Genome-wide identification and analysis of the growth-regulating factor family in tobacco (Nicotiana tabacum)

Accepted Manuscript Genome-wide identification and analysis of the growth-regulating factor family in tobacco (Nicotiana tabacum)

Jianfeng Zhang, Zefeng Li, Jingjing Jin, Xiaodong Xie, Hui Zhang, Qiansi Chen, Zhaopeng Luo, Jun Yang PII: DOI: Reference:

S0378-1119(17)30804-1 doi:10.1016/j.gene.2017.09.070 GENE 42219

To appear in:

Gene

Received date: Revised date: Accepted date:

8 June 2017 22 September 2017 29 September 2017

Please cite this article as: Jianfeng Zhang, Zefeng Li, Jingjing Jin, Xiaodong Xie, Hui Zhang, Qiansi Chen, Zhaopeng Luo, Jun Yang , Genome-wide identification and analysis of the growth-regulating factor family in tobacco (Nicotiana tabacum). The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2017.09.070

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Genome-wide identification and analysis of the growth-regulating factor family in tobacco (Nicotiana tabacum)

Jianfeng Zhang1, Zefeng Li1, Jingjing Jin1, Xiaodong Xie1, Hui Zhang1, Qiansi Chen1, Zhaopeng Luo1, Jun Yang1 China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of

PT

1

CNTC, Zhengzhou 450001, China

RI

Corresponding author: Jun Yang, email address: [email protected]

SC

Abstract

Growth-regulating factors (GRFs) are plant-specific transcription factors that have

NU

important functions in regulating plant growth and development. GRF gene families have been described in several plant species, but a comprehensive analysis of the GRF

MA

gene family in tobacco has not yet been reported. In this study, we identified 25 NtabGRF genes in N. tabacum. The gene structures, motifs, and cis-acting regulatory

D

elements of the NtabGRF genes were analyzed. Phylogenetic analysis divided the

PT E

genes into six clusters. Additionally, highly conserved regions of microsynteny were identified in all of the sequenced tobacco species. Expression analysis showed that NtabGRF genes were highly expressed in actively growing tissues and responded to

CE

various hormone treatments. Our results provide foundational information about the GRF gene family in tobacco species, and open the door for future research on the

AC

functions of these genes.

Keywords: GRF, tobacco, gene structure, phylogenetic, gene duplication, hormone response

ACCEPTED MANUSCRIPT Introduction Growth-regulating factors (GRF) are plant-specific transcription factors that have been shown to play important roles in the regulation of plant growth and development. The first member of the GRF family to be identified was OsGRF1, which plays a regulatory role in gibberellic acid (GA)-induced stem elongation (van der Knaap et al.,

PT

2000). In recent years, with the sequencing of tens of plant genomes, the GRF family has been evaluated in many plant species including Arabidopsis thaliana (Kim et al.,

RI

2003), Oryza sativa (Choi et al., 2004), Zea mays (Zhang et al., 2008), Brassica rapa (Wang et al., 2014), Solanum lycopersicum (Cao et al., 2016a), Pyrus bretschneideri

SC

Rehd, Populous, Vitis vinifera (Cao et al., 2016b), and Brassica napus (Ma et al., 2017). GRF family proteins contain two conserved regions: the QLQ (Gln, Leu, Gln,

NU

IPR014978) and WRC (Trp, Arg, Cys, IPR014977) domains (van der Knaap et al., 2000; Kim et al., 2003; Choi et al., 2004; Omidbakhshfard et al., 2015). The

MA

conserved QLQ domain functions as a protein–protein interaction domain (van der Knaap et al., 2000). It is also present in the SWI2/SNF2 protein in yeast, where it

D

facilitates the interaction with other proteins to form a complex involved in chromatin

PT E

remodeling (Treich et al., 1995). The conserved WRC domain, in combination with a C3H motif, is expected to be relevant for DNA binding and for targeting of the TF to the nucleus (Raventos et al., 1998; van der Knaap et al., 2000). Both the QLQ and

CE

WRC domains are located at the N-terminal part of GRFs. Notably, proteins with a QLQ motif are found in all eukaryotes, while WRC domains are plant-specific

AC

(Omidbakhshfard et al., 2015). Additional less conserved motifs such as TQL, GGPL, and FFD motifs are located within the C-terminal regions of some GRFs (Kim et al., 2003; Choi et al., 2004; Zhang et al., 2008). GRFs are highly expressed in growing zones where cell proliferation occurs, such as shoot tips, flower buds, and growing leaves (Kim et al., 2003; Horiguchi et al., 2005; Kim et al., 2012; Bao et al., 2014; Liang et al., 2014; Pajoro et al., 2014). AtGRF1 to AtGRF3 acts as positive regulators of cell proliferation during leaf development (Kim et al., 2003; Kim and Kende, 2004; Debernardi et al., 2014). Overexpression of AtGRF1 was strongly associated with increases in total seed

ACCEPTED MANUSCRIPT weight and seed size (Van Daele et al., 2012). AtGRF4 is involved in not only cell proliferation in leaves but also the embryonic development of cotyledons and the SAM (Kim and Lee, 2006). AtGRF7 repressed a broad range of osmotic stress– responsive genes to prevent growth inhibition under normal conditions (Kim et al., 2012). ZmGRF10 appears to affect various cellular mechanisms to control growth

PT

(Wu et al., 2014). Overexpression of BrGRF8 in transgenic Arabidopsis plants increased the sizes of the leaves and other organs by regulating cell proliferation

RI

(Wang et al., 2014). Overexpression of BnGRF2 increases seed mass and oil production by upregulating the expression of chloroplast-related genes to enhance

SC

photosynthetic efficiency (Liu et al., 2012). A rare mutation of OsGRF4 in rice cultivars can significantly enhance grain weight and increase grain yield (Hu et al.,

NU

2015). Higher expression of OsGRF4 is correlated with larger grains, longer panicles, and lower seed shattering (Sun et al., 2016).

MA

Tobacco is an important crop that has both agricultural and scientific significance. Nicotiana tabacum (2n=4x=48) is an allotetraploid that was generated

D

through interspecific hybridization by two ancestors, Nicotiana sylvestris (2n=24) and

PT E

Nicotiana tomentosiformis (2n=24) (Leitch et al., 2008; Sierro et al., 2013). A draft of the N. sylvestris, N. tomentosiformis, and N. tabacum genome sequences was completed recently (manuscript under review), which facilitates the characterization

CE

of the GRF gene family in tobacco. In this study, we identified GRF genes in cultival tetraploid tobacco (N. tabacum) and two diploid tobacco species (N. sylvestris and N.

AC

tomentosiformis). We analyzed the gene structures and motifs of the NtabGRF family members, as well as the cis-acting regulatory elements in their promoters. Phylogenetic and microsynteny relationships were also clarified in tobacco GRFs. Furthermore, we characterized their tissue-dependent expression profiles and their potential roles in responses to hormones. Our results provide a foundation for further investigations into the evolution of NtabGRF genes and the specific functions of these genes.

Materials and Methods

ACCEPTED MANUSCRIPT

Identification of tobacco GRF genes The tobacco genome sequence has been completed, and filtered protein and CDS sequences have also become available via the China Tobacco Genome Database. A total of 34 GRF protein sequences were gathered from the three species: 9 from (http://www.arabidopsis.org/),

12

from

rice

PT

Arabidopsis

(http://rice.plantbiology.msu.edu/) and 13 from tomato (https://solgenomics.net/).

RI

These sequences were used as query sequences for a BLASTP analysis of the tobacco genome database. Additionally, Hidden Markov Model profiles of the QLQ (PF08880) WRC

(PF08879)

domains

(obtained

from

the

SC

and

Pfam

database;

http://pfam.xfam.org/) were used to analyze the tobacco genome database. To obtain

NU

potentially overlooked GRFs, all of the GRF candidates that we identified were themselves used as queries to search the draft genome and predicted cDNAs of

MA

tobacco. Subsequently, the Pfam and SMART (http://smart.embl-heidelberg.de/) proteomics servers were used to verify the conserved QLQ and WRC domains of the

PT E

D

putative GRF proteins.

Characterization of tobacco GRFs The molecular masses of the putative GRF proteins were calculated using the

CE

Compute pI/Mw tool of ExPaSy (http://web.expasy.org/compute_pi/). Schematic NtabGRF gene structure diagrams were drawn using the Gene Structure Display

AC

Server (http://gsds.cbi.pku.edu.cn/). Protein sequence motifs were predicted using the MEME program (http://meme.nbcr.net/meme3/mme.html). Sequence identities of the tobacco GRFs were calculated with the DNAMAN algorithm.

Prediction of promoter cis-elements in NtabGRFs The 1500 bp regions upstream of the putative translational initiation sites of all 25 putative NtabGRF loci were retrieved from the tobacco genome and were analyzed to predict

the

presence

of

cis-regulatory

elements

using

the

PlantCARE

(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The motifs putatively

ACCEPTED MANUSCRIPT involved in hormone and stress responsiveness were retained and reported.

Phylogenetic and microsynteny analysis To construct the phylogeny of the GRFs from various species, multiple sequence alignments for all GRF amino acid sequences were conducted using MUSCLE (Edgar,

PT

2004) with default settings. Phylogenetic analyses were carried out with a Neighbor-Joining method using MEGA 7.0 (Kumar et al., 2016) with the following

RI

parameters: pairwise deletion mode, Poisson correction, and bootstrapping (1,000 replicates). The MCScanX program (Wang et al., 2012) was used (with default

SC

settings) to identify syntenic blocks for N.sylvestris, N. tomentosiformis, and N. tabacum genomes, with at least 5 syntenic genes. Chromosomal distributions and

NU

microsynteny relationships were visualized using Circos v0.55 (Krzywinski et al.,

MA

2009).

Expression profiling based on the estimation of expression levels from RNA-Seq

D

data

PT E

The estimated expression levels, as FPKM (fragments per kilobase per million reads) values, for each of the NtabGRFs in 8 different tissues and developmental stages were obtained from the China Tobacco Genome Database. Samples included germinating

CE

seeds, callus, flowering buds, axillary buds, stem, roots, seedling leaves (six leaf stage), and expanded leaves (45 days after seedlings transplanted). A heat map was

AC

generated using MeV software (Saeed et al., 2003) after original the FPKM values were log2-transformed and centered.

Plant materials and hormone treatments Nicotiana tabacum var. Honghua Dajinyuan was used for transcriptional analysis. To verify the gene expression patterns of the NtabGRF genes, samples including germinating seeds, roots, stem, seedling leaves (six leaf stage), flowering buds and axillary buds were collected. For hormone treatments, Seeds were sown on vermiculite supplemented with half-strength Hoagland solution and grown with a

ACCEPTED MANUSCRIPT 16/8h light/dark photoperiod and 60% relative humidity of 28/25℃. Two-week-old seedlings grown on plates were exposed for 5h to 10 μM indole-3-acetic acid (IAA), 10 μM 6-benzylamino purine (6-BA), 10 μM brassinolide (BR), 50 μM gibberellin A3 (GA3), or 50 μM abscisic acid (ABA). The samples were frozen in liquid nitrogen

PT

and then stored at −80 °C for total RNA extraction.

RNA extraction and qRT-PCR

RI

Total RNA was prepared from samples using a Super Pure Plant poly RNA Kit (Gene Answer). RNase-free DNase I (TAKARA) was used in the extraction process to

SC

remove DNA contamination. Both the concentration and the quality of the RNA samples were evaluated with a Nanodrop 2000 spectrophotometer (Thermo). Reverse

NU

transcription of RNA to cDNA was performed using M-MLV Reverse Transcriptase with random primers (TAKARA). qRT-PCR was performed on a CFX ConnectTM

MA

real-time system (BIO-RAD) using iTaqTM Universal SYBR Green Supermix (BIO-RAD). Ribosomal protein gene L25 was used as an internal standard for

D

normalizing cDNA concentration. For the data analyses, the 2–△△CT method was used

PT E

for calculating the relative expression of NtabGRF genes. Sequences of primers used

Results

CE

for qRT-PCR are listed in Supplementary Table S9.

AC

Identification and characteristics of the GRF gene family members in tobacco In order to identify all GRF proteins in tobacco, both BLASTP and Hidden Markov Model (HMM) searches were performed. Initially, 34 amino acid sequences encoding GRF transcription factors from Arabidopsis, rice, and tomato were used as query sequences by performing a BLASTP search at tobacco genome database. Additionally, the Hidden Markov Model (HMM) profiles of the QLQ (PF08880) and WRC (PF08879) domains were searched against the tobacco genome database. Analysis with these two procedures yielded a total of 27 candidate GRF protein sequences in Nicotiana tabacum. Further tobacco database searching using these candidate GRFs

ACCEPTED MANUSCRIPT as queries against the draft genome and predicted mRNAs resulted in no further hits. All of the candidate genes were subjected to InterPro and SMART analysis, and two candidate GRF protein sequences were discarded due to the absence of a QLQ domain (Ntab0739800) and an incomplete WRC domain (Ntab0694830). Thus, in total, 25 proteins from 25 loci were identified as NtabGRFs, and their coding genes

PT

were designated as NtabGRF1 to NtabGRF25, according to their distribution in the Nicotiana tabacum genome (Table 1).

RI

The proteins encoded by the NtabGRFs were predicted to show significant differences in their sizes and their physicochemical properties (Table 1). Their

SC

predicted protein lengths varied from 214 to 610 amino acids. In addition, GRF protein sequences had large variations in predicted isoelectric point (pI) values

NU

(ranging from 5.96 to 10.31) and molecular weights (ranging from 24.32 kDa to 65.53 kDa). For the NtabGRF genes, exon numbers ranged from 2 to 5; 24 could be located

MA

on 15 chromosomes and NtabGRF25 located on Scaffold_3602. Chromosomes 6, 14, 22, and 23 carry three NtabGRFs, chromosome 9 has two, whereas chromosomes 1, 3,

D

5, 8, 10, 11, 13, 15, 17, and 20 each contain only one NtabGRF gene. Using the same

PT E

procedure, 12 GRFs in Nicotiana sylvestris and 13 GRFs in Nicotiana tomentosiformis were identified, respectively. The details of the other predicted properties of GRF protein sequences are summarized in Supplemental Table S1.

CE

Compared with GRF families identified in other species (Supplemental Table S2), the NtabGRF family was the second largest, and the number of genes in the NsylGRF and

AC

NtomGRF families were comparable to the number in rice and tomato.

Gene structure and motif divergence of NtabGRF genes Gene structure and motifs often diverge during the evolution of multi-gene families. To explore the phylogenetic relationships and patterns of the NtabGRF gene structures, we analyzed the intron-exon and motif characteristics of NtabGRFs. As shown in Fig. 1a, the NtabGRFs within each group had similar exon–intron organizations. Most NtabGRF genes harbored 3-4 exons, with the exceptions being NtabGRF9 and NtabGRF11, which have 2 and 5 exons, respectively. All NtabGRFs

ACCEPTED MANUSCRIPT contained the highly-conserved QLQ and WRC domains in their N-terminal regions (Fig. 1b), as in the GRFs identified in other species. It is notable that NtabGRF17 has an extra WRC domain close to its C-terminal region just like AtGRF9 in Arabidopsis and BrGRF12 in Chinese cabbage. The QLQ domains of the NtabGRF proteins are characterized by the conserved Gln-Leu-Gln residues; the exception to this is

PT

NtabGRF23 and NtabGRF24, which have a conservative substitution from Phe to Leu that was also reported for AtGRF9 (Kim et al., 2003). In contrast to the conserved

showed low sequence similarity (data not shown).

RI

N-terminal regions, the C-terminal parts of the NtabGRF proteins are variable, and

SC

Tools of the MEME web server were used to analyze motif distributions and to conduct domain predictions. A total of 20 motifs were identified in the NtabGRFs (Fig.

NU

2). The lengths, conserved sequence, and predicted motif models of each motif are listed in Supplementary Table S4. Motif 1, 2, 3, 6, and 8 were annotated to encode

MA

WRC, QLQ, FFD, TQL, and GGPL domains, respectively, while the remaining motifs did not have functional annotations. Each of the NtabGRF proteins detected carry 1 to

D

9 other motifs in addition to the typical WRC and QLQ domains. The motifs for

PT E

NtabGRFs belonging to the same cluster were conserved. FFD and TQL occurred together in most NtabGRFs, with the exceptions of NtabGRF9, 11, 17, 22, 23, 24, and 25. NtabGRF23 and 24 have an FFD domain but not a TQL domain. NtabGRF2, 3, 5,

CE

7, 8, 10, 12, 13, 14, 15, 16, 18, 23, and 24 have GGPL domain. Among these, NtabGRF2, 18, and 24 carried two GGPL motifs, with one located at the N-terminal

AC

and the other one located at the C-terminal, while NtabGRF5 only had one, located at the N-terminal. Motif 5 was presented in most NtabGRFs except NtabGRF11, 17, and 25. Whether specific functions are associated with these motifs is currently unknown.

Promoter cis-elements analysis of NtabGRF genes Promoter cis-elements play critical roles in the initiation of gene expression. Investigation of cis-regulatory elements in NtabGRF genes was performed within 1500 bp upstream from the putative translation start site using the PlantCARE database. Various cis-elements related to hormone and stress responses were

ACCEPTED MANUSCRIPT identified in the NtabGRF promoters (Fig. 3 and Supplementary Table S5). These cis-elements included: ABRE elements, which are often involved in responses relating to abscisic acid; ERE, ethylene-responsive elements; GARE-motif, TATC-box, and P-box, which are gibberellin-responsive elements; TGA-box, an auxin-responsive element; G-box, CGTCA-motif, and TGACG-motif, involved in the

PT

JA/MeJA-responses; TCA-element, involved in salicylic acid responsiveness. This abundance of hormone-responsive elements indicated that NtabGRFs appear to play roles

low-temperature

in

tobacco

response

hormone

motifs

signal

transduction.

RI

important

(LTR),

heat-shock

Additionally,

elements

(HSE),

SC

drought-inducibility elements (MBS), pathogen response elements (Box-W1), anaerobic induction elements (ARE and GC-motif), wound response elements

NU

(WUN-motif) and defense responsiveness elements (TC-rich repeats) were present in the NtabGRF promoters. This suggested that NtabGRF genes are possibly involved in

MA

responses to both biotic and abiotic stresses and defense signaling transduction in tobacco. Furthermore, the NtabGRF genes in the same phylogenetic cluster only

D

showed moderate consistency in their distributions of the cis-elements, indicating

PT E

that the promoters of these NtabGRFs had diverged.

Phylogenetic analysis of NtabGRF genes

CE

To gain insight into the evolution of GRFs in tobacco, a neighbor-joining phylogenetic tree was constructed using the GRF proteins from representative plant

AC

species, including the monocot Oryza sativa and the dicots Arabidopsis thaliana, Solanum lycopersicum (Supplementary Table S6), Nicotiana sylvestris, Nicotiana tomentosiformis, and Nicotiana tabacum. As shown in Fig. 4, the phylogenetic analysis divided 84 GRF proteins into 6 major clusters, designated as clusters Ⅰ to Ⅵ. In the comparative phylogenetic tree, 5 of the 6 clusters contained GRFs from eudicots and monocots species, whereas clusters Ⅳ had only eudicots species. Detailed inspection revealed that all of the NtabGRFs clearly fell into the Solanaceae groups, and closely clustered with its ancestral GRFs. According to their clustering in the phylogenetic tree and their sequence identities (Supplemental Table S3), most of

ACCEPTED MANUSCRIPT NsylGRFs and NtomGRFs had orthologs in N. tabacum (Fig. 4, indicated by blue boxes). This suggested that most GRF genes were retained after the ancestor’s interspecific hybridization. Considering the evolutionary relationships among the Solanaceae groups, the lack of an ortholog of NtomGRF4 in N. tabacum, and the presence of only one corresponding NsylGRF6/NtomGRF6 ortholog (Fig. 4, indicated

PT

by red dots) in N. tabacum suggests that at least two ancestral GRF genes were lost in cultivated tobacco. However, NtabGRF10 and NtabGRF17 clustered as a singletons, NtabGRF6/NtabGRF21/NsylGRF3

and

NtabGRF4/NtomGRF6/NtomGRF7

RI

and

grouped as a triad (Fig. 4, indicated by green dots), suggesting that gene duplication

SC

may have occurred after the formation of the allotetraploid N. tabacum.

NU

Conserved microsyntenies of tobacco GRF genes

Through pairwise comparisons and comparison of all of the proteins in the genome

MA

regions flanking GRF genes, conserved microsyntenies were identified among the tobacco species (Fig. 5 and Supplementary Table S7). Firstly, the intraspecies

D

microsynteny relationships of the GRF genes were identified. We found 18 collinear

PT E

gene pairs in N. tabacum and 2 in N. tomentosiformis. There were no collinear gene pairs in N.sylvestris. Subsequently, the corresponding interspecies microsynteny relationships of the GRF genes were investigated. 28 conserved syntenic segments

CE

were found between N. tabacum and N. tomentosiformis while 17 between N. tabacum and N.sylvestris, 7 between N. sylvestris and N. tomentosiformis. Notably, 6

AC

GRF genes were not present in any of our microsynteny analyses (NsylGRF2/3/11, NtomGRF4, and NtabGRF10/25). This microsynteny analysis may reflect the phylogenetic relationships between them to a certain extent. Three putative gene duplications were identified in N. tabacum: NtabGRF21 and NtabGRF6 (100% protein sequence identity), NtabGRF10 and NtabGRF12 (92.9% protein sequence identity), NtabGRF17 and NtabGRF11 (69.9% protein sequence identity). As shown in Fig. 5, all of these paralogs are located on different chromosomes, indicating that tandem duplication events appear not to have happened in the NtabGRF family. But NtabGRF17 and NtabGRF11 were identified as a

ACCEPTED MANUSCRIPT syntenic gene pair, suggesting that NtabGRF17 probably originated from segmental duplication, while the other two duplications may originated from dispersed duplications events.

Differential expression profiles of NtabGRF genes

PT

To gain insights into the potential functions of GRF genes in tobacco, RNA-Seq data from expression profiles of 8 different tobacco tissues and developmental stages

RI

corresponding to periods throughout the tobacco lifecycle were used to create a heat map of NtabGRFs expression (Fig. 6). Detailed inspection revealed that almost all of

SC

the NtabGRFs appeared to have relatively high expression throughout the actively growing tissue (such as germinating seeds, callus, and flower buds). All of the

NU

NtabGRFs had relatively higher transcription levels in young seedling leaves (six leaf stage) than in expanded mature leaves, suggesting that NtabGRFs may mainly

MA

function in the early stages of growth and development in leaves. However, NtabGRF3, 7, 18 and 22 in seedling leaves, NtabGRF7 and 18 in axillary buds,

D

NtabGRF4, 7 and 13 in roots, NtabGRF3, 7, 9, 13, 14, and 18 in stems were highly

PT E

expressed, suggesting that these genes may be associated with the regulation of growth and development of these specific tissues. NtabGRF18 was expressed in all of the samples, albeit at varying levels. There were no transcripts of NtabGRF9 in flower

CE

bud. NtabGRF10, 12 and 15 were hardly detected in leaves. Based on the analysis above, it is clear that NtabGRF genes may be expressed

AC

differentially in different organs. On account of this consideration, qRT-PCR was conducted to verify the gene expression patterns of the NtabGRF in six different tissues, including germinating seeds (GS), roots (R), stem (S), leaves (six leaf stage seedling) (L), flowering buds (FB) and axillary buds (AB). As shown in Fig. 7, the expression pattern for each gene detected by qRT-PCR was roughly in consistency with the RNA-seq analysis, which further confirmed their preferential expression. Most NtabGRFs have relatively high expression level in germinating seeds except NtabGRF3 and 14. NtabGRF5, 16, 23, 24, and 25 were mainly expressed in

ACCEPTED MANUSCRIPT germinating seeds. The transcription level of NtabGRF2, 10, 12, and 15 were too low to be detected in leaf, which was consist with results from RNA-seq analysis.

NtabGRFs transcriptional response to exogenous hormones treatments The first GRF gene OsGRF1 ever discovered was a gibberellic acid-induced

PT

gene in rice. Plant hormones help coordinate plant growth and development. Expression of most OsGRFs was enhanced by GA3 treatment while AtGRFs were

RI

mostly not affected by such treatment (Kim et al., 2003; Choi et al., 2004). To characterize the responsiveness of NtabGRF genes to plant hormones, two-week-old

SC

tobacco seedlings were exposed to exogenous GA3, 6-BA, IAA, ABA and BR. Then, quantitative real-time PCR was performed to examine the effects of hormones on the

NU

expression of NtabGRF genes. Comprehensive expression profiles of NtabGRF genes under hormone treatments are shown in Fig. 8. Nearly half of NtabGRFs showed

MA

significantly altered transcript levels after hormones treatments. 12 NtabGRFs in the GA3 treatment, 6 NtabGRFs in the IAA treatment, 9 NtabGRFs in the BR treatment,

D

15 NtabGRFs in the 6-BA treatment, and 11 NtabGRFs in the ABA treatment were

PT E

up-regulated by 2-fold or more. The highest fold inductions in the transcriptional responses to hormones were exhibited by NtabGRF12 (10.4-fold to GA3), NtabGRF1 and NtabGRF3 (2.4-fold to IAA), NtabGRF23 (3.7-fold to BR), and NtabGRF16

CE

(7.8-fold to 6-BA and 5-fold to ABA). Notably, NtabGRF12 and NtabGRF25 accumulated higher transcription levels in response to GA3 treatment but responded

AC

only slightly to other hormones. The expression of NtabGRF2 and NtabGRF10 was suppressed by IAA, their expression was not sensitive to other hormones. The expression of NtabGRF4, NtabGRF16, and NtabGRF20 were elevated more than two fold in all of the hormone treatments. These results indicate that NtabGRF genes likely function in a manner that is responsive to hormone signal transduction.

Discussion As plant-specific transcription factors, growth-regulating factors (GRFs) have been identified in various plants at genome-wide level. Only two GRF genes were found in

ACCEPTED MANUSCRIPT moss, while in other species there are usually eight or more (Supplementary Table S2). It is known that genes with a regulatory function are preferentially retained after large-scale duplications (Maere et al., 2005). Early expansion of the GRF gene family can be linked to the whole-genome triplication that occurred in the common ancestor of eudicots, and further expansions in this family have occurred through several

PT

independent whole-genome duplications in various plant lineages (Omidbakhshfard et al., 2015). It is believed that allotetraploid N. tabacum was formed by an interspecific

RI

hybridization between N. sylvestris and N. tomentosiformis about 200,000 years ago (Leitch et al., 2008; Sierro et al., 2013). In this study, 25 GRFs in N. tabacum, 12 in N.

SC

sylvestris and 13 in N. tomentosiformis were identified. Phylogenetic analysis of the predicted GRFs in different species indicated that the GRFs in tobacco shared the

NU

same ancestor. Each tobacco sub-cluster was closer to tomato than to the Arabidopsis GRFs, which was consistent with the known evolutionary relationships of these

MA

species. The numbers of GRFs in tobacco were larger than in Arabidopsis, but the diploid NsylGRF and NtomGRF families were comparable to the GRF family in

D

tomato. Comparing the phylogenetic relationships and GRF numbers in these species

PT E

suggests that the GRF family of the Solanaceae common ancestor may have experienced an expansion after the split of the Brassicaceae and the Solanaceae. However, the GRF expansions in tobacco have been complex. 22 NtabGRFs can be

CE

tracked origins in the diploid ancestors, based on their phylogenetic and collinearity relationships, suggesting that most GRF genes were well retained after the ancestor’s

AC

interspecific hybridization. Detailed inspection revealed that gene loss and gain events were occurred in the evolution of NtabGRFs. NtomGRF4 and NsylGRF6 appear to have been lost in N. tabacum. 3 gene duplication events in N. tabacum and 1 in N. tomentosiformis occurred after the formation of tetraploid tobacco. Chromosome distribution and microsynteny analysis revealed that both segmental and dispersed duplications had happened but no tandem duplication occurred in the expansion of the NtabGRF family. This is consistent with a previous study showing that tandem duplications have been rather rare in the expansion of GRF families (Omidbakhshfard et al., 2015).

ACCEPTED MANUSCRIPT Analysis of gene expression patterns can be used to some extent to predict the molecular functions of genes involved in different processes. A previous study established that GRFs are expressed in different parts of roots and shoots, often in growing zones where cell proliferation occurs (Kim et al., 2003; Horiguchi et al., 2005; Kim et al., 2012; Bao et al., 2014; Liang et al., 2014; Pajoro et al., 2014). In our

PT

study, NtabGRF genes were highly expressed at actively growing tissues such as germinating seeds, callus, and buds, where cell proliferation takes place vigorously.

RI

GRFs have been shown to positively regulate leaf size by promoting cell expansion and cell proliferation (Kim and Kende, 2004; Horiguchi et al., 2005; Rodriguez et al.,

SC

2010). Furthermore, AtGRF expression levels are known to decrease as the age of the plant increases (Kim et al., 2003; Rodriguez et al., 2010). NtabGRF genes showed

NU

higher expression levels in actively seedling young leaves than in expanded mature leaves, which is in accordance with their reported function in the early stages of the

MA

growth and development in different tissues (Kim et al., 2003; Horiguchi et al., 2005; Rodriguez et al., 2010).

D

Plant hormones regulate many physiological processes that mainly influence

PT E

growth, differentiation, and development. Previous studies revealed that the expression of several rice OsGRF genes and Chinese cabbage BrGRF genes were enhanced by GA3 treatment (van der Knaap et al., 2000; Choi et al., 2004; Wang et al.,

CE

2014), whereas GRFs in Arabidopsis were not obviously affected by GA3 (Kim et al., 2003). KNOX genes are important controllers of meristem development and function,

AC

and they are known to restrict cell differentiation. It is well established that GRFs are upstream repressors of KNOX genes that inhibit GA biosynthesis (Kuijt et al., 2014). It can be inferred that GA3 treatment stimulates the expression of KNOX, and subsequently high KNOX levels led to the up-regulation of GRFs. Here, we tested the responsiveness of NtabGRF genes to various hormones. Nearly half of the NtabGRFs in the GA3 treatment were up-regulated by 2-fold or more. Not only in response to GA3, but also in response to IAA, BR, ABA, and 6-BA treatments, many NtabGRF genes showed significantly changes in expression. The divergent expression of NtabGRF genes in tobacco indicates that these genes may play diverse roles in the

ACCEPTED MANUSCRIPT regulation of hormone feedback. Various cis-elements related to hormone were identified in the NtabGRF promoters. Most of the ABA- or GA3-inducible NtabGRF genes were found to contain ABRE (abscisic acid responsiveness) or GARE-motif, TATC-box, and P-box (gibberellin responsiveness) elements in their promoters. Some of them showed significantly altered transcript levels after related hormones

PT

treatments, such as NtabGRF8, 18, 20, and 22 to ABA and NtabGRF3, 4, 12, 17, 18, 19 to GA3 treatment. However, even IAA treatment induced 6 NtabGRFs only

RI

NtabGRF17 promoter consists of a TGA-box (IAA-responsive). NtabGRF25 accumulated high transcription levels in response to GA3 treatment but there was no

SC

gibberellin-responsive element in its promoter. cis-Element analysis also did not well predict the responsiveness of all NtabGRFs to hormone treatment. Notably, the

NU

expression of both NtabGRF2 and NtabGRF10 was suppressed significantly by IAA, suggesting the possible presence of negative feedback regulation in hormone

MA

signaling in tobacco. More research is needed for a better understanding of transcriptional regulation in tobacco plants, including TFs and their specific

D

cis-elements.

PT E

Our research here identifies and characterizes the tobacco GRF gene family and evaluates their gene and motif structures, their evolutionary histories, and their expression patterns in different tobacco tissues, as well as their expression in response

CE

to hormone treatments. Our findings will be helpful for efforts to further understand the functions of these important transcription factors in various growth and

AC

developmental processes in tobacco.

Acknowledgments This work was supported by a grant (902015CA0270) from Zhengzhou Tobacco Research Institute of CNTC and the Project of Tobacco Transcription Factors Array Design and Expression Regulation of Key Transcription Factors (902014AA0520). We also thank Dr. John Hugh Snyder for his critical reading of this manuscript.

Conflict of interest The authors declare that they have no conflict of interest.

ACCEPTED MANUSCRIPT

Ethical approval This article does not contain any studies with human participants or

AC

CE

PT E

D

MA

NU

SC

RI

PT

animals performed by any of the authors.

ACCEPTED MANUSCRIPT References Bao, M., Bian, H., Zha, Y., Li, F., Sun, Y., Bai, B., Chen, Z., Wang, J., Zhu, M. and Han, N., 2014. miR396a-Mediated basic helix-loop-helix transcription factor bHLH74 repression acts as a regulator for root growth in Arabidopsis seedlings. Plant Cell Physiol 55, 1343-53. Cao, D., Wang, J., Ju, Z., Liu, Q., Li, S., Tian, H., Fu, D., Zhu, H., Luo, Y. and Zhu, B., 2016a. Regulations on growth and development in tomato cotyledon, flower and fruit via destruction of miR396 with short tandem target mimic. Plant Sci 247, 1-12. Cao, Y., Han, Y., Jin, Q., Lin, Y. and Cai, Y., 2016b. Comparative Genomic Analysis of the GRF Genes in Arabidopsis and Rice (Oryza sativa). Front Plant Sci 7, 1750.

PT

Chinese Pear (Pyrus bretschneideri Rehd), Poplar (Populous), Grape (Vitis vinifera), Choi, D., Kim, J.H. and Kende, H., 2004. Whole genome analysis of the OsGRF gene family encoding

RI

plant-specific putative transcription activators in rice (Oryza sativa L.). Plant Cell Physiol 45, 897-904.

SC

Debernardi, J.M., Mecchia, M.A., Vercruyssen, L., Smaczniak, C., Kaufmann, K., Inze, D., Rodriguez, R.E. and Palatnik, J.F., 2014. Post-transcriptional control of GRF transcription factors by microRNA miR396 and GIF co-activator affects leaf size and longevity. Plant J 79, 413-26.

NU

Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32, 1792-7.

Horiguchi, G., Kim, G.T. and Tsukaya, H., 2005. The transcription factor AtGRF5 and the transcription

MA

coactivator AN3 regulate cell proliferation in leaf primordia of Arabidopsis thaliana. Plant J 43, 68-78.

Hu, J., Wang, Y., Fang, Y., Zeng, L., Xu, J., Yu, H., Shi, Z., Pan, J., Zhang, D., Kang, S., Zhu, L., Dong, G., Guo, L., Zeng, D., Zhang, G., Xie, L., Xiong, G., Li, J. and Qian, Q., 2015. A Rare Allele of GS2

D

Enhances Grain Size and Grain Yield in Rice. Mol Plant 8, 1455-65.

PT E

Kim, J. and Lee, B., 2006. GROWTH-REGULATING FACTOR4 ofArabidopsis thaliana is required for development of leaves, cotyledons, and shoot apical meristem. Journal of Plant Biology 49, 463-468.

Kim, J.H., Choi, D. and Kende, H., 2003. The AtGRF family of putative transcription factors is involved in

CE

leaf and cotyledon growth in Arabidopsis. Plant J 36, 94-104. Kim, J.H. and Kende, H., 2004. A transcriptional coactivator, AtGIF1, is involved in regulating leaf growth and morphology in Arabidopsis. Proc Natl Acad Sci U S A 101, 13374-9.

AC

Kim, J.S., Mizoi, J., Kidokoro, S., Maruyama, K., Nakajima, J., Nakashima, K., Mitsuda, N., Takiguchi, Y., Ohme-Takagi,

M.,

Kondou,

Y.,

Yoshizumi,

T.,

Matsui,

M.,

Shinozaki,

K.

and

Yamaguchi-Shinozaki, K., 2012. Arabidopsis growth-regulating factor7 functions as a transcriptional repressor of abscisic acid- and osmotic stress-responsive genes, including DREB2A. Plant Cell 24, 3393-405. Krzywinski, M., Schein, J., Birol, I., Connors, J., Gascoyne, R., Horsman, D., Jones, S.J. and Marra, M.A., 2009. Circos: an information aesthetic for comparative genomics. Genome Res 19, 1639-45. Kuijt, S.J., Greco, R., Agalou, A., Shao, J., t Hoen, C.C., Overnas, E., Osnato, M., Curiale, S., Meynard, D., van Gulik, R., de Faria Maraschin, S., Atallah, M., de Kam, R.J., Lamers, G.E., Guiderdoni, E., Rossini, L., Meijer, A.H. and Ouwerkerk, P.B.,

2014. Interaction between

the

GROWTH-REGULATING FACTOR and KNOTTED1-LIKE HOMEOBOX families of transcription factors. Plant Physiol 164, 1952-66. Kumar, S., Stecher, G. and Tamura, K., 2016. MEGA7: Molecular Evolutionary Genetics Analysis Version

ACCEPTED MANUSCRIPT 7.0 for Bigger Datasets. Mol Biol Evol 33, 1870-4. Leitch, I.J., Hanson, L., Lim, K.Y., Kovarik, A., Chase, M.W., Clarkson, J.J. and Leitch, A.R., 2008. The ups and downs of genome size evolution in polyploid species of Nicotiana (Solanaceae). Ann Bot 101, 805-14. Liang, G., He, H., Li, Y., Wang, F. and Yu, D., 2014. Molecular mechanism of microRNA396 mediating pistil development in Arabidopsis. Plant Physiol 164, 249-58. Liu, J., Hua, W., Yang, H.L., Zhan, G.M., Li, R.J., Deng, L.B., Wang, X.F., Liu, G.H. and Wang, H.Z., 2012. The BnGRF2 gene (GRF2-like gene from Brassica napus) enhances seed oil production through regulating cell number and plant photosynthesis. J Exp Bot 63, 3727-40.

PT

Ma, J.Q., Jian, H.J., Yang, B., Lu, K., Zhang, A.X., Liu, P. and Li, J.N., 2017. Genome-wide analysis and expression profiling of the GRF gene family in oilseed rape (Brassica napus L.). Gene 620,

RI

36-45.

Maere, S., De Bodt, S., Raes, J., Casneuf, T., Van Montagu, M., Kuiper, M. and Van de Peer, Y., 2005.

SC

Modeling gene and genome duplications in eukaryotes. Proc Natl Acad Sci U S A 102, 5454-9. Omidbakhshfard, M.A., Proost, S., Fujikura, U. and Mueller-Roeber, B., 2015. Growth-Regulating Factors (GRFs): A Small Transcription Factor Family with Important Functions in Plant Biology.

NU

Mol Plant.

Pajoro, A., Madrigal, P., Muino, J.M., Matus, J.T., Jin, J., Mecchia, M.A., Debernardi, J.M., Palatnik, J.F., Balazadeh, S., Arif, M., O'Maoileidigh, D.S., Wellmer, F., Krajewski, P., Riechmann, J.L.,

MA

Angenent, G.C. and Kaufmann, K., 2014. Dynamics of chromatin accessibility and gene regulation by MADS-domain transcription factors in flower development. Genome Biol 15, R41.

Raventos, D., Skriver, K., Schlein, M., Karnahl, K., Rogers, S.W., Rogers, J.C. and Mundy, J., 1998. HRT, a

D

novel zinc finger, transcriptional repressor from barley. J Biol Chem 273, 23313-20. Rodriguez, R.E., Mecchia, M.A., Debernardi, J.M., Schommer, C., Weigel, D. and Palatnik, J.F., 2010.

PT E

Control of cell proliferation in Arabidopsis thaliana by microRNA miR396. Development 137, 103-12.

Saeed, A.I., Sharov, V., White, J., Li, J., Liang, W., Bhagabati, N., Braisted, J., Klapa, M., Currier, T., Thiagarajan, M., Sturn, A., Snuffin, M., Rezantsev, A., Popov, D., Ryltsov, A., Kostukovich, E.,

CE

Borisovsky, I., Liu, Z., Vinsavich, A., Trush, V. and Quackenbush, J., 2003. TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34, 374-8. Sierro, N., Battey, J.N., Ouadi, S., Bovet, L., Goepfert, S., Bakaher, N., Peitsch, M.C. and Ivanov, N.V.,

AC

2013. Reference genomes and transcriptomes of Nicotiana sylvestris and Nicotiana tomentosiformis. Genome Biol 14, R60. Sun, P., Zhang, W., Wang, Y., He, Q., Shu, F., Liu, H., Wang, J., Wang, J., Yuan, L. and Deng, H., 2016. OsGRF4 controls grain shape, panicle length and seed shattering in rice. J Integr Plant Biol. Treich, I., Cairns, B.R., de los Santos, T., Brewster, E. and Carlson, M., 1995. SNF11, a new component of the yeast SNF-SWI complex that interacts with a conserved region of SNF2. Mol Cell Biol 15, 4240-8. Van Daele, I., Gonzalez, N., Vercauteren, I., de Smet, L., Inze, D., Roldan-Ruiz, I. and Vuylsteke, M., 2012. A comparative study of seed yield parameters in Arabidopsis thaliana mutants and transgenics. Plant Biotechnol J 10, 488-500. van der Knaap, E., Kim, J.H. and Kende, H., 2000. A novel gibberellin-induced gene from rice and its potential regulatory role in stem growth. Plant Physiol 122, 695-704.

ACCEPTED MANUSCRIPT Wang, F., Qiu, N., Ding, Q., Li, J., Zhang, Y., Li, H. and Gao, J., 2014. Genome-wide identification and analysis of the growth-regulating factor family in Chinese cabbage (Brassica rapa L. ssp. pekinensis). BMC Genomics 15, 807. Wang, Y., Tang, H., Debarry, J.D., Tan, X., Li, J., Wang, X., Lee, T.H., Jin, H., Marler, B., Guo, H., Kissinger, J.C. and Paterson, A.H., 2012. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res 40, e49. Wu, L., Zhang, D., Xue, M., Qian, J., He, Y. and Wang, S., 2014. Overexpression of the maize GRF10, an endogenous truncated growth-regulating factor protein, leads to reduction in leaf size and plant height. J Integr Plant Biol 56, 1053-63.

PT

Zhang, D.-F., Li, B., Jia, G.-Q., Zhang, T.-F., Dai, J.-R., Li, J.-S. and Wang, S.-C., 2008. Isolation and characterization of genes encoding GRF transcription factors and GIF transcriptional

AC

CE

PT E

D

MA

NU

SC

RI

coactivators in Maize (Zea mays L.). Plant science 175, 809-817.

ACCEPTED MANUSCRIPT

Ntab0449590 Ntab0623000 Ntab0213060 Ntab0887310 Ntab0345650 Ntab0544540 Ntab0239680 Ntab0478300 Ntab0375140

Chr06 (-) Chr08 (-) Chr09 (-) Chr09 (+) Chr10 (-) Chr11 (+) Chr13 (+) Chr14 (-) Chr14 (+)

1065 1758 1833 645 1059 1131 1068 1008 1116

354 585 610 214 352 376 355 335 371

Ntab0447180 Ntab0004090 Ntab0574450 Ntab0759910 Ntab0201260 Ntab0128970 Ntab0680170 Ntab0256550

Chr14 (-) Chr15 (+) Chr17 (+) Chr20 (+) Chr22 (+) Chr22 (+) Chr22 (+) Chr23 (+)

1098 1833 1179 1818 1077 1107 1065 648

4 4 3 4 4 3 3 3

365 610 392 605 358 368 354 215

40.18 65.52 44.06 65.51 40.30 41.23 39.66 24.49

9.26 8.31 10.02 8.39 9.27 7.81 7.95 9.54

1413 1410

4 4

470 469

51.37 51.46

7.00 7.12

1209

4

402

44.47

9.45

NU

MA

CE

Chr23 (-) Chr23 (-) Scaffold_3602 NtabGRF25 Ntab0620920 (-)

AC

NtabGRF23 Ntab0095780 NtabGRF24 Ntab0773970

3 4 4 2 3 5 4 3 4

RI

NtabGRF15 NtabGRF16 NtabGRF17 NtabGRF18 NtabGRF19 NtabGRF20 NtabGRF21 NtabGRF22

D

NtabGRF6 NtabGRF7 NtabGRF8 NtabGRF9 NtabGRF10 NtabGRF11 NtabGRF12 NtabGRF13 NtabGRF14

Exon Length No. (aa) 3 403 4 561 4 392 3 356 4 480

PT

Chr01 (-) Chr03 (-) Chr05 (-) Chr06 (+) Chr06 (-)

CDS (bp) 1212 1686 1179 1071 1443

Chr. (strand)

PT E

NtabGRF1 NtabGRF2 NtabGRF3 NtabGRF4 NtabGRF5

Accession No. Ntab0338840 Ntab0254590 Ntab0132150 Ntab0186900 Ntab0514050

Name

SC

Table 1 Characteristics of the GRFs in N.tabacum MW (kDa) 45.17 61.44 42.99 39.64 53.05

7.67 6.05 8.69 7.85 5.96

39.66 63.47 65.53 24.32 40.20 41.84 40.60 38.35 40.53

7.95 8.39 8.49 10.31 8.88 9.47 8.61 9.51 9.29

pI

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 1 Conserved QLQ and WRC domains in NtabGRFs. a. The gene structures and distributions of the conserved domains of NtabGRFs. Exons are represented by

CE

yellow boxes; introns are shown as black lines. QLQ and WRC domains are indicated by red and green boxes, respectively. The sizes of exons/introns and domains are

AC

proportional to their sequence lengths. b. Alignment of the QLQ and WRC domains. Conserved and conservatively substituted amino acids are highlighted.

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

MA

Fig. 2 Motif compositions of NtabGRFs. Conserved motifs in NtabGRFs were detected with MEME. Twenty different motifs are represented by variously colored boxes. The sequences of each putative motif are shown in Supplemental Table 4. The

AC

CE

PT E

D

sizes of motifs are proportional to their sequence lengths.

SC

RI

PT

ACCEPTED MANUSCRIPT

NU

Fig. 3 Promoter cis-elements analysis of NtabGRFs. The distribution of main cis-acting elements and their putative regulating factors were detected in the 1.5 kb

MA

upstream promoter regions of NtabGRF genes. The different types of cis-elements are represented by different shapes and colors. Dark black boxes indicate the ORF of each

AC

CE

PT E

D

NtabGRF gene.

AC

CE

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 4 Phylogenetic relationships among the GRFs of tobacco, Arabidopsis, rice, and tomato. A phylogenetic tree was constructed for 25 Nicotiana tabacum, 12 Nicotiana sylvestris, 13 Nicotiana tomentosiformis, 9 Arabidopsis thaliana, 12 Oryza sativa, and 13 Solanum lycopersicum GRFs. The 6 phylogenetic clusters designated as I–VI are marked with vertical bars. NsylGRFs, NtomGRFs, and their corresponding orthologous genes in N. tabacum are indicated by blue boxes. The predicted gene loss and duplication events are highlighted with red and green dots, respectively. Numbers above branches show bootstrap support values. The scale bar represents 0.1 amino acid changes per site.

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 5 Chromosome distribution and microsynteny relationships among tobacco GRFs.

CE

The N. sylvestris, N. tomentosiformis, and N. tabacum chromosomes are depicted as different color boxes. The distribution of GRF genes on chromosomes are given in the

AC

circle. Microsynteny relationships between GRF regions are represented by different color lines. Blue, NtomGRFs to NtabGRFs; Orange, NsylGRFs to NtabGRFs; Green, NsylGRFs to NtomGRFs; Black lines represent syntenic relationships in each genome.

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

PT E

Fig. 6 Expression heat map of NtabGRF genes in different tobacco tissues. 8 samples were used for expression analysis, including germinating seeds, callus, flowering buds, axillary buds, stems, roots, seedling leaves, and expanded leaves. Expression values

AC

to red.

CE

from RNA-seq data were log2-transformed and are displayed as filled blocks in green

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

MA

Fig. 7 Expression analysis of NtabGRF genes in in different tissues. qRT-PCR analysis of NtabGRFs transcript levels in including germinating seeds (GS), roots (R), stem (S), leaves (six leaf stage seedling) (L), flowering buds (FB) and axillary buds (AB).

AC

CE

PT E

D

Data are presented as means ± SD.

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 8 Expression analysis of NtabGRF genes in response to various hormones.

D

qRT-PCR analysis of NtabGRFs transcript levels in two-week-old seedlings treated

PT E

with distilled water (CK), GA3, 6-BA, IAA, ABA, and BR. Data are presented as

AC

CE

means ± SD.

ACCEPTED MANUSCRIPT Abbreviation GRF, growth regulating-factor; GA, gibberellic acid; IAA, indole-3-acetic acid; 6-BA, 6-benzylamino purine;

PT

BR, brassinolide; ABA, abscisic acid;

AC

CE

PT E

D

MA

NU

SC

RI

NJ, Neighbor Joining method.

ACCEPTED MANUSCRIPT Highlights 

A complete set of 25 members of the GRF family in N. tabacum, 12 in N. sylvestris and 13 in N. tomentosiformis have been identified in tobacco.



The bioinformatic analyses and expression profiles of NtabGRFs in tobacco have been provided.



Most GRF genes were well retained after the ancestor’s interspecific

PT

hybridization, gene loss and duplication were happened after the formation of the allotetraploid N. tabacum.

RI

The NtabGRFs not only in response to GA3, but also in response to IAA, BR,

CE

PT E

D

MA

NU

SC

ABA, and 6-BA.

AC

