Summary: Heavy metal pollution poses a significant risk to human health and wreaks havoc on agricultural productivity. Phytoremediation, a plant‐based, environmentally benign, and cost‐effective method, is employed to remove heavy metals from contaminated soil, particularly in agricultural or heavy metal‐sensitive lands. However, the phytoremediation capacity of various plant species and germplasm resources display significant genetic diversity, and the mechanisms underlying these differences remain hitherto obscure. Given its potential benefits, genetic improvement of plants is essential for enhancing their uptake of heavy metals, tolerance to harmful levels, as well as overall growth and development in contaminated soil. In this study, we uncover a molecular cascade that regulates cadmium (Cd²⁺) tolerance in cotton, involving GhRCD1, GhbHLH12, GhMYB44, and GhHMA1. We identified a Cd²⁺‐sensitive cotton T‐DNA insertion mutant with disrupted GhRCD1 expression. Genetic knockout of GhRCD1 by CRISPR/Cas9 technology resulted in reduced Cd²⁺ tolerance in cotton seedlings, while GhRCD1 overexpression enhanced Cd²⁺ tolerance. Through molecular interaction studies, we demonstrated that, in response to Cd²⁺ presence, GhRCD1 directly interacts with GhbHLH12. This interaction activates GhMYB44, which subsequently activates a heavy metal transporter, GhHMA1, by directly binding to a G‐box cis‐element in its promoter. These findings provide critical insights into a novel GhRCD1–GhbHLH12–GhMYB44–GhHMA1 regulatory module responsible for Cd²⁺ tolerance in cotton. Furthermore, our study paves the way for the development of elite Cd²⁺‐tolerant cultivars by elucidating the molecular mechanisms governing the genetic control of Cd²⁺ tolerance in cotton.

Summary: Verticillium dahliae is among the most destructive plant pathogens, posing a significant threat to global cotton production. Cotton plants have developed sophisticated immune networks to inhibit V. dahliae colonization. Ingeniously, V. dahliae employs numerous virulent effectors to surmount plant immune responses. However, the pathogenic mechanisms of V. dahliae‐derived effectors remain elusive. In this study, we demonstrate that the Vd06254 effector from V. dahliae disrupts the synergistic interaction between jasmonic acid (JA) and strigolactones (SL), thereby suppressing cotton immunity. Ectopic expression of Vd06254 enhanced susceptibility to both viral and V. dahliae infections in Nicotiana benthamiana and cotton, respectively. Vd06254 directly interacts with the JA pathway regulator GhMYC3. The nuclear localization signal (NLS) was found to be essential for the virulence of Vd06254 and its interaction with GhMYC3. Additionally, overexpression and knockout of GhMYC3 in cotton modified the plant's resistance to V. dahliae. Our findings further reveal that GhMYC3 inhibits the expression of GhCCD8 by binding to its promoter, potentially regulating SL homeostasis in cotton through a negative feedback loop. This repression was enhanced by Vd06254, highlighting its crucial role in modulating cotton immunity and illustrating how V. dahliae effectors reprogram cotton transcription to disrupt this regulatory mechanism.

The temporally regulated morphogenesis of plant organs depends critically on the precise differentiation of meristem stem cells. This process is propelled by the orchestrated dynamics of specific stem cell populations within both the apical and lateral meristems, which drive longitudinal elongation and radial thickening. Although considerable progress has been made in elucidating longitudinal growth mediated by apical meristems, the spatiotemporal mechanisms governing radial growth via the vascular cambium remain incompletely characterized. Here, we generated high-resolution single-cell transcriptomes of stem tissues from the cotton cultivar ZM24. Gene regulatory network analysis identified GhXTHB as highly expressed in the vascular cambium region. Overexpression of GhXTHB reduced the number of vascular cambium layers and xylem width compared to WT, whereas CRISPR/Cas9-mediated knockout of GhXTHB increased cambium cell layers without affecting xylem width. Upstream of GhXTHB, we identified GhTALE, whose overexpression promoted cambium cell proliferation and increased the number of cambial layers. In contrast, GhTALE knockout (KO-GhTALE) reduced both cambial proliferation and xylem width. Notably, GhXTHB expression was upregulated in GhTALE-edited lines, indicating that GhTALE acts as a negative regulator of GhXTHB. Further analysis revealed that GhTALE physically interacts with GhOFP1, and GhOFP1 knockout increased the number of vascular cambium cell layers. Mechanistically, GhOFP1 is recruited to the GhXTHB promoter via interaction with GhTALE, modulating vascular cambium development. These findings establish the GhOFP1-GhTALE-GhXTHB module as a potential regulatory factors of vascular cambium proliferation in cotton stems, offering new insight into transcriptional control of stem architecture and biomass accumulation.

Upstream open reading frames (uORFs) are intrinsic regulatory elements that fine-tune gene expression at the translational level without altering genomic sequences or protein structures. Here, we targeted the conserved uORF that resides in the 5'-untranslated region (5'-UTR) of a GDP-galactose phosphorylase gene (GhGGP1) in cotton (Gossypium hirsutum). Regulation of GGP1 is a key control point of plant L-ascorbic acid biosynthesis. Using a dual-luciferase reporter system, we demonstrate that this uORF represses translation of the downstream GGP1 coding sequence in a feedback manner in response to elevated ascorbate, confirming previous findings in other species. Furthermore, CRISPR/Cas9-mediated disruption of the uORFGhGGP1 generated homozygous uorfGhGGP1 mutants with unchanged transcript levels but markedly increased protein abundance and L-ascorbic acid accumulation. Under drought stress, these mutants exhibit reduced malondialdehyde levels, enhanced total antioxidant capacity, and improved drought tolerance. Mechanistically, we further show that under drought conditions, the transcription factor GhBBM directly binds to the uORF-containing cis-regulatory element, enhancing (GhGGP1) transcription and drought resistance. Together, our findings establish the uORFGhGGP1 as a dual-layer regulatory module that integrates transcriptional activation by GhBBM with translational control of the downstream protein encoding open reading frame, thereby fine-tuning drought stress responses in cotton.

Squalene is a high-value triterpenoid. It is increasingly recognized for its regulatory roles in plant signalling. However, the functions of squalene in cotton remain unexplored. Here, we show that exogenous squalene can promote cotton developmental traits (root elongation and ovule development), and identify GhSQS1 as the predominant functional gene encoding squalene synthase. Overexpression of GhSQS1 (GhSQS1-OE) promotes root development, along with metabolic and hormonal reprogramming, including the activation of brassinosteroid (BR)-associated signalling. Furthermore, stable overexpression of GhSQS1 markedly elevates endogenous squalene accumulation, predominantly in leaves. This metabolic enhancement is accompanied by coordinated improvements in root architecture, leaf expansion, fibre elongation, and overall plant vigour. Notably, GhSQS1-OE lines exhibited elevated lignin-associated enzyme activities and increased total lignin content. These changes were correlated with improved tolerance to drought and alkaline stress. Importantly, similar development-promoting phenotypes were reproduced by heterologous expression of a high-activity fungal SQS (sharing only 39.2% amino acid identity with GhSQS1), confirming that enhanced squalene biosynthesis underlies these effects. Collectively, this study establishes squalene biosynthesis as a central metabolic axis coordinating hormonal reprogramming, cotton-specific development and stress adaptation, while concurrently generating squalene-enriched biomass. These findings provide a new paradigm for crop genetic improvement and tolerance enhancement.

Background: Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technology has advanced rapidly; however, current protoplast-based methods for evaluating genome-editing efficiency rely on expensive instruments and are incompatible with certain experimental strategies, highlighting the urgent need for a simple, cost-effective alternative. Results: In this study, we developed a visual system for assessing genome-editing efficiency in cotton using GhF3H , a key gene involved in anthocyanin biosynthesis whose knockout results in distinct changes in tissue coloration. The red-background cotton line RZM24 was obtained via hybridization and backcrossing. Expression vectors harboring Cas9, Cas12a, or CasΦ and their corresponding single-guide RNAs (sgRNAs) targeting GhF3H were constructed and introduced into hypocotyls via Agrobacterium -mediated transformation, followed by callus induction. Successfully edited calli exhibited white or white-and-red chimeric phenotypes, whereas unedited calli remained red. Cas9 showed the highest editing efficiency, followed by Cas12a, with CasΦ showing the lowest. Genotyping confirmed that white and chimeric calli were capable of regenerating plants carrying heritable mutations, and the three Cas systems induced distinct mutation types. Conclusions: This system enables rapid visual screening of editing events within 45 days, facilitating the comparison of CRISPR toolkits, validation of novel nucleases, and optimization of sgRNA delivery strategies. This approach may be adaptable to other crops and offers a practical solution to advance plant genome engineering.