| Global warming has led to frequent high temperature stress,greatly causing yield loss of crops worldwide.Cotton,one of the most important cash crops in the world,has suffered severe yield loss due to male sterility arisen by high temperature stress.Therefore,it is of great importance to study the process of cotton male development and to analyze the molecular mechanism of its response to high temperature for the creation of new germplasm of HT-tolerant cotton.In this study,we performed singlecell transcriptome and single-cell chromatin accessibility sequencing during the most heat-sensitive tetrad stage of cotton anthers,and constructed the first landscape of anther cell type development at the single-cell level under both normal and hightemperature conditions in cotton.The specific expression and epigenetic modification changes of anther cells under high temperature stress were analyzed.We found that different cells in the anther jointly opened the chromatin of the binding motif of stress response-related transcription factors,specifically closed the chromatin of binding motifs of development-related transcription factors,and activated the upregulation of stress response genes and downregulated the anther development genes under high temperature stress,thus enhancing the ability to resist high temperature stress.A new pathway for regulating cotton male fertility under high temperature stress was revealed:the GhMYB66-GhMYB4-GhCKI regulatory pathway,which provides a theoretical basis for breeding new varieties of heat-resistant cotton and has important application value.The specific results of this study are as follows:(1)Construction of single-cell transcriptome and chromatin accessibility maps for cotton anther cells under normal and high temperatures.Firstly,a technique for rapidly extracting high-quality nuclei from cotton anther cells was developed by optimizing the extraction process.Using this technique,nuclei were extracted from the anthers of the heat-tolerant cotton variety "H05" at the tetrad stage under normal and high temperatures,resulting in 17,732 and 15,906 high-quality cells,respectively.Single-cell transcriptome sequencing was performed to construct the expression atlas of cotton anther cells and to identify all cell types within the anther tissue,revealing high expression heterogeneity between cell layers.By analyzing the differentially expressed genes between cell types and conducting RNA in situ hybridization experiments,numerous cell type-specific marker genes were identified,filling the gaps in marker genes for most cell types in the anther.Subsequently,pseudotime analyses were performed on meiotic cells,tapetal cells,and middle layer cells to reconstruct the meiotic process at the single-cell level.It was found that the expression of tapetal cells at the tetrad stage also showed significant heterogeneity,and tapetal cells from different pseudotime clusters had different biological functions.By constructing the regulatory network of transcription factors during tapetal development,some important transcription factors such as MYB4,MYB52,and ZFN1 were identified.In addition,according to the pseudotime analysis and high-precision semi-thin section results of the middle layer,it was proposed that the degradation of middle layer cells in cotton was not completed in the seventh period after meiosis,but was completed in the eleventh period of anther development in coordination with the tapetal cells.Furthermore,through comparative analysis of single-cell transcriptomes under normal conditions and high-temperature stress,it was found that different cell types in the anther have unique response characteristics to high temperature,exhibiting strong heterogeneity.Among them,the tapetal cells responsible for pollen wall synthesis are the most sensitive to high temperature,disappearing completely after being subjected to high-temperature stress.We also discovered that GhNSP and GhCYP703A2,which are highly expressed in these tapetal cells,may be the core key factors in response to high temperature.On the other hand,single-cell multi-omics(transcriptome +chromatin accessibility)sequencing and analysis were completed,identifying chromatin open regions specific to all cell types and discovering that gene expression and corresponding chromatin open activity are highly positively correlated under normal and high temperature.The disappearance of tapetal cells from the transcriptome under high temperature may be due to their chromatin closure.Finally,the chromatin accessibility changes of 465 transcription factor binding motifs in all cell types under high temperature were analyzed,revealing that different transcription factor families have unique high-temperature response mechanisms in different anther cell types,with MYB transcription factors being critical for tapetal cell response to high temperature.(2)GhMYB66-GhMYB4-GhCKI is a key pathway regulating male sterility under high temperature in cotton.Single-cell transcriptome analysis identified a large number of MYB transcription factors that are important for the development of the tapetum layer,and single-cell chromatin accessibility sequencing revealed that MYB transcription factors are involved in high-temperature response in tapetum cells.Further analysis found that one member of the MYB transcription factor family,GhMYB4(Ghir＿A12G003840),was induced by high temperature in tapetum cells.Yeast one-hybrid,dual-luciferase reporter,and gel mobility shift assays showed that GhMYB4 can bind to two MYB binding sites in the promoter sequence of the type I casein kinase gene GhCKI,positively regulating GhCKI transcription.In the early stage of cotton anther development,both high-temperature induction of GhMYB4 expression and overexpression of GhMYB4 under normal temperature conditions lead to severe male sterility phenotypes,accompanied by increased expression of GhCKI.In addition,yeast two-hybrid,bimolecular fluorescence complementation,and GST pulldown assays showed that GhMYB4 interacted with another MYB transcription factor,GhMYB66.GhMYB66 had a similar expression pattern to GhMYB4 under high temperature,but it cannot directly bind to the promoter of GhCKI.Dual-luciferase reporter and gel mobility shift assays found that the heterodimer formed by GhMYB4 and GhMYB66 can enhance the binding ability of GhMYB4 to the GhCKI promoter.Furthermore,analysis found that high temperature reduces CHH-type DNA methylation mediated by si RNA on the GhMYB4 promoter,thereby enhancing GhMYB4 expression at the tetrad stage of anthers under high temperature,promoting the formation of the GhMYB4/GhMYB66 heterodimer,and increasing GhCKI transcription in the tapetum layer,resulting in male sterility.In summary,our results showed that GhMYB66-GhMYB4-GhCKI pathway is a key pathway in response to high temperature stress in cotton anthers.(3)Genome-wide identification,evolutionary analysis and functional characterization of cotton CKI genes.GhCKI is a key hub gene for high-temperature male sterility in cotton,and there have been no reports on the systematic study of the CKI family in cotton.Therefore,identifying the CKI family in cotton and studying the evolutionary history and function of CKI genes are of great theoretical value.In this study,the CKI gene family of five cotton species,G.hirsutum,G.barbadense,G.raimondii,G.arboretum,G.herbaceum were identified,and 58,57,31,29,and 27 CKI genes were obtained,respectively,revealing that the CKI family in cotton is divided into two major classes,type I and type II.Comparative genomic analysis found that the CKI family members amplified with the cotton tetraploid event,and that type I and type II CKI genes may have differentiated during the differentiation period of red and green algae 1.5 billion years ago,suggesting that CKI is an ancient gene involved in some basic biological pathways in plants.Further expression analysis of the upland cotton CKI family found that most CKI genes can respond to light signals and hightemperature stress,and some CKI genes undergo alternative splicing induced by high temperature.This study provides the evolutionary history characteristics of cotton CKI genes at the whole-genome level and lays a theoretical foundation for further studying the functional differentiation of the two types of CKI genes under specific developmental processes and environmental stress conditions.In summary,our study established transcriptomic and open chromatin atlas at the single-cell level of cotton tetrad anthers under normal and high temperature for the first time,depicted the expression and epigenetic changes of cotton male organs at the single-cell level under high temperature stress,identified a large number of core genes involved in anther development and response to high temperature,and unearthed a key pathway regulating male sterility in cotton under high temperature,which has important contributions for cotton functional genomic research and heat-resistant breeding. |
