Isolation of Thiobacillus spp. and its application in the removal of heavy metals from activated sludge

Two strains of Thiobacillus isolated from native excess activated sludge were identified as Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans by 16S rRNA gene sequencing and physiological-biochemical characteristics. Single and mixed cultures of the strains were used to carry out bioleaching for 9 days in order to remove heavy metals from activated sludge. The changes in pH, oxidation-reduction potential, and contents of heavy metals were measured. The results show that the bioleaching effect of the mixed culture was best in all runs, and that the final removals of As, Cr, Cu, Ni, and Zn were 96.09, 93.47, 98.32, 97.88, and 98.60%, respectively, whereas the removals of Cd and Pb decreased rapidly after six days. In addition, we demonstrate for the first time that bioleaching can reduce the pathogenicity of sludge by detecting fecal coliforms before and after bioleaching in order to ensure that the sludge was suitable for agricultural land application.Key words: Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, excess activated sludge, removing heavy metals, sludge pathogenicity

Genome-Wide Identification and Characterization of NODULE-INCEPTION-Like Protein (NLP) Family Genes in Brassica napus

NODULE-INCEPTION-like proteins (NLPs) are conserved, plant-specific transcription factors that play crucial roles in responses to nitrogen deficiency. However, the evolutionary relationships and characteristics of NLP family genes in Brassica napus are unclear. In this study, we identified 31 NLP genes in B. napus, including 16 genes located in the A subgenome and 15 in the C subgenome. Subcellular localization predictions indicated that most BnaNLP proteins are localized to the nucleus. Phylogenetic analysis suggested that the NLP gene family could be divided into three groups and that at least three ancient copies of NLP genes existed in the ancestor of both monocots and dicots prior to their divergence. The ancestor of group III NLP genes may have experienced duplication more than once in the Brassicaceae species. Three-dimensional structural analysis suggested that 14 amino acids in BnaNLP7-1 protein are involved in DNA binding, whereas no binding sites were identified in the two RWP-RK and PB1 domains conserved in BnaNLP proteins. Expression profile analysis indicated that BnaNLP genes are expressed in most organs but tend to be highly expressed in a single organ. For example, BnaNLP6 subfamily members are primarily expressed in roots, while the four BnaNLP7 subfamily members are highly expressed in leaves. BnaNLP genes also showed different expression patterns in response to nitrogen-deficient conditions. Under nitrogen deficiency, all members of the BnaNLP1/4/5/9 subfamilies were upregulated, all BnaNLP2/6 subfamily members were downregulated, and BnaNLP7/8 subfamily members showed various expression patterns in different organs. These results provide a comprehensive evolutionary history of NLP genes in B. napus, and insight into the biological functions of BnaNLP genes in response to nitrogen deficiency

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

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

Genome-Wide Identification, Evolutionary and Expression Analyses of the GALACTINOL SYNTHASE Gene Family in Rapeseed and Tobacco

Galactinol synthase (GolS) is a key enzyme in raffinose family oligosaccharide (RFO) biosynthesis. The finding that GolS accumulates in plants exposed to abiotic stresses indicates RFOs function in environmental adaptation. However, the evolutionary relationships and biological functions of GolS family in rapeseed (Brassica napus) and tobacco (Nicotiana tabacum) remain unclear. In this study, we identified 20 BnGolS and 9 NtGolS genes. Subcellular localization predictions showed that most of the proteins are localized to the cytoplasm. Phylogenetic analysis identified a lost event of an ancient GolS copy in the Solanaceae and an ancient duplication event leading to evolution of GolS4/7 in the Brassicaceae. The three-dimensional structures of two GolS proteins were conserved, with an important DxD motif for binding to UDP-galactose (uridine diphosphate-galactose) and inositol. Expression profile analysis indicated that BnGolS and NtGolS genes were expressed in most tissues and highly expressed in one or two specific tissues. Hormone treatments strongly induced the expression of most BnGolS genes and homologous genes in the same subfamilies exhibited divergent-induced expression. Our study provides a comprehensive evolutionary analysis of GolS genes among the Brassicaceae and Solanaceae as well as an insight into the biological function of GolS genes in hormone response in plants

Genome-Wide Identification of the <i>TIFY</i> Gene Family in <i>Brassiceae</i> and Its Potential Association with Heavy Metal Stress in Rapeseed

The TIFY gene family plays important roles in various plant biological processes and responses to stress and hormones. The chromosome-level genome of the Brassiceae species has been released, but knowledge concerning the TIFY family is lacking in the Brassiceae species. The current study performed a bioinformatics analysis on the TIFY family comparing three diploid (B. rapa, B. nigra, and B. oleracea) and two derived allotetraploid species (B. juncea, and B. napus). A total of 237 putative TIFY proteins were identified from five Brassiceae species, and classified into ten subfamilies (six JAZ types, one PPD type, two TIFY types, and one ZML type) based on their phylogenetic relationships with TIFY proteins in A. thaliana and Brassiceae species. Duplication and synteny analysis revealed that segmental and tandem duplications led to the expansion of the TIFY family genes during the process of polyploidization, and most of these TIFY family genes (TIFYs) were subjected to purifying selection after duplication based on Ka/Ks values. The spatial and temporal expression patterns indicated that different groups of BnaTIFYs have distinct spatiotemporal expression patterns under normal conditions and heavy metal stresses. Most of the JAZIII subfamily members were highest in all tissues, but JAZ subfamily members were strongly induced by heavy metal stresses. BnaTIFY34, BnaTIFY59, BnaTIFY21 and BnaTIFY68 were significantly upregulated mostly under As3+ and Cd2+ treatment, indicating that they could be actively induced by heavy metal stress. Our results may contribute to further exploration of TIFYs, and provided valuable information for further studies of TIFYs in plant tolerance to heavy metal stress

Metabolite Profiling and Transcriptome Analysis Provide Insight into Seed Coat Color in Brassica juncea

The allotetraploid species Brassica juncea (mustard) is grown worldwide as oilseed and vegetable crops; the yellow seed-color trait is particularly important for oilseed crops. Here, to examine the factors affecting seed coat color, we performed a metabolic and transcriptomic analysis of yellow- and dark-seeded B. juncea seeds. In this study, we identified 236 compounds, including 31 phenolic acids, 47 flavonoids, 17 glucosinolates, 38 lipids, 69 other hydroxycinnamic acid compounds, and 34 novel unknown compounds. Of these, 36 compounds (especially epicatechin and its derivatives) accumulated significantly different levels during the development of yellow- and dark-seeded B. juncea. In addition, the transcript levels of BjuDFR, BjuANS,BjuBAN, BjuTT8, and BjuTT19 were closely associated with changes to epicatechin and its derivatives during seed development, implicating this pathway in the seed coat color determinant in B. juncea. Furthermore, we found numerous variations of sequences in the TT8A genes that may be associated with the stability of seed coat color in B. rapa, B. napus, and B. juncea, which might have undergone functional differentiation during polyploidization in the Brassica species. The results provide valuable information for understanding the accumulation of metabolites in the seed coat color of B. juncea and lay a foundation for exploring the underlying mechanism

Comparative genomic analyses reveal the genetic basis of the yellow-seed trait in Brassica napus

Abstract Yellow-seed trait is a desirable breeding characteristic of rapeseed (Brassica napus) that could greatly improve seed oil yield and quality. However, the underlying mechanisms controlling this phenotype in B. napus plants are difficult to discern because of their complexity. Here, we assemble high-quality genomes of yellow-seeded (GH06) and black-seeded (ZY821). Combining in-depth fine mapping of a quantitative trait locus (QTL) for seed color with other omics data reveal BnA09MYB47a, encoding an R2R3-MYB-type transcription factor, as the causal gene of a major QTL controlling the yellow-seed trait. Functional studies show that sequence variation of BnA09MYB47a underlies the functional divergence between the yellow- and black-seeded B. napus. The black-seed allele BnA09MYB47aZY821, but not the yellow-seed allele BnA09MYB47aGH06, promotes flavonoid biosynthesis by directly activating the expression of BnTT18. Our discovery suggests a possible approach to breeding B. napus for improved commercial value and facilitates flavonoid biosynthesis studies in Brassica crops