within last 20 to 9 million years,
after Zizaniinae and Oryzinae were separated, and before Oryza genus was branched off from the emaining genera of Oryzeae (Guo et al., 2005b). Therefore, evolutionary origin of the 135 NAC genes might
undertake 3 evolutionary stages (Table 1).
Genes in different species and related by a speciation event are defined as orthologs. PGDD is a
public database that identifies and catalogs plant genes in terms of cross-genome syntenic relationships
(Tang et al., 2008). As data only of syntenic relationships within the angiosperm are available, 352 NAC
genes from monocots and eudicots (data not shown) were used in this analysis. We found that redundant
63 OsNAC genes containing a NAC domain can be detected in synteny blocks in selected species (Supplementary Table 7). It is suggested that many plant genomes underwent one to several large scale duplication events in their long evolutionary history, in which duplicated functional genes were preferentially
retained. This view provides an explanation for the expansion of many families in the plant kingdom.
Whole genome duplication events within or between species can account for most of the expansion of the
NAC family.
Duplicated pair
Ka
Ks
Os02g06950/Os06g46
270
Os02g34970/Os04g35
660
Os02g36880/Os04g38
720
Os03g03540/Os10g38
834
Os03g21060/Os07g48
450
Os11g03300/Os12g03
040
Os01g01430/Os01g01
470
Os08g02160/Os08g02
300
0.10
3
0.27
3
0.27
3
0.27
2
0.26
3
0.20
1
1.66
1
1.42
9
1.01
7
1.02
9
1.06
9
1.01
9
0.91
3
7.70
3
5.87
7
54.0
3
Ka/K
s
0.103
9
0.265
5
0.275
0
0.255
4
0.287
7
0.026
1
0.282
6
0.026
4
Date (million
years)
Duplicate
type
Purifying Selection
Group
77.08
Segmental
Yes
VI/VI
78.09
Segmental
Yes
IV/IV
77.05
Segmental
Yes
V/V
79.11
Segmental
Yes
VIII/VI
II
60.21
Segmental
Yes
XI/XI
49.26
Segmental
Yes
XI/XI
45.21
Tandem
Yes
XI/V
21.56
Tandem
Yes
X/X
Continued on next page…
…Continued from previous page
Os03g56580/Os07g04
560
Os02g18460/Os02g18
470
0.12
2
1.99
2
0.71
1.84
1
0.171
4
1.081
6
13.18
Segmental
Yes
X/X
14.16
Tandem
No
I/I
Ka/Ks<1= Purifying
λ=6.5x10-9
Table 1: Ka/Ks analysis and estimate of the absolute dates for the duplication events between
the duplicated OsNAC genes.
4
Discussion
The objectives of this study were to determine the putative conserved motifs outside of the NAC domain
and compared among the three species. Gene duplication and purification were performed in the course
of evolution.
4.1
Comparative analysis of the NAC among the three species
Based on the alignment of the amino acid sequences, the OsNAC genes were classified into 16 subgroups
in rice (Nuruzzaman et al., 2010). Ooka et al. (2003) systematically surveyed the gene structures, phylogeny, and conserved motifs of the NAC gene family in rice and Arabidopsis, but relatively few rice
NAC genes (75) and Arabidopsis NAC genes (105) were studied previously. To gain further information
about the NAC family in rice 135, in Arabidopsis 116, and in citrus 26 of the NAC members’ superfamily were selected for this present study, respectively. The structure and phylogeny of the NAC superfamily are similar in the three species (Supplementary Figs 1-5). The presence of a few groups in the three
species also suggests that many of the genes pre-date the species divergence. Likewise, some groups are
present in only one species; for example, groups XVII and XVIII existed only in the Arabidopsis and
citrus NAC family but not in the rice NAC families (Supplementary Figs. 2, 5) suggesting that these
groups had evolved or been lost in one species after this divergence. However, this comparison alone
provides limited functional information, whereas queries with rice, Arabidopsis or citrus NAC genes of
known function could identify candidate rice orthologs with functional similarities. Some incompletely
full-length NAC genes were missed in the present study, decreasing the likely number of NAC family
members in rice from previous finding (Fang et al., 2008; Nuruzzaman et al., 2010). However, a comparative analysis of three species indicated that the phylogenetic analysis in rice was reliable.
Comparative analysis of amino acid residues of the NAC domains in the rice NAC family proteins
with those of Arabidopsis and citrus suggested the NAC domains were well conserved among the three
species (Supplementary Figs 1, 4). These conserved amino acid residues probably indicate crucial roles
for NAC family genes involved in different forms of physical contact with DNA. The conserved motif
analysis of the NAC family demonstrated that most motifs were conserved in rice, Arabidopsis, and citrus (Supplementary Tables 3, 4). Proteins within a group that share these conserved motifs are likely to
have similar functions. For example, the WQ and LP motifs are essential for the transactivational activity
if the C-terminal region (Figure 1 and Figure 2). A study showed that the C-terminal domain of the
AT1G32770/SND1/ANAC012 gene functions as a transcriptional activator (Ko et al., 2007). WQ motif is
found in the C-termini of the CUC-like proteins, which includes NAC2/ORE1 (Kim et al., 2009) from
group VIII/SND. In addition to common conserved motifs in rice, Arabidopsis, and citrus, there are ricespecific NAC family motifs, which may have important roles in regulating biological processes in rice;
those functions need to be demonstrated further. The comparative analysis of conserved motifs in rice,
Arabidopsis, and citrus suggested that protein functions have been both conserved and diverged during
evolution of the NAC gene family.
4.2
NAC genes multiplication and purifying selection
Most gene families have multiple members and the reason for this could be the variable status of whole
genome duplications in plants (Yu et al., 2005). Therefore, we consider the number of NAC genes to
have increased rapidly during the course of evolution that whole genome duplication and tandem/segmental duplication played a key role in the expansion of NAC genes in rice (Figure 3). During
evolution, segmental and tandem gene duplications have also been found in the MYB and F-box gene
families in plants (Cannon et al., 2004; Jain et al., 2007). It is noteworthy that the members of divergent
NAC groups in both rice and Arabidopsis are located within the same chromosomal region, whereas the
members of the same group are distributed in different chromosomal regions, suggesting that NAC genes
were distributed widely in the genome of the common ancestor of monocots and eudicots. Furthermore,
in both species the duplicated pairs of NAC genes appear to have been preferentially retained compared
with other genes, since the density of duplicated genes retained in recently duplicated chromosomal segments was estimated to be only 28% on average (Blanc et al., 2003). This highlights the indispensable
roles of NACs in both monocots and eudicots. We suggested the evolutionary expansion of the OsNAC
gene family in rice genome underwent three stages based on the dates of the duplication events (Table 1).
After the duplication events of these genes, gene retention and loss always occur in the long evolutionary
history. The retention and loss of NAC genes varied in each group (Supplementary Figs. 2, 5). We believe that the genes in the NAC family were under purifying selection for their functional importance.
The Ka/Ks ratio provides a sensitive measure of selective pressure on the protein. Most amino acids in a
functional protein are under affects only a few sites at a few time points. Therefore, positive selection
was thought to be one of the major forces in the emergency of new motifs /functions in protein after gene
duplication (Yang et al., 2006). Several members of group XI/SNAC and other NAC genes play crucial
roles in abiotic and/or biotic stress responses (Supplementary Table 8). These published genes may explain the low success rate of classical forward genetic strategies in the elucidation of the functions of
NAC genes in plants. The common conserved motifs in rice and Arabidopsis suggested that protein functions have been both conserved and diverged during evolution of the NAC gene family. Therefore, the
increased number of genes in these groups might be the evolutionary consequences of adjusting to environmental change.
In conclusion, a comparative genome-wide study of the phylogenetic and motifs among the three
species, and gene duplication relationships between rice and Arabidopsis genes were performed. Most of
these groups are present both in monocot and eudicot, suggesting that the appearance of many of the
genes in these species predates monocot or eudicot divergence but some are specific, suggesting that they
have been lost in one species after this divergence. We indentified conserved motifs outside of the NAC
domain in rice and compared with those of Arabidopsis and citrus. NAC domains were conserved in
three species. The NAC C-terminal domains contain group specific motifs that are characterized by a
high degree of intrinsic disorder. We described potential gene birth-and- death events in the NACs between rice and Arabidopsis. The results suggest that chromosomal segment duplications mainly contributed to the expansion of both OsNACs and AtNACs, whereas tandem or local duplication occurred very
frequently in rice than Arabidopsis. Furthermore, Ka/Ks ratio was used to explore the mechanisms and
found 3 evolutionary stages of the NAC genes divergence following their duplication. The Ka/Ks ratio
provides a sensitive measure of selective pressure on the protein and they are under affects only a few
sites at a few time points. Syntenic information for all examined genes (352) from monocots and eudicots
were used. The results presented here will be helpful for functional studies to unravel their divergent
roles.
Acknowledgements
This work was funded by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN). The funding was awarded to Mohammed Nuruzzaman, Akhter Most Sharoni, and
Shoshi Kikuchi.
Supplementary Tables
•
Supplementary Table 1
Caption:
Summary of conserved motifs (CMs) within OsNAC family.
Download:
http://www.iconceptpress.com/download/paper/12051109453596/supplmentary-table1.pdf
•
Supplementary Table 2
Caption:
All (135) NAC family genes in Oryza sativa ssp. Japonica.
Download:
http://www.iconceptpress.com/download/paper/12051109453596/supplmentary-table2.pdf
•
Supplementary Table 3
Caption:
Summary of conserved motifs (CMs) within OsNAC and AtNAC family.
Download:
http://www.iconceptpress.com/download/paper/12051109453596/supplmentary-table3.pdf
•
Supplementary Table 4
Caption:
Summary of conserved motifs (CMs) within OsNAC family by comparative analysis with citrus.
Download:
http://www.iconceptpress.com/download/paper/12051109453596/supplmentary-table4.pdf
•
Supplementary Table 5
Caption:
AtNAC genes present on duplicated chromosomal segments.
Download:
http://www.iconceptpress.com/download/paper/12051109453596/supplmentary-table5.pdf
•
Supplementary Table 6
Caption:
Tandemly duplicated ATNAC genes.
Download:
http://www.iconceptpress.com/download/paper/12051109453596/supplmentary-table6.pdf
•
Supplementary Table 7
Caption:
Network of the syntenic relationship between NAC members in angiosperm genomes. The
available syntenic relationships between NAC genes were downloaded from the PGDD database. The Ka and Ks values were computed in the PAML program using the maximum likelihood method.
Download:
http://www.iconceptpress.com/download/paper/12051109453596/supplmentary-table7.pdf
•
Supplementary Table 8
Caption:
The NAC genes whose biological function has been characterized in plants.
Download:
http://www.iconceptpress.com/download/paper/12051109453596/supplmentary-table8.pdf
Supplementary Figures
•
Supplementary Figure 1
Caption:
The deduced amino acid sequence alignment of the NAC DNA-binding domains from the 135
rice NAC proteins and 116 Arabidopsis NAC proteins by Clustal W. Classification by Nuruzzaman et al. (2010) is indicated in parentheses.
Download:
http://www.iconceptpress.com/download/paper/12051109453596/supplmentary-figure1.pdf
•
Supplementary Figure 2
Caption:
An unrooted phylogenetic tree of the NAC family of the rice and Arabidopsis. The amino acid
sequences of the NAC domain of 135 rice NAC family proteins and 116 Arabidopsis NAC proteins were aligned by ClustalW and the phylogenetic tree was constructed using MEGA 4.0 and
the NJ method. Bootstrap values from 1000 replicates were used to assess the robustness of the
trees. Classification by Nuruzzaman et al. (2010) is indicated in parentheses.
Download:
http://www.iconceptpress.com/download/paper/12051109453596/supplmentary-figure2.pdf
•
Supplementary Figure 3
Caption:
An unrooted phylogenetic tree of rice NAC proteins. The amino acid sequences of the NAC
domain of 135 rice NAC family proteins were aligned by Clustal W and the phylogenetic tree
was constructed using MEGA 4.0 and the NJ method. Classification by Nuruzzaman et al.
(2010) is indicated in parentheses.
Download:
http://www.iconceptpress.com/download/paper/12051109453596/supplmentary-figure3.pdf
•
Supplementary Figure 4
Caption:
The deduced amino acid sequence alignment of the NAC DNA-binding domains from the 135
rice NAC proteins and 26 citrus NAC proteins by Clustal W. Classification by Nuruzzaman et
al. (2010) is indicated in parentheses.
Download:
http://www.iconceptpress.com/download/paper/12051109453596/supplmentary-figure4.pdf
•
Supplementary Figure 5
Caption:
An unrooted phylogenetic tree of the NAC family of the rice and citrus. The amino acid sequences of the NAC domain of 135 rice NAC family proteins and 26 citrus NAC proteins were
aligned by Clustal W and the phylogenetic tree was constructed using MEGA 4.0 and the NJ
method. Bootstrap values from 1000 replicates were used to assess the robustness of the trees.
Classification by Nuruzzaman et al. (2010) is indicated in parentheses.
Download:
http://www.iconceptpress.com/download/paper/12051109453596/supplmentary-figure5.pdf
•
Supplementary Figure 6
Caption:
An unrooted phylogenetic tree of the NAC family of the rice, Physcomitrella patens, and Selaginella. The amino acid sequences of the NAC domain of 135 rice NAC family proteins, 9 P.
patens NAC proteins and 28 Selaginella NAC proteins were aligned by ClustalW (data not
shown) and the phylogenetic tree was constructed using MEGA 4.0 and the NJ method. Bootstrap values from 1000 replicates were used to assess the robustness of the trees. Classification
by Nuruzzaman et al. (2010) is indicated in parentheses.
Download:
http://www.iconceptpress.com/download/paper/12051109453596/supplmentary-figure6.pdf
References
Bailey, T.L., & Elkan, C. (1994). Fitting a mixture model by expectation maximization to discover motifs in biopolymers.
In R. Altman, D. Brutlog, P. Karp, R. Lathrop, & D. Searls (Eds.), Proceedings of the second international conference on intelligent systems for molecular biology. American Association for Artificial Intelligence Press, Menlo Park,
CA, (pp 28–36).
Blanc, G., Hokamp, K., &Wolfe, K.H. (2003). A recent polyploidy superimposed on older large-scale duplications in the
Arabidopsis genome. Genome Research, 13, 137–144.
Bowers, J.E., Chapman, B.A., Rong, J., & Paterson, A.H. (2003). Unravelling angiosperm genome evolution by phylogenetic analysis of chromosomal duplication events. Nature, 422, 433–438.
Cannon, S.B., Mitra, A., Baumgarten, A., Young, N.D., & May, G. (2004). The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biology, 4, 10.
Christianson, J.A., Wilson, I.W., Llewellyn, D.J., & Dennis, E.S. (2009). The lowoxygen induced NAC domain
transcription factor ANAC102 affects viability of Arabidopsis thaliana seeds following low-oxygen treatment. Plant
Physiology, 149, 1724–1738.
Collinge, M., & Boller, T. (2001). Differential induction of two potato genes, Stprx2 and StNAC, in response to infection
by Phytophthora infestans and to wounding. Plant Molecular Biology, 46, 521–529.
Duval, M., Hsieh, T.F., Kim, S.Y., & Thomas, T.L. (2001). Molecular characterization of AtNAM: a member of the Arabidopsis NAC domain superfamily. Plant Molecular Biology, 50, 237–248.
Eddy, S.R. (1998). Profile hidden Markov models. Bioinformatic,s 14, 755–763.
Fang, Y., You, J., Xie, K., Xie, W., & Xiong, L. (2008). Systematic sequence analysis and identification of tissue-specific
or stress-responsive genes of NAC transcription factor family in rice. Molecular Genetics and Genomics, 280, 547–
563.
Finn, R.D., Mistry, J., Tate, J., Coggill, P., Heger, A., et al. (2010). The Pfam protein families database. Nucleic Acids Research, 38, D211–22.
Fujita, M., Fujita, Y., Maruyama, K., Seki, M., et al., (2004). A dehydration-induced NAC protein, RD26, is involved in a
novel ABA-dependent stress-signaling pathway. Plant Journal, 39, 863–876.
Gaut, B.S. (2002). Evolutionary dynamics of grass genomes. New Phytologist, 154, 15–28.
Greve, K., La Cour, T., Jensen, M.K., Poulsen, F.M., & Skriver, K. (2003). Interactions between plant RING-H2 and plant
specific NAC (NAM/ATAF1/2/CUC2) proteins: RING-H2 molecular specificity and cellular localization. Biochemical Journal, 371, 97–108.
Guo, H.S., Xie, Q., Fei, J.F., & Chua, N.H. (2005a). Micro RNA directs mRNA cleavage of the transcription factor NAC1
to down-regulate auxin signals for Arabidopsis lateral root development. Plant Cell, 17, 1376–86.
Guo, Y.L., & Ge, S. (2005b). Molecular phylogeny of Oryzeae (Poaceae) based on DNA sequences from chloroplast, mitochondrial, and nuclear genomes. American Journal of Botany, 92, 1548–1558.
He, X.J., Mu, R.L., Cao, W.H., Zhang, Z.G., Zhang, J.S., & Chen, S.Y. (2005). AtNAC2, a transcription factor downstream of ethylene and auxin signaling pathways, is involved in salt stress response and lateral root development.
Plant Journal, 44, 903–916.
Hegedus, D., Yu, M., Baldwin, D., Gruber, M., Sharpe, A., Parkin, I., … Lydiate, D. (2003). Molecular characterization of
Brassica napus NAC domain transcriptional activators induced in response to biotic and abiotic stress. Plant Molecular Biology, 53, 383–397.
Hu, H., Dai, M., Yao, J., Xiao, B., Li, X., Zhang, Q., & Xiong, L. (2006). Overexpressing a NAM, ATAF, and CUC
(NAC) transcription factor enhances drought resistance and salt tolerance in rice. Proceedings of the National Academy of Sciences USA, 103, 12987–12992.
Hu, H., You, J., Fang, Y., Zhu, X., Qi, Z., & Xiong, L. (2008). Characterization of transcription factor gene SNAC2
conferring cold and salt tolerance in rice. Plant Molecular Biology, 67, 169–181.
Jain, M., Nijhawan, A., Arora, R., Agarwal, P., Ray, S., Sharma, P., … Khurana, J.P. (2007). F-box proteins in rice. Genome wide analysis, classification, temporal and spatial gene expression during panicle and seed development, and
regulation by light and abiotic stress. Plant Physiology, 143, 1467–1483.
Jensen, M.K., Kjaersgaard, T., Nielsen, M.M., et al., (2010). The Arabidopsis thaliana NAC transcription factor family:
structure-function relationships and determinants of ANAC019 stress signalling. Biochemical Journal, 426,183–196.
Jeong, J.S., Kim, Y.S., Baek, K.H., Jung, H., Ha, S.H., Do Choi, Y., … Kim, J.K. (2010). Root-specific expression of OsNAC10 improves drought tolerance and grain yield in rice under field drought conditions. Plant Physiology, 153,
185–97.
Kim, Y.S., Kim, S.G., Park, J.E., Park, H.Y., Lim, M.H., Chua, N.H., & Park, C.M. (2006). A membrane-bound NAC
transcription factor regulates cell division in Arabidopsis. Plant Cell, 18, 3132–3144.
Kim, H.S., Park, B.O., Yoo, J.H., et al., 2007a. Identification of a calmodulin-binding NAC protein as a transcriptional
repressor in Arabidopsis. Journal of Biological Chemistry, 14, 36292–302.
Kim, S.G., Kim, S.Y., & Park, C.M. (2007b). A membrane-associated NAC transcription factor regulates salt-responsive
flowering via FLOWERING LOCUS T in Arabidopsis. Planta, 226, 647–654.
Kim, S.G., Lee, A.K., Yoon, H.K., & Park, C.M. (2008). A membrane-bound NAC transcription factor NTL8 regulates
gibberellic acid-mediated salt signaling in Arabidopsis seed germination. Plant Journal, 55, 77–88.
Kim J.H., Woo, H.R., Kim, J., Lim, P.O., Lee, I.C., Choi, S.H., … Nam, H.G. (2009). Trifurcate feed-forward regulation
of age-dependent cell death involving miR164 in Arabidopsis. Science, 323, 1053–1057.
Ko, J.H., Yang, S.H., Park, A.H., Lerouxel, O., & Han, K.H. (2007). ANAC012, a member of the plant-specific NAC transcription factor family, negatively regulates xylary fiber development in Arabidopsis thaliana. Plant Journal, 50,
1035–1048.
Kranz, H.D., Denekamp, M., Greco, R., Jin, H., Leyva, A., et al. (1998). Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana. Plant Journal, 16, 263–276.
Liu, L., White, M.J., & MacRae, T.H. (1999). Transcription factors and their genes in higher plants functional domains,
evolution and regulation. European Journal of Biochemistry, 262, 247–257.
Luscombe, N.M., & Thornton, J.M. (2002). Protein-DNA interactions: amino acid conservation and the effects of mutations on binding specificity. Journal of Molecular Biology, 320, 991–1009.
Moore, R.C., & Purugganan, M.D. (2003). The early stages of duplicate gene evolution. Proc. Natl. Acad. Sci. U.S.A. 100,
15682–15687.
Nakashima, K., Tran, L.S., VanNguyen, D., Fujita, M., Maruyama, K., et al. (2003). Functional analysis of a NAC-type
transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant Journal,
51, 617–630.
Nuruzzaman, M., Manimekalai, R., Sharoni, A.M., Satoh, K., Kondoh, H., Ooka, H., & Kikuchi, S. (2010). Genome-wide
analysis of NAC transcription factor family in rice. Gene, 1, 30–44.
Olsen, A.N., Ernst, H.A., Lo Leggio, L., & Skriver, K. (2005). NAC transcription factors: structurally distinct, functionally
diverse. Trends in Plant Science, 10, 1360–1385.
Ooka, H., Satoh, K., Doi, K., Nagata, T., Otomo, Y., Murakami, K., et al., (2003). Comprehensive analysis of NAC family
genes in Oryza sativa and Arabidopsis thaliana. DNA Research,10, 239–247.
Ouyang, S., Zhu, W., Hamilton, J., et al., (2007). The TIGR rice genome annotation resource: improvements and new features. Nucleic Acids Research, 35, D883–D887.
Peng, H., Cheng, H.Y., Chen, C., et al., (2009). A NAC transcription factor gene of chickpea (Cicer arietinum), CarNAC3,
is involved in drought stress response and various developmental processes. Journal of Plant Physiology, 166, 1934–
1945.
Reyes, J.C., Muro-Pastor, M.I., & Florencio, F.J. (2004). The GATA family of transcription factors in Arabidopsis and rice.
Plant Physiology, 134, 1718–1732.
Riano-Pachon, D.M., Ruzicic, S., Dreyer, I., & Mueller-Roeber, B. (2007). PlnTFDB: an integrative plant transcription
factor database. BMC Bioinformatics, 8, 42.
Satoh, K., Shimizu, T., Kondoh, H., Hiraguri, A., Sasaya, T., Choi, I.R. … Kikuchi, S. (2011). Relationship between
symptoms and gene expression induced by the infection of three strains of rice dwarf virus. PLoS ONE, 3, e18094.
Suyama, M., Torrents, D., & Bork, P. (2006). PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic Acids Research, 34, W609–612.
Sperotto, R.A., Ricachenevsky, F.K., Duarte, G.L., Boff, T., Lopes, K.L., Sperb, E.R., … Fett, J.P. (2009). Identification of
up-regulated genes in flag leaves during rice grain filling and characterization of OsNAC5, a new ABA-dependent
transcription factor. Planta, 230, 985–1002.
Tamura, K., Dudley, J., Nei, M., et al. (2007). MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24, 1596–1599.
Tang, H., Bowers, J.E., Wang, X., et al. (2008). Synteny and Collinearity in Plant Genomes, Science 320, 486–488.
Thompson, J.D., Higgins, D.G., & Gibson, T.J. (1994). CLUSTALW: improving the sensitivity of progressive multiple
sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic
Acids Research, 22, 4673–4680.
Tian, C., Wan, P., Sun, S., Li, J., & Che, M. (2004). Genome-wide analysis of the GRAS gene family in rice and Arabidopsis. Plant Molecular Biology, 54, 519–532.
Tian, C., Xiong, Y., Liu, T., Sun, S., Chen, L., & Chen, M. (2005). Evidence for an ancient whole-genome duplication
event in rice and other cereals. Acta Genetica Sinica, 32, 519–527.
Tran, L.S., Nakashima, K., Sakuma, Y., Simpson, S.D., Fujita, Y., Maruyama, K., … Yamaguchi-Shinozaki, K., (2004).
Isolation and functional analysis of Arabidopsis stress inducible NAC transcription factors that bind to a drought responsive cis-element in the early responsive to dehydration stress 1 promoter. Plant Cell, 16, 2481–2498.
Vision, T.J., Brown, D.G., & Tanksley, S.D. (2000). The origins of genomic duplications in Arabidopsis. Science, 290,
2114–2117.
Wray, G.A., Hahn, M.W., Abouheif, E., Balhoff, J.P., Pizer, M., Rockman, M.V., & Romano, L.A. (2003). The evolution
of transcriptional regulation in eukaryotes. Molecular Biology and Evolution, 20, 1377–1419.
Wu, Y., Zhu, Z., Ma, L., & Chen, M. (2008). The preferential retention of starch synthesis genes reveals the impact of
whole-genome duplication on grass evolution. Molecular Biology and Evolution, 25, 1003–1006.
Xie, Q., Frugis, G., Colgan, D., & Chua, N.H. (2000). Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to
promote lateral root development. Genes and Development, 14, 3024–3036.
Yamaguchi-Shinozaki, K., & Shinozaki, K. (2001). Improving plant drought, salt and freezing tolerance by gene transfer
of a single stress-inducible transcription factor. Novartis Found. Symp. 236, 176–186, discussion 186–189.
Yang, X., Tuskan, G.A.,& Cheng, M.Z. (2006). Divergence of the Dof gene families in poplar, Arabidopsis, and rice suggests multiple modes of gene evolution after duplication. Plant Physiology, 142, 820–830.
Yang, Z., Gu, S., et al. (2008). Molecular evolution of the cpp-like gene family in plants: insights from comparative genomics of Arabidopsis and rice. Journal of Molecular Evolution, 67, 266-277.
Yoshii, M., Shimizu, T., Yamazaki, M., Higashi, T., Miyao, A., Hirochika, H., & Omura, T. (2009). Disruption of a novel
gene for a NAC-domain protein in rice confers resistance to rice dwarf virus. Plant Journal, 57, 615–625.
Yoshii, M., Yamazaki, M., Rakwal, R., Kishi-Kaboshi, M., Miyao, A., & Hirochika, H. (2010). The NAC transcription
factor RIM1 of rice is a new regulator of jasmonate signaling. Plant Journal, 61, 804–15.
Yu, J., Wang, J., Lin, W., Li, S., Li, H., et al. (2005). The genomes of Oryza sativa: a history of duplications. PLoS Biology, 3, e38.
Zheng, X., Chen, B., Lu, G., & Han, B. (2009). Overexpression of a NAC transcription factor enhances rice drought and
salt tolerance. Biochemical and Biophysical Research Communications, 379, 985–989.