Cotton, a cornerstone of the global textile industry, relies on two primary species—upland cotton (Gossypium hirsutum) and sea-island cotton (Gossypium barbadense)—to meet over 90% of the world’s fiber demand.
While both species share a common ancestor, they have evolved distinct traits over millennia, with sea-island cotton prized for its luxurious fibers and upland cotton valued for its resilience.
A groundbreaking 2025 study published in the Journal of Advanced Research leverages single-nucleus RNA sequencing (snRNA-seq) to compare leaf cell types between these species, uncovering how cellular and genetic differences drive their unique characteristics.
Critical Role of Cotton Leaves in Fiber Production
Leaves are far more than photosynthetic factories—they are dynamic hubs where critical processes like water regulation, pest defense, and nutrient transport occur. In cotton, leaf health directly impacts fiber yield and quality.
Traditional studies often analyze whole leaves, blending data from diverse cell types and masking their specialized roles. Single-cell RNA sequencing (scRNA-seq) addresses this limitation by profiling gene activity in individual cells, creating a detailed cellular map.
Researchers applied this technique to young leaves of CRI12 (upland cotton) and XH21 (sea-island cotton), sequencing over 18,000 nuclei to identify 22 cell clusters in upland cotton and 20 in sea-island cotton.
These clusters represented six core cell types, including mesophyll cells for photosynthesis, epidermal cells for protection, and pigment gland cells for toxin production.
How Single-Cell RNA Sequencing Reveals Cotton Leaf Diversity
The study’s methodology began with isolating nuclei from leaf tissues, followed by sequencing using the 10x Genomics Chromium platform. This process detected 57,384 genes in upland cotton and 59,459 in sea-island cotton, revealing a wealth of cellular activity.
Advanced algorithms grouped cells into clusters based on gene expression similarities. For example, mesophyll cells showed high activity in photosynthesis-related genes like RBCS and LHCB3, while pigment gland cells expressed toxin-producing genes like CDN and CYP76B6.
To compare species, researchers constructed a Comparative Single-Cell Transcriptomic Map (CSCTM) using 48,000 shared genes. Clusters with a Pearson correlation >0.7 were deemed evolutionarily conserved, while others highlighted species-specific divergence.
Sea-Island Cotton’s Unique Cell Cluster Enhances Disease Resistance
One standout discovery was Gb_cluster14, a sea-island cotton-specific epidermal cell cluster enriched with pathogen resistance genes. This cluster hosted GbNF-YA7, a transcription factor critical for combating Verticillium wilt, a devastating fungal disease.
Silencing GbNF-YA7 via virus-induced gene silencing (VIGS) increased disease severity by 45% and fungal biomass by 2.5-fold.
Other genes in this cluster, like HIPP3 (heavy metal detoxification) and WIN2 (wound signaling), underscored its role in stress adaptation.
Remarkably, 97 genes in Gb_cluster14 lacked homologs in upland cotton, suggesting sea-island cotton evolved unique defenses through natural or human-driven selection.
Divergent Pigment Gland Development
Pigment glands, responsible for producing the pest-deterrent compound gossypol, exhibited developmental differences between the species. Upland cotton had four pigment gland subclusters, while sea-island cotton had three. These subclusters represented distinct stages:
- initiation cells (triggering gland formation),
- secretory cells (producing gossypol),
- dying cells (undergoing programmed cell death).
The gene WRKY15 emerged as a key regulator of gossypol levels. Silencing WRKY15 reduced toxin production by 30% without affecting gland numbers, a finding confirmed by CRISPR-edited upland cotton lines. This highlights WRKY15’s role in fine-tuning chemical defenses rather than gland development.
Genomic Variations Shape Cell-Specific Traits in Cotton Species
The study identified 6,845 structural variations (SVs) between the species, including 487 in promoter regions (DNA segments controlling gene activation). These SVs influenced 434 genes, such as Ghir_A10G023500, a disease resistance gene active only in sea-island cotton’s vascular cells.
Genome-wide association studies (GWAS) linked specific DNA markers to Verticillium wilt resistance, including a region on chromosome A10 and a SNP on chromosome D11. These findings bridge genomic changes to cellular behavior, offering targets for precision breeding.
Vascular Cells Emerge as Key Players
Vascular cells, which transport water and nutrients, were hotspots for disease resistance genes. Fifty out of 60 known resistance genes, including Ghir_A10G023500 and Ghir_D11G033400, showed elevated activity in these cells.
Since Verticillium wilt invades through vascular tissues, this localization explains sea-island cotton’s stronger defense. Companion cells, which support vascular function, also shared genetic links to fiber development, suggesting a trade-off between fiber quality and stress adaptation.
Stress Response Genes Highlight Cellular Adaptation Strategies
Both species shared stress-response genes, but their activity varied across cell types. For example, DUF1230, a cold-responsive gene, was twice as active in sea-island cotton’s mesophyll cells.
Epidermal cells highly expressed GhMLP423, an insect resistance gene, with 20% higher activity in sea-island cotton.
Such cell-specific strategies highlight how plants optimize survival mechanisms across tissues.
Future Applications of Single-Cell Insights in Cotton Breeding
The study’s insights pave the way for breeding cotton varieties with enhanced disease resistance, tailored gossypol levels, and climate resilience. By targeting genes like GbNF-YA7 or WRKY15, breeders can develop crops that balance pest resistance with food safety.
The CSCTM framework also enables predictive breeding, linking DNA changes to cellular outcomes. Future research could expand to roots and fibers, integrate epigenomics, and refine CRISPR tools for cell-type-specific editing.
Conclusion
This research revolutionizes our understanding of cotton biology, revealing how cellular diversity drives species-specific traits. For farmers, it promises hardier, higher-yielding varieties.
For scientists, it offers a blueprint to explore cellular mechanisms in other crops. As climate challenges intensify, such innovations are vital for sustainable agriculture and global food security.
Power Terms
Single-cell transcriptomic map: A detailed “atlas” showing which genes are active in individual cells within a tissue. This helps scientists understand how different cell types work. For example, in cotton leaves, this map showed which cells handle photosynthesis versus disease defense. It’s like a census of cellular jobs.
Orthologous genes: Genes in different species that evolved from the same ancestor gene. For instance, a cotton gene for leaf growth might have a matching “cousin” gene in tomatoes. These help compare species by showing shared biological processes.
Cell clusters: Groups of cells with similar gene activity. In this study, cotton leaf cells formed 22 clusters in one species and 20 in another. These clusters represent cell types like “photosynthesis cells” or “disease-fighting cells.”
Pigment gland cells: Special cotton cells that produce gossypol, a toxic chemical protecting against pests. These cells act like natural pesticide factories. The study found differences in how two cotton species manage these glands.
snRNA-seq (single-nucleus RNA sequencing): A technique to study gene activity by analyzing RNA in individual cell nuclei. It’s like reading the “work orders” inside each cell. Researchers used this to compare leaf cells in two cotton types.
VIGS (Virus-Induced Gene Silencing): A method to “turn off” specific genes using modified viruses. For example, silencing the NF-YA7 gene made cotton more vulnerable to fungal infections, proving its role in disease resistance.
GWAS (Genome-Wide Association Study): A way to link genes to traits by analyzing DNA variations in populations. The study used GWAS to find cotton genes tied to Verticillium wilt resistance, a devastating plant disease.
Mesophyll cells: Leaf cells specialized for photosynthesis. They’re like solar panels converting sunlight into energy. The study showed these cells make up over half of cotton leaf cells and carry many domestication-related genes.
Vascular cells: “Pipeline” cells that transport water and nutrients. In cotton leaves, these cells also play a role in fighting Verticillium wilt, a disease that clogs plant veins.
Pseudotime trajectory analysis: A computational method to guess how cells develop over time. Researchers used this to map how cotton pigment gland cells mature, like tracking a caterpillar turning into a butterfly.
Domestication-related genes: Genes changed during crop breeding. The study found many such genes active in photosynthetic cells, suggesting humans unknowingly selected for better energy production in cotton leaves.
Structural variations (SVs): Large DNA differences between species, like missing or duplicated gene regions. The team found 6,845 SVs between the two cotton types, some affecting gene activity in specific cell types.
Gossypol: A toxic compound in cotton pigment glands that deters pests. The study identified WRKY15 as a gene controlling gossypol levels without altering gland numbers, useful for breeding safer cottonseeds.
Abiotic stress response (ASR) genes: Genes helping plants survive drought, salt, or heat. Researchers found 37 ASR genes shared by both cotton types, but some behaved differently in specific cells, hinting at hidden adaptability.
Verticillium wilt resistance: A plant’s ability to fight a soil-borne fungus. The study linked this trait to genes active in vascular cells, explaining why sea-island cotton resists the disease better than upland cotton.
Companion cells: Helper cells supporting nutrient transport in plant veins. These cells showed unique gene activity differences between cotton species, possibly influencing growth rates.
Epidermal cells: Outer leaf cells forming a protective “skin.” The study found a sea-island cotton-specific epidermal cluster rich in disease-resistance genes, like a specialized security team.
Proliferating cells: Cells actively dividing to grow new tissues. In cotton leaves, these cells had markers similar to human stem cells, driving leaf expansion.
Nuclear Factor-YA7 (NF-YA7): A gene regulating stress responses. Silencing it reduced cotton’s fungal resistance, proving its role in sea-island cotton’s unique defense cluster.
WRKY15: A gene controlling gossypol production. Knocking it out lowered toxin levels without affecting gland numbers, offering a way to breed cotton with edible seeds.
Homologous clusters: Cell groups in different species with similar gene activity. Most cotton leaf cell clusters (like vascular cells) were homologous, showing evolutionary conservation.
Co-expression networks: Gene groups working together like teammates. The study built these networks to find key players in gossypol production, such as ERF105 and WRKY15.
Programmed cell death (PCD): A controlled cell suicide process. In cotton pigment glands, PCD helps create hollow spaces to store gossypol, like carving compartments in a storage room.
Bulk RNA-seq: A technique measuring average gene activity in a tissue mix. Unlike single-cell methods, it can’t detect cell-specific patterns. The study used both to validate findings.
Cell type plasticity: A cell’s ability to change gene activity under stress. The DUF1230 gene showed this in cotton, behaving differently in the same cell types across species without stress.
Reference:
Cheng, H., Liu, S., Zhang, Y., Zuo, D., Wang, Q., Lv, L., Yang, Y., Hao, L., Zhang, X., Zhang, S., & Song, G. (2025). Comparative single-cell transcriptomic map reveals divergence in leaves between two cotton species at cell type resolution. Journal of Advanced Research. Advance online publication. https://doi.org/10.1016/j.jare.2025.04.012
