Characterization of Cotton ARF Factors and the Role of GhARF2b in Fiber Development

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At present, our understanding of cotton fiber development and the regulatory network is still primitive. Results: Here, we identify auxin response factor (ARF) genes in three cotton species: the tetraploid upland cotton G. hirsutum , which has 73 ARF genes, and its putative extent parental diploids G. arboreum and G. raimondii , which have 36 and 35 ARFs, respectively. Ka and Ks analyses revealed that in G. hirsutum ARF genes have undergone asymmetric evolution in the two subgenomes. The cotton ARFs can be classified into four phylogenetic clades and are actively expressed in young tissues. We demonstrate that GhARF2b , a homolog of the Arabidopsis AtARF2 , was preferentially expressed in developing ovules and fibers. Overexpression of GhARF2b by a fiber specific promoter inhibited fiber cell elongation but promoted initiation and, conversely, its downregulation by RNAi of resulted in fewer but longer fiber. Conclusion: Our results uncover an important role of the ARF factor in modulating cotton fiber development at the early stage. Epigenetics & Genomics Cotton GhARF2b Fiber elongation Fiber initiation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Cotton is the most important natural and renewable material for the textile industry in the world [ 1 ]. The primary cultivated species upland cotton ( G. hirsutum L.) is grown in over 80 countries and accounts for more than 90% of global cotton fiber output. Cotton fibers are unusually long, single-celled epidermal seed trichomes and a model for plant cell growth research [ 2 ]. Fiber development can be divided into four overlapping stages: initiation, elongation, secondary cell wall biosynthesis and maturation [ 3 ]. The fiber length and density are both key traits that determine cotton quality and yield. The study of cotton fiber development regulation provides not only valuable knowledge to understanding plant cell growth and cell wall biosynthesis, but also candidate genes for cotton molecular breeding. To date a number of genes that function in cotton fiber cells have been identified, including homeodomain transcription factor GaHOX1, GhHOX3 and GhHD1 [ 4 – 6 ], bHLH transcription factor GhPRE1 [ 7 ], KNOX transcription factor knl1 [ 8 ], the sterol carrier gene [ 9 ], MYB transcription factors GhMYB25, GhMYB25-like, GhMML3 and GhMML4 [ 10 – 13 ], NAC transcription factor fsn1 [ 14 ], transcription factor WLIM1a gene [ 15 ], sucrose synthase gene [ 16 ], cotton actin1 gene [ 17 ], cotton BURP domain protein GhRDL1 [ 18 ], ethylene pathway related genes [ 19 ], fasciclin-like arabinogalactan protein, Ghfla1 [ 20 ], and TCP transcription factor GhTCP4 [ 21 ] etc. Among recent progresses are the characterizations of transcription factors which regulate the major events of cotton fiber development, such as MYBs and HD-ZIP IVs involved in cotton fiber initiation and elongation, as well as a number of other types of factors. The MIXTA type MYB transcription factors (GhMYB25, GhMYB25-like and GhMML4_D12) are master regulators of cotton fiber initiation [ 10 , 12 , 13 ] and lint fiber development [ 11 ], whereas the HD-ZIP IV transcription factor GhHOX3 plays a pivotal role in controlling fiber elongation [ 4 ], whose activity is regulated by the phytohormone gibberellin. In addition, NAC (GhFSN1) and TCP4 transcription factors positively regulates secondary cell wall biosynthesis [ 14 , 21 ]. However, cotton fiber growth and development are complex processes involving cell differentiation, cell skeleton orientation growth, cell wall synthesis, and so on [ 22 ]. Currently the picture of the regulation network of cotton fiber is far from complete. Auxin response factors (ARFs), a group of plant transcription factors, are composed of a conserved N-terminal DNA binding domain (DBD), a most case conserved C-terminal dimerization domain (CTD) and a non-conserved middle region (MR) [ 23 ]. The MR region has been proposed to function as a repression or an activation domain [ 24 ]. Arabidopsis thaliana contains 23 ARF genes and Oryza sativa has 25 [ 25 , 26 ]. It has been reported that ARF2 negatively modulates plant growth in A. thaliana [ 25 , 27 – 29 ] and tomato [ 30 ], yet functions of transcription factors can vary with tissues and more diversified in polyploid species, to date the role ARF2 in cotton has not been explored. In this study, we conducted a genome-wide analysis ARF genes in three cotton species ( G. hirsutum , G. arboreum and G. raimondii ), and classified them into four clades. In G. hirsutum most ARF genes were expressed in multiple cotton tissues, among which GhARF2b exhibited a preferential expression in developing cotton fiber cells, and it negatively affects cotton fiber elongation but plays a role in promoting fiber initiation. Results ARF transcription factors in G. arboreum and G . hirsutum The genome sequences of G. raimondii and G. arboreum provide us data resources to conduct a genome-wide screen of the ARF genes in the extent diploid progenitors of the allotetraploid G. hirsutum . In the previous studies, Sun et al., (2015) identified 35 ARF genes in G. raimondii [ 31 ]. To mine more ARF transcription factors in cottons the conserved domain (Pfam ID: PF06507) was used to hmmersearch against the G. arboreum and G. hirsutum genome databases, which resulted in 36 and 73 genes in G. arboreum and G. hirsutum genomes, respectively. The 36 G. arboreum ARF genes were designated GaARF1 – GaARF20 , and the 73 G. hirsutum ARF genes in A- and D-subgenomes were designated as GhARF1A/D – GhARF21A/D (Table 1 ). As those of Arabidopsis, cotton ARF proteins are composed of three domain regions, including DBD (DNA-binding Domain), MI (Middle Region) and CTD (C-terminal Domain) (Additional file 1: Figure S1). Phylogenetic analysis of Gossypium ARF proteins To illustrate the evolutionary relationships among the cotton ARFs, a phylogenetic tree was constructed using the protein sequences of 144 cotton ARFs, which were clustered into four clades (I–IV). The highest number of Gossypium ARFs are found in clade III and I, followed by clade IV and II (Fig. 1 ). Overall, the expected diploid-polyploid topology is reflected in the tree for each set of orthologous/homoeologous genes, indicating general preservation during divergence of diploids and through the polyploid formation. We found that the number of ARF genes in G. hirsutum are approximately twice that in G. raimondii and G. arboreum , with one A t or D t homoeologous copy corresponding to one ortholog in each of the diploid cottons. Further, as shown in Fig. 1 , the orthologous paired genes of the A genome ( G. arboreum ) and A t sub-genome, or from the D genome ( G. raimondii ) and D t sub-genome, tend to be clustered together and share a sister relationship. Divergence of ARF genes in allotetraploid G. hirsutum and its diploid progenitors The ARF genes in the two diploid species were then compared with G. hirsutum A t - and D t -subgenome homoeologs (Table 1 ). To explore the evolutionary relationship and possible functional divergence of ARF genes between the allotetraploid cotton and its extend diploid progenitors, the nonsynonymous substitution ( Ka ) and synonymous substitution values ( Ks ) and the K a/ K s ratios for each pair of the genes were calculated (Table 1 ). By comparing the Ka and Ks values of 66 orthologous gene sets between the allotetraploid and its diploid progenitor genomes, we found that the Ka and Ks values are higher in the D t subgenome than in the A t subgenome (Fig. 2 ). These results indicate that GhARF genes in the D t subgenome tend to have experienced faster sequence divergence than their A t counterparts, suggesting an inconsistent evolution of ARF genes in the two subgenomes (Fig. 2 ). In addition, the Ka/Ks ratios of one D t -subgenome genes ( GhARF3b_D ) and five A t -subgenome gene ( GhARF2e_A , GhARF3c_A , GhARF4b_A , GhARF16b_A and GhARF17b_A ) are greater than 1 (Table 1 ), suggesting that these genes have under positive selections after divergence of G. hirsutum from diploid ancestors, and may have gained new functions. Expression analysis of GhARF genes in different cotton tissues The expression profile of a gene family can provide valuable clues to possible functions of each genes. Analysis of 73 GhARF genes showed that most genes have different spatial expression patterns. For instance, GhARF1, GhARF2a, GhARF2b and GhARF2c were expressed in all the tissues of cotton examined (Additional file 2: Figure S2), whereas GhARF3a and GhARF3c were expressed preferentially in the pistils and ovules. Compared to GhARF5b , GhARF5a showed higher expressions in the root, pistil and ovule organs. Transcripts of GhARF3c and GhARF4a , GhARF9a and GhARF9b were most abundant in stem and root, respectively. Over half of GhARF genes showed a relatively high level of transcript accumulation in leaf. Notably, there are more than ten genes (including GhARF1 , GhARF2a , GhARF2b , GhARF8a , GhARF9a , GhARF10b , GhARF11 , GhARF16a , GhARF18 and GhARF19 ) that were highly expressed in cotton fiber cells at the fast elongation stage (5 dpa). In summary, most of the GhARF genes were up-regulated in ovule, fiber, vegetative and other tissues (Additional file 2: Figure S2). GhARF2 and GhARF18 showed the highest expression in fiber (5 dpa) and both were located in the Clade I (Fig. 1 ), suggesting that they may function in cotton fiber development. Previous studies have demonstrated that ARF2 plays a role in transcriptional regulation in auxin-mediated cell division [ 29 ], leaf longevity [ 32 ], response to stress [ 33 ], regulation of fruit ripening [ 30 ] and so on. As GhARF2s shown pleiotropic effects on plant development [ 34 ], we decided to identify the major GhARF2s in regulation of cotton fiber elongation in subsequent experiments. GhARF2 had a high expression pattern during fiber elongation process There are nine ARF2 genes in G. hirsutum ( GhARF2c_At not annotated), we first examined their expression profiles in different tissues in cotton (Fig. 3 ). Based on the published RNA-seq data (Zhang et al., 2015) GhARF2a , GhARF2b and GhARF2c genes had higher expression levels in various tissues than GhARF2d or GhARF2e (Fig. 3 a). The transcripts of GhARF2b homoeologs ( GhARF2b_At and GhARF2b_Dt ) were enriched and abundant in cotton fiber cells (Fig. 3 a), subsequent quantitative RT-PCR (qRT-PCR) confirmed the expression pattern (Fig. 3 b). The highly up-regulated expression in fiber cell suggested that GhARF2b has been recruited to act primarily in cotton fiber. GhARF2b overexpression represses cotton fiber elongation To test the function of GhARF2b , we constructed the vectors to over-express and down-regulate GhARF2b_Dt in G. hirsutum by using the fiber-specific GhRDL1 promoter [ 7 , 18 , 35 ]. The expression levels of GhARF2b in transgenic cotton were clearly elevated in the overexpression lines according to qRT-PCR analysis; for example, the GhARF2b transcript abundance was about two-fold higher in the OE-3 than in the wild-type cotton fiber cells (Fig. 4 a). However, GhARF2b did not stimulate fiber cell elongation, rather, it resulted in shorter fiber (Fig. 4 b,c). On the contrary, suppressing GhARF2b expression by RNAi resulted in longer fibers (Fig. 5 a,b). The expression levels of GhARF2b in RNAi cottons in the RNAi lines were about 3 ~ 5-fold down-regulated in cotton fiber of 0DPA, 6DPA and 12DPA (Fig. 5 c-e). Together, these data suggest that GhARF2b acted as a negative regulator of fiber cell elongation, at least when its expression exceeded the threshold. Alternatively, it may function in other aspects of cotton fiber development. GhARF2b overexpression enhances cotton fiber initiation Next, we examined the effects of GhARF2b up-regulation on cotton fiber initiation. The over-expression line OE-3 and RNAi line ds-2 were selected for analyses. The SEM with 60 × magnification of ovules of WT-R15, OE-3 and ds-2 collected at -1, 0, 1 DPA were observed (Fig. 6 ). The cotton fiber initiation of the − 1-DPA ovules did not present differences among the three types of cottons, however, the 0- and 1-DPA ovules of OE-3 and ds-2 lines showed higher and lower densities of fiber initials compared to the wild-type control (Fig. 6 ). Further, we magnified the SEM views of ovules to 500–700× (Fig. 7 ). Obviously, at the fiber initiation stage (0, 1 DPA), the fiber initial density of the OE-3 was increased by about 1.5-fold compared with that of the wild-type, in contrast, the fiber initial density of the ds-2 line was reduced (Fig. 7 a-c). These results support a role of GhARF2b in promoting cotton fiber cell initiation. Discussion Previous studies showed that ARF family genes have been identified in many plant species, including 23 ARF genes in Arabidopsis thaliana [ 25 ], 25 in Oryza sativa [ 26 ], 39 in Populus trichocarpa [ 36 ], 31 in Zea mays [ 37 ], 15 in Cucumis sativus [ 38 ] and 35 in G. raimondii [ 31 ]. Here, we additionally characterized 36 ARF genes in G. arboreum and 73 in G. hirsutum , adding valuable data to understanding the distribution and evolution of ARF genes in plants. After whole genome duplication, the amplified genes generally undergo the events of functional loss, or neofunctionalization or subfunctionalization [ 39 ]. In this study, we found that six GhARF genes (five from A t subgenome) have experienced relatively faster positive selection compared to its diploid progenitors. Thus, duplicated genes from A t and D t subgenomes might be functionally diverged in the allotetraploid cotton after the merge of the two genomes. Auxin response factors (ARFs) are important in plant development as they play crucial roles in regulating a variety of signaling pathways [ 23 , 24 ]. According to their functions, ARF proteins are divided into two classes: transcriptional activators and transcriptional repressors [ 23 ]. Many studies have revealed their regulatory roles in regulating various aspects of cellular activities [ 34 , 40 – 42 ]. As transcriptional repressors, ARF2 was involved in the regulation of K + uptake by repressing HAK5 transcription in Arabidopsis [ 33 ]. In addition, ARF2 is regulated by a variety of upstream factors at the transcription and protein levels, and participated in the pathways of auxin, gibberellin, oleoresin, ethylene and abscisic acid [ 30 , 43 – 45 ]. Studies showed that G. hirsutum ARF genes promoted the trichome initiation in transgenic Arabidopsis plants [ 46 ] and was related with fiber quality in cotton [ 47 ]. Further, expression of the IAA biosynthetic gene, iaaM , can significantly increase IAA levels in the epidermis of cotton ovules at the fiber initiation stage, subsequently, increased the number of lint fibers and lint percentage in a 4-year field trial [ 48 ]. In this study, GhARF2b was shown to promote the production of fiber initials, suggesting that auxin is an important player in controlling cotton fiber initiation. The auxin signaling pathways in developing cotton fiber cells deserve further investigation. Materials And Methods Identification of Gossypium species ARF factors G. raimondii [49], G. arboreum [50], G. hirsutum [51] genome sequences were acquired from the CottonGen database [52]. We developed a Hidden Markov Model [53] profile matrix of ARF factors (Pfam ID: PF06507) via the hmmbuild program [54] with default parameters to identify Gossypium ARF transcription factor proteins. SMART conserved domain search tool [55] and Pfam databases [56] were used to identify the conserved domain. Sequence alignment, Ka, Ks analyses and phylogenetic analyses Complete ARF protein-coding sequences and inferred nucleotide sequences for each of these Gossypium genes were aligned using MAFFT with the G-INS-i algorithm [57]. Ka, Ks and Ka/Ks values for each gene pairs between diploid and allotetraploid were evaluated with DnaSP v5 [58]. The Neighbor-Joining (NJ) phylogenetic tree was drawn by MEGA 5.03 [59] by sampling 1000 bootstrap replicates based on the ARF whole protein sequences. Gene expression analyses based on transcriptome Raw RNA-Seq data were downloaded from the NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA248163) [51], including G. hirsutum seed, root, stem, leaf, torus, petal, stamen, ovary, calyx, ovule (-3 dpa, -1 dpa, 0 dpa, 1 dpa, 3 dpa, 5 dpa, 10 dpa, 20 dpa, 25dpa, 35dpa) and fiber (5 dpa, 10 dpa, 20 dpa, 25dpa). The method of gene expression analyses based on transcriptome was same to our previous study [60]. Genes were considered differentially expressed if expression varied more than two-fold change with a p-value of less than 0.05. Multiple Experiment Viewer (MeV) [61] was used to display the gene expression values. Cotton cultivation and treatment Upland cotton R15 plants and its transgenic lines were grown in a greenhouse in a controlled environment at 28 °C day/20 °C night, a 16-h light/8-h dark photoperiod. Roots, stems, leaves, cotyledon and hypocotyl were collected from three-week-old seedlings. The following samples, including petal, stamen, pistil, sepals, torus, ovules (-3, -1, 0 and 6 dpa) and fiber (3, 6, 12 and 18 dpa) were then collected in a continuing growing cotton plant. Fibers were collected by scraping the ovule in liquid nitrogen. All plant tissues were frozen in liquid nitrogen immediately after collection and stored at -80 °C until RNA extraction. All treatments were sampled at least three times. qRT-PCR analyses All cotton samples were ground in liquid nitrogen and total RNAs of these cotton tissues were extracted using the RNAprep pure plant kit (TIANGEN, Shanghai, China) following the manufacturer’s protocol. We treated the resulting RNAs with DNase I prior to synthesizing cDNA with oligo (dT) primers and M-MLV Reverse Transcriptase (Invitrogen); and these products were diluted 5-fold before use. The forward and reverse primers of specific gene for quantitative real-time PCR (qRT-PCR) analyses, were designed using the Primer5 software (Additional file 3: Table S1). Analyses were performed with SYBR-Green PCR Mastermix (TaKaRa) on a cycler (Mastercycler RealPlex; Eppendorf Ltd, Shanghai, China). The G. hirsutum histone-3 ( GhHIS3 , AF024716) gene were used as internal references, and the relative amount of amplified product was calculated following the 2-∆∆Ct method [62]. Relative expression levels among different organs of G. hirsutum samples were normalized by calibrating with the WT samples. Cotton transformation and phenotypic analysis The open reading frame (ORF) of GhARF2b was PCR-amplified from a G. hirsutum cv R15 fiber cDNA library with PrimeSTAR HS DNA polymerase (Takara Biomedical Technology Co. Ltd, Beijing, China) and inserted into the pCAMBIA2301 vector to construct RDL1::GhARF2b . For 35S::dsGhARF2b , sense and antisense :GhARF2b fragments, separated by a 120-bp intron of the RTM1 gene from A. thaliana , were cloned into pCAMBIA2301 . Primers used in this investigation are listed in Additional file 3: Table S1. The binary constructs were transferred into Agrobacterium tumefaciens . Cotton transformation was performed with the same protocol in previous study [63]. Briefly, hypocotyl segments of the 5- to 7-d-old cultivated G. hirsutum cv R15 seedlings were used as explants for A. tumefaciens infection, calluses were induced and proliferated, and plantlets were then regenerated. Transgenic cotton plants were grown in glasshouse or field. For T 0 and subsequent generations, b-glucuronidase (GUS) histochemistry staining and PCR were carried out to identify the transgenic lines. 30 seeds from each plant were harvested to statistics fiber length. Microscope observation Images were generated with an optical microscope (BX51, Olympus). For scanning electron microscope images, cotton ovules (0 DPA) were attached with colloidal graphite to a copper stub, frozen under vacuum and visualized with a scanning electron microscope (JSM-6360LV, JEOL). Abbreviations ARFs: Auxin response factors; Dpa: Days post anthesis; FPKM: Fragments per kilobase of transcript per million mapped fragments; G. arboreum : Gossypium arboreum ; G. hirsutum : Gossypium hirsutum ; G. raimondii : Gossypium raimondii ; WT: wild type; OE: Overexpression; ds: RNAi; qRT-PCR: Quantitative real-time polymerase chain reaction Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The genome sequences of three cotton species and the genome annotation gff3 file were downloaded from the CottonGen database (https://www.cottongen.org/data/download) [61]. Raw RNA-Seq data for G. hirsutum seed, root, stem, leaf, torus, petal, stamen, ovary, calyx, ovule and fiber were downloaded from the NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA248163) (NCBI Sequence Read Archive SRR1695173, SRR1695174, SRR1695175, SRR1695177, SRR1695178, SRR1695179, SRR1695181, SRR1695182, SRR1695183, SRR1695184, SRR1695185, SRR1695191, SRR1695192, SRR1695193,SRR1695194, SRR1768504, SRR1768505, SRR1768506, SRR1768507, SRR1768508, SRR1768509, SRR1768510, SRR1768511, SRR1768512, SRR1768513, SRR1768514, SRR1768515, SRR1768516, SRR1768517, SRR1768518 and SRR1768519) [30]. The G. hirsutum histone-3 ( GhHIS3 , AF024716) gene was downloaded from the National Center for Biotechnology Information (NCBI) database, which were used as internal references. All other data generated or analyzed during this study are included in this published article and its Additional files. Competing interests The authors declare that they have no competing interests. Funding This work reported in this publication was supported by the National Natural Science Foundation of China through the Awards Nos. 31690092, 31571251, 31788103, the National Key R&D Program of China (2016YFD0100500) and the Ministry of Agriculture of China (2016ZX08005-003), the China Postdoctoral Science Foundation through the Awards Nos. 2017M621546 and 2018T110411. The funding bodies did not participate in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Authors’ contributions ZWC, XFZ and LJW designed the research. ZWC, XFZ, JFC, CCH, XL and YGZ performed the experiments. XFZ, ZSZ, XXSG, LJW and ZWC contributed materials and analyzed data. ZWC wrote and revised the manuscript. All the authors read and approved the final version of the manuscript. 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Promoter of a cotton fibre MYB gene functional in trichomes of Arabidopsis and glandular trichomes of tobacco. J Exp Bot. 2008;59(13):3533–42. Tables TABLE 1 | Ka , Ks and K a/ K s analyses of GhARF from the A and D subgenomes compared with their corresponding progenitor homoeologs. Locus Name Gene Name Chrom Locus Name Gene Name Chrom Ka Ks Ka/Ks Gh_A10G1402 GhARF1_A A10 Cotton_A_31395 GaARF1 CA_chr1 0.0147 0.0261 0.5632 Gh_D10G0803 GhARF1_D D10 Gorai.011G091100.1 GrARF1 Chr11 0.0019 0.0108 0.1759 Gh_A07G0411 GhARF2a_A A07 Cotton_A_03644 GaARF2a CA_chr1 0.0035 0.0068 0.5147 Gh_D07G0476 GhARF2a_D D07 Gorai.001G054600.1 GrARF2a Chr1 0.0061 0.0169 0.3609 Gh_A11G0358 GhARF2b_A A11 Cotton_A_01955 GaARF2b CA_chr6 0.0015 0.0151 0.0993 Gh_D11G0416 GhARF2b_D D11 Gorai.007G044900.1 GrARF2b Chr7 0.0028 0.0045 0.6222 Gh_D12G1909 GhARF2c_D D12 Gorai.008G210200.1 GrARF2c Chr8 0.0083 0.0263 0.3156 Gh_A11G1082 GhARF2d_A A11 Cotton_A_08273 GaARF2d CA_chr4 0.0012 0.0021 0.5714 Gh_D11G1233 GhARF2d_D D11 Gorai.007G131900.1 GrARF2d Chr7 0.0058 0.0181 0.3204 Gh_A08G0656 GhARF2e_A A08 Cotton_A_22543 GaARF2e CA_chr10 0.0032 0.0000 2.0000 Gh_D08G0758 GhARF2e_D D08 Gorai.004G085400.1 GrARF2e Chr4 0.0098 0.0213 0.4601 Gh_A10G0266 GhARF3a_A A10 Cotton_A_03933 GaARF3a CA_chr9 0.0096 0.0167 0.5749 Gh_D10G0266 GhARF3a_D D10 Gorai.011G030900.1 GrARF3a Chr11 0.0038 0.0125 0.3040 Gh_A06G2038 GhARF3b_A A06 Cotton_A_40208 GaARF3b CA_chr8 0.0019 0.0060 0.3167 Gh_D06G1415 GhARF3b_D D06 Gorai.010G157400.1 GrARF3b Chr10 0.0089 0.0077 1.1558 Gh_A05G1337 GhARF3c_A A05 Cotton_A_11311 GaARF3c CA_chr10 0.0165 0.0140 1.1786 Gh_D05G1506 GhARF3c_D D05 Gorai.009G166100.1 GrARF3c Chr9 0.0018 0.0076 0.2368 Gh_A09G0993 GhARF4a_A A09 Cotton_A_01738 GaARF4a CA_chr11 0.0017 0.0116 0.1466 Gh_A05G3908 GhARF4b_A A05 Cotton_A_11048 GaARF4b CA_chr10 0.0027 0.0018 1.5000 Gh_A01G0908 GhARF5a_A A01 Cotton_A_27669 GaARF5a CA_chr13 0.0009 0.0110 0.0818 Gh_D01G0951 GhARF5a_D D01 Gorai.002G124400.1 GrARF5a Chr2 0.0032 0.0094 0.3404 Gh_A05G1607 GhARF5b_A A05 Cotton_A_16408 GaARF5b CA_chr8 0.0046 0.0079 0.5823 Gh_D05G1792 GhARF5b_D D05 Gorai.009G196100.1 GrARF5b Chr9 0.0067 0.0172 0.3895 Gh_A10G0412 GhARF6a_A A10 Cotton_A_02933 GaARF6a CA_chr9 0.0038 0.0159 0.2390 Gh_D10G0426 GhARF6a_D D10 Gorai.011G048200.1 GrARF6a Chr11 0.0019 0.0047 0.4043 Gh_A05G1225 GhARF6b_A A05 Cotton_A_26156 GaARF6b CA_chr10 0.0034 0.0095 0.3579 Gh_D05G3848 GhARF6b_D D05 Gorai.009G152700.1 GrARF6b Chr9 0.0107 0.0205 0.5220 Gh_D07G1785 GhARF8a_D D07 Gorai.001G204500.1 GrARF8a Chr1 0.0028 0.0091 0.3077 Gh_A12G0813 GhARF8b_A A12 Cotton_A_35443 GaARF8b CA_chr6 0.0017 0.0054 0.3148 Gh_D12G0831 GhARF8b_D D12 Gorai.008G097200.1 GrARF8b Chr8 0.0039 0.0049 0.7959 Gh_A12G0483 GhARF8c_A A12 Cotton_A_21333 GaARF8c CA_chr6 0.0235 0.0303 0.7756 Gh_D12G0491 GhARF8c_D D12 Gorai.008G054600.1 GrARF8c Chr8 0.0061 0.0167 0.3653 Gh_A09G0074 GhARF8d_A A09 Cotton_A_14740 GaARF8d CA_chr11 0.0030 0.0131 0.2290 Gh_D09G0071 GhARF8d_D D09 Gorai.006G008700.1 GrARF8d Chr6 0.0031 0.0150 0.2067 Gh_A11G0231 GhARF9a_A A11 Cotton_A_18937 GaARF9a CA_chr10 0.0108 0.0301 0.3588 Gh_D11G0245 GhARF9a_D D11 Gorai.007G026900.1 GrARF9a Chr7 0.0098 0.0198 0.4949 Gh_A02G0979 GhARF9b_A A02 Cotton_A_36154 GaARF9b CA_chr7 0.0013 0.0044 0.2955 Gh_D03G0771 GhARF9b_D D03 Gorai.003G078000.1 GrARF9b Chr3 0.0019 0.0064 0.2969 Gh_A03G0274 GhARF10a_A A03 Cotton_A_04263 GaARF10a CA_chr7 0.0013 0.0062 0.2097 Gh_D03G1293 GhARF10a_D D03 Gorai.003G142500.1 GrARF10a Chr3 0.0058 0.0171 0.3392 Gh_A05G0895 GhARF10b_A A05 Cotton_A_07064 GaARF10b CA_chr10 0.0038 0.0102 0.3725 Gh_D05G0978 GhARF10b_D D05 Gorai.009G107800.1 GrARF10b Chr9 0.0025 0.0185 0.1351 Gh_A07G1254 GhARF11_A A07 Cotton_A_31049 GaARF11 CA_chr1 0.0044 0.0105 0.4190 Gh_A10G1836 GhARF16a_A A10 Cotton_A_23397 GaARF16a CA_chr9 0.0019 0.0150 0.1267 Gh_D10G2093 GhARF16a_D D10 Gorai.011G238900.1 GrARF16a Chr11 0.0063 0.0193 0.3264 Gh_A05G3576 GhARF16b_A A05 Cotton_A_06107 GaARF16b CA_chr12 0.0097 0.0066 1.4697 Gh_D04G0030 GhARF16b_D D04 Gorai.012G004800.1 GrARF16b Chr12 0.0051 0.0087 0.5862 Gh_A09G1401 GhARF16c_A A09 Cotton_A_24047 GaARF16c CA_chr10 0.0031 0.0103 0.3010 Gh_D09G1405 GhARF16c_D D09 Gorai.006G166400.1 GrARF16c Chr6 0.0025 0.0081 0.3086 Gh_A13G2013 GhARF16d_A A13 Cotton_A_10518 GaARF16d CA_chr8 0.0080 0.0109 0.7339 Gh_D13G2411 GhARF16d_D D13 Gorai.013G267100.1 GrARF16d Chr13 0.0041 0.0223 0.1839 Gh_A05G1991 GhARF17a_A A05 Cotton_A_16138 GaARF17a CA_chr10 0.0030 0.0243 0.1235 Gh_D05G3805 GhARF17a_D D05 Gorai.009G241900.1 GrARF17a Chr9 0.0015 0.0121 0.1240 Gh_A06G0332 GhARF17b_A A06 Cotton_A_18446 GaARF17b CA_chr8 0.0030 0.0025 1.2000 Gh_D06G0360 GhARF17b_D D06 Gorai.010G046000.1 GrARF17b Chr10 0.0076 0.0099 0.7677 Gh_A11G0886 GhARF18a_A A11 Cotton_A_14407 GaARF18a CA_chr4 0.0041 0.0074 0.5541 Gh_D11G1034 GhARF18a_D D11 Gorai.007G109500.1 GrARF18a Chr7 0.0057 0.0133 0.4286 Gh_A12G1016 GhARF18b_A A12 Cotton_A_25871 GaARF18b CA_chr6 0.0045 0.0150 0.3000 Gh_D12G1134 GhARF18b_D D12 Gorai.008G126200.1 GrARF18b Chr8 0.0026 0.0201 0.1294 Gh_A06G0710 GhARF19.1a_A A06 Cotton_A_38575 GaARF19.1a CA_chr13 0.0048 0.0130 0.3692 Gh_D06G0818 GhARF19.1a_D D06 Gorai.010G091300.1 GrARF19.1a Chr10 0.0293 0.0343 0.8542 Gh_A07G2353 GhARF19.1b_A A07 Cotton_A_05677 GaARF19.1b CA_chr1 0.0042 0.0218 0.1927 Gh_D07G0132 GhARF19.1b_D D07 Gorai.001G017000.1 GrARF19.1b Chr1 0.0032 0.0113 0.2832 Gh_A05G3541 GhARF19.2_A A05 Cotton_A_06071 GaARF19.2 CA_chr12 0.0148 0.0254 0.5827 Gh_D04G0067 GhARF19.2_D D04 Gorai.012G009000.1 GrARF19.2 Chr12 0.0039 0.0040 0.9750 Gh_A05G0264 GhARF20_A A05 Cotton_A_27843 GaARF20 CA_chr9 0.0020 0.0121 0.1653 Gh_D08G1407 GhARF21_D D08 Gorai.006G045300.1 GrARF21 Chr6 0.1826 0.2293 0.7963 Supplementary Files Additionalfile1FigureS1.tif Additional file 1: Figure S1. Multiple alignment of GrARF2 (Gossypium raimondii ARF2) and AtARF2 protein sequences. Additionalfile2FigureS2.pdf Additional file 2: Figure S2. Expression patterns of ARF genes in G. hirsutum based on RNA-seq data. FPKM represents fragments per kilobase of exon model per million mapped reads. DPA, days post-anthesis. Additionalfile3TableS1.docx Additional file 3: Table S1. List of forward and reverse primers used for this study. Cite Share Download PDF Status: Published Journal Publication published 22 Mar, 2021 Read the published version in BMC Genomics → Version 1 posted Review # 3 received at journal 23 Dec, 2020 Editorial decision: Major revision 23 Dec, 2020 Review # 1 received at journal 09 Dec, 2020 Review # 2 received at journal 09 Dec, 2020 Reviewer # 3 agreed at journal 06 Dec, 2020 Reviewer # 2 agreed at journal 03 Dec, 2020 Reviewer # 1 agreed at journal 03 Dec, 2020 Reviewers invited by journal 02 Dec, 2020 Editor invited by journal 29 Nov, 2020 First submitted to journal 15 Nov, 2020 Editor assigned by journal 15 Nov, 2020 Submission checks completed at journal 15 Nov, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-119017","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":5585832,"identity":"c1935647-83cf-4c13-b089-fe8e95d2d970","order_by":0,"name":"Xiufang Zhang","email":"","orcid":"","institution":"Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiufang","middleName":"","lastName":"Zhang","suffix":""},{"id":5585833,"identity":"19cceddc-46ca-451a-a8e4-ffaa0c3c7335","order_by":1,"name":"Junfeng Cao","email":"","orcid":"","institution":"Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Junfeng","middleName":"","lastName":"Cao","suffix":""},{"id":5585834,"identity":"2a18c4a6-4daf-4ca6-9b6f-491f96e57471","order_by":2,"name":"Chaochen Huang","email":"","orcid":"","institution":"Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chaochen","middleName":"","lastName":"Huang","suffix":""},{"id":5585835,"identity":"eb5e3f6d-fe16-4c0c-ac6c-74f409ac5434","order_by":3,"name":"Zishou Zheng","email":"","orcid":"","institution":"Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zishou","middleName":"","lastName":"Zheng","suffix":""},{"id":5585836,"identity":"b379fb39-cb50-4b62-99b6-c3b46dc2cec9","order_by":4,"name":"Xia Liu","email":"","orcid":"","institution":"Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xia","middleName":"","lastName":"Liu","suffix":""},{"id":5585837,"identity":"4ac112d7-ec9a-477c-b200-ed1b6a89fdc5","order_by":5,"name":"Xiaoxia Shangguan","email":"","orcid":"","institution":"Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoxia","middleName":"","lastName":"Shangguan","suffix":""},{"id":5585838,"identity":"ba52b058-ab0f-4fd7-a3da-e030f00a3a7c","order_by":6,"name":"Lingjian Wang","email":"","orcid":"","institution":"Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lingjian","middleName":"","lastName":"Wang","suffix":""},{"id":5585839,"identity":"ea4437b2-d8b8-401d-90d0-cbfba175fffb","order_by":7,"name":"Yugao Zhang","email":"","orcid":"","institution":"Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yugao","middleName":"","lastName":"Zhang","suffix":""},{"id":5585840,"identity":"a9f4308e-1319-4195-9f2a-72b396a18e7b","order_by":8,"name":"Zhiwen Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIie3QMQrCMBSA4ZSAWR50jaB3iARSi4JXSRZdxMWlQ4dAoY6uXsMbRArtEnfHTs5O4iQGdHGwxs0hP2QIvI8XglAo9IcxTIyRLIf4eccehIBq26we9LU3iREftRZPmfElCUZzqsoe8KY6U5RNlCZH00nSIqodGYCwc0GRXSgNK9n9sOq1RZyQoFFZKU2BfSPCEQx8R640uvsRzqTFwCi4LdqDpIX7ZJnVQO1yPZb1gpew7CZJ3JjDjeWzeNPsT5d8MtwS203ek+70fpgPhUKh0IceUq9AUGSXZ2sAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-0268-5942","institution":"Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Zhiwen","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2020-11-30 21:20:56","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-119017/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-119017/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-021-07504-6","type":"published","date":"2021-03-22T15:00:30+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":3991975,"identity":"7f7a0213-7c77-42ab-997d-f33594706cc7","added_by":"auto","created_at":"2020-12-03 17:02:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":106906,"visible":true,"origin":"","legend":"Phylogenetic trees of Gossypium ARFs family. 144 Gossypium ARFs were divided into four clades. Black dots represent the ARF2b genes in three Gossypium species.","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-119017/v1/5cf8079295a88f2c1b5a244e.png"},{"id":3991977,"identity":"79990c9e-e128-4202-8fb2-ea66a5aaa1a3","added_by":"auto","created_at":"2020-12-03 17:02:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":14839,"visible":true,"origin":"","legend":"Distribution of Ka and Ks values of ARF genes between the A and D subgenomes versus their corresponding diploid progenitor homoeologs.","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-119017/v1/d22c5189e646df743b698428.png"},{"id":3991979,"identity":"54e6bd85-654d-493d-aa5e-8413194a891d","added_by":"auto","created_at":"2020-12-03 17:02:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":854484,"visible":true,"origin":"","legend":"Expression patterns of GhARF2 in different cotton tissues and fiber cells of different stages. (A) Expression profiles of nine GhARF2 genes based on the RPKM values of RNA-seq data. GhARF2b was highlighted in yellow box. (B) qRT-PCR analyses of GhARF2b expression across different cotton tissues. The expression is relative to GhHIS3. Error bar indicates stdev.s level of three qRT-PCR assays.","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-119017/v1/92040ac3bdf8546ce921283a.png"},{"id":3991981,"identity":"ee88483d-dc2e-4189-b415-af91dac56652","added_by":"auto","created_at":"2020-12-03 17:02:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":448116,"visible":true,"origin":"","legend":"GhARF2b affects fiber length and fiber related gene expression in RDL1::GhARF2b transgenic cotton. (A) Expression of GhARF2b in 3, 6 and 9 dpa fibers in the RDL1::GhPRE1 overexpression (OE) lines compared to wild-type (WT). (B) Fiber phenotype of RDL1::GhARF2b and wild-type (WT) cotton cultivated in farm on shanghai, bar=10mm. (C) Statistical analysis of RDL1::GhARF2b and wild-type (WT) mature fiber length. Error bar indicates standard deviation; *** denotes significant difference from wild type (Student’s t-test，P\u003c0.001, n=30). ","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-119017/v1/bcaeb495b616b4b4661a4846.png"},{"id":3991982,"identity":"2b7ce54f-3acf-4831-8093-b795e9a068b6","added_by":"auto","created_at":"2020-12-03 17:02:25","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":416397,"visible":true,"origin":"","legend":"GhARF2b affects fiber length and gene expression in 35S::GhARF2b RNAi transgenic cotton. (A) Fiber phenotype of RDL1::GhARF2b (OE), 35S::GhARF2b RNAi (ds) and wild-type (R15) cotton cultivated in farm on shanghai, bar=10mm. (B) Statistical analysis of 35S::GhARF2b RNAi (ds) lines and wild-type (WT) mature fiber length. Error bar indicates standard deviation; *** denotes significant difference from wild type (Student’s t-test，P\u003c0.001, n=30). (C) Expression of GhARF2b in 0 dpa ovules in the overexpression (OE), RNAi (ds) lines compared to wild-type (WT). (D) Expression of GhARF2b in 6-dpa fibers in the overexpression (OE), RNAi (ds) lines compared to wild-type (WT). (E) Expression of GhARF2b in 12-dpa fibers in the overexpression (OE) and the RNAi (ds) lines compared to the wild-type (WT). Error bar indicates stdev.s level of three qRT-PCR assays.","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-119017/v1/8118b76a0cc475b0eeff0bcd.png"},{"id":3991983,"identity":"664ff63c-9771-48d0-9350-121c8af949c2","added_by":"auto","created_at":"2020-12-03 17:02:25","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2103240,"visible":true,"origin":"","legend":"SEM images of WT-R15, OE-3 (over-expression line) and ds-2 (RNAi line) ovules at -1, 0 and 1 DPA. Bar=100μm [60×magnification]. ","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-119017/v1/9382df9a63f84face4f53f54.png"},{"id":3991984,"identity":"a7bf52c8-a96e-4368-a3f2-eeb017669328","added_by":"auto","created_at":"2020-12-03 17:02:25","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2176374,"visible":true,"origin":"","legend":"GhARF2b enhances the fiber initiation. (A) SEM images of WT-R15, OE-3 (over-expression line) and ds-2 (RNAi line) ovules at -1, 0, 1 DPA. Bar=100μm [700×magnification for ovules of -1 and 0 DPA, 500×magnification for 1-DPA ovules]. (B) Number of fiber cells per 0.01 mm2 of 0-DPA ovules in the overexpression (OE-3) and RNAi (ds-2) cotton lines compared to wild-type (WT-R15). Error bar indicates standard deviation; *** denotes significant difference from wild-type (Student’s t-test，P\u003c0.001, n=30); * denotes significant difference from wild type (Student’s t-test，P\u003c0.05, n=30). (C) Number of fiber cells per 0.01 mm2 of 1-DPA ovules in the overexpression (OE-3) and RNAi (ds-2) cotton lines compared to wild-type (WT-R15). Error bar indicates standard deviation; *** denotes significant difference from wild type (Student’s t-test，P\u003c0.001, n=30).","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-119017/v1/ef1199e4a9a8a73d7eeae381.png"},{"id":13621562,"identity":"b2adc4d2-0f7f-4397-a9ac-d5bf7d96a60b","added_by":"auto","created_at":"2021-09-17 07:11:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3576792,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-119017/v1/22bbda17-dbf5-4f69-b7ef-57df24c6d64b.pdf"},{"id":3991976,"identity":"c353479b-d81b-42ac-a4d7-60873a77b9af","added_by":"auto","created_at":"2020-12-03 17:02:24","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8124704,"visible":true,"origin":"","legend":"Additional file 1: Figure S1. Multiple alignment of GrARF2 (Gossypium raimondii ARF2) and AtARF2 protein sequences.","description":"","filename":"Additionalfile1FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-119017/v1/c6a6e9a427957670241b8ddc.tif"},{"id":3991978,"identity":"c36fd84e-206a-4e3f-89b6-e4560e68d965","added_by":"auto","created_at":"2020-12-03 17:02:24","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":904522,"visible":true,"origin":"","legend":"Additional file 2: Figure S2. Expression patterns of ARF genes in G. hirsutum based on RNA-seq data. FPKM represents fragments per kilobase of exon model per million mapped reads. DPA, days post-anthesis.","description":"","filename":"Additionalfile2FigureS2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-119017/v1/273973573ffa01394d3b7f66.pdf"},{"id":3991980,"identity":"0ead26ab-0bb5-40f6-bbd5-32168f7194de","added_by":"auto","created_at":"2020-12-03 17:02:24","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":16561,"visible":true,"origin":"","legend":"Additional file 3: Table S1. List of forward and reverse primers used for this study.","description":"","filename":"Additionalfile3TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-119017/v1/3a788cee96193d5d630ad8b3.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eCharacterization of Cotton ARF Factors and the Role of GhARF2b in Fiber Development\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eCotton is the most important natural and renewable material for the textile industry in the world [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The primary cultivated species upland cotton (\u003cem\u003eG. hirsutum\u003c/em\u003e L.) is grown in over 80 countries and accounts for more than 90% of global cotton fiber output. Cotton fibers are unusually long, single-celled epidermal seed trichomes and a model for plant cell growth research [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Fiber development can be divided into four overlapping stages: initiation, elongation, secondary cell wall biosynthesis and maturation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The fiber length and density are both key traits that determine cotton quality and yield.\u003c/p\u003e \u003cp\u003eThe study of cotton fiber development regulation provides not only valuable knowledge to understanding plant cell growth and cell wall biosynthesis, but also candidate genes for cotton molecular breeding. To date a number of genes that function in cotton fiber cells have been identified, including homeodomain transcription factor GaHOX1, GhHOX3 and GhHD1 [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], bHLH transcription factor GhPRE1 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], KNOX transcription factor knl1 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], the sterol carrier gene [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], MYB transcription factors GhMYB25, GhMYB25-like, GhMML3 and GhMML4 [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], NAC transcription factor fsn1 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], transcription factor WLIM1a gene [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], sucrose synthase gene [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], cotton actin1 gene [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], cotton BURP domain protein GhRDL1 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], ethylene pathway related genes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], fasciclin-like arabinogalactan protein, Ghfla1 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and TCP transcription factor GhTCP4 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] etc. Among recent progresses are the characterizations of transcription factors which regulate the major events of cotton fiber development, such as MYBs and HD-ZIP IVs involved in cotton fiber initiation and elongation, as well as a number of other types of factors. The MIXTA type MYB transcription factors (GhMYB25, GhMYB25-like and GhMML4_D12) are master regulators of cotton fiber initiation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and lint fiber development [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], whereas the HD-ZIP IV transcription factor GhHOX3 plays a pivotal role in controlling fiber elongation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], whose activity is regulated by the phytohormone gibberellin. In addition, NAC (GhFSN1) and TCP4 transcription factors positively regulates secondary cell wall biosynthesis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, cotton fiber growth and development are complex processes involving cell differentiation, cell skeleton orientation growth, cell wall synthesis, and so on [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Currently the picture of the regulation network of cotton fiber is far from complete.\u003c/p\u003e \u003cp\u003eAuxin response factors (ARFs), a group of plant transcription factors, are composed of a conserved N-terminal DNA binding domain (DBD), a most case conserved C-terminal dimerization domain (CTD) and a non-conserved middle region (MR) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The MR region has been proposed to function as a repression or an activation domain [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. \u003cem\u003eArabidopsis thaliana\u003c/em\u003e contains 23 ARF genes and \u003cem\u003eOryza sativa\u003c/em\u003e has 25 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. It has been reported that ARF2 negatively modulates plant growth in \u003cem\u003eA. thaliana\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and tomato [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], yet functions of transcription factors can vary with tissues and more diversified in polyploid species, to date the role ARF2 in cotton has not been explored.\u003c/p\u003e \u003cp\u003eIn this study, we conducted a genome-wide analysis ARF genes in three cotton species (\u003cem\u003eG. hirsutum\u003c/em\u003e, \u003cem\u003eG. arboreum\u003c/em\u003e and \u003cem\u003eG. raimondii\u003c/em\u003e), and classified them into four clades. In \u003cem\u003eG. hirsutum\u003c/em\u003e most \u003cem\u003eARF\u003c/em\u003e genes were expressed in multiple cotton tissues, among which \u003cem\u003eGhARF2b\u003c/em\u003e exhibited a preferential expression in developing cotton fiber cells, and it negatively affects cotton fiber elongation but plays a role in promoting fiber initiation.\u003c/p\u003e "},{"header":"Results","content":" \u003cp\u003e \u003cb\u003eARF transcription factors in\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eG. arboreum\u003c/span\u003e \u003cb\u003eand G\u003c/b\u003e. \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ehirsutum\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe genome sequences of \u003cem\u003eG. raimondii\u003c/em\u003e and \u003cem\u003eG. arboreum\u003c/em\u003e provide us data resources to conduct a genome-wide screen of the \u003cem\u003eARF\u003c/em\u003e genes in the extent diploid progenitors of the allotetraploid \u003cem\u003eG. hirsutum\u003c/em\u003e. In the previous studies, Sun et al., (2015) identified 35 \u003cem\u003eARF\u003c/em\u003e genes in \u003cem\u003eG. raimondii\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. To mine more ARF transcription factors in cottons the conserved domain (Pfam ID: PF06507) was used to hmmersearch against the \u003cem\u003eG. arboreum\u003c/em\u003e and \u003cem\u003eG. hirsutum\u003c/em\u003e genome databases, which resulted in 36 and 73 genes in \u003cem\u003eG. arboreum\u003c/em\u003e and \u003cem\u003eG. hirsutum\u003c/em\u003e genomes, respectively. The 36 \u003cem\u003eG. arboreum ARF\u003c/em\u003e genes were designated \u003cem\u003eGaARF1\u003c/em\u003e\u0026ndash;\u003cem\u003eGaARF20\u003c/em\u003e, and the 73 \u003cem\u003eG. hirsutum ARF\u003c/em\u003e genes in A- and D-subgenomes were designated as \u003cem\u003eGhARF1A/D\u003c/em\u003e\u0026ndash;\u003cem\u003eGhARF21A/D\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). As those of Arabidopsis, cotton ARF proteins are composed of three domain regions, including DBD (DNA-binding Domain), MI (Middle Region) and CTD (C-terminal Domain) (Additional file 1: Figure S1).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic analysis of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eGossypium\u003c/span\u003e \u003cb\u003eARF proteins\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo illustrate the evolutionary relationships among the cotton ARFs, a phylogenetic tree was constructed using the protein sequences of 144 cotton ARFs, which were clustered into four clades (I\u0026ndash;IV). The highest number of \u003cem\u003eGossypium\u003c/em\u003e ARFs are found in clade III and I, followed by clade IV and II (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, the expected diploid-polyploid topology is reflected in the tree for each set of orthologous/homoeologous genes, indicating general preservation during divergence of diploids and through the polyploid formation. We found that the number of ARF genes in \u003cem\u003eG. hirsutum\u003c/em\u003e are approximately twice that in \u003cem\u003eG. raimondii\u003c/em\u003e and \u003cem\u003eG. arboreum\u003c/em\u003e, with one A\u003csub\u003et\u003c/sub\u003e or D\u003csub\u003et\u003c/sub\u003e homoeologous copy corresponding to one ortholog in each of the diploid cottons. Further, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the orthologous paired genes of the A genome (\u003cem\u003eG. arboreum\u003c/em\u003e) and A\u003csub\u003et\u003c/sub\u003e sub-genome, or from the D genome (\u003cem\u003eG. raimondii\u003c/em\u003e) and D\u003csub\u003et\u003c/sub\u003e sub-genome, tend to be clustered together and share a sister relationship.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDivergence of ARF genes in allotetraploid\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eG. hirsutum\u003c/span\u003e \u003cb\u003eand its diploid progenitors\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eARF\u003c/em\u003e genes in the two diploid species were then compared with \u003cem\u003eG. hirsutum\u003c/em\u003e A\u003csub\u003et\u003c/sub\u003e- and D\u003csub\u003et\u003c/sub\u003e-subgenome homoeologs (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). To explore the evolutionary relationship and possible functional divergence of \u003cem\u003eARF\u003c/em\u003e genes between the allotetraploid cotton and its extend diploid progenitors, the nonsynonymous substitution (\u003cem\u003eKa\u003c/em\u003e) and synonymous substitution values (\u003cem\u003eKs\u003c/em\u003e) and the \u003cem\u003eK\u003c/em\u003ea/\u003cem\u003eK\u003c/em\u003es ratios for each pair of the genes were calculated (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). By comparing the \u003cem\u003eKa\u003c/em\u003e and \u003cem\u003eKs\u003c/em\u003e values of 66 orthologous gene sets between the allotetraploid and its diploid progenitor genomes, we found that the \u003cem\u003eKa\u003c/em\u003e and \u003cem\u003eKs\u003c/em\u003e values are higher in the D\u003csub\u003et\u003c/sub\u003e subgenome than in the A\u003csub\u003et\u003c/sub\u003e subgenome (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results indicate that \u003cem\u003eGhARF\u003c/em\u003e genes in the D\u003csub\u003et\u003c/sub\u003e subgenome tend to have experienced faster sequence divergence than their A\u003csub\u003et\u003c/sub\u003e counterparts, suggesting an inconsistent evolution of \u003cem\u003eARF\u003c/em\u003e genes in the two subgenomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the \u003cem\u003eKa/Ks\u003c/em\u003e ratios of one D\u003csub\u003et\u003c/sub\u003e-subgenome genes (\u003cem\u003eGhARF3b_D\u003c/em\u003e) and five A\u003csub\u003et\u003c/sub\u003e-subgenome gene (\u003cem\u003eGhARF2e_A\u003c/em\u003e, \u003cem\u003eGhARF3c_A\u003c/em\u003e, \u003cem\u003eGhARF4b_A\u003c/em\u003e, \u003cem\u003eGhARF16b_A\u003c/em\u003e and \u003cem\u003eGhARF17b_A\u003c/em\u003e) are greater than 1 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), suggesting that these genes have under positive selections after divergence of \u003cem\u003eG. hirsutum\u003c/em\u003e from diploid ancestors, and may have gained new functions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression analysis of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eGhARF\u003c/span\u003e \u003cb\u003egenes in different cotton tissues\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe expression profile of a gene family can provide valuable clues to possible functions of each genes. Analysis of 73 \u003cem\u003eGhARF\u003c/em\u003e genes showed that most genes have different spatial expression patterns. For instance, \u003cem\u003eGhARF1, GhARF2a, GhARF2b\u003c/em\u003e and \u003cem\u003eGhARF2c\u003c/em\u003e were expressed in all the tissues of cotton examined (Additional file 2: Figure S2), whereas \u003cem\u003eGhARF3a\u003c/em\u003e and \u003cem\u003eGhARF3c\u003c/em\u003e were expressed preferentially in the pistils and ovules. Compared to \u003cem\u003eGhARF5b\u003c/em\u003e, \u003cem\u003eGhARF5a\u003c/em\u003e showed higher expressions in the root, pistil and ovule organs. Transcripts of \u003cem\u003eGhARF3c\u003c/em\u003e and \u003cem\u003eGhARF4a\u003c/em\u003e, \u003cem\u003eGhARF9a\u003c/em\u003e and \u003cem\u003eGhARF9b\u003c/em\u003e were most abundant in stem and root, respectively. Over half of \u003cem\u003eGhARF\u003c/em\u003e genes showed a relatively high level of transcript accumulation in leaf. Notably, there are more than ten genes (including \u003cem\u003eGhARF1\u003c/em\u003e, \u003cem\u003eGhARF2a\u003c/em\u003e, \u003cem\u003eGhARF2b\u003c/em\u003e, \u003cem\u003eGhARF8a\u003c/em\u003e, \u003cem\u003eGhARF9a\u003c/em\u003e, \u003cem\u003eGhARF10b\u003c/em\u003e, \u003cem\u003eGhARF11\u003c/em\u003e, \u003cem\u003eGhARF16a\u003c/em\u003e, \u003cem\u003eGhARF18\u003c/em\u003e and \u003cem\u003eGhARF19\u003c/em\u003e) that were highly expressed in cotton fiber cells at the fast elongation stage (5 dpa). In summary, most of the \u003cem\u003eGhARF\u003c/em\u003e genes were up-regulated in ovule, fiber, vegetative and other tissues (Additional file 2: Figure S2).\u003c/p\u003e \u003cp\u003e \u003cem\u003eGhARF2\u003c/em\u003e and \u003cem\u003eGhARF18\u003c/em\u003e showed the highest expression in fiber (5 dpa) and both were located in the Clade I (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), suggesting that they may function in cotton fiber development. Previous studies have demonstrated that ARF2 plays a role in transcriptional regulation in auxin-mediated cell division [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], leaf longevity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], response to stress [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], regulation of fruit ripening [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and so on. As GhARF2s shown pleiotropic effects on plant development [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], we decided to identify the major GhARF2s in regulation of cotton fiber elongation in subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eGhARF2\u003c/span\u003e \u003cb\u003ehad a high expression pattern during fiber elongation process\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThere are nine \u003cem\u003eARF2\u003c/em\u003e genes in \u003cem\u003eG. hirsutum\u003c/em\u003e (\u003cem\u003eGhARF2c_At\u003c/em\u003e not annotated), we first examined their expression profiles in different tissues in cotton (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Based on the published RNA-seq data (Zhang et al., 2015) \u003cem\u003eGhARF2a\u003c/em\u003e, \u003cem\u003eGhARF2b\u003c/em\u003e and \u003cem\u003eGhARF2c\u003c/em\u003e genes had higher expression levels in various tissues than \u003cem\u003eGhARF2d\u003c/em\u003e or \u003cem\u003eGhARF2e\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The transcripts of \u003cem\u003eGhARF2b\u003c/em\u003e homoeologs (\u003cem\u003eGhARF2b_At and GhARF2b_Dt\u003c/em\u003e) were enriched and abundant in cotton fiber cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), subsequent quantitative RT-PCR (qRT-PCR) confirmed the expression pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). The highly up-regulated expression in fiber cell suggested that GhARF2b has been recruited to act primarily in cotton fiber.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eGhARF2b\u003c/span\u003e \u003cb\u003eoverexpression represses cotton fiber elongation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo test the function of \u003cem\u003eGhARF2b\u003c/em\u003e, we constructed the vectors to over-express and down-regulate \u003cem\u003eGhARF2b_Dt\u003c/em\u003e in \u003cem\u003eG. hirsutum\u003c/em\u003e by using the fiber-specific \u003cem\u003eGhRDL1\u003c/em\u003e promoter [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The expression levels of \u003cem\u003eGhARF2b\u003c/em\u003e in transgenic cotton were clearly elevated in the overexpression lines according to qRT-PCR analysis; for example, the \u003cem\u003eGhARF2b\u003c/em\u003e transcript abundance was about two-fold higher in the OE-3 than in the wild-type cotton fiber cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). However, \u003cem\u003eGhARF2b\u003c/em\u003e did not stimulate fiber cell elongation, rather, it resulted in shorter fiber (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb,c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the contrary, suppressing \u003cem\u003eGhARF2b\u003c/em\u003e expression by RNAi resulted in longer fibers (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b). The expression levels of \u003cem\u003eGhARF2b\u003c/em\u003e in RNAi cottons in the RNAi lines were about 3\u0026thinsp;~\u0026thinsp;5-fold down-regulated in cotton fiber of 0DPA, 6DPA and 12DPA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-e). Together, these data suggest that GhARF2b acted as a negative regulator of fiber cell elongation, at least when its expression exceeded the threshold. Alternatively, it may function in other aspects of cotton fiber development.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eGhARF2b\u003c/span\u003e \u003cb\u003eoverexpression enhances cotton fiber initiation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNext, we examined the effects of \u003cem\u003eGhARF2b\u003c/em\u003e up-regulation on cotton fiber initiation. The over-expression line OE-3 and RNAi line ds-2 were selected for analyses. The SEM with 60\u0026thinsp;\u0026times;\u0026thinsp;magnification of ovules of WT-R15, OE-3 and ds-2 collected at -1, 0, 1 DPA were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The cotton fiber initiation of the \u0026minus;\u0026thinsp;1-DPA ovules did not present differences among the three types of cottons, however, the 0- and 1-DPA ovules of OE-3 and ds-2 lines showed higher and lower densities of fiber initials compared to the wild-type control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Further, we magnified the SEM views of ovules to 500\u0026ndash;700\u0026times; (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Obviously, at the fiber initiation stage (0, 1 DPA), the fiber initial density of the OE-3 was increased by about 1.5-fold compared with that of the wild-type, in contrast, the fiber initial density of the ds-2 line was reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-c). These results support a role of GhARF2b in promoting cotton fiber cell initiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"Discussion","content":" \u003cp\u003ePrevious studies showed that ARF family genes have been identified in many plant species, including 23 ARF genes in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], 25 in \u003cem\u003eOryza sativa\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], 39 in \u003cem\u003ePopulus trichocarpa\u003c/em\u003e [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], 31 in \u003cem\u003eZea mays\u003c/em\u003e [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], 15 in \u003cem\u003eCucumis sativus\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and 35 in \u003cem\u003eG. raimondii\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Here, we additionally characterized 36 ARF genes in \u003cem\u003eG. arboreum\u003c/em\u003e and 73 in \u003cem\u003eG. hirsutum\u003c/em\u003e, adding valuable data to understanding the distribution and evolution of ARF genes in plants.\u003c/p\u003e \u003cp\u003eAfter whole genome duplication, the amplified genes generally undergo the events of functional loss, or neofunctionalization or subfunctionalization [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this study, we found that six \u003cem\u003eGhARF\u003c/em\u003e genes (five from A\u003csub\u003et\u003c/sub\u003e subgenome) have experienced relatively faster positive selection compared to its diploid progenitors. Thus, duplicated genes from A\u003csub\u003et\u003c/sub\u003e and D\u003csub\u003et\u003c/sub\u003e subgenomes might be functionally diverged in the allotetraploid cotton after the merge of the two genomes.\u003c/p\u003e \u003cp\u003eAuxin response factors (ARFs) are important in plant development as they play crucial roles in regulating a variety of signaling pathways [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. According to their functions, ARF proteins are divided into two classes: transcriptional activators and transcriptional repressors [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Many studies have revealed their regulatory roles in regulating various aspects of cellular activities [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. As transcriptional repressors, ARF2 was involved in the regulation of K\u003csup\u003e+\u003c/sup\u003e uptake by repressing \u003cem\u003eHAK5\u003c/em\u003e transcription in Arabidopsis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In addition, ARF2 is regulated by a variety of upstream factors at the transcription and protein levels, and participated in the pathways of auxin, gibberellin, oleoresin, ethylene and abscisic acid [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Studies showed that \u003cem\u003eG. hirsutum ARF\u003c/em\u003e genes promoted the trichome initiation in transgenic Arabidopsis plants [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and was related with fiber quality in cotton [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Further, expression of the IAA biosynthetic gene, \u003cem\u003eiaaM\u003c/em\u003e, can significantly increase IAA levels in the epidermis of cotton ovules at the fiber initiation stage, subsequently, increased the number of lint fibers and lint percentage in a 4-year field trial [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In this study, GhARF2b was shown to promote the production of fiber initials, suggesting that auxin is an important player in controlling cotton fiber initiation. The auxin signaling pathways in developing cotton fiber cells deserve further investigation.\u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eIdentification of \u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eGossypium\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e species ARF factors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eG. raimondii\u003c/em\u003e [49],\u003cem\u003e G. arboreum\u003c/em\u003e [50], \u003cem\u003eG. hirsutum \u003c/em\u003e[51] genome sequences were acquired from the CottonGen database [52]. We developed a Hidden Markov Model [53] profile matrix of ARF factors (Pfam ID: PF06507) via the hmmbuild program [54] with default parameters to identify \u003cem\u003eGossypium\u003c/em\u003e ARF transcription factor proteins. SMART conserved domain search tool [55] and Pfam databases [56] were used to identify the conserved domain.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequence alignment, Ka, Ks analyses\u003c/strong\u003e\u003cstrong\u003e and phylogenetic analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComplete ARF protein-coding sequences and inferred nucleotide sequences for each of these \u003cem\u003eGossypium\u003c/em\u003e genes were aligned using MAFFT with the G-INS-i algorithm [57]. Ka, Ks and Ka/Ks values for each gene pairs between diploid and allotetraploid were evaluated with DnaSP v5 [58]. The Neighbor-Joining (NJ) phylogenetic tree was drawn by MEGA 5.03 [59] by sampling 1000 bootstrap replicates based on the ARF whole protein sequences.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene expression analyses based on transcriptome\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw RNA-Seq data were downloaded from the NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA248163) [51], including \u003cem\u003eG. hirsutum\u003c/em\u003e seed, root, stem, leaf, torus, petal, stamen, ovary, calyx, ovule (-3 dpa, -1 dpa, 0 dpa, 1 dpa, 3 dpa, 5 dpa, 10 dpa, 20 dpa, 25dpa, 35dpa) and fiber (5 dpa, 10 dpa, 20 dpa, 25dpa). The method of gene expression analyses based on transcriptome was same to our previous study [60]. Genes were considered differentially expressed if expression varied more than two-fold change with a p-value of less than 0.05. Multiple Experiment Viewer (MeV) [61] was used to display the gene expression values.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCotton cultivation and treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUpland cotton R15 plants and its transgenic lines were grown in a greenhouse in a controlled environment at 28 \u0026deg;C day/20 \u0026deg;C night, a 16-h light/8-h dark photoperiod. Roots, stems, leaves, cotyledon and hypocotyl were collected from three-week-old seedlings. The following samples, including petal, stamen, pistil, sepals, torus, ovules (-3, -1, 0 and 6 dpa) and fiber (3, 6, 12 and 18 dpa) were then collected in a continuing growing cotton plant. Fibers were collected by scraping the ovule in liquid nitrogen. All plant tissues were frozen in liquid nitrogen immediately after collection and stored at -80 \u0026deg;C until RNA extraction. All treatments were sampled at least three times.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll cotton samples were ground in liquid nitrogen and total RNAs of these cotton tissues were extracted using the RNAprep pure plant kit (TIANGEN, Shanghai, China) following the manufacturer\u0026rsquo;s protocol. We treated the resulting RNAs with DNase I prior to synthesizing cDNA with oligo (dT) primers and M-MLV Reverse Transcriptase (Invitrogen); and these products were diluted 5-fold before use. The forward and reverse primers of specific gene for quantitative real-time PCR (qRT-PCR) analyses, were designed using the Primer5 software (Additional file 3: Table S1). Analyses were performed with SYBR-Green PCR Mastermix (TaKaRa) on a cycler (Mastercycler RealPlex; Eppendorf Ltd, Shanghai, China). The \u003cem\u003eG. hirsutum\u003c/em\u003e\u003cem\u003ehistone-3 \u003c/em\u003e(\u003cem\u003eGhHIS3\u003c/em\u003e, AF024716) gene were used as internal references, and the relative amount of amplified product was calculated following the 2-∆∆Ct method [62]. Relative expression levels among different organs of \u003cem\u003eG. hirsutum\u003c/em\u003e samples were normalized by calibrating with the WT samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCotton transformation and phenotypic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe open reading frame (ORF) of \u003cem\u003eGhARF2b \u003c/em\u003ewas PCR-amplified from a \u003cem\u003eG. hirsutum \u003c/em\u003ecv R15 fiber cDNA library with PrimeSTAR HS DNA polymerase (Takara Biomedical Technology Co. Ltd, Beijing, China) and inserted into the \u003cem\u003epCAMBIA2301 \u003c/em\u003evector to construct \u003cem\u003eRDL1::GhARF2b\u003c/em\u003e. For \u003cem\u003e35S::dsGhARF2b\u003c/em\u003e, sense and antisense \u003cem\u003e:GhARF2b\u003c/em\u003e fragments, separated by a 120-bp intron of the \u003cem\u003eRTM1\u003c/em\u003e gene from \u003cem\u003eA. thaliana\u003c/em\u003e, were cloned into \u003cem\u003epCAMBIA2301\u003c/em\u003e. Primers used in this investigation are listed in Additional file 3: Table S1.\u003c/p\u003e\n\u003cp\u003eThe binary constructs were transferred into\u003cem\u003e Agrobacterium tumefaciens\u003c/em\u003e. Cotton transformation was performed with the same protocol in previous study [63]. Briefly, hypocotyl segments of the 5- to 7-d-old cultivated \u003cem\u003eG. hirsutum\u003c/em\u003e cv R15 seedlings were used as explants for \u003cem\u003eA. tumefaciens\u003c/em\u003e infection, calluses were induced and proliferated, and plantlets were then regenerated. Transgenic cotton plants were grown in glasshouse or field. For T\u003csub\u003e0\u003c/sub\u003e and subsequent generations, b-glucuronidase (GUS) histochemistry staining and PCR were carried out to identify the transgenic lines. 30 seeds from each plant were harvested to statistics fiber length.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscope observation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImages were generated with an optical microscope (BX51, Olympus). For scanning electron microscope images, cotton ovules (0 DPA) were attached with colloidal graphite to a copper stub, frozen under vacuum and visualized with a scanning electron microscope (JSM-6360LV, JEOL).\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eARFs: Auxin\u0026nbsp;response\u0026nbsp;factors; Dpa: Days post anthesis; FPKM: Fragments per kilobase of transcript per million mapped fragments; \u003cem\u003eG. arboreum\u003c/em\u003e: \u003cem\u003eGossypium arboreum\u003c/em\u003e; \u003cem\u003eG. hirsutum\u003c/em\u003e: \u003cem\u003eGossypium hirsutum\u003c/em\u003e; \u003cem\u003eG. raimondii\u003c/em\u003e: \u003cem\u003eGossypium raimondii\u003c/em\u003e; WT: wild type; OE: Overexpression; ds: RNAi; qRT-PCR: Quantitative real-time polymerase chain reaction\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genome sequences of three cotton species and the genome annotation gff3 file were downloaded from the CottonGen database (https://www.cottongen.org/data/download) [61]. Raw RNA-Seq data for \u003cem\u003eG. hirsutum\u003c/em\u003e seed, root, stem, leaf, torus, petal, stamen, ovary, calyx, ovule and fiber were downloaded from the NCBI Sequence Read Archive (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA248163) (NCBI Sequence Read Archive SRR1695173, SRR1695174, SRR1695175, SRR1695177, SRR1695178, SRR1695179, SRR1695181, SRR1695182, SRR1695183, SRR1695184, SRR1695185, SRR1695191, SRR1695192, SRR1695193,SRR1695194, SRR1768504, SRR1768505, SRR1768506, SRR1768507, SRR1768508, SRR1768509, SRR1768510, SRR1768511, SRR1768512, SRR1768513, SRR1768514, SRR1768515, SRR1768516, SRR1768517, SRR1768518 and SRR1768519) [30]. The \u003cem\u003eG. hirsutum\u003c/em\u003e\u003cem\u003ehistone-3 \u003c/em\u003e(\u003cem\u003eGhHIS3\u003c/em\u003e, AF024716) gene was downloaded from the National Center for Biotechnology Information (NCBI) database, which were used as internal references. All other data generated or analyzed during this study are included in this published article and its Additional files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work reported in this publication was supported by the National Natural Science Foundation of China through the Awards Nos. 31690092, 31571251, 31788103, the National Key R\u0026amp;D Program of China (2016YFD0100500) and the Ministry of Agriculture of China (2016ZX08005-003), the China Postdoctoral Science Foundation through the Awards Nos. 2017M621546 and 2018T110411. The funding bodies did not participate in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZWC, XFZ and LJW designed the research. ZWC, XFZ, JFC, CCH, XL and YGZ performed the experiments. XFZ, ZSZ, XXSG, LJW and ZWC contributed materials and analyzed data. ZWC wrote and revised the manuscript. All the authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Tian-Zhen Zhang for providing the RNA-seq data and calculating the RPKM values and Prof. Xiao-Ya Chen participating in discussion and revising the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSchell J. 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J Exp Bot. 2008;59(13):3533\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTABLE 1 |\u003c/strong\u003e \u003cem\u003eKa\u003c/em\u003e, \u003cem\u003eKs\u003c/em\u003e and \u003cem\u003eK\u003c/em\u003ea/\u003cem\u003eK\u003c/em\u003es analyses of \u003cem\u003eGhARF\u003c/em\u003e from the A and D subgenomes compared with their corresponding progenitor homoeologs.\u003c/p\u003e\n\u003ctable border=\"1\" width=\"100%\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003e\u003cstrong\u003eLocus Name\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"11%\"\u003e\n\u003cp\u003e\u003cstrong\u003eGene Name\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003e\u003cstrong\u003eChrom\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003e\u003cstrong\u003eLocus Name\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003e\u003cstrong\u003eGene Name\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003e\u003cstrong\u003eChrom\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e\u003cstrong\u003eKa\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e\u003cstrong\u003eKs\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e\u003cstrong\u003eKa/Ks\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003eGh_A10G1402\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"11%\"\u003e\n\u003cp\u003eGhARF1_A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003eA10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003eCotton_A_31395\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003eGaARF1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003eCA_chr1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0147\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0261\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.5632\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003eGh_D10G0803\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"11%\"\u003e\n\u003cp\u003eGhARF1_D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003eD10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003eGorai.011G091100.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003eGrARF1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003eChr11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0019\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0108\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.1759\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003eGh_A07G0411\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"11%\"\u003e\n\u003cp\u003eGhARF2a_A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003eA07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003eCotton_A_03644\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003eGaARF2a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003eCA_chr1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0035\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0068\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.5147\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003eGh_D07G0476\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"11%\"\u003e\n\u003cp\u003eGhARF2a_D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003eD07\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003eGorai.001G054600.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003eGrARF2a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003eChr1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0061\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0169\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.3609\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003eGh_A11G0358\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"11%\"\u003e\n\u003cp\u003eGhARF2b_A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003eA11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003eCotton_A_01955\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003eGaARF2b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003eCA_chr6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0015\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0151\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0993\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003eGh_D11G0416\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"11%\"\u003e\n\u003cp\u003eGhARF2b_D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003eD11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003eGorai.007G044900.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003eGrARF2b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003eChr7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0028\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0045\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.6222\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003eGh_D12G1909\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"11%\"\u003e\n\u003cp\u003eGhARF2c_D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003eD12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003eGorai.008G210200.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003eGrARF2c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003eChr8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0083\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0263\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.3156\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003eGh_A11G1082\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"11%\"\u003e\n\u003cp\u003eGhARF2d_A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003eA11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003eCotton_A_08273\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003eGaARF2d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003eCA_chr4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0012\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0021\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.5714\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003eGh_D11G1233\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"11%\"\u003e\n\u003cp\u003eGhARF2d_D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003eD11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003eGorai.007G131900.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003eGrARF2d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003eChr7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0058\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0181\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.3204\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003eGh_A08G0656\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"11%\"\u003e\n\u003cp\u003eGhARF2e_A\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003eA08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003eCotton_A_22543\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003eGaARF2e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003eCA_chr10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0032\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0000\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e2.0000\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003eGh_D08G0758\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd 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width=\"14%\"\u003e\n\u003cp\u003eCotton_A_03933\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003eGaARF3a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"17%\"\u003e\n\u003cp\u003eCA_chr9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0096\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.0167\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"6%\"\u003e\n\u003cp\u003e0.5749\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"10%\"\u003e\n\u003cp\u003eGh_D10G0266\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"11%\"\u003e\n\u003cp\u003eGhARF3a_D\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"16%\"\u003e\n\u003cp\u003eD10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"14%\"\u003e\n\u003cp\u003eGorai.011G030900.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"9%\"\u003e\n\u003cp\u003eGrARF3a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd 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width=\"6%\"\u003e\n\u003cp\u003e0.7963\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cotton, GhARF2b, Fiber elongation, Fiber initiation","lastPublishedDoi":"10.21203/rs.3.rs-119017/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-119017/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Cotton fiber is a model system for studying plant cell development. At present, our understanding of cotton fiber development and the regulatory network is still primitive. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eHere, we identify auxin response factor (ARF) genes in three cotton species: the tetraploid upland cotton \u003cem\u003eG. hirsutum\u003c/em\u003e, which has 73 ARF genes, and its putative extent parental diploids \u003cem\u003eG. arboreum\u003c/em\u003e and \u003cem\u003eG. raimondii\u003c/em\u003e, which have 36 and 35 ARFs, respectively. Ka and Ks analyses revealed that in \u003cem\u003eG. hirsutum ARF\u003c/em\u003e genes have undergone asymmetric evolution in the two subgenomes. The cotton ARFs can be classified into four phylogenetic clades and are actively expressed in young tissues. We demonstrate that\u003cem\u003e GhARF2b\u003c/em\u003e, a homolog of the Arabidopsis \u003cem\u003eAtARF2\u003c/em\u003e, was preferentially expressed in developing ovules and fibers. Overexpression of \u003cem\u003eGhARF2b\u003c/em\u003e by a fiber specific promoter inhibited fiber cell elongation but promoted initiation and, conversely, its downregulation by RNAi of resulted in fewer but longer fiber. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eOur results uncover an important role of the ARF factor in modulating cotton fiber development at the early stage.\u003c/p\u003e","manuscriptTitle":"Characterization of Cotton ARF Factors and the Role of GhARF2b in Fiber Development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-12-03 17:02:21","doi":"10.21203/rs.3.rs-119017/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2020-12-24T00:00:00+00:00","index":3,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"decision","content":"Major revision","date":"2020-12-24T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-12-10T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"editorInvitedReview","content":"","date":"2020-12-10T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2020-12-07T00:00:00+00:00","index":3,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-12-04T01:00:00+00:00","index":2,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-12-04T00:00:00+00:00","index":1,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-12-03T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-11-30T00:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-11-16T00:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2020-11-16T00:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-11-15T23:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"12d4b8a2-fffe-435a-89e5-0b0daf6233b9","owner":[],"postedDate":"December 3rd, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":1311027,"name":"Epigenetics \u0026 Genomics"}],"tags":[],"updatedAt":"2021-03-29T15:00:44+00:00","versionOfRecord":{"articleIdentity":"rs-119017","link":"https://doi.org/10.1186/s12864-021-07504-6","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2021-03-22 15:00:30","publishedOnDateReadable":"March 22nd, 2021"},"versionCreatedAt":"2020-12-03 17:02:21","video":"","vorDoi":"10.1186/s12864-021-07504-6","vorDoiUrl":"https://doi.org/10.1186/s12864-021-07504-6","workflowStages":[]},"version":"v1","identity":"rs-119017","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-119017","identity":"rs-119017","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}