# Genome-Wide Analysis of theTCP Gene Family in Switchgrass (Panicum virgatum L.)

Authors: Yuzhu Huo; Wangdan Xiong; Kunlong Su; Yu Li; Yawen Yang; Chunxiang Fu; Zhenying Wu; Zhen Sun
Journal: International Journal of Genomics
 (2019)

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## Abstract

The plant-specific transcription factor TCPs play multiple roles in plant growth, development, and stress responses. However, a genome-wide analysis of TCP proteins and their roles in salt stress has not been declared in switchgrass (Panicum virgatum L.). In this study, 42 PvTCP genes (PvTCPs) were identified from the switchgrass genome and 38 members can be anchored to its chromosomes unevenly. Nine PvTCPs were predicted to be microRNA319 (miR319) targets. Furthermore, PvTCPs can be divided into three clades according to the phylogeny and conserved domains. Members in the same clade have the similar gene structure and motif localization. Although all PvTCPs were expressed in tested tissues, their expression profiles were different under normal condition. The specific expression may indicate their different roles in plant growth and development. In addition, approximately 20 cis-acting elements were detected in the promoters of PvTCPs, and 40% were related to stress response. Moreover, the expression profiles of PvTCPs under salt stress were also analyzed and 29 PvTCPs were regulated after NaCl treatment. Taken together, the PvTCP gene family was analyzed at a genome-wide level and their possible functions in salt stress, which lay the basis for further functional analysis of PvTCPs in switchgrass.


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## Body

## 1. Introduction

TheTCP gene family is a class of plant-specific genes encoding proteins with the conserved TCP domain, a 59 amino acid motif that allows DNA binding and protein interaction. The so-called “TCP” is named from four initially identified transcription factors: TEOSINTE BRANCHED1 (TB1) from maize (Zea mays), which involved in apical dominance regulation [1, 2]; CYCLOIDEA (CYC) from snapdragon (Antirrhinum majus), which controlled floral asymmetry [3]; and the PROLIFERATING CELL FACTORS 1 and 2 (PCF1 and PCF2) from rice (Oryza sativa), which are essential for meristematic tissue-specific expression [4]. To date, TCP genes have been identified in a number of plant species. For example, there are 24 TCP members that were found in Arabidopsis (Arabidopsis thaliana) genome, and 28 in rice genome, 30 in tomato (Solanum lycopersicum), 21 in medicago (Medicago truncatula), 36 in poplar (Populus trichocarpa), and 39 in turnips (Brassica rapa ssp. rapa) [5–10]. TCP proteins can be divided into two main classes according to their sequences of TCP conserved domain and phylogenetic relationships, which were referred to as class I (also called PCF class or TCP-P class) and class II (also named as TCP-C class) [11, 12]. In angiosperms, class II can be further classified into two clades based on their differences within the TCP domain, which were named as clade CYC/TB1 and clade CIN (CINCINNATA) [5].TCP proteins play vital roles in plant growth, development, and responses to biotic/abiotic stresses [5, 13]. Class I TCP members were mainly involved in promoting cell proliferation and differentiation by regulating plant hormone signaling, such as gibberellin, auxin, cytokinin, and abscisic acid [13–18]. Class II TCP members were approximately reported to participate in lateral organ development. Furthermore, the origin of clade CYC/TB1 members has occurred later than clade CIN members in angiosperms, and they are primarily involved in shoot branching and apical dominance regulation [5]. TB1 functions as a transcriptional regulator of strong apical dominance and controls the tillering in maize [2]. AtTCP18 (BRANCHED1, BRC1) and AtTCP12 (BRANCHEND2, BRC2) in Arabidopsis, two orthologs of maize TB1, are highly expressed in axillary buds and negatively regulate shoot branching [19, 20]. Additionally, jaw-TCPs, the targets of miR319 are almost a cluster of CIN members, and microRNA319- (miR319-) targeted TCPs take part in plant cell wall biosynthesis, abiotic stress response, and flowering time regulation in Arabidopsis and rice [21–23]. It was also reported that miR319-targeted TCPs play a role in plant response to salt stress in bentgrass [24, 25]. Besides, some of the TCP genes in Phaseolus vulgaris which are identified can respond under salt stress [26]. However, the regulation mechanism of TCP transcriptional factors involved in the salt stress has not been elucidated.Switchgrass (Panicum virgatum L.) is a perennial C4 warm-season tall grass used as a bioenergy and animal feedstock for its impressive biomass yield and can confer tolerance to drought, salinity, and poor nutrition [27]. Due to the recent study on high-throughput genome sequencing and assembling, establishment of gene expression atlas, genetic-linkage mapping, and high-efficiency transformation system [28, 29], switchgrass has been developed into the model species as energy grass. The function of numerous genes in switchgrass has been gradually clarified, especially on stress response and development regulation. Until now, WRKY, CCCH, SPL, and ARF gene families had been comprehensively analyzed at the whole-genome level in switchgrass [30–33]. Furthermore, transcriptome microRNAs and long noncoding RNAs exposed to drought stress had been sequenced and analyzed to study the systematic regulatory mechanism of drought response in switchgrass [34–36].Large amounts of switchgrass will be cultivated on marginal land to avoid competing with food crops for the use of arable fields. Thus, switchgrass regularly faces adverse growth conditions, such as salinity, drought, and extreme temperatures. Analysis has indicated that theTCP gene family can respond to salt tolerance, while still little is known about the response of TCP genes in switchgrass under a salt stress condition [24, 25]. In this study, a total of 42 TCP members were identified in the switchgrass genome. Genome-wide analysis was carried out, including biochemical characterization, phylogenetic analysis, gene structure arrangement, chromosome location, expression profiles of tissue-specific pattern, and responsive pattern under salt stress. Therefore, this work would help us to study the profound functions of the PvTCPs in the future.


## 2. Materials and Methods

### 2.1. Sequence Retrieval and Identification of PvTCPs

The hidden Markov model (HMM) profile of the conserved TCP domain (pfam06507) was retrieved from the Pfam protein family database (http://pfam.sanger.ac.uk/) and used as a query for BLAST searches against the switchgrass genome database in Phytozome v12.0 (Panicum virgatum v4.0, DOE-JGI, http://phytozome.jgi.doe.gov/). The candidates were selected for further analysis if the E value was less than 1e-10. Subsequently, we corrected some errors in annotation of TCP coding sequences on the basis of the switchgrass unitranscript (PviUTs) database (https://switchgrassgenomics.noble.org/) [28]. Finally, all putative PvTCPs were confirmed to be TCP proteins by the Pfam program (http://pfam.xfam.org/), and the peptide length, molecular weight, and isoelectric point parameters of each PvTCP were calculated by the online ExPASy program (https://www.expasy.org/tools/).


### 2.2. Chromosomal Location and Gene Duplication ofPvTCPs

The lowland switchgrass cultivar, Alamo, is allotetraploid (2n=4x=36) and consists of two highly homologous subgenomes, designated as ChrN and ChrK (Panicum virgatum v4.0, DOE-JGI, http://phytozome.jgi.doe.gov/). The chromosomal location of each PvTCP was completed using MapChart2.2 based on the physical map in Phytozome v12.0 [37]. Tandem gene duplication was defined as paralogous genes located within 50 kb in tandem and was separated by fewer than five nonhomologous spacer genes [38].


### 2.3. Phylogenetic Analysis of the TCP Proteins

To comprehensively analyze the evolutionary relationships of the TCP proteins in switchgrass, we used putative PvTCPs along with TCP proteins fromArabidopsis (model species of dicots) and rice (model species of monocots) to construct a phylogenetic tree. Sequences of the Arabidopsis and rice TCP proteins were retrieved from TAIR (https://www.arabidopsis.org/) and rice genome database (http://rice.plantbiology.msu.edu/), respectively. Clustal X1.83 was used to do the multiple alignment of the selected TCPs [39]. The neighbor-joining tree (bootstrap value=1000) was constructed using MEGA5.0 [40] and then manually improved by the online program EvolView (http://www.evolgenius.info/evolview/).


### 2.4. Gene Structure, Conserved Motif, andcis-Acting DNA Element Analysis

The exon/intron structure ofPvTCPs was determined by comparing the coding sequences and corresponding genomic sequences in the Gene Structure Display Server (GSDS, http://gsds1.cbi.pku.edu.cn/) [41]. Conserved motifs were analyzed using the MEME program (http://meme-suite.org/) [42]. The cis-acting DNA element analysis was performed in the promoter sequences (2 kb upstream region) of the PvTCPs using the online program PLACE (a database of plant cis-acting regulatory DNA elements, https://sogo.dna.affrc.go.jp/). Ka/Ks calculation was analyzed by PAL2NAL [43].


### 2.5. Preparation for Plant Materials

Switchgrass cultivar the lowland Alamo (introduced from the USA and domesticated at Qingdao, China) was used as inbred line for the study. Tissue-cultured seedlings of switchgrass, which can eliminate the interference of genetic background, were subjected to salt stress (about vegetative 3 stage) [44, 45]. During the treatment, 1/2 MS medium supplied with 250 mM NaCl was irrigated [31]. The seedlings irrigated with 1/2 MS medium were regarded as control. Shootings were harvested from three seedlings for each point, and the collection was repeated three times as biological replicates. Samples were frozen immediately in liquid nitrogen and stored at −80°C prior to analysis.


### 2.6. Expression Pattern Analysis ofPvTCPs

Each of the PvTCPs’ transcript sequence was used as a query to blast against the public database of switchgrass (https://switchgrassgenomics.noble.org/). The expression data of spatiotemporal patterns were retrieved, and pretty heatmap was constructed using the online program ImageGP (http://www.ehbio.com/ImageGP/). Total RNA of samples were extracted using the TRIzol method (Invitrogen Life Technologies, USA). The isolated RNA was subsequently treated with RNase-Free DNase I (Roche, http://www.roche.com). The first-strand cDNA was synthesized from 1 μg of total RNA of each sample, using M-MLV reverse transcriptase (TaKaRa, http://www.takarabiomed.com.cn/) according to the protocol. The primers used in this study were showed in Table S1. PvUBQ (GenBank accession number: HM209468) was used as the reference gene. qRT-PCR was performed with real-time PCR system (LightCycler 480) using TB Green Premix EX Taq II kit (TaKaRa, Japan) and the methods described in the previous study [32]. Each PCR assay was run in triplicate for three independent biological repeats.


## 2.1. Sequence Retrieval and Identification of PvTCPs

The hidden Markov model (HMM) profile of the conserved TCP domain (pfam06507) was retrieved from the Pfam protein family database (http://pfam.sanger.ac.uk/) and used as a query for BLAST searches against the switchgrass genome database in Phytozome v12.0 (Panicum virgatum v4.0, DOE-JGI, http://phytozome.jgi.doe.gov/). The candidates were selected for further analysis if the E value was less than 1e-10. Subsequently, we corrected some errors in annotation of TCP coding sequences on the basis of the switchgrass unitranscript (PviUTs) database (https://switchgrassgenomics.noble.org/) [28]. Finally, all putative PvTCPs were confirmed to be TCP proteins by the Pfam program (http://pfam.xfam.org/), and the peptide length, molecular weight, and isoelectric point parameters of each PvTCP were calculated by the online ExPASy program (https://www.expasy.org/tools/).


## 2.2. Chromosomal Location and Gene Duplication ofPvTCPs

The lowland switchgrass cultivar, Alamo, is allotetraploid (2n=4x=36) and consists of two highly homologous subgenomes, designated as ChrN and ChrK (Panicum virgatum v4.0, DOE-JGI, http://phytozome.jgi.doe.gov/). The chromosomal location of each PvTCP was completed using MapChart2.2 based on the physical map in Phytozome v12.0 [37]. Tandem gene duplication was defined as paralogous genes located within 50 kb in tandem and was separated by fewer than five nonhomologous spacer genes [38].


## 2.3. Phylogenetic Analysis of the TCP Proteins

To comprehensively analyze the evolutionary relationships of the TCP proteins in switchgrass, we used putative PvTCPs along with TCP proteins fromArabidopsis (model species of dicots) and rice (model species of monocots) to construct a phylogenetic tree. Sequences of the Arabidopsis and rice TCP proteins were retrieved from TAIR (https://www.arabidopsis.org/) and rice genome database (http://rice.plantbiology.msu.edu/), respectively. Clustal X1.83 was used to do the multiple alignment of the selected TCPs [39]. The neighbor-joining tree (bootstrap value=1000) was constructed using MEGA5.0 [40] and then manually improved by the online program EvolView (http://www.evolgenius.info/evolview/).


## 2.4. Gene Structure, Conserved Motif, andcis-Acting DNA Element Analysis

The exon/intron structure ofPvTCPs was determined by comparing the coding sequences and corresponding genomic sequences in the Gene Structure Display Server (GSDS, http://gsds1.cbi.pku.edu.cn/) [41]. Conserved motifs were analyzed using the MEME program (http://meme-suite.org/) [42]. The cis-acting DNA element analysis was performed in the promoter sequences (2 kb upstream region) of the PvTCPs using the online program PLACE (a database of plant cis-acting regulatory DNA elements, https://sogo.dna.affrc.go.jp/). Ka/Ks calculation was analyzed by PAL2NAL [43].


## 2.5. Preparation for Plant Materials

Switchgrass cultivar the lowland Alamo (introduced from the USA and domesticated at Qingdao, China) was used as inbred line for the study. Tissue-cultured seedlings of switchgrass, which can eliminate the interference of genetic background, were subjected to salt stress (about vegetative 3 stage) [44, 45]. During the treatment, 1/2 MS medium supplied with 250 mM NaCl was irrigated [31]. The seedlings irrigated with 1/2 MS medium were regarded as control. Shootings were harvested from three seedlings for each point, and the collection was repeated three times as biological replicates. Samples were frozen immediately in liquid nitrogen and stored at −80°C prior to analysis.


## 2.6. Expression Pattern Analysis ofPvTCPs

Each of the PvTCPs’ transcript sequence was used as a query to blast against the public database of switchgrass (https://switchgrassgenomics.noble.org/). The expression data of spatiotemporal patterns were retrieved, and pretty heatmap was constructed using the online program ImageGP (http://www.ehbio.com/ImageGP/). Total RNA of samples were extracted using the TRIzol method (Invitrogen Life Technologies, USA). The isolated RNA was subsequently treated with RNase-Free DNase I (Roche, http://www.roche.com). The first-strand cDNA was synthesized from 1 μg of total RNA of each sample, using M-MLV reverse transcriptase (TaKaRa, http://www.takarabiomed.com.cn/) according to the protocol. The primers used in this study were showed in Table S1. PvUBQ (GenBank accession number: HM209468) was used as the reference gene. qRT-PCR was performed with real-time PCR system (LightCycler 480) using TB Green Premix EX Taq II kit (TaKaRa, Japan) and the methods described in the previous study [32]. Each PCR assay was run in triplicate for three independent biological repeats.


## 3. Results

### 3.1. Identification and Chromosomal Location ofPvTCPs

To identify TCP proteins in switchgrass, the hidden Markov model (HMM) profile of the conserved TCP domain (pfam03634) was used as a blast query to search against the public available switchgrass genome database (Phytozome v12). A total of 42 putative TCP members were identified, which were named as PvTCP1 to PvTCP42 according to their chromosomal location (Figure1; Table 1). In general, 90.5% (38 out of 42) of PvTCPs are anchored onto the chromosomes, while the other four genes are located on an unmapped region. The distribution and density of PvTCPs on chromosomes were not uniform (Figure 1). Since switchgrass experienced a whole-genome allotetraploidization (2n=4x=36), the PvTCPs exist as paralogous gene pairs in the genome, and the sequence similarity between the gene pairs was larger than 90% (data not shown). 15 pairs of PvTCPs are putatively distributed on the ChrN and ChrK, respectively (Figure 1). The numbers for PvTCPs on Chr 2, 5, 6, and 7 are two pairs of PvTCPs. Chr 1 has three pairs, while Chr 3, 4, 8, and 9 each only has one pair of PvTCPs (Figure 1). In addition, according to the results of the specific location of each PvTCP, no tandem repeat gene was detected in switchgrass (Figure 1; Table 1).Figure 1
Chromosomal localization of switchgrassTCP genes. Chromosomal localization of PvTCPs was based on the physical map described in Phytozome v12.0. A total of 38 PvTCPs were anchored onto the chromosomes. ChrK and ChrN are two sets of subgenomes of switchgrass (2n=4x=36). The scale on the left represented the physical length of the chromosomes; Mb = million base pair. The red line represented a pair of paralogous TCP genes. The green character style represented putative gene pairs.Table 1
Overview ofTCP genes in switchgrass.
Gene namea
Gene IDb
ORF length (bp)
Deduced polypeptide
Chr
Chr location
Length (aa)
MW (kDa)
pI
PvTCP1
Pavir.1KG397100
663
220
22.53
9.83
01K
63645349-63646113
PvTCP2
Pavir.1KG510200
1188
395
39.93
9.42
01K
75765762-75768115
PvTCP3
Pavir.1KG552700
681
226
23.00
9.79
01K
79521775-79523415
PvTCP4
Pavir.1NG030900
426
141
14.49
10.42
01N
3887213-3889118
PvTCP5
Pavir.1NG539700
1206
401
40.27
9.42
01N
89471749-89473804
PvTCP6
Pavir.1NG547900
663
220
22.50
10.09
01N
96987327-96989924
PvTCP7
Pavir.2KG036700
957
318
33.85
6.29
02K
5031244-5036367
PvTCP8
Pavir.2KG296300
801
266
28.62
6.05
02K
65347281-65349763
PvTCP9
Pavir.2NG040500
1293
430
45.36
9.32
02N
5718258-5719550
PvTCP10
Pavir.2NG168500
933
310
32.42
10.10
02N
32012380-32013416
PvTCP11
Pavir.2NG320400
795
264
28.39
6.21
02N
59626967-59628392
PvTCP12
Pavir.2NG441900
990
329
33.63
4.95
02N
81045305-81046294
PvTCP13
Pavir.3KG357500
870
289
30.21
8.93
03K
28804800-28807508
PvTCP14
Pavir.3KG547300
870
289
30.66
6.38
03K
69673046-69678099
PvTCP15
Pavir.3NG031100
1200
399
40.43
5.99
03N
2368139-2370112
PvTCP16
Pavir.3NG279000
864
287
30.06
5.96
03N
52604278-52608164
PvTCP17
Pavir.4KG172900
1197
398
40.38
8.97
04K
10928170-10929640
PvTCP18
Pavir.4NG098900
1173
389
39.50
7.83
04N
13829961-13832629
PvTCP19
Pavir.4NG231900
978
325
34.15
6.29
04N
19989691-19991909
PvTCP20
Pavir.5KG544700
1251
416
44.33
6.38
05K
94391370-94392775
PvTCP21
Pavir.5KG556600
837
278
29.62
8.08
05K
95365227-95369286
PvTCP22
Pavir.5KG742600
978
325
33.98
6.37
05K
113279411-113281724
PvTCP23
Pavir.5NG501800
1272
423
45.00
6.41
05N
86933569-86934999
PvTCP24
Pavir.5NG508900
531
176
18.87
9.75
05N
87491419-87493406
PvTCP25
Pavir.6KG270000
849
282
30.48
6.80
06K
55905938-55907215
PvTCP26
Pavir.6KG395100
1065
354
36.50
5.51
06K
70566109-70567758
PvTCP27
Pavir.6NG051800
1215
404
42.02
9.02
06N
10745904-10747230
PvTCP28
Pavir.6NG140000
711
236
25.40
5.61
06N
58122711-58123421
PvTCP29
Pavir.6NG344700
1329
442
45.95
8.82
06N
77613527-77615377
PvTCP30
Pavir.7KG023900
525
174
18.14
8.25
07K
23221010-23224633
PvTCP31
Pavir.7KG255700
606
201
20.79
10.01
07K
56383609-56384723
PvTCP32
Pavir.7NG066100
285
94
9.70
4.59
07N
15663289-15674390
PvTCP33ζ
Pavir.7NG333200
603
200
20.79
9.82
07N
55200631-55201233
PvTCP34
Pavir.8KG079400
1209
402
41.22
8.77
08K
9383778-9386025
PvTCP35
Pavir.8NG062800
1191
396
40.65
9.13
08N
8490834-8493093
PvTCP36
Pavir.9KG031700
1110
369
39.15
8.55
09K
2411064-2412737
PvTCP37
Pavir.9NG079800
1158
385
39.42
9.32
09N
5336448-5340260
PvTCP38
Pavir.9NG142700
1116
371
39.78
8.39
09N
13496559-13498279
PvTCP39
Pavir.J125500
582
193
19.25
10.19
scaffold14987
1395-1995
PvTCP40
Pavir.J227000
888
295
31.09
8.91
scaffold20
54419-56973
PvTCP41
Pavir.J362100
1335
444
46.34
6.78
scaffold276
1-2111
PvTCP42
Pavir.J675700
1353
450
46.78
6.67
scaffold7087
83-2442
aGene name referred to the identified PvTCP genes in switchgrass in this study. bGene ID in Phytozome v12.0 database. ζCorrected TCP genes by PCR and PviUTs database (https://switchgrassgenomics.noble.org/).Biochemical properties of PvTCP members were globally analyzed. Based on the detailed information, lengths of these predicted PvTCP peptides ranged from 94 (PvTCP32) to 450 (PvTCP42) amino acids and molecular weight from 9.70 (PvTCP32) to 46.79 (PvTCP42) KDa (Table1). The isoelectric point varied from 4.59 (PvTCP32) to 10.42 (PvTCP4) (Table 1).


### 3.2. Phylogenetic Analysis ofTCPs

In order to comprehensively dissect the function ofPvTCPs, phylogenetic relationships were firstly analyzed. An unrooted phylogenetic neighbor-joining (NJ) tree was constructed based on the multiple sequence alignments of TCP proteins from switchgrass, Arabidopsis, (model species of dicots) and rice (model species of monocots). Two main classical subfamilies were obviously distinguished according to the NJ tree topology and bootstrap values (higher than 50%), which were referred to as class clades I and II. 23 PvTCPs are classified into clade I (PCF), and the rest 19 members are classified into class II (Figure 2; Table S2). The class II group is further divided into clade CIN (13 members) and clade CYC/TB1 (six members) (Figure 2). For the paralogous gene pairs, like PvTCP1/4, PvTCP2/5, and PvTCP3/6, they are all clustered together in the phylogenetic tree, indicating the phylogenetic signature of allotetraploidization (Figure 2). The sequence alignment analysis shows that almost all PvTCP proteins contain the conserved basic helix-loop-helix (bHLH) domain, and the members that belonged to clade I (PCF) have a four amino acid deletions in the bHLH domain compared with class II (CYC/TB1 and CIN) (Figure 3). This result was consistent with the phylogenetic analysis.Figure 2
Phylogenetic analysis of TCP proteins in switchgrass,Arabidopsis, and rice. An unrooted neighbor-joining (NJ) tree was constructed using MEGA5.0 (bootstrap value=1,000) after the multiple alignment of peptide sequences. All sequences used in this project were retrieved from the public genome database Phytozome v12.0 (https://phytozome.jgi.doe.gov/pz/portal.html#). The detailed information was listed in Table S2.Figure 3
Alignment of the predicted conserved basic helix-loop-helix domain sequence of switchgrass TCP members. Amino acids are expressed in the standard single-letter code. (a) Three clades were classified according to an unrooted NJ tree, which were constructed using PvTCP peptides. (b) Multiple sequence alignment was generated by GenDoc.PvTCPs in both class I and II gathered closely with the counterparts in rice, rather than Arabidopsis, which might imply that TCP genes were duplicated after the diversification of dicot and monocot species in angiosperms (Figure 2). Ka/Ks ratios were subsequently calculated between PvTCPs and OsTCPs (Table 2). The results showed that about 1/3 orthologous genes belonged to purifying selection between the evolution of switchgrass and rice; the other 2/3 orthologous genes belonged to positive selection.Table 2
Ka/Ks ratio ofTCP orthologous genes between switchgrass and rice.
Orthologous genes
Ka/Ks ratio
Selection pattern
PvTCP1/4 vs. OsTCP7
0.026
Purifying selection
PvTCP39 vs. OsTCP7
99.000
Positive selection
PvTCP31/33 vs. OsTCP17
99.000
Positive selection
PvTCP15 vs. OsTCP28
99.000
Positive selection
PvTCP27 vs. OsPCF3
99.000
Positive selection
PvTCP34/35 vs. OsPCF3
99.000
Positive selection
PvTCP30/32 vs. OsPCF1
99.000
Positive selection
PvTCP2/5 vs. OsTCP9
99.000
Positive selection
PvTCP17/18 vs. OsTCP19
1.250
Positive selection
PvTCP19/22 vs. OsTCP6
26.467
Positive selection
PvTCP12 vs. OsTCP25
99.000
Positive selection
PvTCP26/29 vs. OsPCF2
0.552
Purifying selection
PvTCP36/38 vs. OsTB1
0.847
Purifying selection
PvTCP8/11 vs. OsTCP24
0.474
Purifying selection
PvTCP25/28 vs. OsTCP22
0.516
Purifying selection
PvTCP41/42 vs. OsPCF5
99.000
Positive selection
PvTCP21/24 vs. OsTCP5
99.000
Positive selection
PvTCP13/40 vs. OsTCP18
0.665
Purifying selection
PvTCP14/16 vs. OsPCF8
99.000
Positive selection
PvTCP37 vs. OsPCF6
0.032
Purifying selection
PvTCP7/9 vs. OsTCP21
99.000
Positive selection


### 3.3. Gene Structure, Conserved Motifs, and Recognition Sites ofmiR319

To understand the evolution ofPvTCP gene family, introns in TCP genes and conserved motifs of their coding proteins were analyzed (Figure 4). All members in the CYC/TB1 group contain no introns. The intron/exon organization in the PCF clade was relatively conserved, with 14 of 23 members that had no introns, four that had one intron in the coding sequence (CDS) region, one that had two introns in the CDS region, and four that contained one or three introns in the untranslated region (UTR). Introns of PvTCPs in clade CIN was not conserved as those in other clades: three contain one intron in the CDS region, nine possessed one or two introns in the UTR region, and only one gene contain no intron (Figure 4). The conserved motifs were also analyzed and ten motifs were identified in PvTCPs using the MEME tool (Figure 4). Motifs 1 and 2 are conserved in PvTCPs except for PvTCP7, PvTCP10, PvTCP32, and PvTCP33. Proteins in the same clade of the phylogenetic tree contain similar motif arrangement. Motif 3 was conserved in all PvTCP proteins of clade PCF except for PvTCP10. Proteins in the other two clades, except for PvTCP7, PvTCP20, and PvTCP23, did not harbor motif 3. This is the same case for motifs 6 and 10. Most PvTCP proteins in clade PCF contain motifs 6 and 10, but not for proteins in clades CYC/TB1 and CIN. Motif 4 was only conserved in clades PCF and CIN, and motif 5 was conserved in clades CYC/TB1 and CIN. Only proteins in clade CIN contain the motif 7. These results implied that TCP transcription factors might take diverse roles in switchgrass due to their structure diversity.Figure 4
Gene structures and motif locations of switchgrassTCP genes. (a) Three clades were classified according to an unrooted NJ tree, which were constructed using PvTCP peptides. (b) Exon/intron arrangements of the PvTCP gene. Exons, introns, and untranslated region (UTR) were represented by green boxes, black lines, and blue boxes, respectively. Nucleic acid lengths are indicated by the scale at the bottom; bp = base pair. (c) Schematic representation of conserved motifs in the PvTCP proteins predicted by the MEME program. Each motif is represented by a number in the colored box. The black lines represented the nonconserved sequences. Lengths of motifs for each PvTCP protein were displayed proportionally. aa = amino acid.It was reported thatTCP genes can be posttranscriptionally regulated by miR319 [25]. Similarly, nine PvTCP genes contain miR319 binding sites, which were located in the CDS, and all of these miR319-targeted PvTCPs were CIN family members (Figure 5).Figure 5
PutativemicroRNA319-targeted binding sites of the PvTCPgenes. Alignment of complementary pairing bases was generated by GenDoc. Targeted sites were retrieved from the coding sequences of PvTCP genes, while mature sequence of miR319 was rice miR319b from miRBase (http://www.mirbase.org/).


### 3.4. Tissue Expression Profiles of thePvTCPs

To roundly speculate the function of PvTCP proteins,cis-acting DNA elements in the promoter of each PvTCPs were retrieved and analyzed (Table S4). The results showed that 18, 15, and 13 elements were, respectively, shared in clades PCF, CYC/TB1, and CIN (Table 3). Obviously, photosynthesis, environmental stress response, and phytohormone regulation were the three major aspects in which TCP proteins were involved. In order to deeply analyze the tissue expression profiles of the PvTCP family, microarray data was obtained from the public database. As expected, both PvTCPs of the gene pair share one probe (Table S3). All PvTCPs were expressed in the examined tissues (leaf, node, internode, root, flower, and seed) (Figure 6). Part of the genes in the same clade exhibited similar expression mode. For example, members in clade CIN (PvTCP37, PvTCP13/40, PvTCP14/16, and PvTCP21/24) predominantly expressed in flowers, which might take roles in pollen development. Genes in clade PCF, like PvTCP1/4, PvTCP39, PvTCP15, and PvTCP27, represented a high expression level in flowers, node, and seed of E4 stage. Besides, PvTCP26/29, PvTCP19/22, and PvTCP17/18 displayed high expression levels in all tested tissues, while PvTCP10, PvTCP25/28, PvTCP36/38, PvTCP30/32, PvTCP7/9, and PvTCP41/42 were expressed relatively low in all tested tissues.Table 3
Putativecis-acting DNA elements in the promoter of PvTCP genes.
Clade name
Element no.a
Element nameb
Signal sequencec
Putative functiond
FOe
PCF
S000449
CACTFTPPCA1
YACT
Photosynthesis
237
S000265
DOFCOREZM
AAAG
Photosynthesis; leaf and shoot development
213
S000454
ARR1AT
NGATT
Cytokinin response
161
S000198
GT1CONSENSUS
GRWAAW
HR reaction
f
; systemic acquired resistance
146
S000407
MYCCONSENSUSAT
CANNTG
Abiotic stress; salinity stress
143
S000144
EBOXBNNAPA
CANNTG
Salinity stress; phenylpropanoid biosynthesis
143
S000501
CGCGBOXAT
VCGCGB
Calmodulin; auxin response
108
S000447
WRKY71OS
TGAC
Biotic and abiotic stress; GA response
97
S000378
GTGANTG10
GTGA
Pollen development; pectin regulation
95
S000493
CURECORECR
GTAC
Copper; oxygen; hypoxic reaction
92
S000245
POLLEN1LELAT52
AGAAA
Pollen development
91
S000415
ACGTATERD1
ACGT
Photosynthesis
74
S000462
NODCON2GM
CTCTT
Root nodulin
66
S000203
TATABOX5
TTATTT
Glutamine synthetase
45
S000457
WBOXNTERF3
TGACY
Jasmonic acid response
43
S000179
MYBPZM
CCWACC
Flavonoid biosynthesis; seed development
37
S000176
MYBCORE
CNGTTR
Abiotic stress; salinity; flavonoid biosynthesis
35
CYC/TB1
S000449
CACTFTPPCA1
YACT
Photosynthesis
87
S000265
DOFCOREZM
AAAG
Photosynthesis; leaf and shoot development
53
S000407
MYCCONSENSUSAT
CANNTG
Abiotic stress; salinity stress
48
S000144
EBOXBNNAPA
CANNTG
Salinity stress; phenylpropanoid biosynthesis
48
S000198
GT1CONSENSUS
GRWAAW
HR reaction; systemic acquired resistance
38
S000454
ARR1AT
NGATT
Cytokinin response
33
S000378
GTGANTG10
GTGA
Pollen development; pectin regulation
32
S000447
WRKY71OS
TGAC
Biotic and abiotic stress; GA response
21
S000482
SORLIP1AT
GCCAC
phyA; phytochrome; light response
17
S000203
TATABOX5
TTATTT
Glutamine synthetase
14
S000030
CCAATBOX1
CCAAT
Heat shock response
13
S000103
SEF4MOTIFGM7S
RTTTTTR
Seed globulin
10
CIN
S000449
CACTFTPPCA1
YACT
Photosynthesis
153
S000454
ARR1AT
NGATT
Cytokinin response
132
S000265
DOFCOREZM
AAAG
Photosynthesis; leaf and shoot dvelopment
103
S000407
MYCCONSENSUSAT
CANNTG
Abiotic stress; salinity stress
77
S000144
EBOXBNNAPA
CANNTG
Salinity stress; phenylpropanoid biosynthesis
77
S000501
CGCGBOXAT
VCGCGB
Calmodulin; auxin response
69
S000198
GT1CONSENSUS
GRWAAW
HR reaction; systemic acquired resistance
64
S000447
WRKY71OS
TGAC
Biotic and abiotic stress; GA response
49
S000493
CURECORECR
GTAC
Copper; oxygen; hypoxic reaction
48
S000378
GTGANTG10
GTGA
Photosynthesis; leaf and shoot development
43
S000203
TATABOX5
TTATTT
Glutamine synthetase
27
S000103
SEF4MOTIFGM7S
RTTTTTR
Seed globulin
26
S000245
POLLEN1LELAT52
AGAAA
Pollen development
21
a-cThe ID number, name, and signal sequences of the element in the online PLACE program (https://sogo.dna.affrc.go.jp/). dThe putative function of each element predicted by the online PLACE program and references from NCBI. eFrequency of occurrence in the promoters of TCP genes in each clade. fCharacters in bold represent those functions related to stress response.Figure 6
Heatmap of expression profiles of switchgrassTCP gene pairs in different tested tissues. The detailed microarray data were obtained from switchgrass gene atlas database (https://switchgrassgenomics.noble.org/). Clustering analysis was carried out using the online program pretty heatmap (http://www.ehbio.com/ImageGP/index.php/). The detailed information was listed in Table S3.


### 3.5. Gene Expression Response ofPvTCPs under Salinity Condition

Based on the statistical results fromcis-acting DNA elements, about 40% were showed to respond to environmental stress, especially to salinity (Table 3). To explore the expression profiles of PvTCPs under salinity condition, 42 PvTCPs were analyzed by qRT-PCR (Figure 7). 29 PvTCPs were regulated under salinity condition, and the other 13 PvTCPs were not statistically significant after 6 h salt stress (Figure 7). 14 out of 23 (about 60.8%) PvTCPs in clade PCF were upregulated during the 6 h salinity treatment. Of these genes, PvTCP27 and PvTCP39 were showed upregulated in all three treatment points. PvTCP3/6, PvTCP30/32, and PvTCP34/35 were upregulated at 0.5 h and exposed to salt stress for 2 h, and recovered to the normal expression level at 6 h treatment point. PvTCP10, PvTCP12, and PvTCP17/18 were upregulated at 6 h treatment point. PvTCP31/33 was induced after 2 h treatment. All PvTCPs in clade CYC/TB1 were upregulated. Similarly, nine out of 13 PvTCP genes in clade CIN were upregulated after 6 h treatment. These results showed that a large number of PvTCPs were response to salt stress and displayed different expression profiles when exposed to salinity condition.Figure 7
The expression ofPvTCP genes in response to treatment with 250 mM NaCl for 0.5, 2, and 6 hours in seedlings. Control plants were collected before the treatment by NaCl solution. Error bars represented variability of three independent replicates. Statistically significant differences were assessed using Student’s t-tests (∗∗represented p≤0.01).


## 3.1. Identification and Chromosomal Location ofPvTCPs

To identify TCP proteins in switchgrass, the hidden Markov model (HMM) profile of the conserved TCP domain (pfam03634) was used as a blast query to search against the public available switchgrass genome database (Phytozome v12). A total of 42 putative TCP members were identified, which were named as PvTCP1 to PvTCP42 according to their chromosomal location (Figure1; Table 1). In general, 90.5% (38 out of 42) of PvTCPs are anchored onto the chromosomes, while the other four genes are located on an unmapped region. The distribution and density of PvTCPs on chromosomes were not uniform (Figure 1). Since switchgrass experienced a whole-genome allotetraploidization (2n=4x=36), the PvTCPs exist as paralogous gene pairs in the genome, and the sequence similarity between the gene pairs was larger than 90% (data not shown). 15 pairs of PvTCPs are putatively distributed on the ChrN and ChrK, respectively (Figure 1). The numbers for PvTCPs on Chr 2, 5, 6, and 7 are two pairs of PvTCPs. Chr 1 has three pairs, while Chr 3, 4, 8, and 9 each only has one pair of PvTCPs (Figure 1). In addition, according to the results of the specific location of each PvTCP, no tandem repeat gene was detected in switchgrass (Figure 1; Table 1).Figure 1
Chromosomal localization of switchgrassTCP genes. Chromosomal localization of PvTCPs was based on the physical map described in Phytozome v12.0. A total of 38 PvTCPs were anchored onto the chromosomes. ChrK and ChrN are two sets of subgenomes of switchgrass (2n=4x=36). The scale on the left represented the physical length of the chromosomes; Mb = million base pair. The red line represented a pair of paralogous TCP genes. The green character style represented putative gene pairs.Table 1
Overview ofTCP genes in switchgrass.
Gene namea
Gene IDb
ORF length (bp)
Deduced polypeptide
Chr
Chr location
Length (aa)
MW (kDa)
pI
PvTCP1
Pavir.1KG397100
663
220
22.53
9.83
01K
63645349-63646113
PvTCP2
Pavir.1KG510200
1188
395
39.93
9.42
01K
75765762-75768115
PvTCP3
Pavir.1KG552700
681
226
23.00
9.79
01K
79521775-79523415
PvTCP4
Pavir.1NG030900
426
141
14.49
10.42
01N
3887213-3889118
PvTCP5
Pavir.1NG539700
1206
401
40.27
9.42
01N
89471749-89473804
PvTCP6
Pavir.1NG547900
663
220
22.50
10.09
01N
96987327-96989924
PvTCP7
Pavir.2KG036700
957
318
33.85
6.29
02K
5031244-5036367
PvTCP8
Pavir.2KG296300
801
266
28.62
6.05
02K
65347281-65349763
PvTCP9
Pavir.2NG040500
1293
430
45.36
9.32
02N
5718258-5719550
PvTCP10
Pavir.2NG168500
933
310
32.42
10.10
02N
32012380-32013416
PvTCP11
Pavir.2NG320400
795
264
28.39
6.21
02N
59626967-59628392
PvTCP12
Pavir.2NG441900
990
329
33.63
4.95
02N
81045305-81046294
PvTCP13
Pavir.3KG357500
870
289
30.21
8.93
03K
28804800-28807508
PvTCP14
Pavir.3KG547300
870
289
30.66
6.38
03K
69673046-69678099
PvTCP15
Pavir.3NG031100
1200
399
40.43
5.99
03N
2368139-2370112
PvTCP16
Pavir.3NG279000
864
287
30.06
5.96
03N
52604278-52608164
PvTCP17
Pavir.4KG172900
1197
398
40.38
8.97
04K
10928170-10929640
PvTCP18
Pavir.4NG098900
1173
389
39.50
7.83
04N
13829961-13832629
PvTCP19
Pavir.4NG231900
978
325
34.15
6.29
04N
19989691-19991909
PvTCP20
Pavir.5KG544700
1251
416
44.33
6.38
05K
94391370-94392775
PvTCP21
Pavir.5KG556600
837
278
29.62
8.08
05K
95365227-95369286
PvTCP22
Pavir.5KG742600
978
325
33.98
6.37
05K
113279411-113281724
PvTCP23
Pavir.5NG501800
1272
423
45.00
6.41
05N
86933569-86934999
PvTCP24
Pavir.5NG508900
531
176
18.87
9.75
05N
87491419-87493406
PvTCP25
Pavir.6KG270000
849
282
30.48
6.80
06K
55905938-55907215
PvTCP26
Pavir.6KG395100
1065
354
36.50
5.51
06K
70566109-70567758
PvTCP27
Pavir.6NG051800
1215
404
42.02
9.02
06N
10745904-10747230
PvTCP28
Pavir.6NG140000
711
236
25.40
5.61
06N
58122711-58123421
PvTCP29
Pavir.6NG344700
1329
442
45.95
8.82
06N
77613527-77615377
PvTCP30
Pavir.7KG023900
525
174
18.14
8.25
07K
23221010-23224633
PvTCP31
Pavir.7KG255700
606
201
20.79
10.01
07K
56383609-56384723
PvTCP32
Pavir.7NG066100
285
94
9.70
4.59
07N
15663289-15674390
PvTCP33ζ
Pavir.7NG333200
603
200
20.79
9.82
07N
55200631-55201233
PvTCP34
Pavir.8KG079400
1209
402
41.22
8.77
08K
9383778-9386025
PvTCP35
Pavir.8NG062800
1191
396
40.65
9.13
08N
8490834-8493093
PvTCP36
Pavir.9KG031700
1110
369
39.15
8.55
09K
2411064-2412737
PvTCP37
Pavir.9NG079800
1158
385
39.42
9.32
09N
5336448-5340260
PvTCP38
Pavir.9NG142700
1116
371
39.78
8.39
09N
13496559-13498279
PvTCP39
Pavir.J125500
582
193
19.25
10.19
scaffold14987
1395-1995
PvTCP40
Pavir.J227000
888
295
31.09
8.91
scaffold20
54419-56973
PvTCP41
Pavir.J362100
1335
444
46.34
6.78
scaffold276
1-2111
PvTCP42
Pavir.J675700
1353
450
46.78
6.67
scaffold7087
83-2442
aGene name referred to the identified PvTCP genes in switchgrass in this study. bGene ID in Phytozome v12.0 database. ζCorrected TCP genes by PCR and PviUTs database (https://switchgrassgenomics.noble.org/).Biochemical properties of PvTCP members were globally analyzed. Based on the detailed information, lengths of these predicted PvTCP peptides ranged from 94 (PvTCP32) to 450 (PvTCP42) amino acids and molecular weight from 9.70 (PvTCP32) to 46.79 (PvTCP42) KDa (Table1). The isoelectric point varied from 4.59 (PvTCP32) to 10.42 (PvTCP4) (Table 1).


## 3.2. Phylogenetic Analysis ofTCPs

In order to comprehensively dissect the function ofPvTCPs, phylogenetic relationships were firstly analyzed. An unrooted phylogenetic neighbor-joining (NJ) tree was constructed based on the multiple sequence alignments of TCP proteins from switchgrass, Arabidopsis, (model species of dicots) and rice (model species of monocots). Two main classical subfamilies were obviously distinguished according to the NJ tree topology and bootstrap values (higher than 50%), which were referred to as class clades I and II. 23 PvTCPs are classified into clade I (PCF), and the rest 19 members are classified into class II (Figure 2; Table S2). The class II group is further divided into clade CIN (13 members) and clade CYC/TB1 (six members) (Figure 2). For the paralogous gene pairs, like PvTCP1/4, PvTCP2/5, and PvTCP3/6, they are all clustered together in the phylogenetic tree, indicating the phylogenetic signature of allotetraploidization (Figure 2). The sequence alignment analysis shows that almost all PvTCP proteins contain the conserved basic helix-loop-helix (bHLH) domain, and the members that belonged to clade I (PCF) have a four amino acid deletions in the bHLH domain compared with class II (CYC/TB1 and CIN) (Figure 3). This result was consistent with the phylogenetic analysis.Figure 2
Phylogenetic analysis of TCP proteins in switchgrass,Arabidopsis, and rice. An unrooted neighbor-joining (NJ) tree was constructed using MEGA5.0 (bootstrap value=1,000) after the multiple alignment of peptide sequences. All sequences used in this project were retrieved from the public genome database Phytozome v12.0 (https://phytozome.jgi.doe.gov/pz/portal.html#). The detailed information was listed in Table S2.Figure 3
Alignment of the predicted conserved basic helix-loop-helix domain sequence of switchgrass TCP members. Amino acids are expressed in the standard single-letter code. (a) Three clades were classified according to an unrooted NJ tree, which were constructed using PvTCP peptides. (b) Multiple sequence alignment was generated by GenDoc.PvTCPs in both class I and II gathered closely with the counterparts in rice, rather than Arabidopsis, which might imply that TCP genes were duplicated after the diversification of dicot and monocot species in angiosperms (Figure 2). Ka/Ks ratios were subsequently calculated between PvTCPs and OsTCPs (Table 2). The results showed that about 1/3 orthologous genes belonged to purifying selection between the evolution of switchgrass and rice; the other 2/3 orthologous genes belonged to positive selection.Table 2
Ka/Ks ratio ofTCP orthologous genes between switchgrass and rice.
Orthologous genes
Ka/Ks ratio
Selection pattern
PvTCP1/4 vs. OsTCP7
0.026
Purifying selection
PvTCP39 vs. OsTCP7
99.000
Positive selection
PvTCP31/33 vs. OsTCP17
99.000
Positive selection
PvTCP15 vs. OsTCP28
99.000
Positive selection
PvTCP27 vs. OsPCF3
99.000
Positive selection
PvTCP34/35 vs. OsPCF3
99.000
Positive selection
PvTCP30/32 vs. OsPCF1
99.000
Positive selection
PvTCP2/5 vs. OsTCP9
99.000
Positive selection
PvTCP17/18 vs. OsTCP19
1.250
Positive selection
PvTCP19/22 vs. OsTCP6
26.467
Positive selection
PvTCP12 vs. OsTCP25
99.000
Positive selection
PvTCP26/29 vs. OsPCF2
0.552
Purifying selection
PvTCP36/38 vs. OsTB1
0.847
Purifying selection
PvTCP8/11 vs. OsTCP24
0.474
Purifying selection
PvTCP25/28 vs. OsTCP22
0.516
Purifying selection
PvTCP41/42 vs. OsPCF5
99.000
Positive selection
PvTCP21/24 vs. OsTCP5
99.000
Positive selection
PvTCP13/40 vs. OsTCP18
0.665
Purifying selection
PvTCP14/16 vs. OsPCF8
99.000
Positive selection
PvTCP37 vs. OsPCF6
0.032
Purifying selection
PvTCP7/9 vs. OsTCP21
99.000
Positive selection


## 3.3. Gene Structure, Conserved Motifs, and Recognition Sites ofmiR319

To understand the evolution ofPvTCP gene family, introns in TCP genes and conserved motifs of their coding proteins were analyzed (Figure 4). All members in the CYC/TB1 group contain no introns. The intron/exon organization in the PCF clade was relatively conserved, with 14 of 23 members that had no introns, four that had one intron in the coding sequence (CDS) region, one that had two introns in the CDS region, and four that contained one or three introns in the untranslated region (UTR). Introns of PvTCPs in clade CIN was not conserved as those in other clades: three contain one intron in the CDS region, nine possessed one or two introns in the UTR region, and only one gene contain no intron (Figure 4). The conserved motifs were also analyzed and ten motifs were identified in PvTCPs using the MEME tool (Figure 4). Motifs 1 and 2 are conserved in PvTCPs except for PvTCP7, PvTCP10, PvTCP32, and PvTCP33. Proteins in the same clade of the phylogenetic tree contain similar motif arrangement. Motif 3 was conserved in all PvTCP proteins of clade PCF except for PvTCP10. Proteins in the other two clades, except for PvTCP7, PvTCP20, and PvTCP23, did not harbor motif 3. This is the same case for motifs 6 and 10. Most PvTCP proteins in clade PCF contain motifs 6 and 10, but not for proteins in clades CYC/TB1 and CIN. Motif 4 was only conserved in clades PCF and CIN, and motif 5 was conserved in clades CYC/TB1 and CIN. Only proteins in clade CIN contain the motif 7. These results implied that TCP transcription factors might take diverse roles in switchgrass due to their structure diversity.Figure 4
Gene structures and motif locations of switchgrassTCP genes. (a) Three clades were classified according to an unrooted NJ tree, which were constructed using PvTCP peptides. (b) Exon/intron arrangements of the PvTCP gene. Exons, introns, and untranslated region (UTR) were represented by green boxes, black lines, and blue boxes, respectively. Nucleic acid lengths are indicated by the scale at the bottom; bp = base pair. (c) Schematic representation of conserved motifs in the PvTCP proteins predicted by the MEME program. Each motif is represented by a number in the colored box. The black lines represented the nonconserved sequences. Lengths of motifs for each PvTCP protein were displayed proportionally. aa = amino acid.It was reported thatTCP genes can be posttranscriptionally regulated by miR319 [25]. Similarly, nine PvTCP genes contain miR319 binding sites, which were located in the CDS, and all of these miR319-targeted PvTCPs were CIN family members (Figure 5).Figure 5
PutativemicroRNA319-targeted binding sites of the PvTCPgenes. Alignment of complementary pairing bases was generated by GenDoc. Targeted sites were retrieved from the coding sequences of PvTCP genes, while mature sequence of miR319 was rice miR319b from miRBase (http://www.mirbase.org/).


## 3.4. Tissue Expression Profiles of thePvTCPs

To roundly speculate the function of PvTCP proteins,cis-acting DNA elements in the promoter of each PvTCPs were retrieved and analyzed (Table S4). The results showed that 18, 15, and 13 elements were, respectively, shared in clades PCF, CYC/TB1, and CIN (Table 3). Obviously, photosynthesis, environmental stress response, and phytohormone regulation were the three major aspects in which TCP proteins were involved. In order to deeply analyze the tissue expression profiles of the PvTCP family, microarray data was obtained from the public database. As expected, both PvTCPs of the gene pair share one probe (Table S3). All PvTCPs were expressed in the examined tissues (leaf, node, internode, root, flower, and seed) (Figure 6). Part of the genes in the same clade exhibited similar expression mode. For example, members in clade CIN (PvTCP37, PvTCP13/40, PvTCP14/16, and PvTCP21/24) predominantly expressed in flowers, which might take roles in pollen development. Genes in clade PCF, like PvTCP1/4, PvTCP39, PvTCP15, and PvTCP27, represented a high expression level in flowers, node, and seed of E4 stage. Besides, PvTCP26/29, PvTCP19/22, and PvTCP17/18 displayed high expression levels in all tested tissues, while PvTCP10, PvTCP25/28, PvTCP36/38, PvTCP30/32, PvTCP7/9, and PvTCP41/42 were expressed relatively low in all tested tissues.Table 3
Putativecis-acting DNA elements in the promoter of PvTCP genes.
Clade name
Element no.a
Element nameb
Signal sequencec
Putative functiond
FOe
PCF
S000449
CACTFTPPCA1
YACT
Photosynthesis
237
S000265
DOFCOREZM
AAAG
Photosynthesis; leaf and shoot development
213
S000454
ARR1AT
NGATT
Cytokinin response
161
S000198
GT1CONSENSUS
GRWAAW
HR reaction
f
; systemic acquired resistance
146
S000407
MYCCONSENSUSAT
CANNTG
Abiotic stress; salinity stress
143
S000144
EBOXBNNAPA
CANNTG
Salinity stress; phenylpropanoid biosynthesis
143
S000501
CGCGBOXAT
VCGCGB
Calmodulin; auxin response
108
S000447
WRKY71OS
TGAC
Biotic and abiotic stress; GA response
97
S000378
GTGANTG10
GTGA
Pollen development; pectin regulation
95
S000493
CURECORECR
GTAC
Copper; oxygen; hypoxic reaction
92
S000245
POLLEN1LELAT52
AGAAA
Pollen development
91
S000415
ACGTATERD1
ACGT
Photosynthesis
74
S000462
NODCON2GM
CTCTT
Root nodulin
66
S000203
TATABOX5
TTATTT
Glutamine synthetase
45
S000457
WBOXNTERF3
TGACY
Jasmonic acid response
43
S000179
MYBPZM
CCWACC
Flavonoid biosynthesis; seed development
37
S000176
MYBCORE
CNGTTR
Abiotic stress; salinity; flavonoid biosynthesis
35
CYC/TB1
S000449
CACTFTPPCA1
YACT
Photosynthesis
87
S000265
DOFCOREZM
AAAG
Photosynthesis; leaf and shoot development
53
S000407
MYCCONSENSUSAT
CANNTG
Abiotic stress; salinity stress
48
S000144
EBOXBNNAPA
CANNTG
Salinity stress; phenylpropanoid biosynthesis
48
S000198
GT1CONSENSUS
GRWAAW
HR reaction; systemic acquired resistance
38
S000454
ARR1AT
NGATT
Cytokinin response
33
S000378
GTGANTG10
GTGA
Pollen development; pectin regulation
32
S000447
WRKY71OS
TGAC
Biotic and abiotic stress; GA response
21
S000482
SORLIP1AT
GCCAC
phyA; phytochrome; light response
17
S000203
TATABOX5
TTATTT
Glutamine synthetase
14
S000030
CCAATBOX1
CCAAT
Heat shock response
13
S000103
SEF4MOTIFGM7S
RTTTTTR
Seed globulin
10
CIN
S000449
CACTFTPPCA1
YACT
Photosynthesis
153
S000454
ARR1AT
NGATT
Cytokinin response
132
S000265
DOFCOREZM
AAAG
Photosynthesis; leaf and shoot dvelopment
103
S000407
MYCCONSENSUSAT
CANNTG
Abiotic stress; salinity stress
77
S000144
EBOXBNNAPA
CANNTG
Salinity stress; phenylpropanoid biosynthesis
77
S000501
CGCGBOXAT
VCGCGB
Calmodulin; auxin response
69
S000198
GT1CONSENSUS
GRWAAW
HR reaction; systemic acquired resistance
64
S000447
WRKY71OS
TGAC
Biotic and abiotic stress; GA response
49
S000493
CURECORECR
GTAC
Copper; oxygen; hypoxic reaction
48
S000378
GTGANTG10
GTGA
Photosynthesis; leaf and shoot development
43
S000203
TATABOX5
TTATTT
Glutamine synthetase
27
S000103
SEF4MOTIFGM7S
RTTTTTR
Seed globulin
26
S000245
POLLEN1LELAT52
AGAAA
Pollen development
21
a-cThe ID number, name, and signal sequences of the element in the online PLACE program (https://sogo.dna.affrc.go.jp/). dThe putative function of each element predicted by the online PLACE program and references from NCBI. eFrequency of occurrence in the promoters of TCP genes in each clade. fCharacters in bold represent those functions related to stress response.Figure 6
Heatmap of expression profiles of switchgrassTCP gene pairs in different tested tissues. The detailed microarray data were obtained from switchgrass gene atlas database (https://switchgrassgenomics.noble.org/). Clustering analysis was carried out using the online program pretty heatmap (http://www.ehbio.com/ImageGP/index.php/). The detailed information was listed in Table S3.


## 3.5. Gene Expression Response ofPvTCPs under Salinity Condition

Based on the statistical results fromcis-acting DNA elements, about 40% were showed to respond to environmental stress, especially to salinity (Table 3). To explore the expression profiles of PvTCPs under salinity condition, 42 PvTCPs were analyzed by qRT-PCR (Figure 7). 29 PvTCPs were regulated under salinity condition, and the other 13 PvTCPs were not statistically significant after 6 h salt stress (Figure 7). 14 out of 23 (about 60.8%) PvTCPs in clade PCF were upregulated during the 6 h salinity treatment. Of these genes, PvTCP27 and PvTCP39 were showed upregulated in all three treatment points. PvTCP3/6, PvTCP30/32, and PvTCP34/35 were upregulated at 0.5 h and exposed to salt stress for 2 h, and recovered to the normal expression level at 6 h treatment point. PvTCP10, PvTCP12, and PvTCP17/18 were upregulated at 6 h treatment point. PvTCP31/33 was induced after 2 h treatment. All PvTCPs in clade CYC/TB1 were upregulated. Similarly, nine out of 13 PvTCP genes in clade CIN were upregulated after 6 h treatment. These results showed that a large number of PvTCPs were response to salt stress and displayed different expression profiles when exposed to salinity condition.Figure 7
The expression ofPvTCP genes in response to treatment with 250 mM NaCl for 0.5, 2, and 6 hours in seedlings. Control plants were collected before the treatment by NaCl solution. Error bars represented variability of three independent replicates. Statistically significant differences were assessed using Student’s t-tests (∗∗represented p≤0.01).


## 4. Discussion

TheTCP gene family is a cluster of plant-specific transcription factors, which play pivotal roles in plant growth, development, and stress response [1]. In switchgrass, 42 TCP genes were identified from the genome and they were unevenly distributed on the chromosomes. The number of TCP genes in switchgrass is approximately twice that in Arabidopsis and rice, which have 24 and 21 TCP members, respectively [5]. No tandem repeats occurred in the evolutionary process in switchgrass TCP genes. So, large enrichment of switchgrass TCP genes was presumably due to the allotetraploid event. Furthermore, Ka/Ks analysis between the PvTCPs and OsTCPs was carried out, and the results that showed approximately 2/3 orthologous PvTCP genes, compared to OsTCP genes, are selected by natural selection pressure (Table 2), which might be due to the divergency between rice and switchgrass, at least 50 Mya [31]. As reported previously in PvC3H genes, the two sets of subgenomes of switchgrass originated from two closely diploid progenitors [31]. So, we speculated that PvTCP genes existed as paralogous gene pairs, which evolutionarily derived from the two sets of subgenomes, respectively. These results were also supported by previous studies in PvSPL genes and PvARF genes [32, 33].TheTCP gene family was classified into three clades, named as clade PCF, CYC/TB1, and CIN [5]. Similarly, PvTCP proteins were phylogenetically divided into those three clades in our study as well. Members that belonged to clade PCF have a four amino acid deletion in the basic helix-loop-helix (bHLH) conserved domain compared with clades CYC/TB1 and CIN (Figure 3). Exon/intron arrangement and motif location of PvTCP members were roughly conserved in the same clade but showed significant distinction among different clades (Figure 4). High similarity of the TCP members in switchgrass to other species, such as Arabidopsis and rice, suggested that TCP genes were highly conserved in plants, although there are great differences in gene numbers among different species [5]. Therefore, PvTCP genes would share similar functions with their orthologs in other species.Previous reports about TCP roles mainly focused on cell cycle-mediated regulation of growth. TB1 is a major contributor to regulate apical dominance in maize [2]. PCF1 and PCF2 participate in DNA replication and repair, maintenance of chromatin structure, chromosome segregation, and cell-cycle progression by means of binding the promoter of the rice PROLIFERATING CELL NUCLEAR ANTIGEN (PCNA) gene, and CYC participates in the control of floral asymmetry in snapdragon [3, 4]. AtTCP4, a member in clade CIN, is critical in Arabidopsis floral organs [4]. Moreover, AtTCP4 can activate secondary cell wall biosynthesis and programmed cell death [22]. For those flower that predominantly expressed PvTCP genes in clade CIN, PvTCP37, PvTCP13/40, and PvTCP14/16, they may also take an important role in floral development, such as anther and pollen development. Not only the genes in clade CIN, but also the TCP genes belonged to clade CYC/TB1 can also control the floral asymmetry in Lotus japonicus (LjCYC2 and LjCYC3) and Pisum sativum (PsCYC2 and PsCYC3) [46, 47]. The expression levels of CYC/TB1 genes PvTCP8/11 were relatively high in flower, which may affect the flower shape. Additionally, five PCF clade genes (PvTCP1/4, PvTCP15, PvTCP27, and PvTCP39) were predominantly high in flower and stem, which indicated that they might have a special function in floral development and cell wall biosynthesis. The expression profiles in different tissues of the PvTCP genes can help us study the detailed functions during switchgrass growth and development accurately in the future.Several studies on the relationship between TCP proteins and plant abiotic stress have been reported [24, 25]. In Agrostis stolonifera, miR319-targeted TCP genes can respond to salt and dehydration stress and Osa-miR319 overexpression transgenic creeping bentgrass improves salt and drought resistance [3]. AsTCP5 transcript increased after 0.5 h salinity stress and then decreased at 6 h treatment point [3]. OsTCP19 in shoots was upregulated under salt and drought stress in rice, and overexpression of OsTCP19 in Arabidopsis can improve the abiotic tolerance of the transgenic plants [2]. In our study, we firstly analyzed the cis-acting DNA elements of the PvTCPs’ promoters. It is revealed that a lot of photosynthesis, plant hormone signaling, and organ development regulatory elements were accumulated, such as S000449, S000265, and S000454 (Table 3). In addition, about 40% of cis-acting DNA elements were related to biotic and abiotic stress response, especially to salt and drought stress, such as S000407, S000144, S000447, and S000174. Subsequently, the expression pattern of PvTCPs was tested in switchgrass seedling when exposed to 250 mM NaCl. As described here, 29 out of 42 PvTCPs showed a trend of regulation under salt treatment but seemed to follow the different response patterns. PvTCP17/18, the homologous gene of OsTCP19 in switchgrass, also can be induced under salinity condition, and their expression levels were nearly 2.3-fold higher than the control. Besides, about 69% of PvTCPs were response to salt stress, but the regulatory mechanism was not elucidated. Our study would provide great assistance for establishing the regulatory network about salt tolerance based on the transcription level in switchgrass.


## 5. Conclusion

In this study, we conducted a genome-wide analysis for the switchgrassTCP gene family to reveal their genome organization, phylogeny, gene structure, motif localization, function prediction, and expression profiles in different tissues and when exposed to salt treatment. A total of 42 TCP proteins were identified and phylogenetically divided into three clades; 29 of the PvTCP genes respond to salt treatment. It will provide us not only an insight of prediction and selection for TCP gene functions but also an information to exploit much more important gene resource for creating new germplasm in the future.



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*Source: 8514928-2019-04-09.xml*