Systematic identification and functional analysis of root meristem growth factors (RGFs) reveals role of PgRGF1 in modulation of root development and ginsenoside production in Panax ginseng.

Panax ginseng C.A. Mey., known for its medicinal and dietary supplement properties, primarily contains pharmacologically active ginsenosides. However, the regulatory mechanisms linking ginseng root development with ginsenoside biosynthesis are still unclear. Root meristem growth factors (RGFs) are crucial for regulating plant root growth. In our study, we identified five ginseng RGF peptide sequences from the ginseng genome and transcriptome libraries. We treated Arabidopsis and ginseng adventitious roots with exogenous Panax ginseng RGFs (PgRGFs) to assess their activities. Our results demonstrate that PgRGF1 influences gravitropic responses and reduces lateral root formation in Arabidopsis. PgRGF1 has been found to restrict the number and length of ginseng adventitious root branches in ginseng. Given the medicinal properties of ginseng, We determined the ginsenoside content and performed transcriptomic analysis of PgRGF1-treated ginseng adventitious roots. Specifically, the total ginsenoside content in ginseng adventitious roots decreased by 19.98 % and 63.71 % following treatments with 1 µM and 10 µM PgRGF1, respectively, compared to the control. The results revealed that PgRGF1 affects the accumulation of ginsenosides by regulating the expression of genes associated with auxin transportation and ginsenoside biosynthesis. These findings suggest that PgRGF1, as a peptide hormone regulator in ginseng, can modulate adventitious root growth and ginsenoside accumulation.

A dominant negative mutation of GhMYB25-like alters cotton fiber initiation, reducing lint and fuzz.

Cotton (Gossypium hirsutum) fibers, vital natural textile materials, are single-cell trichomes that differentiate from the ovule epidermis. These fibers are categorized as lint (longer fibers useful for spinning) or fuzz (shorter, less useful fibers). Currently, developing cotton varieties with high lint yield but without fuzz remains challenging due to our limited knowledge of the molecular mechanisms underlying fiber initiation. This study presents the identification and characterization of a naturally occurring dominant negative mutation GhMYB25-like_AthapT, which results in a reduced lint and fuzzless phenotype. The GhMYB25-like_AthapT protein exerts its dominant negative effect by suppressing the activity of GhMYB25-like during lint and fuzz initiation. Intriguingly, the negative effect of GhMYB25-like_AthapT could be alleviated by high expression levels of GhMYB25-like. We also uncovered the role of GhMYB25-like in regulating the expression of key genes such as GhPDF2 (PROTODERMAL FACTOR 2), CYCD3; 1 (CYCLIN D3; 1) and PLD (Phospholipase D), establishing its significance as a pivotal transcription factor in fiber initiation. We identified other genes within this regulatory network, expanding our understanding of the determinants of fiber cell fate. These findings offer valuable insights for cotton breeding and contribute to our fundamental understanding of fiber development.

GhWRKY41 forms a positive feedback regulation loop and increases cotton defence response against Verticillium dahliae by regulating phenylpropanoid metabolism.

Despite the established significance of WRKY proteins and phenylpropanoid metabolism in plant immunity, how WRKY proteins modulate aspects of the phenylpropanoid pathway remains undetermined. To understand better the role of WRKY proteins in plant defence, we identified a cotton (Gossypium hirsutum) protein, GhWRKY41, that is, universally and rapidly induced in three disease-resistant cotton cultivars following inoculation with the plant pathogenic fungus, Verticillium dahliae. We show that overexpression of GhWRKY41 in transgenic cotton and Arabidopsis enhances resistance to V. dahliae, while knock-down increases cotton more susceptibility to the fungus. GhWRKY41 physically interacts with itself and directly activates its own transcription. A genome-wide chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), in combination with RNA sequencing (RNA-seq) analyses, revealed that 43.1% of GhWRKY41-binding genes were up-regulated in cotton upon inoculation with V. dahliae, including several phenylpropanoid metabolism master switches, receptor kinases, and disease resistance-related proteins. We also show that GhWRKY41 homodimer directly activates the expression of GhC4H and Gh4CL, thereby modulating the accumulation of lignin and flavonoids. This finding expands our understanding of WRKY-WRKY protein interactions and provides important insights into the regulation of the phenylpropanoid pathway in plant immune responses by a WRKY protein.

Single-cell RNA-seq reveals fate determination control of an individual fibre cell initiation in cotton (Gossypium hirsutum).

Cotton fibre is a unicellular seed trichome, and lint fibre initials per seed as a factor determines fibre yield. However, the mechanisms controlling fibre initiation from ovule epidermis are not understood well enough. Here, with single-cell RNA sequencing (scRNA-seq), a total of 14 535 cells were identified from cotton ovule outer integument of Xu142_LF line at four developmental stages (1.5, 1, 0.5 days before anthesis and the day of anthesis). Three major cell types, fibre, non-fibre epidermis and outer pigment layer were identified and then verified by RNA in situ hybridization. A comparative analysis on scRNA-seq data between Xu142 and its fibreless mutant Xu142 fl further confirmed fibre cluster definition. The developmental trajectory of fibre cell was reconstructed, and fibre cell was identified differentiated at 1 day before anthesis. Gene regulatory networks at four stages revealed the spatiotemporal pattern of core transcription factors, and MYB25-like and HOX3 were demonstrated played key roles as commanders in fibre differentiation and tip-biased diffuse growth respectively. A model for early development of a single fibre cell was proposed here, which sheds light on further deciphering mechanism of plant trichome and the improvement of cotton fibre yield.

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.

Phosphate deficiency enhances cotton resistance to Verticillium dahliae through activating jasmonic acid biosynthesis and phenylpropanoid pathway.

Living in natural environment, plants often suffer from various biotic and abiotic stresses. Phosphate deficiency is a common factor affecting crop production in field, while pathogen invasion is another serious problem. Here we report that Pi-deficient cotton plants exhibit enhanced resistance to Verticillium dahliae. Transcriptomic and histochemical analysis revealed that cotton phenylpropanoid pathway was activated under phosphate deficiency, including lignin and flavonoid biosynthesis. Metabolomic data showed that Pi-deficient cotton accumulates many flavonoids metabolites and displays obvious anti-fungi activity in terms of methanolic extract. Additionally, JA biosynthesis was activated under phosphate deficiency and the Pi-deficiency induced disease resistance was significantly attenuated in GhAOS knock down plants. Taken together, our study demonstrated that phosphate deficiency enhanced cotton resistance to V. dahliae through activating phenylpropanoid pathway and JA biosynthesis, providing new insights into how phosphate deficiency affects plant disease resistance.

Cytosolic Ascorbate Peroxidases Plays a Critical Role in Photosynthesis by Modulating Reactive Oxygen Species Level in Stomatal Guard Cell.

Photosynthetic rate is one of the key factors limiting yield of cotton. Reactive oxygen species (ROS) generated by abiotic stress imposes numerous detrimental effects and causes tremendous loss of yield. It is worth to study whether ROS scavenging enzymes could affect yield through regulating photosynthetic rate in cotton. In this study, we created transgenic cotton with changes of endogenous ROS by overexpressing or suppressing the expression of cytosolic ascorbate peroxidases (APXs), which are hydrogen peroxide (H2O2) scavenging enzymes in plants. The suppression of cytosolic APXs by RNAi brings about a great influence on plant growth and development. Plant height and leaf size declined, and yield-related traits including single boll weight, seed weight, seed size, and lint weight dropped significantly, in IAO lines (cytosolic APX-suppressed lines). The stunted plant growth was due to the decrease of plant photosynthetic rate. The evidences showed that increased ROS level in guard cells inhibited stomatal opening and suppressed the absorption of CO2 and H2O in IAO line. The decrease of water content and the increase of water loss rate in leaf exacerbated the decline of photosynthetic rate in cytosolic APX-suppressed lines. Based on these results, it implies that cytosolic APXs as a whole play an important role in maintaining REDOX balance to regulate photosynthetic rate and yield in cotton.

Combined GWAS and eQTL analysis uncovers a genetic regulatory network orchestrating the initiation of secondary cell wall development in cotton.

The cotton fibre serves as a valuable experimental system to study cell wall synthesis in plants, but our understanding of the genetic regulation of this process during fibre development remains limited. We performed a genome-wide association study (GWAS) and identified 28 genetic loci associated with fibre quality in allotetraploid cotton. To investigate the regulatory roles of these loci, we sequenced fibre transcriptomes of 251 cotton accessions and identified 15 330 expression quantitative trait loci (eQTL). Analysis of local eQTL and GWAS data prioritised 13 likely causal genes for differential fibre quality in a transcriptome-wide association study (TWAS). Characterisation of distal eQTL revealed unequal genetic regulation patterns between two subgenomes, highlighted by an eQTL hotspot (Hot216) that established a genome-wide genetic network regulating the expression of 962 genes. The primary regulatory role of Hot216, and specifically the gene encoding a KIP-related protein, was found to be the transcriptional regulation of genes responsible for cell wall synthesis, which contributes to fibre length by modulating the developmental transition from rapid cell elongation to secondary cell wall synthesis. This study uncovered the genetic regulation of fibre-cell development and revealed the molecular basis of the temporal modulation of secondary cell wall synthesis during plant cell elongation.

Reference genome sequences of two cultivated allotetraploid cottons, Gossypium hirsutum and Gossypium barbadense.

Allotetraploid cotton species (Gossypium hirsutum and Gossypium barbadense) have long been cultivated worldwide for natural renewable textile fibers. The draft genome sequences of both species are available but they are highly fragmented and incomplete1-4. Here we report reference-grade genome assemblies and annotations for G. hirsutum accession Texas Marker-1 (TM-1) and G. barbadense accession 3-79 by integrating single-molecule real-time sequencing, BioNano optical mapping and high-throughput chromosome conformation capture techniques. Compared with previous assembled draft genomes1,3, these genome sequences show considerable improvements in contiguity and completeness for regions with high content of repeats such as centromeres. Comparative genomics analyses identify extensive structural variations that probably occurred after polyploidization, highlighted by large paracentric/pericentric inversions in 14 chromosomes. We constructed an introgression line population to introduce favorable chromosome segments from G. barbadense to G. hirsutum, allowing us to identify 13 quantitative trait loci associated with superior fiber quality. These resources will accelerate evolutionary and functional genomic studies in cotton and inform future breeding programs for fiber improvement.

Evolutionary dynamics of 3D genome architecture following polyploidization in cotton.

The formation of polyploids significantly increases the complexity of transcriptional regulation, which is expected to be reflected in sophisticated higher-order chromatin structures. However, knowledge of three-dimensional (3D) genome structure and its dynamics during polyploidization remains poor. Here, we characterize 3D genome architectures for diploid and tetraploid cotton, and find the existence of A/B compartments and topologically associated domains (TADs). By comparing each subgenome in tetraploids with its extant diploid progenitor, we find that genome allopolyploidization has contributed to the switching of A/B compartments and the reorganization of TADs in both subgenomes. We also show that the formation of TAD boundaries during polyploidization preferentially occurs in open chromatin, coinciding with the deposition of active chromatin modification. Furthermore, analysis of inter-subgenomic chromatin interactions has revealed the spatial proximity of homoeologous genes, possibly associated with their coordinated expression. This study advances our understanding of chromatin organization in plants and sheds new light on the relationship between 3D genome evolution and transcriptional regulation.

A global survey of alternative splicing in allopolyploid cotton: landscape, complexity and regulation.

Alternative splicing (AS) is a crucial regulatory mechanism in eukaryotes, which acts by greatly increasing transcriptome diversity. The extent and complexity of AS has been revealed in model plants using high-throughput next-generation sequencing. However, this technique is less effective in accurately identifying transcript isoforms in polyploid species because of the high sequence similarity between coexisting subgenomes. Here we characterize AS in the polyploid species cotton. Using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq), we developed an integrated pipeline for Iso-Seq transcriptome data analysis (https://github.com/Nextomics/pipeline-for-isoseq). We identified 176 849 full-length transcript isoforms from 44 968 gene models and updated gene annotation. These data led us to identify 15 102 fibre-specific AS events and estimate that c. 51.4% of homoeologous genes produce divergent isoforms in each subgenome. We reveal that AS allows differential regulation of the same gene by miRNAs at the isoform level. We also show that nucleosome occupancy and DNA methylation play a role in defining exons at the chromatin level. This study provides new insights into the complexity and regulation of AS, and will enhance our understanding of AS in polyploid species. Our methodology for Iso-Seq data analysis will be a useful reference for the study of AS in other species.

Asymmetric subgenome selection and cis-regulatory divergence during cotton domestication.

Comparative population genomics offers an excellent opportunity for unraveling the genetic history of crop domestication. Upland cotton (Gossypium hirsutum) has long been an important economic crop, but a genome-wide and evolutionary understanding of the effects of human selection is lacking. Here, we describe a variation map for 352 wild and domesticated cotton accessions. We scanned 93 domestication sweeps occupying 74 Mb of the A subgenome and 104 Mb of the D subgenome, and identified 19 candidate loci for fiber-quality-related traits through a genome-wide association study. We provide evidence showing asymmetric subgenome domestication for directional selection of long fibers. Global analyses of DNase I-hypersensitive sites and 3D genome architecture, linking functional variants to gene transcription, demonstrate the effects of domestication on cis-regulatory divergence. This study provides new insights into the evolution of gene organization, regulation and adaptation in a major crop, and should serve as a rich resource for genome-based cotton improvement.

A cotton fiber-preferential promoter, PGbEXPA2, is regulated by GA and ABA in Arabidopsis.

KEY MESSAGE: PGbEXPA2 (Promoter of GbEXPA2 ) was preferentially and strongly expressed during cotton fiber development, and the 461-bp PGbEXPA2 fragment was essential for responding to exogenous GA and ABA in Arabidopsis. Cotton fibers are highly elongated single-cell, unbranched and non-glandular seed trichomes. Previous studies have reported that the transcript level of GbEXPA2 is significantly up-regulated during fiber cell elongation, suggesting that GbEXPA2 has an important function in fiber development. In this study, the promoter of GbEXPA2 (839 bp) from the D(T) sub-genome was isolated from Gossypium barbadense 3-79. Consistent with the expression pattern of GbEXPA2, the promoter PGbEXPA2 was able to express GUS to high levels in elongating fibers, but not in the root, stem, or leaf. In Arabidopsis, GUS activity was only found in the rosette leaf trichomes and rosette leaf vascular tissue, indicating that the transcription factors which bind to PGbEXPA2 in the leaf trichomes of transgenic Arabidopsis were similar to those found in cotton fiber. A deletion analysis of PGbEXPA2 revealed that a 461-bp fragment was sufficient to drive GUS expression in cotton fibers and Arabidopsis rosette leaf trichomes. Exogenous phytohormonal treatments on transgenic Arabidopsis with different promoter lengths (P-839, P-705, P-588 and P-461) showed that GUS activity in Arabidopsis trichomes could be strongly up-regulated by GA and, in contrast, down-regulated by ABA.