Genome-wide characterization, expression analyses, and functional prediction of the NPF family in Brassica napus.

BACKGROUND: NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER (NRT1/PTR) family (NPF) members are essential transporters for many substrates in plants, including nitrate, hormones, peptides, and secondary metabolites. Here, we report the global characterization of NPF in the important oil crop Brassica napus, including that for phylogeny, gene/protein structures, duplications, and expression patterns. RESULTS: A total of 199 B. napus (BnaNPFs) NPF-coding genes were identified. Phylogenetic analyses categorized these genes into 11 subfamilies, including three new ones. Sequence feature analysis revealed that members of each subfamily contain conserved gene and protein structures. Many hormone-/abiotic stress-responsive cis-acting elements and transcription factor binding sites were identified in BnaNPF promoter regions. Chromosome distribution analysis indicated that BnaNPFs within a subfamily tend to cluster on one chromosome. Syntenic relationship analysis showed that allotetraploid creation by its ancestors (Brassica rapa and Brassica oleracea) (57.89%) and small-scale duplication events (39.85%) contributed to rapid BnaNPF expansion in B. napus. A genome-wide spatiotemporal expression survey showed that NPF genes of each Arabidopsis and B. napus subfamily have preferential expression patterns across developmental stages, most of them are expressed in a few organs. RNA-seq analysis showed that many BnaNPFs (32.66%) have wide exogenous hormone-inductive profiles, suggesting important hormone-mediated patterns in diverse bioprocesses. Homologs in a clade or branch within a given subfamily have conserved organ/spatiotemporal and hormone-inductive profiles, indicating functional conservation during evolution. qRT-PCR-based comparative expression analysis of the 12 BnaNPFs in the NPF2-1 subfamily between high- and low-glucosinolate (GLS) content B. napus varieties revealed that homologs of AtNPF2.9 (BnaNPF2.12, BnaNPF2.13, and BnaNPF2.14), AtNPF2.10 (BnaNPF2.19 and BnaNPF2.20), and AtNPF2.11 (BnaNPF2.26 and BnaNPF2.28) might be involved in GLS transport. qRT-PCR further confirmed the hormone-responsive expression profiles of these putative GLS transporter genes. CONCLUSION: We identified 199 B. napus BnaNPFs; these were divided into 11 subfamilies. Allopolyploidy and small-scale duplication events contributed to the immense expansion of BnaNPFs in B. napus. The BnaNPFs had preferential expression patterns in different tissues/organs and wide hormone-induced expression profiles. Four BnaNPFs in the NPF2-1 subfamily may be involved in GLS transport. Our results provide an abundant gene resource for further functional analysis of BnaNPFs.

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.

Effects of oxygen tension on the microbial community and functional gene expression of aerobic methane oxidation coupled to denitrification systems.

Aerobic CH4 oxidation coupled to denitrification (AME-D) can not only mitigate the emission of greenhouse gas (e.g., CH4) to the atmosphere, but also reduce NO3- and/or NO2- and alleviate nitrogen pollution. The effects of O2 tension on the community and functional gene expression of methanotrophs and denitrifiers were investigated in this study. Although higher CH4 oxidation occurred in the AME-D system with an initial O2 concentration of 21% (i.e., the O2-sufficient condition), more NO3--N was removed at the initial O2 concentration of 10% (i.e., the O2-limited environment). Type I methanotrophs, including Methylocaldum, Methylobacter, Methylococcus, Methylomonas, and Methylomicrobium, and type II methanotrophs, including Methylocystis and Methylosinus, dominated in the AME-D systems. Compared with type II methanotrophs, type I methanotrophs were more abundant in the AME-D systems. Proteobacteria and Actinobacteria were the main denitrifiers in the AME-D systems, and their compositions varied with the O2 tension. Quantitative PCR of the pmoA, nirS, and 16S rRNA genes showed that methanotrophs and denitrifiers were the main microorganisms in the AME-D systems, accounting for 46.4% and 24.1% in the O2-limited environment, respectively. However, the relative transcripts of the functional genes including pmoA, mmoX, nirK, nirS, and norZ were all less than 1%, especially the functional genes involved in denitrification under the O2-sufficient condition, likely due to the majority of the denitrifiers being dormant or even nonviable. These findings indicated that an optimal O2 concentration should be used to optimize the activity and functional gene expression of aerobic methanotrophs and denitrifiers in AME-D systems.