Summary: Long hairpin RNA (hpRNA) transgenes are a powerful tool for gene function studies in plants, but a genomewide RNAi mutant library using hpRNA transgenes has not been reported for plants. Here, we report the construction of a hpRNA library for the genomewide identification of gene function in rice using an improved rolling circle amplification‐mediated hpRNA (RMHR) method. Transformation of rice with the library resulted in thousands of transgenic lines containing hpRNAs targeting genes of various function. The target mRNA was down‐regulated in the hpRNA lines, and this was correlated with the accumulation of siRNAs corresponding to the double‐stranded arms of the hpRNA. Multiple members of a gene family were simultaneously silenced by hpRNAs derived from a single member, but the degree of such cross‐silencing depended on the level of sequence homology between the members as well as the abundance of matching siRNAs. The silencing of key genes tended to cause a severe phenotype, but these transgenic lines usually survived in the field long enough for phenotypic and molecular analyses to be conducted. Deep sequencing analysis of small RNAs showed that the hpRNA‐derived siRNAs were characteristic of Argonaute‐binding small RNAs. Our results indicate that RNAi mutant library is a high‐efficient approach for genomewide gene identification in plants.

In this study, the tetrad stage of microspore development in a new cotton (Gossypium barbadense L.) cytoplasmic male sterility (CMS) line, H276A, was identified using paraffin sections at the abortion stage. To explore the molecular mechanism underlying CMS in cotton, a comparative transcriptome analysis between the CMS line H276A and its maintainer line H276B at the tetrad stage was conducted using an Illumina HiSeq 4000 platform. The comparison of H276A with H276B revealed a total of 64,675 genes, which consisted of 59,255 known and 5420 novel genes. An analysis of the two libraries with a given threshold yielded a total of 3603 differentially expressed genes (DEGs), which included 1363 up- and 2240 down-regulated genes. Gene Ontology (GO) annotation showed that 2171 DEGs were distributed into 38 categories, and a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 2683 DEGs were classified into 127 groups. Thirteen DEGs were randomly selected and detected by quantitative reverse-transcribed PCR (qRT-PCR), and the results indicated that the transcriptome sequencing results were reliable. The bioinformatic analysis results in conjunction with previously reported data revealed key DEGs that might be associated with the male sterility features of H276A. Our results provide a comprehensive foundation for understanding anther development and will accelerate the study of the molecular mechanisms of CMS in cotton.

Low temperature is a common biological abiotic stress in major cotton-growing areas. Cold stress significantly affects the growth, yield, and yield quality of cotton. Therefore, it is important to develop more robust and cold stress-resilient cotton germplasms. In response to climate change and erratic weather conditions, plants have evolved various survival mechanisms, one of which involves the induction of various stress responsive transcript factors, of which the C-repeat-binding factors (CBFs) have a positive effect in enhancing plants response to cold stress. In this study, genomewide identification and functional characterization of the cotton CBFs were carried out. A total of 29, 28, 25, 21, 30, 26, and 15 proteins encoded by the CBF genes were identified in seven Gossypium species. A phylogenetic evaluation revealed seven clades, with Clades 1 and 6 being the largest. Moreover, the majority of the proteins encoded by the genes were predicted to be located within the nucleus, while some were distributed in other parts of the cell. Based on the transcriptome and RT-qPCR analysis, Gthu17439 (GthCBF4) was highly upregulated and was further validated through forward genetics. The Gthu17439 (GthCBF4) overexpressed plants exhibited significantly higher tolerance to cold stress, as evidenced by the higher germination rate, increased root growth, and high-induction levels of stress-responsive genes. Furthermore, the overexpressed plants under cold stress had significantly reduced oxidative damage due to a reduction in hydrogen peroxide (H₂O₂) production. Moreover, the overexpressed plants under cold stress had minimal cell damage compared to the wild types, as evidenced by the Trypan and 3,3′-Diaminobenzidine (DAB) staining effect. The results showed that the Gthu17439 (GthCBF4) could be playing a significant role in enhancing cold stress tolerance in cotton and can be further exploited in developing cotton germplasm with improved cold-stress tolerance.