Genome-Wide Characterization of the PIFs Family in Sweet Potato and Functional Identification of IbPIF3.1 under Drought and Fusarium Wilt Stresses.

Phytochrome-interacting factors (PIFs) are essential for plant growth, development, and defense responses. However, research on the PIFs in sweet potato has been insufficient to date. In this study, we identified PIF genes in the cultivated hexaploid sweet potato (Ipomoea batatas) and its two wild relatives, Ipomoea triloba, and Ipomoea trifida. Phylogenetic analysis revealed that IbPIFs could be divided into four groups, showing the closest relationship with tomato and potato. Subsequently, the PIFs protein properties, chromosome location, gene structure, and protein interaction network were systematically analyzed. RNA-Seq and qRT-PCR analyses showed that IbPIFs were mainly expressed in stem, as well as had different gene expression patterns in response to various stresses. Among them, the expression of IbPIF3.1 was strongly induced by salt, drought, H2O2, cold, heat, Fusarium oxysporum f. sp. batatas (Fob), and stem nematodes, indicating that IbPIF3.1 might play an important role in response to abiotic and biotic stresses in sweet potato. Further research revealed that overexpression of IbPIF3.1 significantly enhanced drought and Fusarium wilt tolerance in transgenic tobacco plants. This study provides new insights for understanding PIF-mediated stress responses and lays a foundation for future investigation of sweet potato PIFs.

Integrated analysis of metabolome and transcriptome reveals the cytoplasmic effects of CMS-D2 on pollen fertility resulting from disrupted lipid metabolism.

Using cytoplasmic male sterility of Gossypium harknesii (CMS-D2) is an economical and effective method to produce cotton hybrids. However, the detrimental cytoplasmic effects of CMS-D2 on pollen fertility and fiber yields greatly limit the further development of three-line hybrid cotton in China. In this study, an integrated non-targeted metabolomics and transcriptome analysis was performed on mature pollens of maintainer line NB, isonuclear alloplasmic near-isogenic restorer lines NH and SH under two environments. A total of 820 metabolites were obtained, of which lipids and lipid-like molecules were the most, followed by organic acids derivatives, phenylpropanoids, and polyketides. Transcriptome analysis revealed significantly more differentially expressed genes (DEGs) in SH versus NH both in Anyang and Jiujiang, and most of the DEGs were significantly upregulated. Further KEGG analysis showed that most DEGs were enriched in the biosynthesis of unsaturated fatty acids, phenylalanine metabolism, and phagosome. Based on the weighted gene co-expression network analysis, totally 74 hub genes were also identified, of which three transcription factors, i.e., WRKY22, WRKY53, and ARF18 were significantly upregulated in SH and may play a negative regulatory role in pollen development by directly or indirectly regulating the jasmonic acid synthesis and signal transduction. Moreover, we found that the negative effects of CMS-D2 cytoplasm on pollen fertility were mainly due to disturbed lipid metabolism, especially the metabolic balance of unsaturated fatty acids, ultimately resulting in the decline of pollen fertility. Meanwhile, in the presence of CMS-D2 sterile cytoplasm, the cytoplasmic-nucleus interaction effects generated a substantial quantity of flavonoids involved in the fertility restoration process. This study preliminarily clarified some of the reasons for the negative effects of CMS-D2 cytoplasm on pollen fertility, and our results will provide an important theoretical reference for further breeding and improvement of three-line hybrid cotton in the future.

Single-molecule real-time transcript sequencing of developing cotton anthers facilitates genome annotation and fertility restoration candidate gene discovery.

Heterosis refers to the superior phenotypes observed in hybrids. Cytoplasmic male sterility (CMS) system plays an important role in cotton heterosis utilization. However, the global gene expression patterns of CMS-D2 and its interaction with the restorer gene Rf1 remain unclear. Here, the full-length transcript sequencing was performed in anthers of the CMS-D2 restorer line using PacBio single-molecule real-time sequencing technology. Combining PacBio SMRT long-read isoforms and Illumina RNA-seq data, 107,066 isoforms from 44,338 loci were obtained, including 10,086 novel isoforms of novel genes and 66,419 new isoforms of known genes. Totally 56,572 alternative splicing (AS) events, 1146 lncRNAs, 61 fusion transcripts and 10,466 genes exhibited alternative polyadenylation (APA), and 60,995 novel isoforms with predicted open reading frames (ORFs) were further identified. Furthermore, the specifically expressed genes in restorer line were selected and confirmed by qRT-PCR. These findings provide a basis for upland cotton genome annotation and transcriptome research, and will help to reveal the molecular mechanism of interaction between Rf1 and CMS-D2 cytoplasm.

Integrated Methylome and Transcriptome Analysis between the CMS-D2 Line ZBA and Its Maintainer Line ZB in Upland Cotton.

DNA methylation is an important epigenetic modification involved in multiple biological processes. Altered methylation patterns have been reported to be associated with male sterility in some plants, but their role in cotton cytoplasmic male sterility (CMS) remains unclear. Here, integrated methylome and transcriptome analyses were conducted between the CMS-D2 line ZBA and its near-isogenic maintainer line ZB in upland cotton. More methylated cytosine sites (mCs) and higher methylation levels (MLs) were found among the three sequence contexts in ZB compared to ZBA. A total of 4568 differentially methylated regions (DMRs) and 2096 differentially methylated genes (DMGs) were identified. Among the differentially expressed genes (DEGs) associated with DMRs (DMEGs), 396 genes were upregulated and 281 genes were downregulated. A bioinformatics analysis of these DMEGs showed that hyper-DEGs were significantly enriched in the "oxidative phosphorylation" pathway. Further qRT-PCR validation indicated that these hypermethylated genes (encoding the subunits of mitochondrial electron transport chain (ETC) complexes I and V) were all significantly upregulated in ZB. Our biochemical data revealed a higher extent of H2O2 production but a lower level of adenosine triphosphate (ATP) synthesis in CMS-D2 line ZBA. On the basis of the above results, we propose that disrupted DNA methylation in ZBA may disrupt the homeostasis of reactive oxygen species (ROS) production and ATP synthesis in mitochondria, triggering a burst of ROS that is transferred to the nucleus to initiate programmed cell death (PCD) prematurely, ultimately leading to microspore abortion. This study illustrates the important role of DNA methylation in cotton CMS.