Promoter editing enables researchers to develop heat-tolerant cotton germplasms in response to global warming

Recently, the cotton genetic improvement team at Huazhong Agricultural University successfully developed new heat-resistant cotton lines by precisely editing the promoter region of the key high-temperature-responsive gene GhCKI. This breakthrough provides novel genetic resources and molecular breeding technologies for improving cotton's heat tolerance.

In earlier studies, the research team identified GhCKI as a key gene negatively regulating male fertility in cotton under high temperatures. Both overexpression and knockdown of GhCKI resulted in severe male sterility, limiting its application in breeding for heat tolerance. To overcome this limitation, the researchers shifted their focus to promoter editing, aiming to fine-tune the expression level or pattern of GhCKI. Using single-cell ATAC-seq data, they conducted an in-depth analysis of the chromatin accessibility in the promoter region of GhCKI. Combining this with the identification of two critical MYB transcription factor binding sites in the GhCKI promoter responsive to heat stress, the researchers designed 12 sgRNAs. They then applied CRISPR/Cas9 and CRISPR/Cpf1 genome editing technologies to precisely edit and delete specific regions of the GhCKI promoter.

Editing analysis revealed that most editing events resulted in large fragment deletions, and the edited plants were categorized into eight major genotypes (GhCKI-pro1 to GhCKI-pro8) based on their promoter modifications (Figure 1). These editing events reduced GhCKI expression levels, and further phenotypic analyses showed that mutants with excessively reduced GhCKI expression exhibited significant male sterility under normal temperatures. However, mutants with moderately reduced expression displayed normal anther development. Under high-temperature stress, two mutants (GhCKI-pro5 and GhCKI-pro6) maintained moderate GhCKI expression levels, showing normal anther development, significantly higher pollen viability, and improved anther dehiscence rates compared to wild-type plants, demonstrating a clear heat-tolerant phenotype (Figure 2).

Further investigations into the molecular regulatory mechanisms underlying the heat tolerance of GhCKI-pro5 and GhCKI-pro6 revealed that MYB transcription factors GhMYB73 and GhMYB4 bind to two MYB binding sites in the GhCKI promoter, positively regulating GhCKI expression under heat stress. When the MYB binding sites or their flanking sequences were deleted, the ability of GhMYB73 and GhMYB4 to activate GhCKI expression under high temperatures was hindered. This alteration allowed GhCKI-pro5 and GhCKI-pro6 to maintain normal anther development under extreme heat conditions (Figure 3).

This research not only highlights the critical role of the GhCKI gene in breeding heat-tolerant cotton but also lays a solid foundation for developing high-yield, high-quality, and heat-resistant cotton varieties in the future. Moreover, it offers a new strategy for enhancing heat tolerance in other crops by editing promoter regions of key genes, providing technical support to address agricultural challenges posed by global climate change.

This breakthrough represents another significant advancement by the Huazhong Agricultural University cotton team in the field of cotton heat tolerance research. In previous studies, the team utilized multi-omics technologies and molecular biology approaches to uncover the mechanisms of heat-induced sterility in cotton and identify heat-tolerant genes, providing theoretical, technical, and resource support for breeding heat-tolerant cotton varieties (Li et al., 2024a, Science China Life Sciences; Li et al., 2024b, Advanced Science; Li et al., 2023, Plant Communications; Khan et al., 2023, Plant Biotechnology Journal; Khan et al., 2023, Crop Journal; Ma et al., 2022, JIPB; Li et al., 2022, Plant Physiology; Ma et al., 2021, New Phytologist; Ma et al., 2018, Plant Cell).

---

---