An insight into the gene expression evolution in Gossypium species based on the leaf transcriptomes.

BACKGROUND: Gene expression pattern is associated with biological phenotype and is widely used in exploring gene functions. Its evolution is also crucial in understanding species speciation and divergence. The genus Gossypium is a bona fide model for studying plant evolution and polyploidization. However, the evolution of gene expression during cotton species divergence has yet to be extensively discussed. RESULTS: Based on the seedling leaf transcriptomes, this work analyzed the transcriptomic content and expression patterns across eight cotton species, including six diploids and two natural tetraploids. Our findings indicate that, while the biological function of these cotton transcriptomes remains largely conserved, there has been significant variation in transcriptomic content during species divergence. Furthermore, we conducted a comprehensive analysis of expression distances across cotton species. This analysis lends further support to the use of G. arboreum as a substitute for the A-genome donor of natural cotton polyploids. Moreover, our research highlights the evolution of stress-responsive pathways, including hormone signaling, fatty acid degradation, and flavonoid biosynthesis. These processes appear to have evolved under lower selection pressures, presumably reflecting their critical role in the adaptations of the studied cotton species to diverse environments. CONCLUSIONS: In summary, this study provided insights into the gene expression variation within the genus Gossypium and identified essential genes/pathways whose expression evolution was closely associated with the evolution of cotton species. Furthermore, the method of characterizing genes and pathways under unexpected high or slow selection pressure can also serve as a new strategy for gene function exploration.

The functions of laccase gene GhLAC15 in fiber colouration and development in brown-colored cotton.

The monotonicity of color type in naturally colored cottons (NCCs) has become the main limiting factor to their widespread use, simultaneously coexisting with poor fiber quality. The synchronous improvement of fiber quality and color become more urgent and crucial as the demand for sustainable development increases. The homologous gene of wild cotton Gossypium stocksii LAC15 in G. hirsutum, GhLAC15, was also dominantly expressed in the developing fibers of brown cotton XC20 from 5 DPA (day post anthesis) to 25 DPA, especially at the secondary cell wall thickening stage (20 DPA and 25 DPA). In XC20 plants with downregulated GhLAC15 (GhLAC15i), a remarkable reduction in proanthocyanidins (PAs) and lignin contents was observed. Some of the key genes in the phenylpropane and flavonoid biosynthesis pathway were down-regulated in GhLAC15i plants. Notably, the fiber length of GhLAC15i plants showed an obvious increase and the fiber color was lightened. Moreover, we found that the thickness of cotton fiber cell wall was decreased in GhLAC15i plants and the fiber surface became smoother compared to that of WT. Taken together, this study revealed that GhLAC15 played an important role in PAs and lignin biosynthesis in naturally colored cotton fibers. It might mediate fiber color and fiber quality by catalyzing PAs oxidation and lignin polymerization, ultimately regulating fiber colouration and development.

A study of the effect of natural brown cotton gauze on the healing of infected wounds.

BACKGROUND: Medical dressings are designed to promote wound healing and reduce infection. The aim of project is to investigate the effect of natural brown colored cotton dressings on the healing of infected wounds in E.coli animals. MATERIALS AND METHODS: In this study, degreased white cotton gauze was used as the control group, with degreased brown cotton gauze and degreased bleached brown cotton gauze as the experimental group 1 and experimental group 2, to investigate the effect on the repair of post-infectious wound damage in animals by establishing an infected wound model in rats with E.coli as the infecting organism. RESULTS: The ability to promote healing of infected wounds was investigated by analyzing the wound healing status, macroscopic wound healing rate, hematoxylin-eosin staining, Masson staining, secretion of inflammatory factors by Elisa assay. The result showed that at day 14 of wound healing, the macroscopic wound healing rate was greater than 98% for all three groups of dressings; the collagen content reached 49.85 ± 5.84% in the experimental group 1 and 53.48 ± 5.32% in the experimental group 2, which was higher than the control group; brown cotton gauze promotes skin wound healing by shortening the inflammatory period in both groups. The expression of three inflammatory factors THF-α, IL-2, and IL-8 and three cytokines MMP-3, MMP-8, and MMP-9 were lower than that of the control group. CONCLUSIONS: It was found that natural brown cotton gauze has better repairing and promoting healing effect on infected wounds. It opens up the application of natural brown cotton gauze in the treatment of infected wounds.

The cotton pectin methyl esterase gene GhPME21 functions in microspore development and fertility in Gossypium hirsutum L.

Pectin widely exists in higher plants' cell walls and intercellular space of higher plants and plays an indispensable role in plant growth and development. We identified 55 differentially expressed genes related to pectin degradation by transcriptomic analysis in the male sterile mutant, ms1. A gene encoding pectin methylesterase (GhPME21) was found to be predominantly expressed in the developing stamens of cotton but was significantly down-regulated in ms1 stamens. The tapetal layer of GhPME21 interfered lines (GhPME21i) was significantly thickened compared to that of WT at the early stage; anther compartment morphology of GhPME21i lines was abnormal, and the microspore wall was broken at the middle stage; Alexander staining showed that the pollen grains of GhPME21i lines differed greatly in volume at the late stage. The mature pollen surfaces of GhPME21i lines were deposited with discontinuous and broken sheets and prickles viewed under SEM. Fewer pollen tubes were observed to germinate in vitro in GhPME21i lines, while tiny of those in vivo were found to elongate to the ovary. The seeds harvested from GhPME21i lines as pollination donors were dry and hollow. The changes of phenotypes in GhPME21i lines at various stages illustrated that the GhPME21 gene played a vital role in the development of cotton stamens and controlled plant fertility by affecting stamen development, pollen germination, and pollen tube elongation. The findings of this study laid the groundwork for further research into the molecular mechanisms of PMEs involved in microspore formation and the creation of cotton male sterility materials.

Cloning and functional analysis of GhDFR1, a key gene of flavonoid synthesis pathway in naturally colored cotton.

BACKGROUND: The naturally colored brown cotton fiber is the most widely used environmentally friendly textile material, which primarily contains proanthocyanidins and their derivatives. Many structural genes in the flavonoid synthesis pathway are known to improve the genetic resources of naturally colored cotton. Among them, DFR is a crucial late enzyme to synthesis both anthocyanins and proanthocyanidins in the plant flavonoid pathway. METHODS: The protein sequences of GhDFRs were analyzed using bioinformatic tools. The expression levels of GhDFRs in various tissues and organs of upland cotton Zongxu1 (ZX1), were analyzed by quantitative real-time PCR, and the expression pattern of GhDFR1 during fiber development of white cotton and brown cotton was analyzed further. The function of GhDFR1 in NCC ZX1 was preliminarily analyzed by virus induced gene silencing (VIGS) technology. RESULTS: Bioinformatic analysis revealed that GhDFRs sequences in upland cotton genome were extremely conserved. Furthermore, evolutionary tree analysis revealed that the functions of GhDFR1 and GhDFR2, and GhDFR3 and GhDFR4, presented different and shared some similarities. Our study showed GhDFR1 and GhDFR2 were specifically expressed in fibers, while GhDFR3 and GhDFR4 were specifically expressed in petals. GhDFR1 was exclusively expressed in brown cotton fiber at various stages of development and progressively increased with the growth of fiber, but the trend of expression in white cotton was quite the opposite. We silenced GhDFR1 expression in brown cotton fiber using VIGS technology, and observed the VIGS-interference plants. After reducing the expression level of GhDFR1, the period for significant GhDFR1 expression in the developing fibers changed, reducing the content of anthocyanins, and lightening the color of mature cotton fibers. CONCLUSION: GhDFR1 was preferentially expressed in brown cotton during fiber development. The timing of GhDFR1 expression for flavonoid synthesis altered, resulting in anthocyanin contents reduced and the fiber color of the GhDFR1i lines lightened. These findings showed the role of GhDFR1 in fiber coloration of NCC and provided a new candidate for NCC genetic improvement.

Function deficiency of GhOMT1 causes anthocyanidins over-accumulation and diversifies fibre colours in cotton (Gossypium hirsutum).

Naturally coloured cotton (NCC) fibres need little or no dyeing process in textile industry to low-carbon emission and are environment-friendly. Proanthocyanidins (PAs) and their derivatives were considered as the main components causing fibre coloration and made NCCs very popular and healthy, but the monotonous fibre colours greatly limit the wide application of NCCs. Here a G. hirsutum empurpled mutant (HS2) caused by T-DNA insertion is found to enhance the anthocyanidins biosynthesis and accumulate anthocyanidins in the whole plant. HPLC and LC/MS-ESI analysis confirmed the anthocyanidins methylation and peonidin, petunidin and malvidin formation are blocked. The deficiency of GhOMT1 in HS2 was associated with the activation of the anthocyanidin biosynthesis and the altered components of anthocyanidins. The transcripts of key genes in anthocyanidin biosynthesis pathway are significantly up-regulated in HS2, while transcripts of the genes for transport and decoration were at similar levels as in WT. To investigate the potential mechanism of GhOMT1 deficiency in cotton fibre coloration, HS2 mutant was crossed with NCCs. Surprisingly, offsprings of HS2 and NCCs enhanced PAs biosynthesis and increased PAs levels in their fibres from the accumulated anthocyanidins through up-regulated GhANR and GhLAR. As expected, multiple novel lines with improved fibre colours including orange red and navy blue were produced in their generations. Based on this work, a new strategy for breeding diversified NCCs was brought out by promoting PA biosynthesis. This work will help shed light on mechanisms of PA biosynthesis and bring out potential molecular breeding strategy to increase PA levels in NCCs.

A novel multimodal needs assessment to inform the longitudinal education program for an international interprofessional critical care team.

BACKGROUND: The current global pandemic has caused unprecedented strain on critical care resources, creating an urgency for global critical care education programs. Learning needs assessment is a core element of designing effective, targeted educational interventions. In theory, multimodal methods are preferred to assess both perceived and unperceived learning needs in diverse, interprofessional groups, but a robust design has rarely been reported. Little is known about the best approach to determine the learning needs of international critical care professionals. METHOD: We conducted multimodal learning needs assessment in a pilot group of critical care professionals in China using combined quantitative and qualitative methods. The assessments consisted of three phases: 1) Twenty statements describing essential entrustable professional activities (EPAs) were generated by a panel of critical care education experts using a Delphi method. 2) Eleven Chinese critical care professionals participating in a planned education program were asked to rank-order the statements according to their perceived learning priority using Q methodology. By-person factor analysis was used to study the typology of the opinions, and post-ranking focus group interviews were employed to qualitatively explore participants' reasoning of their rankings. 3) To identify additional unperceived learning needs, daily practice habits were audited using information from medical and nursing records for 3 months. RESULTS: Factor analysis of the rank-ordered statements revealed three learning need patterns with consensual and divergent opinions. All participants expressed significant interest in further education on organ support and disease management, moderate interest in quality improvement topics, and relatively low interest in communication skills. Interest in learning procedure/resuscitation skills varied. The chart audit revealed suboptimal adherence to several evidence-based practices and under-perceived practice gaps in patient-centered communication, daily assessment of antimicrobial therapy discontinuation, spontaneous breathing trial, and device discontinuation. CONCLUSIONS: We described an effective mixed-methods assessment to determine the learning needs of an international, interprofessional critical care team. The Q survey and focus group interviews prioritized and categorized perceived learning needs. The chart audit identified additional practice gaps that were not identified by the learners. Multimodal methods can be employed in cross-cultural scenarios to customize and better target medical education curricula.

Biochemical and Expression Analyses Revealed the Involvement of Proanthocyanidins and/or Their Derivatives in Fiber Pigmentation of Gossypium stocksii.

The wild cotton species Gossypium stocksii produces a brown fiber that provides a valuable resource for the color improvement of naturally colored cotton (NCC) fiber. However, the biochemical basis and molecular mechanism of its fiber pigmentation remain unclear. Herein, we analyzed the dynamics of proanthocyanidins (PAs) accumulation in developing the fiber of G. stocksii, which suggested a similar role of PAs and/or their derivatives in the fiber coloration of G. stocksii. In addition, comparative transcriptomics analyses revealed that the PA biosynthetic genes were expressed at higher levels and for a longer period in developing fibers of G. stocksii than G. arboreum (white fiber), and the transcription factors, such as TT8, possibly played crucial regulatory roles in regulating the PA branch genes. Moreover, we found that the anthocyanidin reductase (ANR) was expressed at a higher level than the leucoanthocyanidin reductases (LARs) and significantly upregulated during fiber elongation, suggesting a major role of ANR in PA synthesis in G. stocksii fiber. In summary, this work revealed the accumulation of PAs and the expression enhancement of PA biosynthetic genes in developing fibers of G. stocksii. We believe this work will help our understanding of the molecular mechanisms of cotton fiber coloration and further promote the future breeding of novel NCCs.

Integrative Analysis of Expression Profiles of mRNA and MicroRNA Provides Insights of Cotton Response to Verticillium dahliae.

Cotton Verticillium wilt, caused by the notorious fungal phytopathogen Verticillium dahliae (V. dahliae), is a destructive soil-borne vascular disease and severely decreases cotton yield and quality worldwide. Transcriptional and post-transcriptional regulation of genes responsive to V. dahliae are crucial for V. dahliae tolerance in plants. However, the specific microRNAs (miRNAs) and the miRNA/target gene crosstalk involved in cotton resistance to Verticillium wilt remain largely limited. To investigate the roles of regulatory RNAs under V. dahliae induction in upland cotton, mRNA and small RNA libraries were constructed from mocked and infected roots of two upland cotton cultivars with the V. dahliae-sensitive cultivar Jimian 11 (J11) and the V. dahliae-tolerant cultivar Zhongzhimian 2 (Z2). A comparative transcriptome analysis revealed 8330 transcripts were differentially expressed under V. dahliae stress and associated with several specific biological processes. Moreover, small RNA sequencing identified a total of 383 miRNAs, including 330 unique conserved miRNAs and 53 novel miRNAs. Analysis of the regulatory network involved in the response to V. dahliae stress revealed 31 differentially expressed miRNA−mRNA pairs, and the up-regulation of GhmiR395 and down-regulation of GhmiR165 were possibly involved in the response to V. dahliae by regulating sulfur assimilation through the GhmiR395-APS1/3 module and the establishment of the vascular pattern and secondary cell wall formation through GhmiR165-REV module, respectively. The integrative analysis of mRNA and miRNA expression profiles from upland cotton lays the foundation for further investigation of regulatory mechanisms of resistance to Verticillium wilt in cotton and other crops.

Cloning and characterization of the MYB transcription factor gene GhTT2 in Gossypium hirsutum.

As one of the important secondary metabolites, proanthocyanidins (PAs) are not only a defense mechanism for plants to cope with biotic and abiotic stresses, but also a key factor affecting the development and quality of plants. Although the biosynthetic and metabolic pathways of proanthocyanidins have been basically clarified in the model plants, the regulatory mechanism in cotton has not been fully elucidated. In this work, a transcription factor gene GhTT2 (transparent testa 2) was cloned from Gossypium hirsutum. Its gene structure, expression pattern, subcellular localization, and function were further analyzed. The results show that the GhTT2 has a typical MYB domain and is predominantly expressed in fibers. Its transcription level was negatively correlated with anthocyanin content. The GhTT2-GFP fusion protein is located in the nucleus. Moreover, yeast transformation results show that GhTT2 has obvious transcriptional activation characteristics. Furthermore, the content of proanthocyanidins in GhTT2-silenced cottons is significantly reduced, indicating that GhTT2 may be involved in regulation of the proanthocyanidins biosynthesis in Gossypium hirsutum. These results provide a reference for further elucidating the molecular mechanisms of MYB transcription factors involved in the regulation of the biosynthetic pathway of PAs.

Roles of the 14-3-3 gene family in cotton flowering.

BACKGROUND: In plants, 14-3-3 proteins, also called GENERAL REGULATORY FACTORs (GRFs), encoded by a large multigene family, are involved in protein-protein interactions and play crucial roles in various physiological processes. No genome-wide analysis of the GRF gene family has been performed in cotton, and their functions in flowering are largely unknown. RESULTS: In this study, 17, 17, 31, and 17 GRF genes were identified in Gossypium herbaceum, G. arboreum, G. hirsutum, and G. raimondii, respectively, by genome-wide analyses and were designated as GheGRFs, GaGRFs, GhGRFs, and GrGRFs, respectively. A phylogenetic analysis revealed that these proteins were divided into ε and non-ε groups. Gene structural, motif composition, synteny, and duplicated gene analyses of the identified GRF genes provided insights into the evolution of this family in cotton. GhGRF genes exhibited diverse expression patterns in different tissues. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that the GhGRFs interacted with the cotton FLOWERING LOCUS T homologue GhFT in the cytoplasm and nucleus, while they interacted with the basic leucine zipper transcription factor GhFD only in the nucleus. Virus-induced gene silencing in G. hirsutum and transgenic studies in Arabidopsis demonstrated that GhGRF3/6/9/15 repressed flowering and that GhGRF14 promoted flowering. CONCLUSIONS: Here, 82 GRF genes were identified in cotton, and their gene and protein features, classification, evolution, and expression patterns were comprehensively and systematically investigated. The GhGRF3/6/9/15 interacted with GhFT and GhFD to form florigen activation complexs that inhibited flowering. However, GhGRF14 interacted with GhFT and GhFD to form florigen activation complex that promoted flowering. The results provide a foundation for further studies on the regulatory mechanisms of flowering.

Identification and functional analysis of a pollen fertility-associated gene GhGLP4 of Gossypium hirsutum L.

KEY MESSAGE: Cotton male fertility-associated gene GhGLP4, encoding a germin-like protein, is essential for anthers development by keeping ROS homeostasis through reducing H2O2 level. Utilization of heterosis is an important way to increase cotton yield and improve fiber quality in hybrid cotton development programs. Male sterility is used in the development of cotton hybrids to reduce the cost of hybrid seed production by eliminating the process of emasculation. From the transcriptome analysis of genic male sterile mutant (ms1) and its background C312 of G. hirsutum, a gene encoding germin-like protein (GhGLP4) was found significantly down-regulated in different developmental stages of ms1 anthers. To explore the gene function in cotton fertility, GhGLP4 was further studied and interfered by virus-induced gene silencing. In the GhGLP4 interfered cotton lines, the expression level of GhGLP4 was significantly decreased in the stamens, and the down-regulation of GhGLP4 resulted in pollen sac closure, stigma exertion, filament shortening, decrease in the number of anthers and complete male sterility. The expression levels of respiratory burst oxidase homologs (Rboh, NADPH oxidase) were significantly altered. Further investigation showed that the SOD activity decreased while the H2O2 content increased in the atypical stamens. These results indicated that GhGLP4 gene affected the cotton anther development through maintenance of ROS homeostasis by H2O2 reduction.

Transcriptomic analysis of gene expression of Verticillium dahliae upon treatment of the cotton root exudates.

BACKGROUND: Cotton Verticillium wilt is one of the most devastating diseases for cotton production in the world. Although this diseases have been widely studied at the molecular level from pathogens, the molecular basis of V. dahliae interacted with cotton has not been well examined. RESULTS: In this study, RNA-seq analysis was carried out on V. dahliae samples cultured by different root exudates from three cotton cultivars (a susceptible upland cotton cultivar, a tolerant upland cotton cultivar and a resistant island cotton cultivar) and water for 0 h, 6 h, 12 h, 24 h and 48 h. Statistical analysis of differentially expressed genes revealed that V. dahliae responded to all kinds of root exudates but more strongly to susceptible cultivar than to tolerant and resistant cultivars. Go analysis indicated that 'hydrolase activity, hydrolyzing O-glycosyl compounds' related genes were highly enriched in V. dahliae cultured by root exudates from susceptible cotton at early stage of interaction, suggesting genes related to this term were closely related to the pathogenicity of V. dahliae. Additionally, 'transmembrane transport', 'coenzyme binding', 'NADP binding', 'cofactor binding', 'oxidoreductase activity', 'flavin adenine dinucleotide binding', 'extracellular region' were commonly enriched in V. dahliae cultured by all kinds of root exudates at early stage of interaction (6 h and 12 h), suggesting that genes related to these terms were required for the initial steps of the roots infections. CONCLUSIONS: Based on the GO analysis results, the early stage of interaction (6 h and 12 h) were considered as the critical stage of V. dahliae-cotton interaction. Comparative transcriptomic analysis detected that 31 candidate genes response to root exudates from cotton cultivars with different level of V. dahliae resistance, 68 response to only susceptible cotton cultivar, and 26 genes required for development of V. dahliae. Collectively, these expression data have advanced our understanding of key molecular events in the V. dahliae interacted with cotton, and provided a framework for further functional studies of candidate genes to develop better control strategies for the cotton wilt disease.

Genome-wide identification of cold responsive transcription factors in Brassica napus L.

BACKGROUND: Cold stress is one of the primary environmental factors that affect plant growth and productivity, especially for crops like Brassica napus that live through cold seasons. Till recently, although a number of genes and pathways involved in B. napus cold response have been revealed by independent studies, a genome-wide identification of the key regulators and the regulatory networks is still lack. In this study, we investigated the transcriptomes of cold stressed semi-winter and winter type rapeseeds in short day condition, mainly with the purpose to systematically identify the functional conserved transcription factors (TFs) in cold response of B. napus. RESULTS: Global modulation of gene expression was observed in both the semi-winter type line (158A) and the winter type line (SGDH284) rapeseeds, in response to a seven-day chilling stress in short-day condition. Function analysis of differentially expressed genes (DEGs) revealed enhanced stresses response mechanisms and inhibited photosynthesis in both lines, as well as a more extensive inhibition of some primary biological processes in the semi-winter type line. Over 400 TFs were differentially expressed in response to cold stress, including 56 of them showed high similarity to the known cold response TFs and were consistently regulated in 158A and SGDH284, as well as 25 TFs which targets were over-represented in the total DEGs. A further investigation based on their interactions indicated the critical roles of several TFs in cold response of B. napus. CONCLUSION: In summary, our results revealed the alteration of gene expression in cold stressed semi-winter and winter ecotype B. napus lines and provided a valuable collection of candidate key regulators involved in B. napus response to cold stress, which could expand our understanding of plant stress response and benefit the future improvement of the breed of rapeseeds.

Correction to: Functional analysis of GhCHS, GhANR and GhLAR in colored fiber formation of Gossypium hirsutum L.

In the original publication of this article [1], the authors pointed out the Fig. 4b was same with Fig. 4c. The correct Fig. 4b should be below.

Functional analysis of GhCHS, GhANR and GhLAR in colored fiber formation of Gossypium hirsutum L.

BACKGROUND: The formation of natural colored fibers mainly results from the accumulation of different anthocyanidins and their derivatives in the fibers of Gossypium hirsutum L. Chalcone synthase (CHS) is the first committed enzyme of flavonoid biosynthesis, and anthocyanidins are transported into fiber cells after biosynthesis mainly by Anthocyanidin reductase (ANR) and Leucoanthocyanidin reductase (LAR) to present diverse colors with distinct stability. The biochemical and molecular mechanism of pigment formation in natural colored cotton fiber is not clear. RESULTS: The three key genes of GhCHS, GhANR and GhLAR were predominantly expressed in the developing fibers of colored cotton. In the GhCHSi, GhANRi and GhLARi transgenic cottons, the expression levels of GhCHS, GhANR and GhLAR significantly decreased in the developing cotton fiber, negatively correlated with the content of anthocyanidins and the color depth of cotton fiber. In colored cotton Zongxu1 (ZX1) and the GhCHSi, GhANRi and GhLARi transgenic lines of ZX1, HZ and ZH, the anthocyanidin contents of the leaves, cotton kernels, the mixture of fiber and seedcoat were all changed and positively correlated with the fiber color. CONCLUSION: The three genes of GhCHS, GhANR and GhLAR were predominantly expressed early in developing colored cotton fibers and identified to be a key genes of cotton fiber color formation. The expression levels of the three genes affected the anthocyanidin contents and fiber color depth. So the three genes played a crucial part in cotton fiber color formation and has important significant to improve natural colored cotton quality and create new colored cotton germplasm resources by genetic engineering.

Characterization and Activity Analyses of the FLOWERING LOCUS T Promoter in Gossypium Hirsutum.

Flowering transition is a crucial development process in cotton (Gossypium hirsutum L.), and the flowering time is closely correlated with the timing of FLOWERING LOCUS T (FT) expression. However, the mechanism underlying the coordination of various cis-regulatory elements in the FT promoter of cotton has not been determined. In this study, a 5.9-kb promoter of FT was identified from cotton. A bioinformatics analysis showed that multiple insertion-deletion sites existed in the 5.9-kb promoter. Different expression levels of a reporter gene, and the induction by sequential deletions in GhFT promoter, demonstrated that 1.8-kb of the GhFT promoter was stronger than 4.2-, 4.8-, and 5.9-kb promoter fragments. The binding sites of the CONSTANS (CO) and NUCLEAR FACTOR Y transcription factors were located within the 1.0-kb sequence upstream of the FT transcription start site. A large number of repeat segments were identified in proximal promoter regions (-1.1 to -1.4 kb). A complementation analysis of deletion constructs between 1.0 and 1.8 kb of G. hirsutum, Gossypium arboretum, and Gossypium raimondii FT promoters revealed that the 1.0-kb fragment significantly rescued the late-flowering phenotype of the Arabidopsis FT loss-of-function mutant ft-10, whereas the 1.8-kb promoter only slightly rescued the late-flowering phenotype. Furthermore, the conserved CORE motif in the cotton FT promoter is an atypical TGTG(N2-3)ATG, but the number of arbitrary bases between TGTG and ATG is uncertain. Thus, the proximal FT promoter region might play an important role affecting the activity levels of FT promoters in cotton flowering.

iTRAQ-based comparative proteomic analysis provides insights into somatic embryogenesis in Gossypium hirsutum L.

KEY MESSAGE: iTRAQ based proteomic identified key proteins and provided new insights into the molecular mechanisms underlying somatic embryogenesis in cotton. Somatic embryogenesis, which involves cell dedifferentiation and redifferentiation, has been used as a model system for understanding molecular events of plant embryo development in vitro. In this study, we performed comparative proteomics analysis using samples of non-embryogenic callus (NEC), embryogenic callus (EC) and somatic embryo (SE) using the isobaric tags for relative and absolute quantitation (iTRAQ) technology. In total, 5892 proteins were identified amongst the three samples. The majority of these proteins (93.4%) were found to have catalytic activity, binding activity, transporter activity or structural molecular activity. Of these proteins, 1024 and 858 were differentially expressed in NEC versus EC and EC versus SE, respectively. Compared to NEC, EC had 452 and 572 down- and up-regulated proteins, respectively, and compared to EC, SE had 647 and 221 down- and up-regulated proteins, respectively. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis indicated that genetic information transmission, plant hormone transduction, glycolysis, fatty acid biosynthesis and metabolism, galactose metabolism were the top pathways involved in somatic embryogenesis. Our proteomics results not only confirmed our previous transcriptomic results on the role of the polyamine metabolic pathways and stress responses in cotton somatic embryogenesis, but identified key proteins important for cotton somatic embryogenesis and provided new insights into the molecular mechanisms underlying somatic embryogenesis in cotton.

Highly Efficient Targeted Gene Editing in Upland Cotton Using the CRISPR/Cas9 System.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system has been shown to be able to induce highly efficient mutagenesis in the targeted DNA of many plants, including cotton, and has become an important tool for investigation of gene function and crop improvement. Here, we developed a simple and easy to operate CRISPR/Cas9 system and demonstrated its high editing efficiency in cotton by targeting-ALARP, a gene encoding alanine-rich protein that is preferentially expressed in cotton fibers. Based on sequence analysis of the target site in the 10 transgenic cottons containing CRISPR/Cas9, we found that the mutation frequencies of GhALARP-A and GhALARP-D target sites were 71.4⁻100% and 92.9⁻100%, respectively. The most common editing event was deletion, but deletion together with large insertion was also observed. Mosaic mutation editing events were detected in most transgenic plants. No off-target mutation event was detected in any the 15 predicted sites analyzed. This study provided mutants for further study of the function of GhALARP in cotton fiber development. Our results further demonstrated the feasibility of use of CRISPR/Cas9 as a targeted mutagenesis tool in cotton, and provided an efficient tool for targeted mutagenesis and functional genomics in cotton.

De novo transcriptome analysis reveals insights into dynamic homeostasis regulation of somatic embryogenesis in upland cotton (G. hirsutum L.).

Plant regeneration via somatic embryogenesis (SE) is the key step for genetic improvement of cotton (Gossypium hirsutum L.) through genetic engineering mediated by Agrobacteria, but the molecular mechanisms underlying SE in cotton is still unclear. Here, RNA-Sequencing was used to analyze the genes expressed during SE and their expression dynamics using RNAs isolated from non-embryogenic callus (NEC), embryogenic callus (EC) and somatic embryos (SEs). A total of 101, 670 unigenes were de novo assembled. The genes differentially expressed (DEGs) amongst NEC, EC and SEs were identified, annotated and classified. More DEGs were found between SEs and EC than between EC and NEC. A significant number of DEGs were related to hormone homeostasis, stress and ROS responses, and metabolism of polyamines. To confirm the expression dynamics of selected DEGs involved in various pathways, experiments were set up to investigate the effects of hormones (Indole-3-butytric acid, IBA; Kinetin, KT), polyamines, H2O2 and stresses on SE. Our results showed that exogenous application of IBA and KT positively regulated the development of EC and SEs, and that polyamines and H2O2 promoted the conversion of EC into SEs. Furthermore, we found that low and moderate stress is beneficial for proliferation of EC and SEs formation. Together, our global analysis of transcriptomic dynamics reveals that hormone homeostasis, polyamines, and stress response synergistically regulating SE in cotton.