Real-time quantitative polymerase chain reaction (qPCR) is one of the most important methods for analyzing the expression patterns of target genes. However, successful qPCR experiments rely heavily on the use of high-quality primers. Various qPCR primer databases have been developed to address this issue, but these databases target only a few important organisms. Here, we developed the qPrimerDB database, founded on an automatic gene-specific qPCR primer design and thermodynamics-based validation workflow. The qPrimerDB database is the most comprehensive qPCR primer database available to date, with a web front-end providing gene-specific and pre-computed primer pairs across 147 important organisms, including human, mouse, zebrafish, yeast, thale cress, rice and maize. In this database, we provide 3331426 of the best primer pairs for each gene, based on primer pair coverage, as well as 47760359 alternative gene-specific primer pairs, which can be conveniently batch downloaded. The specificity and efficiency was validated for qPCR primer pairs for 66 randomly selected genes, in six different organisms, through qPCR assays and gel electrophoresis. The qPrimerDB database represents a valuable, timesaving resource for gene expression analysis. This resource, which will be routinely updated, is publically accessible at http://biodb.swu.edu.cn/qprimerdb.

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.

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.

Leaf senescence is a vital aspect of plant physiology and stress responses and is induced by endogenous factors and environmental cues.. The plant-specific NAC (NAM, ATAF1/2, CUC2) transcription factor family influences growth, development, and stress responses in Arabidopsis (Arabidopsis thaliana) and other species. However, the roles of NACs in tobacco (Nicotiana tabacum) leaf senescence are still unclear. Here, we report that NtNAC56 regulates leaf senescence in tobacco. Transgenic plants overexpressing NtNAC56 (NtNAC56-OE) showed induction of senescence-related genes and exhibited early senescence and lower chlorophyll content compared to wild-type (WT) plants and the Ntnac56-19 mutant. In addition, root development and seed germination were inhibited in the NtNAC56-OE lines. Transmission electron microscopy observations accompanied by physiological and biochemical assays revealed that NtNAC56 overexpression triggers chloroplast degradation and reactive oxygen species accumulation in tobacco leaves. Transcriptome analysis demonstrated that NtNAC56 activates leaf senescence-related genes and jasmonic acid (JA) biosynthesis pathway genes. In addition, the JA content of NtNAC56-OE plants was higher than in WT plants, and JA treatment induced NtNAC56 expression. We performed DNA affinity purification sequencing to identify direct targets of NtNAC56, among which we focused on LIPOXYGENASE 5 (NtLOX5), a key gene in JA biosynthesis. A dual-luciferase reporter assay and a yeast one-hybrid assay confirmed that NtNAC56 directly binds to the TTTCTT motif in the NtLOX5 promoter. Our results reveal a mechanism whereby NtNAC56 regulates JA-induced leaf senescence in tobacco and provide a strategy for genetically manipulating leaf senescence and plant growth.

Hypersensitive-induced response protein (HIR) is a class of plant immune proteins that play pivotal roles in Sclerotinia sclerotiorum (Lib.) de Bary resistance. However, there has been no systematic investigation and identification of HIR genes in rapeseed (Brassica napus L.). Hence, we identified 50 BnHIR genes and classified them into four groups. Subcellular localization prediction suggested that HIR proteins are mainly localized in the mitochondria. Cis-acting elements involved in light and diverse abiotic stress were found in the promoter regions of BnHIR. The majority of BnHIR genes in Groups 1/3/4 were expressed in most examined tissues, especially in leaves and siliques pericarp, while the BnHIR genes in Group 2 were not or had low expression in all detected tissues. In the case of S. sclerotiorum inoculation, HIR genes in Groups 1/3/4 were strongly induced, especially homologous genes in Group 1, which exhibited different expression patterns. Moreover, overexpression of BnHIR2.7 in Arabidopsis thaliana illustrated its prominent resistance to S. sclerotiorum. Our study provides insight into the evolutionary relationships of the HIR family genes in B. napus and lays the foundation for their resistance to S. sclerotiorum in B. napus.