The GDSL esterase/lipase (GELP) family comprises a group of multifunctional hydrolases with broad substrate specificity and catalytic versatility, playing an essential role in plant growth and developmental processes. The GDSL lipase gene GhGELP23D from Gossypium hirsutum is highly expressed during cotton fiber elongation development. This study aims to investigate its function in plant cell elongation and provides references for the mechanism elucidation of GELP-mediated regulation of cell growth.

The GhGELP23D was cloned from upland cotton fibers. Bioinformatic analyses were performed to characterize its physicochemical properties, structural features, and phylogenetic relationships. The promoter region was analyzed with PlantCARE to identify putative cis-elements. Expression patterns were detected based on public transcriptomic data and RT-qPCR validation. A GUS reporter construct driven by the GhGELP23D promoter was generated to examine the tissue-specific expression of GhGELP23D in stably transformed Arabidopsis. Subcellular localization of GhGELP23D was determined through transient expression in Nicotiana benthamiana leaves. Transgenic Arabidopsis lines heterologously expressing GhGELP23D (GhGELP23D-OE) were obtained using the floral-dip method, and phenotypic analyses were conducted. Additionally, virus-induced gene silencing (VIGS) was employed to suppress GhGELP23D expression in cotton. Silencing efficiency was verified by RT-qPCR, and the resulting changes in fiber length were assessed in GhGELP23D-VIGS plants.

GhGELP23D encodes a 356-amino-acid protein that is stable, weakly basic, and hydrophilic, containing a typical signal peptide but no transmembrane domain. Transcriptomic analysis and RT-qPCR detection showed that GhGELP23D was highly expressed during cotton fiber elongation. Subcellular localization analysis revealed that GhGELP23D is predominantly localized to the extracellular space. Analysis of the GhGELP23D promoter indicated the presence of multiple cis-elements related to light responsiveness, hormone signaling, and stress response. Histochemical GUS staining showed that the GhGELP23D promoter can drive reporter gene expression in various Arabidopsis tissues, indicating broad tissue expression activity. Heterologous expression of GhGELP23D in Arabidopsis (GhGELP23D-OE) significantly enhanced plant growth and development, resulting in increased plant height and longer primary roots and root hairs. Furthermore, complementation of GhGELP23D in the mutant resulted in a pronounced recovery of primary root and root hair length similar to that of the wild type (WT). In cotton, the fiber length of GhGELP23D-VIGS lines was significantly reduced compared with that of empty-vector control plants, with an average decrease of approximately 13.14%.

GhGELP23D encodes an extracellular GDSL lipase that is highly expressed during the fiber elongation stage of upland cotton and plays an important role in plant cell elongation development.

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin synthesis pathway, which plays an important role in enhancing plant mechanical strength and resisting pathogen invasion. The aim of this study is to identify CAD and CAD-Like (CADL) gene family members in Gossypium barbadense and to analyze their expression characteristics and their role in Verticillium wilt resistance, which provides reference for the mechanism elucidation and disease resistance breeding of cotton against Verticillium wilt.

The CAD and CADL gene family members in G. barbadense genome were identified by bioinformatics method, and their chromosomal location, phylogenetic relationship, gene structure and promoter cis-element prediction were systematically analyzed. The expression characteristics of GbCAD and GbCADL were analyzed by obtaining publicly released transcriptome data and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). Functional analysis of GbCAD and GbCADL genes was performed by viral-induced gene silencing (VIGS) technique.

A total of 25 GbCAD and 34 GbCADL genes were identified from G. barbadense and distributed on 10 and 17 different chromosomes, respectively. GbCAD and GbCADL genes are divided into 3 and 4 subgroups, respectively. The genes in the same group contain similar exon-intron structures and conserved domains. GbCAD and GbCADL genes have different transcriptional expression characteristics, and the promoters of GbCAD and GbCADL genes contain various hormone response elements and stress response elements. Transcriptome data and qRT-PCR showed that the expressions of GbCAD10A/D, GbCADL4A/D, GbCADL5A/D, GbCADL6A/D, and GbCADL7A/D were induced by Verticillium dahliae, especially the GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, and GbCADL7A/D indicated significant increased expressions under V. dahliae treatment. The genes of GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, and GbCADL7A/D were respectively silenced in cotton by virus-induced gene silencing (VIGS) technology, to analyze the changes of VIGS plant lines against V. dahliae treatment. The results showed that, compared with the control plants, the VIGS plant lines indicated significant decreased resistance to V. dahliae. The results of diaminobenzidine (DAB) histochemical stain displayed that, both control and VIGS plants showed similar normal phenotype without V. dahliae addition; after 6 h treatment of V. dahliae, the VIGS plant lines silencing GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, GbCADL7A/D expressions demonstrated a deeper brown coloring, indicating a higher reactive oxygen species (ROS) accumulation in the VIGS plant lines. The results of stem sectioning showed that, the stem vascular tissues of VIGS plant lines TRV:GbCAD10A/D, TRV:GbCADL4A/D, TRV:GbCADL6A/D, and TRV:GbCADL7A/D showed obvious dark brown enrichment after V. dahliae treatment, indicating the significant decreased resistance to V. dahliae.

Suppressing the expressions of GbCAD10A/D, GbCADL4A/D, GbCADL6A/D, GbCADL7A/D could significantly reduce the cotton resistance to V. dahliae.