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

Increasing soil salinization has led to severe losses of plant yield and quality. Researching the formation of plant salt tolerance and the molecular mechanism of the salt stress response is therefore urgent. We here take advantage of recent progress in single-cell transcriptomics technology to systematically analyze plant roots response to salt stress, and 57,185 high-quality cells were totally obtained from 5-day-old lateral root tips of Gossypium arboreum under natural growth and different salt-treatment conditions. Eleven cell types with an array of novel marker genes were identified and confirmed with RNA in situ hybridization. Pseudotime analysis on epidermal and root hair cells indicated the differentiation trajectory, and differential root-type cells responding to salt stress resulted in more epidermal and less endodermal cells. Abundant differentially expressed genes (DEGs) were found to be engaged in glycometabolism, reactive oxygen species hemeostasis, and phosphatidylinositol and MAKP signal pathways through functional enrichment analyses. Some candidate DEGs related with transcription factors and plant hormones responding to salt stress were also screened, which requires further functional verification to reveal the regulatory model of the plant roots response to salt stress. Combined with bulk RNA-seq data, a common DEG (Ga08G2497) annotated as Aldo-keto reductase-1 (AKR1) was selected to perform virus-induced gene silencing verification, and the silencing of GaAKR1 gene in G. arboreum resulted in severe stress-susceptibility phenotype, shorter root length, and less number of lateral roots. Physiological and biochemical detection also demonstrated that GaAKR1-silenced plants suffered more serious oxidative damages and, indicating that GaAKR1 might participates in salt tolerance of cotton by oxidation-reduction process. For the first time, we characterized a transcriptional atlas of plant roots under salt stress at a single-cell resolution, which explored the cellular heterogeneity, differential root-type cells, and differentiation trajectory, while providing valuable insights into the molecular mechanism underlying stress tolerance in plants. After being sterilized with 0.1% HgCl2 for 15 min, the full-grained seeds of G. arboreum SXY1 were rinsed with deionized water no less than three times and then germinated on ½ Murashige and Skoog (MS) solid medium with 15-g glucose and 2.6 g/L phytagel, adjusted to pH 5.7, and grown in an illumination incubator at 28°C/light for 16 h and 25°C/dark for 8 h. Subsequently, 5-day-old cotton seedlings were uniformly collected and randomly separated into four groups, which were differentially immersed into 40 mL MS liquid medium for only 0.5 h (natural growth control), MS liquid medium with 100 mM NaCl solution for 0.5 h (salt1) and 1 h (salt2), and 150 mM NaCl solution for 0.5 h (salt3). Two biological replications were conducted to ensure the statistical significance of this study, and numerous lateral roots were separately collected for further protoplast dissociation.