Metabolism, antioxidant and immunity in acute and chronic hypoxic stress and the improving effect of vitamin C in the channel catfish (Ictalurus punctatus).

Hypoxia is the most significant factor that threatens the health and even survival of freshwater and marine fish. Priority should be given to the investigation of hypoxia adaptation mechanisms and their subsequent modulation. Acute and chronic studies were designed for the current study. Acute hypoxia comprised of normoxia dissolved oxygen (DO) 7.0 ± 0.5 mg/mL (N0), low-oxygen 5.0 ± 0.5 mg/mL(L0), and hypoxia 1.0 ± 0.1 mg/mL (H0) and 300 mg/L Vc for hypoxia regulation (N300, L300, H300). Chronic hypoxia comprised of normoxia (DO 7.0 ± 0.5 mg/mL) with 50 mg/kg Vc in the diet (N50) and low oxygen (5.0 ± 0.5 mg/mL) with 50, 250, 500 mg/kg Vc in the diet (L50, L250, L500) to assess the effect of Vc in hypoxia. The growth, behavior, hematological parameters, metabolism, antioxidants, and related inflammatory factors of channel catfish were investigated, and it was found that channel catfish have a variety of adaptive mechanisms in response to acute and chronic hypoxia. Under acute 5 mg/mL DO, the body color lightened (P < 0.05) and reverted to normal with 300 mg/mL Vc. PLT was significantly elevated after 300 mg/L Vc (P < 0.05), indicating that Vc can effectively restore hemostasis following oxygen-induced tissue damage. Under acute hypoxia, the significantly increased of cortisol, blood glucose, the gene of pyruvate kinase (pk), and phosphofructokinase (pfk), together with the decreased expression of fructose1,6-bisphosphatase (fbp) and the reduction in myoglycogen, suggested that Vc might enhance the glycolytic ability of the channel catfish. And the enzyme activities of superoxide dismutase (SOD) and catalase (CAT) and the gene expression of sod rose significantly, showing that Vc might improve the antioxidant capacity of the channel catfish. The significant up-regulation of tumor necrosis factor-alpha (tnf-α), interleukin-1ß (il-1ß), and cd68 under acute hypoxia implies that hypoxia may generate inflammation in channel catfish, whereas the addition of Vc and down-regulation of these genes suggests that Vc suppresses inflammation under acute hypoxia. We found that the final weight, WGR, FCR, and FI of channel catfish were significantly reduced under chronic hypoxia, and that feeding 250 mg/kg of Vc in the diet was effective in alleviating the growth retardation caused by hypoxia. The significant increase in cortisol, blood glucose, myoglycogen, and the expression of tnf-α, il-1ß, and cd68 (P < 0.05) and the significant decrease in lactate (P < 0.05) under chronic hypoxia indicated that the channel catfish had gradually adapted to the survival threat posed by hypoxia and no longer relied on carbohydrates as their primary source of energy. While the addition of Vc did not appear to increase the energy supply of the fish under hypoxia in terms of glucose metabolism, but the significantly decreased expression of tnf-α, il-1ß, and cd68 (P < 0.05) also were found, indicating that chronic hypoxia, similar acute hypoxia, may increase inflammation in the channel catfish. This study indicates that under acute stress, channel catfish withstand stress by raising energy supply through glycolysis, and acute hypoxic stress significantly promotes inflammation in channel catfish, but Vc assists the channel catfish resist stress by raising glycolysis, antioxidant capacity, and decreasing the production of inflammatory markers. Under chronic hypoxia, the channel catfish no longer utilize carbohydrates as their primary energy source, and Vc may still effectively reduce inflammation in the channel catfish under hypoxia.

Dietary Leucine Supplementation Improves Muscle Fiber Growth and Development by Activating AMPK/Sirt1 Pathway in Blunt Snout Bream (Megalobrama amblycephala).

This research is aimed at evaluating the effects of leucine supplementation on muscle fibers growth and development of blunt snout bream through a feeding trial and a primary muscle cells treatment. An 8-week trial with diets containing 1.61% leucine (LL) or 2.15% leucine (HL) was conducted in blunt snout bream (mean initial weight = 56.56 ± 0.83 g). Results demonstrated that the specific gain rate and the condition factor of fish in the HL group were the highest. The essential amino acids content of fish fed HL diets was significantly higher than that fed LL diets. The texture (hardness, springiness, resilience, and chewiness), the small-sized fiber ratio, fibers density, and sarcomere lengths in fish all obtained the highest in the HL group. Additionally, the proteins expression related with the activation of the AMPK pathway (p-Ampk, Ampk, p-Ampk/Ampk, and Sirt1) and the expression of genes (myogenin (myog), myogenic regulatory factor 4 (mrf4) and myoblast determination protein (myod), and protein (Pax7) related to muscle fiber formation were significantly upregulated with increasing level of dietary leucine. In vitro, the muscle cells were treated with 0, 40 and 160 mg/L leucine for 24 h. The results showed that treated with 40 mg/L leucine significantly raised the protein expressions of BCKDHA, Ampk, p-Ampk, p-Ampk/Ampk, Sirt1, and Pax7 and the gene expressions of myog, mrf4, and myogenic factor 5 (myf5) in muscle cells. In summary, leucine supplementation promoted muscle fibers growth and development, which may be related to the activation of BCKDH and AMPK.

Interactive effects of dietary leucine and isoleucine affect amino acid profile and metabolism through AKT/TOR signaling pathways in blunt snout bream (Megalobrama amblycephala).

The purpose of this research is to explore the interaction between dietary leucine and isoleucine levels on whole-body composition, plasma and liver biochemical indexes, amino acids deposition in the liver, and amino acid metabolism of blunt snout bream (Megalobrama amblycephala). The test fish (average weight: 56.00 ± 0.55 g) were fed one of six diets at random containing two leucine levels (1.70% and 2.50%) and three isoleucine levels (1.00%, 1.20%, and 1.40%) for 8 weeks. The results showed that the final weight and weight gain rate were the highest in the fish fed low-level leucine and high-level isoleucine diets (P > 0.05). Furthermore, the crude lipid content was significantly adjusted by diets with diverse levels of leucine and isoleucine (P < 0.05). In addition, interactive effects of these two branched-chain amino acids (BCAAs) were found on plasma total protein, blood ammonia, and blood urea nitrogen of test fish (P < 0.05). Additionally, the liver amino acid profiles were significantly influenced by the interactive effects of the two BCAAs (P < 0.05). Moreover, interactive effects of dietary leucine and isoleucine were significantly observed in the expressions of amino acid metabolism-related genes (P < 0.05). These findings suggested that dietary leucine and isoleucine had interaction. Meanwhile, the interaction between them was more conducive to the growth and quality improvement of blunt snout bream when the dietary leucine level was 1.70% and isoleucine level was 1.40%.

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.

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.