Genome-wide identification and functional analysis of the SiCIN gene family in foxtail millet (Setaria italica L.).

Cell wall invertase (CIN) is a vital member of plant invertase (INV) and plays a key role in the breakdown of sucrose. This enzyme facilitates the hydrolysis of sucrose into glucose and fructose, which is crucial for various aspects of plant growth and development. However, the function of CIN genes in foxtail millet (Setaria italica) is less studied. In this research, we used the blast-p of NCBI and TBtools for bidirectional comparison, and a total of 13 CIN genes (named SiCINs) were identified from foxtail millet by using Arabidopsis and rice CIN sequences as reference sequences. The phylogenetic tree analysis revealed that the CIN genes can be categorized into three subfamilies: group 1, group 2, and group 3. Furthermore, upon conducting chromosomal localization analysis, it was observed that the 13 SiCINs were distributed unevenly across five chromosomes. Cis-acting elements of SiCIN genes can be classified into three categories: plant growth and development, stress response, and hormone response. The largest number of cis-acting elements were those related to light response (G-box) and the cis-acting elements related to seed-specific regulation (RY-element). qRT-PCR analysis further confirmed that the expression of SiCIN7 and SiCIN8 in the grain was higher than that in any other tissues. The overexpression of SiCIN7 in Arabidopsis improved the grain size and thousand-grain weight, suggesting that SiCIN7 could positively regulate grain development. Our findings will help to further understand the grain-filling mechanism of SiCIN and elucidate the biological mechanism underlying the grain development of SiCIN.

Widespread incomplete lineage sorting and introgression shaped adaptive radiation in the Gossypium genus.

Cotton (Gossypium) stands as a crucial economic crop, serving as the primary source of natural fiber for the textile sector. However, the evolutionary mechanisms driving speciation within the Gossypium genus remain unresolved. In this investigation, we leveraged 25 Gossypium genomes and introduced four novel assemblies-G. harknessii, G. gossypioides, G. trilobum, and G. klotzschianum (Gklo)-to delve into the speciation history of this genus. Notably, we encountered intricate phylogenies potentially stemming from introgression. These complexities are further compounded by incomplete lineage sorting (ILS), a factor likely to have been instrumental in shaping the swift diversification of cotton. Our focus subsequently shifted to the rapid radiation episode during a concise period in Gossypium evolution. For a recently diverged lineage comprising G. davidsonii, Gklo, and G. raimondii, we constructed a finely detailed ILS map. Intriguingly, this analysis revealed the non-random distribution of ILS regions across the reference Gklo genome. Moreover, we identified signs of robust natural selection influencing specific ILS regions. Noteworthy variations pertaining to speciation emerged between the closely related sister species Gklo and G. davidsonii. Approximately 15.74% of speciation structural variation genes and 12.04% of speciation-associated genes were estimated to intersect with ILS signatures. These findings enrich our understanding of the role of ILS in adaptive radiation, shedding fresh light on the intricate speciation history of the Gossypium genus.

Genome-Wide Identification and Functional Analysis of RF2 Gene Family and the Critical Role of GhRF2-32 in Response to Drought Stress in Cotton.

Cotton is an important natural fiber crop. The RF2 gene family is a member of the bZIP transcription factor superfamily, which plays an important role in plant resistance to environmental stresses. In this paper, the RF2 gene family of four cotton species was analyzed genome-wide, and the key gene RF2-32 was cloned for functional verification. A total of 113 RF2 genes were identified in the four cotton species, and the RF2 family was relatively conserved during the evolution of cotton. Chromosome mapping and collinear analysis indicated that fragment replication was the main expansion mode of RF2 gene family during evolution. Cis-element analysis showed that there were many elements related to light response, hormone response and abiotic stress response in the promoters of RF2 genes. The transcriptome and qRT-PCR analysis of RF2 family genes in upland cotton showed that RF2 family genes responded to salt stress and drought stress. GhRF2-32 protein was localized in the cell nucleus. Silencing the GhRF2-32 gene showed less leaf wilting and increased total antioxidant capacity under drought and salt stress, decreased malondialdehyde content and increased drought and salt tolerance. This study revealed the evolutionary and functional diversity of the RF2 gene family, which laid a foundation for the further study of stress-resistant genes in cotton.

Genome-wide chromatin accessibility landscape and dynamics of transcription factor networks during ovule and fiber development in cotton.

BACKGROUND: The development of cotton fiber is regulated by the orchestrated binding of regulatory proteins to cis-regulatory elements associated with developmental genes. The cis-trans regulatory dynamics occurred throughout the course of cotton fiber development are elusive. Here we generated genome-wide high-resolution DNase I hypersensitive sites (DHSs) maps to understand the regulatory mechanisms of cotton ovule and fiber development. RESULTS: We generated DNase I hypersensitive site (DHS) profiles from cotton ovules at 0 and 3 days post anthesis (DPA) and fibers at 8, 12, 15, and 18 DPA. We obtained a total of 1185 million reads and identified a total of 199,351 DHSs through ~ 30% unique mapping reads. It should be noted that more than half of DNase-seq reads mapped multiple genome locations and were not analyzed in order to achieve a high specificity of peak profile and to avoid bias from repetitive genomic regions. Distinct chromatin accessibilities were observed in the ovules (0 and 3 DPA) compared to the fiber elongation stages (8, 12, 15, and 18 DPA). Besides, the chromatin accessibility during ovules was particularly elevated in genomic regions enriched with transposable elements (TEs) and genes in TE-enriched regions were involved in ovule cell division. We analyzed cis-regulatory modules and revealed the influence of hormones on fiber development from the regulatory divergence of transcription factor (TF) motifs. Finally, we constructed a reliable regulatory network of TFs related to ovule and fiber development based on chromatin accessibility and gene co-expression network. From this network, we discovered a novel TF, WRKY46, which may shape fiber development by regulating the lignin content. CONCLUSIONS: Our results not only reveal the contribution of TEs in fiber development, but also predict and validate the TFs related to fiber development, which will benefit the research of cotton fiber molecular breeding.

Genome-wide characterization of trichome birefringence-like genes provides insights into fiber yield improvement.

Cotton is an important fiber crop. The cotton fiber is an extremely long trichome that develops from the epidermis of an ovule. The trichome is a general and multi-function plant organ, and trichome birefringence-like (TBL) genes are related to trichome development. At the genome-wide scale, we identified TBLs in four cotton species, comprising two cultivated tetraploids (Gossypium hirsutum and G. barbadense) and two ancestral diploids (G. arboreum and G. raimondii). Phylogenetic analysis showed that the TBL genes clustered into six groups. We focused on GH_D02G1759 in group IV because it was located in a lint percentage-related quantitative trait locus. In addition, we used transcriptome profiling to characterize the role of TBLs in group IV in fiber development. The overexpression of GH_D02G1759 in Arabidopsis thaliana resulted in more trichomes on the stems, thereby confirming its function in fiber development. Moreover, the potential interaction network was constructed based on the co-expression network, and it was found that GH_D02G1759 may interact with several genes to regulate fiber development. These findings expand our knowledge of TBL family members and provide new insights for cotton molecular breeding.

Large-scale metabolome analysis reveals dynamic changes of metabolites during foxtail millet grain filling.

Compared with traditional staple crops, foxtail millet grain is rich in nutrition and beneficial to human health. Foxtail millet is also tolerance to various abiotic stresses, including drought, making it a good plant for growing in barren land. The study on the composition of metabolites and its dynamics changes during grain development is helpful to understand the process of foxtail millet grain formation. In our study, metabolic and transcriptional analysis were used to uncover the metabolic processes that could influence grain filling in foxtail millet. A total of 2104 known metabolites, belonging to 14 categories, were identified during grain filling. Functional analysis of DAMs and DEGs revealed a stage-specific metabolic properties in foxtail millet grain filling. Some important metabolic processes, such as flavonoid biosynthesis, glutathione metabolism, linoleic acid metabolism, starch and sucrose metabolism and valine, leucine and isoleucine biosynthesis were co-mapped for DEGs and DAMs. Thus, we constructed a gene-metabolite regulatory network of these metabolic pathways to explain their potential functions during grain filling. Our study showed the important metabolic processes during grain filling and focused on the dynamic changes of related metabolites and genes at different stages, which provided a reference for us to better understand and improve foxtail millet grain development and yield.

In Vitro Inhibition of Growth, Biofilm Formation, and Persisters of Staphylococcus aureus by Pinaverium Bromide.

Biofilm or persister cells formed by Staphylococcus aureus are closely related to pathogenicity. However, no antimicrobials exist to inhibit biofilm formation or persister cells induced by S. aureus in clinical practice. This study found that pinaverium bromide had antibacterial activity against S. aureus, with the MIC50/MIC90 at 12.5/25 µM, respectively. Pinaverium bromide (at 4 × MIC) showed a rapid bactericidal effect on S. aureus planktonic cells, and it was more effective (at least 1-log10 cfu/mL) than linezolid, vancomycin, and ampicillin at 4 h of the time-killing test. Pinaverium bromide (at 10 × MIC) significantly inhibited the formation of S. aureus persister cells (at least 3-log10 cfu/mL) than linezolid, vancomycin, and ampicillin at 24, 48, 72, 96, and 120 h of the time-killing test. Biofilm formation and adherent cells of S. aureus isolates were significantly inhibited by pinaverium bromide (at 1/2 or 1/4 × MICs). The fluorescence intensity of the membrane polarity of S. aureus increased with the treatment of pinaverium bromide (≥1 × MIC), and the MICs of pinaverium bromide increased by 4 times with the addition of cell membrane phospholipids, phosphatidyl glycerol and cardiolipin. The cell viabilities of human hepatocellular carcinoma cells HepG2 and Huh7, mouse monocyte-macrophage cells J774, and human hepatic stellate cells LX-2 were slightly inhibited by pinaverium bromide (<50 µM). There were 54 different abundance proteins detected in the pinaverium bromide-treated S. aureus isolate by proteomics analysis, of which 33 proteins increased, whereas 21 proteins decreased. The abundance of superoxide dismutase sodM and ica locus proteins icaA and icaB decreased. While the abundance of global transcriptional regulator spxA and Gamma-hemolysin component B increased. In conclusion, pinaverium bromide had an antibacterial effect on S. aureus and significantly inhibited the formation of biofilm and persister cells of S. aureus.

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

KEY MESSAGE: 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.

Genome-Wide Identification and Characterization of the PPO Gene Family in Cotton (Gossypium) and Their Expression Variations Responding to Verticillium Wilt Infection.

Polyphenol oxidases (PPOs) are copper-binding metalloproteinases encoded by nuclear genes, ubiquitously existing in the plastids of microorganisms, plants, and animals. As one of the important defense enzymes, PPOs have been reported to participate in the resistant processes that respond to diseases and insect pests in multiple plant species. However, PPO gene identification and characterization in cotton and their expression patterns under Verticillium wilt (VW) treatment have not been clearly studied. In this study, 7, 8, 14, and 16 PPO genes were separately identified from Gossypium arboreum, G. raimondii, G. hirsutum, and G. barbadense, respectively, which were distributed within 23 chromosomes, though mainly gathered in chromosome 6. The phylogenetic tree manifested that all the PPOs from four cotton species and 14 other plants were divided into seven groups, and the analyses of the conserved motifs and nucleotide sequences showed highly similar characteristics of the gene structure and domains in the cotton PPO genes. The dramatically expressed differences were observed among the different organs at various stages of growth and development or under the diverse stresses referred to in the published RNA-seq data. Quantitative real-time PCR (qRT-PCR) experiments were also performed on the GhPPO genes in the roots, stems, and leaves of VW-resistant MBI8255 and VW-susceptible CCRI36 infected with Verticillium dahliae V991, proving the strong correlation between PPO activity and VW resistance. A comprehensive analysis conducted on cotton PPO genes contributes to the screening of the candidate genes for subsequent biological function studies, which is also of great significance for the in-depth understanding of the molecular genetic basis of cotton resistance to VW.

Overexpression of cotton GhNAC072 gene enhances drought and salt stress tolerance in transgenic Arabidopsis.

BACKGROUND: Crops face several environmental stresses (biotic and abiotic), thus resulting in severe yield losses. Around the globe abiotic stresses are the main contributors of plant damages, primarily drought and salinity. Many genes and transcription factors are involved in abiotic and biotic stress responses. NAC TF (Transcription Factors) improves tolerance to stresses by controlling the physiological and enzyme activities of crops. RESULTS: In current research, GhNAC072 a highly upregulated TF in RNA-Seq was identified as a hub gene in the co-expression network analysis (WGCNA). This gene was transformed to Arabidopsis thaliana to confirm its potential role in drought and salt stress tolerance. Significant variations were observed in the morpho-physiological traits with high relative leaf water contents, chlorophyll contents, higher germination and longer root lengths of the overexpressed lines and low excised leaf loss and ion leakage as compared to the wildtype plants. Besides, overexpressed lines have higher amounts of antioxidants and low oxidant enzyme activities than the wildtype during the period of stress exposure. CONCLUSIONS: In summary, the above analysis showed that GhNAC072 might be the true candidate involved in boosting tolerance mechanisms under drought and salinity stress.

Evolutionary divergence of duplicated genomes in newly described allotetraploid cottons.

Allotetraploid cotton (Gossypium) species represents a model system for the study of plant polyploidy, molecular evolution, and domestication. Here, chromosome-scale genome sequences were obtained and assembled for two recently described wild species of tetraploid cotton, Gossypium ekmanianum [(AD)6, Ge] and Gossypium stephensii [(AD)7, Gs], and one early form of domesticated Gossypium hirsutum, race punctatum [(AD)1, Ghp]. Based on phylogenomic analysis, we provide a dated whole-genome level perspective for the evolution of the tetraploid Gossypium clade and resolved the evolutionary relationships of Gs, Ge, and domesticated G. hirsutum. We describe genomic structural variation that arose during Gossypium evolution and describe its correlates-including phenotypic differentiation, genetic isolation, and genetic convergence-that contributed to cotton biodiversity and cotton domestication. Presence/absence variation is prominent in causing cotton genomic structural variations. A presence/absence variation-derived gene encoding a phosphopeptide-binding protein is implicated in increasing fiber length during cotton domestication. The relatively unimproved Ghp offers the potential for gene discovery related to adaptation to environmental challenges. Expanded gene families enoyl-CoA δ isomerase 3 and RAP2-7 may have contributed to abiotic stress tolerance, possibly by targeting plant hormone-associated biochemical pathways. Our results generate a genomic context for a better understanding of cotton evolution and for agriculture.

Homoeolog gene expression analysis reveals novel expression biases in upland hybrid cotton under intraspecific hybridization.

Hybridization is useful to enhance the yield potential of agronomic crops in the world. Cotton has genome doubling due to the allotetraploid process and hybridization in coordination with duplicated genome can produce more yield and adaptability. Therefore, the expression of homoeologous gene pairs between hybrids and inbred parents is vital to characterize the genetic source of heterosis in cotton. Investigation results of homoeolog gene pairs between two contrasting hybrids and their respective inbred parents identified 36853 homoeolog genes in hybrids. It was observed both high and low hybrids had similar trends in homoeolog gene expression patterns in each tissue under study. An average of 96% of homoeolog genes had no biased expression and their expressions were derived from the equal contribution of both parents. Besides, very few homoeolog genes (an average of 1%) showed no biased or novel expression in both hybrids. The functional analysis described secondary metabolic pathways had a majority of novel biased homoeolog genes in hybrids. These results contribute preliminary knowledge about how hybridization affects expression patterns of homoeolog gene pairs in upland cotton hybrids. Our study also highlights the functional genomics of metabolic genes to explore the genetic mechanism of heterosis in cotton.

Conservation and Divergence of Phosphoenolpyruvate Carboxylase Gene Family in Cotton.

Phosphoenolpyruvate carboxylase (PEPC) is an important enzyme in plants, which regulates carbon flow through the TCA cycle and controls protein and oil biosynthesis. Although it is important, there is little research on PEPC in cotton, the most important fiber crop in the world. In this study, a total of 125 PEPCs were identified in 15 Gossypium genomes. All PEPC genes in cotton are divided into six groups and each group generally contains one PEPC member in each diploid cotton and two in each tetraploid cotton. This suggests that PEPC genes already existed in cotton before their divergence. There are additional PEPC sub-groups in other plant species, suggesting the different evolution and natural selection during different plant evolution. PEPC genes were independently evolved in each cotton sub-genome. During cotton domestication and evolution, certain PEPC genes were lost and new ones were born to face the new environmental changes and human being needs. The comprehensive analysis of collinearity events and selection pressure shows that genome-wide duplication and fragment duplication are the main methods for the expansion of the PEPC family, and they continue to undergo purification selection during the evolutionary process. PEPC genes were widely expressed with temporal and spatial patterns. The expression patterns of PEPC genes were similar in G. hirsutum and G. barbadense with a slight difference. PEPC2A and 2D were highly expressed in cotton reproductive tissues, including ovule and fiber at all tested developmental stages in both cultivated cottons. However, PEPC1A and 1D were dominantly expressed in vegetative tissues. Abiotic stress also induced the aberrant expression of PEPC genes, in which PEPC1 was induced by both chilling and salinity stresses while PEPC5 was induced by chilling and drought stresses. Each pair (A and D) of PEPC genes showed the similar response to cotton development and different abiotic stress, suggesting the similar function of these PEPCs no matter their origination from A or D sub-genome. However, some divergence was also observed among their origination, such as PEPC5D was induced but PEPC5A was inhibited in G. barbadense during drought treatment, suggesting that a different organized PEPC gene may evolve different functions during cotton evolution. During cotton polyploidization, the homologues genes may refunction and play different roles in different situations.

Protoplast Dissociation and Transcriptome Analysis Provides Insights to Salt Stress Response in Cotton.

As one of the pioneer crops widely planted in saline-alkaline areas, Gossypium provides daily necessities, including natural fiber, vegetable proteins, and edible oils. However, cotton fiber yield and quality are highly influenced by salt stress. Therefore, elucidating the molecular mechanisms of cotton in response to salinity stress is importance to breed new cultivars with high tolerance. In this study, we first developed a method for single-cell RNA-seq based on isolating protoplast from cotton root tips; then, we studied the impact of salinity stress on gene expression profiling and their dynamic changes using the developed high-efficiency method for protoplast dissociation suitable for single-cell RNA-seq. A total of 3391 and 2826 differentially expressed genes (DEGs) were identified in salt-treated samples before and after protoplast dissociation, respectively, which were enriched into several molecular components, including response to stimulus, response to stress, and cellular macromolecule metabolic process by gene ontology (GO) analysis. Plant hormone signal transduction, phenylpropanoid biosynthesis, and MAPK signaling pathway were found to be enriched via Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Twenty-two and nine salinity-responsive DEGs participated in plant hormone signaling and MAPK signaling in roots, before and after protoplast dissociation, respectively; six upregulated DEGs were involved in ABA signaling transduction, namely, Ga04G2111, Ga07G0142, Ga09G2061, Ga10G0262, Ga01G0063, and Ga08G1915 which indicates their potential functions on plants adapting to salt stress. Additionally, 384 and 257 transcription factors (TFs) were differentially expressed in salt-stress roots before and after protoplast dissociation, respectively, of which significantly up-regulated TFs mainly belonged to the AP2/ERF-ERF family, which implied their potential roles responding to salt stress. These results not only provide novel insights to reveal the regulatory networks in plant's root response to salt stress, but also lay the solid foundation for further exploration on cellular heterogeneity by single-cell transcriptome sequencing.

Identification of Candidate Cotton Genes Associated With Fiber Length Through Quantitative Trait Loci Mapping and RNA-Sequencing Using a Chromosome Segment Substitution Line.

Fiber length is an important determinant of fiber quality, and it is a quantitative multi-genic trait. Identifying genes associated with fiber length is of great importance for efforts to improve fiber quality in the context of cotton breeding. Integrating transcriptomic information and details regarding candidate gene regions can aid in candidate gene identification. In the present study, the CCRI45 line and a chromosome segment substitution line (CSSL) with a significantly higher fiber length (MBI7747) were utilized to establish F2 and F2:3 populations. Using a high-density genetic map published previously, six quantitative trait loci (QTLs) associated with fiber length and two QTLs associated with fiber strength were identified on four chromosomes. Within these QTLs, qFL-A07-1, qFL-A12-2, qFL-A12-5, and qFL-D02-1 were identified in two or three environments and confirmed by a meta-analysis. By integrating transcriptomic data from the two parental lines and through qPCR analyses, four genes associated with these QTLs including Cellulose synthase-like protein D3 (CSLD3, GH_A12G2259 for qFL-A12-2), expansin-A1 (EXPA1, GH_A12G1972 for qFL-A12-5), plasmodesmata callose-binding protein 3 (PDCB3, GH_A12G2014 for qFL-A12-5), and Polygalacturonase (At1g48100, GH_D02G0616 for qFL-D02-1) were identified as promising candidate genes associated with fiber length. Overall, these results offer a robust foundation for further studies regarding the molecular basis for fiber length and for efforts to improve cotton fiber quality.

Identification of the Golden-2-like transcription factors gene family in Gossypium hirsutum.

BACKGROUND: Golden2-Like (GLK) transcription factors are a type of transcriptional regulator in plants. They play a pivotal role in the plant physiological activity process and abiotic stress response. METHODS: In this study, the potential function of GLK family genes in Gossypium hirsutum was studied based on genomic identification, phylogenetic analysis, chromosome mapping and cis-regulatory elements prediction. Gene expression of nine key genes were analyzed by qRT-PCR experiments. RESULTS: Herein, we identified a total of 146 GhGLK genes in Gossypium hirsutum, which were unevenly distributed on each of the chromosomes. There were significant differences in the number and location of genes between the At sub-genome and the Dt sub-genome. According to the phylogenetic analysis, they were divided into ten subgroups, each of which had very similar number and structure of exons and introns. Some cis-regulatory elements were identified through promoter analysis, including five types of elements related to abiotic stress response, five types of elements related to phytohormone and five types of elements involved in growth and development. Based on public transcriptome data analysis, we identified nine key GhGLKs involved in salt, cold, and drought stress. The qRT-PCR results showed that these genes had different expression patterns under these stress conditions, suggesting that GhGLK genes played an important role in abiotic stress response. This study laid a theoretical foundation for the screening and functional verification of genes related to stress resistance of GLK gene family in cotton.

DNA Methylation and RNA-Sequencing Analysis Show Epigenetic Function During Grain Filling in Foxtail Millet (Setaria italica L.).

Grain filling is a crucial process for crop yield and quality. Certain studies already gained insight into the molecular mechanism of grain filling. However, it is unclear whether epigenetic modifications are associated with grain filling in foxtail millet. Global DNA methylation and transcriptome analysis were conducted in foxtail millet spikelets during different stages to interpret the epigenetic effects of the grain filling process. The study employed the whole-genome bisulfite deep sequencing and advanced bioinformatics to sequence and identify all DNA methylation during foxtail millet grain filling; the DNA methylation-mediated gene expression profiles and their involved gene network and biological pathway were systematically studied. One context of DNA methylation, namely, CHH methylation, was accounted for the largest percentage, and it was gradually increased during grain filling. Among all developmental stages, the methylation levels were lowest at T2, followed by T4, which mainly occurred in CHG. The distribution of differentially methylated regions (DMR) was varied in the different genetic regions for three contexts. In addition, gene expression was negatively associated with DNA methylation. Evaluation of the interconnection of the DNA methylome and transcriptome identified some stage-specific differentially expressed genes associated with the DMR at different stages compared with the T1 developmental stage, indicating the potential function of epigenetics on the expression regulation of genes related to the specific pathway at different stages of grain development. The results demonstrated that the dynamic change of DNA methylation plays a crucial function in gene regulation, revealing the potential function of epigenetics in grain development in foxtail millet.

Functional Characterization of GhACX3 Gene Reveals Its Significant Role in Enhancing Drought and Salt Stress Tolerance in Cotton.

The acyl-coenzyme A oxidase 3 (ACX3) gene involved in the ß-oxidation pathway plays a critical role in plant growth and development as well as stress response. Earlier on, studies focused primarily on the role of ß-oxidation limited to fatty acid breakdown. However, ACX3 peroxisomal ß-oxidation pathways result in a downstream cascade of events that act as a transduction of biochemical and physiological responses to stress. A role that is yet to be studied extensively. In this study, we identified 20, 18, 22, 23, 20, 11, and 9 proteins in Gossypium hirsutum, G. barbadense, G. tomentosum, G. mustelinum, G. darwinii, G. arboretum, and G. raimondii genomes, respectively. The tetraploid cotton genome had protein ranging between 18 and 22, while diploids had between 9 and 11. After analyzing the gene family evolution or selection pressure, we found that this gene family undergoes purely segmental duplication both in diploids and tetraploids. W-Box (WRKY-binding site), ABRE, CAAT-Box, TATA-box, MYB, MBS, LTR, TGACG, and CGTCA-motif are abiotic stress cis-regulatory elements identified in this gene family. All these are the binding sites for abiotic stress transcription factors, indicating that this gene is essential. Genes found in G. hirsutum showed a clear response to drought and salinity stress, with higher expression under drought and salt stress, particularly in the leaf and root, according to expression analysis. We selected Gh_DO1GO186, one of the highly expressed genes, for functional characterization. We functionally characterized the GhACX3 gene through overexpression and virus-induced gene silencing (VIGS). Overexpression of this gene enhanced tolerance under stress, which was exhibited by the germination assay. The overexpressed seed growth rate was faster relative to control under drought and salt stress conditions. The survival rate was also higher in overexpressed plants relative to control plants under stress. In contrast, the silencing of the GhACX3 gene in cotton plants resulted in plants showing the stress susceptibility phenotype and reduced root length compared to control. Biochemical analysis also demonstrated that GhACX3-silenced plants experienced oxidative stress while the overexpressed plants did not. This study has revealed the importance of the ACX3 family during stress tolerance and can breed stress-resilient cultivar.

Genome-wide identification of the MIOX gene family and their expression profile in cotton development and response to abiotic stress.

The enzyme myo-inositol oxygenase (MIOX) catalyzes the myo-inositol into glucuronic acid. In this study, 6 MIOX genes were identified from all of the three diploid cotton species (Gossypium arboretum, Gossypium herbaceum and Gossypium raimondii) and Gossypioides kirkii, 12 MIOX genes were identified from two domesticated tetraploid cottons Gossypium hirsutum, Gossypium barbadense, and 11 MIOX genes were identified from three wild tetraploid cottons Gossypium tomentosum, Gossypium mustelinum and Gossypium darwinii. The number of MIOX genes in tetraploid cotton genome is roughly twice that of diploid cotton genome. Members of MIOX family were classified into six groups based on the phylogenetic analysis. Integrated analysis of collinearity events and chromosome locations suggested that both whole genome duplication and segmental duplication events contributed to the expansion of MIOX genes during cotton evolution. The ratios of non-synonymous (Ka) and synonymous (Ks) substitution rates revealed that purifying selection was the main force driving the evolution of MIOX genes. Numerous cis-acting elements related to light responsive element, defense and stress responsive element were identified in the promoter of the MIOX genes. Expression analyses of MIOX genes based on RNA-seq data and quantitative real time PCR showed that MIOX genes within the same group shared similar expression patterns with each other. All of these results provide the foundation for further study of the biological functions of MIOX genes in cotton environmental adaptability.

Micro-FTIR combined with curve fitting method to study cellulose crystallinity of developing cotton fibers.

This study aimed to use micro-FTIR with transmission mode to investigate cellulose crystallinity of developing cotton fibers. Compared with ATR-FTIR method, we found that micro-FTIR can obtain more information of cellulose inside of the developing cotton fibers, especially in high wavenumber of 2800-3000 cm-1 region. Combined with curve fitting method, a new IR crystallinity index (CI) method named wax crystallinity index (WCI) was introduced to evaluate the cellulose crystallinity in the development of cotton fibers based on the peak and area ratios of 2900 cm-1/2850 cm-1 and 2900 cm-1/2920 cm-1. The obtained WCI values demonstrated an excellent coefficient of determination with X-ray diffraction (XRD) CI method with the value up to 0.99. This study suggested that micro-FTIR was an effective technique to qualitatively analyze the crystallinity in developing cotton fibers combined with curve fitting method.