Dynamic N6 -methyladenosine RNA modification regulates peanut resistance to bacterial wilt.

N6 -methyladenosine (m6 A) is the most abundant mRNA modification in eukaryotes and is an important regulator of gene expression as well as many other critical biological processes. However, the characteristics and functions of m6 A in peanut (Arachis hypogea L.) resistance to bacterial wilt (BW) remain unknown. Here, we analyzed the dynamic of m6 A during infection of resistant (H108) and susceptible (H107) peanut accessions with Ralstonia solanacearum (R. solanacearum), the causative agent of BW. Throughout the transcriptome, we identified 'URUAY' as a highly conserved motif for m6 A in peanut. The majority of differential m6 A located within the 3' untranslated region (UTR) of the transcript, with fewer in the exons. Integrative analysis of RNA-Seq and m6 A methylomes suggests the correlation between m6 A and gene expression in peanut R. solanacearum infection, and functional analysis reveals that m6 A-associated genes were related to plant-pathogen interaction. Our experimental analysis suggests that AhALKBH15 is an m6 A demethylase in peanut, leading to decreased m6 A levels and upregulation of the resistance gene AhCQ2G6Y. The upregulation of AhCQ2G6Y expression appears to promote BW resistance in the H108 accession.

Genome-wide survey of the bHLH super gene family in Brassica napus.

BACKGROUND: The basic helix-loop-helix (bHLH) gene family is one of the largest transcription factor families in plants and is functionally characterized in diverse species. However, less is known about its functions in the economically important allopolyploid oil crop, Brassica napus. RESULTS: We identified 602 potential bHLHs in the B. napus genome (BnabHLHs) and categorized them into 35 subfamilies, including seven newly separated subfamilies, based on phylogeny, protein structure, and exon-intron organization analysis. The intron insertion patterns of this gene family were analyzed and a total of eight types were identified in the bHLH regions of BnabHLHs. Chromosome distribution and synteny analyses revealed that hybridization between Brassica rapa and Brassica oleracea was the main expansion mechanism for BnabHLHs. Expression analyses showed that BnabHLHs were widely in different plant tissues and formed seven main patterns, suggesting they may participate in various aspects of B. napus development. Furthermore, when roots were treated with five different hormones (IAA, auxin; GA3, gibberellin; 6-BA, cytokinin; ABA, abscisic acid and ACC, ethylene), the expression profiles of BnabHLHs changed significantly, with many showing increased expression. The induction of five candidate BnabHLHs was confirmed following the five hormone treatments via qRT-PCR. Up to 246 BnabHLHs from nine subfamilies were predicted to have potential roles relating to root development through the joint analysis of their expression profiles and homolog function. CONCLUSION: The 602 BnabHLHs identified from B. napus were classified into 35 subfamilies, and those members from the same subfamily generally had similar sequence motifs. Overall, we found that BnabHLHs may be widely involved in root development in B. napus. Moreover, this study provides important insights into the potential functions of the BnabHLHs super gene family and thus will be useful in future gene function research.

Genome-Wide Survey and Expression Analysis of the KT/HAK/KUP Family in Brassica napus and Its Potential Roles in the Response to K+ Deficiency.

The KT/HAK/KUP (HAK) family is the largest potassium (K+) transporter family in plants, which plays key roles in K+ uptake and homeostasis, stress resistance, and root and embryo development. However, the HAK family has not yet been characterized in Brassica napus. In this study, 40 putative B. napus HAK genes (BnaHAKs) are identified and divided into four groups (Groups I-III and V) on the basis of phylogenetic analysis. Gene structure analysis revealed 10 conserved intron insertion sites across different groups. Collinearity analysis demonstrated that both allopolyploidization and small-scale duplication events contributed to the large expansion of BnaHAKs. Transcription factor (TF)-binding network construction, cis-element analysis, and microRNA prediction revealed that the expression of BnaHAKs is regulated by multiple factors. Analysis of RNA-sequencing data further revealed extensive expression profiles of the BnaHAKs in groups II, III, and V, with limited expression in group I. Compared with group I, most of the BnaHAKs in groups II, III, and V were more upregulated by hormone induction based on RNA-sequencing data. Reverse transcription-quantitative polymerase reaction analysis revealed that the expression of eight BnaHAKs of groups I and V was markedly upregulated under K+-deficiency treatment. Collectively, our results provide valuable information and key candidate genes for further functional studies of BnaHAKs.

Global Survey and Expressions of the Phosphate Transporter Gene Families in Brassica napus and Their Roles in Phosphorus Response.

Phosphate (Pi) transporters play critical roles in Pi acquisition and homeostasis. However, currently little is known about these genes in oil crops. In this study, we aimed to characterize the five Pi transporter gene families (PHT1-5) in allotetraploid Brassica napus. We identified and characterized 81 putative PHT genes in B. napus (BnaPHTs), including 45 genes in PHT1 family (BnaPHT1s), four BnaPHT2s, 10 BnaPHT3s, 13 BnaPHT4s and nine BnaPHT5s. Phylogenetic analyses showed that the largest PHT1 family could be divided into two groups (Group I and II), while PHT4 may be classified into five, Groups I-V. Gene structure analysis revealed that the exon-intron pattern was conservative within the same family or group. The sequence characteristics of these five families were quite different, which may contribute to their functional divergence. Transcription factor (TF) binding network analyses identified many potential TF binding sites in the promoter regions of candidates, implying their possible regulating patterns. Collinearity analysis demonstrated that most BnaPHTs were derived from an allopolyploidization event (~40.7%) between Brassica rapa and Brassica oleracea ancestors, and small-scale segmental duplication events (~39.5%) in the descendant. RNA-Seq analyses proved that many BnaPHTs were preferentially expressed in leaf and flower tissues. The expression profiles of most colinearity-pairs in B. napus are highly correlated, implying functional redundancy, while a few pairs may have undergone neo-functionalization or sub-functionalization during evolution. The expression levels of many BnaPHTs tend to be up-regulated by different hormones inductions, especially for IAA, ABA and 6-BA treatments. qRT-PCR assay demonstrated that six BnaPHT1s (BnaPHT1.11, BnaPHT1.14, BnaPHT1.20, BnaPHT1.35, BnaPHT1.41, BnaPHT1.44) were significantly up-regulated under low- and/or rich- Pi conditions in B. napus roots. This work analyzes the evolution and expression of the PHT family in Brassica napus, which will help further research on their role in Pi transport.

Genome-wide survey and expression analyses of the GRAS gene family in Brassica napus reveals their roles in root development and stress response.

MAIN CONCLUSION: Genome-wide identification, classification, expression analyses, and functional characterization of GRAS genes in oil crop, Brassica napus, indicate their importance in root development and stress response. GRAS proteins are a plant-specific transcription factor gene family involved in tissues development and stress response. We classified 87 putative GRAS genes in the Brassica napus genome (BnGRASs) into 13 subfamilies by phylogenetic analysis. The C-terminal GRAS domains of Brassica napus (B. napus) proteins were less conserved among subfamilies, but were conserved within each subfamily. A series of analyses revealed that 89.7% of the BnGRASs did not have intron insertions, and 24 specific-motifs were found at the N-terminal. A highly conserved microRNA 171 (miRNA171) target was observed specifically in the HAM subfamily across land plants. A total of 868 pairs of interaction proteins were predicted, the primary of which were transcription factors involved in transcriptional regulation and signal transduction. Integrated comparative analysis of GRAS genes across 26 species of algae, mosses, ferns, gymnosperms, and angiosperms revealed that this gene family originated in early mosses and was classified into 19 subfamilies, 14 of which may have originated prior to bryophyte evolution. RNA-Seq analysis demonstrated that most BnGRASs were widely expressed in different tissues/organs at different stages in B. napus, and 24 BnGRASs were highly/specifically expressed in roots. Results from a qRT-PCR analysis suggested that two BnGRASs belonging to SCR and LISCL subfamilies potentially have important roles in the stress response of roots.

Evolutionary and Comparative Expression Analyses of TCP Transcription Factor Gene Family in Land Plants.

The plant-specific Teosinte-branched 1/Cycloidea/Proliferating (TCP) transcription factor genes are involved in plants' development, hormonal pathways, and stress response but their evolutionary history is uncertain. The genome-wide analysis performed here for 47 plant species revealed 535 TCP candidates in terrestrial plants and none in aquatic plants, and that TCP family genes originated early in the history of land plants. Phylogenetic analysis divided the candidate genes into Classes I and II, and Class II was further divided into CYCLOIDEA (CYC) and CINCINNATA (CIN) clades; CYC is more recent and originated from CIN in angiosperms. Protein architecture, intron pattern, and sequence characteristics were conserved in each class or clade supporting this classification. The two classes significantly expanded through whole-genome duplication during evolution. Expression analysis revealed the conserved expression of TCP genes from lower to higher plants. The expression patterns of Class I and CIN genes in different stages of the same tissue revealed their function in plant development and their opposite effects in the same biological process. Interaction network analysis showed that TCP proteins tend to form protein complexes, and their interaction networks were conserved during evolution. These results contribute to further functional studies on TCP family genes.

Global Analysis of WOX Transcription Factor Gene Family in Brassica napus Reveals Their Stress- and Hormone-Responsive Patterns.

The plant-specific WUSCHEL-related homeobox (WOX) transcription factor gene family is important for plant growth and development but little studied in oil crops. We identified and characterized 58 putative WOX genes in Brassica napus (BnWOXs), which were divided into three major clades and nine subclades based on the gene structure and conserved motifs. Collinearity analysis revealed that most BnWOXs were the products of allopolyploidization and segmental duplication events. Gene structure analysis indicated that introns/exons and protein motifs were conserved in each subclade and RNA sequencing revealed that BnWOXs had narrow expression profiles in major tissues and/or organs across different developmental stages. The expression pattern of each clade was highly conserved and similar to that of the sister and orthologous pairs from Brassica rapa and Brassica oleracea. Quantitative real-time polymerase chain reaction showed that members of the WOX4 subclade were induced in seedling roots by abiotic and hormone stresses, indicating their contribution to root development and abiotic stress responses. 463 proteins were predicted to interact with BnWOXs, including peptides regulating stem cell homeostasis in meristems. This study provides insights into the evolution and expression of the WOX gene family in B. napus and will be useful in future gene function research.

MYB Superfamily in Brassica napus: Evidence for Hormone-Mediated Expression Profiles, Large Expansion, and Functions in Root Hair Development.

MYB proteins are involved in diverse important biological processes in plants. Herein, we obtained the MYB superfamily from the allotetraploid Brassica napus, which contains 227 MYB-related (BnMYBR/Bn1R-MYB), 429 R2R3-MYB (Bn2R-MYB), 22 R1R2R3-MYB (Bn3R-MYB), and two R1R2R2R1/2-MYB (Bn4R-MYB) genes. Phylogenetic analysis classified the Bn2R-MYBs into 43 subfamilies, and the BnMYBRs into five subfamilies. Sequence characteristics and exon/intron structures within each subfamily of the Bn2R-MYBs and BnMYBRs were highly conserved. The whole superfamily was unevenly distributed on 19 chromosomes and underwent unbalanced expansion in B. napus. Allopolyploidy between B. oleracea and B. rapa mainly contributed to the expansion in their descendent B. napus, in which B. rapa-derived genes were more retained. Comparative phylogenetic analysis of 2R-MYB proteins from nine Brassicaceae and seven non-Brassicaceae species identified five Brassicaceae-specific subfamilies and five subfamilies that are lacking from the examined Brassicaceae species, which provided an example for the adaptive evolution of the 2R-MYB gene family alongside angiosperm diversification. Ectopic expression of four Bn2R-MYBs under the control of the viral CaMV35S and/or native promoters could rescue the lesser root hair phenotype of the Arabidopsis thaliana wer mutant plants, proving the conserved negative roles of the 2R-MYBs of the S15 subfamily in root hair development. RNA-sequencing data revealed that the Bn2R-MYBs and BnMYBRs had diverse transcript profiles in roots in response to the treatments with various hormones. Our findings provide valuable information for further functional characterizations of B. napusMYB genes.

The auxin response factor gene family in allopolyploid Brassica napus.

Auxin response factor (ARF) is a member of the plant-specific B3 DNA binding superfamily. Here, we report the results of a comprehensive analysis of ARF genes in allotetraploid Brassica napus (2n = 38, AACC). Sixty-seven ARF genes were identified in B. napus (BnARFs) and divided into four subfamilies (I-IV). Sixty-one BnARFs were distributed on all chromosomes except C02; the remaining were on Ann and Cnn. The full length of the BnARF proteins was highly conserved especially within each subfamily with all members sharing the N-terminal DNA binding domain (DBD) and the middle region (MR), and most contained the C-terminal dimerization domain (PBI). Twenty-one members had a glutamine-rich MR that may be an activator and the remaining were repressors. Accordingly, the intron patterns are highly conserved in each subfamily or clade, especially in DBD and PBI domains. Several members in subfamily III are potential targets for miR167. Many putative cis-elements involved in phytohormones, light signaling responses, and biotic and abiotic stress were identified in BnARF promoters, implying their possible roles. Most ARF proteins are likely to interact with auxin/indole-3-acetic acid (Aux/IAA) -related proteins, and members from different subfamilies generally shared many common interaction proteins. Whole genome-wide duplication (WGD) by hybridization between Brassica rapa and Brassica oleracea and segmental duplication led to gene expansion. Gene loss following WGD is biased with the An-subgenome retaining more ancestral genes than the Cn-subgenome. BnARFs have wide expression profiles across vegetative and reproductive organs during different developmental stages. No obvious expression bias was observed between An- and Cn-subgenomes. Most synteny-pair genes had similar expression patterns, indicating their functional redundancy. BnARFs were sensitive to exogenous IAA and 6-BA treatments especially subfamily III. The present study provides insights into the distribution, phylogeny, and evolution of ARF gene family.

Evolution and expression analyses of the MADS-box gene family in Brassica napus.

MADS-box transcription factors are important for plant growth and development, and hundreds of MADS-box genes have been functionally characterized in plants. However, less is known about the functions of these genes in the economically important allopolyploid oil crop, Brassica napus. We identified 307 potential MADS-box genes (BnMADSs) in the B. napus genome and categorized them into type I (Mα, Mß, and Mγ) and type II (MADS DNA-binding domain, intervening domain, keratin-like domain, and C-terminal domain [MIKC]c and MIKC*) based on phylogeny, protein motif structure, and exon-intron organization. We identified one conserved intron pattern in the MADS-box domain and seven conserved intron patterns in the K-box domain of the MIKCc genes that were previously ignored and may be associated with function. Chromosome distribution and synteny analysis revealed that hybridization between Brassica rapa and Brassica oleracea, segmental duplication, and homologous exchange (HE) in B. napus were the main BnMADSs expansion mechanisms. Promoter cis-element analyses indicated that BnMADSs may respond to various stressors (drought, heat, hormones) and light. Expression analyses showed that homologous genes in a given subfamily or sister pair are highly conserved, indicating widespread functional conservation and redundancy. Analyses of BnMADSs provide a basis for understanding their functional roles in plant development.