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analyses of GATA transcription
factors among seven Populus
Mangi Kim1,2, Hong Xi1,2, Suhyeon Park1,2, Yunho Yun1,2 & Jongsun Park1,2*
GATA transcription factors (TFs) are widespread eukaryotic regulators whose DNA‑binding domain is
a class IV zinc nger motif (CX2CX17–20CX2C) followed by a basic region. We identied 262 GATA genes
(389 GATA TFs) from seven Populus genomes using the pipeline of GATA‑TFDB. Alternative splicing
forms of Populus GATA genes exhibit dynamics of GATA gene structures including partial or full loss
of GATA domain and additional domains. Subfamily III of Populus GATA genes display lack CCT and/
or TIFY domains. 21 Populus GATA gene clusters (PCs) were dened in the phylogenetic tree of GATA
domains, suggesting the possibility of subfunctionalization and neofunctionalization. Expression
analysis of Populus GATA genes identied the ve PCs displaying tissue‑specic expression, providing
the clues of their biological functions. Amino acid patterns of Populus GATA motifs display well
conserved manner of Populus GATA genes. The ve Populus GATA genes were predicted as membrane‑
bound GATA TFs. Biased chromosomal distributions of GATA genes of three Populus species. Our
comparative analysis approaches of the Populus GATA genes will be a cornerstone to understand
various plant TF characteristics including evolutionary insights.
A transcription factor (TF) is a protein that controls the rate of transcription by binding to specic DNA
sequences, including promoter regions. TF can also combine and interact with cis-acting elements in the pro-
moter region as well as interact with other proteins to regulate the start site of transcription1. In plant, TF plays
important roles such as controlling ower developments2, 3, circadian clock4, carbon and nitrogen regulatory
networks5, protein–protein interaction6, cell dierentiation7, pathogen and hormone responses8, and disease
Due to a large number of plant genomes available (2,220 genomes from 725 species; Plant Genome Database
Release 2.75; http:// www. plant genome. info/; Park etal., in preparation), many genome-wide analyses of plant
TFs have been conducted10–15. One of the genome-wide TF databases is the plantTFDB which identies 58 TF
families from 165 plant species11. Some of these TF families are plant-specic, including AP2/ERF16, NAC17,
WRKY18, and GRAS19, 20, and some are general in eukaryotic such as bHLH (basic helix-loop-helix)21, 22, bZIP
(basic leucine-zipper)23, and GATA
24–27. With the published plant genomes, various genome-wide analyses of
TF families have been conducted; AP2/ERF, NAC28–31, bHLH32–34, bZIP23, 35–37, GRAS20, 38, 39, and GATA
25, 40, 41
TF families displaying their features in various aspects including evolutionary aspect. Genome-wide analyses of
TF families in Arabidopsis thaliana have also been studied, presenting 122 AP2/ERF genes42, 105 NAC genes28,
162 bHLH genes34, 75 bZIP genes23, 32 GRAS genes20, 29 GATA genes25 as well as 566 GATA genes from 19 A.
GATA TFs contain more than one highly conserved type IV zinc nger motifs (CX2X17–20CX2C) followed
by a basic region that can bind to a consensus DNA sequence, WGA TAR (W means T or A; R indicates G
or A)25, 44, 45. Most plant GATA TFs contain a single GATA domain of which pattern is CX2CX18CX2C (type
IVb) or CX2CX20CX2C (type IVc)27. Except for these known types, additional patterns were also identied: e.g.,
CX4CX18CX2X, which have four amino acids in the rst Cysteine-Cysteine, named as type IV443.
Plant GATA TFs have various roles like the chloroplast development46, photosynthesis and growth47, epithe-
lial innate immune responses48, seed germination49, hypocotyl and petiole elongation50, and cryptochrome1-
dependent response51. Genome-wide analyses and/or expression analyses of GATA TFs have been reported in
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21 plant species25, 40, 41, 43, 52–66 (TableS1), including P. trichocarpa of which genome-wide identication of GATA
genes was conducted based on the old gene model (Version 3.0; TableS1).
Populus genus is a model system for investigating the wood development, crown formation, and disease
resistance in perennial plants67, which has several advantages including rapid growth, ease of cloning, and small
genome68. Owing to it, its genome was sequenced as a rst wood plant genome69, and then additional genome
sequences of Populus species have been sequenced and analyzed55, 70–75 (Table1), which is an excellent resource
to identify genus-wide analyses of Populus GATA TFs. ese species were classied into independent three clades
based on phylogenetic studies using single-copy76 nuclear genes and whole chloroplast genome sequences77, 78:
(i) P. tremuloides, P. tremula, and P. tremula x alba, (ii) P. pruinosa and P. euphratica, and (iii) P. trichocarpa and
P. deltoides. In spite of abundant resources of Salicaceae genomes including Salix purpurea79, there is only one
study for characterizing the biological function of Populus GATA gene (PdGNC), which regulates chloroplast
ultrastructure, photosynthesis, and vegetative growth in Arabidopsis80, suggesting genome-wide identication
of Populus GATA genes are strongly required.
Here, we conducted genome-wide identication, phylogenetic analyses, expression level analysis, identica-
tion of amino acid patterns and transmembrane helix of GATA TFs in seven Populus genomes with the GATA-
TFDB (http:// gata. genef amily. info/; Park etal., in preparation). Our comparative and comprehensive analyses
conducted with the integrated bioinformatic pipeline provided by the GATA-TFDB will be a cornerstone to
understand various plant TF characteristics including evolutionary insights.
Results and discussions
Identication of GATA TFs from seven Populus genomes. We identied 262 GATA genes (389
GATA TFs) from seven Populus genomes available in public using the pipeline of the GATA-TFDB (http:// gata.
genef amily. info/; TableS2). e number of GATA genes for each Populus genome ranges from 33 to 40 (Table1),
which is larger than those of A. thaliana (29 to 30 GATA genes)25, 43, V. vinifera (19 GATA genes), and O. sativa
(28 GATA genes)81; while is smaller than that of G. max (64 GATA genes)40. e phylogenetic relationship of
the seven Populus species inferred from the complete chloroplast genomes (Fig.1a), congruent to the previous
studies76, 78, shows no correlation with the number of GATA genes. It can be explained that the number of GATA
genes is rather aected by the accuracy of the gene model (e.g., the largest number of GATA TFs is not from P.
trichocarpa, which is the model Populus species; Fig.1b). e proportion of Populus GATA genes against whole
genes ranges from 0.08% (P. deltoides) to 0.13% (P. euphratica; Table1), which is similar to that of A. thaliana
(0.11%) and is slightly higher than those of V. vinifera (0.07%), G. max (0.06%), and O. sativa (0.05%).
Among Populus genomes, P. euphratica has the largest number of GATA genes (40); while P. tremula contains
the smallest (33; Fig.1b): dierence of the number of GATA genes between the largest and the smallest is seven.
Similarity, three Arabidopsis genomes, Arabidopsis halleri, Arabidopsis lyrata, and A. thaliana, contain 22, 28, and
30 GATA genes, respectively (Fig. S1) and four Oryza genomes, O. sativa, Oryza glaberrima, Oryza brachyantha,
and Oryza rupogon, showed 28, 25, 24, and 28 GATA genes, respectively25 (Fig.S1), displaying similar interspe-
cies dierences. While, Gossypium raimondii, Gossypium arboretum, and Gossypium hirsutum presented 46, 46,
and 87, respectively because G. hirsutum is a tetraploid species52. ese interspecic dierences of GATA genes
indicate that many evolutionary events including the gain and loss of GATA genes were occurred in three genera.
Except P. pruinosa genome not containing alternative splicing forms, numbers of GATA TFs are larger than
those of GATA genes (Table1). Numbers of GATA genes which have alternative splicing forms range from 7 to
16 (Table1 and TableS3), accounting for 30.22% of Populus GATA genes, which is similar that of A. thaliana (9
out of 30 GATA genes; 30.00%). PdGATA6 from P. deltoides contains nine alternative splicing forms, which is the
largest number. In addition, P. tremula (PtaGATA26) and P. trichocarpa (PtrGATA12) show seven, P. euphratica
(PeGATA35) displays six, P. tremula x alba (PtaaGATA36) presents ve, and P. tremuloides (PtsGATA3, 17, 22,
27, 30, 35, and 36) has two. Average numbers of alternative splicing forms of GATA genes range from 2.00 (P.
tremuloides) to 3.43 (P. deltoides). ese dierences can be partially explained by that alternative splicing forms
are controlled via multilayered regulatory network82, however, further studies are required.
Dierences in the number of alternative splicing forms of GATA genes in Populus genus can be caused by
(i) dierent gene prediction programs83–85 and (ii) amount of evidence transcript sequences, covering fully
Table 1. Characteristics of identied GATA TFs from seven Populus genomes. *It shows that total ratio (B/A)
of Populus genomes except P. pruinosa.
Populu s species name Ver sion # of GATA genes(A) # of GATA TFs
# of GATA genes
# of GATA TFs having
forms # of genes # of proteins Ratio (B/A) (%)
Populus trichocarpa 3.1 39 67 13 41 42,950 63,498 33.33
Populus pruinosa 1 37 37 0 0 35,131 35,131 0.00
Populus euphratica 1 40 55 9 24 30,688 49,676 22.50
Populus deltoides 2.1 38 55 7 24 44,853 57,249 18.42
Populus tremuloides 1.1 37 44 7 14 36,830 48,320 18.92
Populus tremula 1.1 33 60 16 43 35,309 83,720 48.48
Populus tremula x alba 1.1 38 71 16 49 41,335 73,013 42.11
Tot a l 262 389 68 195 267,096 410,607 30.22*
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characterized genes, expressed sequences tags (ESTs), and/or RNA-Seq data. In the gene prediction process,
evidence sequences are essential to achieve accurate prediction of genes as well as alternative splicing forms.
More available EST or RNA-Seq sequences will bring plentiful alternative splicing forms of GATA genes: e.g.,
the human genome contains around averagely of 10 alternative splicing forms per one gene, predicted from a
large amount of transcript sequences86. Available RNA-Seq data of Populus genus (As of 2018 Jun) deposited in
NCBI Short Read Archive show that P. trichocarpa and P. tremula presenting a large proportion of alternative
splicing forms of GATA genes contain a large amount of RNA-Seq data (TableS4).
In‑depth investigations of alternative splicing forms of Populus GATA genes. We identied the
phenomena that some alternative splicing forms originated from one Populus GATA gene encode the same
amino acids. Each of the nine alternative splicing forms of PdGATA6, a typical example of this phenomenon, is
composed of 232 aa, 295 aa, and 301 aa in protein length and exon is composed of between 3 and 5. Among the
nine alternative splicing forms of PdGATA6, all except PdGATA6f and 6h present the same start and end posi-
tions of ORFs. e rst ORF exons of the eight alternative splicing forms except PdGATA6f contain start methio-
nine without stop codon are classied into two types: one is 627bp and the other is 645bp. It results in two types
of amino acid sequences from the eight alternative splicing forms, indicating that most of alternative splicing
events are occurred in 5’ and 3’ UTR regions (Fig.2). In addition, GATA domain sequences of eight alternative
splicing forms of PdGATA6 are identical, suggesting that the GATA domain is important to bind DNA.
Interestingly, some of alternative splicing forms of Populus GATA genes display the same amino acids: one
GATA gene from P. tremuloides, four from P. euphratica and P. deltoides, six from P. trichocarpa and P. tremula
x alba, and eight from P. tremula. One of known roles of untranslated regions of messenger RNA is changing
the amount of translated proteins87. e number of TFs will increase or decrease the transcription amount of
target genes, so that these alternative splicing forms may be important to the regulatory network of GATA TFs.
We also identied that some alternative splicing forms of the twelve GATA genes of three Populus species
(P. tremula x alba, P. tremula, and P. tremuloides; TableS5) missed GATA domain which was not included in
the Populus GATA TFs list. Interestingly, GATA TFs without GATA domain can negatively regulate the target
genes by competing with normal GATA TFs88, indicating that these twelve GATA genes containing alternative
splicing forms without domain can also play a role of negative regulators. In addition, one Populus GATA gene,
PtaGATA28 (from P. tremula), has ve out of six alternative splicing forms that missed GATA domain, suggest-
ing that this gene might have a dominant role of negative regulation in contrast to the normal GATA genes even
NC 009143P. trichocarpav3.1
NC 040929 P. deltoides v2.1
NC 024747 P. euphratica v1
NC 037417 P. pruinosa v1
MN561844 P. tremuloidesv1.1
NC 027425 P. tremula v1.1
NC 028504 P. tremula x P. alba v1.1
NC 043878 Salix gracilistyla
# of GATA genes
# of GATA TFs
Figure1. Phylogenetic tree of seven Populus species in complete chloroplast genomes. (a) shows a phylogenetic
tree of seven Populus species in complete chloroplast genomes. Bootstrap analyses with 1000 pseudo-replicates
were conducted with the same options. S. gracilistyla is aligned to the seven Populus genomes as an outgroup.
Light blue and green lines indicate the gene loss of GATA TFs in P. deltoides and P. tremuloides lineage,
respectively. Yellow star means gene duplication events of the 10 paralogous pairs. Black, green, blue letters of
7 Populus genomes mean independent three clades based on phylogenetic studies. (b) presents the number of
GATA genes and GATA TFs for each genome.
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though additional research such as expression level of each alternative splicing forms in various conditions are
required. PtaGATA33 from P. tremula has two out of three, and the rest ten GATA genes have one.
Identication and investigation characteristics of Populus GATA subfamilies. We constructed
a neighbor-joining phylogenetic tree based on amino acid sequences of GATA domains of 389 Populus and 41
Arabidopsis GATA TFs together to identify subfamilies (Fig.3a), resulting those four subfamilies (I to IV; see
Material and Methods; TableS2) were successfully identied. Subfamily I has the largest number of Populus
GATA genes; while subfamily IV contains the smallest number (TableS6) as same as A. thaliana25, V. vinifera89,
and G. max40 except O. sativa81, monocot species. Subfamily III presents the largest average number of alterna-
tive splicing forms (1.81) and subfamily II displays the lowest (1.15; TableS6). GATA domains belonging to sub-
families I, II, and III are located adjacent to the C-terminal; however, those in subfamily IV are at the N-terminal
as same as A. thaliana25, V. vinifera89, G. max40, and O. sativa81.
Amino acid lengths of Populus GATA TFs in each subfamily present a wider range than those of A. thaliana
(Fig.3b). However, three Populus GATA TFs display extremely short lengths: PdGATA18 belonging to subfamily
I is 82 aa, PtsGATA29 and PdGATA36 from subfamily III are 46 and 86 aa, respectively (TableS2). Interest-
ingly, some of GATA TFs of the other plant species including A. thaliana also display the short GATA TFs: 120
aa (AtGATA23) in A. thaliana25, 109 aa (VvGATA13) in V. vinifera89, 80 aa (GmGATA10) in G. max40, and 101
aa (OsGATA8b) in O. sativa81. It indicates that the three shortest Populus GATA TFs may be functional GATA
TFs, suspecting that the gene prediction program can miss some of exons nearby the exon containing the GATA
Two Populus GATA TFs in subfamily II have unique domains in comparison to those of A. thaliana;
PpGATA21 contains NIR domain (IPR005343) found in the Noc2 gene family in Arabidopsis. is domain seems
to be involved in protein–protein interaction, indicating that PpGATA21 may have partner protein for forming
protein complex to regulate target genes. In addition, PpGATA23 covers two HMA domains (IPR006121), which
can bind heavy metal ions90. ese two Populus GATA genes will have unknown additional functions like WC1,
which is involved in circadian clock mechanism ofNeurospora crassa with light-sensing domain91.
: Untranslated region
: Coding region
Chromosome 4 : 21,942,354bp –21,948,242bp
Figure2. Diagram of alternative splicing forms of PdGATA6. It shows the gene structure of the PdGATA6
gene (P. deltoides). Orange thick boxes indicate translated regions and black thick boxes display untranslated
regions. Black thin lines mean intron and black dotted lines are intergenic regions. Green dotted and solid lines
indicate the conserved and dierent structure of GATA genes including exon, intron, and,untranslated regions,
respectively. e number around the boxes display relative positions of translated, untranslated, and exons based
on the start position of PdGATA6b. Names of alternative splicing forms of the PdGATA6 gene are displayed in
the le part of each gene diagram.
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A. thaliana GATA TFs in subfamily III contain two known additional domains: CCT domain (IPR010402)
found in circadian clock and a owering control gene (CONSTANS92) and TIFY domain (IPR010399) to medi-
ate homo- and heteromeric interactions between TIFY proteins and other specic TFs93, 94. In contrast to A.
thaliana25 and G. max40, fourteen GATA TFs in six Populus species except P. euphratica lack CCT and/or TIFY
domains. Some of GATA TFs of O. sativa also presented the same phenomenon81. In detail, nine of the 14 GATA
TFs are the unique transcript, indicating that they completely lost CCT and/or TIFY domains during evolution,
similar to those of V. vinifera89. e remaining ve GATA TFs have other alternative splicing forms, presenting
the selective loss of these domains.
Comparisons of the principal component analysis of seven Populus species. In a previous study,
six Populus species except P. tremula x alba were used for conducting the phylogenetic analysis based on a total
of 76 morphological properties for buds, leaves, inorescences, owers, and fruits95, which is congruent to the
chloroplast phylogenetic tree (Fig.1), except P. trichocarpa and P. deltoides. e incongruency of the two species
is caused by limited species in Fig.1. However, the principal component analysis of Populus GATA genes shows
one cluster covering P. euphratica, P. tremula x alba, P. tremuloides, and P. trichocarpa (Fig.S2), which is incon-
VIylimafbuSIylimafbuSSubfamily IIISubfamily II
Populus A. thaliana Populus A. thaliana Populus A. thaliana PopulusA. thaliana
Unique amino acid
two different amino acids
three different amino acids
four different amino acids
1 5 10 15 20 25 30
P. tremula x
Figure3. Sequence characteristics of Populus GATA TFs. (a) shows that the phylogenetic tree of GATA domain
sequences constructed by the neighbor-joining method. Black triangles on the tree indicate a group of Populus
GATA domains. Names of the Populus GATA genes not condensed were displayed with blue colors. Bootstrap
values calculated from 10,000 replicates are shown on the node except that those are lower than 50. e scale
bar corresponds to 0.050 estimated amino acid substitutions per site. Ranges of subfamilies were presented with
lines on the right side. (b) displays distribution of amino acid length of Populus and A. thaliana GATA TFs. e
length distribution of GATA genes in each subfamily was plotted separated with the dotted lines. e Y-axis
represents an amino acid length of GATA TF. Bold lines mean average length of GATA genes. in lines present
maximum or minimum the length of GATA genes. (c) provides the information of conserved and diversity
splicing forms of GATA genes among six Populus. e X-axis displays a list of PCs. Y-axis indicates Populus
species name. Z-axis shows the ratio of the number of GATA TF per GATA gene. (d) presents the pattern
of amino acid sequence of GATA motif (CX2-4CX18-20CX2C) along with Populus gene clusters (PCs). Yellow,
light green, and dark green shaded alphabets mean two, three, and four dierent amino acids in that position,
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gruent to the aforementioned two phylogenetic trees. It reects that GATA TFs may not evolve in the similar to
species evolution due to their widely regulatory roles96.
Identication of Populus GATA gene clusters (PCs) on phylogenetic tree of Populus GATA
genes. To understand the phylogenetic relationship of Populus GATA genes clearly, we clustered them in the
phylogenetic tree (Fig.3a), resulting in 21 distinct Populus GATA gene clusters (PCs; Fig.3a and TableS7). All
PCs except PC11 containing ve species except P. tremuloides and P. tremula covers all seven Populus species,
displaying conserveness of Populus GATA genes. Eleven of the 21 PCs (52.38%) contain the same amount of
GATA genes for each Populus species, while the rest 9 PCs (42.86%) show dierent numbers (TableS8). PC03,
PC05, and PC14 lack of only one GATA gene from P. pruinosa, P. tremula, and P. deltoides, respectively; while
PC12 and PC20 have additional GATA gene of P. deltoides and P. euphratica, respectively. e remaining 4 PCs
display a complex pattern of the number of GATA genes for each Populus species (TableS8), indicating complex
history of gain and loss of GATA genes in the Populus genus.
Subfamily I, containing the largest Populus GATA genes, displays the most complex structure with the largest
number of PCs (Fig.3a). Interestingly, AtGATA3, AtGATA10, AtGATA11, AtGATA13, AtGATA14, and PC12
do not show neighbor GATA genes like an independent clade (Fig.3a). Subfamily II shows the largest ratio of
the number of PCs to the number of GATA genes, implying faster evolution might be occurred. In addition,
four Populus GATA genes (PpGATA21, PpGATA23, PeGATA19, and PeGATA23) are not clustered into PCs
(Fig.3a), presenting species-specic GATA genes. In subfamily III, PC01, containing four Populus GATA genes
per species, might be experienced a gene duplication event in comparison to the other PCs. In addition, PC01
and PC02 seem to be independent of three Arabidopsis GATA genes (Fig.3a), suggesting Populus-specic GATA
genes, while PC03 and PC04 have their partner Arabidopsis GATA genes (Fig.3a). Subfamily IV covers only one
PC and two Arabidopsis GATA genes, the smallest subfamily (Fig.3a).
Among six Populus species except P. pruinosa, PCs that have a relatively high average of the ratio of the num-
ber of GATA TF per GATA gene are PC01 (2.58), PC12 (2.36), and PC21 (2.17) (Fig.S3), suggesting that GATA
TFs in these PCs may have diverse biological functions, similar to the case of OsGATA2381. In the species level,
PC01 in P. euphratica (3.50) and P. tremula x alba (4.00), PC12 in P. deltoides (3.67) and P. trichocarpa (6.00),
PC04 in P. tremuloides (2.00), and PC18 in P. tremula (4.00) display the high ratio, while ve PCs (PC07, PC10,
PC11, PC15, and PC19) have one GATA TF per GATA gene (Fig.3c). We suspected that GATA TFs of each
Populus species might have dynamic features of their functional diversication, including subfunctionalization
In total, 23 out of 556 (4.14%) amino acid positions in the CX2-4CX18-20CX2C region (inter-species variations)
show more than one amino acid (Fig.3d), which is larger than that of 19 A. thaliana genomes43 (intraspecic
variations; 0.93%). Positions of variable amino acids in the CX2-4CX18-20CX2C region are scattered throughout
this region along with PCs (Fig.3d). Most variable positions are 9th in PC01 (four amino acids) and 21st in
PC15 (three amino acids; Fig.3d); while the maximum number of dierent amino acids in Arabidopsis GATA
genes is 243.
Genome‑wide inference of GATA TF functions based on characterized A. thaliana GATA
TFs. Till now, biological functions of one Populus80 and 15 A. thaliana GATA TFs have been characterized43.
Nine of 15 characterized Arabidopsis GATA genes were also successfully mapped based on the PCs (Fig.3a and
Table2) and similarity of amino acids, resulting in that seven PCs are related to the characterized Arabidopsis
GATA genes. 129 Populus GATA TFs in the seven PCs are candidates for deducing their functional roles in
Populus. In addition, PdGNC from P. n ig ra x (P. deltoides x P. n ig ra), a uniquely characterized GATA TF, known
to regulate chloroplast ultrastructure, photosynthesis, and vegetative growth in Arabidopsis80 is successfully
mapped to PtrGATA19 (PC09) with 98.02% amino acid similarity. It supports this inference method because
both GNC in Arabidopsis and PdGNC are in the same PC with the similar functions even though Arabidopsis
and Populus belong to Brassicaceae and Salicaceae and are an herb and a tree species, respectively. Moreover,
OsGATA12 involved in the seedling stage based on expression prole, is similar to that of BME3 in A. thaliana81.
Based on this result, researchers can eciently and systematically identify the biological functions of Populus
GATA genes in the near future.
Expression level analysis of GATA genes in P. deltoides and P. pruinosa. Based on available RNA-
Seq raw reads obtained from leaf, phloem, xylem, and root tissues of P. deltoides and P. pruinosa (TableS9),
expression levels of Populus GATA TFs were calculated (Fig.4). In the four tissues, three GATA genes in the PC9
were well clustered displaying leaf and phloem specic expressions (Fig.4a), which is congruent to the putative
functions of GATA genes in PC9, such as regulation of chloroplast development, growth, and divisions (Table2).
In addition, four clusters covering GATA genes in PC13, PC1, PC6, and PC5, also presented similar expression
patterns across the tissues, but these clusters did not cover all members in each PC. PC1 and PC5 showed high
expression in all four tissues; while PC13 was leaf and phloem specic and PC6 was expressed lowly, especially
in xylem, which can be a clue to understand their biological functions due to lack of homologous genes of which
biological functions were characterized. Expression proles of each tissue exhibited that some clustered GATA
genes from the same PC were same as those in the four tissues and the rest were not (Fig.4b–e), showing that
expression level of Populus GATA genes partially reects their conserveness across the species.
Domain types of Populus GATA genes. e DNA-binding motif of GATA TFs was classied into three
types designated as type IVa (CX2CX17CX2C), IVb (CX2CX18CX2C), and IVc (CX2CX20CX2C) among which Type
IVb and IVc are common in plants25, 40, 81, 89. Additional types, including type IVp (p indicates partial; mentioned
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Table 2. Function inference of Populus GATA gene clusters (PCs) based on characterized A. thaliana GATA
PC name GATA gene Biological functions Subfamily References
PC03 AtGATA25 Hypocotyl and petiole elongation III 50
PC04 AtGATA28 (ZML2) Mediation of cryptochrome1-dependent response 133
PC08 AtGATA15 (GATA15) Cytokinin-regulated development, including greening, hypocotyl elongation, phyllotaxy, oral organ initiation,
accessory meristem formation, owering time, and senescence
PC09 AtGATA21 (GNC)
a nitrate‐inducible member important for chlorophyll synthesis and glucose sensitivity 136
Modulation of chlorophyll biosynthesis (greening) and glutamate synthase (GLU1/Fd-GOGAT) expression 137, 138
Downstream eectors of oral homeotic gene action by controlling two MADS-box TFs 139
Control of convergence of auxin and gibberellin signaling 140, 141
Control of greening, cold tolerance, and owering time 142
Regulation of chloroplast development, growth, and division as well as photosynthetic activities 143, 144
Cytokinin-regulated development, including greening, hypocotyl elongation, phyllotaxy, oral organ initiation,
accessory meristem formation, owering time, and senescence 135
PIF- and light-regulated stomata formation in hypocotyls 145
PC10 AtGATA18 (HAN)
Regulation of shoot apical meristem and ower development 145–149
Stable establishment of cotyledon identity during embryogenesis 148
Position the proembryo boundary in the early Arabidopsis embryo 149
PC19 AtGATA2 (GATA-2)
Regulation of light-responsive genes I 45
PC20 AtGATA1 (GATA-1)
Figure4. Heatmap of GATA genes in four tissues of P. deltoides and P. pruinosa. Dendrograms at the le side of
heatmaps are the result of hierarchical clustering of each expression data using hclust function in R package stats
version 4.0.3. Heatmaps present expression levels based on FPKM values calculated by cuink with gradient
colors from blue to red displayed in the legend on the right side. Dendrograms at the le side of heatmaps
are the result of hierarchical clustering of each expression data. Labels consist of GATA gene and PC names
separated with ‘:’. Green dotted boxes indicate the case that some of members in the same PC were clustered.
Blue dotted boxes mean that all members in the PC were clustered together. (a) displays heatmap of GATA
genes of two Populus species in the four tissues, (b)–(e) are for heatmaps in leaf, phloem, xylem, root tissues.
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in Arabidopsis43 and G. max GATA TF analyses25) and type IV4 (four amino acids between the rst two cysteines,
which seems to be functionally active43), were also found.
In Populus GATA TFs, type IVb is the most abundant (272 of 389; 69.92%), and type IVc is the second (102;
26.22%). Type IVb, a common type of DNA-binding motifs of plant GATA TFs, occupies the largest proportion
in Populus GATA TFs and is found in subfamilies I, II, and IV and Type IVc, the second largest, is a common in
subfamily III of Populus GATA TFs, which is similar to those of A. thaliana, G. max, V. vinifera, and O. sativa.
Type IV4 is found only in eight Populus GATA TFs (2.06%) belonging to subfamily II. It was also identied in
some species including A. thaliana and G. max, suggesting that this type was independently occurred during
evolution by adding two amino acids only in the rst two cysteines of subfamily II. In contrast, type IVp is found
in all subfamilies of Populus GATA TFs as well as those of A. thaliana, G. max, and O. sativa, suggesting that
random modications of GATA domains of all subfamilies have been occurred during evolution. Interestingly,
type IVp can be generated by alternative splicing forms: PtaaGATA36d is GATA TF displaying type IVp; while
the rest four alternative splicing forms of PtaaGATA36 show type IVc domain, which is similar to the cases of
OsGATA881 and AtGATA26. Type IVp was also considered as an ancestral form of GATA zinc nger40, requiring
more research of Type IVp with additional GATA genes from many plant genomes.
Amino acid patterns of GATA domains in seven Populus species and A. thaliana. Amino acid
sequences of 422 GATA domain from 382 Populus and 40 A. thaliana GATA TFs excluding seven type IVp
domains of Populus and one Arabidopsis were used for multiple sequence alignment (Fig.5a). Subfamily IV of
Populus displays the most conserved manner (49 of 55 conserved amino acids are identical.) in their domains,
while subfamily II of Populus shows the least conserveness (18 of 66 conserved amino acids). Considering with
the number of Populus GATA genes in each subfamily, subfamily I, the largest subfamily, is more conserved than
subfamilies II and III. Some of dominant amino acids in GATA domain are dierent among Populus species (e.g.,
the 5’ region of GATA domains; Fig.5a).
Subfamily IV displays incongruent of conserved amino acids between Populus and A. thaliana at 8th and 47th
(Fig.5a; blue-colored transparent boxes), indicating that subfamily IV was evolved and stabilized in early stage.
In addition, six, one, and two amino acids which are 100% conserved in Populus genus but not in A. thaliana are
found in subfamilies I, II, and IV, respectively (Fig.5a; red-colored transparent boxes), suggesting that subfamily
I has been most diversied in the lineage of A. thaliana.
Twelve conserved amino acids of Populus and A. thaliana belonging to the zinc finger motif
(CX2-4CX18-20CX2C) are identical in all subfamilies (Fig.5a) suggesting that the zinc nger motif is the most
conserved and important region in the GATA domain. 22nd and 28th conserved amino acids in subfamilies I,
II, and IV are Tryptophan and Glycine, respectively; while subfamily III displays methionine and glutamic acid
(Fig.5a). ese dierences can be key factors to classify four subfamilies.
As expected, the zinc nger motif, which can bind to DNA and is the most important region in GATA domain,
contains a smaller number of dierent amino acids (Fig.5b). Despite of large number of species in Populus genus
used in this study, four positions in this region show a high number of amino acids in A. thaliana (Fig.5b), sug-
gesting that selection pressures have been dierently applied in the two lineages. is phenomenon is also found
outside this region (Fig.5b). It is congruent to the ndings described in the previous paragraph. Once more
plant genomes including the large number of resequencing data of A. thaliana and P. trichocarpa are analyzed,
the detailed evolutionary history of the GATA domain will be uncovered.
Identication of transmembrane helix (TMH) of GATA gene family in seven Populus
genomes. Membrane-bound transcription factors (MTFs) are docked in cellular membranes using their
transmembrane domains100. MTFs have usually been found in plant species101 of which are related to seed
germination102, cell division101, heat stress103, and salt stress104. Mechanisms of MTFs are well known in two
major plant TF families: NAC TF family and bZIP TF family105 (Fig.S4). NTL6 (NAC TF) of A. thaliana is
localized in the plasma membrane under normal conditions; while under stress conditions, NTL6 is processed
by an as-yet-unidentied intramembrane protease and SnRK2.8 kinase phosphorylates NTL6 and facilitates its
nuclear import105. (ii) Intracellular movement of Arabidopsis bZIP60 and bZIP28 was characterized105. bZIP60
and bZIP28, which are other MTFs in Arabidopsis, were localized on the membrane of the endoplasmic reticu-
lum and then transported to nucleus by cleaving TMH.
Five Populus GATA TFs were identified with TMHs predicted by TMHMM106. PtrGATA14b, 14c (P.
trichocarpa) in subfamily I, PpGATA21, 25 (P. pruinosa), and PtaaGATA23 (P. tremula x alba; TableS10), belong-
ing to subfamily II. ese putative GATA MTFs have one TMH, which is the same as the previously characterized
Arabidopsis MTFs107. As far as we know, this is the rst time to report putative GATA MTFs in plant species;
additional putative GATA MTFs were also identied in other plant species using our pipeline: VvGATA19 (V.
vinifera; subfamily IV)89, GmGATA39 (G. max; subfamily I)40, OsGATA14 (O. sativa; subfamily II)81 with one
TMH, while no GATA MTF was found in A. thaliana25. It is interesting that there are no common subfamilies
containing putative GATA MTFs along with dierent species. In addition, some alternative splicing forms of
PtrGATA14 (P. trichocarpa) have TMH, indicating that truncation of TMH region by alternative splicing may
switch their functions by changing subcellular localization of GATA TFs, similar to the bZIP60 in A. thaliana105.
With accumulating more data including expression proles and subcellular localization, roles of these putative
GATA MTFs can be uncovered. Moreover, these results can be a corner stone to understand plant GATA MTFs
together with a large number of plant genomes available now108–110 in a broad taxonomic range of plant species.
Chromosomal distribution of P. trichocarpa, P. tremula x alba, and P. deltoides GATA gene
family. Chromosomal distribution of Populus GATA genes from the three species, P. trichocarpa, P. tremula x
Scientic Reports | (2021) 11:16578 |
alba, and P. deltoides belonging to the same clade (Fig.6), presents several important features: (i) 38 of 39 GATA
genes in P. trichocarpa, 37 out of 38 in P. tremula x alba, and 36 of 38 in P. deltoides were distributed on 15 of
19 chromosomes (Fig.6), presenting similar chromosomal distribution among three species. (ii) Chromosome
5 in both species contains the largest number of GATA genes; while chromosomes 9, 13, and 19 in both spe-
cies contain the smallest (Fig.6). is biased chromosomal distribution was also found in many plant species
including A. thaliana25, V. vinifera89, G. max40, O. sativa81. (iii) In the three species, chromosome 7 shows the
highest density of GATA genes in both species; and chromosome 5 is a second rank. (iv) Most of GATA genes of
three species are in the same PCs and in similar chromosomal position (Fig.6) except the four genes of which
chromosomal positions are not assigned (See ChrUn in Fig.6). It indicates that there might be no chromosomal
rearrangement events and biological functions of GATA genes may have similar functions among the three spe-
cies. Interestingly, three of the four genes are additional copy of GATA TFs in PC12 and PC17, which is the result
of independent gene gain events.
Based on paralogous GATA TFs of P. trichocarpa identied in the previous study111, 10 paralogous pairs were
successfully mapped to GATA TFs from three Populus species (Fig.6), displaying that all paralogous pairs contain
three GATA TF from each species in the similar chromosomal positions, indicating that gene duplication events
of the 10 paralogous pairs were occurred before speciation of three Populus species (see yellow star in Fig.1a).
1 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75
Number of diﬀerent amino acids
Locaon that Arabidopsis has more form than Populus
CX2-4CX18-20CX2C region Amino acid
Amino acid conserved: 100%
Amino acid conserved: less than 100%
β2 β3 β4
Figure5. Conserved amino acids of GATA domain along with subfamilies and genera (A. thaliana and
Populus). (a) shows the conserved amino acid in each position of the GATA domain. Yellow shaded characters
mean 100% conserved amino acids and gray background color in amino acid indicate that there are gaps
in the position. SI, SII, SIII, and SIV are shortened forms of subfamily I, II, III, and IV, respectively. Blue-
colored transparent boxes show incongruent of conserved amino acids between the two species. Red-colored
transparent boxes present amino acids which are 100% conserved in the Populus genus but not in A. thaliana.
(b) e X-axis indicates each amino acid position of the aligned amino acids of GATA domain and Y-axis
displays the number of dierent amino acids in the specic position of aligned GATA domain of Populus (blue
line) and A. thaliana (orange line). Red dots on the graph mean position where A. thaliana has more dierent
amino acids than that of Populus. e blue-colored transparent box presents the CX2CX18-20CX2C region.
Scientic Reports | (2021) 11:16578 |
In addition, three PCs, PC14 (PtrGATA15 and PtaaGATA8 in chromosome 5), PC02 (PtrGATA35 and
PdGATA36 in chromosome 17), and PC09 (PtrGATA37 and PtaaGATA28 in chromosome 18), have not complete
set of Populus GATA TFs. PC14 and PC09 suggest the loss event of GATA genes in the lineage of P. deltoides
(see the light blue line in Fig.1). PC02 indicates another loss event occurred in the lineage of P. tremuloides (See
green lines in Fig.1). Taken together with the incongruency inferred from the PCA, GATA TFs were evolved
with the events occurred in various lineages in Populus species, which is independent to their species evolution.
Using the identication pipeline of GATA TFs in the GATA-TFDB, we successfully identied 262 GATA genes
(389 GATA TFs) from seven Populus species. Alternative splicing forms of Populus GATA genes display the
high number of alternative splicing forms (nine is maximum) with only changes in untranslated regions and
loss of DNA-binding motif or additional domains. Populus GATA genes were classied into the four subfamilies,
same to Arabidopsis GATA genes, except that some genes in subfamily III lack CCT and/or TIFY domains. 21
Populus GATA gene clusters (PCs) were identied from the phylogenetic tree of GATA domain sequences and
20 of 21 PCs cover the seven Populus species, displaying the conserveness of Populus GATA genes. Distribution
of alternative splicing forms in the PCs exhibits the possibility of subfunctionalization and neofunctionalization
of Populus GATA genes. rough the expression analysis of GATA genes of two Populus species, the ve PCs
which display similar expression patterns across the four tissues were identied for predicting their biological
functions. Populus-specic conserved amino acids in the GATA domain were discovered in comparison to A.
thaliana, suggesting a complex evolutionary history of the GATA domain. Five Populus GATA TFs contain one
transmembrane helix (TMH), which is the rst report of membrane-bound GATA TFs. Together with the biased
distribution of GATA genes across the chromosomes, paralogous pairs of GATA genes suggested several gene
duplication events in the lineages of Populus genus. Taken together, our rst comprehensive analyses of genus-
wide GATA TFs in plants successfully provide characteristics of Populus GATA genes across the seven species
as well as their putative functions and evolutionary traits of Populus GATA genes.
Figure6. Chromosomal distribution of P. trichocarpa, P. tremula x alba, and P. deltoides GATA genes. Black,
purple, and yellow bars indicate P. trichocarpa, P. tremula x alba, and P. deltoides chromosomes, respectively.
Black, purple, yellow letters mean GATA gene names of P. trichocarpa, P. tremula x alba, and P. deltoides. ChrUn
present scaold sequences which are not assigned to any chromosomes. Yellow-colored transparent boxes
indicate 10 paralogous pairs.
Scientic Reports | (2021) 11:16578 |
Materials and methods
Collecting Populus genome sequences from various sources. We utilized the seven Populus
genomes sequences deposited from the Populus Comparative Genome Database108–110 (http:// www. popul usgen
ome. info/), which adopted-whole genome sequences from the Plant Genome Database (http:// www. plant
genome. info/; Park etal., in preparation). ese genomes were originated from the NCBI genome database
(http:// genome. ncbi. nlm. nih. gov/) and Phytozome (http:// www. phyot ozome. info/)112 in the standardized form
of genome sequences provided by the GenomeArchive® (http:// www. genom earch ive. info/)113.
Identifying GATA TFs from whole Populus genome sequences. Amino acid sequences from seven
Populus genomes were subjected to InterProScan114 to identify GATA TFs. e pipeline for identifying Populus
GATA TFs implemented at the GATA-TFDB (http:// gata. genef amily. info/; Park etal., in preparation), which is
an automated pipeline for identifying GATA TFs with GATA DNA-binding motif InterPro term (IPR000679)
and post-process to lter-out false positive results and for analyzing various analyses including domain sequence
analysis, gene family analysis, as well as phylogenetic analysis. GATA-TFDB was constructed and maintained as
one of members of the Gene Family Database (http:// www. genef amily. info/; Park etal., in preparation).
Exon structure and alternative splicing forms of Populus GATA TFs. Based on the Populus Com-
parative Genome Database (http:// www. popul usgen ome. info/; Park etal., in preparation), exon structure and
alternative splicing forms of GATA TFs were retrieved. Diagrams of exon structure and alternative splicing
forms of GATA TFs were drawn primarily based on the diagram generated by the GATA-TFDB (http:// gata.
genef amily. info; Park etal., in preparation) with adding additional information manually.
Construction of phylogenetic tree of Populus GATA TFs. Phylogenetic trees were constructed with
a Neighbor-joining method with bootstrap option (10,000 repeats) by ClustalW 2.1115 based on the alignment
of amino acids of GATA domains obtained from the GATA-TFDB (http:// gata. genef amily. info; Park etal., in
preparation) also by ClustalW 2.1115.
Chromosomal distribution of Populus GATA TFs. We drew the chromosomal distribution map of Pop-
ulus GATA genes from three species based on the chromosomal coordination from their pseudo-molecule level
assemblies deposited in the Plant Genome Database (http:// www. plant genome. info/).
Prediction of transmembrane helixes on Populus GATA TFs. Transmembrane helixes on Populus
GATA TFs were predicted by TMHMM106 under the environment of the Plant Genome Database (http:// www.
plant genome. info/).
Principal component analysis of Populus GATA TFs. Principal component analysis (PCA) was con-
ducted based on 19 characteristics of GATA genes using prcomp function in R package stats version 4.0.3116. e
result was visualized into a scatterplot including variance, with the rst two principal components.
Expression analysis of GATA TFs based on Populus RNA‑seq data. Raw reads of RNA-Seq experi-
ments of P. pruinosa and P. deltoides were downloaded from NCBI (TableS9). RNA-Seq raw reads were aligned
against the whole genome of P. pruinosa and P. deltoides with hisat2 v2.2.0117 aer generating datasets of each
Populus genome. Aer generating bam le for each SRA raw reads, bam les from the same experiments were
merged using samtools v1.9118. Expression levels of the merged bam les were calculated by cuink v2.2.1119.
Hierarchical clustering was conducted for the ve datasets: one covers four dierent conditions (four tissues)
and the last four contain each condition (Fig.4) using hclust function in R package stats version 4.0.3116.
Construction of phylogenetic tree of seven Populus species based on complete chloroplast
genomes. Complete chloroplast genomes of seven Populus chloroplast genomes69, 120–123 and Salix gracil-
istyla78, used as an outgroup, were aligned using MAFFT v7.450124. All chloroplast genome sequences were
retrieved from the PCD (http:// www. cp- genome. net; Park etal., in preparation). e maximum-likelihood trees
were reconstructed in MEGA X125. During the ML analysis, a heuristic search was used with nearest-neighbor
interchange branch swapping, the Tamura-Nei model, and uniform rates among sites. All other options were set
to their default values. Bootstrap analyses with 1,000 pseudoreplicates were conducted with the same options.
All bioinformatic processes were conducted under the environment of the Genome Information System (GeIS)
used in the various previous studies43, 126–134.
All GATA TFs identied in this study can be accessed at the Populus Comparative Genome Database (http://
www. popul usgen ome. info/).
Received: 2 March 2021; Accepted: 2 August 2021
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is study was supported by InfoBoss Research Grant (IBB-001) to JP.
J.P. designed this manuscript and M.K. and H.X. identied and GATA TFs and analyzed them. M.K. curated
GATA TFs. M.K., S.P., and Y.Y. visualized the analyzed data. M.K. and J.P. wrote the original manuscript and all
authors improved the manuscript. All authors read and approved the nal dra of the manuscript.
e authors declare no competing interests.
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 021- 95940-5.
Correspondence and requests for materials should be addressed to J.P.
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