Transcriptional Landscape of Cotton Roots in Response to Salt Stress at Single-cell Resolution

Increasing soil salinization has led to severe losses of plant yield and quality. Thus, it is urgent to investigate the molecular mechanism of the salt stress response. In this study, we took systematically analyzed cotton root response to salt stress by single-cell transcriptomics technology; 56,281 high-quality cells were totally obtained from 5-days-old lateral root tips of Gossypium arboreum under natural growth and different salt-treatment conditions. Ten cell types with an array of novel marker genes were synthetically identified and confirmed with in situ RNA hybridization, and some specific-type cells of pesudotime analysis also pointed out their potential differentiation trajectory. The prominent changes of cell numbers responding to salt stress were observed on outer epidermal and inner endodermic cells, which were significantly enriched in response to stress, amide biosynthetic process, glutathione metabolism, and glycolysis/gluconeogenesis. Other functional aggregations were concentrated on plant-type primary cell wall biogenesis, defense response, phenylpropanoid biosynthesis and metabolic pathways by analyzing the abundant differentially expressed genes (DEGs) identified from multiple comparisons. Some candidate DEGs related with transcription factors and plant hormones responding to salt stress were also identified, of which the function of Ga03G2153, an annotated auxin-responsive GH3.6, was confirmed by using virus-induced gene silencing (VIGS). The GaGH3.6-silenced plants presented severe stress-susceptive phenotype, and suffered more serious oxidative damages by detecting some physiological and biochemical indexes, indicating that GaGH3.6 might participate in salt tolerance in cotton through regulating oxidation-reduction process. For the first time, a transcriptional atlas of cotton roots under salt stress were characterized at a single-cell resolution, which explored the cellular heterogeneityand differentiation trajectory, providing valuable insights into the molecular mechanism underlying stress tolerance in plants.