Transcriptional landscape of cotton roots in response to salt stress at single-cell resolution

Abstract

Increasing soil salinization has led to severe reductions in plant yield and quality, and investigating the molecular mechanism of salt stress response is therefore an urgent priority. In this study, we systematically analyzed the response of cotton roots to salt stress using single-cell transcriptomics technology; 56 281 high-quality cells were obtained from 5-day-old lateral root tips of Gossypium arboreum under natural growth conditions and different salt treatments. Ten cell types with an array of novel marker genes were identified and confirmed by in situ RNA hybridization, and pseudotime analysis of some specific cell types revealed their potential differentiation trajectories. Prominent changes in cell numbers under salt stress were observed for outer epidermal and inner endodermal cells, which were significantly enriched in response to stress, amide biosynthetic process, glutathione metabolism, and glycolysis/gluconeogenesis. Analysis of differentially expressed genes identified in multiple comparisons revealed other functional aggregations concentrated on plant-type primary cell wall biogenesis, defense response, phenylpropanoid biosynthesis, and metabolic pathways. Some candidate differentially expressed genes encoding transcription factors or associated with plant hormones also responsive to salt stress were identified, and the function of Ga03G2153, annotated as auxin-responsive GH3.6, was confirmed by virus-induced gene silencing. The GaGH3.6-silenced plants showed a severe stress-susceptible phenotype, and physiological and biochemical measurements indicated that they suffered more significant oxidative damage. These results suggest that GaGH3.6 might participate in cotton salt tolerance by regulating redox processes. We thus construct a transcriptional atlas of salt-stressed cotton roots at single-cell resolution, enabling us to explore cellular heterogeneity and differentiation trajectories and providing valuable insights into the molecular mechanisms that underlie plant stress tolerance.