Single‐Cell Transcriptome Atlas and Regulatory Dynamics in Developing Cotton Anthers

Plant anthers are composed of different specialized cell types with distinct roles in plant reproduction. High temperature (HT) stress causes male sterility, resulting in crop yield reduction. However, the spatial expression atlas and regulatory dynamics during anther development and in response to HT remain largely unknown. Here, the first single‐cell transcriptome atlas and chromatin accessibility survey in cotton anther are established, depicting the specific expression and epigenetic landscape of each type of cell in anthers. The reconstruction of meiotic cells, tapetal cells, and middle layer cell developmental trajectories not only identifies novel expressed genes, but also elucidates the precise degradation period of middle layer and reveals a rapid function transition of tapetal cells during the tetrad stage. By applying HT, heterogeneity in HT response is shown among cells of anthers, with tapetal cells responsible for pollen wall synthesis are most sensitive to HT. Specifically, HT shuts down the chromatin accessibility of genes specifically expressed in the tapetal cells responsible for pollen wall synthesis, such as QUARTET 3 (QRT3) and CYTOCHROME P450 703A2 (CYP703A2), resulting in a silent expression of these genes, ultimately leading to abnormal pollen wall and male sterility. Collectively, this study provides substantial information on anthers and provides clues for heat‐tolerant crop creation

High Efficient Multisites Genome Editing in Allotetraploid Cotton (Gossypium Hirsutum) Using CRISPR/Cas9 System

Gossypium hirsutum is an allotetraploid with a complex genome. Most genes have multiple copies that belong to At and Dt subgenomes. Sequence similarity is also very high between gene homologues. To efficiently achieve site/gene‐specific mutation is quite needed. Due to its high efficiency and robustness, the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has exerted broad site‐specific genome editing from prokaryotes to eukaryotes. In this study, we utilized a CRISPR/Cas9 system to generate two sgRNAs in a single vector to conduct multiple sites genome editing in allotetraploid cotton. An exogenously transformed gene Discosoma red fluorescent protein2(DsRed2) and an endogenous gene GhCLA1 were chosen as targets. The DsRed2‐edited plants in T0 generation reverted its traits to wild type, with vanished red fluorescence the whole plants. Besides, the mutated phenotype and genotype were inherited to their T1 progenies. For the endogenous gene GhCLA1, 75% of regenerated plants exhibited albino phenotype with obvious nucleotides and DNA fragments deletion. The efficiency of gene editing at each target site is 66.7–100%. The mutation genotype was checked for both genes with Sanger sequencing. Barcode‐based high‐throughput sequencing, which could be highly efficient for genotyping to a population of mutants, was conducted in GhCLA1‐edited T0 plants and it matched well with Sanger sequencing results. No off‐target editing was detected at the potential off‐target sites. These results prove that the CRISPR/Cas9 system is highly efficient and reliable for allotetraploid cotton genome editing

Regulatory controls of duplicated gene expression during fiber development in allotetraploid cotton.

Polyploidy complicates transcriptional regulation and increases phenotypic diversity in organisms. The dynamics of genetic regulation of gene expression between coresident subgenomes in polyploids remains to be understood. Here we document the genetic regulation of fiber development in allotetraploid cotton Gossypium hirsutum by sequencing 376 genomes and 2,215 time-series transcriptomes. We characterize 1,258 genes comprising 36 genetic modules that control staged fiber development and uncover genetic components governing their partitioned expression relative to subgenomic duplicated genes (homoeologs). Only about 30% of fiber quality-related homoeologs show phenotypically favorable allele aggregation in cultivars, highlighting the potential for subgenome additivity in fiber improvement. We envision a genome-enabled breeding strategy, with particular attention to 48 favorable alleles related to fiber phenotypes that have been subjected to purifying selection during domestication. Our work delineates the dynamics of gene regulation during fiber development and highlights the potential of subgenomic coordination underpinning phenotypes in polyploid plants. [Abstract copyright: © 2023. The Author(s).

Nanopore-Based Comparative Transcriptome Analysis Reveals the Potential Mechanism of High-Temperature Tolerance in Cotton (<i>Gossypium hirsutum</i> L.)

Extreme high temperatures are threatening cotton production around the world due to the intensification of global warming. To cope with high-temperature stress, heat-tolerant cotton cultivars have been bred, but the heat-tolerant mechanism remains unclear. This study selected heat-tolerant (‘Xinluzao36′) and heat-sensitive (‘Che61-72′) cultivars of cotton treated with high-temperature stress as plant materials and performed comparative nanopore sequencing transcriptome analysis to reveal the potential heat-tolerant mechanism of cotton. Results showed that 120,605 nonredundant sequences were generated from the raw reads, and 78,601 genes were annotated. Differentially expressed gene (DEG) analysis showed that a total of 19,600 DEGs were screened; the DEGs involved in the ribosome, heat shock proteins, auxin and ethylene signaling transduction, and photosynthesis pathways may be attributed to the heat tolerance of the heat-tolerant cotton cultivar. This study also predicted a total of 5118 long non-coding RNAs (lncRNAs)and 24,462 corresponding target genes. Analysis of the target genes revealed that the expression of some ribosomal, heat shock, auxin and ethylene signaling transduction-related and photosynthetic proteins may be regulated by lncRNAs and further participate in the heat tolerance of cotton. This study deepens our understandings of the heat tolerance of cotton

Rapid Identification of Pollen- and Anther-Specific Genes in Response to High-Temperature Stress Based on Transcriptome Profiling Analysis in Cotton

Anther indehiscence and pollen sterility caused by high temperature (HT) stress have become a major problem that decreases the yield of cotton. Pollen- and anther-specific genes play a critical role in the process of male reproduction and the response to HT stress. In order to identify pollen-specific genes that respond to HT stress, a comparative transcriptome profiling analysis was performed in the pollen and anthers of Gossypium hirsutum HT-sensitive Line H05 against other tissue types under normal temperature (NT) conditions, and the analysis of a differentially expressed gene was conducted in the pollen of H05 under NT and HT conditions. In total, we identified 1111 pollen-specific genes (PSGs), 1066 anther-specific genes (ASGs), and 833 pollen differentially expressed genes (DEGs). Moreover, we found that the late stage of anther included more anther- and pollen-specific genes (APSGs). Stress-related cis-regulatory elements (CREs) and hormone-responsive CREs are enriched in the promoters of APSGs, suggesting that APSGs may respond to HT stress. However, 833 pollen DEGs had only 10 common genes with 1111 PSGs, indicating that PSGs are mainly involved in the processes of pollen development and do not respond to HT stress. Promoters of these 10 common genes are enriched for stress-related CREs and MeJA-responsive CREs, suggesting that these 10 common genes are involved in the process of pollen development while responding to HT stress. This study provides a pathway for rapidly identifying cotton pollen-specific genes that respond to HT stress

Nanopore-Based Comparative Transcriptome Analysis Reveals the Potential Mechanism of High-Temperature Tolerance in Cotton (Gossypium hirsutum L.)

Extreme high temperatures are threatening cotton production around the world due to the intensification of global warming. To cope with high-temperature stress, heat-tolerant cotton cultivars have been bred, but the heat-tolerant mechanism remains unclear. This study selected heat-tolerant (‘Xinluzao36′) and heat-sensitive (‘Che61-72′) cultivars of cotton treated with high-temperature stress as plant materials and performed comparative nanopore sequencing transcriptome analysis to reveal the potential heat-tolerant mechanism of cotton. Results showed that 120,605 nonredundant sequences were generated from the raw reads, and 78,601 genes were annotated. Differentially expressed gene (DEG) analysis showed that a total of 19,600 DEGs were screened; the DEGs involved in the ribosome, heat shock proteins, auxin and ethylene signaling transduction, and photosynthesis pathways may be attributed to the heat tolerance of the heat-tolerant cotton cultivar. This study also predicted a total of 5118 long non-coding RNAs (lncRNAs)and 24,462 corresponding target genes. Analysis of the target genes revealed that the expression of some ribosomal, heat shock, auxin and ethylene signaling transduction-related and photosynthetic proteins may be regulated by lncRNAs and further participate in the heat tolerance of cotton. This study deepens our understandings of the heat tolerance of cotton

N6‐methyladenosine RNA modification regulates cotton drought response in a Ca2+ and ABA‐dependent manner

N6-methyladenosine (m6A) is the most prevalent internal modification present in mRNAs, and is considered to participate in a range of developmental and biological processes. Drought response is highly regulated at the genomic, transcriptional and post-transcriptional levels. However, the biological function and regulatory mechanism of m6A modification in the drought stress response is still poorly understood. We generated a transcriptome-wide m6A map using drought-resistant and drought-sensitive varieties of cotton under different water deficient conditions to uncover patterns of m6A methylation in cotton response to drought stress. The results reveal that m6A represents a common modification and exhibit dramatic changes in distribution during drought stress. More 5'UTR m6A was deposited in the drought-resistant variety and was associated with a positive effect on drought resistance by regulating mRNA abundance. Interestingly, we observed that increased m6A abundance was associated with increased mRNA abundance under drought, contributing to drought resistance, and vice versa. The demethylase GhALKBH10B was found to decrease m6A levels, facilitating the mRNA decay of ABA signal-related genes (GhZEP, GhNCED4 and GhPP2CA) and Ca2+ signal-related genes (GhECA1, GhCNGC4, GhANN1 and GhCML13), and mutation of GhALKBH10B enhanced drought resistance at seedling stage in cotton. Virus-induced gene silencing (VIGS) of two Ca2+-related genes, GhECA1 and GhCNGC4, reduced drought resistance with the decreased m6A enrichment on silenced genes in cotton. Collectively, we reveal a novel mechanism of post-transcriptional modification involved in affecting drought response in cotton, by mediating m6A methylation on targeted transcripts in the ABA and Ca2+ signalling transduction pathways

High-temperature stress suppresses allene oxide cyclase 2 and causes male sterility in cotton by disrupting jasmonic acid signaling

Cotton (Gossypium spp.) yield is reduced by stress. In this study, high temperature (HT) suppressed the expression of the jasmonic acid (JA) biosynthesis gene allene oxide cyclase 2 (GhAOC2), reducing JA content and causing male sterility in the cotton HT-sensitive line H05. Anther sterility was reversed by exogenous application of methyl jasmonate (MeJA) to early buds. To elucidate the role of GhAOC2 in JA biosynthesis and identify its putative contribution to the anther response to HT, we created gene knockout cotton plants using the CRISPR/Cas9 system. Ghaoc2 mutant lines showed male-sterile flowers with reduced JA content in the anthers at the tetrad stage (TS), tapetum degradation stage (TDS), and anther dehiscence stage (ADS). Exogenous application of MeJA to early mutant buds (containing TS or TDS anthers) rescued the sterile pollen and indehiscent anther phenotypes, while ROS signals were reduced in ADS anthers. We propose that HT downregulates the expression of GhAOC2 in anthers, reducing JA biosynthesis and causing excessive ROS accumulation in anthers, leading to male sterility. These findings suggest exogenous JA application as a strategy for increasing male fertility in cotton under HT