NtGCN2 confers cadmium tolerance in Nicotiana tabacum L. by regulating cadmium uptake, efflux, and subcellular distribution.

General control non-derepressible-2 (GCN2) is widely expressed in eukaryotes and responds to biotic and abiotic stressors. However, the precise function and mechanism of action of GCN2 in response to cadmium (Cd) stress in Nicotiana tabacum L. (tobacco) remains unclear. We investigated the role of NtGCN2 in Cd tolerance and explored the mechanism by which NtGCN2 responds to Cd stress in tobacco by exposing NtGCN2 transgenic tobacco lines to different concentrations of CdCl2. NtGCN2 was activated under 50 µmol·L-1 CdCl2 stress and enhanced the Cd tolerance and photosynthetic capacities of tobacco by increasing chlorophyll content and antioxidant capacity by upregulating NtSOD, NtPOD, and NtCAT expression and corresponding enzyme activities and decreasing malondialdehyde and O2·- contents. NtGCN2 enhanced the osmoregulatory capacity of tobacco by elevating proline (Pro) and soluble sugar contents and maintaining low levels of relative conductivity. Finally, NtGCN2 enhanced Cd tolerance in tobacco by reducing Cd uptake and translocation, promoting Cd efflux, and regulating Cd subcellular distribution. In conclusion, NtGCN2 improves the tolerance of tobacco to Cd through a series of mechanisms, namely, increasing antioxidant, photosynthetic, and osmoregulation capacities and regulating Cd uptake, translocation, efflux, and subcellular distribution. This study provides a scientific basis for further exploration of the role of NtGCN2 in plant responses to Cd stress and enhancement of the Cd stress signaling network in tobacco.

Taurine Inhibits Lung Metastasis in Triple-Negative Breast Cancer by Modulating Macrophage Polarization Through PTEN-PI3K/Akt/mTOR Pathway.

SUMMARY: Taurine (Tau) has been found to inhibit triple-negative breast cancer (TNBC) invasion and metastasis. However, its effect on tumor-promoting macrophages and tumor suppressor macrophages in breast cancer progression remains unknown. In this study, we investigated the effects of Tau on macrophage polarization and its role in TNBC cell growth, invasion, and metastasis. We induced human THP-1 monocytes to differentiate into M2 macrophages through exogenous addition of interleukin-4. We used the TNBC cell lines MDA-MB-231 and BT-549 cultured in a conditioned medium from M2 macrophages to investigate the effect of Tau on tumor growth and invasion. We analyzed macrophage subset distribution, M1 and M2 macrophage-associated markers, and mRNA expression by quantitative polymerase chain reaction. We also detected the PTEN-PI3K/Akt/mTOR signaling pathway that mediates M1 macrophage to suppress tumor invasion using western blotting. Our results showed that Tau inhibits breast cancer metastasis to the lungs in vivo and cell invasion by altering the polarization of tumor-associated macrophage in vitro. In addition, Tau can up-regulate PTEN expression, suppress the PI3K-Akt signaling pathway, and promote the M1 polarization of macrophages, which ultimately inhibits the metastasis of TNBC cells. Our findings suggest that Tau inhibits the activation of the PI3K-Akt-mTOR signaling pathway by up-regulating PTEN, promotes the proportion of M1 macrophages in tumor-associated macrophage, and suppresses the invasion and metastasis of TNBC. This provides a potential therapeutic approach to influence cancer progression and metastasis.

Molybdenum inhibited the growth of Phytophthora nicotiana and improved the resistance of Nicotiana tabacum L. against tobacco black shank.

Tobacco black shank (TBS) is a soil-borne fungal disease caused by Phytophthora nicotiana (P. nicotianae), significantly impeding the production of high-quality tobacco. Molybdenum (Mo), a crucial trace element for both plants and animals, plays a vital role in promoting plant growth, enhancing photosynthesis, bolstering antioxidant capacity, and maintaining ultrastructural integrity. However, the positive effect of Mo on plant biotic stress is little understood. This study delves into the inhibitory effects of Mo on P. nicotianae and seeks to unravel the underlying mechanisms. The results showed that 16.32 mg/L of Mo significantly inhibited mycelial growth, altered mycelial morphological structure, damaged mycelial cell membrane, and ultimately led to the leakage of cell inclusions. In addition, 0.6 mg/kg Mo applied in soil significantly reduced the severity of TBS. Mo increased photosynthetic parameters and photosynthetic pigment contents of tobacco leaves, upregulated expression of NtPAL and NtPPO resistance genes, as well as improved activities of SOD, POD, CAT, PPO, and PAL in tobacco plants. Furthermore, Mo could regulate nitrogen metabolism and amino acids metabolism to protect tobacco plants against P. nicotianae infection. These findings not only present an ecologically sound approach to control TBS but also contribute valuable insights to the broader exploration of the role of microelements in plant disease management.

Characterization of the DUF868 gene family in Nicotiana and functional analysis of NtDUF868-E5 involved in pigment metabolism.

Domains of unknown function (DUF) proteins represent a large group of uncharacterized protein families. The DUF868 gene family in Nicotiana has not yet been described. In the present study, we identified 12, 11, and 25 DUF868 family members in the genome of Nicotiana sylvestris, N. tomentosiformis, and N. tabacum, respectively. Based on phylogenetic analysis, these were categorized into five groups (A-E). Within each group, the gene structures, motifs, and tertiary structures showed high similarity. NtDUF868 family expansion during evolution was mainly driven by segmental duplication events. MicroRNA (miRNA) target site prediction identified 12 miRNA members that target 16 NtDUF868 family genes. The promoters of these genes contain cis-regulatory elements responsive to light, phytohormones, and abiotic stresses. Expression profiling revealed their tissue- and stage-specific expression patterns. RNA-sequencing and quantitative reverse transcription PCR revealed that the NtDUF868 family genes are potentially involved in the response to abiotic and biotic stresses, particularly drought and hormone stresses, and in the resistance to black shank and bacterial wilt. We generated transformed plants using NtDUF868-E5 overexpression and gene-editing vectors. NtDUF868-E5 overexpression resulted in enhanced tobacco plant growth and development, leading to increased leaf photosynthetic capacity and higher chlorophyll and carotenoid contents. This study provided a comprehensive genome-wide analysis of the DUF868 gene family, shedding light on their potential roles in plant growth and stress responses.

A non-specific lipid transfer protein, NtLTPI.38, positively mediates heat tolerance by regulating photosynthetic ability and antioxidant capacity in tobacco.

Non-specific lipid transfer proteins (nsLTPs) play an important role in plant growth and stress resistance; however, their function in tobacco remains poorly understood. Therefore, to explore the function of NtLTP in response to high temperature, we identified an NtLTPI.38 from tobacco, obtained its overexpression and knockout transgenic plants, and further studied their response to heat stress (42 °C). The results showed that NtLTPI.38 overexpression in tobacco reduced chlorophyll degradation, alleviated the high temperature damage to photosynthetic organs, and enhanced the photosynthetic capacity of tobacco under heat stress. NtLTPI.38 overexpression in heat-stressed tobacco increased the contents of soluble sugar and protein, proline, and flavonoid substances, reduced the relative conductivity, and decreased H2O2, O2•-, and MDA accumulation, and increased the enzymatic antioxidant activities, such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), compared to wild type (WT) and knockout mutant plants. RT-PCR confirmed that the expression levels of antioxidant enzymes and thermal stress-related genes were significantly upregulated under thermal stress in overexpression plants. Therefore, NtLTPI.38 enhanced heat tolerance in tobacco by mitigating photosynthetic damage and improving osmoregulation and antioxidant capacity. These results provided the theoretical basis and a potential resource for further breeding projects to improve heat tolerance in plants.

NtNCED3 regulates responses to phosphate deficiency and drought stress in Nicotiana tabacum.

Plants are sessile and encounter to abiotic environmental stressors, such as nutrient deficiency and drought stress. Identifying stress tolerance genes and their mechanisms is vital to ensuring plant survival. In this study, we characterized NCED3 in the tobacco plant Nicotiana tabacum, a key enzyme in the biosynthesis of abscisic acid that is widely involved in abiotic stress responses, using overexpression and RNA interference knockdown. Overexpression of NtNCED3 promoted primary root development, leading to increased dry weight, root-to-shoot ratio, photosynthetic capacity, and acid phosphatase activity, coinciding with highly increased phosphate uptake capability under low phosphate conditions. Under both drought and extreme phosphate deficiency conditions, the phosphate starvation response preceded the drought stress response. However, under high phosphate conditions, the drought stress phenotype emerged before the symptoms of phosphate deficiency. Plants overexpressing NtNCED3 grew better than the wild-type and NtNCED3 knockdown plants, with more developed root systems and higher biomass, phosphorus content, and hormone content. This study provides evidence that NtNCED3 enzyme participates in plant responses to phosphate deficiency and drought stress in N. tabacum, and NtNCED3 may serve as a potentially valuable gene for genetic modification of plant tolerance to both drought stress and phosphate starvation.

NtLTPI.38, a plasma membrane-localized protein, mediates lipid metabolism and salt tolerance in Nicotiana tabacum.

Non-specific lipid transfer proteins (nsLTPs) typically have conserved structural resemblance, low sequence identity, and broad biological functions in plant growth and stress resistance. Here, a plasma membrane-localized nsLTP, NtLTPI.38, was identified in tobacco plants. Multi-omics integrated analysis revealed that NtLTPI.38 overexpression or knock out significantly changed glycerophospholipid and glycerolipid metabolism pathways. NtLTPI.38 overexpression remarkably increased phosphatidylcholine, phosphatidylethanolamine, triacylglycerol, and flavonoid levels, but decreased ceramides compared to wild type and mutant lines. Differentially expressed genes were associated with lipid metabolite and flavonoid synthesis. Many genes related to Ca2+ channels, abscisic acid (ABA) signal transduction, and ion transport pathways were upregulated in overexpressing plants. NtLTPI.38 overexpression in salt-stressed tobacco triggered a Ca2+ and K+ influx in leaves, increased the contents of chlorophyll, proline, flavonoids, and osmotic tolerance, and raised enzymatic antioxidant activities as well as the expression level of related genes. However, mutants accumulated more O2- and H2O2, exhibited ionic imbalance, gathered excess Na+, Cl-, and malondialdehyde, with more severe ion leakage. Therefore, NtLTPI.38 enhanced salt tolerance in tobacco by regulating lipid and flavonoid synthesis, antioxidant activity, ion homeostasis, and ABA signaling pathways.

Overexpression of NtGCN2 improves drought tolerance in tobacco by regulating proline accumulation, ROS scavenging ability, and stomatal closure.

Drought stress is a severe threat to plants. Genes that respond to drought stress are essential for plant growth and development. General control nonderepressible 2 (GCN2) encodes a protein kinase that responds to various biotic and abiotic stresses. However, the mechanism of GCN2 in plant drought tolerance remains unclear. In the present study, the promoters of NtGCN2 from Nicotiana tabacum K326, which contained a drought-responsive Cis-acting element MYB that can be activated by drought stress, were cloned. Furthermore, the drought tolerance function of NtGCN2 was investigated using NtGCN2-overexpressed transgenic tobacco plants. NtGCN2-overexpressed transgenic plants were more tolerant to drought stress than wild-type (WT) plants. The transgenic tobacco plants exhibited higher proline and abscisic acid (ABA) contents, antioxidant enzyme activities, leaf relative water content, and expression levels of genes encoding key antioxidant enzymes and proline synthase, but lower levels of malondialdehyde and reactive oxygen species, and reduced stomatal apertures, stomatal densities, and stomatal opening rates compared to WT plants under drought stress. These results indicated that overexpression of NtGCN2 conferred drought tolerance in transgenic tobacco plants. RNA-seq analysis showed that overexpression of NtGCN2 responded to drought stress by regulating the expression of genes related to proline synthesis and catabolism, abscisic acid synthesis and catabolism, antioxidant enzymes, and ion channels in guard cells. These results showed that NtGCN2 might regulate drought tolerance by regulating proline accumulation, reactive oxygen species (ROS) scavenging, and stomatal closure in tobacco and may have the potential for application in the genetic modification of crop drought tolerance.

NtRAV4 negatively regulates drought tolerance in Nicotiana tabacum by enhancing antioxidant capacity and defence system.

KEY MESSAGE: NtRAV4 is a nucleus-localised protein and no self-activation effect. ntrav4 mutants maintain the steady state of the ROS system under drought stress by enhancing antioxidant capacity and defence system. The APETALA2/ethylene response factor (AP2/ERF) transcription factor (TF) family plays an important role in plant responses to environmental stresses. In this study, we identified a novel NtRAV4 TF, a member of RAV subfamily among AP2/ERF gene family, which have AP2 and B3 domain in its N- and C-terminus, respectively. Subcellular localisation and self-activation activity analysis revealed that NtRAV4 localised in the nucleus and had no self-activation effect. The overexpression and gene editing vectors of NtRAV4 were constructed by homologous recombination and CRISPR/Cas9 gene editing methods, and transformed into tobacco by agrobacterium-mediated method. ntrav4 led to the appearance of termination codon in advance and lacked the unique B3 domain of RAV subfamily protein. Further analysis displayed that knockout of the NtRAV4 in tobacco increased drought tolerance with high relative water content, accompanied by reduced stomatal aperture, density, and stomatal opening ratio compared to overexpression lines and WT. Moreover, ntrav4 knockout plants also exhibited increased osmotic tolerance with low malondialdehyde (MDA) and ion leakage (EL), less accumulation of O2•- and H2O2, and high enzymatic antioxidant (SOD, POD, CAT) activities, non-enzymatic antioxidant (AsA-GSH cycle) contents and hormone (IAA, ABA, GA3, and ZR) levels under drought stress. Furthermore, ntrav4 mutants in tobacco improved the expression levels of ROS-related proline synthesis and stress-responsive genes under osmotic stress. Our results indicate that NtRAV4 negatively regulates plant tolerance to drought stress by reducing water loss and activating the antioxidant system and stress-related gene expression to maintain the steady state of the ROS system.

Methyl Gallate Suppresses the Migration, Invasion, and Epithelial-Mesenchymal Transition of Hepatocellular Carcinoma Cells via the AMPK/NF-κB Signaling Pathway in vitro and in vivo.

Methyl gallate (MG), a polyphenolic compound found in plants, is widely used in traditional Chinese medicine. MG is known to alleviate several cancer symptoms. However, most studies that have reported the antitumor effects of MG have done so at the cellular level, and the inhibitory effect and therapeutic mechanism of MG in hepatocellular carcinoma (HCC) have not been extensively explored in vivo. We aimed to understand the therapeutic mechanism of MG in HCC in vitro and in vivo. MTT and colony formation assays were used to determine the impact of MG on the proliferation of a human HCC cell line, BEL-7402; wound healing and transwell assays were used to quantify the migration and invasion of HCC cells. Western blotting was used to quantify the expression of the AMPK/NF-κB signaling pathway proteins. In vivo tumor growth was measured in a xenograft tumor nude mouse model treated with MG, and hematoxylin-eosin staining and immunohistochemistry (IHC) were used to visualize the histological changes in the tumor tissue. We found that MG showed anti-proliferative effects both in vitro and in vivo. MG downregulated the protein expression of AMPK, NF-κB, p-NF-κB, and vimentin and upregulated the expression of E-cadherin in a dose-dependent manner. Additionally, MG inhibited the migration and invasion of HCC cells by decreasing MMP9 and MMP2 expression and increasing TIMP-2 expression. These were consistent with the results of IHC in vivo. MG inhibited the proliferation, migration, and invasion of HCC cells. This effect potentially involves the regulation of the AMPK/NF-κB pathway, which in turn impacts epithelial-mesenchymal transition and MMP expression.

MicorRNA-195 links long non-coding RNA SEMA3B antisense RNA 1 (head to head) and cyclin D1 to regulate the proliferation of glioblastoma cells.

Long non-coding RNA (lncRNA) SEMA3B antisense RNA 1 (head to head) (SEMA3B-AS1) is a recently identified tumor suppressor in gastric cancer. However, its role in glioblastoma (GBM) is unclear. This study was conducted to explore the role of SEMA3B-AS1 in GBM. In this study, the expression of SEMA3B-AS1, cyclin D1 and miR-195 were determined by RT-qPCR. Gene interactions were evaluated by dual-luciferase assay and overexpression experiments. BrdU assay was performed to monitor cell proliferation. We observed downregulation of SEMA3B-AS1 in GBM. The expression of SEMA3B-AS1 was inversely correlated with the expression of cyclin D1 but positively correlated with the expression of miR-195. In GBM cells, overexpression of SEMA3B-AS1 and miR-195 caused reduced expression levels of cyclin D1. MiR-195 inhibitor reduced the effects of overexpression of SEMA3B-AS1 on the expression of cyclin D1. Moreover, overexpression of SEMA3B-AS1 increased the expression levels of miR-195. Cell proliferation data showed that, SEMA3B-AS1 and miR-195 decreased cell proliferation, while overexpression of cyclin D1 increased GBM cell proliferation. In addition, miR-195 inhibitor inhibited the role of overexpression of SEMA3B-AS1 in cancer cell proliferation. Moreover, miR-195 interacted with cyclin D1, but not SEMA3B-AS1. Furthermore, SEMA3B-AS1 decreased the methylation of the promoter region of miR-195. Therefore, we concluded that miR-195 links lncRNA SEMA3B-AS1 and cyclin D1 to regulate the proliferation of GBM cells.

An integrated metabonomics study to reveal the inhibitory effect and metabolism regulation of taurine on breast cancer.

Breast cancer is a common metastatic malignant tumor in women. Taurine has been found to have anti-tumor effects on a variety of cancers. However, to the best of our knowledge, the role of taurine in the metastasis of breast cancer has not been reported. Thus, this study examined the effects of taurine on the growth and lung metastasis of breast cancer. Furthermore, the metabolism of serum, tumor tissue, and lung metastasis tissue were studied in a 4T1 subcutaneously transplanted breast cancer model through the integration of a 1H NMR-based metabonomics approach and histopathological assessments. The results showed that taurine significantly attenuated the tumor growth and lung metastasis, improved the pathological structure of tumor and lung tissue, and improved the metabolic disorders in 4T1 breast cancer mice. Additionally, taurine reversed the changes in serum lactate, creatine, and choline caused by the progression of breast cancer tumors. The levels of leucine/isoleucine, valine, alanine, arginine, methionine, glutamate, histidine, trimethylamine oxide (TMAO), taurine, and glucose in tumor tissues decreased, with an increment in lipids, lactate, and N-acetyl glycoprotein. Also, there was a reversal of leucine/isoleucine, valine, lactate, arginine, N-acetyl glycoprotein, glutamate, histidine, choline, and glycerophosphocholine/phosphocholine (GPC/PC) in the lung tissues. These metabolites changes were involved in the metabolic pathways of glycolysis, choline, amino acid, and lipid, suggesting that taurine exerted anti-breast cancer effects through the regulation of the underlying metabolism. This study provides a scientific basis for the adoption of taurine in the treatment of breast cancer metastasis.

Systematic analysis of the non-specific lipid transfer protein gene family in Nicotiana tabacum reveal its potential roles in stress responses.

Plant non-specific lipid transfer proteins (nsLTPs) are characterized by an eight-cysteine motif backbone stabilized by four disulfide bonds; these proteins can bind or transfer lipids. NsLTPs play important roles in plant growth and development, and in the responses to abiotic and biotic stresses. In this study, 50, 51, and 100 nsLTPs from Nicotiana sylvestris, N. tomentosiformis, and their descendant N. tabacum, respectively, were identified and classified into six types (I, II, IV, V, VII, and VIII). The phylogeny, gene structures, motifs, tertiary structures, gene duplications and expression patterns were systematically analyzed. The intron/exon patterns and the conserved motifs were highly similar among the same types of nsLTP genes. Purifying selection and segmental duplication dominated the expansion of the nsLTPs family during evolution. Cis-regulatory elements of the NtLTP promoters were involved in light responsiveness, abiotic stress, and phytohormone responsiveness. Expression pattern analysis using RNA-seq and qPCR revealed that NtLTP family genes exhibited tissue-specific expression patterns and they have potential roles in response to abiotic and biotic stresses, especially drought stress, and resistance to black shank and bacterial wilt. Furthermore, overexpression of NtLTPI.38 in tobacco increased drought tolerance by improving the antioxidant defense ability, through reducing O2•- and H2O2 accumulation and increasing the number of lateral roots. These results provide a comprehensive overview of this gene family and provide valuable insights for the functional characterization of nsLTP family genes.

1H-NMR based metabolic study of MMTV-PyMT mice along with pathological progress to screen biomarkers for the early diagnosis of breast cancer.

A 1H NMR-based metabonomic approach was applied to monitor the alterations of serum metabolic profiles in MMTV-PyMT transgenic mice to detect the dynamic changes associated with the pathological process and explore the early-stage biomarkers. The 1H NMR spectra of sera samples from four different stages in MMTV-PyMT mice including hyperplasia, adenoma, early carcinoma and late carcinoma stages were recorded and analyzed using multivariate statistical techniques. The results showed that the increased levels of lipid and lactate, and decreased leucine/isoleucine, valine, methionine, glutamine, creatine, PC/GPC, taurine and glucose were of significance for the early carcinoma stage. As the disease progressed (late carcinoma stage), the metabolic profiles changed significantly; some were negatively regulated compared with those at the early carcinoma stage, such as lipid, leucine/isoleucine, methionine and creatine, accompanied by other new metabolite changes of alanine, pyruvate, glutamate, citrate, aspartate, myo-inositol, 3-methylhistidine and formate. It is important to note that breast cancer patients and the early carcinoma stage of MMTV-PyMT mice had some similar metabolite changes, including lipid, lactate, glutamine, creatine, taurine and glucose, which were determined to be of great value for the early clinical diagnosis of breast cancer. The findings from this study provided valuable biomarkers for the early clinical diagnosis of breast cancer, and showed the potential power of integrating NMR techniques and pattern recognition methods for the analysis of the biochemical changes under certain pathophysiological conditions.

Decylubiquinone Inhibits Colorectal Cancer Growth Through Upregulating Sirtuin2.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. Decylubiquinone (DUb), a coenzyme Q10 analog, was reported to inhibit breast cancer growth and metastasis by us. However, the influence of DUb on CRC remains unclear. Herein, we found that DUb significantly inhibited CRC growth in the patient-derived xenograft (PDX) and CT26 xenograft models. DUb was further identified to significantly suppress CRC cell proliferation, colony formation, migration and invasion in a dose-dependent manner, while not inhibiting CRC cell apoptosis from flow cytometry assay. Sirtuin2 (SIRT2), a member of the sirtuin protein family, plays a critical role in growth and metastasis in various cancers. Moreover, DUb inhibited CRC progression by upregulating SIRT2. These findings reveal that DUb has the potential to a novel drug for the treatment of CRC by inhibiting CRC cell proliferation.

Molecular characterization and systematic analysis of NtAP2/ERF in tobacco and functional determination of NtRAV-4 under drought stress.

The APETALA2/ethylene response factor (AP2/ERF) transcription factor (TF) superfamily play crucial roles in plant growth and development as well as biotic and abiotic stresses response. Here, we systematically characterized 375 AP2/ERF TFs in the Nicotiana tabacum genome. Phylogenetic tree topology and conserved domain number allowed TF classifications into three families of 29 AP2, 341 ERF, and 5 RAV genes, which were unevenly distributed throughout 24 tobacco chromosomes. Gene family expansions were retained from whole genome or segmental duplications followed by tandem duplication. Gene structure and motif analysis revealed intra-group conservation. MicroRNA target site prediction identified nine miR172 family members targeting six NtAP2-family genes; 41 NtAP2/ERFs participated in protein co-regulatory networks. NtAP2/ERF gene global expression profiles ascertained by RNA-seq displayed diverse expression patterns across tissues and under different abiotic and biotic stresses (including drought, cold, and Phytopthora parasitica inoculation). As determined by qRT-PCR, the expression of NtAP2/ERF were induced by five hormone and four abiotic stress. RNA interference of NtRAV-4 in tobacco accelerates seed germination, enhance root development and leaf photosynthetic ability. Suppression of NtRAV-4 increases drought tolerance by improving antioxidant defense ability and reduced relative electrolyte leakage under drought stress. These results enhance understanding of NtAP2/ERF gene function and will facilitate genetic improvement of tobacco stress tolerance.

Carotenoid Cleavage Dioxygenases: Identification, Expression, and Evolutionary Analysis of This Gene Family in Tobacco.

Carotenoid cleavage dioxygenases (CCDs) selectively catalyze carotenoids, forming smaller apocarotenoids that are essential for the synthesis of apocarotenoid flavor, aroma volatiles, and phytohormone ABA/SLs, as well as responses to abiotic stresses. Here, 19, 11, and 10 CCD genes were identified in Nicotiana tabacum, Nicotiana tomentosiformis, and Nicotiana sylvestris, respectively. For this family, we systematically analyzed phylogeny, gene structure, conserved motifs, gene duplications, cis-elements, subcellular and chromosomal localization, miRNA-target sites, expression patterns with different treatments, and molecular evolution. CCD genes were classified into two subfamilies and nine groups. Gene structures, motifs, and tertiary structures showed similarities within the same groups. Subcellular localization analysis predicted that CCD family genes are cytoplasmic and plastid-localized, which was confirmed experimentally. Evolutionary analysis showed that purifying selection dominated the evolution of these genes. Meanwhile, seven positive sites were identified on the ancestor branch of the tobacco CCD subfamily. Cis-regulatory elements of the CCD promoters were mainly involved in light-responsiveness, hormone treatment, and physiological stress. Different CCD family genes were predominantly expressed separately in roots, flowers, seeds, and leaves and exhibited divergent expression patterns with different hormones (ABA, MeJA, IAA, SA) and abiotic (drought, cold, heat) stresses. This study provides a comprehensive overview of the NtCCD gene family and a foundation for future functional characterization of individual genes.

Antisense of survivin inhibits cervical cancer growth in mice.

INTRODUCTION: It is proposed that survivin plays a critical role in the pathogenesis of cancer. Immune regulatory cells are associated with the growth of cancer in the body. Antisenses for the key molecules can suppress tumor growth. This study tests the hypothesis that the antisense of survivin can inhibit cervical cancer. RESULTS: The results showed that human cervical cancer cells expressed high levels of survivin. The levels of survivin in cervical cancer positively correlated with the frequency of interleukin (IL)-10-producing B cells (B10 cells) in the cancer tissue. Survivin increased the expression of IL-10 in B cells. Exposure to survivin antisense efficiently decreased IL-10 expression in B cells. Administration of antisense of survivin inhibited cervical cancer growth and reduced the frequency of B10 cells in tumor-bearing mice. CONCLUSIONS: The results suggest that the survivin antisense has the potential to be used in the treatment of cervical cancer.

Expression, purification, and in vitro characterization of kinase domain of NtGCN2 from tobacco.

General control nonderepressible 2 (GCN2) can phosphorylate the α subunit of eukaryotic initiation factor eIF2 (eukaryotic translation initiation factor 2) to down-regulateprotein synthesis in response to various biotic and abiotic stresses. However, the kinase activity of plant GCN2 has not been well-characterized in vitro. In this study, the kinase domain of Nicotiana tabacum GCN2 (NtGCN2) was inserted into the pET15b vector for prokaryotic expressionin Escherichia coli BL21-CodonPlus-(DE3)-RIPL after induction by 0.5 mmol L-1 IPTG for 13 h at 16 °C. The soluble protein was collected and purified by Ni2+-NTA agarose column, anion exchange, and molecular sieve, and the purified proteinwas used for kinase assays and the preparation of a polyclonal antibody. Enzyme-linked immunosorbent assay results showed that the titer of the antiserum was 1:520K. Western blot analysis showed that the prepared antibody reacted with GCN2 in tobacco. Additionally, the kinase activity of NtGCN2 was characterized by using recombinant NteIF2α protein as a substrate in vitro. The results showed that NtGCN2 phosphorylated NteIF2α in vitro, with the level of phosphorylation positively correlated with the NtGCN2 concentration and reaction time. Our study has prepared a specific antibody, and proves NtGCN2 can phosphorylate NteIF2α in vitro, which lays a foundation for further study of the function and interaction network of NtGCN2.

Overexpression of Tobacco GCN2 Stimulates Multiple Physiological Changes Associated With Stress Tolerance.

General control non-derepressible-2 (GCN2) is a ubiquitous protein kinase that phosphorylates the α subunit of the eukaryotic initiation factor, eIF2, preventing the initiation of a new cycle of protein synthesis, subsequently reducing the global protein biosynthesis. GCN2 can also regulate the response of plants to biotic and abiotic stresses. In this study, two GCN2 homologs, NtGCN2-1 and NtGCN2-2, were cloned from Nicotiana tabacum, and were predicted to have been derived from their progenitors in N. tomentosiformis and N. sylvestris, respectively. The phosphorylation of NteIF2α could be activated by promoting the expression of NtGCN2 with plant hormones, including salicylic acid (SA), azelaic acid (AZA), methyl jasmonate (MeJA), and by imposition of different stresses (Bemisia tabaci infection, drought, and cold), indicating that NtGCN2 is involved in the response of plants to multiple biotic and abiotic stresses. We also observed that the overexpression of NtGCN2-1 significantly influenced different physiological processes. It promoted seed germination and root elongation. The content of total soluble sugars and reducing sugars were decreased, whereas those of chlorophyll a and b were increased in the GCN2 overexpressing plants. In addition, the overexpressing plants had lower content of reactive oxygen species and exhibited higher antioxidant activities. These physiological alterations could be attributed to the changes in the endogenous phytohormones, decrease in the SA and abscisic acid content, and accumulation of MeJA and AZA. It indicated that the overexpression of NtGCN2 in tobacco, stimulated the plant defense responses via phosphorylation of NteIF2α and regulation of plant hormones, and changes in the antioxidant ability and plant nutrient status.