Background: The family of NAC proteins (NAM, ATAF1/2, and CUC2) represent a class of large plant-specific transcription factors. However, identification and functional surveys of NAC genes of tomato (Solanum lycopersicum) remain unstudied, despite the tomato genome being decoded for several years. This study aims to identify the NAC gene family and investigate their potential roles in responding to Al stress. Results: Ninety-three NAC genes were identified and named in accordance with their chromosome location. Phylogenetic analysis found SlNACs are broadly distributed in 5 groups. Gene expression analysis showed that SlNACs had different expression levels in various tissues and at different fruit development stages. Cycloheximide treatment and qRT-PCR analysis indicated that SlNACs may aid regulation of tomato in response to Al stress, 19 of which were significantly up-or down-regulated in roots of tomato following Al stress. Conclusion: This work establishes a knowledge base for further studies on biological functions of SlNACs in tomato and will aid in improving agricultural traits of tomato in the future.

Accurate analysis of gene expression requires selection of appropriate reference genes. In this study, we report analysis of eight candidate reference genes (ACTIN, UBQ, EF-1α, UBC, IF-4α, TUB, PP2A, and HIS), which were screened from the genome and transcriptome data in Brassica juncea. Four statistical analysis softwares geNorm, NormFinder, BestKeeper, and RefFinder were used to test the reliability and stability of gene expression of the reference genes. To further validate the stability of reference genes, the expression levels of two CYCD3 genes (BjuB045330 and BjuA003219) were studied. In addition, all genes in the xyloglucan endotransglucosylase/hydrolase (XTH) family were identified in B. juncea and their patterns at different periods of stem enlargement were analyzed. Results indicated that UBC and TUB genes showed stable levels of expression and are recommended for future research. In addition, XTH genes were involved in regulation of stem enlargement expression. These results provide new insights for future research aiming at exploring important functional genes, their expression patterns and regulatory mechanisms for mustard development.

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Heavy metal lead, which enters the human body through food intake, endangers human health. Microbe has the ability of adsorbing heavy metal, among which lactic acid bacteria are promising microbes to adsorb and remove Pb 2+. The purpose of this study was to screen lactic acid bacteria from Ya'an pickle water to effectively remove Pb 2+. The 7 strains having strong ability to effectively remove Pb 2+ were detected. These strains were identified by microscopic examination and 16S rDNA sequencing, 4 strains of Lactobacillus plantarum and 3 strains of Lactobacillus brevis were obtained. Then the bacteria had a blind adsorption effect on Pb 2+. After microwave digestion, the Pb 2+ concentration was measured by flame atomic absorption spectrometry. The highest removal reached 82.25%. The adsorption mechanism of lactic acid bacteria was mainly divided into biosorption and bioaccumulation. The 7 strains of lactic acid bacteria could provide potential for detoxification of contaminated foods and reduction of the Pb 2+ accumulation in the human diet and animal feed. At the same time, this study was helpful to further understand the mechanism of Pb 2+ being adsorbed by lactic acid bacteria.

Aluminum (Al) toxicity constitutes one of the major limiting factors of plant growth and development on acid soils, which comprises approximately 50% of potentially arable lands worldwide. When suffering Al toxicity, plants reprogram the transcription of genes, which activates physiological and metabolic pathways to deal with the toxicity. Here, we report the role of a NAM, ATAF1, 2 and CUC2 (NAC) transcription factor (TF) in tomato Al tolerance. Among 53 NAC TFs in tomatoes, SlNAC063 was most abundantly expressed in root apex and significantly induced by Al stress. Furthermore, the expression of SlNAC063 was not induced by other metals. Meanwhile, the SlNAC063 protein was localized at the nucleus and has transcriptional activation potentials in yeast. By constructing CRISPR/Cas9 knockout mutants, we found that slnac063 mutants displayed increased sensitivity to Al compared to wild-type plants. However, the mutants accumulated even less Al than wild-type (WT) plants, suggesting that internal tolerance mechanisms but not external exclusion mechanisms are implicated in SlNAC063-mediated Al tolerance in tomatoes. Further comparative RNA-sequencing analysis revealed that only 45 Al-responsive genes were positively regulated by SlNAC063, although the expression of thousands of genes (1,557 upregulated and 636 downregulated) was found to be affected in slnac063 mutants in the absence of Al stress. The kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed that SlNAC063-mediated Al-responsive genes were enriched in “phenylpropanoid metabolism,” “fatty acid metabolism,” and “dicarboxylate metabolism,” indicating that SlNAC063 regulates metabolisms in response to Al stress. Quantitative real-time (RT)-PCR analysis showed that the expression of SlAAE3-1 was repressed by SlNAC063 in the absence of Al. However, the expression of SlAAE3-1 was dependent on SlNAC063 in the presence of Al stress. Taken together, our results demonstrate that a NAC TF SlNAC063 is involved in tomato Al tolerance by regulating the expression of genes involved in metabolism, and SlNAC063 is required for Al-induced expression of SlAAE3-1.

In response to changing environments, plants regulate gene expression and subsequent metabolism to acclimate and survive. A superfamily of acyl-activating enzymes (AAEs) has been observed in every class of creatures on planet. Some of plant AAE genes have been identified and functionally characterized to be involved in growth, development, biotic, and abiotic stresses via mediating diverse metabolic pathways. However, less information is available about AAEs superfamily in tomato (Solanum lycopersicum), the highest value fruit and vegetable crop globally. In this study, we aimed to identify tomato AAEs superfamily and investigate potential functions with respect to aluminum (Al) stress that represents one of the major factors limiting crop productivity on acid soils worldwide. Fifty-three AAE genes of tomato were identified and named on the basis of phylogenetic relationships between Arabidopsis and tomato. The phylogenetic analysis showed that AAEs could be classified into six clades; however, clade III contains no AAE genes of tomato. Synteny analyses revealed tomato vegetable paralogs and Arabidopsis orthologs. The RNA-seq and quantitative reverse-transcriptase PCR (qRT-PCR) analysis indicated that 9 out of 53 AAEs genes were significantly up- or downregulated by Al stress. Numerous cis-acting elements implicated in biotic and abiotic stresses were detected in the promoter regions of SlAAEs. As the most abundantly expressed gene in root apex and highly induced by Al, there are many potential STOP1 cis-acting elements present in the promoter of SlAAE3-1, and its expression in root apex was specific to Al. Finally, transgenic tobacco lines overexpressing SlAAE3-1 displayed increased tolerance to Al. Altogether, our results pave the way for further studies on the functional characterization of SlAAE genes in tomato with a wish of improvement in tomato crop in the future.

The SQUAMOSA promoter binding protein (SBP)-box consists of a family of transcription factors unique to plants and mediates important roles in plant growth and development. We identified 57 SBP genes in the Brassica juncea (swollen-stem mustard) genome, and analyzed the structural characteristics, evolutionary relationships, as well as their involvement in regulation and response to environmental stress. By the help of phylogenetic analysis, the SBP genes were divided into seven subgroups (G1-G7). Interestingly, most members of the same group shared conserved gene structure and motifs as well as gene duplications. This suggests that their functions are redundant highlighting evolutionary relatedness. Identification of the cis-acting elements of the promoter revealed multiple regulatory elements that responded to phytohormone or abiotic stresses. In addition, data from GO annotation indicated that BjuSBPs play a role in biological processes. Further interrogation of the expression of BjuSBPs in different stages of swollen-stem mustard showed spatiotemporal expression patterns. On the other hand, quantitative real-time polymerase chain reaction (qRT-PCR) showed that the expression levels of SBP genes at the development stage correlated with their transcriptome data but treatment with phytohormones affected their expression levels. Our study showed that the SBP-box responds to phytohormone signals and the genes might confer different functions in the development of the mustard plant. Our data lay the foundation for further studies evaluating the regulatory mechanisms of SBP-box family of genes.

Brain endothelial cells (ECs) are an important component of the blood-brain barrier (BBB) and play key roles in restricting entrance of possible toxic components and pathogens into the brain. However, identifying endothelial genes that regulate BBB homeostasis remains a time-consuming process. Although somatic genome editing has emerged as a powerful tool for discovery of essential genes regulating tissue homeostasis, its application in brain ECs is yet to be demonstrated in vivo. Here, we used an adeno-associated virus targeting brain endothelium (AAV-BR1) combined with the CRISPR/Cas9 system (AAV-BR1-CRISPR) to specifically knock out genes of interest in brain ECs of adult mice. We first generated a mouse model expressing Cas9 in ECs (Tie2 Cas9 ). We selected endothelial β-catenin (Ctnnb1) gene, which is essential for maintaining adult BBB integrity, as the target gene. After intravenous injection of AAV-BR1-sgCtnnb1-tdTomato in 4-week-old Tie2 Cas9 transgenic mice resulted in mutation of 36.1% of the Ctnnb1 alleles, thereby leading to a dramatic decrease in the level of CTNNB1 in brain ECs. Consequently, Ctnnb1 gene editing in brain ECs resulted in BBB breakdown. Taken together, these results demonstrate that the AAV-BR1-CRISPR system is a useful tool for rapid identification of endothelial genes that regulate BBB integrity in vivo.