Differential proteomics analysis to identify proteins and pathways associated with male sterility of soybean using iTRAQ-based strategy.

To further elucidate the molecular mechanism of cytoplasmic male sterility (CMS) in soybean, a differential proteomic analysis was completed between the CMS line NJCMS1A and its maintainer NJCMS1B using iTRAQ-based strategy. As a result, 180 differential abundance proteins (DAPs) were identified, of which, 60 were down-regulated and 120 were up-regulated in NJCMS1A compared with NJCMS1B. Bioinformatic analysis showed that 167 DAPs were annotated in 41 Gene Ontology functional groups, 106 DAPs were classified into 20 clusters of orthologous groups of protein categories, and 128 DAPs were enrichment in 53 KEGG pathways. Fifteen differential level proteins/genes with the same expression pattern were identified in the further conjoint analysis of DAPs and the previously reported differential expression genes. Moreover, multiple reaction monitoring test, qRT-PCR analysis and enzyme activity assay validated that the iTRAQ results were reliable. Based on functional analysis of DAPs, we concluded that male sterility in NJCMS1A might be related to insufficiencies in energy supply, unbalance of protein synthesis and degradation, disruption of flavonoid synthesis, programmed cell death, abnormalities of substance metabolism, etc. These results might facilitate our understanding of the molecular mechanisms behind CMS in soybean. BIOLOGICAL SIGNIFICANCE: Soybean is an important global crop that provides protein and oil. Heterosis is a significantly potential approach to increase the yield of soybean. Cytoplasmic male sterility (CMS) plays a vital role in the production of hybrid seeds. However, the genetic and molecular mechanisms of male sterility in soybean still need to be further elucidated. In the present paper, a differential proteomic analysis was carried out and the results showed that several key proteins involved in key pathways were associated with male sterility in soybean. This work provides a new insight to understand the genetic and molecular mechanisms underlying CMS in soybean.

Identification of miRNAs and their targets by high-throughput sequencing and degradome analysis in cytoplasmic male-sterile line NJCMS1A and its maintainer NJCMS1B of soybean.

BACKGROUND: Cytoplasmic male sterility (CMS) provides crucial breeding materials that facilitate hybrid seed production in various crops, and thus plays an important role in the study of hybrid vigor (heterosis), in plants. However, the CMS regulatory network in soybean remains unclear. MicroRNAs (miRNAs) play crucial roles in flower and pollen development by targeting genes that regulate their expression in plants. To identify the miRNAs and their targets that exist in the soybean CMS line NJCMS1A and its maintainer NJCMS1B, high-throughput sequencing and degradome analysis were conducted in this study. RESULTS: Two small RNA libraries were constructed from the flower buds of the soybean CMS line NJCMS1A and its maintainer NJCMS1B. A total of 105 new miRNAs present on the other arm of known pre-miRNAs, 23 new miRNA members, 158 novel miRNAs and 160 high-confidence soybean miRNAs were identified using high-throughput sequencing. Among the identified miRNAs, 101 differentially expressed miRNAs with greater than two-fold changes between NJCMS1A and NJCMS1B were discovered. The different expression levels of selected miRNAs were confirmed by stem-loop quantitative real-time PCR. A degradome analysis showed that 856 targets were predicted to be targeted by 296 miRNAs, including a squamosa promoter-binding protein-like transcription factor family protein, a pentatricopeptide repeat-containing protein, and an auxin response factor, which were previously shown to be involved in floral organ or anther development in plants. Additionally, some targets, including a MADS-box transcription factor, NADP-dependent isocitrate dehydrogenase and NADH-ubiquinone oxidoreductase 24 kDa subunit, were identified, and they may have some relationship with the programmed cell death, reactive oxygen species accumulation and energy deficiencies, which might lead to soybean male sterility. CONCLUSIONS: The present study is the first to use deep sequencing technology to identify miRNAs and their targets in the flower buds of the soybean CMS line NJCMS1A and its maintainer NJCMS1B. The results revealed that the miRNAs might participate in flower and pollen development, which could facilitate our understanding of the molecular mechanisms behind CMS in soybean.

Comparative Transcriptome Analysis between the Cytoplasmic Male Sterile Line NJCMS1A and Its Maintainer NJCMS1B in Soybean (Glycine max (L.) Merr.).

BACKGROUND: The utilization of soybean heterosis is probably one of the potential approaches in future yield breakthrough as was the situation in rice breeding in China. Cytoplasmic male sterility (CMS) plays an important role in the production of hybrid seeds. However, the molecular mechanism of CMS in soybean remains unclear. RESULTS: The comparative transcriptome analysis between cytoplasmic male sterile line NJCMS1A and its near-isogenic maintainer NJCMS1B in soybean was conducted using Illumina sequencing technology. A total of 88,643 transcripts were produced in Illumina sequencing. Then 56,044 genes were obtained matching soybean reference genome. Three hundred and sixty five differentially expressed genes (DEGs) between NJCMS1A and NJCMS1B were screened by threshold, among which, 339 down-regulated and 26 up-regulated in NJCMS1A compared to in NJCMS1B. Gene Ontology (GO) annotation showed that 242 DEGs were annotated to 19 functional categories. Clusters of Orthologous Groups of proteins (COG) annotation showed that 265 DEGs were classified into 19 categories. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that 46 DEGs were assigned to 33 metabolic pathways. According to functional and metabolic pathway analysis combined with reported literatures, the relations between some key DEGs and the male sterility of NJCMS1A were discussed. qRT-PCR analysis validated that the gene expression pattern in RNA-Seq was reliable. Finally, enzyme activity assay showed that energy supply was decreased in NJCMS1A compared to in NJCMS1B. CONCLUSIONS: We concluded that the male sterility of NJCMS1A might be related to the disturbed functions and metabolism pathways of some key DEGs, such as DEGs involved in carbohydrate and energy metabolism, transcription factors, regulation of pollen development, elimination of reactive oxygen species (ROS), cellular signal transduction, and programmed cell death (PCD) etc. Future research will focus on cloning and transgenic function validation of possible candidate genes associated with soybean CMS.