Regulating Cotton Fiber Research Articles

The DUF579 proteins GhIRX15s regulate cotton fiber development by interacting with proteins involved in xylan synthesis

Cotton provides the most abundant natural fiber for the textile industry. The mature cotton fiber largely consists of secondary cell walls with the highest proportion of cellulose and a small amount of hemicellulose and lignin. To dissect the roles of hemicellulosic polysaccharides during fiber development, four IRREGULAR XYLEM 15 (IRX15) genes, GhIRX15-1/-2/-3/-4, were functionally characterized in cotton. These genes encode DUF579 domain-containing proteins, which are homologs of AtIRX15 involved in xylan biosynthesis. The four GhIRX15 genes were predominantly expressed during fiber secondary wall thickening, and the encoded proteins were localized to the Golgi apparatus. Each GhIRX15 gene could restore the xylan deficient phenotype in the Arabidopsis irx15irx15l double mutant. Silencing of GhIRX15s in cotton resulted in shorter mature fibers with a thinner cell wall and reduced cellulose content as compared to the wild type. Intriguingly, GhIRX15-2 and GhIRX15-4 formed homodimers and heterodimers. In addition, the GhIRX15s showed physical interaction with glycosyltransferases GhGT43C, GhGT47A and GhGT47B, which are responsible for synthesis of the xylan backbone and reducing end sequence. Moreover, the GhIRX15s can form heterocomplexes with enzymes involved in xylan modification and side chain synthesis, such as GhGUX1/2, GhGXM1/2 and GhTBL1. These findings suggest that GhIRX15s participate in fiber xylan biosynthesis and modulate fiber development via forming large multiprotein complexes.

GhMAX2 Contributes to Auxin-Mediated Fiber Elongation in Cotton (Gossypium hirsutum).

Strigolactones (SLs) represent a new group of phytohormones that play a pivotal role in the regulation of plant shoot branching and the development of adventitious roots. In cotton (Gossypium hirsutum, Gh), SLs play a crucial role in the regulation of fiber cell elongation and secondary cell wall thickness. However, the underlying molecular mechanisms of SL signaling involved in fiber cell development are largely unknown. In this study, we report two SL-signaling genes, GhMAX2-3 and GhMAX2-6, which positively regulate cotton fiber elongation. Further protein-protein interaction and degradation assays showed that the repressor of the auxin cascade GhIAA17 serves as a substrate for the F-box E3 ligase GhMAX2. The in vivo ubiquitination assay suggested that GhMAX2-3 and GhMAX2-6 ubiquitinate GhIAA17 and coordinately degrade GhIAA17 with GhTIR1. The findings of this investigation offer valuable insights into the roles of GhMAX2-mediated SL signaling in cotton and establish a solid foundation for future endeavors aimed at optimizing cotton plant cultivation.

Strigolactone-induced degradation of SMXL7 and SMXL8 contributes to gibberellin- and auxin-mediated fiber cell elongation in cotton.

Cotton (Gossypium) fiber length, a key trait determining fiber yield and quality, is highly regulated by a class of recently identified phytohormones, strigolactones (SLs). However, the underlying molecular mechanisms of SL signaling involved in fiber cell development are largely unknown. Here, we show that the SL signaling repressors MORE AXILLARY GROWTH2-LIKE7 (GhSMXL7) and GhSMXL8 negatively regulate cotton fiber elongation. Specifically, GhSMXL7 and GhSMXL8 inhibit the polyubiquitination and degradation of the gibberellin (GA)-triggered DELLA protein (GhSLR1). Biochemical analysis revealed that GhSMXL7 and GhSMXL8 physically interact with GhSLR1, which interferes with the association of GhSLR1 with the E3 ligase GA INSENSITIVE2 (GhGID2), leading to the repression of GA signal transduction. GhSMXL7 also interacts with the transcription factor GhHOX3, preventing its binding to the promoters of essential fiber elongation regulatory genes. Moreover, both GhSMXL7 and GhSMXL8 directly bind to the promoter regions of the AUXIN RESPONSE FACTOR (ARF) genes GhARF18-10A, GhARF18-10D, and GhARF19-7D to suppress their expression. Cotton plants in which GhARF18-10A, GhARF18-10D, and GhARF19-7D transcript levels had been reduced by virus-induced gene silencing (VIGS) displayed reduced fiber length compared with control plants. Collectively, our findings reveal a mechanism illustrating how SL integrates GA and auxin signaling to coordinately regulate plant cell elongation at the single-cell level.

Auxin resistant 2 and short hypocotyl 2 regulate cotton fiber initiation and elongation.

Auxin, a pivotal regulator of diverse plant growth processes, remains central to development. The auxin-responsive genes auxin/indole-3-acetic acids (AUX/IAAs) are indispensable for auxin signal transduction, which is achieved through intricate interactions with auxin response factors (ARFs). Despite this, the potential of AUX/IAAs to govern the development of the most fundamental biological unit, the single cell, remains unclear. In this study, we harnessed cotton (Gossypium hirsutum) fiber, a classic model for plant single-cell investigation, to determine the complexities of AUX/IAAs. Our research identified 2 pivotal AUX/IAAs, auxin resistant 2 (GhAXR2) and short hypocotyl 2 (GhSHY2), which exhibit opposite control over fiber development. Notably, suppressing GhAXR2 reduced fiber elongation, while silencing GhSHY2 fostered enhanced fiber elongation. Investigating the mechanistic intricacies, we identified specific interactions between GhAXR2 and GhSHY2 with distinct ARFs. GhAXR2's interaction with GhARF6-1 and GhARF23-2 promoted fiber cell development through direct binding to the AuxRE cis-element in the constitutive triple response 1 promoter, resulting in transcriptional inhibition. In contrast, the interaction of GhSHY2 with GhARF7-1 and GhARF19-1 exerted a negative regulatory effect, inhibiting fiber cell growth by activating the transcription of xyloglucan endotransglucosylase/hydrolase 9 and cinnamate-4-hydroxylase. Thus, our study reveals the intricate regulatory networks surrounding GhAXR2 and GhSHY2, elucidating the complex interplay of multiple ARFs in AUX/IAA-mediated fiber cell growth. This work enhances our understanding of single-cell development and has potential implications for advancing plant growth strategies and agricultural enhancements.

GhWDL3 is involved in the formation and development of fiber cell morphology in upland cotton (Gossypium hirsutum L.)

BackgroundCotton fiber is a model tissue for studying microtubule-associated proteins (MAPs). The Xklp2 (TPX2) proteins that belong to the novel MAPs member mainly participate in the formation and development of microtubule (MT). However, there is a lack of studies concerning the systematic characterization of the TPX2 genes family in cotton. Therefore, the identification and portrayal of G. hirsutum TPX2 genes can provide key targets for molecular manipulation in the breeding of cotton fiber improvement.ResultIn this study, TPX2 family genes were classified into two distinct subclasses TPXLs and MAP genes WAVE DAMPENED2-LIKE (WDLs) and quite conservative in quantity. GhWDL3 was significantly up-regulated in 15 days post anthesis fibers of ZRI-015 (an upland cotton with longer and stronger fiber). GhWDL3 promotes all stem hairs to become straight when overexpressed in Arabidopsis, which may indirectly regulate cotton fiber cell morphology during fiber development. Virus induced gene silencing (VIGS) results showed that GhWDL3 inhibited fiber cell elongation at fiber development periods through regulating the expression of cell wall related genes.ConclusionThese results reveal that GhWDL3 regulated cotton fiber cell elongation and provide crucial information for the further investigation in the regulatory mechanisms/networks of cotton fiber length.

Systematically and Comprehensively Understanding the Regulation of Cotton Fiber Initiation: A Review.

Cotton fibers provide an important source of raw materials for the textile industry worldwide. Cotton fiber is a kind of single cell that differentiates from the epidermis of the ovule and provides a perfect research model for the differentiation and elongation of plant cells. Cotton fiber initiation is the first stage throughout the entire developmental process. The number of fiber cell initials on the seed ovule epidermis decides the final fiber yield. Thus, it is of great significance to clarify the mechanism underlying cotton fiber initiation. Fiber cell initiation is controlled by complex and interrelated regulatory networks. Plant phytohormones, transcription factors, sugar signals, small signal molecules, functional genes, non-coding RNAs, and histone modification play important roles during this process. Here, we not only summarize the different kinds of factors involved in fiber cell initiation but also discuss the mechanisms of these factors that act together to regulate cotton fiber initiation. Our aim is to synthesize a systematic and comprehensive review of different factors during fiber initiation that will provide the basics for further illustrating these mechanisms and offer theoretical guidance for improving fiber yield in future molecular breeding work.

Mapping intron retention events contributing to complex traits using splice quantitative trait locus

BackgroundAlternative splicing (AS) of mRNA plays an important roles in transcriptome diversity, involving regulation of plant growth and stress response. Understanding the variation of AS events underlying GWAS loci in a crop population can provide insight into the molecular mechanisms of complex agronomic traits. To date, genome-wide association studies relating AS events to agronomic traits have rarely been conducted at the population level in crops.ResultsHere, a pipeline was constructed to identify candidate AS events related to complex traits. Firstly, ovule transcriptome data were used to characterize intron retention (IR), the predominant type of AS in plants, on a genome-wide scale. This was done in a natural population consisting of 279 upland cotton lines. Secondly, splice quantitative trait locus (sQTL) analysis was carried out, which yielded a total of 2295 sQTLs involving 1607 genes. Of these, 14.25% (n = 427) were cis-regulatory loci. Integration with expression quantitative trait loci (eQTL) revealed that 53 (21.4%) cis-sGenes were regulated by both cis-sQTLs and cis-eQTLs. Finally, co-localization analysis integrated with GWAS loci in this population showed 32 cis-QTLs to be co-located with genetic regulatory loci related to fiber yield and quality traits, indicating that sQTLs are likely to participate in regulating cotton fiber yield and quality. An in-depth evaluation confirmed that differences in the IR rates of sQTL-regulated candidate genes such as GhLRRK1 and GhGC1 are associated with lint percentage (LP), which has potential in breeding applications.ConclusionThis study provides a clue that AS of mRNA has an impact on crop yield, along with functional sQTLs are new genetic resources for cotton precision breeding.

Brassinosteroids regulate cotton fiber elongation by modulating very-long-chain fatty acid biosynthesis.

Brassinosteroid (BR), a growth-promoting phytohormone, regulates many plant growth processes including cell development. However, the mechanism by which BR regulates fiber growth is poorly understood. Cotton (Gossypium hirsutum) fibers are an ideal single-cell model in which to study cell elongation due to their length. Here we report that BR controls cotton fiber elongation by modulating very-long-chain fatty acid (VLCFA) biosynthesis. BR deficiency reduces the expression of 3-ketoacyl-CoA synthases (GhKCSs), the rate-limiting enzymes involved in VLCFA biosynthesis, leading to lower saturated VLCFA contents in pagoda1 (pag1) mutant fibers. In vitro ovule culture experiments show that BR acts upstream of VLCFAs. Silencing of BRI1-EMS-SUPPRESOR 1.4 (GhBES1.4), encoding a master transcription factor of the BR signaling pathway, significantly reduces fiber length, whereas GhBES1.4 overexpression produces longer fibers. GhBES1.4 regulates endogenous VLCFA contents and directly binds to BR RESPONSE ELEMENTS (BRREs) in the GhKCS10_At promoter region, which in turn regulates GhKCS10_At expression to increase endogenous VLCFA contents. GhKCS10_At overexpression promotes cotton fiber elongation, whereas GhKCS10_At silencing inhibits cotton fiber growth, supporting a positive regulatory role for GhKCS10_At in fiber elongation. Overall, these results uncover a mechanism of fiber elongation through crosstalk between BR and VLCFAs at the single-cell level.

Fine mapping and candidate gene analysis of qFL-A12-5: a fiber length-related QTL introgressed from Gossypium barbadense into Gossypium hirsutum.

The fiber length-related qFL-A12-5 identified in CSSLs introgressed from Gossypium barbadense into Gossypium hirsutum was fine-mapped to an 18.8 kb region on chromosome A12, leading to the identification of the GhTPR gene as a potential regulator of cotton fiber length. Fiber length is a key determinant of fiber quality in cotton, and it is a key target of artificial selection for breeding and domestication. Although many fiber length-related quantitative trait loci have been identified, there are few reports on their fine mapping or candidate gene validation, thus hampering efforts to understand the mechanistic basis of cotton fiber development. Our previous study identified the qFL-A12-5 associated with superior fiber quality on chromosome A12 in the chromosome segment substitution line (CSSL) MBI7747 (BC4F3:5). A single segment substitution line (CSSL-106) screened from BC6F2 was backcrossed to construct a larger segregation population with its recurrent parent CCRI45, thus enabling the fine mapping of 2852 BC7F2 individuals using denser simple sequence repeat markers to narrow the qFL-A12-5 to an 18.8 kb region of the genome, in which six annotated genes were identified in Gossypium hirsutum. Quantitative real-time PCR and comparative analyses led to the identification of GH_A12G2192 (GhTPR) encoding a tetratricopeptide repeat-like superfamily protein as a promising candidate gene for qFL-A12-5. A comparative analysis of the protein-coding regions of GhTPR among Hai1, MBI7747, and CCRI45 revealed two non-synonymous mutations. The overexpression of GhTPR resulted in longer roots in Arabidopsis, suggesting that GhTPR may regulate cotton fiber development. These results provide a foundation for future efforts to improve cotton fiber length.

Co-expression networks regulating cotton fiber initiation generated by comparative transcriptome analysis between fiberless XZ142FLM and GhVIN1i

Cotton produces natural fiber as crucial raw material for textile industry and thus is economically one of the most important crops worldwide. Currently the mechanisms that control fiber initiation on ovules are still not well understood. In this study, we performed comparative transcriptome analysis between the natural fiberless mutant XZ142FLM and transgenic fiberless line GhVIN1i generated by RNAi silencing of GhVIN1 to identify molecular regulatory networks controlling fiber initiation. It was found that XZ142FLM and GhVIN1i shared many common differentially expressed genes (DEGs) in ovules during fiber initiation, and their respective DEGs were enriched in several identical pathways related to fiber initiation. Many reported fiber initiation or elongation related genes including GhMML3, GhVIN1, GhMYB25, GhHD-1, GhHOX3 exhibited similar expression patterns in XZ142FLM and GhVIN1i during fiber initiation, suggesting similar mechanisms of fiber initiation operating in the two fiberless lines, which was further verified by comparative temporal expression pattern analysis between TM-1/XZ142FLM and Coker 312/GhVIN1i. Co-expression regulatory networks for controlling fiber initiation were constructed for XZ142FLM and GhVIN1i, respectively, which centered on GhMML3 or GhVIN1 with many known fiber initiation/elongation related genes. Several transcription factors and hub genes were identified in the co-expression networks generated, which may facilitate future research in identification of novel candidate genes regulating fiber initiation and elongation. Overall, the results obtained in this study shed light on the regulatory networks mediated by GhMML3 and GhVIN1 for controlling fiber initiation in cotton.

A Novel Tandem Zinc Finger Protein in Gossypium hirsutum, GhTZF2, Interacts with GhMORF8 to Regulate Cotton Fiber Cell Development

Arginine-rich Tandem Zinc Finger (RR-TZF) proteins make up a plant-specific superfamily that participates in plant development, while their roles in cotton fiber development remain to be explored. In this study, we identified an RR-TZF protein-coding gene, GhTZF2, containing two CCCH domains (C-X7-C-X5-C-X3-H-X16-C-X5-C-X4-C-X3-H) and one RR domain at the N-terminus of the two CCCH domains, by comparing the differences of chromatin H3K4me3 modifications between wild-type upland cotton (WT) and the fuzzless-lintless mutant (fl) ovules. GhTZF2 was highly expressed in ovule cells near anthesis, and multiple experiments revealed that GhTZF2 could interact directly with GhMORF8. Homozygotic GhTZF2-knockout cotton lines produced significantly shorter fibers with thinner cell walls. Additionally, comparative transcriptome analysis confirmed that many differentially expressed transcripts contain adenine- and uridine-rich (AU-rich) elements (AREs) in their 3’ untranslated regions (UTR). Together, this study indicated that GhTZF2 may regulate cotton fiber cell development through interacting with GhMORF8, or may be involved in mRNA turnover.

Molecular Regulation of Cotton Fiber Development: A Review.

Cotton (Gossypium spp.) is an economically important natural fiber crop. The quality of cotton fiber has a substantial effect on the quality of cotton textiles. The identification of cotton fiber development-related genes and exploration of their biological functions will not only enhance our understanding of the elongation and developmental mechanisms of cotton fibers but also provide insights that could aid the cultivation of new cotton varieties with improved fiber quality. Cotton fibers are single cells that have been differentiated from the ovule epidermis and serve as a model system for research on single-cell differentiation, growth, and fiber production. Genes and fiber formation mechanisms are examined in this review to shed new light on how important phytohormones, transcription factors, proteins, and genes linked to fiber development work together. Plant hormones, which occur in low quantities, play a critically important role in regulating cotton fiber development. Here, we review recent research that has greatly contributed to our understanding of the roles of different phytohormones in fiber development and regulation. We discuss the mechanisms by which phytohormones regulate the initiation and elongation of fiber cells in cotton, as well as the identification of genes involved in hormone biosynthetic and signaling pathways that regulate the initiation, elongation, and development of cotton fibers.

A cotton germin-like protein GbGLP2 controls fiber length via regulating genes involved in secondary cell wall synthesis

Cotton is not only a natural fiber crop, but also an important economic crop in China. The cultivation of cotton with longer, stronger, and finer fiber has been the goal of cotton breeders. Germin-like proteins (GLPs) is a group of protein family with cupin domain, which mainly acts as an enzyme. However, the function of GLPs involved in regulating cotton fiber development is rarely understood. Here, we identified a gene named GbGLP2 from the G. barbadense cDNA library and elucidated its function negatively regulating fiber elongation that resulted in shorter fiber. The transcripts of GbGLP2 were mainly abundant in the elongating fiber of 10 days post-anthesis (DPA). GbGLP2 shared 99.8% similarity with GhGLP1. RNAi silencing of the homolog GhGLP1 in G. hirsutum accession YZ-1 produced longer fiber relative to the control. Enhancing the expression of GbGLP2 significantly reduced fiber length and strength. Through RNA-seq analysis between RNAi and WT lines, 566 genes differentially expressed were identified, of which 113 genes were downregulated and 453 genes were upregulated in the RNAi lines. Surprisingly, most of the downregulated genes participated in secondary cell wall biosynthesis, including cellulose synthase genes (CESA4, CESA7, and CESA8), hemicellulose biosynthesis genes (IRX9, COBL4) and transcription factors (MYB46, MYB86). Simultaneously, the transcriptome comparison between 30 cotton varieties with much longer fiber and 30 varieties with much shorter fiber revealed that the above downregulated genes were markedly decreased in long fiber. Taken together, these results suggest that knockdown of GhGLP1 promotes fiber elongation via delaying secondary cell wall thickening stage.

Comparative phosphoproteomic analysis of BR-defective mutant reveals a key role of GhSK13 in regulating cotton fiber development.

Brassinosteroid (BR), a steroid phytohormone, whose signaling transduction pathways include a series of phosphorylation and dephosphorylation events, and GSK3s are the main negative regulator kinases. BRs have been shown to play vital roles in cotton fiber elongation. However, the underlying mechanism is still elusive. In this study, fibers of a BR-defective mutant Pagoda 1 (pag1), and its corresponding wild-type (ZM24) were selected for a comparative global phosphoproteome analysis at critical developmental time points: fast-growing stage (10 days after pollination (DPA)) and secondary cell wall synthesis stage (20 DPA). Based on the substrate characteristics of GSK3, 900 potential substrates were identified. Their GO and KEGG annotation results suggest that BR functions in fiber development by regulating GhSKs (GSK3s of Gossypium hirsutum L.) involved microtubule cytoskeleton organization, and pathways of glucose, sucrose and lipid metabolism. Further experimental results revealed that among the GhSK members identified, GhSK13 not only plays a role in BR signaling pathway, but also functions in developing fiber by respectively interacting with an AP2-like ethylene-responsive factor GhAP2L, a nuclear transcription factor Gh_DNF_YB19, and a homeodomain zipper member GhHDZ5. Overall, our phosphoproteomic research advances the understanding of fiber development controlled by BR signal pathways especially through GhSKs, and also offers numbers of target proteins for improving cotton fiber quality.

Heterologous Expression of GbTCP4, a Class II TCP Transcription Factor, Regulates Trichome Formation and Root Hair Development in Arabidopsis.

Two class I family teosinte branched1/cycloidea/proliferating cell factor1 (TCP) proteins from allotetraploid cotton are involved in cotton fiber cell differentiation and elongation and root hair development. However, the biological function of most class II TCP proteins is unclear. This study sought to reveal the characteristics and functions of the sea-island cotton class II TCP gene GbTCP4 by biochemical, genetic, and molecular biology methods. GbTCP4 protein localizes to nuclei, binding two types of TCP-binding cis-acting elements, including the one in its promoter. Expression pattern analysis revealed that GbTCP4 is widely expressed in tissues, with the highest level in flowers. GbTCP4 is expressed at different fiber development stages and has high transcription in fibers beginning at 5 days post anthesis (DPA). GbTCP4 overexpression increases primary root hair length and density and leaf and stem trichomes in transgenic Arabidopsis relative to wild-type plants (WT). GbTCP4 binds directly to the CAPRICE (CPC) promoter, increasing CPC transcript levels in roots and reducing them in leaves. Compared with WT plants, lignin content in the stems of transgenic Arabidopsis overexpressing GbTCP4 increased, and AtCAD5 gene transcript levels increased. These results suggest that GbTCP4 regulates trichome formation and root hair development in Arabidopsis and may be a candidate gene for regulating cotton fiber elongation.

A Pivotal Role of Hormones in Regulating Cotton Fiber Development.

Cotton is the main source of renewable fiber in the world and is primarily used for textile production. Cotton fibers are single cells differentiated from the ovule epidermis and are an excellent model system for studying cell elongation, polyploidization, and cell wall biosynthesis. Plant hormones, which are present in relatively low concentrations, play important roles in various developmental processes, and recently, multiple reports have revealed the pivotal roles of hormones in regulating cotton fiber development. For example, exogenous application of hormones has been shown to promote the initiation and growth of fiber cells. However, a comprehensive understanding about phytohormone regulating fiber development is still unknown. Here, we focus on recent advances in elucidating the roles of multiple phytohormones in the control of fiber development, namely auxin, gibberellin, brassinosteroid, ethylene, cytokinin, abscisic acid, and strigolactones. We not only review the identification of genes involved in hormone biosynthetic and signaling pathways but also discuss the mechanisms of these phytohormones in regulating the initiation and elongation of fiber cells in cotton. Auxin, gibberellin, brassinosteroid, ethylene, jasmonic acid, and strigolactones play positive roles in fiber development, whereas cytokinin and abscisic acid inhibit fiber growth. Our aim is to provide a comprehensive review of the role of phytohormones in cotton fiber development that will serve as the basis for further elucidation of the mechanisms by which plant hormones regulate fiber growth.

A Genome-Scale Analysis of the PIN Gene Family Reveals Its Functions in Cotton Fiber Development.

The PIN-FORMED (PIN) protein, the most important polar auxin transporter, plays a critical role in the distribution of auxin and controls multiple biological processes. However, characterizations and functions of this gene family have not been identified in cotton. Here, we identified the PIN family in Gossypium hirsutum, Gossypium arboreum, and Gossypium raimondii. This gene family was divided into seven subgroups. A chromosomal distribution analysis showed that GhPIN genes were evenly distributed in eight chromosomes and that the whole genome and dispersed duplications were the main duplication events for GhPIN expansion. qRT-PCR analysis showed a tissue-specific expression pattern for GhPIN. Likely due to the cis-element variations in their promoters, transcripts of PIN6 and PIN8 genes from the At (tetraploid genome orginated from G. arboreum) subgenome and PIN1a from the Dt (tetraploid genome orginated from G. raimondii) subgenome in G. hirsutum was significantly increased compared to the transcripts in the diploids. The differential regulation of these PIN genes after the polyploidization may be conducive to fiber initiation and elongation. Exogenously applied auxin polar transport inhibitor significantly suppressed fiber growth, which is consistent with the essential function of these PIN genes for regulating cotton fiber development. Furthermore, the overexpression of GhPIN1a_Dt, GhPIN6_At, and GhPIN8_At in Arabidopsis promoted the density and length of trichomes in leaves.

Differential expression of microRNAs during fiber development between fuzzless-lintless mutant and its wild-type allotetraploid cotton

Cotton is one of the most important textile crops but little is known how microRNAs regulate cotton fiber development. Using a well-studied cotton fiberless mutant Xu-142-fl, we compared 54 miRNAs for their expression between fiberless mutant and its wildtype. In wildtype Xu-142, 26 miRNAs are involved in cotton fiber initiation and 48 miRNAs are related to primary wall synthesis and secondary wall thickening. Thirty three miRNAs showed different expression in fiber initiation between Xu-142 and Xu-142-fl. These miRNAs potentially target 723 protein-coding genes, including transcription factors, such as MYB, ARF, and LRR. ARF18 was newly predicted targets of miR160a, and miR160a was expressed at higher level in −2DPA of Xu-142-fl compared with Xu-142. Furthermore, the result of Gene Ontology-based term classification (GO), EuKaryotic Orthologous Groups (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis shows that miRNA targets were classified to 222 biological processes, 64 cellular component and 42 molecular functions, enriched in 22 KOG groups, and classified into 28 pathways. Together, our study provides evidence for better understanding of miRNA regulatory roles in the process of fiber development, which is helpful to increase fiber yield and improve fiber quality.

GhCaM7-like, a calcium sensor gene, influences cotton fiber elongation and biomass production

Calcium signaling regulates many developmental processes in plants. Calmodulin (CaM) is one of the most conserved calcium sensors and has a flexible conformation in eukaryotes. The molecular functions of CaM are unknown in cotton, which is a major source of natural fiber. In this study, a Gossypium hirsutum L.CaM7-like gene was isolated from upland cotton. Bioinformatics analysis indicated that the GhCaM7-like gene was highly conserved as compared with Arabidopsis AtCaM7. The GhCaM7-like gene showed a high expression level in elongating fibers. Expression of β-glucuronidase was observed in trichomes on the stem, leaf and root in transgenic Arabidopsis plants of a PROGhCaM7-like:GUS fusion. Silencing of the GhCaM7-like gene resulted in decreased fiber length, but also caused reduction in stem height, leaf dimensions, seed length and 100-seed weight, in comparison with those of the control. Reduced expression of the GhCaM7-like gene caused decreased Ca2+ influx in cells of the leaf hypodermis and stem apex, and down-regulation of GhIQD1 (IQ67-domain containing protein), GhAnn2 (Annexins) and GhEXP2 (Expansin). These results indicate that the GhCaM7-like gene plays a vital role in calcium signaling pathways, and may regulate cotton fiber elongation and biomass production by affecting Ca2+ signatures and downstream signaling pathways of CaM.

Cotton cytosolic pyruvate kinase GhPK6 participates in fast fiber elongation regulation in a ROS-mediated manner.

Cotton cytosolic pyruvate kinase GhPK6 is preferentially expressed in the late stage of fiber elongation process, transgenic experiments indicated that its expression level was negatively correlated to cell expansion rate. Pyruvate kinase (PK) plays vital regulatory roles in rapid cell growth in mammals. However, the function of PK in plant cell growth remains unclear. In allotetraploid upland cotton (Gossypium hirsutum L.), a total of 33 PK genes are encoded by the genome. Analysis of the transcriptome data indicated that only two cytosolic PK genes, GhPK6 and its duplicated gene GhPK26, are preferentially expressed in elongating cotton fiber cells. RT-qPCR and western blot analyses revealed that the expression of GhPK6 was negatively correlated with fiber elongation rate, which well explains the observed sharp increase of cytosolic PK activity at the end of fast fiber elongation process. Furthermore, virus-induced gene silencing of GhPK6 in cotton plants resulted in increased fiber cell elongation and reduced reactive oxygen species (ROS) accumulation. On the contrary, Arabidopsis plants ectopically expressing GhPK6 exhibited ROS-mediated growth inhibition, whereas the addition of ROS scavenging reagents could partly rescue this inhibition. These data collectively suggested that GhPK6 might play an important role in regulating cotton fiber elongation in a ROS-dependent inhibition manner.