Decoding the guardians of cotton resilience: A comprehensive exploration of the ßCA genes and its role in Verticillium dahliae resistance.

Plant Carbonic anhydrases (Cas) have been shown to be stress-responsive enzymes that may play a role in adapting to adverse conditions. Cotton is a significant economic crop in China, with upland cotton (Gossypium hirsutum) being the most widely cultivated species. We conducted genome-wide identification of the ßCA gene in six cotton species and preliminary analysis of the ßCA gene in upland cotton. In total, 73 ßCA genes from six cotton species were identified, with phylogenetic analysis dividing them into five subgroups. GHßCA proteins were predominantly localized in the chloroplast and cytoplasm. The genes exhibited conserved motifs, with motifs 1, 2, and 3 being prominent. GHßCA genes were unevenly distributed across chromosomes and were associated with stress-responsive cis-regulatory elements, including those responding to light, MeJA, salicylic acid, abscisic acid, cell cycle regulation, and defence/stress. Expression analysis indicated that GHßCA6, GHßCA7, GHßCA10, GHßCA15, and GHßCA16 were highly expressed under various abiotic stress conditions, whereas GHßCA3, GHßCA9, GHßCA10, and GHßCA18 had higher expression patterns under Verticillium dahliae infection at different time intervals. In Gossypium thurberi, GthßCA1, GthßCA2, and GthßCA4 showed elevated expression across stress conditions and tissues. Silencing GHßCA10 through VIGS increased Verticillium wilt severity and reduced lignin deposition compared to non-silenced plants. GHßCA10 is crucial for cotton's defense against Verticillium dahliae. Further research is needed to understand the underlying mechanisms and develop strategies to enhance resistance against Verticillium wilt.

Genome-wide identification and functional analysis of ICE genes reveal that Gossypium thurberi "GthICE2" is responsible for cold and drought stress tolerance.

Cold stress has been found to have a negative impact on cotton growth and annual production. To address this issue, the utilization of cold-tolerant gene resources from wild species of Gossypium is crucial for genetic improvements in cultivated cotton. ICE (inducer of CBF expression) are the key regulators of cold tolerance in plants, however, there is relatively little information on ICE genes in cotton. Herein, we performed comprehensive bioinformatics analyses of the ICE gene family in eight cotton species. Phylogenetic analysis showed that 52 ICE genes were clustered into four subgroups. Cis-regulatory elements analysis suggests that the expression of ICE genes might be regulated by light, plant hormones, and various environment stresses. Higher expression of GthICE2 was observed in leaves as compared to roots and stems, in response to cold, drought, and exogenous hormone ABA. Furthermore, overexpression of GthICE2 in A. thaliana led to higher germination and survival rates, longer root length, lower ion leakage, and induction under cold and drought stress. Histochemical staining showed that oxidative damage in transgenic lines was much lower compared to wild-type plants. Lower MDA contents and higher SOD and POD activities were observed in overexpressed plants. Y1H and LUC assays revealed that GthICE2 might activate the expression of GthCBF4, a cold-responsive gene, by connecting with the MYC cis-element present in the promoter of GthCBF4. GthICE2 confers cold and drought stress tolerance in cotton. Our findings add significantly to the existing knowledge regarding cold stress tolerance and helps to elucidate cold response mechanisms in cotton.

Protoplast Dissociation and Transcriptome Analysis Provides Insights to Salt Stress Response in Cotton.

As one of the pioneer crops widely planted in saline-alkaline areas, Gossypium provides daily necessities, including natural fiber, vegetable proteins, and edible oils. However, cotton fiber yield and quality are highly influenced by salt stress. Therefore, elucidating the molecular mechanisms of cotton in response to salinity stress is importance to breed new cultivars with high tolerance. In this study, we first developed a method for single-cell RNA-seq based on isolating protoplast from cotton root tips; then, we studied the impact of salinity stress on gene expression profiling and their dynamic changes using the developed high-efficiency method for protoplast dissociation suitable for single-cell RNA-seq. A total of 3391 and 2826 differentially expressed genes (DEGs) were identified in salt-treated samples before and after protoplast dissociation, respectively, which were enriched into several molecular components, including response to stimulus, response to stress, and cellular macromolecule metabolic process by gene ontology (GO) analysis. Plant hormone signal transduction, phenylpropanoid biosynthesis, and MAPK signaling pathway were found to be enriched via Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Twenty-two and nine salinity-responsive DEGs participated in plant hormone signaling and MAPK signaling in roots, before and after protoplast dissociation, respectively; six upregulated DEGs were involved in ABA signaling transduction, namely, Ga04G2111, Ga07G0142, Ga09G2061, Ga10G0262, Ga01G0063, and Ga08G1915 which indicates their potential functions on plants adapting to salt stress. Additionally, 384 and 257 transcription factors (TFs) were differentially expressed in salt-stress roots before and after protoplast dissociation, respectively, of which significantly up-regulated TFs mainly belonged to the AP2/ERF-ERF family, which implied their potential roles responding to salt stress. These results not only provide novel insights to reveal the regulatory networks in plant's root response to salt stress, but also lay the solid foundation for further exploration on cellular heterogeneity by single-cell transcriptome sequencing.