Genome-wide identification of the restorer-of-fertility-like (RFL) gene family in Brassica napus and expression analysis in Shaan2A cytoplasmic male sterility.

BACKGROUND: Cytoplasmic male sterility (CMS) is very important in hybrid breeding. The restorer-of-fertility (Rf) nuclear genes rescue the sterile phenotype. Most of the Rf genes encode pentatricopeptide repeat (PPR) proteins. RESULTS: We investigated the restorer-of-fertility-like (RFL) gene family in Brassica napus. A total of 53 BnRFL genes were identified. While most of the BnRFL genes were distributed on 10 of the 19 chromosomes, gene clusters were identified on chromosomes A9 and C8. The number of PPR motifs in the BnRFL proteins varied from 2 to 19, and the majority of BnRFL proteins harbored more than 10 PPR motifs. An interaction network analysis was performed to predict the interacting partners of RFL proteins. Tissue-specific expression and RNA-seq analyses between the restorer line KC01 and the sterile line Shaan2A indicated that BnRFL1, BnRFL5, BnRFL6, BnRFL8, BnRFL11, BnRFL13 and BnRFL42 located in gene clusters on chromosomes A9 and C8 were highly expressed in KC01. CONCLUSIONS: In the present study, identification and gene expression analysis of RFL gene family in the CMS system were conducted, and seven BnRFL genes were identified as candidates for the restorer genes in Shaan2A CMS. Taken together, this method might provide new insight into the study of Rf genes in other CMS systems.

The initial deficiency of protein processing and flavonoids biosynthesis were the main mechanisms for the male sterility induced by SX-1 in Brassica napus.

BACKGROUND: Rapeseed (Brassica napus) is an important oil seed crop in the Brassicaceae family. Chemical induced male sterility (CIMS) is one of the widely used method to produce the hybrids in B. napus. Identification of the key genes and pathways that involved in CIMS were important to understand the underlying molecular mechanism. In the present report, a multi-omics integrative analysis, including of the proteomic, transcriptomic and miRNAs, combined with morphological and physiological analysis were conducted. RESULTS: Earlier degeneration of the tapetosomes and elaioplasts, aberrantly stacking in tapetal cells and incompletely deposition in tryphine of pollen wall were observed in chemical hybridization agent (CHA) of SX-1 treated B. napus through SEM and TEM analysis. It was revealed that the deficiencies in protein processing in endoplasmic reticulum (ER) and flavonoids biosynthesis were occurred at early stage in the SX-1 treated materials. Subsequently, plant hormone signal transduction, biosynthesis of amino acids, fatty acids and steroid in anther at later stages were identified down-regulated after SX-1 treatment. 144 transcript factors (TFs) were also indentified to down-regulated at early stage, which suggested the early regulation in anther and pollen wall development were disordered in CHA treated B. napus. In addition, 7 important miRNAs were identified and 2 of the predicted target genes of miRNAs were Rf-like genes. CONCLUSIONS: Taken together, an interaction network of candidate genes and the putative metabolism pathways were constructed based on the multi-omics integrative analysis, it provided a new insight into the male sterility induced by CHA of SX-1 in B. napus.

Transcriptomic and Proteomic Analysis of Shaan2A Cytoplasmic Male Sterility and Its Maintainer Line in Brassica napus.

Cytoplasmic male sterility (CMS) lines are widely used for hybrid production in Brassica napus. The Shaan2A CMS system is one of the most important in China and has been used for decades; however, the male sterility mechanism underlying Shaan2A CMS remains unknown. Here, we performed transcriptomic and proteomic analysis, combined with additional morphological observation, in the Shaan2A CMS. Sporogenous cells, endothecium, middle layer, and tapetum could not be clearly distinguished in Shaan2A anthers. Furthermore, Shaan2A anther chloroplasts contained fewer starch grains than those in Shaan2B (a near-isogenic line of Shaan2A), and the lamella structure of chloroplasts in Shaan2A anther wall cells was obviously aberrant. Transcriptomic analysis revealed differentially expressed genes (DEGs) mainly related to carbon metabolism, lipid and flavonoid metabolism, and the mitochondrial electron transport/ATP synthesis pathway. Proteomic results showed that differentially expressed proteins were mainly associated with carbohydrate metabolism, energy metabolism, and genetic information processing pathways. Importantly, nine gene ontology categories associated with anther and pollen development were enriched among down-regulated DEGs at the young bud (YB) stage, including microsporogenesis, sporopollenin biosynthetic process, and tapetal layer development. Additionally, 464 down-regulated transcription factor (TF) genes were identified at the YB stage, including some related to early anther differentiation such as SPOROCYTELESS (SPL, also named NOZZLE, NZZ), DYSFUNCTIONAL TAPETUM 1 (DYT1), MYB80 (formerly named MYB103), and ABORTED MICROSPORES (AMS). These results suggested that the sterility gene in the Shaan2A mitochondrion might suppress expression of these TF genes in the nucleus, affecting early anther development. Finally, we constructed an interaction network of candidate proteins based on integrative analysis. The present study provides new insights into the molecular mechanism of Shaan2A CMS in B. napus.

Quantitative Proteomic Analysis Provides Novel Insights into Cold Stress Responses in Petunia Seedlings.

Low temperature is a major adverse environmental factor that impairs petunia growth and development. To better understand the molecular mechanisms of cold stress adaptation of petunia plants, a quantitative proteomic analysis using iTRAQ technology was performed to detect the effects of cold stress on protein expression profiles in petunia seedlings which had been subjected to 2°C for 5 days. Of the 2430 proteins whose levels were quantitated, a total of 117 proteins were discovered to be differentially expressed under low temperature stress in comparison to unstressed controls. As an initial study, 44 proteins including well known and novel cold-responsive proteins were successfully annotated. By integrating the results of two independent Gene Ontology (GO) enrichment analyses, seven common GO terms were found of which "oxidation-reduction process" was the most notable for the cold-responsive proteins. By using the subcellular localization tool Plant-mPLoc predictor, as much as 40.2% of the cold-responsive protein group was found to be located within chloroplasts, suggesting that the chloroplast proteome is particularly affected by cold stress. Gene expression analyses of 11 cold-responsive proteins by real time PCR demonstrated that the mRNA levels were not strongly correlated with the respective protein levels. Further activity assay of anti-oxidative enzymes showed different alterations in cold treated petunia seedlings. Our investigation has highlighted the role of antioxidation mechanisms and also epigenetic factors in the regulation of cold stress responses. Our work has provided novel insights into the plant response to cold stress and should facilitate further studies regarding the molecular mechanisms which determine how plant cells cope with environmental perturbation. The data have been deposited to the ProteomeXchange with identifier PXD002189.

Transcriptional profiling of Petunia seedlings reveals candidate regulators of the cold stress response.

Petunias are important ornamentals with the capacity for cold acclimation. So far, there is limited information concerning gene regulation and signaling pathways associated with the cold stress response in petunias. A custom-designed petunia microarray representing 24816 genes was used to perform transcriptome profiling in petunia seedlings subjected to cold at 2°C for 0.5 h, 2 h, 24 h, and 5 d. A total of 2071 transcripts displayed differential expression patterns under cold stress, of which 1149 were up-regulated and 922 were down-regulated. Gene ontology enrichment analysis demarcated related biological processes, suggesting a possible link between flavonoid metabolism and plant adaptation to low temperatures. Many novel stress-responsive regulators were revealed, suggesting that diverse regulatory pathways may exist in petunias in addition to the well-characterized CBF pathway. The expression changes of selected genes under cold and other abiotic stress conditions were confirmed by real-time RT-PCR. Furthermore, weighted gene co-expression network analysis divided the petunia genes on the array into 65 modules that showed high co-expression and identified stress-specific hub genes with high connectivity. Our identification of these transcriptional responses and groups of differentially expressed regulators will facilitate the functional dissection of the molecular mechanism in petunias responding to environment stresses and extend our ability to improve cold tolerance in plants.