Cloning and functional mechanism of the dwarf gene gba affecting stem elongation and cellulose biosynthesis in jute (Corchorus olitorius).

Plant height (PH) is an important factor affecting bast fiber yield in jute. Here, we report the mechanism of dwarfism in the 'Guangbaai' (gba) of jute. The mutant gba had shorter internode length and cell length compared to the standard cultivar 'TaiZi 4' (TZ4). Exogenous GA3 treatment indicated that gba is a GA-insensitive dwarf mutant. Quantitative trait locus (QTL) analysis of three PH-related traits via a high-density genetic linkage map according to re-seq showed that a total of 25 QTLs were identified, including 13 QTLs for PH, with phenotypic variation explained ranging from 2.42 to 74.16%. Notably, the functional mechanism of the candidate gene CoGID1a, the gibberellic acid receptor, of the major locus qPHIL5 was evaluated by transgenic analysis and virus-induced gene silencing. A dwarf phenotype-related single nucleotide mutation in CoGID1a was identified in gba, which was also unique to the dwarf phenotype of gba among 57 cultivars. Cogid1a was unable to interact with the growth-repressor DELLA even in the presence of highly accumulated gibberellins in gba. Differentially expressed genes between transcriptomes of gba and TZ4 after GA3 treatment indicated up-regulation of genes involved in gibberellin and cellulose synthesis in gba. Interestingly, it was found that up-regulation of CoMYB46, a key transcription factor in the secondary cell wall, by the highly accumulated gibberellins in gba promoted the expression of cellulose synthase genes CoCesA4 and CoCesA7. These findings provide valuable insights into fiber development affected by endogenous gibberellin accumulation in plants.

Coordination of floral and fiber development in cotton (Gossypium) by hormone- and flavonoid-signalling associated regulatory miRNAs.

Various plant development activities and stress responses are tightly regulated by various microRNAs (miRNA) and their target genes, or transcription factors in a spatiotemporal manner. Here, to exemplify how flowering-associated regulatory miRNAs synchronize their expression dynamics during floral and fiber development in cotton, constitutive expression diminution transgenic lines of auxin-signaling regulatory Gh-miR167 (35S-MIM167) were developed through target mimicry approach. 'Moderate' (58% to 80%)- and 'high' (> 80%)-Gh-miR167 diminution mimic lines showed dosage-dependent developmental deformities in anther development, pollen maturation, and fruit (= boll) formation. Cross pollination of 'moderate' 35S-MIM167 mimic lines with wild type (WT) plant partially restored boll formation and emergence of fiber initials on the ovule surface. Gh-miR167 diminution favored organ-specific transcription biases in miR159, miR166 as well as miR160, miR164, and miR172 along with their target genes during anther and petal development, respectively. Similarly, accumulative effect of percent Gh-miR167 diminution, cross regulation of its target ARF6/8 genes, and temporal mis-expression of hormone signaling- and flavonoid biosynthesis-associated regulatory miRNAs at early fiber initiation stage caused irregular fiber formation. Spatial and temporal transcription proportions of regulatory miRNAs were also found crucial for the execution of hormone- and flavonoid-dependent progression of floral and fiber development. These observations discover how assorted regulatory genetic circuits get organized in response to Gh-miR167 diminution and converge upon ensuing episodes of floral and fiber development in cotton.

Comparative phosphoproteomic analysis reveals that phosphorylation of sucrose synthase GhSUS2 by Ca2+-dependent protein kinases GhCPK84/93 affects cotton fiber development.

Cotton fiber elongation is a critical growth phase that affects final fiber length. Morphological analysis indicated an asynchronous fiber elongation pattern between two cotton varieties, J7-1 and J14-1. Through phosphoproteomic analysis, a total of 89 differentially-phosphorylated proteins (DPPs) were identified in elongating fibers between J7-1 and J14-1. Gene ontology (GO) analysis showed that these DPPs were mainly enriched in sucrose synthase activity, transferase activity, and UDP-glycosyltransferase activity. In J14-1, the phosphorylation level of GhSUS2, a key sucrose synthase in the sucrose metabolism pathway, was significantly higher than that in J7-1. We further revealed that GhSUS2 positively regulates fiber elongation, and GhSUS2-silenced transgenic cotton displayed the phenotype of 'short fibers' compared with the controls. During fiber development, the residue Ser11 in the GhSUS2 protein is phosphorylated by the Ca2+-dependent protein kinases GhCPK84 and GhCPK93. Phosphorylated GhSUS2 is localized in the cytoplasm, whereas unphosphorylated GhSUS2 is localized in the plasma membrane. Moreover, abscisic acid (ABA) could promote the transcription and translation of GhCPK84 and GhCPK93, thereby enhancing the phosphorylation of GhSUS2 to impede fiber elongation. Thus, our data demonstrates that GhSUS2 plays a positive role in fiber development, but its phosphorylation by GhCPK84 and GhCPK93 hinders fiber elongation of cotton.

The Landscape of Accessible Chromatin and Developmental Transcriptome Maps Reveal a Genetic Mechanism of Skeletal Muscle Development in Pigs.

The epigenetic regulation mechanism of porcine skeletal muscle development relies on the openness of chromatin and is also precisely regulated by transcriptional machinery. However, fewer studies have exploited the temporal changes in gene expression and the landscape of accessible chromatin to reveal the underlying molecular mechanisms controlling muscle development. To address this, skeletal muscle biopsy samples were taken from Landrace pigs at days 0 (D0), 60 (D60), 120 (D120), and 180 (D180) after birth and were then analyzed using RNA-seq and ATAC-seq. The RNA-seq analysis identified 8554 effective differential genes, among which ACBD7, TMEM220, and ATP1A2 were identified as key genes related to the development of porcine skeletal muscle. Some potential cis-regulatory elements identified by ATAC-seq analysis contain binding sites for many transcription factors, including SP1 and EGR1, which are also the predicted transcription factors regulating the expression of ACBD7 genes. Moreover, the omics analyses revealed regulatory regions that become ectopically active after birth during porcine skeletal muscle development after birth and identified 151,245, 53,435, 30,494, and 40,911 peaks. The enriched functional elements are related to the cell cycle, muscle development, and lipid metabolism. In summary, comprehensive high-resolution gene expression maps were developed for the transcriptome and accessible chromatin during postnatal skeletal muscle development in pigs.

Transcriptome, Ectopic Expression and Genetic Population Analysis Identify Candidate Genes for Fiber Quality Improvement in Cotton.

Comparative transcriptome analysis of fiber tissues between Gossypium barbadense and Gossypium hirsutum could reveal the molecular mechanisms underlying high-quality fiber formation and identify candidate genes for fiber quality improvement. In this study, 759 genes were found to be strongly upregulated at the elongation stage in G. barbadense, which showed four distinct expression patterns (I-IV). Among them, the 346 genes of group IV stood out in terms of the potential to promote fiber elongation, in which we finally identified 42 elongation-related candidate genes by comparative transcriptome analysis between G. barbadense and G. hirsutum. Subsequently, we overexpressed GbAAR3 and GbTWS1, two of the 42 candidate genes, in Arabidopsis plants and validated their roles in promoting cell elongation. At the secondary cell wall (SCW) biosynthesis stage, 2275 genes were upregulated and exhibited five different expression profiles (I-V) in G. barbadense. We highlighted the critical roles of the 647 genes of group IV in SCW biosynthesis and further picked out 48 SCW biosynthesis-related candidate genes by comparative transcriptome analysis. SNP molecular markers were then successfully developed to distinguish the SCW biosynthesis-related candidate genes from their G. hirsutum orthologs, and the genotyping and phenotyping of a BC3F5 population proved their potential in improving fiber strength and micronaire. Our results contribute to the better understanding of the fiber quality differences between G. barbadense and G. hirsutum and provide novel alternative genes for fiber quality improvement.

Alternative Splicing during Fiber Development in G. hirsutum.

Cotton is a valuable cash crop in many countries. Cotton fiber is a trichome that develops from a single epidermal cell and serves as an excellent model for understanding cell differentiation and other life processes. Alternative splicing (AS) of genes is a common post-transcriptional regulatory process in plants that is essential for plant growth and development. The process of AS during cotton fiber formation, on the other hand, is mainly unknown. A substantial number of multi-exon genes were discovered to be alternatively spliced during cotton fiber formation in this study, accounting for 23.31% of the total number of genes in Gossypium hirsutum. Retention intron (RI) is not necessarily the most common AS type, indicating that AS genes and processes during fiber development are very temporal and tissue-specific. When compared to fiber samples, AS is more prevalent at the fiber initiation stages and in the ovule, indicating that development stages and tissues use different AS strategies. Genes involved in fiber development have gone through stage-specific AS, demonstrating that AS regulates cotton fiber development. Furthermore, AS can be regulated by trans-regulation elements such as splicing factor and cis-regulation elements such as gene length, exon numbers, and GC content, particularly at exon-intron junction sites. Our findings also suggest that increased DNA methylation may aid in the efficiency of AS, and that gene body methylation is key in AS control. Finally, our research will provide useful information about the roles of AS during the cotton fiber development process.

RNA-Seq with a novel glabrous-ZM24fl reveals some key lncRNAs and the associated targets in fiber initiation of cotton.

BACKGROUND: Cotton fiber is an important natural resource for textile industry and an excellent model for cell biology study. Application of glabrous mutant cotton and high-throughput sequencing facilitates the identification of key genes and pathways for fiber development and cell differentiation and elongation. LncRNA is a type of ncRNA with more than 200 nt in length and functions in the ways of chromatin modification, transcriptional and post-transcriptional modification, and so on. However, the detailed lncRNA and associated mechanisms for fiber initiation are still unclear in cotton. RESULTS: In this study, we used a novel glabrous mutant ZM24fl, which is endowed with higher somatic embryogenesis, and functions as an ideal receptor for cotton genetic transformation. Combined with the high-throughput sequencing, fatty acid pathway and some transcription factors such as MYB, ERF and bHLH families were identified the important roles in fiber initiation; furthermore, 3,288 lncRNAs were identified, and some differentially expressed lncRNAs were also analyzed. From the comparisons of ZM24_0 DPA vs ZM24_-2 DPA and fl_0 DPA vs ZM24_0 DPA, one common lncRNA MSTRG 2723.1 was found that function upstream of fatty acid metabolism, MBY25-mediating pathway, and pectin metabolism to regulate fiber initiation. In addition, other lncRNAs MSTRG 3390.1, MSTRG 48719.1, and MSTRG 31176.1 were also showed potential important roles in fiber development; and the co-expression analysis between lncRNAs and targets showed the distinct models of different lncRNAs and complicated interaction between lncRNAs in fiber development of cotton. CONCLUSIONS: From the above results, a key lncRNA MSTRG 2723.1 was identified that might mediate some key genes transcription of fatty acid metabolism, MYB25-mediating pathway, and pectin metabolism to regulate fiber initiation of ZM24 cultivar. Co-expression analysis implied that some other important lncRNAs (e.g., MSTRG 3390.1, MSTRG 48719.1, and MSTRG 31176.1) were also showed the different regulatory model and interaction between them, which proposes some valuable clues for the lncRNAs associated mechanisms in fiber development.

Identification, characterization and expression profiling of circular RNAs in the early cotton fiber developmental stages.

Circular RNA is one of the endogenous non-coding RNAs with a covalently closed loop structure and largely involved in regulating gene expression. However, the abundance of circular RNAs and their regulatory functions during the early stages of fiber development are still not known. In this work, we conducted high-throughput sequencing of the Ligonlintless-1 and its wild-type at 0 DPA, 8 DPA and stem. A total of 2811 circular RNAs were identified and unevenly distributed across cotton chromosomes. We found 34, 142 and 27 circular RNAs were differentially expressed between Ligonlintless-1 and wild-type at 0 DPA, 8 DPA and stem, respectively. Both circular RNA-microRNA-mRNA network and MeJA treatment results in Ligonlintless-1 and wild-type might provide a strong indication of four circular RNAs and ghr_miR169b being important biological molecular associating with fiber development. The results provide new insight into the putative molecular function of circular RNAs in the regulation of fiber development.

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.

Transcriptional loss of domestication-driven cytoskeletal GhPRF1 gene causes defective floral and fiber development in cotton (Gossypium).

KEY MESSAGE: Constitutive- and fiber-specific RNAi of GhPRF1 gene illustrated strong correlation between domestication-driven profilin genes and floral/fiber architecture in cotton. During morpho-transformation of short-fuzz of wild cotton into the elongating spinnable fibers under the millennia of human selection, actin-polymerizing cytoskeletal profilin genes had undergone significant sequence alterations and spatiotemporal shift in their transcription levels. To comprehend the expression dynamics of profilin genes with their phenotypic implications, transgenic expression modulation of cotton profilin 1 (GhPRF1) gene was performed in the constitutive- and fiber-specific manner in Coker 310FR cotton cultivar. The constitutive GhPRF1-RNAi lines (35S:GhPRF1-RNAi) exhibited distorted 'monadelphous' staminal-tube, reduced pollen-viability and poorly developed fibers, whereas floral and fiber development of fiber-specific GhPRF1-RNAi lines showed no abnormalities. Moreover, the fiber-specific GhPRF1 overexpression lines (FBP7:GhPRF1-Ox) showed increased emergence of fiber-initials on the ovule surface, on the contrary to no fiber-initials in fiber-specific RNAi lines (FBP7:GhPRF1-RNAi). Interestingly, the average seed weight and fiber weight of FBP7:GhPRF1-Ox lines increased > 60% and > 38%, respectively, compared with FBP7:GhPRF1-RNAi lines and untransformed control seeds. On a molecular basis, the aberrant floral and fiber development of 35S:GhPRF1-RNAi lines was largely associated with sugar metabolism and hormone-signaling mechanisms. These observations illustrated the strong correlation between domestication-driven GhPRF genes, and floral/fiber development in cotton. Also, the enhanced agronomic traits in GhPRF1-Ox lines of cotton empowered us to recognize their imperative roles, and their future deployment for the sustainable cotton crop improvement.

Multi-responses of O-methyltransferase genes to salt stress and fiber development of Gossypium species.

BACKGROUND: O-methyltransferases (OMTs) are an important group of enzymes that catalyze the transfer of a methyl group from S-adenosyl-L-methionine to their acceptor substrates. OMTs are divided into several groups according to their structural features. In Gossypium species, they are involved in phenolics and flavonoid pathways. Phenolics defend the cellulose fiber from dreadful external conditions of biotic and abiotic stresses, promoting strength and growth of plant cell wall. RESULTS: An OMT gene family, containing a total of 192 members, has been identified and characterized in three main Gossypium species, G. hirsutum, G. arboreum and G. raimondii. Cis-regulatory elements analysis suggested important roles of OMT genes in growth, development, and defense against stresses. Transcriptome data of different fiber developmental stages in Chromosome Substitution Segment Lines (CSSLs), Recombination Inbred Lines (RILs) with excellent fiber quality, and standard genetic cotton cultivar TM-1 demonstrate that up-regulation of OMT genes at different fiber developmental stages, and abiotic stress treatments have some significant correlations with fiber quality formation, and with salt stress response. Quantitative RT-PCR results revealed that GhOMT10_Dt and GhOMT70_At genes had a specific expression in response to salt stress while GhOMT49_At, GhOMT49_Dt, and GhOMT48_At in fiber elongation and secondary cell wall stages. CONCLUSIONS: Our results indicate that O-methyltransferase genes have multi-responses to salt stress and fiber development in Gossypium species and that they may contribute to salt tolerance or fiber quality formation in Gossypium.

Evolution, expression and functional analysis of cultivated allotetraploid cotton DIR genes.

BACKGROUND: Dirigent (DIR) proteins mediate regioselectivity and stereoselectivity during lignan biosynthesis and are also involved in lignin, gossypol and pterocarpan biosynthesis. This gene family plays a vital role in enhancing stress resistance and in secondary cell-wall development, but systematical understanding is lacking in cotton. RESULTS: In this study, 107 GbDIRs and 107 GhDIRs were identified in Gossypium barbadense and Gossypium hirsutum, respectively. Most of these genes have a classical gene structure without intron and encode proteins containing a signal peptide. Phylogenetic analysis showed that cotton DIR genes were classified into four distinct subfamilies (a, b/d, e, and f). Of these groups, DIR-a and DIR-e were evolutionarily conserved, and segmental and tandem duplications contributed equally to their formation. In contrast, DIR-b/d mainly expanded by recent tandem duplications, accompanying with a number of gene clusters. With the rapid evolution, DIR-b/d-III was a Gossypium-specific clade involved in atropselective synthesis of gossypol. RNA-seq data highlighted GhDIRs in response to Verticillium dahliae infection and suggested that DIR gene family could confer Verticillium wilt resistance. We also identified candidate DIR genes related to fiber development in G. barbadense and G. hirsutum and revealed their differential expression. To further determine the involvement of DIR genes in fiber development, we overexpressed a fiber length-related gene GbDIR78 in Arabidopsis and validated its function in trichomes and hypocotyls. CONCLUSIONS: These findings contribute novel insights towards the evolution of DIR gene family and provide valuable information for further understanding the roles of DIR genes in cotton fiber development as well as in stress responses.

Ubiquitylome analysis reveals the involvement of ubiquitination in the bast fiber growth of ramie.

MAIN CONCLUSION: A total of 249 sites from 197 proteins showed a differential ubiquitination level in the fiber development of ramie barks. The function of two differentially ubiquitinated proteins for fiber growth was demonstrated. Ubiquitination is one of the most common post-translational modifications of proteins, and it plays essential roles in plant growth and development. However, the involvement of ubiquitination in the growth of plant fibers remains largely unknown. We compared the ubiquitylome of the top and middle stems of ramie bark, with different fiber growth stages. We identified 249 differentially ubiquitinated sites in 197 proteins in fiber-developing barks in the stems and found that seven were homologs of Arabidopsis proteins associated with fiber growth. Overexpression of the differentially ubiquitinated proteins, RWA3 homolog whole_GLEAN_10024150 and MYB protein whole_GLEAN_10015497, significantly promoted fiber growth in transgenic Arabidopsis, indicating their involvement in this process. We also found that the abundance of these proteins decreased when their ubiquitination levels increased and vice versa in the fiber-developing bark. These results indicated that the abundance of these two proteins was adjusted through ubiquitin-dependent degradation. Collectively, our findings provide important insights into the involvement of ubiquitination in the growth of ramie fibers.

Genome-wide identification of cotton GRAM family proteins reveals that GRAM31 regulates fiber length.

The glucosyltransferases, Rab-like GTPase activators and myotubularins (GRAM) domain is highly conserved in eukaryotic cells and is found in proteins involved in membrane-associated processes. GRAM domain proteins have not yet been functionally characterized in cotton. In this study, we identified 164 genes encoding GRAM domain proteins in four cotton species, comprising two subfamilies. In Gossypium hirsutum, our transcriptome data showed that GhGRAM31 was predominantly expressed during the rapid elongation stage of fiber development and that it might control fiber length. GhGRAM31-RNAi transgenic cotton lines showed inhibition of fiber elongation and produced shorter mature fibers, and this was coupled with expression changes of genes related to fiber development. In addition, lint percentage and seed size were also decreased in the RNAi lines. Further examination revealed that GhGRAM31 directly interacts with two other GRAM-domain proteins, GhGRAM5 and GhGRAM35. GhGRAM5 also interacts with the transcription factor GhTTG1, while GhGRAM35 interacts with the transcription factors GhHOX1 and GhHD1. Co-expression of GhGRAM31 and GhGRAM35 was able to promote GhHD1 transcription activity in cotton protoplasts. Our results provide new insights into the biological function of the GRAM-domain protein family in cotton, and selected genes have the potential to be utilized in future programs for the genetic improvement of fibers.

Genome-wide identification and expression analysis of R2R3, 3R- and 4R-MYB transcription factors during lignin biosynthesis in flax (Linum usitatissimum).

MYB transcription factors (TFs) have vital roles in regulating lignin or fiber development. Flax (Linum usitatissimum) is known as one of the plants with high fiber production capacity. However, no studies have been conducted to identify and characterize MYB TFs in the flax genome. Results showed that flax genome harbours 167 R2R3, seven 3R, and one 4R-type MYB TFs. 22 MYB genes (%13) were estimated to be tandem duplicated dated around 13.3-86.98 Mya. 130 flax MYB members have apparent orthologous with Arabidopsis, in which 17 R2R3 MYBs are associated with lignin biosynthesis. MYB062, MYB072, MYB096, MYB141, and MYB146 genes were up-regulated in tissues having higher lignin production capacity. In opposite, MYB012 and MYB113 genes were down-regulated which points out the involvement of those genes in the lignin biosynthesis mechanism. This comprehensive study can provide a basis for understanding the role of MYBs in fiber or lignin production in flax.

A cotton α1,3-/4-fucosyltransferase-encoding gene, FucT4, plays an important role in cell elongation and is significantly associated with fiber quality.

Fucosylation, one of the key posttranslational modifications, plays an important role in plants. It is involved in the development, signal transduction, reproduction, and disease resistance. α1,3-/4-Fucosyltransferase is responsible for transferring L-fucose from GDP-L-fucose to the N-glycan to exert fucosylational functions. However, the roles of the fucosyltransferase gene in cotton remain unknown. This study provided a comprehensive investigation of its possible functions. A genome-wide analysis identified four, four, eight, and eight FucT genes presented in the four sequenced cotton species, diploid Gossypium raimondii, G. arboreum, tetraploid G. hirsutum acc. TM-1, and G. barbadense cv. H7124, respectively. These FucTs were classified into two groups, with FucT4 homologs alone as a group. We isolated FucT4 in TM-1 and H7124, and named it GhFucT4 and GbFucT4, respectively. Quantitative RT-PCR and transcriptome data demonstrated that GhFucT4 had the highest expression levels in fibers among all GhFucT genes. Association studies and QTL co-localization supported the possible involvement of GhFucT4 in cotton fiber development. GhFucT4 and GbFucT4 shared high sequence identities, and FucT4 had higher expression in H7124 fiber tissues compared with TM-1. Furthermore, ectopic expression of FucT4 in transgenic Arabidopsis promoted root cell elongation, upregulated expression of genes related to cell wall loosening, and led to longer primary root. These results collectively indicate that FucT4 plays an important role in promoting cell elongation and modulating fiber development, which could be utilized to improve fiber quality traits in cotton breeding.

A genome-wide analysis of pentatricopeptide repeat (PPR) protein-encoding genes in four Gossypium species with an emphasis on their expression in floral buds, ovules, and fibers in upland cotton.

Cotton is the most important natural fiber used in textiles. Breeding for "three-lines", i.e., cytoplasmic male sterility (CMS)-based sterile (A), maintainer (B), and restorer (R) line, is a promising approach to harness hybrid vigor in cotton. Pentatricopeptide repeat (PPR) protein-encoding genes play an important role in plant growth and development including restoration of CMS plants to male fertility. However, PPRs, especially those contributing to CMS and fiber development, remain largely unknown in cotton. In this study, a genome-wide identification and characterization of PPR gene family in four Gossypium species with genome sequences (G. arboreum, G. raimondii, G. hirsutum, and G. barbadense) were performed, and expressed PPR genes in developing floral buds, ovules, and fibers were compared to identify possible PPRs related to CMS restoration and fiber development. A total of 539, 558, 1032, and 1055 PPRs were predicted in the above four species, respectively, which were further mapped to chromosomes for a synteny analysis. Through an RNA-seq analysis, 86% (882) PPRs were expressed in flowering buds of upland cotton (G. hirsutum); however, only 11 and 6 were differentially expressed (DE) between restorer R and its near-isogenic (NI) B and between R and its NI A line, respectively. Another RNA-seq analysis identified the expression of only 54% (556) PPRs in 0 and 3 day(s) post-anthesis (DPA) ovules and 24% (247) PPRs in 10 DPA fibers; however, only 59, 6, and 27 PPRs were DE in 0 and 3 DPA ovules, and 10 DPA fibers between two backcross inbred lines (BILs) with differing fiber length, respectively. Only 2 PPRs were DE between Xuzhou 142 and its fiberless and fuzzless mutant. Quantitative RT-PCR analysis confirmed the validity of the RNA-seq results for the gene expression pattern. Therefore, only a very small number of PPRs may be associated with fertility restoration of CMS and genetic differences in fiber initiation and elongation. These results lay a foundation for understanding the roles of PPR genes in cotton, and will be useful in the prioritization of candidate PPR gene functional validation for cotton CMS restoration and fiber development.

The transcription factor MML4_D12 regulates fiber development through interplay with the WD40-repeat protein WDR in cotton.

In planta, a vital regulatory complex, MYB-basic helix-loop-helix (bHLH)-WD40 (MBW), is involved in trichome development and synthesis of anthocyanin and proanthocyanin in Arabidopsis. Usually, WD40 proteins provide a scaffold for protein-protein interaction between MYB and bHLH proteins. Members of subgroup 9 of the R2R3 MYB transcription factors, which includes MYBMIXTA-Like (MML) genes important for plant cell differentiation, are unable to interact with bHLH. In this study, we report that a cotton (Gossypium hirsutum) seed trichome or lint fiber-related GhMML factor, GhMML4_D12, interacts with a diverged WD40 protein (GhWDR) in a process similar to but different from that of the MBW ternary complex involved in Arabidopsis trichome development. Amino acids 250-267 of GhMML4_D12 and the first and third WD40 repeat domains of GhWDR determine their interaction. GhWDR could rescue Arabidopsis ttg1 to its wild type, confirming its orthologous function in trichome development. Our findings shed more light towards understanding the key role of the MML and WD40 families in plants and in the improvement of cotton fiber production.

Long non-coding RNAs and their potential functions in Ligon-lintless-1 mutant cotton during fiber development.

BACKGROUND: Long non-coding RNAs (LncRNAs) are part of genes, which are not translated into proteins and play a vital role in plant growth and development. Nevertheless, the presence of LncRNAs and how they functions in Ligon-lintless-1 mutant during the early cessation of cotton fiber development are still not well understood. In order to investigate the function of LncRNAs in cotton fiber development, it is necessary and important to identify LncRNAs and their potential roles in fiber cell development. RESULTS: In this work, we identified 18,333 LncRNAs, with the proportion of long intergenic noncoding RNAs (LincRNAs) (91.5%) and anti-sense LncRNAs (8.5%), all transcribed from Ligon-lintless-1 (Li1) and wild-type (WT). Expression differences were detected between Ligon-lintless-1 and wild-type at 0 and 8 DPA (day post anthesis). Pathway analysis and Gene Ontology based on differentially expressed LncRNAs on target genes, indicated fatty acid biosynthesis and fatty acid elongation being integral to lack of fiber in mutant cotton. The result of RNA-seq and RT-qPCR clearly singles out two potential LncRNAs, LNC_001237 and LNC_017085, to be highly down-regulated in the mutant cotton. The two LncRNAs were found to be destabilized or repressed by ghr-miR2950. Both RNA-seq analysis and RT-qPCR results in Ligon-lintless-1 mutant and wild-type may provide strong evidence of LNC_001237, LNC_017085 and ghr-miR2950 being integral molecular elements participating in various pathways of cotton fiber development. CONCLUSION: The results of this study provide fundamental evidence for the better understanding of LncRNAs regulatory role in the molecular pathways governing cotton fiber development. Further research on designing and transforming LncRNAs will help not only in the understanding of their functions but will also in the improvement of fiber quality.

Genome-Wide Identification of R2R3-MYB Transcription Factors Regulating Secondary Cell Wall Thickening in Cotton Fiber Development.

MYB proteins represent one of the largest transcription factor (TF) families in plants, some of which act as key transcriptional regulators of secondary cell wall (SCW) biosynthesis. Cotton (Gossypium hirsutum) fiber is thought to be an ideal single-cell model to study cell elongation and SCW biosynthesis. However, little knowledge regarding the TFs controlling fiber SCW biosynthesis, particularly for R2R3-MYBs is known. By far, no comprehensive genome-wide analysis of the secondary wall-associated R2R3-MYBs has been reported in cultivated tetraploid upland cotton. In this study, we identified 419 R2R3-MYB genes by systematically examining the cotton genome. A combination of phylogenetic, RNA-seq and co-expression analyses indicated that 36 R2R3-MYBs were either preferentially or highly expressed in 20 day post anthesis (dpa) fibers and are putative SCW regulators. Among these MYB genes, 22 MYBs are homologs of known SCW MYB proteins and the other 14 MYBs are novel proteins without prior reported SCW biosynthesis-related functions. Finally, we highlighted on the roles of two MYBs named GhMYB46_D13 and GhMYB46_D9, both of which displayed the highest expression in 20 dpa fibers. Expression of GhMYB46_D13 or GhMYB46_D9 individually in Arabidopsis resulted in ectopic SCW deposition in transgenic plants. Furthermore, both GhMYB46_D13 and GhMYB46_D9 were able to activate the cotton fiber SCW cellulose synthase gene promoters. Thus, we have identified 36 R2R3-MYBs as potential SCW regulators in cotton fibers that represent strong candidates for further functional studies during fiber development and SCW thickening.