Comparative transcriptome analysis uncovers cell wall reorganization and repressed cell division during cotton fiber initiation

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Although various omics studies have revealed molecular basis for fiber development, a better understanding of transcriptional regulation mechanism regulating lint fiber initiation is necessary to meet global natural fiber demand. Conclusions Here, we aimed to perform transcriptome sequencing to identify DEGs (differentially expressed genes) in ovules of the cotton variety Xu142 and its fibreless mutant Xu142fl during early lint fiber initiation period. Totally, 5516 DEGs including 1840 upregulated and 3676 downregulated were identified. GO enrichment analysis revealed that the downregulated DEGs mainly associated with biological processes such as transcription related biosynthesis and metabolism, organic cyclic compound biosynthesis and metabolism, photosynthesis, and plant cell wall organization, with molecular functions involving transcription related binding, organic cyclic compound binding, and dioxygenase activity, while the upregulated DEGs were associated with DNA replication and phospholipid biosynthetic related processes. Among the 490 DEGs annotated as transcription factor genes 86.5% were downregulated in the mutant including the Malvaceae -specific MMLs , expression patterns of which were confirmed during the central period of lint fiber initiation. Investigation of the 20 genes enriched in the cell wall organization revealed that 17 were EXPA coding genes. Overall, we suggest that lint fiber initiation is a complicated process involving cooperation of multiple transcription factor families, which might ultimately lead to the reorganization of the cell wall and terminated cell division of the differentiating fiber initials. Developmental Biology Lint fiber initiation Transcriptome sequencing Xuzhou142 fibreless mutant (Xu142fl) MML EXPA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Cotton plants serve as the largest natural fiber source for the global textile industry [ 1 ]. Mature cotton seeds are covered with adherent fuzz and spinnable lint fibers. Cotton fibers development can be classified into four overlapping stages: initiation, elongation, thickening of the secondary cell wall, and maturation [ 2 ]. Lint fibers starts initiation from − 3dpa and continue to 3 dpa [ 3 ]. The initiation of lint fiber involves multiple regulators such as the upstream transcription factors and the downstream expansins proteins [ 4 – 6 ]. The transcriptional mechanisms have been widely explored by various researchers, which established the model in cotton that fiber initiation mimics the hair trichome initiation in Arabidopsis involving the MBW complex consisting of the R2R3 MYB protein GL1, the bHLH protein GL3, and the WD-repeat containing protein TTG1, which controls the expression of the downstream HD-ZIP transcription factor gene GL2 [ 7 ]. In cotton, the GL1 homologous R2R3-MYB transcription factor genes GhMYB25-like and GhMYB25 have been implicated in regulating fiber initiation and elongation respectively [ 8 , 9 ], and renamed as MYB-MIXTA-like 3 ( MML3 ) and MML7 respectively lately [ 10 ]. Previously, the N1 gene ( GhMML3_A12 ) in N1 naked seed mutant ( N1NSM ) and the fi3 gene ( GhMML4_D12 ) in Xuzhou142 fibreless mutant ( Xu142fl ) have been isolated through a map based cloning method respectively [ 11 , 12 ]. Totally 10 MMLs in G.raimondii have been classified into one lineage as the Malvaceae -specific 9th subfamily R2R3-MYBs that regulates epidermal cell differentiation [ 12 ], different from the 15th subfamily which regulates leaf hair trichome development in Arabidopsis according to evolution analysis [ 13 ], and they contain a signature protein motif and are highly expressed during the lint fiber initiation period [ 10 ]. Other homologous transcription factor genes in the MBW pathway such as GL3 homolog bHLH transcription factor gene GhDEL65 [ 14 ] and GL2 homologs GaHOX1 and GhHOX3 also contribute to lint fiber development [ 15 , 16 ]. The down-stream biological events regulating cell wall reorganization and biosynthesis employ various proteins like expansins, sucrose synthases, and tubulins [ 17 – 19 ]. Expansins are the first identified cell-wall-loosening proteins [ 20 ], which function by weakening the noncovalent bonds between cell wall matrix polymers to promote slippage of cellulose microfibrils and cause cell wall relaxation and cell extension [ 21 ], and constitute a large multigene family of four groups: alpha-expansin (EXPA), beta-expansin (EXPB), expansin-like A and expansin-like B. EXPAs were firstly speculated to involve in fiber development since the isolation of two alpha-expansin cDNAs from the developing fiber of G.hirsutum [ 22 ], then two homologous fiber-specific EXPAs coding genes- GbEXPA2 from the D subgenome and GbEXPATR from the A subgenome of G.barbadense were cloned and genetically characterized to participate in the process of lint fiber elongation, evidenced by the facts that silencing of GbEXPA in G.hirsutum resulted into shorter fibers with thicker cell walls while overexpressing GbEXPATR induced longer, finer, and stronger fibers coupled with significantly thinner cell walls [ 23 ]. It has been reported that overexpression of GhEXPA8 significantly improves fiber length and micronaire value [ 24 ], while co-expression of GhRDL1 and GhEXPA1 led to longer fiber [ 25 ]. However, the correlation between expansins proteins and lint fiber initiation at the transcriptome level still need to be investigated. Despite various studies strongly suggest the key role of Xu142 and Xu142fl in investigating lint fiber initiation; however, transcriptional regulation mechanisms of Xu142 and Xu142fl regulating fiber initiation, in particular, with a focus on the changing upstream transcription regulations and the downstream cell wall reorganization during early lint fiber initiation in cotton are poorly understood. In this study, we compared the whole genome transcription changes between Xu142 and Xu142fl at early fiber initiation stages and identified 3676 downregulated and 1840 upregulated genes. Gene Ontology (GO) enrichment analysis revealed that the downregulated genes were mainly involved in the biological processes related to transcription, organic cyclic compound biosynthesis and metabolism, photosynthesis, response to chitin and plant cell wall organization, and had molecular functions such as transcription related binding, organic cyclic compound binding and dioxygenase activity, while the upregulated DEGs were enriched in DNA replication and phospholipid biosynthesis related processes. Classification of the transcription factor genes revealed that various transcription factor families involved in lint fiber initiation, including the members belonging to the MBW complex. Examination of the cell wall organization related genes revealed 17 EXPA coding genes, one pectinesterase (PE) coding gene, and two COBL10 coding genes, which may contribute to cell loosening and reorganization during lint fiber initiation. Overall, these findings provide preliminary data which suggest importance of introducing the essential transcription factor genes or EXPAs for improving lint fiber initiation rate. Results Overview of the comparative transcriptome sequencing using ovules of Xu142fl and Xu142 at early fiber initiation stage In order to study Xu142fl and Xu142 in the context of fiber initiation, we first performed transcriptome sequencing by mixing ovules at -3 and -1 DPA of Xu142fl and Xu142 respectively, before obvious fiber initials could be observed from the epidermis of the wild type cotton seeds under optical microscope, by setting 3 biological replicates for each variety. After removing adapter contamination and low quality tags, a total of 66.12-72.19 million clean reads were generated from each library, with clean read ratios between 92.51%-93.74%, and ~ 95% of the clean reads can be mapped to cotton TM-1 genome (Table 1). Besides this, 49381 novel transcripts were identified, including 36093 candidate protein coding and 13288 noncoding transcripts and 5604 novel genes were predicted. After calculating the expression levels of each gene in each sample; Pearson correlation (R 2 ) was calculated based on the whole gene expression profile between each sample pair among the total 6 samples. The result showed that the correlations between biological repeats were 0.966-0.993 for the mutant pairs, and 0.971-0.996 for the wild type pairs, but lower (0.921-0.960) between mutant and wild type pairs (Additional file 1 Figure 1). The results indicated high uniformity between biological repeats. Finally, 5516 DEGs including 1840 upregulated and 3676 downregulated genes were identified in the mutant compared with the wild type. Table 1. Overview of the data quality and genome mapping of the transcriptome sequencing of Xu142 and Xu142fl . Ovule Sample Total Raw Reads (M) Total Clean Reads (M) Clean Reads Q20(%) Clean Reads Ratio (%) Total Mapped (%) Uniquely Mapped (%) Xu142-1 70.82 66.39 98.56 93.74 95.89 76.20 Xu142-2 75.39 70.42 98.59 93.41 95.55 75.11 Xu142-3 73.31 68.7 98.59 93.71 95.95 75.99 Xu142fl -1 70.61 66.12 98.53 93.64 95.75 74.58 Xu142fl -2 75.72 70.78 98.55 93.48 95.85 75.43 Xu142fl -3 78.04 72.19 98.6 92.51 95.87 75.52 Note: M means megabase. Complicated upstream transcription and downstream biosynthesis and metabolism events occurred during early lint fiber initiation To investigate the biological processes and functions attributed to the DEGs, GO enrichment analysis of the downregulated and upregulated DEGs were conducted respectively. The results showed that the downregulated genes were enriched in 15 level 3 biological process (BP) terms including transcription (262 genes), RNA (288 genes) and nucleic acid metabolism (339 genes), nucleobase-containing compound biosynthesis (267 genes) and metabolism (358 genes), heterocycle biosynthesis (275 genes) and metabolism (377 genes), aromatic compound biosynthesis (273 genes) and metabolism (383 genes) and organic cyclic compound biosynthesis (276 genes) and metabolism (380 genes), photosynthesis (42 genes) and light harvesting (15 genes), respond to chitin (7 genes), and plant cell wall organization (20 genes) (Fig. 1a; Additional file 2 Table S1), with molecular functions (MF) including DNA binding transcription factor activity (205 genes), transcription regulator activity (208 genes) and dioxygenase activity (32 genes), and DNA (404 genes), nucleic acid (585 genes), organic cyclic compound and heterocyclic compound binding activities (1008 genes) (Fig. 1b; Additional file 3 Table S2). These results demonstrated that the essential transcriptional regulations in Xu142fl were impaired and led to the aborted lint fiber initiation. Compared with the significantly enriched GO terms for the downregulated DEGs, the enriched GO terms for the upregulated DEGs were fewer on the BP level, and no GO terms were found on the MF level. The significantly enriched level 3 terms was DNA replication (21 genes), DNA-dependent DNA replication (11 genes), DNA replication initiation (8 genes) and phospholipid biosynthesis (19 genes) (Fig. 1c; Additional file 4 Table S3). Overall, these findings suggest that DNA replication and phospholipid biosynthesis in the process of cell mitosis division, were inhibited in the fiber initials of wild type during lint fiber initiation. Transcription factor expression dynamics during early fiber initiation Next, to characterize the complicated transcription regulations during lint fiber initiation, the TF coding genes were firstly predicted and then filtered to obtain the differentially expressed TF genes closely related to lint fiber initiation (only TF family containing more than two genes were considered), and finally 490 genes belonging to 26 TF families were identified, consisting of 424 downregulated genes and 66 upregulated genes. Moreover, most TF families consisted more downregulated genes (57.14%-100%), except for LOB domain-containing and MADS domain-containing families which contained more up-regulated genes (76.92% and 71.43% respectively) (Table 2). As shown in Table 2, among the down-regulated TF families containing members more than 20, the biggest TF family was AP2-EREBP, which contained 123 DEGs, followed by MYB (54 DEGs), WRKY (41 DEGs), NAC(33 DEGs), C2C2-Dof (33 DEGs), bHLH (29 DEGs) and GRAS (23 DEGs). Subsequently, we chose six TF families including MYB, bHLH, NAC, C2C2 Dof, GRAS, and WRKY containing members ranging from 23-54 to plot expression heat maps. The results showed that 6 Malvaceae -specific MML homologs (Wu et al., 2018) including two MML3 , one MML4 , two MML8 and one MML9 were all downregulated. The MML3 on chromosome D12 chromosome (Gh_D12G1628) had higher expression level in the wild type and were much more downregulated in the mutant than the one on A12 chromosome (Gh_A12G1503) (Fig. 2a). Although most members were downregulated in the identified TF families, subclasses of members were still upregulated for 5 TF families, including the MYB family which contains one RL6 , 3 MYB44 and 4 GAM1 (Fig. 3a), bHLH family which contains 5 members including BHLH82 , BHLH130 , BEE3 and two novel genes (Fig. 2b), NAC family which contains 6 NAC genes, three of which annotated as NAC100 had dominant expression in both varieties (Fig. 2c), the GRAS family which contains 2 members (Fig. 2d), and WRKY family which contains 7 members, with the genes annotated WRKY48 and WRKY65 had higher expression levels while expression of the other five genes were lower in both varieties (Fig. 2e), except for the C2C2 Dof family genes which were all down-regulated (Fig. 2f). Table 2 Statistics of the differentially expressed transcription factor genes. TF Family T otal DEGs Upreg Downreg U preg ( % ) D ownreg ( % ) ABI3VP1 7 1 6 14.29 85.71 AP2-EREBP 123 4 119 3.25 96.75 bHLH 29 9 20 31.03 68.97 C2C2-Dof 33 0 33 0 100 C2C2-GATA 6 0 6 0 100 C2H2 17 2 15 11.76 88.24 C3H 11 0 11 0 100 FAR1 5 1 4 20 80 G2-like 7 2 5 28.57 71.43 GRAS 23 2 21 8.7 91.3 GRF 4 0 4 0 100 HSF 12 1 11 8.33 91.67 LIM 3 1 2 33.33 66.67 LOB 13 10 3 76.92 23.08 MADS 7 5 2 71.43 28.57 mTERF 7 1 6 14.29 85.71 MYB 54 8 46 14.81 85.19 NAC 33 6 27 18.18 81.82 OFP 11 0 11 0 100 PLATZ 4 0 4 0 100 SBP 4 1 3 25 75 TCP 5 0 5 0 100 Tify 14 0 14 0 100 Trihelix 7 3 4 42.86 57.14 WRKY 41 7 34 17.07 82.93 zf-HD 10 2 8 20 80 Total 490 66 424 Note: Upreg means the upregulated genes; Downreg means the downregulated genes. Main GhMMLs contributed to lint fiber initiation The 9th subfamily R2R3-MYB transcription factors MMLs is considered as Malvaceae -specific through evolutionary analysis [ 12 ] and among the 10 GhMMLs from GhMML1 to GhMML10, GhMML3 and GhMML4 had been demonstrated responsible for fuzz and lint fiber initiation respectively [ 11 , 12 ] . Our TF classification had revealed that GhMML3 , GhMML4 , GhMML8 , and GhMML9 were involved in lint fiber initiation, to confirm that, we conducted RT-PCR of all ten GhMMLs in n2NSM and Xu142fl during early lint fiber initiation period from -1 DPA to 1 DPA, given the fact that n2NSM and Xu142fl are all naked seed mutants and the only difference is whether the lint fiber initiates or not [ 12 ]. The results showed that 8 MMLs can be detected except Gh MML8 and Gh MML9 (Fig. 3), and 4 were down-regulated including GhMML1 , Gh MML3 , Gh MML4 and Gh MML7 in Xu142fl compared with n2NSM ,. Further investigation of their expression patterns by qRT-PCR confirmed that Gh MML3 , Gh MML4 and Gh MML7 were significantly down-regulated in Xu142fl compared with n2NSM at all three time points while no obvious expression differences were observed for GhMML1 (Fig. 4). We also found that expression levels of Gh MML3 and Gh MML4 in n2NSM were decreased, while that of Gh MML7 were increased from -1 DPA to 1 DPA, implying different mechanisms between GhMML7, and GhMML3 and GhMML4 (Fig. 4). Expansins enriched in cell wall organization may contributed to early lint fiber initiation Cell wall reorganization is an essential event during fiber development involving multiple enzymes and wall proteins [ 5 ]. Here, we had a detailed investigation of the GO term-plant cell wall organization which contains 20 genes, and the results showed that 17 were EXPA encoding genes, including 4 EXPA1 , 1 EXPA2 , 6 EXPA4 , 3 EXPA8 and 3 EXPA15 , one was Pectinesterase (PE) encoding gene, and 2 were COBL10 (protein transport protein SEC61 subunit alpha) encoding genes, and they were all downregulated in Xu142fl comparing with that in the wild type (Table 3). It was worth noting that the EXPA2 gene, one EXPA4 encoding gene (Gh_A10G2323) and the two COBL10 encoding genes had very low expression levels in the wild type and hardly detectable in the mutant, implying that they might not the dominant genes for early lint fiber initiation. Of special note, data indicated that EXPAs might be the most important cell wall proteins for the early fiber cell initiation. Table 3. Expression differences of the cell wall organization related genes. Gene ID Gene Annotation Xu142_RPKM Xu142fl _RPKM log2FoldChange ( Xu142fl /Xu142) Gh_A06G0018 EXPA1 8.39 3.13 -1.41 Gh_D05G1754 EXPA1 35.18 13.74 -1.36 Gh_A05G1576 EXPA1 27.35 11.60 -1.25 Gh_D12G1759 EXPA1 19.72 9.50 -1.06 Gh_A13G0672 EXPA15 16.72 4.63 -1.86 Gh_D13G0786 EXPA15 20.71 6.30 -1.71 Gh_A03G0885 EXPA15 48.83 21.23 -1.21 Gh_D10G1145 EXPA2 0.69 0.00 -6.20 Gh_A10G2323 EXPA4 0.32 0.00 -5.12 Gh_A07G0902 EXPA4 30.35 9.97 -1.59 Gh_D09G1463 EXPA4 125.90 43.37 -1.54 Gh_A04G0707 EXPA4 27.78 11.04 -1.35 Gh_A09G1454 EXPA4 63.47 27.08 -1.23 Gh_D07G0974 EXPA4 28.64 13.56 -1.01 Gh_A05G3493 EXPA8 17.43 2.33 -2.93 Gh_D04G1924 EXPA8 3.52 1.60 -1.17 Gh_D10G1861 EXPA8 11.23 5.25 -1.11 Gh_A10G1502 Pectinesterase 26.62 10.09 -1.40 Gh_D06G1606 COBL10 0.27 0.02 -3.70 Gh_A06G1281 COBL10 0.78 0.09 -3.11 Note: COBL10 encodes a protein transport protein SEC61 subunit alpha Discussion Lint fiber initiation is a complicated morphogenesis process involving complex metabolite biosynthesis and metablism In this study, through GO analysis of the downregulated and upregulated DEGs, multiple enriched biological processes were identified which were associated lint fiber initiation (Fig. 1). Based on our findings, we speculated that the downregulated DEGs mainly function as positive regulatory factors for lint fiber initiation, while the upregulated DEGs mainly function to inhibit lint fiber initiation. For the downregulated DEGs, more than 200 genes were associated DNA-tempated transcription (Fig. 1a), which have transcription associated molecular functions as DNA binding transcription factor activity (Fig. 1b). These biological processes related genes may construct the upstream regulation networks during lint fiber initiation, and the downstream regulation involves complicated metabolic cascades associated with organic compound biosynthesis and metabolism including nucleobase-containing compound, organic cyclic compound, heterocyclic compound and aromatic compound such as flavonoid, sugar and Phytohormones biosynthesis and metabolism (Fig. 1a,b; ). Several NCEDs (data not shown) which encode the rate-limiting dioxygenases controlling ABA biosynthesis [ 26 ], were found in the GO term of dioxygenase (Fig. 1b), implying that ABA might be a positive regulator during lint fiber initiation. This can be supported by the gradually accumulation of endogenous ABA content during the fiber cell initiation and elongation stages [ 27 ] although ABA was considered as a negative regulator of fiber initiation [ 28 ]. Chitin oligosaccharides can induce various defense responses in a wide range of plant cells including both monocots and dicots [ 29 ]. However, do defense responses related to fiber initiation is still not clear. Immature fiber (im) mutant with thinner fiber cell wall compared to the isogenic wild type (TM-1) with fiber of normal thickness revealed that the mutant had lower net photosynthesis, because of the lower chlorophyll content per unit leaf area due to less chlorophyll a levels than the wild type [ 30 ], however how the photosynthesis related genes were changed in Xu142fl need further investigation (Fig. 1a). The upregulated DEGs were mainly enriched in DNA replication and phospholipid biosynthetic processes, which were closely related to cell division. This is reasonable because fiber cells are unable to undergo cell division during fiber development. Before fiber cell differentiation, the ovular epidermal cells are closely packed, cuboidal, and rich in cytoplasm containing a large nucleus, which represents a status of rapid cell division [ 31 ]. The early development of fibers consists of two intergrading steps-spherical expansion above the ovular epidermis and elongation [ 32 ]. The morphological differentiation of a fiber occurs when an epidermal cell balloons above the epidermal surface, followed by transition to elongation phase and stopping division [ 33 ]. Collectively, cell division should be stopped to initiate fiber development in the wild type while rapid cell division continues which inhibit cell differentiation into fiber cells in Xu142fl mutant. Complicated transcription regulation during lint fiber initiation In this study, through TF annotation and expression pattern profiling, many down-regulated TF families were identified, including known fiber development related MYBs , bHLHs and TCPs [ 6 ]. Other TF families such as NAC, WRKY, GRAS and Dof identified here were also detected in another study exploring fiber elongation related pathways by transcriptome analysis of a short fiber mutant and a wild type [ 34 ], suggesting that some TF family genes might have dual role both in fiber initiation and elongation. For example, fiber cell expansion and elongation can be mediated by a homeodomain leucine zipper gene, GhHD-1 , through a WRKY transcription factor by regulating the levels of ethylene and reactive oxidation species (ROS) [ 35 ]. Because some lint fiber initiation related TFs which were genetically characterized as positive regulators for fiber development were firstly found downregulated in fiber related mutants, including R2R3 MYB proteins [ 8 , 9 , 36 , 37 ], HD-ZIP proteins [ 15 , 16 ] and bHLH proteins [ 14 ], so the other downregulated TF family genes found in this study deserve further investigation of their roles in regulating lint fiber initiation. Besides this, two class of TF families involved more upregulated DEGs than the downregulate DEGs (Table 2), including LOB (Lateral Organ Boundaries) family which are essential in the regulation of plant lateral organ development [ 38 ] and MADS which regulate floral organ differentiation and development [ 39 ], and small group of genes in other TF families were also up-regulated (Fig. 2, Table 2), which might be negative regulators of fiber initiation and contribute to the lintless ovules of Xu142fl due to their upregulation. Dynamic expression of Malvaceae -specific MMLs during early lint fiber initiation Among the 10 pair of Malvaceae -specific MYB MIXTA-like ( GhMML ) homoeologs at least one copy on one sub-genome in allotetraploid cotton were predominantly expressed during fiber initiation in the wild type TM-1 [ 10 ], and MML3-A12 and MML4-D12 were demonstrated contributing to the fuzz and lint fiber initiation respectively [ 11 , 12 ], implying the subfunctionalization of MML genes. Combined our transcriptome analysis and RT-PCR analysis, MML3 , MML4 and MML7 should be the MMLs most related to lint fiber initiation (Fig. 2a, Fig. 3, 4) . However, it was contrary in that GhMML3-D12 (Gh_D12G1628) and GhMML4-A12 (Gh_A12G1504) might be more important for lint fiber initiation, because GhMML3-D12 had higher expression level in the wild type and was more downregulated in the mutant than GhMML3-A12, while GhMML4-D12 was not detected as DEG in our study (Fig. 2a). Different from GhMML3 and GhMML4 which were downregulated in the fuzzless-linted mutant n2NSM from -1 DPA to 1 DPA, GhMML7 was upregulated and had highest expression level in ovules at 1 DPA (Fig. 4). This phenomenon indicated that, except for the role in fiber initiation, GhMML7 may also involve in fiber elongation, which can be assisted by the evidences that GhMML7/GhMYB25 expressed in the epidermis of ovules, developing fiber initials and fibers, and GhMML7/GhMYB25 -silenced cotton produced shorter fiber, while overexpression of GhMYB25 promoted fiber initiation [ 8 ]. Cell wall organization initiates from the very beginning during lint fiber initiation Previously, Stage-specific developmental markers such as EXPAs, xyloglucan endo-transglycosylases (XETs) and PEs have been reported to regulate fiber cell expansion in cotton [ 4 , 5 , 40 ]. Here, we also identified many EXPA encoding genes and a PE encoding gene, indicated that EXPAs are more important during fiber initiation and elongation. Taken together, upstream regulators of EXPAs will further facilitate our understanding underlying mechanisms of lint fiber initiation and elongation. Conclusion In this study, we compared the whole genome transcription changes between Xu142 and Xu142fl at early fiber initiation stages and identified 3676 downregulated and 1840 upregulated genes. Gene Ontology (GO) enrichment analysis revealed that the downregulated genes were mainly involved in the biological processes related to transcription, organic cyclic compound biosynthesis and metabolism, photosynthesis, response to chitin and plant cell wall organization, and had molecular functions such as transcription related binding, organic cyclic compound binding and dioxygenase activity, while the upregulated DEGs were enriched in DNA replication and phospholipid biosynthesis related processes. Classification of the transcription factor genes revealed that various transcription factor families involved in lint fiber initiation, including the members belonging to the MBW complex. Examination of the cell wall organization related genes revealed 17 EXPA coding genes, one pectinesterase (PE) coding gene, and two COBL10 coding genes, which may contribute to cell loosening and reorganization during lint fiber initiation. Overall, these findings provide preliminary data which suggest importance of introducing the essential transcription factor genes or EXPAs for improving lint fiber initiation rate. In conclusion, this study provides new information for lint fiber initiation mechanism, which might involve dynamic expression of multiple types of TF family genes, which mediate the complicated downstream organic compound biosynthesis and metabolism resulting into the termination of cell division and cell wall reorganization of the expanding fiber cells during lint fiber initiation (Fig. 5). Methods Plant materials One cotton variety Gossypium hirsutum cv. Xu142 and two fibreless cotton mutants Xu142fl and n2NSM were selected in this study. The seeds of these lines were retrieved from National Medium-term Gene Bank of Cotton in China and National cotton germplasm resources platform. The seeds were grown in the experimental field of Linyi University in April 14, 2018. Ovules at -3, -1, 0 and 1 DPA were collected between July and August during the flowering peak, frozen immediately in liquid nitrogen and stored at -80℃. Transcriptome sequencing and bioinformatics analysis High-quality RNA extraction was performed from the frozen ovules tissues collected at -3 DPA and -1 DPA of Xu142 and Xu142fl respectively as previously reported [ 41 ]. Subsequent cDNA libraries were constructed and sequenced with BGI-SEQ500 at the Beijing Genomics Institute (BGI, Shenzhen, China). The raw reads were filtered first to get clean data, and then aligned to the genome of G. hirsutum L. (https://cottonfgd.org/about/download/assembly/genome.Ghir.NAU.fa.gz) to reconstruct transcripts through String Tie ( http://ccb.jhu.edu/software/stringtie , v1.0.4), identify known genes by Bowtie2 (http://bowtie-bio.sourceforge.net/Bowtie2, v2.2.5) [ 42 ], and predict new transcripts using HISAT2 (http://www.ccb.jhu.edu/software/hisat, v2.0.4) [ 43 ]. Expression levels of genes and transcripts were calculated using RSEM [ 44 ], and expression corrections were calculated by Cor package of R (v3.6.2). DEGs (filtered by fold change >= 2, Q-value <= 0.001) between Xu142 and Xu142fl were identified through DEGseq [ 45 ], after normalizing raw reads of each gene as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). GO enriched terms were determined by Q-value <= 0.001 using the phyper package of R. The transcription factor (TF) coding genes were predicted by using Getorf (http://emboss.sourceforge.net/apps/cvs/emboss/apps/getorf.html) to get the ORF of all unigenes first, and then aligned to TF protein structure domain using hmmsearch (http://hmmer.org, v3.0) to annotate TF through the property of specific TF family described in PlantTFDB (http://planttfdb.cbi.pku.edu.cn, v5.0). Heatmaps of TF proteins were generated by Genesis v1.7.6 [ 46 ]. RT-PCR and RT-qPCR analyses RT-PCR and qRT-PCR were used to evaluate expression levels of GhMMLs . Total RNA from ovules at -1, 0 and 1 DPA of n2NSM and Xu142fl was extracted as previously reported [ 41 ]. Subsequent cDNA was synthesized using a Thermos Scientific RevertAid First Strand cDNA Synthesis Kit according to the manufacture’s instruction. Total 20-µl-reaction volume was applied for RT-PCR analysis to evaluate expression levels of GhMMLs . After this, PCR reaction mixture was subjected to 95°C denaturation for 3 min, then 29 cycles of amplification of the endogenous reference gene GhUbq7 or 38 cycles for GhMMLs at 95°C for 30 sec, 55°C annealing for 30 sec and 72°C extension for 30 sec, plus a final extension at 72°C for 5 min. Quantitative real-time PCR (qRT-PCR) was carried out using Hieff qPCR SYBR Green Master Mix (No Rox) (Yeason, Shanghai, China). The calculation of the relative expression levels of each gene and statistical analysis were determined as previously reported [ 47 ]. Primers were designed by Primer 5.0 and synthesized commercially (Genscript Bioscience, Nanjing, China). The sequences of all the primers sequences are listed in Additional file 5 Table 4. Declarations Acknowledgements We thank Kashif Rafiq for critiquing the manuscript. We thank National Medium-term Gene Bank of Cotton in China and National cotton germplasm resources platform for providing the cotton seeds used in this study. Authors’ contributions L. W.: project design, transcriptome data mining, writing the manuscript. Y. L., X. L. and Z. F.: collecting the ovules and extracting the total RNA. W. L: experiments shown in Figure 3 and Figure 4. All authors read and approved the final manuscript. Funding The authors are sponsored by State Key Laboratory of Cotton Biology Open Fund (Grant No. CB2018A20), and Linyi University PhD Fund for Scientific Start-up (LYDX2018BS029). Availability of data and materials Raw data for the transcriptomes are available on the GEO platform as series GSE176384. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests No competing interests declared. References Hu Q, Xiao S, Guan Q, Tu L, Sheng F, Du X, Zhang X: The laccase gene GhLac1 modulates fiber initiation and elongation by coordinating jasmonic acid and flavonoid metabolism . The Crop Journal 2020, 8 (4):522–533. Qin Y-M, Zhu Y-X: How cotton fibers elongate: a tale of linear cell-growth mode . Current opinion in plant biology 2011, 14 (1):106–111. Graves D, Stewart J: Chronology of the differentiation of cotton (Gossypium hirsutum L.) fiber cells . Planta 1988, 175 (2):254–258. 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Walford SA, Wu Y, Llewellyn DJ, Dennis ES: GhMYB25-like: a key factor in early cotton fibre development . The Plant journal: for cell and molecular biology 2011, 65 (5):785–797. Zhang T, Hu Y, Jiang W, Fang L, Guan X, Chen J, Zhang J, Saski CA, Scheffler BE, Stelly DM et al : Sequencing of allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement . Nature biotechnology 2015, 33 (5):531–537. Wan Q, Guan X, Yang N, Wu H, Pan M, Liu B, Fang L, Yang S, Hu Y, Ye W et al : Small interfering RNAs from bidirectional transcripts of GhMML3_A12 regulate cotton fiber development . The New phytologist 2016, 210 (4):1298–1310. Wu H, Tian Y, Wan Q, Fang L, Guan X, Chen J, Hu Y, Ye W, Zhang H, Guo W et al : Genetics and evolution of MIXTA genes regulating cotton lint fiber development . The New phytologist 2018, 217 (2):883–895. Stracke R, Werber M, Weisshaar B: The R2R3-MYB gene family in Arabidopsis thaliana . Current opinion in plant biology 2001, 4 (5):447–456. Shangguan XX, Yang CQ, Zhang XF, Wang LJ: Functional characterization of a basic helix - loop - helix ( bHLH ) transcription factor GhDEL65 from cotton ( Gossypium hirsutum ). Physiologia plantarum 2016, 158 (2):200–212. Guan XY, Li QJ, Shan CM, Wang S, Mao YB, Wang LJ, Chen XY: The HD - Zip IV gene GaHOX1 from cotton is a functional homologue of the Arabidopsis GLABRA2 . Physiologia plantarum 2008, 134 (1):174–182. Shan CM, Shangguan XX, Zhao B, Zhang XF, Chao LM, Yang CQ, Wang LJ, Zhu HY, Zeng YD, Guo WZ et al : Control of cotton fibre elongation by a homeodomain transcription factor GhHOX3 . Nature communications 2014, 5 :5519. Shi YH, Zhu SW, Mao XZ, Feng JX, Qin YM, Zhang L, Cheng J, Wei LP, Wang ZY, Zhu YX: Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation . The Plant cell 2006, 18 (3):651–664. Lv L, Zuo D, Wang X, Cheng H, Ma Z: Genome - wide identification of expansin gene family reveals expansin genes are involved in fibre cells growth in cotton . 2020. Mukhtar A, Ali SA, Sidra A, Ayesha L, Ud DS, Ma F, Rao AQ, Bilal SM, Tayyab H, Wang X: Sucrose synthase genes: a way forward for cotton fiber improvement . Biologia 2018, 73 :1–11. McQueen-Mason, S.: Two endogenous proteins that induce cell wall extension in plants . The Plant cell 1992, 4 (11):1425–1433. Sampedro J, Cosgrove DJ: The expansin superfamily . Genome biology 2005, 6 (12):242. Harmer S, Orford S, Timmis J: Characterisation of six alpha - expansin genes in Gossypium hirsutum ( upland cotton ). Molecular Genetics & Genomics 2002, 268 (1):1–9. Li Y, Tu L, Pettolino FA, Ji S, Hao J, Yuan D, Deng F, Tan J, Hu H, Wang Q et al : GbEXPATR, a species-specific expansin, enhances cotton fibre elongation through cell wall restructuring . Plant biotechnology journal 2016, 14 (3):951–963. Bajwa KS, Shahid AA, Rao AQ, Bashir A, Aftab A, Husnain T: Stable transformation and expression of GhEXPA8 fiber expansin gene to improve fiber length and micronaire value in cotton . Frontiers in plant science 2015, 6 :838. Xu B, Gou JY, Li FG, Shangguan XX, Zhao B, Yang CQ, Wang LJ, Yuan S, Liu CJ, Chen XY: A cotton BURP domain protein interacts with alpha-expansin and their co-expression promotes plant growth and fruit production . Molecular plant 2013, 6 (3):945–958. Matilla AJ, Carrillo-Barral N, Rodríguez-Gacio M: An Update on the Role of NCED and CYP707A ABA Metabolism Genes in Seed Dormancy Induction and the Response to After-Ripening and Nitrate . Journal of Plant Growth Regulation 2015, 34 (2):274–293. Davis LA, Addicott FT: Abscisic Acid: Correlations with Abscission and with Development in the Cotton Fruit . Plant physiology 1972, 49 (4):644–648. Xiao G, Zhao P, Zhang Y: A Pivotal Role of Hormones in Regulating Cotton Fiber Development . Frontiers in plant science 2019, 10 :87. Shibuya N: Oligosaccharide signalling for defence responses in plant . Physiol Mol Plant Pathol 2001, 59 . Gordon M, Arasah A, Stern K, Kim HJ, Ratnayaka H: Photosynthesis and growth characteristics of cotton immature fiber mutant ( im ) in comparison with isogenic wild type ( TM - 1 ). In: Plant Biology 2016, American Society of Plant Biologists: 2016 . Joshi PC, Wadhwani AM, Johri BM: Morphological and embryological studies of Gossypium L . 1967. Stewart JM: FIBER INITIATION ON THE COTTON OVULE (GOSSYPIUM HIRSUTUM) . American Journal of Botany 1975, 62 (7). Basra AS, Malik CP: Development of the Cotton Fiber . International review of cytology 1984, 89 (6):65–113. Kang LA, Jing SA, Ly A, Yy B: Transcriptome analysis reveals critical genes and key pathways for early cotton fiber elongation in Ligon lintless-1 mutant . Genomics 2012, 100 (1):42–50. Walford SA, Wu Y, Llewellyn DJ, Dennis ES: Epidermal cell differentiation in cotton mediated by the homeodomain leucine zipper gene, GhHD-1 . The Plant journal: for cell and molecular biology 2012, 71 (3):464–478. Huang Y, Liu X, Tang K, Zuo K: Functional analysis of the seed coat-specific gene GbMYB2 from cotton . Plant physiology and biochemistry: PPB 2013, 73 :16–22. Pu L, Li Q, Fan X, Yang W, Xue Y: The R2R3 MYB transcription factor GhMYB109 is required for cotton fiber development . Genetics 2008, 180 (2):811–820. Majer C, Hochholdinger F: Defining the boundaries: structure and function of LOB domain proteins . Trends in plant science 2011, 16 (1):47–52. Theissen G: Development of floral organ identity: stories from the MADS house . Current opinion in plant biology 2001, 4 (1):75–85. Al-Ghazi Y, Bourot S, Arioli T, Dennis ES, Llewellyn DJ: Transcript profiling during fiber development identifies pathways in secondary metabolism and cell wall structure that may contribute to cotton fiber quality . Plant & cell physiology 2009, 50 (7):1364–1381. Wang L, Liu N, Wang T, Li J, Wen T, Yang X, Lindsey K, Zhang X: The GhmiR157a-GhSPL10 regulatory module controls initial cellular dedifferentiation and callus proliferation in cotton by modulating ethylene-mediated flavonoid biosynthesis . Journal of experimental botany 2018, 69 (5):1081–1093. Langmead B, Salzberg SL: Fast gapped-read alignment with Bowtie 2 . Nature methods 2012, 9 (4):357–359. Kim D, Langmead B, Salzberg SL: HISAT: a fast spliced aligner with low memory requirements . Nature methods 2015, 12 (4):357–360. Li B, Dewey CN: RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome . BMC bioinformatics 2011, 12 :323. Wang L, Feng Z, Wang X, Zhang X: DEGseq: an R package for identifying differentially expressed genes from RNA-seq data . Bioinformatics 2010, 26 (1):136–138. Sturn A, Quackenbush J, Trajanoski Z: Genesis: cluster analysis of microarray data . Bioinformatics 2002, 18 (1):207–208. Yang X, Wang L, Yuan D, Lindsey K, Zhang X: Small RNA and degradome sequencing reveal complex miRNA regulation during cotton somatic embryogenesis . Journal of experimental botany 2013, 64 (6):1521–1536. Additional Declarations No competing interests reported. Supplementary Files AdditionalFiles.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 05 Aug, 2021 Reviews received at journal 23 Jul, 2021 Reviewers agreed at journal 02 Jul, 2021 Reviewers invited by journal 29 Jun, 2021 Editor assigned by journal 25 Jun, 2021 Editor invited by journal 25 Jun, 2021 Submission checks completed at journal 25 Jun, 2021 First submitted to journal 09 Jun, 2021 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-607693","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":33815245,"identity":"1c5810e7-8534-4659-9149-e0bb4b46d7bb","order_by":0,"name":"Wenyuan Liu","email":"","orcid":"","institution":"Linyi University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wenyuan","middleName":"","lastName":"Liu","suffix":""},{"id":33815246,"identity":"a2b0c129-931a-46f5-9540-2abb2c710654","order_by":1,"name":"Yanjia Lv","email":"","orcid":"","institution":"Linyi University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yanjia","middleName":"","lastName":"Lv","suffix":""},{"id":33815247,"identity":"a28f6873-270a-4fc2-801d-87ef7064f080","order_by":2,"name":"Xiaoyue Li","email":"","orcid":"","institution":"Linyi University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoyue","middleName":"","lastName":"Li","suffix":""},{"id":33815249,"identity":"66884e6d-3338-41d6-b958-420ce887efcb","order_by":3,"name":"Zongqin Feng","email":"","orcid":"","institution":"Linyi University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zongqin","middleName":"","lastName":"Feng","suffix":""},{"id":33815253,"identity":"9690d8af-4d31-45e6-9a21-fb0f0e2486f9","order_by":4,"name":"Lichen Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYBACPmYGNiiT+cCBDxVEaGFDaGFLPDjjDDFaGOBaeIwP87YQo4Wd+dmDjzsOy5vzr/lwgLeBQZ5f7AAhh7GZG848c9hw54y3Gw5I7mAwnDk7gZAWHjZp3rbbjBtunN1wwPAMQ4LBbWK0/G27bb/hxpkHBxLbiNXC2HY7ccP5HoYDB4nTwmYm2dv2P3nDDTaDgw1nJAj7hZ//8DOJn21pthvOH378+U+FjTy/NAEtCCABVilBrHKwfQdIUT0KRsEoGAUjCQAAfYpGsrwEwpMAAAAASUVORK5CYII=","orcid":"","institution":"Linyi University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Lichen","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2021-06-10 02:29:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-607693/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-607693/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":10507981,"identity":"ebc5e2d9-35f9-4a5d-aef7-f51b1693ce3c","added_by":"auto","created_at":"2021-06-17 20:28:14","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":109689,"visible":true,"origin":"","legend":"Significantly enriched GO terms on level 3 of the down- and upregulted genes respectively\nGraphs showing functional annotation GO terms and gene number for the downregulated genes on the level of biological process (a) and molecular function (b), and for the upregulated genes on the level of biological process (c). The blue bars showing -log10(Q value) of the enrichment analysis of each term, and orange dots showing the gene number contained by each GO term. Upreg: upregulated genes; Downreg: downregulated genes; BP: biological process; MF: molecular function.\n","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-607693/v1/377b0bff762b51fbf1786b3b.jpeg"},{"id":10507765,"identity":"7c0608a8-72ab-4d05-b8ab-e0d8a431231e","added_by":"auto","created_at":"2021-06-17 20:25:14","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":248279,"visible":true,"origin":"","legend":"Expression patterns of 6 transcription factor family genes in ovules of Xu142 and Xu142fl\nHeat maps of MYB (a), bHLH (b), NAC (c), GRAS (d), WRKY (e), and DOF (f) domain containing transcription factor genes. The graphs were generated by Genesis which showing the hierarchical clusters of each type of genes. MMLs homologs in (a) were indicated with black dots, and the upregulated genes in (a)-(e) were marked using a bracket. The log2 values of the RPKM of each gene were used to plot the map, which were indicated by the gradient color bars (red to green reflecting the expression levels from high to low).\n","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-607693/v1/5e79613a691d12b12d31f66e.jpeg"},{"id":10507980,"identity":"44b890db-5681-49e6-ac2a-be0037aa1ef4","added_by":"auto","created_at":"2021-06-17 20:28:14","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":98603,"visible":true,"origin":"","legend":"Expression patterns of 8 GhMMLs in ovules of n2NSM and Xu142fl during lint fiber initiation\nThe transcripts of GhMML1-7 and GhMML10 in -1, 0 and 1 DPA ovules of n2NSM and Xu142fl were detected by RT-PCR. GhUbq7 was used as an endogenous reference gene.\n","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-607693/v1/d4a34f959eeffc16ae5a4326.jpeg"},{"id":10508216,"identity":"2f1f081c-fa4b-410c-82e1-265f1c9f7bc7","added_by":"auto","created_at":"2021-06-17 20:31:14","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":212898,"visible":true,"origin":"","legend":"Expression patterns of GhMML1/3/4/7 in ovules of n2NSM and Xu142fl during lint fiber initiation\nqRT-PCR analysis showing relative expression levels of GhMML1, GhMML3, GhMML4 and GhMML7 in -1, 0 and 1 DPA ovules of n2NSM and Xu142fl. GhUbq7 was used as an endogenous reference gene, and the data represents the mean ± SD of three biological replicates. “*” represent p \u003c 0.05.\n","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-607693/v1/54747d54fd73ed1bf57437d7.jpeg"},{"id":10507983,"identity":"b646d044-c781-4c90-99bd-b3861c7a132a","added_by":"auto","created_at":"2021-06-17 20:28:14","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":32929,"visible":true,"origin":"","legend":"Transcription regulation model of lint fiber initiation in cotton.\nMultiple TF family proteins are implicated during lint fiber initiation to inhibit DNA replication and phospholipid biosynthesis to terminate cell division, and activate expression of EXPAs and PEs to cause cell wall reorganization and fiber cell expansion. EXPAs: Alpha-expansin encoding genes; PEs: Pectinesterase.\n","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-607693/v1/aed7a7423294f115ef4d5e6d.jpeg"},{"id":13699475,"identity":"63d88c1d-7489-451c-9f96-6a4bdfe25540","added_by":"auto","created_at":"2021-09-17 13:19:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1968947,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-607693/v1/a7a6c598-3270-4cbf-a5ea-7a8b22b67ec8.pdf"},{"id":10507761,"identity":"b148cda4-cd6a-4f5c-adae-15bd06f46a4b","added_by":"auto","created_at":"2021-06-17 20:25:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":85205,"visible":true,"origin":"","legend":"","description":"","filename":"AdditionalFiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-607693/v1/99f7aa611475efaa93978634.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative transcriptome analysis uncovers cell wall reorganization and repressed cell division during cotton fiber initiation","fulltext":[{"header":"Background","content":" \u003cp\u003eCotton plants serve as the largest natural fiber source for the global textile industry [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Mature cotton seeds are covered with adherent fuzz and spinnable lint fibers. Cotton fibers development can be classified into four overlapping stages: initiation, elongation, thickening of the secondary cell wall, and maturation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Lint fibers starts initiation from \u0026minus;\u0026thinsp;3dpa and continue to 3 dpa [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The initiation of lint fiber involves multiple regulators such as the upstream transcription factors and the downstream expansins proteins [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe transcriptional mechanisms have been widely explored by various researchers, which established the model in cotton that fiber initiation mimics the hair trichome initiation in \u003cem\u003eArabidopsis\u003c/em\u003e involving the MBW complex consisting of the R2R3 MYB protein GL1, the bHLH protein GL3, and the WD-repeat containing protein TTG1, which controls the expression of the downstream HD-ZIP transcription factor gene \u003cem\u003eGL2\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In cotton, the \u003cem\u003eGL1\u003c/em\u003e homologous R2R3-MYB transcription factor genes \u003cem\u003eGhMYB25-like\u003c/em\u003e and \u003cem\u003eGhMYB25\u003c/em\u003e have been implicated in regulating fiber initiation and elongation respectively [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and renamed as \u003cem\u003eMYB-MIXTA-like 3\u003c/em\u003e (\u003cem\u003eMML3\u003c/em\u003e) and \u003cem\u003eMML7\u003c/em\u003e respectively lately [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Previously, the \u003cem\u003eN1\u003c/em\u003e gene (\u003cem\u003eGhMML3_A12\u003c/em\u003e) in \u003cem\u003eN1 naked seed mutant\u003c/em\u003e (\u003cem\u003eN1NSM\u003c/em\u003e) and the \u003cem\u003efi3\u003c/em\u003e gene (\u003cem\u003eGhMML4_D12\u003c/em\u003e) in \u003cem\u003eXuzhou142 fibreless mutant\u003c/em\u003e (\u003cem\u003eXu142fl\u003c/em\u003e) have been isolated through a map based cloning method respectively [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Totally 10 MMLs in \u003cem\u003eG.raimondii\u003c/em\u003e have been classified into one lineage as the \u003cem\u003eMalvaceae\u003c/em\u003e-specific 9th subfamily R2R3-MYBs that regulates epidermal cell differentiation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], different from the 15th subfamily which regulates leaf hair trichome development in \u003cem\u003eArabidopsis\u003c/em\u003e according to evolution analysis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and they contain a signature protein motif and are highly expressed during the lint fiber initiation period [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Other homologous transcription factor genes in the MBW pathway such as \u003cem\u003eGL3\u003c/em\u003e homolog bHLH transcription factor gene \u003cem\u003eGhDEL65\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and \u003cem\u003eGL2\u003c/em\u003e homologs \u003cem\u003eGaHOX1\u003c/em\u003e and \u003cem\u003eGhHOX3\u003c/em\u003e also contribute to lint fiber development [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe down-stream biological events regulating cell wall reorganization and biosynthesis employ various proteins like expansins, sucrose synthases, and tubulins [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Expansins are the first identified cell-wall-loosening proteins [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], which function by weakening the noncovalent bonds between cell wall matrix polymers to promote slippage of cellulose microfibrils and cause cell wall relaxation and cell extension [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and constitute a large multigene family of four groups: alpha-expansin (EXPA), beta-expansin (EXPB), expansin-like A and expansin-like B. EXPAs were firstly speculated to involve in fiber development since the isolation of two alpha-expansin cDNAs from the developing fiber of \u003cem\u003eG.hirsutum\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], then two homologous fiber-specific EXPAs coding genes-\u003cem\u003eGbEXPA2\u003c/em\u003e from the D subgenome and \u003cem\u003eGbEXPATR\u003c/em\u003e from the A subgenome of \u003cem\u003eG.barbadense\u003c/em\u003e were cloned and genetically characterized to participate in the process of lint fiber elongation, evidenced by the facts that silencing of \u003cem\u003eGbEXPA\u003c/em\u003e in \u003cem\u003eG.hirsutum\u003c/em\u003e resulted into shorter fibers with thicker cell walls while overexpressing \u003cem\u003eGbEXPATR\u003c/em\u003e induced longer, finer, and stronger fibers coupled with significantly thinner cell walls [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It has been reported that overexpression of \u003cem\u003eGhEXPA8\u003c/em\u003e significantly improves fiber length and micronaire value [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], while co-expression of \u003cem\u003eGhRDL1\u003c/em\u003e and \u003cem\u003eGhEXPA1\u003c/em\u003e led to longer fiber [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the correlation between expansins proteins and lint fiber initiation at the transcriptome level still need to be investigated.\u003c/p\u003e \u003cp\u003eDespite various studies strongly suggest the key role of Xu142 and \u003cem\u003eXu142fl\u003c/em\u003e in investigating lint fiber initiation; however, transcriptional regulation mechanisms of Xu142 and \u003cem\u003eXu142fl\u003c/em\u003e regulating fiber initiation, in particular, with a focus on the changing upstream transcription regulations and the downstream cell wall reorganization during early lint fiber initiation in cotton are poorly understood. In this study, we compared the whole genome transcription changes between Xu142 and \u003cem\u003eXu142fl\u003c/em\u003e at early fiber initiation stages and identified 3676 downregulated and 1840 upregulated genes. Gene Ontology (GO) enrichment analysis revealed that the downregulated genes were mainly involved in the biological processes related to transcription, organic cyclic compound biosynthesis and metabolism, photosynthesis, response to chitin and plant cell wall organization, and had molecular functions such as transcription related binding, organic cyclic compound binding and dioxygenase activity, while the upregulated DEGs were enriched in DNA replication and phospholipid biosynthesis related processes. Classification of the transcription factor genes revealed that various transcription factor families involved in lint fiber initiation, including the members belonging to the MBW complex. Examination of the cell wall organization related genes revealed 17 EXPA coding genes, one pectinesterase (PE) coding gene, and two COBL10 coding genes, which may contribute to cell loosening and reorganization during lint fiber initiation. Overall, these findings provide preliminary data which suggest importance of introducing the essential transcription factor genes or \u003cem\u003eEXPAs\u003c/em\u003e for improving lint fiber initiation rate.\u003c/p\u003e "},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eOverview of the comparative transcriptome sequencing using ovules of \u003cem\u003eXu142fl\u0026nbsp;\u003c/em\u003eand Xu142 at early fiber initiation stage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to study \u003cem\u003eXu142fl\u003c/em\u003e and\u0026nbsp;Xu142 in the context of fiber initiation, we first performed transcriptome sequencing by mixing ovules at -3 and -1 DPA of \u003cem\u003eXu142fl\u0026nbsp;\u003c/em\u003eand Xu142 respectively, before obvious fiber initials could be observed from the epidermis of the wild type cotton seeds under optical microscope, by setting 3 biological replicates for each variety. After removing adapter contamination and low quality tags, a total of 66.12-72.19 million clean reads were generated from each library, with clean read ratios between 92.51%-93.74%, and ~ 95% of the clean reads can be mapped to cotton TM-1 genome (Table 1). Besides this, 49381 novel transcripts were identified, including 36093 candidate protein coding and 13288 noncoding transcripts and 5604 novel genes were predicted.\u003c/p\u003e\n\u003cp\u003eAfter calculating the expression levels of each gene in each sample; Pearson correlation (R\u003csup\u003e2\u003c/sup\u003e) was calculated based on the whole gene expression profile between each sample pair among the total 6 samples. The result showed that the correlations between biological repeats were 0.966-0.993 for the mutant pairs, and 0.971-0.996 for the wild type pairs, but lower (0.921-0.960) between mutant and wild type pairs (Additional file 1 Figure 1). The results indicated high uniformity between biological repeats. Finally, 5516 DEGs including 1840 upregulated and 3676 downregulated genes were identified in the mutant compared with the wild type.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:200%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eTable 1. Overview of the data quality and genome mapping of the transcriptome sequencing of Xu142 and\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eXu142fl\u003c/span\u003e\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003e.\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\n \u003ctable style=\"width: 4.2e+2pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 70.9pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eOvule Sample\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eTotal Raw Reads (M)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.45pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eTotal Clean Reads (M)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eClean Reads Q20(%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.75pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eClean Reads Ratio (%)\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eTotal Mapped (%)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-color: windowtext currentcolor;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0in 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style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e70.82\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.45pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e66.39\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp 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4.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e66.12\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border: medium none;padding: 0in 5.4pt;height: 4.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e98.53\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.75pt;border: medium none;padding: 0in 5.4pt;height: 4.5pt;vertical-align: top;\"\u003e\n \u003cp 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none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e75.72\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.45pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e70.78\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border: medium none;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp 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5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cem\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eXu142fl\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e-3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e78.04\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.45pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e72.19\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.8pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e98.6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 63.75pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e92.51\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49.65pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e95.87\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 56.7pt;border-color: currentcolor currentcolor windowtext;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0in 5.4pt;height: 13.5pt;vertical-align: top;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e75.52\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:200%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size: 16px; line-height: 200%; font-family: \"Times New Roman\", serif; color: rgb(0, 0, 0);'\u003eNote: M means megabase.\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:200%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComplicated upstream transcription and downstream biosynthesis and metabolism events occurred during early lint fiber initiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the biological processes and functions attributed to the DEGs, \u0026nbsp; GO enrichment analysis of the downregulated and upregulated DEGs were conducted respectively. The results showed that the downregulated genes were enriched in 15 level 3 biological process (BP) terms including transcription (262 genes), RNA (288 genes) and nucleic acid metabolism (339 genes), nucleobase-containing compound biosynthesis (267 genes) and metabolism (358 genes), heterocycle biosynthesis (275 genes) and metabolism (377 genes), aromatic compound biosynthesis (273 genes) and metabolism (383 genes) and organic cyclic compound biosynthesis (276 genes) and metabolism (380 genes), photosynthesis (42 genes) and light harvesting (15 genes), respond to chitin (7 genes), and plant cell wall organization (20 genes) (Fig. 1a; Additional file 2 Table S1), with molecular functions (MF) including DNA binding transcription factor activity (205 genes), transcription regulator activity (208 genes) and dioxygenase activity (32 genes), and DNA (404 genes), nucleic acid (585 genes), organic cyclic compound and heterocyclic compound binding activities (1008 genes) (Fig. 1b; Additional file 3 Table S2). These results demonstrated that the essential transcriptional regulations in \u003cem\u003eXu142fl\u003c/em\u003e were impaired and led to the aborted lint fiber initiation.\u003c/p\u003e\n\u003cp\u003eCompared with the significantly enriched GO terms for the downregulated DEGs, the enriched GO terms for the upregulated DEGs were fewer on the BP level, and no GO terms were found on the MF level. The significantly enriched level 3 terms was DNA replication (21 genes), DNA-dependent DNA replication (11 genes), DNA replication initiation (8 genes) and phospholipid biosynthesis (19 genes) (Fig. 1c; Additional file 4 Table S3). Overall, these findings suggest that DNA replication and phospholipid biosynthesis in the process of cell mitosis division, were inhibited in the fiber initials of wild type during lint fiber initiation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscription factor expression dynamics during early fiber initiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, to characterize the complicated transcription regulations during lint fiber initiation, the TF coding genes were firstly predicted and then filtered to obtain the differentially expressed TF genes closely related to lint fiber initiation (only TF family containing more than two genes were considered), and finally 490 genes belonging to 26 TF families were identified, consisting of 424 downregulated genes and 66 upregulated genes. Moreover, most TF families consisted more downregulated genes (57.14%-100%), except for LOB domain-containing\u0026nbsp;and MADS domain-containing\u0026nbsp;families which contained more up-regulated genes (76.92% and 71.43% respectively) (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs shown in Table 2, among the down-regulated TF families containing members more than 20, the biggest TF family was AP2-EREBP, which contained 123 DEGs, followed by MYB (54 DEGs), WRKY (41 DEGs), NAC(33 DEGs), C2C2-Dof (33 DEGs), bHLH (29 DEGs) and GRAS (23 DEGs). Subsequently, we chose six TF families including MYB, bHLH, NAC, C2C2 Dof, GRAS, and WRKY containing members ranging from 23-54 to plot expression heat maps. The results showed that 6 \u003cem\u003eMalvaceae\u003c/em\u003e-specific \u003cem\u003eMML homologs\u0026nbsp;\u003c/em\u003e(Wu et al., 2018) including two \u003cem\u003eMML3\u003c/em\u003e, one \u003cem\u003eMML4\u003c/em\u003e, two \u003cem\u003eMML8\u003c/em\u003e and one \u003cem\u003eMML9\u003c/em\u003e were all downregulated. The \u003cem\u003eMML3\u0026nbsp;\u003c/em\u003eon chromosome D12 chromosome (Gh_D12G1628) had higher expression level in the wild type and were much more downregulated in the mutant than the one on A12 chromosome (Gh_A12G1503) (Fig. 2a). Although most members were downregulated in the identified TF families, subclasses of members were still upregulated for 5 TF families, including the MYB family which contains one \u003cem\u003eRL6\u003c/em\u003e, 3 \u003cem\u003eMYB44\u003c/em\u003e and 4 \u003cem\u003eGAM1\u0026nbsp;\u003c/em\u003e(Fig. 3a), bHLH family which contains 5 members including \u003cem\u003eBHLH82\u003c/em\u003e, \u003cem\u003eBHLH130\u003c/em\u003e, \u003cem\u003eBEE3\u003c/em\u003e and two novel genes (Fig. 2b), NAC family which contains 6 \u003cem\u003eNAC\u003c/em\u003e genes, three of which annotated as \u003cem\u003eNAC100\u003c/em\u003e had dominant expression in both varieties (Fig. 2c), the GRAS family which contains 2 members (Fig. 2d), and WRKY family which contains 7 members, with the genes annotated \u003cem\u003eWRKY48\u003c/em\u003e and \u003cem\u003eWRKY65\u003c/em\u003e had higher expression levels while expression of the other five genes were lower in both varieties (Fig. 2e), except for the C2C2 Dof\u003cem\u003e\u0026nbsp;\u003c/em\u003efamily genes which were all down-regulated (Fig. 2f).\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:200%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eTable 2 Statistics of the differentially expressed transcription factor genes.\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"border: none;width:99.96%;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26%;border-color: black currentcolor windowtext;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eTF Family\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.98%;border-color: black currentcolor windowtext;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eT\u003c/span\u003e\u003cspan style='font-family:\"Times New Roman\",serif;color:black;text-decoration:none;'\u003e\u003cspan style=\"font-size:16px;line-height:107%;\"\u003eotal DEGs\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.26%;border-color: black currentcolor windowtext;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eUpreg\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.96%;border-color: black currentcolor windowtext;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp 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style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e42.86\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.96%;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e57.14\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26%;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eWRKY\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.98%;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e41\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.26%;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.96%;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e34\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.82%;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e17.07\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.96%;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e82.93\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26%;padding: 0.75pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003ezf-HD\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.98%;padding: 0.75pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 12.26%;padding: 0.75pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.96%;padding: 0.75pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.82%;padding: 0.75pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e20\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.96%;padding: 0.75pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e80\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26%;border-color: currentcolor currentcolor black;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0.75pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eTotal\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:11.98%;border:none;border-bottom:solid black 1.5pt;padding:.75pt .75pt .75pt .75pt;height:12.75pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e490\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:12.26%;border:none;border-bottom:solid black 1.5pt;padding:.75pt .75pt .75pt .75pt;height:12.75pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e66\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:17.96%;border:none;border-bottom:solid black 1.5pt;padding:.75pt .75pt .75pt .75pt;height:12.75pt;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e424\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.82%;border-color: currentcolor currentcolor black;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0.75pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.96%;border-color: currentcolor currentcolor black;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0.75pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:200%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eNote: Upreg means the upregulated genes; Downreg means the downregulated genes.\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:200%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMain \u003cem\u003eGhMMLs\u0026nbsp;\u003c/em\u003econtributed to\u003cem\u003e\u0026nbsp;\u003c/em\u003elint fiber initiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 9th subfamily R2R3-MYB transcription factors MMLs is\u0026nbsp;considered as \u003cem\u003eMalvaceae\u003c/em\u003e-specific through evolutionary analysis\u0026nbsp;[\u003ca href=\"#_ENREF_12\" title=\"Wu, 2018 #70\"\u003e12\u003c/a\u003e]\u0026nbsp;and among the 10 GhMMLs\u0026nbsp;from GhMML1 to\u0026nbsp;GhMML10, GhMML3 and GhMML4 had been demonstrated responsible for fuzz and lint fiber initiation respectively\u0026nbsp;[\u003ca href=\"#_ENREF_11\" title=\"Wan, 2016 #60\"\u003e11\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_12\" title=\"Wu, 2018 #70\"\u003e12\u003c/a\u003e]\u0026nbsp;. Our TF classification had revealed that \u003cem\u003eGhMML3\u003c/em\u003e,\u0026nbsp;\u003cem\u003eGhMML4\u003c/em\u003e,\u0026nbsp;\u003cem\u003eGhMML8\u003c/em\u003e, and\u0026nbsp;\u003cem\u003eGhMML9\u003c/em\u003e were involved in lint fiber initiation, to confirm that, we conducted RT-PCR of all ten \u003cem\u003eGhMMLs\u0026nbsp;\u003c/em\u003ein \u003cem\u003en2NSM\u003c/em\u003e and \u003cem\u003eXu142fl\u0026nbsp;\u003c/em\u003eduring early lint fiber initiation period from -1 DPA to 1 DPA, given the fact that \u003cem\u003en2NSM\u003c/em\u003e and \u003cem\u003eXu142fl\u0026nbsp;\u003c/em\u003eare all naked seed mutants and the only difference is whether the lint fiber initiates or not\u0026nbsp;[\u003ca href=\"#_ENREF_12\" title=\"Wu, 2018 #70\"\u003e12\u003c/a\u003e].\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eThe results showed that 8 \u003cem\u003eMMLs\u0026nbsp;\u003c/em\u003ecan be detected except \u003cem\u003eGh\u003c/em\u003e\u003cem\u003eMML8\u003c/em\u003e and \u003cem\u003eGh\u003c/em\u003e\u003cem\u003eMML9\u0026nbsp;\u003c/em\u003e(Fig. 3), and 4 were down-regulated including \u003cem\u003eGhMML1\u003c/em\u003e, \u003cem\u003eGh\u003c/em\u003e\u003cem\u003eMML3\u003c/em\u003e, \u003cem\u003eGh\u003c/em\u003e\u003cem\u003eMML4\u003c/em\u003e and \u003cem\u003eGh\u003c/em\u003e\u003cem\u003eMML7\u0026nbsp;\u003c/em\u003ein \u003cem\u003eXu142fl\u0026nbsp;\u003c/em\u003ecompared with \u003cem\u003en2NSM\u003c/em\u003e,. Further investigation of their expression patterns by qRT-PCR confirmed that \u003cem\u003eGh\u003c/em\u003e\u003cem\u003eMML3\u003c/em\u003e, \u003cem\u003eGh\u003c/em\u003e\u003cem\u003eMML4\u003c/em\u003e and \u003cem\u003eGh\u003c/em\u003e\u003cem\u003eMML7\u003c/em\u003e were significantly down-regulated in \u003cem\u003eXu142fl\u0026nbsp;\u003c/em\u003ecompared with \u003cem\u003en2NSM\u003c/em\u003e at all three time points while no obvious expression differences were observed for \u003cem\u003eGhMML1\u0026nbsp;\u003c/em\u003e(Fig. 4). We also found that expression levels of \u003cem\u003eGh\u003c/em\u003e\u003cem\u003eMML3\u0026nbsp;\u003c/em\u003eand \u003cem\u003eGh\u003c/em\u003e\u003cem\u003eMML4\u003c/em\u003e in \u003cem\u003en2NSM\u003c/em\u003e were decreased, while that of \u003cem\u003eGh\u003c/em\u003e\u003cem\u003eMML7\u0026nbsp;\u003c/em\u003ewere increased from -1 DPA to 1 DPA, implying different mechanisms between GhMML7, and GhMML3 and GhMML4\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Fig. 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpansins enriched in cell wall organization may contributed to early lint fiber initiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell wall reorganization is an essential event during fiber development involving multiple enzymes and wall proteins\u0026nbsp;[\u003ca href=\"#_ENREF_5\" title=\"Ruan, 2007 #3172\"\u003e5\u003c/a\u003e]. Here, we had a detailed investigation of the GO term-plant cell wall organization which contains 20 genes, and the results showed that 17 were EXPA encoding genes, including 4 \u003cem\u003eEXPA1\u003c/em\u003e, 1 \u003cem\u003eEXPA2\u003c/em\u003e, 6 \u003cem\u003eEXPA4\u003c/em\u003e, 3 \u003cem\u003eEXPA8\u0026nbsp;\u003c/em\u003eand 3 \u003cem\u003eEXPA15\u003c/em\u003e, one was Pectinesterase (PE) encoding gene, and 2 were COBL10 (protein transport protein SEC61 subunit alpha) encoding genes, and they were all downregulated in \u003cem\u003eXu142fl\u003c/em\u003e comparing with that in the wild type (Table 3). It was worth noting that the \u003cem\u003eEXPA2\u0026nbsp;\u003c/em\u003egene, one EXPA4 encoding gene (Gh_A10G2323) and the two COBL10 encoding genes had very low expression levels in the wild type and hardly detectable in the mutant, implying that they might not the dominant genes for early lint fiber initiation. Of special note, data indicated that EXPAs might be the most important cell wall proteins for the early fiber cell initiation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:200%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cstrong\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eTable 3. Expression differences of the cell wall organization related genes.\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\n \u003ctable style=\"border: none;width:100.0%;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21.1%;border-color: black currentcolor windowtext;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eGene ID\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.82%;border-color: black currentcolor windowtext;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eGene Annotation\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.1%;border-color: black currentcolor windowtext;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eXu142_RPKM\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.46%;border-color: black currentcolor windowtext;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cem\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eXu142fl\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e_RPKM\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.5%;border-color: black currentcolor windowtext;border-style: solid none;border-width: 1.5pt medium 1pt;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003elog2FoldChange\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e(\u003cem\u003eXu142fl\u003c/em\u003e/Xu142)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21.1%;border: medium none;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp 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style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003ePectinesterase\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.1%;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e26.62\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.46%;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e10.09\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.5%;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e-1.40\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21.1%;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eGh_D06G1606\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.82%;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eCOBL10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.1%;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0.27\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.46%;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0.02\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.5%;padding: 0.75pt;height: 14.25pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e-3.70\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 21.1%;border-color: currentcolor currentcolor black;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0.75pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eGh_A06G1281\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.82%;border-color: currentcolor currentcolor black;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0.75pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003eCOBL10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.1%;border-color: currentcolor currentcolor black;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0.75pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0.78\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.46%;border-color: currentcolor currentcolor black;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0.75pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e0.09\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.5%;border-color: currentcolor currentcolor black;border-style: none none solid;border-width: medium medium 1.5pt;border-image: none 100% / 1 / 0 stretch;padding: 0.75pt;height: 12.75pt;vertical-align: bottom;\"\u003e\n \u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:center;line-height:107%;font-size:14px;font-family:\"Calibri\",sans-serif;vertical-align:bottom;'\u003e\u003cspan style='font-size:16px;line-height:107%;font-family:\"Times New Roman\",serif;'\u003e-3.11\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:8.0pt;margin-left:0in;text-align:justify;line-height:200%;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eNote:\u0026nbsp;\u003c/span\u003e\u003cem\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eCOBL10\u0026nbsp;\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eencodes a\u0026nbsp;\u003c/span\u003e\u003cspan style='font-size:16px;line-height:200%;font-family:\"Times New Roman\",serif;'\u003eprotein transport protein SEC61 subunit alpha\u003c/span\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eLint fiber initiation is a complicated\u0026nbsp;\u003c/strong\u003e\u003ca href=\"javascript%3A;\"\u003e\u003cstrong\u003emorphogenesis\u003c/strong\u003e\u003c/a\u003e\u003cstrong\u003e\u0026nbsp;process involving complex metabolite biosynthesis and metablism\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, through GO analysis of the downregulated and upregulated DEGs, multiple enriched biological processes were identified which were associated lint fiber initiation (Fig. 1). Based on our findings, we speculated that the downregulated DEGs mainly function as positive regulatory factors for lint fiber initiation, while the upregulated DEGs mainly function to inhibit lint fiber initiation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the downregulated DEGs, more than 200 genes were associated DNA-tempated transcription (Fig. 1a), which have transcription associated molecular functions as DNA binding transcription factor activity (Fig. 1b). These biological processes related genes may construct the upstream regulation networks during lint fiber initiation, and the downstream regulation involves complicated metabolic cascades associated with organic compound biosynthesis and metabolism including nucleobase-containing compound, organic cyclic compound, heterocyclic compound and aromatic compound such as flavonoid, sugar and Phytohormones biosynthesis and metabolism (Fig. 1a,b; ). Several \u003cem\u003eNCEDs\u003c/em\u003e (data not shown) which encode the rate-limiting dioxygenases controlling ABA biosynthesis\u0026nbsp;[\u003ca href=\"#_ENREF_26\" title=\"Matilla, 2015 #3760\"\u003e26\u003c/a\u003e], were found in the GO term of dioxygenase (Fig. 1b), implying that ABA might be a positive regulator during lint fiber initiation. This can be supported by the gradually accumulation of endogenous ABA content during the fiber cell initiation and elongation stages\u0026nbsp;[\u003ca href=\"#_ENREF_27\" title=\"Davis, 1972 #3761\"\u003e27\u003c/a\u003e]\u0026nbsp;although ABA was considered as a negative regulator of fiber initiation\u0026nbsp;[\u003ca href=\"#_ENREF_28\" title=\"Xiao, 2019 #3211\"\u003e28\u003c/a\u003e]. Chitin oligosaccharides can induce various defense responses in a wide range of plant cells including both monocots and dicots\u0026nbsp;[\u003ca href=\"#_ENREF_29\" title=\"Shibuya, 2001 #3762\"\u003e29\u003c/a\u003e]. However, do defense responses related to fiber initiation is still not clear. Immature fiber (im) mutant with thinner fiber cell wall compared to the isogenic wild type (TM-1) with fiber of normal thickness revealed that the mutant had lower net photosynthesis, because of the lower chlorophyll content per unit leaf area due to less chlorophyll a levels than the wild type\u0026nbsp;[\u003ca href=\"#_ENREF_30\" title=\"Gordon, 2016 #3763\"\u003e30\u003c/a\u003e], however how the photosynthesis related genes were changed in \u003cem\u003eXu142fl\u003c/em\u003e need further investigation (Fig. 1a).\u003c/p\u003e\n\u003cp\u003eThe upregulated DEGs were mainly enriched in DNA replication and phospholipid biosynthetic processes, which were closely related to cell division. This is reasonable because fiber cells are unable to undergo cell division during fiber development. Before fiber cell differentiation, the ovular epidermal cells are closely packed, cuboidal, and rich in cytoplasm containing a large nucleus, which represents a status of rapid cell division\u0026nbsp;[\u003ca href=\"#_ENREF_31\" title=\"Joshi, 1967 #3764\"\u003e31\u003c/a\u003e]. The early development of fibers consists of two intergrading steps-spherical expansion above the ovular epidermis and elongation\u0026nbsp;[\u003ca href=\"#_ENREF_32\" title=\"Stewart, 1975 #3765\"\u003e32\u003c/a\u003e]. The morphological differentiation of a fiber occurs when an epidermal cell balloons above the epidermal surface, followed by transition to elongation phase and stopping division\u0026nbsp;[\u003ca href=\"#_ENREF_33\" title=\"Basra, 1984 #3766\"\u003e33\u003c/a\u003e]. Collectively, cell division should be stopped to initiate fiber development in the wild type while rapid cell division continues which inhibit cell differentiation into fiber cells in \u003cem\u003eXu142fl\u0026nbsp;\u003c/em\u003emutant.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComplicated transcription regulation during lint fiber initiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, through TF annotation and expression pattern profiling, many down-regulated TF families were identified, including known fiber development related MYBs , bHLHs \u0026nbsp;and TCPs\u0026nbsp;[\u003ca href=\"#_ENREF_6\" title=\"Wang, 2020 #3095\"\u003e6\u003c/a\u003e]. Other TF families such as NAC, WRKY, GRAS and Dof identified here were also detected in another study exploring fiber elongation related pathways by transcriptome analysis of a short fiber mutant and a wild type\u0026nbsp;[\u003ca href=\"#_ENREF_34\" title=\"Kang, 2012 #3771\"\u003e34\u003c/a\u003e], suggesting that some TF family genes might have dual role both in fiber initiation and elongation. For example, fiber cell expansion and elongation can be mediated by a homeodomain leucine zipper gene, \u003cem\u003eGhHD-1\u003c/em\u003e, through a WRKY transcription factor by regulating the levels of ethylene and reactive oxidation species (ROS)\u0026nbsp;[\u003ca href=\"#_ENREF_35\" title=\"Walford, 2012 #2169\"\u003e35\u003c/a\u003e]. Because some lint fiber initiation related TFs which were genetically characterized as positive regulators for fiber development were firstly found downregulated in fiber related mutants, including R2R3 MYB proteins\u0026nbsp;[\u003ca href=\"#_ENREF_8\" title=\"Machado, 2009 #34\"\u003e8\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_9\" title=\"Walford, 2011 #58\"\u003e9\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_36\" title=\"Huang, 2013 #18\"\u003e36\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_37\" title=\"Pu, 2008 #104\"\u003e37\u003c/a\u003e], HD-ZIP proteins\u0026nbsp;[\u003ca href=\"#_ENREF_15\" title=\"Guan, 2008 #94\"\u003e15\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_16\" title=\"Shan, 2014 #48\"\u003e16\u003c/a\u003e]\u0026nbsp;and bHLH proteins\u0026nbsp;[\u003ca href=\"#_ENREF_14\" title=\"Shangguan, 2016 #50\"\u003e14\u003c/a\u003e], so the other downregulated TF family genes found in this study deserve further investigation of their roles in regulating lint fiber initiation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBesides this, two class of TF families involved more upregulated DEGs than the downregulate DEGs (Table 2), including LOB (Lateral Organ Boundaries) family which are essential in the regulation of plant lateral organ development\u0026nbsp;[\u003ca href=\"#_ENREF_38\" title=\"Majer, 2011 #3772\"\u003e38\u003c/a\u003e]\u0026nbsp;and MADS which regulate floral organ differentiation and development\u0026nbsp;[\u003ca href=\"#_ENREF_39\" title=\"Theissen, 2001 #1795\"\u003e39\u003c/a\u003e], and small group of genes in other TF families were also up-regulated (Fig. 2, Table 2), which might be negative regulators of fiber initiation and contribute to the lintless ovules of \u003cem\u003eXu142fl\u0026nbsp;\u003c/em\u003edue to\u0026nbsp;their upregulation.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDynamic expression of \u003cem\u003eMalvaceae\u003c/em\u003e-specific \u003cem\u003eMMLs\u003c/em\u003e during early lint fiber initiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the 10 pair of \u003cem\u003eMalvaceae\u003c/em\u003e-specific \u003cem\u003eMYB MIXTA-like\u0026nbsp;\u003c/em\u003e(\u003cem\u003eGhMML\u003c/em\u003e) homoeologs at least one copy on one sub-genome in allotetraploid cotton were predominantly expressed during fiber initiation in the wild type TM-1\u0026nbsp;[\u003ca href=\"#_ENREF_10\" title=\"Zhang, 2015 #88\"\u003e10\u003c/a\u003e], and \u003cem\u003eMML3-A12\u0026nbsp;\u003c/em\u003eand \u003cem\u003eMML4-D12\u0026nbsp;\u003c/em\u003ewere demonstrated contributing to the fuzz and lint fiber initiation respectively\u0026nbsp;[\u003ca href=\"#_ENREF_11\" title=\"Wan, 2016 #60\"\u003e11\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_12\" title=\"Wu, 2018 #70\"\u003e12\u003c/a\u003e], implying the subfunctionalization of \u003cem\u003eMML\u0026nbsp;\u003c/em\u003egenes. Combined our transcriptome analysis and RT-PCR analysis, \u003cem\u003eMML3\u003c/em\u003e, \u003cem\u003eMML4\u003c/em\u003e and \u003cem\u003eMML7 should be the MMLs most related to lint fiber initiation\u003c/em\u003e (Fig. 2a, Fig. 3, 4)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eHowever, it was contrary in that\u003cem\u003e\u0026nbsp;GhMML3-D12\u0026nbsp;\u003c/em\u003e(Gh_D12G1628) and\u0026nbsp;\u003cem\u003eGhMML4-A12\u0026nbsp;\u003c/em\u003e(Gh_A12G1504) might be more important for lint fiber initiation, because \u003cem\u003eGhMML3-D12\u0026nbsp;\u003c/em\u003ehad higher expression level in the wild type and was more downregulated in the mutant than \u003cem\u003eGhMML3-A12,\u0026nbsp;\u003c/em\u003ewhile\u0026nbsp;\u003cem\u003eGhMML4-D12\u0026nbsp;\u003c/em\u003ewas\u0026nbsp;not detected as DEG in our study\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Fig. 2a).\u003c/p\u003e\n\u003cp\u003eDifferent from\u003cem\u003e\u0026nbsp;GhMML3\u003c/em\u003e and \u003cem\u003eGhMML4\u0026nbsp;\u003c/em\u003ewhich were downregulated in the fuzzless-linted mutant\u0026nbsp;\u003cem\u003en2NSM\u0026nbsp;\u003c/em\u003efrom -1 DPA to 1 DPA,\u003cem\u003e\u0026nbsp;GhMML7\u0026nbsp;\u003c/em\u003ewas upregulated and had highest expression level in ovules at 1 DPA (Fig. 4). This phenomenon indicated that, except for the role in fiber initiation,\u0026nbsp;\u003cem\u003eGhMML7\u0026nbsp;\u003c/em\u003emay also involve in fiber elongation, which can be assisted by the evidences that\u0026nbsp;\u003cem\u003eGhMML7/GhMYB25\u003c/em\u003e expressed in the epidermis of ovules, developing fiber initials and fibers, and \u003cem\u003eGhMML7/GhMYB25\u003c/em\u003e-silenced cotton produced shorter fiber, while overexpression of \u003cem\u003eGhMYB25\u003c/em\u003e promoted fiber initiation\u0026nbsp;[\u003ca href=\"#_ENREF_8\" title=\"Machado, 2009 #34\"\u003e8\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell wall organization initiates from the very beginning during lint fiber initiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePreviously, Stage-specific developmental markers such as EXPAs, xyloglucan endo-transglycosylases (XETs) and PEs have been reported to regulate fiber cell expansion in cotton\u0026nbsp;[\u003ca href=\"#_ENREF_4\" title=\"Lee, 2007 #3171\"\u003e4\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_5\" title=\"Ruan, 2007 #3172\"\u003e5\u003c/a\u003e,\u0026nbsp;\u003ca href=\"#_ENREF_40\" title=\"Al-Ghazi, 2009 #3170\"\u003e40\u003c/a\u003e]. Here, we also identified many EXPA encoding genes and a PE encoding gene, indicated that EXPAs are more important during fiber initiation and elongation. Taken together, upstream regulators of EXPAs will further facilitate our understanding underlying mechanisms of lint fiber initiation and elongation.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we compared the whole genome transcription changes between Xu142 and \u003cem\u003eXu142fl\u0026nbsp;\u003c/em\u003eat early fiber initiation stages and\u0026nbsp;identified 3676 downregulated and 1840 upregulated genes. Gene Ontology (GO) enrichment analysis revealed that the downregulated genes were mainly involved in the biological processes related to\u0026nbsp;transcription, organic cyclic compound biosynthesis and metabolism, photosynthesis, response to chitin and plant cell wall organization, and had molecular functions such as transcription related binding, organic cyclic compound binding and dioxygenase activity, while the upregulated DEGs were enriched in DNA replication and phospholipid biosynthesis related processes. Classification of the transcription factor genes revealed that various transcription factor families involved in lint fiber initiation, including the members belonging to the MBW complex. Examination of the cell wall organization related genes revealed 17 EXPA\u003cem\u003e\u0026nbsp;\u003c/em\u003ecoding genes, one\u0026nbsp;pectinesterase (PE) coding gene, and two COBL10 coding genes, which may contribute to cell loosening and reorganization during lint fiber initiation. Overall, these findings provide preliminary data which suggest importance of introducing the essential transcription factor genes or\u0026nbsp;\u003cem\u003eEXPAs\u003c/em\u003e for improving lint fiber initiation rate.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study provides new information for lint fiber initiation mechanism, which might involve dynamic expression of multiple types of TF family genes, which mediate the complicated downstream organic compound biosynthesis and metabolism resulting into the termination of cell division and cell wall reorganization of the expanding fiber cells during lint fiber initiation (Fig. 5).\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOne cotton variety \u003cem\u003eGossypium hirsutum\u003c/em\u003e cv. Xu142 and two \u0026nbsp;fibreless cotton mutants \u003cem\u003eXu142fl\u0026nbsp;\u003c/em\u003eand \u003cem\u003en2NSM\u003c/em\u003e were selected in this study. The seeds of these lines were retrieved from National Medium-term Gene Bank of Cotton in China and National cotton germplasm resources platform. The seeds were grown in the experimental field of Linyi University in April 14, 2018. Ovules at -3, -1, 0 and 1 DPA were collected between July and August during the flowering peak, frozen immediately in liquid nitrogen and stored at -80℃.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptome sequencing and bioinformatics analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh-quality RNA extraction was performed from the frozen ovules tissues collected at -3 DPA and -1 DPA of Xu142 and \u003cem\u003eXu142fl\u0026nbsp;\u003c/em\u003erespectively as previously reported\u0026nbsp;[\u003ca href=\"#_ENREF_41\" title=\"Wang, 2018 #3110\"\u003e41\u003c/a\u003e]. Subsequent cDNA libraries were constructed and sequenced with BGI-SEQ500 at the Beijing Genomics Institute (BGI, Shenzhen, China).\u003c/p\u003e\n\u003cp\u003eThe raw reads were filtered first to get clean data, and then aligned to the genome of \u003cem\u003eG. hirsutum\u003c/em\u003e L. (https://cottonfgd.org/about/download/assembly/genome.Ghir.NAU.fa.gz) to reconstruct transcripts through String Tie (\u003ca href=\"http://ccb.jhu.edu/software/stringtie\"\u003ehttp://ccb.jhu.edu/software/stringtie\u003c/a\u003e,\u0026nbsp;v1.0.4), identify known genes by Bowtie2 (http://bowtie-bio.sourceforge.net/Bowtie2, v2.2.5)\u0026nbsp;[\u003ca href=\"#_ENREF_42\" title=\"Langmead, 2012 #3184\"\u003e42\u003c/a\u003e], and predict new transcripts using HISAT2 (http://www.ccb.jhu.edu/software/hisat, v2.0.4)\u0026nbsp;[\u003ca href=\"#_ENREF_43\" title=\"Kim, 2015 #1815\"\u003e43\u003c/a\u003e].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExpression levels of genes and transcripts were calculated using RSEM\u0026nbsp;[\u003ca href=\"#_ENREF_44\" title=\"Li, 2011 #1820\"\u003e44\u003c/a\u003e], and expression corrections were calculated by Cor package of R (v3.6.2). DEGs (filtered by fold change \u0026gt;= 2, Q-value \u0026lt;= 0.001) between Xu142 and \u003cem\u003eXu142fl\u0026nbsp;\u003c/em\u003ewere identified through DEGseq\u0026nbsp;[\u003ca href=\"#_ENREF_45\" title=\"Wang, 2010 #2206\"\u003e45\u003c/a\u003e], after normalizing raw reads of each gene as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). GO enriched terms were determined by Q-value \u0026lt;= 0.001 using the phyper package of R.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe transcription factor (TF) coding genes were predicted by using Getorf (http://emboss.sourceforge.net/apps/cvs/emboss/apps/getorf.html) to get the ORF of all unigenes first, and then aligned to TF protein structure domain using hmmsearch (http://hmmer.org, v3.0) to annotate TF through the property of specific TF family described in PlantTFDB (http://planttfdb.cbi.pku.edu.cn, v5.0). Heatmaps of TF proteins were generated by Genesis v1.7.6\u0026nbsp;[\u003ca href=\"#_ENREF_46\" title=\"Sturn, 2002 #3185\"\u003e46\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT-PCR and RT-qPCR analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRT-PCR and qRT-PCR were used to evaluate expression levels of \u003cem\u003eGhMMLs\u003c/em\u003e. Total RNA from ovules at -1, 0 and 1 DPA of \u003cem\u003en2NSM\u003c/em\u003e and \u003cem\u003eXu142fl\u0026nbsp;\u003c/em\u003ewas extracted as previously reported\u0026nbsp;[\u003ca href=\"#_ENREF_41\" title=\"Wang, 2018 #3110\"\u003e41\u003c/a\u003e]. Subsequent cDNA was synthesized using a Thermos Scientific RevertAid First Strand cDNA Synthesis Kit according to the manufacture\u0026rsquo;s instruction. Total 20-\u0026micro;l-reaction volume was applied for RT-PCR analysis to evaluate expression levels of \u003cem\u003eGhMMLs\u003c/em\u003e. After this, PCR reaction mixture was subjected to 95\u0026deg;C\u0026nbsp;denaturation for 3 min, then 29 cycles of amplification of the endogenous reference gene\u0026nbsp;\u003cem\u003eGhUbq7\u003c/em\u003e or 38 cycles for \u003cem\u003eGhMMLs\u003c/em\u003e at 95\u0026deg;C for 30 sec, 55\u0026deg;C annealing for 30 sec and 72\u0026deg;C extension for 30 sec, plus a final extension at 72\u0026deg;C for 5 min. Quantitative real-time PCR (qRT-PCR) was carried out using Hieff qPCR SYBR Green Master Mix (No Rox) (Yeason, Shanghai, China). The calculation of the relative expression levels of each gene and statistical analysis were determined as previously reported\u0026nbsp;[\u003ca href=\"#_ENREF_47\" title=\"Yang, 2013 #478\"\u003e47\u003c/a\u003e]. Primers were designed by Primer 5.0 and synthesized commercially (Genscript Bioscience, Nanjing, China). The sequences of all the primers sequences are listed in Additional file 5 Table 4.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Kashif Rafiq for critiquing the manuscript. We thank\u0026nbsp;National Medium-term Gene Bank of Cotton in China and National cotton germplasm resources platform for providing the cotton seeds used in this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL. W.: project design, transcriptome data mining, writing the manuscript. Y. L., X. L. and Z. F.: collecting the ovules and extracting the total RNA. W. L: experiments shown in Figure 3 and Figure 4. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are sponsored by State Key Laboratory of Cotton Biology Open Fund (Grant No. CB2018A20), and Linyi University PhD Fund for Scientific Start-up (LYDX2018BS029).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw data for the transcriptomes are available on the GEO platform as series GSE176384.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo competing interests declared.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHu Q, Xiao S, Guan Q, Tu L, Sheng F, Du X, Zhang X: \u003cb\u003eThe laccase gene GhLac1 modulates fiber initiation and elongation by coordinating jasmonic acid and flavonoid metabolism\u003c/b\u003e. \u003cem\u003eThe Crop Journal\u003c/em\u003e 2020, \u003cb\u003e8\u003c/b\u003e(4):522\u0026ndash;533.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin Y-M, Zhu Y-X: \u003cb\u003eHow cotton fibers elongate: a tale of linear cell-growth mode\u003c/b\u003e. \u003cem\u003eCurrent opinion in plant biology\u003c/em\u003e 2011, \u003cb\u003e14\u003c/b\u003e(1):106\u0026ndash;111.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGraves D, Stewart J: \u003cb\u003eChronology of the differentiation of cotton (Gossypium hirsutum L.) fiber cells\u003c/b\u003e. \u003cem\u003ePlanta\u003c/em\u003e 1988, \u003cb\u003e175\u003c/b\u003e(2):254\u0026ndash;258.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee JJ, Woodward AW, Chen ZJ: \u003cb\u003eGene expression changes and early events in cotton fibre development\u003c/b\u003e. \u003cem\u003eAnnals of botany\u003c/em\u003e 2007, \u003cb\u003e100\u003c/b\u003e(7):1391\u0026ndash;1401.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuan YL: \u003cb\u003eRapid cell expansion and cellulose synthesis regulated by plasmodesmata and sugar: insights from the single-celled cotton fibre\u003c/b\u003e. \u003cem\u003eFunctional plant biology: FPB\u003c/em\u003e 2007, \u003cb\u003e34\u003c/b\u003e(1):1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Wang G, Long L, Altunok S, Feng Z, Wang D, Khawar KM, Mujtaba M: \u003cb\u003eUnderstanding the role of phytohormones in cotton fiber development through omic approaches; recent advances and future directions\u003c/b\u003e. \u003cem\u003eInternational journal of biological macromolecules\u003c/em\u003e 2020, \u003cb\u003e163\u003c/b\u003e:1301\u0026ndash;1313.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzymanski DB, Lloyd AM, Marks MD: \u003cb\u003eProgress in the molecular genetic analysis of trichome initiation and morphogenesis in Arabidopsis\u003c/b\u003e. \u003cem\u003eTrends in plant science\u003c/em\u003e 2000, \u003cb\u003e5\u003c/b\u003e(5):214\u0026ndash;219.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMachado A, Wu Y, Yang Y, Llewellyn DJ, Dennis ES: \u003cb\u003eThe MYB transcription factor GhMYB25 regulates early fibre and trichome development\u003c/b\u003e. \u003cem\u003eThe Plant journal: for cell and molecular biology\u003c/em\u003e 2009, \u003cb\u003e59\u003c/b\u003e(1):52\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalford SA, Wu Y, Llewellyn DJ, Dennis ES: \u003cb\u003eGhMYB25-like: a key factor in early cotton fibre development\u003c/b\u003e. \u003cem\u003eThe Plant journal: for cell and molecular biology\u003c/em\u003e 2011, \u003cb\u003e65\u003c/b\u003e(5):785\u0026ndash;797.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang T, Hu Y, Jiang W, Fang L, Guan X, Chen J, Zhang J, Saski CA, Scheffler BE, Stelly DM \u003cem\u003eet al\u003c/em\u003e: \u003cb\u003eSequencing of allotetraploid cotton (Gossypium hirsutum L. acc. 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Although various omics studies have revealed molecular basis for fiber development, a better understanding of transcriptional regulation mechanism regulating lint fiber initiation is necessary to meet global natural fiber demand.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eHere, we aimed to perform transcriptome sequencing to identify DEGs (differentially expressed genes) in ovules of the cotton variety Xu142 and its fibreless mutant \u003cem\u003eXu142fl\u003c/em\u003e during early lint fiber initiation period. Totally, 5516 DEGs including 1840 upregulated and 3676 downregulated were identified. GO enrichment analysis revealed that the downregulated DEGs mainly associated with biological processes such as transcription related biosynthesis and metabolism, organic cyclic compound biosynthesis and metabolism, photosynthesis, and plant cell wall organization, with molecular functions involving transcription related binding, organic cyclic compound binding, and dioxygenase activity, while the upregulated DEGs were associated with DNA replication and phospholipid biosynthetic related processes. Among the 490 DEGs annotated as transcription factor genes 86.5% were downregulated in the mutant including the \u003cem\u003eMalvaceae\u003c/em\u003e-specific \u003cem\u003eMMLs\u003c/em\u003e, expression patterns of which were confirmed during the central period of lint fiber initiation. Investigation of the 20 genes enriched in the cell wall organization revealed that 17 were EXPA coding genes. Overall, we suggest that lint fiber initiation is a complicated process involving cooperation of multiple transcription factor families, which might ultimately lead to the reorganization of the cell wall and terminated cell division of the differentiating fiber initials.\u003c/p\u003e","manuscriptTitle":"Comparative transcriptome analysis uncovers cell wall reorganization and repressed cell division during cotton fiber initiation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-06-17 20:25:12","doi":"10.21203/rs.3.rs-607693/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-08-05T09:02:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-07-23T16:53:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"91460fc3-f475-43ed-b213-5341ddc8cba5","date":"2021-07-02T11:15:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-06-30T02:29:20+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-06-25T14:22:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-06-25T05:17:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-06-25T05:09:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Developmental Biology","date":"2021-06-10T02:18:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-developmental-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"debo","sideBox":"Learn more about [BMC Developmental Biology](http://bmcdevbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/debo","title":"BMC Developmental Biology","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9539d0d8-e998-4405-ad0b-7dadc2cba3fd","owner":[],"postedDate":"June 17th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":5100094,"name":"Developmental Biology"}],"tags":[],"updatedAt":"2021-10-19T10:14:08+00:00","versionOfRecord":[],"versionCreatedAt":"2021-06-17 20:25:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-607693","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-607693","identity":"rs-607693","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}