Overexpression of SQUAMOSA promoter binding protein-like 4a (NtSPL4a) alleviates Cd toxicity in Nicotiana tabacum.

Squamosa Promoter Binding Protein-Like (SPL) plays a crucial role in regulating plant development and combating stress, yet its mechanism in regulating resistance to Cd toxicity remains unclear. In this study, we cloned a nuclear-localized transcription factor, NtSPL4a, from the tobacco cultivar TN90. Transient co-expression results showed that miR156 significantly reduced the expression of NtSPL4a by binding to the 3'-UTR of its transcript. We obtained transgenic tobacco overexpressing NtSPL4a (including the 3'-UTR) and NtSPL4aΔ (lacking the 3'-UTR) through Agrobacterium-mediated genetic transformation. Compared to the wild type (WT), overexpression of NtSPL4a/NtSPL4aΔ shortened the flowering time and exhibited a more developed root system. The transgenic tobacco showed significantly reduced Cd content, being 85.1% (OE-NtSPL4a) and 46.7% (OE-NtSPL4aΔ) of WT, respectively. Moreover, the upregulation of NtSPL4a affected the mineral nutrient homeostasis in transgenic tobacco. Additionally, overexpression of NtSPL4a/NtSPL4aΔ effectively alleviated leaf chlorosis and oxidative stress induced by Cd toxicity. One possible reason is that the overexpression of NtSPL4a/NtSPL4aΔ can effectively promote the accumulation of non-enzymatic antioxidants. A comparative transcriptomic analysis was performed between transgenic tobacco and WT to further unravel the global impacts brought by NtSPL4a. The tobacco overexpressing NtSPL4a had 183 differentially expressed genes (77 upregulated, 106 downregulated), while the tobacco overexpressing NtSPL4aΔ had 594 differentially expressed genes (244 upregulated, 350 downregulated) compared to WT. These differentially expressed genes mainly included transcription factors, metal transport proteins, flavonoid biosynthesis pathway genes, and plant stress-related genes. Our study provides new insights into the role of the transcript factor SPL in regulating Cd tolerance.

Comparative Transcriptome Analysis Reveals the Effect of miR156a Overexpression on Mineral Nutrient Homeostasis in Nicotiana tabacum.

Mineral nutrition plays an important role in crop growth, yield and quality. MiR156 is a regulatory hub for growth and development. To date, the understanding of miR156-mediated mineral homeostasis is limited. In this study, we overexpressed Nta-miR156a in the tobacco cultivar TN90 and analyzed the effects of miR156 on mineral element homeostasis in tobacco by comparative transcriptome analysis. The results showed that the overexpression of miR156a caused significant morphological changes in transgenic tobacco. Chlorophyll and three anti-resistance markers, proline, total phenolics, and total flavonoids, were altered due to increased miR156 expression levels. Interestingly, the distribution of Cu, Mn, Zn, and Fe in different tissues of transgenic tobacco was disordered compared with that of the wild type. Comparative transcriptome analysis showed that the overexpression of miR156 resulted in 2656 significantly differentially expressed genes. The expression levels of several metal-transport-related genes, such as NtABC, NtZIP, NtHMA, and NtCAX, were significantly increased or decreased in transgenic tobacco. These results suggest that miR156 plays an essential role in regulating mineral homeostasis. Our study provides a new perspective for the further study of mineral nutrient homeostasis in plants.

Heterologous expression of the tobacco metallothionein gene NtMT2F confers enhanced tolerance to Cd stress in Escherichia coli and Arabidopsis thaliana.

Heavy metal pollution in the soil is a serious threat to crop growth and human health. Metallothionein (MT) is a low molecular weight protein that is rich in cysteine, which can effectively alleviate the toxicity of heavy metals in plants. In this study, a novel metallothionein encoding gene, NtMT2F, was cloned from the Cd-hyperaccumulator tobacco and heterologously expressed in E. coli and A. thaliana to verify its biological function. Recombinant E. coli incubated with NtMT2F effectively resisted heavy metal stress, particularly Cd. The recombinant strain grew significantly faster and had a higher content of Cd than the control. Mutations in the C-terminal Cys residues of NtMT2F significantly reduced its ability to chelate heavy metals. The overexpression of NtMT2F significantly enhanced resistance to Cd toxicity in transgenic A. thaliana. The germination rate, root length, and fresh weight of transgenic plants under Cd stress were higher than those of the wild type (WT). The contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) were lower than those of the WT. In addition, the activities of anti-peroxidase enzymes including glutathione reductase (GR), catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), were significantly increased in the transgenic plants. The results of this study indicate that NtMT2F significantly improved the tolerance of microorganisms and plants to Cd and could be an important candidate protein for phytoremediation.

The NtSPL Gene Family in Nicotiana tabacum: Genome-Wide Investigation and Expression Analysis in Response to Cadmium Stress.

The SQUAMOSA promoter binding protein-like (SPL)SPL family genes play an important role in regulating plant growth and development, synthesis of secondary metabolites, and resistance to stress. Understanding of the role of the SPL family in tobacco is still limited. In this study, 42 NtSPL genes were identified from the genome of the tobacco variety TN90. According to the results of the conserved motif and phylogenetic tree, the NtSPL genes were divided into eight subgroups, and the genes in the same subgroup showed similar gene structures and conserved domains. The cis-acting element analysis of the NtSPL promoters showed that the NtSPL genes were regulated by plant hormones and stresses. Twenty-eight of the 42 NtSPL genes can be targeted by miR156. Transcriptome data and qPCR results indicated that the expression pattern of miR156-targeted NtSPL genes was usually tissue specific. The expression level of miR156 in tobacco was induced by Cd stress, and the expression pattern of NtSPL4a showed a significant negative correlation with that of miR156. These results suggest that miR156-NtSPL4a may mediate the tobacco response to Cd stress. This study lays a foundation for further research on the function of the NtSPL gene and provides new insights into the involvement of NtSPL genes in the plant response to heavy metal stress.

The Superoxide Dismutase Gene Family in Nicotiana tabacum: Genome-Wide Identification, Characterization, Expression Profiling and Functional Analysis in Response to Heavy Metal Stress.

Superoxide dismutases (SODs) play an important role in protecting plants against ROS toxicity induced by biotic and abiotic stress. Recent studies have shown that the SOD gene family is involved in plant growth and development; however, knowledge of the SOD gene family in tobacco is still limited. In the present study, the SOD gene family was systematically characterized in the tobacco genome. Based on the conserved motif and phylogenetic tree, 15 NtSOD genes were identified and classified into three subgroups, including 5 NtCSDs, 7 NtFSDs and 3 NtMSDs. The predicted results of the transport peptide or signal peptide were consistent with their subcellular localization. Most NtSOD genes showed relatively well-maintained exon-intron and motif structures in the same subgroup. An analysis of cis-acting elements in SOD gene promoters showed that NtSOD expression was regulated by plant hormones, defense and stress responses, and light. In addition, multiple transcription factors and miRNAs are predicted to be involved in the regulation of NtSOD gene expression. The qPCR results indicated specific spatial and temporal expression patterns of the NtSOD gene family in different tissues and developmental stages, and this gene family played an important role in protecting against heavy metal stress. The results of functional complementation tests in the yeast mutant suggested that NtCSD1a, NtFSD1e and NtMSD1b scavenge ROS produced by heavy metal stress. This study represents the first genome-wide analysis of the NtSOD gene family, which lays a foundation for a better understanding of the function of the NtSOD gene family and improving the tolerance of plants to heavy metal toxicity.

The NtNRAMP1 transporter is involved in cadmium and iron transport in tobacco (Nicotiana tabacum).

Plant natural resistance-associated macrophage protein (NRAMP) plays an important role in maintaining intracellular metal homeostasis and coping with environmental heavy metal stress. Until now, studies on NRAMP in tobacco have been limited. In this study, NtNRAMP1 was cloned from tobacco cultivar TN90, and the highest expression level was observed in the roots, which was strongly induced by Fe deficiency. Heterologously expressed NtNRAMP1 significantly increased the Cd sensitivity of the yeast Δycf1 mutant. Three overexpressed NtNRAMP1 lines were generated to reveal the biofunction of NtNRAMP1. In the soil pot experiments under natural conditions, the contents of Fe and total chlorophyll were increased in the leaves of transgenic tobacco compared with the WT. To reveal the characteristics of NtNRAMP1 in metal transport, transgenic plants were cultured in hydroponic solution with 50 µM Cd and 200 µM Fe. Compared with the WT, the Cd concentrations in transgenic plants increased by 1.26-2.02-fold in the roots. Interestingly, the Cd content in the shoots of transgenic plants was slightly reduced compared with that of the WT. Overexpression of NtNRAMP1 did not promote Fe uptake from the external environment into the roots but enhanced the transfer of Fe from the roots to shoots. Additionally, Fe overload in the leaves of transgenic tobacco resulted in increased levels of MDA and H2O2 while Fe toxicity may be relieved by POD. In conclusion, overexpression of NtNRAMP1 in tobacco could promote Cd uptake and Fe transport from the roots to shoots while disturbing Fe homeostasis in the leaves of transgenic tobacco.

Ferroptosis: A Novel Mechanism of Artemisinin and its Derivatives in Cancer Therapy.

BACKGROUND: Artemisinin is a sesquiterpene lactone compound with a special peroxide bridge that is tightly linked to the cytotoxicity involved in fighting malaria and cancer. Artemisinin and its derivatives (ARTs) are considered to be potential anticancer drugs that promote cancer cell apoptosis, induce cell cycle arrest and autophagy, inhibit cancer cell invasion and migration. Additionally, ARTs significantly increase intracellular Reactive Oxygen Species (ROS) in cancer cells, which result in ferroptosis, a new form of cell death, depending on the ferritin concentration. Ferroptosis is regarded as a cancer suppressor and as well as considered a new mechanism for cancer therapy. METHODS: The anticancer activities of ARTs and reference molecules were compared by literature search and analysis. The latest research progress on ferroptosis was described, with a special focus on the molecular mechanism of artemisinin-induced ferroptosis. RESULTS: Artemisinin derivatives, artemisinin-derived dimers, hybrids and artemisinin-transferrin conjugates, could significantly improve anticancer activity, and their IC50 values are lower than those of reference molecules such as doxorubicin and paclitaxel. The biological activities of linkers in dimers and hybrids are important in the drug design processes. ARTs induce ferroptosis mainly by triggering intracellular ROS production, promoting the lysosomal degradation of ferritin and regulating the System Xc-/Gpx4 axis. Interestingly, ARTs also stimulate the feedback inhibition pathway. CONCLUSION: Artemisinin and its derivatives could be used in the future as cancer therapies with broader applications due to their induction of ferroptosis. Meanwhile, more attention should be paid to the development of novel artemisinin-related drugs based on the mechanism of artemisinininduced ferroptosis.