Transcriptome-wide m6A methylation profile reveals its potential role underlying drought response in wheat (Triticum aestivum L.).

MAIN CONCLUSION: This study revealed the transcriptome-wide m6A methylation profile under drought stress and found that TaETC9 might regulate drought tolerance through mediating RNA methylation in wheat. Drought is one of the most destructive environmental constraints limiting crop growth and development. N6-methyladenosine (m6A) is a prevalent and important post-transcriptional modification in various eukaryotic RNA molecules, playing the crucial role in regulating drought response in plants. However, the significance of m6A in wheat (Triticum aestivum L.), particularly its involvment in drought response, remains underexplored. In this study, we investigated the transcriptome-wide m6A profile under drought stress using parallel m6A immunoprecipitation sequencing (MeRIP-seq). Totally, 4221 m6A peaks in 3733 m6A-modified genes were obtained, of which 373 methylated peaks exhibited differential expression between the control (CK) and drought-stressed treatments. These m6A loci were significantly enriched in proximity to stop codons and within the 3'-untranslated region. Integration of MeRIP-seq and RNA-seq revealed a positive correlation between m6A methylation and mRNA abundance and the genes displaying both differential methylation and expression were obtained. Finally, qRT-PCR analyses were further performed and the results found that the m6A-binding protein (TaETC9) showed significant up-regulation, while the m6A demethylase (TaALKBH10B) was significantly down-regulated under drought stress, contributing to increased m6A levels. Furthermore, the loss-of-function mutant of TaECT9 displayed significantly higher drought sensitivity compared to the wild type, highlighting its role in regulating drought tolerance. This study reported the first wheat m6A profile associated with drought stress, laying the groundwork for unraveling the potential role of RNA methylation in drought responses and enhancing stress tolerance in wheat through epigenetic approaches.

Combined GWAS and eGWAS reveals the genetic basis underlying drought tolerance in emmer wheat (Triticum turgidum L.).

Drought is one of the major environmental constraints for wheat production world-wide. As the progenitor and genetic reservoir of common wheat, emmer wheat is considered as an invaluable gene pool for breeding drought-tolerant wheat. Combining GWAS and eGWAS analysis of 107 accessions, we identified 86 QTLs, 105 462 eQTLs as well as 68 eQTL hotspots associating with drought tolerance (DT) in emmer wheat. A complex regulatory network composed of 185 upstream regulator and 2432 downstream drought-responsive candidates was developed, of which TtOTS1 was found to play a negative effect in determining DT through affecting root development. This study sheds light on revealing the genetic basis underlying DT, which will provide the indispensable genes and germplasm resources for elite drought tolerance wheat improvement and breeding.

BarleyExpDB: an integrative gene expression database for barley.

BACKGROUND: RNA-sequencing (RNA-seq) has been widely used to study the dynamic expression patterns of transcribed genes, which can lead to new biological insights. However, processing and analyzing these huge amounts of histological data remains a great challenge for wet labs and field researchers who lack bioinformatics experience and computational resources. RESULTS: We present BarleyExpDB, an easy-to-operate, free, and web-accessible database that integrates transcriptional profiles of barley at different growth and developmental stages, tissues, and stress conditions, as well as differential expression of mutants and populations to build a platform for barley expression and visualization. The expression of a gene of interest can be easily queried by searching by known gene ID or sequence similarity. Expression data can be displayed as a heat map, along with functional descriptions as well as Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Proteins Families Database, and Simple Modular Architecture Research Tool annotations. CONCLUSIONS: BarleyExpDB will serve as a valuable resource for the barley research community to leverage the vast publicly available RNA-seq datasets for functional genomics research and crop molecular breeding.

Multi-omics reveals the key and specific miRNA-mRNA modules underlying salt tolerance in wild emmer wheat (Triticum dicoccoides L.).

BACKGROUND: Salt stress is one of the most destructive environmental factors limiting crop growth and development. MicroRNAs (miRNAs) are a class of conserved endogenous small non-coding RNAs, playing the crucial role in regulating salt response and tolerance in plants. However, the miRNAs in wild emmer wheat, especially the key and specific salt-responsive miRNAs are not well studied. RESULTS: Here, we performed small RNA, transcriptome, and degradome sequencing of both of salt-tolerance (ST) and salt-sensitive (SS) wild emmer genotypes to identify the miRNA-mRNA modules associating with salt tolerance. Totally, 775 miRNAs, including 361 conserved known miRNAs and 414 novel miRNAs were detected. Differential expression analysis identified 93 salt-responsive miRNAs under salt stress. Combined with RNA-seq and degradome sequencing analysis, 224 miRNA-mRNA modules displayed the complete opposite expression trends between ST and SS genotypes, most of which functionally enriched into ROS homeostasis maintaining, osmotic pressure modulating, and root growth and development. Finally, the qRT-PCR and a large-scale yeast functional screening were also performed to initially validate the expression pattern and function of candidate genes. CONCLUSIONS: This study reported the key and specific miRNA-mRNA modules associated with salt tolerance in wild emmer, which lay the foundation for improving the salt tolerance in cultivated emmer and bread wheat through miRNA engineering approach.

Population transcriptomic analysis identifies the comprehensive lncRNAs landscape of spike in wheat (Triticum aestivum L.).

BACKGROUND: Long noncoding RNAs (lncRNAs) are emerging as the important regulators involving in growth and development as well as stress response in plants. However, current lncRNA studies were mainly performed at the individual level and the significance of it is not well understood in wheat. RESULTS: In this study, the lncRNA landscape of wheat spike was characterized through analysing a total of 186 spike RNA-seq datasets from 93 wheat genotypes. A total of 35,913 lncRNAs as well as 1,619 lncRNA-mRNA pairs comprised of 443 lncRNAs and 464 mRNAs were obtained. Compared to coding genes, these lncRNAs displayed rather low conservation among wheat and other gramineous species. Based on re-sequencing data, the genetic variations of these lncRNA were investigated and obvious genetic bottleneck were found on them during wheat domestication process. Furthermore, 122 lncRNAs were found to act as ceRNA to regulate endogenous competition. Finally, association and co-localization analysis of the candidate lncRNA-mRNA pairs identified 170 lncRNAs and 167 target mRNAs significantly associated with spike-related traits, including lncRNA.127690.1/TraesCS2A02G518500.1 (PMEI) and lncRNA.104854.1/TraesCS6A02G050300.1 (ATG5) associated with heading date and spike length, respectively. CONCLUSIONS: This study reported the lncRNA landscape of wheat spike through the population transcriptome analysis, which not only contribute to better understand the wheat evolution from the perspective of lncRNA, but also lay the foundation for revealing roles of lncRNA playing in spike development.

BGFD: an integrated multi-omics database of barley gene families.

BACKGROUND: A gene family comprises a group of genes with similar functional domains that play various roles in plant growth, development, and responses to environmental stimuli. Barley (Hordeum vulgare L.) is the fourth most cultivated cereal crop worldwide, and it is an important model species for genetic studies. Systematic identification and annotation of gene families are key for studies of molecular function and evolutionary history. RESULTS: We constructed a multi-omics database containing 5593 genes of 77 gene families called the Barley Gene Family Database (BGFD: http://barleygfdb.com ). BGFD is a free, user-friendly, and web-accessible platform that provides data on barley family genes. BGFD provides intuitive visual displays to facilitate studies of the physicochemical properties, gene structure, phylogenetic relationships, and motif organization of genes. Massive multi-omics datasets have been acquired and processed to generate an atlas of expression pattern profiles and genetic variation in BGFD. The platform offers several practical toolkits to conduct searches, browse, and employ BLAST functions, and the data are downloadable. CONCLUSIONS: BGFD will aid research on the domestication and adaptive evolution of barley; it will also facilitate the screening of candidate genes and exploration of important agronomic traits in barley.

Genome-wide identification of PYL gene family in wheat: Evolution, expression and 3D structure analysis.

Here, 38 wheat PYL genes (TaPYLs) belonging to 13 homoeologous groups were identified using the genome-search method, with 26 and 12 PYL genes identified in Triticum dicoccoides and Aegilops tauschii, respectively. Phylogenetic relationship, conserved domain and molecular evolution analysis revealed that PYL genes showed highly conservative between wheat and theprogenitors. Interaction network and miRNA target prediction found that TaPYLs could interact with the important components of ABA signaling pathway and Tae-miR966b-3p might be a hub regulator mediating wheat ABA signal network. Furthermore, the tissue-specific and stress-responsive TaPYLs were detected through RNA-seq analysis. Expressions of 10 TaPYLs were validated by QPCR analysis and the homoeologous genes showed significantly differential expression, suggesting subfunctionalization of them has occurred. Finally, 3D structures of the TaPYL proteins were predicted by homology modeling. This study lays the foundation for further functional study of PYL genes for development and stress tolerance improvement in wheat and beyond.

Genome-Wide Identification and Characterization of RNA/DNA Differences Associated with Drought Response in Wheat.

RNA/DNA difference (RDD) is a post-transcriptional RNA modification to enrich genetic information, widely involved in regulating diverse biological processes in eukaryotes. RDDs in the wheat nuclear genome, especially those associated with drought response or tolerance, were not well studied up to now. In this study, we investigated the RDDs related to drought response based on the RNA-seq data of drought-stressed and control samples in wheat. In total, 21,782 unique RDDs were identified, of which 265 were found to be drought-induced, representing the first drought-responsive RDD landscape in the wheat nuclear genome. The drought-responsive RDDs were located in 69 genes, of which 35 were differentially expressed under drought stress. Furthermore, the effects of RNA/DNA differences were investigated, showing that they could result in changes of RNA secondary structure, miRNA-target binding as well as protein conserved domains in the RDD-containing genes. In particular, the A to C mutation in TraesCS2A02G053100 (orthology to OsRLCK) led to the loss of tae-miR9657b-5p targeting, indicating that RNA/DNA difference might mediate miRNA to regulate the drought-response process. This study reported the first drought-responsive RDDs in the wheat nuclear genome. It sheds light on the roles of RDD in drought tolerance, and may also contribute to wheat genetic improvement based on epi-transcriptome methods.

Integrate Small RNA and Degradome Sequencing to Reveal Drought Memory Response in Wheat (Triticum aestivum L.).

Drought has gradually become one of the most severe abiotic stresses on plants. Plants that experience stress training can exhibit enhanced stress tolerance. According to MicroRNA (miRNA) sequencing data, this study identified 195 candidate drought memory-related miRNAs in wheat, and targets of 64 (32.8%) candidate miRNAs were validated by degradome sequencing. Several drought memory-related miRNAs such as tae-miR9676-5p, tae-MIR9676-p3_1ss21GA, tae-miR171a, tae-miR531_L-2, tae-miR408_L-1, PC-3p-5049_3565, tae-miR396c-5p, tae-miR9778, tae-miR164a-5p, and tae-miR9662a-3p were validated as having a strong response to drought memory by regulating the expression of their target genes. In addition, overexpression of drought memory-related miRNA, tae-miR531_L-2, can remarkably improve the drought tolerance of transgenic Arabidopsisthaliana. Drought memory can regulate plant cellular signal transduction, plant biosynthetic processes, and other biological processes to cope with drought via transcriptional memory. In addition, drought memory-related miRNAs can promote starch and sucrose catabolism and soluble sugar accumulation and regulate proline homeostasis to improve plant drought resistance. Our results could contribute to an understanding of drought memory in wheat seedlings and may provide a new strategy for drought-resistant breeding.

Genome-Wide Identification and Characterization of RNA/DNA Differences Associated with Fusarium graminearum Infection in Wheat.

RNA/DNA difference (RDD) is a post-transcriptional modification playing a crucial role in regulating diverse biological processes in eukaryotes. Although it has been extensively studied in plant chloroplast and mitochondria genomes, RDDs in plant nuclear genomes are not well studied at present. Here, we investigated the RDDs associated with fusarium head blight (FHB) through a novel method by comparing the RNA-seq data between Fusarium-infected and control samples of four wheat genotypes. A total of 187 high-confidence unique RDDs in 36 genes were identified, representing the first landscape of the FHB-responsive RDD in wheat. The majority (26) of these 36 RDD genes were correlated either positively or negatively with FHB levels. Effects of these RDDs on RNA and protein sequences have been identified, their editing frequency and the expression level of the corresponding genes provided, and the prediction of the effect on the minimum folding free energy of mRNA, miRNA binding, and colocation of RDDs with conserved domains presented. RDDs were predicted to induce modifications in the mRNA and protein structures of the corresponding genes. In two genes, TraesCS1B02G294300 and TraesCS3A02G263900, editing was predicted to enhance their affinity with tae-miR9661-5p and tae-miR9664-3p, respectively. To our knowledge, this study is the first report of the association between RDD and FHB in wheat; this will contribute to a better understanding of the molecular basis underlying FHB resistance, and potentially lead to novel strategies to improve wheat FHB resistance through epigenetic methods.