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

Abstract

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.

Establishment of an efficient cotton root protoplast isolation protocol suitable for single-cell RNA sequencing and transient gene expression analysis

“…For example, root age influences the quantity and quality of protoplasts. Liu [ 12 ] compared the yield and viability of protoplasts isolated from 5-, 7-, 9- and 10-day-old root tips. They found that 5-day-old root-generated protoplasts exhibited high viability (~ 85%) and were suitable for transcriptome sequencing.…”

“…The developmental stage of the materials used is critical for successful protoplast isolation [ 12 ]. During seed germination, the radicle breaks through the seed coat to form a primary root and begin primary growth.…”

Transcriptome and Metabolome Analysis of Upland Cotton (Gossypium hirsutum) Seed Pretreatment with MgSO4 in Response to Salinity Stress

“…Multiple studies have been conducted on cotton based on its salt tolerance ability by utilizing quantitative trait loci (QTL) by linkage mapping and genome-wide association studies. Little work on the transcriptomic and metabolomic aspects has been conducted yet [56][57][58]. Several QTL or genes were discovered by scientists in previous studies on the salt tolerance mechanism of cotton [55,59,60].…”

Opportunities and challenges in the application of single-cell and spatial transcriptomics in plants

“…It is worth mentioning that they also revealed that external stimuli, such as sucrose, can influence the developmental process, causing changes in sucrose response-related cells. In a similar case, in 2022, a group studied another external stimulus, salt stress, using cotton as their object and observed the influence of external stimuli on the profile and dynamic changes in gene expression ( Liu et al., 2022b ). For further research on the exploration of cellular heterogeneity, it is essential to compare gene expression profiles before and after external environmental stimuli by studying single-cell transcriptome sequencing results and constructing an atlas.…”