The Integration of Metabolomics and Transcriptomics Provides New Insights for the Identification of Genes Key to Auxin Synthesis at Different Growth Stages of Maize.

As a staple food crop, maize is widely cultivated worldwide. Sex differentiation and kernel development are regulated by auxin, but the mechanism regulating its synthesis remains unclear. This study explored the influence of the growth stage of maize on the secondary metabolite accumulation and gene expression associated with auxin synthesis. Transcriptomics and metabonomics were used to investigate the changes in secondary metabolite accumulation and gene expression in maize leaves at the jointing, tasseling, and pollen-release stages of plant growth. In total, 1221 differentially accumulated metabolites (DAMs) and 4843 differentially expressed genes (DEGs) were screened. KEGG pathway enrichment analyses of the DEGs and DAMs revealed that plant hormone signal transduction, tryptophan metabolism, and phenylpropanoid biosynthesis were highly enriched. We summarized the key genes and regulatory effects of the tryptophan-dependent auxin biosynthesis pathways, giving new insights into this type of biosynthesis. Potential MSTRG.11063 and MSTRG.35270 and MSTRG.21978 genes in auxin synthesis pathways were obtained. A weighted gene co-expression network analysis identified five candidate genes, namely TSB (Zm00001d046676 and Zm00001d049610), IGS (Zm00001d020008), AUX2 (Zm00001d006283), TAR (Zm00001d039691), and YUC (Zm00001d025005 and Zm00001d008255), which were important in the biosynthesis of both tryptophan and auxin. This study provides new insights for understanding the regulatory mechanism of auxin synthesis in maize.

Overexpression of ZmDHN15 Enhances Cold Tolerance in Yeast and Arabidopsis.

Maize (Zea mays L.) originates from the subtropical region and is a warm-loving crop affected by low-temperature stress. Dehydrin (DHN) protein, a member of the Group 2 LEA (late embryogenesis abundant proteins) family, plays an important role in plant abiotic stress. In this study, five maize DHN genes were screened based on the previous transcriptome sequencing data in our laboratory, and we performed sequence analysis and promoter analysis on these five DHN genes. The results showed that the promoter region has many cis-acting elements related to cold stress. The significantly upregulated ZmDHN15 gene has been further screened by expression pattern analysis. The subcellular localization results show that ZmDHN15 fusion protein is localized in the cytoplasm. To verify the role of ZmDHN15 in cold stress, we overexpressed ZmDHN15 in yeast and Arabidopsis. We found that the expression of ZmDHN15 can significantly improve the cold resistance of yeast. Under cold stress, ZmDHN15-overexpressing Arabidopsis showed lower MDA content, lower relative electrolyte leakage, and less ROS (reactive oxygen species) when compared to wild-type plants, as well as higher seed germination rate, seedling survival rate, and chlorophyll content. Furthermore, analysis of the expression patterns of ROS-associated marker genes and cold-response-related genes indicated that ZmDHN15 genes play an important role in the expression of these genes. In conclusion, the overexpression of the ZmDHN15 gene can effectively improve the tolerance to cold stress in yeast and Arabidopsis. This study is important for maize germplasm innovation and the genetic improvement of crops.

Overexpression of ZmSRG7 Improves Drought and Salt Tolerance in Maize (Zea mays L.).

Osmotic stress caused by drought and high salinity is the key factor limiting plant growth. However, its underlying molecular regulatory mechanism remains unclear. In this study, we found the stress-related gene Zm00001d019704 (ZmSRG7) based on transcriptome sequencing results previously obtained in the laboratory and determined its biological function in maize. We found that ZmSRG7 was significantly expressed in both roots and leaves under 10% PEG6000 or 150 mM NaCl. Subcellular localization showed that the gene was localized in the nucleus. The germination rate and root length of the ZmSRG7 overexpressing lines were significantly increased under drought or salt stress compared with the control. However, after drought stress, the survival rate and relative water content of maize were increased, while the water loss rate was slowed down. Under salt stress, the Na+ concentration and Na+: K+ ratio of maize was increased. In addition, the contents of antioxidant enzymes and proline in maize under drought or salt stress were higher than those in the control, while the contents of MDA, H2O2 and O2- were lower than those in the control. The results showed that the ZmSRG7 gene played its biological function by regulating the ROS signaling pathway. An interaction between ZmSRG7 and the Zmdhn1 protein was found using a yeast two-hybrid experiment. These results suggest that the ZmSRG7 gene can improve maize tolerance to drought or salt by regulating hydrogen peroxide homeostasis.

A Novel Senescence-Specific Gene (ZmSAG39) Negatively Regulates Darkness and Drought Responses in Maize.

The papain-like cysteine proteases (PLCPs) is a subfamily of cysteine proteases that plays an important role in leaf senescence, and some of its members are involved in the regulation of plant growth and development under stress. In this study, we cloned a new gene, ZmSAG39, from maize. Expression profile analysis showed that ZmSAG39 was induced by darkness and drought treatments. In addition, the ZmSAG39 overexpression in maize accelerated the senescence of maize leaves under darkness and drought treatments. However, the knockout of ZmSAG39 in maize enhanced the resistance of maize to darkness and drought stresses and reduced the degree of senescence of maize leaves. Under drought stress, compared with WT plants, the knockout lines had a higher seed germination rate, seedling survival rate and chlorophyll content, and lower reactive oxygen species (ROS) level and malondialdehyde (MDA) content. In addition, quantitative real-time PCR (qRT-PCR) analysis showed that ZmSAG39 negatively regulated some stress-related genes but positively regulated senescence-related genes under darkness and drought stress conditions. To summarize, these results indicate that ZmSAG39 is a senescence-related gene and plays a negative role in response to darkness and drought stresses. This study laid a theoretical foundation for the innovation of maize germplasm resources with high quality, high yield and strong stress resistance.

miR-221 Mediates Chemoresistance of Esophageal Adenocarcinoma by Direct Targeting of DKK2 Expression.

BACKGROUND: Chemoresistance is a main obstacle to effective esophageal cancer (EC) therapy. We hypothesize that altered expression of microRNAs (miRNAs) play a role in EC cancer progression and resistance to 5-fluorouracil (5-FU) based chemotherapeutic strategies. METHODS: Four pairs of esophageal adenocarcinoma (EAC) cell lines and corresponding 5-FU resistant variants were established. The expression levels of miRNAs previously shown to be involved in the general regulation of stem cell pathways were analyzed by qRT-PCR. The effects of selected miRNAs on proliferation, apoptosis, and chemosensitivity were evaluated both in vitro and in vivo. We identified a particular miRNA and analyzed its putative target genes in 14 pairs of human EC tumor specimens with surrounding normal tissue by qRT-PCR as well as Wnt pathway associated genes by immunohistochemistry in another 45 EAC tumor samples. RESULTS: MiR-221 was overexpressed in 5-FU resistant EC cell lines as well as in human EAC tissue. DKK2 was identified as a target gene for miR-221. Knockdown of miR-221 in 5-FU resistant cells resulted in reduced cell proliferation, increased apoptosis, restored chemosensitivity, and led to inactivation of the Wnt/ß-catenin pathway mediated by alteration in DKK2 expression. Moreover, miR-221 reduction resulted in alteration of EMT-associated genes such as E-cadherin and vimentin as well as significantly slower xenograft tumor growth in nude mice. RT profiler analysis identified a substantial dysregulation of 4 Wnt/ß-catenin signaling and chemoresistance target genes as a result of miR-221 modulation: CDH1, CD44, MYC, and ABCG2. CONCLUSION: MiR-221 controls 5-FU resistance of EC partly via modulation of Wnt/ß-catenin-EMT pathways by direct targeting of DKK2 expression. MiR-221 may serve as a prognostic marker and therapeutic target for patients with 5-FU resistant EAC.

Zmhdz9, an HD-Zip transcription factor, promotes drought stress resistance in maize by modulating ABA and lignin accumulation.

Maize is the largest crop in the world in terms of both planting area and total yield, and it plays a crucial role in ensuring global food and feed security. However, in recent years, with climate deterioration, environmental changes, and the scarcity of freshwater resources, drought has become a serious limiting factor for maize yield and quality. Drought stress-induced signals undergo a series of transmission processes to regulate the expression of specific genes, thereby affecting the drought tolerance of plants at the tissue, cellular, physiological and biochemical levels. Therefore, in this study we investigated the HD-Zip transcription factor gene Zmhdz9, and yeast activation experiments demonstrated that Zmhdz9 exhibited transcriptional activation activity. Under drought stress, high abscisic acid (ABA) and lignin levels significantly improved drought resistance in maize. Yeast two-hybrid, bimolecular fluorescence complementation (BIFC) and pull-down experiments showed that Zmhdz9 interacted with ZmWRKY120 and ZmTCP9, respectively. Overexpression of Zmhdz9 and gene editing of ZmWRKY120 or ZmTCP9 improved maize drought resistance, indicating their importance in the drought stress response. Furthermore, Zmhdz9 promoted the direct transcription of ZmWRKY120 in the W-box, activating elements of the ZmNCED1 promoter, which encodes a key enzyme in ABA biosynthesis. Additionally, Zmhdz9 promoted direct transcription of ZmTCP9 in the GGTCA motif, activating elements of the ZmKNOX8 promoter, which encodes a key enzyme in lignin synthesis. This study showed that the regulation of ABA and lignin by Zmhdz9 is essential for drought stress resistance in maize.

Cloning and functional analysis of ZmMADS42 gene in maize.

Maize (Zea mays L.) is the most important cereal crop in the world. Flowering period and photoperiod play important roles in the reproductive development of maize. This study, investigated ZmMADS42, a gene that is highly expressed in the shoot apical meristem. Agrobacterium infection was used to successfully obtain overexpressed ZmMADS42 plants. Fluorescence quantitative PCR revealed that the expression of the ZmMADS42 gene in the shoot apical meristem of transgenic plants was 2.8 times higher than that of the wild-type(WT). In addition, the expression of the ZmMADS42 gene in the endosperm was 2.4 times higher than that in the wild-type. The seed width of the T2 generation increased by 5.35%, whereas the seed length decreased by 7.78% compared with that of the wild-type. Dissection of the shoot tips of transgenic and wild-type plants from the 7-leaf stage to the 9-leaf stage revealed that the transgenic plants entered the differentiation stage earlier and exhibited more tassel meristems during their vegetative growth period. The mature transgenic plants were approximately 20 cm shorter in height and had a lower panicle position than the wild-type plants. Comparing the flowering period, the tasseling, powdering, and silking stages of the transgenic plants occurred 10 days earlier than those of the wild-type plants. The results showed that the ZmMADS42 gene played a significant role in regulating the flowering period and plant height of maize.

Analysis of the mechanism of action of Euphorbia fischeriana Steud on cirrhosis based on network pharmacology.

This study aimed to employ network pharmacology to elucidate the mechanism by which Euphorbia fischeriana Steud (EFS) exhibits the efficacy on cirrhosis. The compounds and targets of EFS were retrieved from Traditional Chinese Medicine Integrated Database and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Next, these compounds and targets were analyzed based on protein-protein interaction (PPI) network. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling network was established based on KEGG database. We constructed a compound-compound target-intersection target-pathway PPI network, including 20 compounds, 19 intersection targets between compound targets and EFS targets. Among the 20 compounds, 8-Isopentenyl-kaempferol has the most targets, with 27 targets, followed by 3,4',5-Trihydroxy-7-methoxy-8-isopentenylflavone, Formononetin, Isoxanthohumol, and Isokurarinone with potential targets of 26, 22, 18, and 14, respectively. Top 5 targets are HSP90AA1, PTGS2, NOS2, MAPK14, and PPARG. KEGG pathway enrichment analysis showed that pathways such as Hepatitis B, Hepatitis C, Lipid and atherosclerosis, and AGE-RAGE signaling pathway in diabetic complications were closely related to the infection and abnormal metabolism of the liver. The application of network pharmacology could identify potential targets of EFS with a low false-positive rate and provide novel insight into the mechanism of action of EFS on cirrhosis.

Overexpression of the homeobox-leucine zipper protein ATHB-6 improves the drought tolerance of maize (Zea mays L.).

Homeo-Leucine Zipper (HD-Zip) proteins are a class of transcription factors unique to higher plants and are involved in plant stress responses and regulation of growth and development. However, the function of maize HD-Zip genes in enhancing drought tolerance is unknown. Here, Sub-Cellular Localization results showed that ATHB-6 fusion proteins were only localized in the nucleus. The malondialdehyde content was lower than the wild type under drought tolerance, proving that the introduction of the ATHB-6 gene can improve the drought tolerance of plants. Follow-up analysis showed that ATHB-6 could promote root growth and activities of a series of ROS-scavenging enzymes in maize. Moreover, overexpression of ATHB-6 in maize activated the expression of critical genes in the ROS signals pathway and ABA-dependent pathway under drought tolerance.Our results provides a significant advancement in undestanding the functions of HD-Zip transcription factors in maize.

ZmLBD2 a maize (Zea mays L.) lateral organ boundaries domain (LBD) transcription factor enhances drought tolerance in transgenic Arabidopsis thaliana.

Maize (Zea mays L.) is an annual gramineous herb and is among the world's most important crop species. Drought is the main factor contributing to maize yield reduction. The lateral organ boundaries domain (LBD) proteins belong to a class of higher-plant-specific transcription factors. LBD proteins usually include the highly conserved lateral organ boundaries (LOB) domains that play essential roles in plant growth and response to biotic stresses. However, few studies have addressed the biological functions of LBD genes associated with maize response to drought. Here we cloned the ZmLBD2 gene from maize and described its role in combating drought. Investigating ZmLBD2 subcellular localization, we show that it localizes to the cell nucleus and can specifically bind with inverted repeats of "GCGGCG". Under drought stress, Arabidopsis thaliana overexpressing ZmLBD2 performed better than the wild-type plants in terms of seed germination rates, root length, relative water content, fresh weight, chlorophyll content, proline content, and antioxidant enzyme content. Arabidopsis overexpressing ZmLBD2 contained less MDA, H2O2, and O 2 - than the wild-type plants. Our protein-protein interaction results indicate an interaction between the ZmLBD2 and ZmIAA5 genes. In conclusion, the ZmLBD2 gene positively regulates H2O2 homeostasis in plants, strengthening drought resistance.

Increased CD4(+) CD69(+) CD25(-) T cells in patients with hepatocellular carcinoma are associated with tumor progression.

BACKGROUND AND AIM: A new subset of Treg cells, CD4(+) CD69(+) CD25(-) T cells, has been identified in mice. Herein, we aimed to identify this subset of T cells and to evaluate its function in patients with hepatocellular carcinoma (HCC). METHODS: We detected CD4(+) CD69(+) CD25(-) T cells and its expression of CCR6 and transforming growth factor-ß1 (TGF-ß1) in peripheral blood of 91 HCC patients, 38 chronic hepatitis patients and 34 healthy donors by flow cytometry. CD4(+) CD69(+) CD25(-) T cells in HCC tissues were also analyzed. RESULTS: CD4(+) CD69(+) CD25(-) T cells were significantly increased in peripheral blood of HCC patients compared with healthy persons and chronic hepatitis patients (8.74% ± 0.42% vs 4.55% ± 0.33% and 5.15% ± 0.36%, P < 0.0001). The percentage of peripheral CD4(+) CD69(+) CD25(-) T cells was significantly higher in HCC patients with Tumor Node Metastasis (TNM) stage III plus IV (P < 0.05). Patients with large tumor size and tumor vascular invasion were inclined to obtain high percentage of CD4(+) CD69(+) CD25(-) T cells (P < 0.05). The frequency of membrane-bound TGF-ß1 positive cells in CD4(+) CD69(+) CD25(-) T cells from HCC patients was higher than that from the other two groups (P < 0.0001). A considerable proportion of CD4(+) CD69(+) CD25(-) T cells were present in HCC tissues, which has significant correlation with tumor size and TNM stage. Few CD4(+) CD69(+) CD25(-) T cells express CCR6 both in peripheral blood and tumor tissues from HCC patients. CONCLUSIONS: Increased CD4(+) CD69(+) CD25(-) T cells in HCC patients are significantly correlated with tumor size, vascular invasion and TNM stage. Thus, increased CD4(+) CD69(+) CD25(-) T cells exert a critical role in HCC progression and might be a clinically aggressive phenotype of HCC.

[Application of portal vein chemoembolization in major hepatectomy for initially unresectable primary and secondary hepatic tumors].

OBJECTIVE: To investigate the surgical treatment of initially unresectable primary and secondary hepatic tumors. METHODS: For the patients with multiple and bilobar colonic hepatic metastases, a first-stage hepatectomy consisted in a radical resection of sigmoid colonic carcinoma and left lateral hepatic segment. Subsequently, under the guidance of ultrasonography and radiography, a right portal vein chemoembolization (PVCE) was performed via a percutaneous approach through left portal branch to induce the atrophy of right hemiliver and hypertrophy of left hemiliver. At Week 5 post-PVCE, a second-stage hepatectomy was planned to resect the right hemiliver. For patients with huge hepatocellular carcinoma (HCC), transcatheter arterial chemoembolization (TACE) were performed and it was followed by PVCE 1 week later. At Week 4 post-PVCE, a right trisegmentectomy was attempted to resect the right liver tumor. The volume of liver was evaluated with three-dimensional CT scan at Weeks 2 and 4 weeks post-PVCE. RESULTS: At Week 4 post-PVCE, the atrophy of right lobe was induced and the left lobe underwent compensatory hypertrophy. The remnant volumes of right lobe and right trisegmentectomy for HCC decreased from 1380.0 cm(3), 1685.4 cm(3) at pre-PVCE to 740.2 cm(3), 1228.1 cm(3) at post-PVCE. The values increased from pre-PVCE 435.1 cm(3), 151.5 cm(3) to post-PVCE 624.4 cm(3), 560.2 cm(3) for left hepatic lobe remnant of colonic liver metastases and left lateral segment for HCC. The ratios of liver remnant to estimated total liver volume increased from 25.6%, 13.6% at pre-PVCE to 50.0%, 43.1% post-PVCE respectively. The postoperative course was uneventful. The liver function, serum CEA and AFP decreased to the normal levels. Two patients were followed up for 18 and 8 months respectively. There was no tumor recurrence. CONCLUSION: PVCE prevents the hepatic function failure after a major hepatectomy. And it may benefit more patients with previously unresectable liver tumors.

Characterization of the complete chloroplast genome of Alsophila spinulosa, an endangered species endemic to China.

Tree fern Alsophila spinulosa is an endangered relic plant in the world. It is currently on the International Union for Conservation of Nature (IUCN) red list of threatened species. In this study, we first assembled the complete chloroplast (cp) genome of A. spinulosa by Illumina paired-end reads data. The whole genome was 156,661 bp, consisting of a pair of inverted repeats of 24,364 bp, large single copy region and a small single copy region (70,352 and 21,624 bp in length, respectively). The cp genome contained 133 genes, including 92 protein-coding genes, 33 trRNA genes, and eight rRNA genes. The overall GC content of the whole genome was 40.4%. A neighbour-joining phylogenetic analysis demonstrated a close relationship between A. spinulosa and Cystoathyrium chinense Ching.

The complete chloroplast genome sequence of Camellia granthamiana.

Camellia granthamiana is a rare and endangered plant peculiar to China, and a total of 5 plants have been found at present. Based on the next generation sequencing, the whole chloroplast (Cp) genome of (Camellia granthamiana Sealy) of Camellia oleifera was constructed.In this study, the complete chloroplast (cp) genome of Camellia granthamiana was assembled based on next generation sequencing.The cp genome was 157,001 bp in length, including a large single copy (LSC) region of 70,387 bp, a small single copy (SSC) region of 18,296 bp and a pair of inverted repeats (IRs) of 52,082 bp. The genome contained 135 genes, including 90 protein-coding genes, 37 tRNA genes and 8 ribosomal RNA genes. The majority of these gene species occurred as a single copy.

Characterization of the complete chloroplast genome of Ginkgo biloba L. (Ginkgoaceae), an endangered species endemic to China.

Ginkgo biloba L. is the oldest relict plant among the gymnosperms, left after the quaternary glacial movement. There are few living G. biloba and few old trees over a hundred years old. It is currently on the International Union for Conservation of Nature (IUCN) red list of threatened species. In this study, we first assembled the complete chloroplast (cp) genome of G. biloba L. by Illumina paired-end reads data. The whole genome was 156,988 bp, consisting of a pair of inverted repeats of 34,056 bp, large and small single copy regions of 56,819 and 22,763 bp in length, respectively. The cp genome contained 131 genes, including 84 protein-coding genes, 31 trRNA genes and 5 rRNA genes. The overall GC content of the whole genome was 39.6%. A neighbour-joining phylogenetic analysis demonstrated a close relationship between G. biloba L. and Cycas revoluta.

Characterization of the complete chloroplast genome of Davidia involucrata Baill. (Nysssaceae), an endangered species endemic to China.

Davidia involucrata Baill. is a kind of tertiary paleotropical plant floristic relic species unique to China. This rare plant is disappearing due to poor adaptability and serious poaching. We first assembled the complete chloroplast (cp) genome of Davidia involucrata Baill. by Illumina paired-end reads data. The whole genome was 169,085 bp, consisting of a pair of inverted repeats of 169,379 bp, large single copy region and a small single copy region (96,712 and 67,667 bp in length, respectively). The cp genome contained 90 genes, including 64 protein-coding genes, 22 trRNA genes and 4 rRNA genes. The overall GC content of the whole genome was 38.04%. A neighbor-joining phylogenetic analysis demonstrated a close relationship between Davidia involucrata Baill. and Nyssa yunnanensis.

MicroRNA-34c targets TGFB-induced factor homeobox 2, represses cell proliferation and induces apoptosis in hepatitis B virus-related hepatocellular carcinoma.

MicroRNAs (miRs) are short, non-coding RNAs with post-transcriptional regulatory functions. Previous studies have demonstrated that miR-34c is involved in diverse biological processes, including carcinogenesis. The aim of the present study was to investigate the role of miR-34c and its target genes in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Expression levels of miR-34c and its predicted target genes were measured. The target genes were validated by a luciferase assay. The effects of miR-34c restoration were evaluated by the detection of HBV antigens, cell proliferation and apoptosis in vitro, in addition to the tumor growth in vivo. The data demonstrated that miR-34c was downregulated in HBV-associated HCC clinical tissues and HCC cell lines compared with their corresponding controls. transforming growth factor-ß-induced factor homeobox 2 (TGIF2), a transcription factor repressing transforming growth factor-ß (TGFß) signaling, was observed to be upregulated and was identified as a target gene of miR-34c. The restoration of miR-34c in HepG2.2.15 cells suppressed TGIF2 expression, HBV replication and viral antigen synthesis; inhibited cell proliferation; and induced apoptosis. miR-34c also inhibited tumor growth in a mouse model. The present study indicates that miR-34c may act as a tumor suppressor by targeting TGIF2 during HBV-associated hepatocellular carcinogenesis. miR-34c and TGIF2 may represent key regulatory factors, diagnostic markers and therapeutic targets for the prevention and treatment of HBV-associated HCC.