Genome-Wide Analysis of Phosphorus Transporter Genes in Brassica and Their Roles in Heavy Metal Stress Tolerance.

Phosphorus transporter (PHT) genes encode H2PO4-/H+ co-transporters that absorb and transport inorganic nutrient elements required for plant development and growth and protect plants from heavy metal stress. However, little is known about the roles of PHTs in Brassica compared to Arabidopsis thaliana. In this study, we identified and extensively analyzed 336 PHTs from three diploid (B. rapa, B. oleracea, and B. nigra) and two allotetraploid (B. juncea and B. napus) Brassica species. We categorized the PHTs into five phylogenetic clusters (PHT1-PHT5), including 201 PHT1 homologs, 15 PHT2 homologs, 40 PHT3 homologs, 54 PHT4 homologs, and 26 PHT5 homologs, which are unevenly distributed on the corresponding chromosomes of the five Brassica species. All PHT family genes from Brassica are more closely related to Arabidopsis PHTs in the same vs. other clusters, suggesting they are highly conserved and have similar functions. Duplication and synteny analysis revealed that segmental and tandem duplications led to the expansion of the PHT gene family during the process of polyploidization and that members of this family have undergone purifying selection during evolution based on Ka/Ks values. Finally, we explored the expression profiles of BnaPHT family genes in specific tissues, at various developmental stages, and under heavy metal stress via RNA-seq analysis and qRT-PCR. BnaPHTs that were induced by heavy metal treatment might mediate the response of rapeseed to this important stress. This study represents the first genome-wide analysis of PHT family genes in Brassica species. Our findings improve our understanding of PHT family genes and provide a basis for further studies of BnaPHTs in plant tolerance to heavy metal stress.

KIF20B and MET, hub genes of DIAPHs, predict poor prognosis and promote pancreatic cancer progression.

BACKGROUND: The DIAPHs (DIAPH1, DIAPH2, and DIAPH3) are members of the diaphanous subfamily of the formin family. KIF20B and MET, hub genes of DIAPHs, play crucial roles in cytoskeletal remodeling, cell migration, and adhesion. However, their combined prognostic and treatment value in pancreatic adenocarcinoma (PC) warrants further investigation. METHODS: Multiomics analysis tools were used to comprehensively assess the genomic expression and prognostic value of KIF20B and MET in PC. Immune cell infiltration, functional enrichment, single-cell RNA-seq (scRNA) analysis, potential therapeutic drugs, and nomograms were established and analyzed. CCK-8 levels, transwell assay, Co-IP assay, mass spectrometry, and western blotting were performed to assess the role of KIF20B and MET as modulators of ß-catenin and Lactate Dehydrogenase A (LDHA) in vitro. Xenograft tumor models were used to evaluate the anti-tumor effects in vivo. RESULTS: DIAPHs, KIF20B, and MET were overexpressed and functioned as poor prognostic markers of PC. Immunoinfiltration analysis revealed that pDC and NK cells were enriched with low expression levels of KIF20B and MET, whereas Th2 cells were enriched with high expression levels of these two genes. The copy number variations (CNVs) in KIF20B and MET were positively correlated with B cell and CD4 + T cell infiltration. Immunological checkpoints NT5E and CD44 were positively correlated with KIF20B and MET expression. Moreover, the nomogram constructed based on KIF20B and MET demonstrated predictive value for overall survival. scRNA-Seq analysis indicated that KIF20B and MET were enriched in endothelial, malignant, B, T, and CD8 + T cells, which correlated with glycolysis and the epithelial-mesenchymal transition (EMT). The interactions of KIF20B and MET with ß-catenin and LDHA were verified by Co-IP assay and mass spectrometry. Knockdown of KIF20B and MET downregulates ß-catenin and LDHA in vitro. Furthermore, dual knockdown of KIF20B and MET exhibited a synergistic suppressive effect on PC progression in vitro and in vivo. CONCLUSION: DIAPHs, KIF20B, and MET are promising candidates for the prognosis and treatment of PC. More importantly, downregulation of KIF20B and MET inhibited pancreatic cancer progression by regulating LDHA and EMT.

EIF2Ss, a Novel c-Myc-Correlated Gene Family, is Associated with Poor Prognosis and Immune Infiltration in Pancreatic Adenocarcinoma.

BACKGROUND: Pancreatic adenocarcinoma (PAAD) is a highly malignant tumor in urgent need of novel diagnostics, prognostic markers, and treatments. Eukaryotic translation initiation factor 2 subunits (EIF2Ss), comprising Eukaryotic translation initiation factor 2 subunit alpha (EIF2S1), Eukaryotic translation initiation factor 2 subunit beta (EIF2S2), and Eukaryotic translation initiation factor 2 subunit gamma (EIF2S3), is a family of eukaryotic initiation factors that participate in early protein synthesis and are crucial for tumor initiation and progression. However, the role of EIF2Ss in PAAD has yet to be reported. The aim of this study was therefore to analyze EIF2Ss in relation to the diagnosis, prognosis, and treatment of PAAD. METHODS: The cancer genome atlas (TCGA) database was used to investigate gene expression and patient survival. Gene alterations, immune cell infiltration, and immune checkpoints in PAAD were also evaluated. Univariate and multivariate analysis, nomograms, calibration curves, and Decision Curve Analysis (DCA) diagrams were used to develop and evaluate a prediction model for patient outcome. Single-cell RNA-seq (scRNA) analysis, functional enrichment, co-IP assay, mass spectrometry, and western blot were used to study the relationship between EIF2Ss and c-myc in PAAD. RESULTS: EIF2Ss are over-expressed in PAAD tissue and are associated with poor prognosis. The frequency of EIF2S1, EIF2S2, and EIF2S3 gene alteration in PAAD was 0.2%, 0.4%, and 0.2%, respectively. High EIF2Ss expression was associated with Th2 cell infiltration, whereas low expression was associated with pDC infiltration. Moreover, EIF2Ss expression was positively correlated with the expression of the NT5E, ULBP1, PVR, CD44, IL10RB, and CD276 checkpoints. A prediction model developed using EIF2Ss and important clinicopathologic features showed good predictive value for the overall survival of PAAD patients. ScRNA-Seq data showed that EIF2Ss was associated with enrichment for endothelial cells, fibroblasts, malignant cells, and ductal cells. EIF2Ss expression was also correlated with adipogenesis, interferon-alpha response, epithelial-mesenchymal transition, myc targets, G2M checkpoint, oxidative phosphorylation, and hypoxia. Functional enrichment analysis of EIF2Ss showed a close correlation with the myc pathway, and interactions between EIF2Ss and c-myc were confirmed by co-IP assay and mass spectrometry. Importantly, knockdown of c-myc decreased the expression of EIF2S1, EIF2S2, and EIF2S3 in PAAD cells. CONCLUSIONS: EIF2Ss were found to have significant clinical implications for the prognosis and treatment of PAAD. Inhibition of c-myc caused the downregulation of EIF2S1, EIF2S2, and EIF2S3 expression.

Genome-Wide Association Study of Glucosinolate Metabolites (mGWAS) in Brassica napus L.

Glucosinolates (GSLs) are secondary plant metabolites that are enriched in rapeseed and related Brassica species, and they play important roles in defense due to their anti-nutritive and toxic properties. Here, we conducted a genome-wide association study of six glucosinolate metabolites (mGWAS) in rapeseed, including three aliphatic glucosinolates (m145 gluconapin, m150 glucobrassicanapin and m151 progoitrin), one aromatic glucosinolate (m157 gluconasturtiin) and two indole glucosinolates (m165 indolylmethyl glucosinolate and m172 4-hydroxyglucobrassicin), respectively. We identified 113 candidate intervals significantly associated with these six glucosinolate metabolites. In the genomic regions linked to the mGWAS peaks, 187 candidate genes involved in glucosinolate biosynthesis (e.g., BnaMAM1, BnaGGP1, BnaSUR1 and BnaMYB51) and novel genes (e.g., BnaMYB44, BnaERF025, BnaE2FC, BnaNAC102 and BnaDREB1D) were predicted based on the mGWAS, combined with analysis of differentially expressed genes. Our results provide insight into the genetic basis of glucosinolate biosynthesis in rapeseed and should facilitate marker-based breeding for improved seed quality in Brassica species.

Comparative transcriptome analysis identifies candidate genes related to seed coat color in rapeseed.

Yellow seed coat in rapeseed (Brassica napus) is a desirable trait that can be targeted to improve the quality of this oilseed crop. To better understand the inheritance mechanism of the yellow-seeded trait, we performed transcriptome profiling of developing seeds in yellow- and black-seeded rapeseed with different backgrounds. The differentially expressed genes (DEGs) during seed development showed significant characteristics, these genes were mainly enriched for the Gene Ontology (GO) terms carbohydrate metabolic process, lipid metabolic process, photosynthesis, and embryo development. Moreover, 1206 and 276 DEGs, which represent candidates to be involved in seed coat color, were identified between yellow- and black-seeded rapeseed during the middle and late stages of seed development, respectively. Based on gene annotation, GO enrichment analysis, and protein-protein interaction network analysis, the downregulated DEGs were primarily enriched for the phenylpropanoid and flavonoid biosynthesis pathways. Notably, 25 transcription factors (TFs) involved in regulating flavonoid biosynthesis pathway, including known (e.g., KNAT7, NAC2, TTG2 and STK) and predicted TFs (e.g., C2H2-like, bZIP44, SHP1, and GBF6), were identified using integrated gene regulatory network (iGRN) and weight gene co-expression networks analysis (WGCNA). These candidate TF genes had differential expression profiles between yellow- and black-seeded rapeseed, suggesting they might function in seed color formation by regulating genes in the flavonoid biosynthesis pathway. Thus, our results provide in-depth insights that facilitate the exploration of candidate gene function in seed development. In addition, our data lay the foundation for revealing the roles of genes involved in the yellow-seeded trait in rapeseed.

A Genome-Wide Survey of MATE Transporters in Brassicaceae and Unveiling Their Expression Profiles under Abiotic Stress in Rapeseed.

The multidrug and toxic compound extrusion (MATE) protein family is important in the export of toxins and other substrates, but detailed information on this family in the Brassicaceae has not yet been reported compared to Arabidopsis thaliana. In this study, we identified 57, 124, 81, 85, 130, and 79 MATE genes in A. thaliana, Brassica napus, Brassica oleracea, Brassica rapa, Brassica juncea, and Brassica nigra, respectively, which were unevenly distributed on chromosomes owing to both tandem and segmental duplication events. Phylogenetic analysis showed that these genes could be classified into four subgroups, shared high similarity and conservation within each group, and have evolved mainly through purifying selection. Furthermore, numerous B. napusMATE genes showed differential expression between tissues and developmental stages and between plants treated with heavy metals or hormones and untreated control plants. This differential expression was especially pronounced for the Group 2 and 3 BnaMATE genes, indicating that they may play important roles in stress tolerance and hormone induction. Our results provide a valuable foundation for the functional dissection of the different BnaMATE homologs in B. napus and its parental lines, as well as for the breeding of more stress-tolerant B. napus genotypes.

Genome-wide identification AINTEGUMENTA-like (AIL) genes in Brassica species and expression patterns during reproductive development in Brassica napus L.

The AINTEGUMENTA-like (AIL) proteins, which belong to the AP2 family, play important roles in regulating the growth and development of plant organs. The AIL family has not yet been comprehensively studied in rapeseed (Brassica napus), an allotetraploid and model organism for the study of polyploid evolution. In the present study, 99 AIL family genes were identified and characterized from B. rapa, B. oleracea, B. napus, B. juncea, and B. nigra using a comprehensive genome-wide study, including analyses of phylogeny, gene structure, chromosomal localization, and expression pattern. Using a phylogenetic analysis, the AIL genes were divided into eight groups, which were closely related to the eight AtAIL genes, and which shared highly conserved structural features within the same subfamily. The non-synonymous/synonymous substitution ratios of the paralogs and orthologs were less than 1, suggesting that the AIL genes mainly experienced purifying selection during evolution. In addition, the RNA sequencing data and qRT-PCR analysis revealed that the B. napus AIL genes exhibited organ- and developmental stage-specific expression patterns. Certain genes were highly expressed in the developing seeds (BnaAIL1, BnaAIL2, BnaAIL5, and BnaAIL6), the roots (BnaANT, BnaAIL5, and BnaAIL6), and the stem (BnaAIL7B). Our results provide valuable information for further functional analysis of the AIL family in B. napus and related Brassica species.

Association Mapping Analysis of Fatty Acid Content in Different Ecotypic Rapeseed Using mrMLM.

Brassica napus L. is a widely cultivated oil crop and provides important resources of edible vegetable oil, and its quality is determined by fatty acid composition and content. To explain the genetic basis and identify more minor loci for fatty acid content, the multi-locus random-SNP-effect mixed linear model (mrMLM) was used to identify genomic regions associated with fatty acid content in a genetically diverse population of 435 rapeseed accessions, including 77 winter-type, 55 spring-type, and 303 semi-winter-type accessions grown in different environments. A total of 149 quantitative trait nucleotides (QTNs) were found to be associated with fatty acid content and composition, including 34 QTNs that overlapped with the previously reported loci, and 115 novel QTNs. Of these, 35 novel QTNs, located on chromosome A01, A02, A03, A05, A06, A09, A10, and C02, respectively, were repeatedly detected across different environments. Subsequently, we annotated 95 putative candidate genes by BlastP analysis using sequences from Arabidopsis thaliana homologs of the identified regions. The candidate genes included 34 environmentally-insensitive genes (e.g., CER4, DGK2, KCS17, KCS18, MYB4, and TT16) and 61 environment-sensitive genes (e.g., FAB1, FAD6, FAD7, KCR1, KCS9, KCS12, and TT1) as well as genes invloved in the fatty acid biosynthesis. Among these, BnaA08g08280D and BnaC03g60080D differed in genomic sequence between the high- and low-oleic acid lines, and might thus be the novel alleles regulating oleic acid content. Furthermore, RT-qPCR analysis of these genes showed differential expression levels during seed development. Our results highlight the practical and scientific value of mrMLM or QTN detection and the accuracy of linking specific QTNs to fatty acid content, and suggest a useful strategy to improve the fatty acid content of B. napus seeds by molecular marker-assisted breeding.

Genome-Wide Identification and Structural Analysis of bZIP Transcription Factor Genes in Brassica napus.

The basic region/leucine zipper motif (bZIP) transcription factor family is one of the largest families of transcriptional regulators in plants. bZIP genes have been systematically characterized in some plants, but not in rapeseed (Brassica napus). In this study, we identified 247 BnbZIP genes in the rapeseed genome, which we classified into 10 subfamilies based on phylogenetic analysis of their deduced protein sequences. The BnbZIP genes were grouped into functional clades with Arabidopsis genes with similar putative functions, indicating functional conservation. Genome mapping analysis revealed that the BnbZIPs are distributed unevenly across all 19 chromosomes, and that some of these genes arose through whole-genome duplication and dispersed duplication events. All expression profiles of 247 bZIP genes were extracted from RNA-sequencing data obtained from 17 different B. napus ZS11 tissues with 42 various developmental stages. These genes exhibited different expression patterns in various tissues, revealing that these genes are differentially regulated. Our results provide a valuable foundation for functional dissection of the different BnbZIP homologs in B. napus and its parental lines and for molecular breeding studies of bZIP genes in B. napus.