The Brachypodium distachyon BdWRKY36 gene confers tolerance to drought stress in transgenic tobacco plants.

KEY MESSAGE: The expression of BdWRKY36 was upregulated by drought treatment. BdWRKY36 -overexpressing transgenic tobacco increased drought tolerance by controlling ROS homeostasis and regulating transcription of stress related genes. WRKY transcription factor plays important roles in plant growth, development and stress response. However, the function of group IIe WRKYs is less known. In this study, we cloned and characterized a gene of group IIe WRKY, designated as BdWRKY36, from Brachypodium distachyon. Transient expression of BdWRKY36 in onion epidermal cell suggested its localization in the nucleus. Transactivation assay revealed that the C-terminal region, instead of full length BdWRKY36, had transcriptional activity. BdWRKY36 expression was upregulated by drought. Overexpression of BdWRKY36 in transgenic tobacco plants resulted in enhanced tolerance to drought stress. Physiological-biochemical indices analyses showed that BdWRKY36-overexpressing tobacco lines had lesser ion leakage (IL) and reactive oxygen species (ROS) accumulation, but higher contents of chlorophyll, relative water content (RWC) and activities of antioxidant enzyme than that in control plants under drought condition. Meanwhile expression levels of some ROS-scavenging and stress-responsive genes were upregulated in BdWRKY36-overexpressing tobacco lines under drought stress. These results demonstrate that BdWRKY36 functions as a positive regulator of drought stress response by controlling ROS homeostasis and regulating transcription of stress related genes.

A rice calcium-dependent protein kinase OsCPK9 positively regulates drought stress tolerance and spikelet fertility.

BACKGROUND: In plants, calcium-dependent protein kinases (CDPKs) are involved in tolerance to abiotic stresses and in plant seed development. However, the functions of only a few rice CDPKs have been clarified. At present, it is unclear whether CDPKs also play a role in regulating spikelet fertility. RESULTS: We cloned and characterized the rice CDPK gene, OsCPK9. OsCPK9 transcription was induced by abscisic acid (ABA), PEG6000, and NaCl treatments. The results of OsCPK9 overexpression (OsCPK9-OX) and OsCPK9 RNA interference (OsCPK9-RNAi) analyses revealed that OsCPK9 plays a positive role in drought stress tolerance and spikelet fertility. Physiological analyses revealed that OsCPK9 improves drought stress tolerance by enhancing stomatal closure and by improving the osmotic adjustment ability of the plant. It also improves pollen viability, thereby increasing spikelet fertility. In OsCPK9-OX plants, shoot and root elongation showed enhanced sensitivity to ABA, compared with that of wild-type. Overexpression and RNA interference of OsCPK9 affected the transcript levels of ABA- and stress-responsive genes. CONCLUSIONS: Our results demonstrated that OsCPK9 is a positive regulator of abiotic stress tolerance, spikelet fertility, and ABA sensitivity.

Sesamin induces cell cycle arrest and apoptosis through the inhibition of signal transducer and activator of transcription 3 signalling in human hepatocellular carcinoma cell line HepG2.

Sesamin, one of the most abundant lignans in sesame seeds, has been shown to exhibit various pharmacological effects. The aim of this study was to elucidate whether sesamin promotes cell cycle arrest and induces apoptosis in HepG2 cells and further to explore the underlying molecular mechanisms. Here, we found that sesamin inhibited HepG2 cell growth by inducing G2/M phase arrest and apoptosis. Furthermore, sesamin suppressed the constitutive and interleukin (IL)-6-induced signal transducer and activator of transcription 3 (STAT3) signalling pathway in HepG2 cells, leading to regulate the downstream genes, including p53, p21, cyclin proteins and the Bcl-2 protein family. Our studies showed that STAT3 signalling played a key role in sesamin-induced G2/M phase arrest and apoptosis in HepG2 cells. These findings provided a molecular basis for understanding of the effects of sesamin in hepatocellular carcinoma tumour cell proliferation. Therefore, sesamin may thus be a potential chemotherapy drug for liver cancer.

Germacrone induces apoptosis in human hepatoma HepG2 cells through inhibition of the JAK2/STAT3 signalling pathway.

Previous studies have shown that STAT3 plays a vital role in the genesis and progression of cancer. In this study, we investigated the relationship between the JAK2/STAT3 signalling pathway and germacrone-induced apoptosis in HepG2 cells. HepG2 cells were incubated with germacrone for 24 h, the protein expression of p-STAT3, STAT3, p-JAK2 and JAK2 was detected by Western Blotting, and RT-PCR was used to determine the expression of STAT3, p53, Bcl-2 and Bax at transcriptional levels. Besides that, HepG2 cells were pre-treated with AG490 or IL-6 for 2 h, and then incubated with germacrone for 24 h. The expression of p-JAK2, JAK2, p-STAT3, STAT3, p53, Bax and Bcl-2 was detected by Western blotting. The activity of HepG2 cells was tested by MTT assay. The apoptosis of HepG2 cells and levels of reactive oxygen species (ROS) were flow cytometrically measured. The results showed that germacrone exposure decreased p-STAT3 and p-JAK2 and regulated expression of p53 and Bcl-2 family members at the same time. Moreover, IL-6 enhanced the activation of the JAK2/STAT3 signalling pathway and therefore attenuated the germacrone-induced apoptosis. Suppression of JAK2/STAT3 signalling pathway by AG490, an inhibitor of JAK2, resulted in apoptosis and an increase in ROS in response to germacrone exposure. We therefore conclude that germacrone induces apoptosis through the JAK2/STAT3 signalling pathway.

Anti-tumor effect of germacrone on human hepatoma cell lines through inducing G2/M cell cycle arrest and promoting apoptosis.

Germacrone is one of the main bioactive components in the traditional Chinese medicine Rhizoma curcuma. In this study, the anti-proliferative effect of germacrone on the human hepatoma cell lines and the molecular mechanism underlying the cytotoxicity of germacrone were investigated. Treatment of human hepatoma cell lines HepG2 and Bel7402 with germacrone resulted in cell cycle arrest and apoptosis in a dose-dependent manner as measured by MTT assay, flow cytometric and fluorescent microscopy analysis, while much lower effect on normal human liver cell L02 was observed. Flow cytometric analysis revealed that germacrone induced G2/M arrest in the cell cycle progression that was associated with an obvious decrease in the protein expression of cyclin B1 and its activating partner CDK1 with concomitant inductions of p21. Hoechst 33258 and Annexin V/PI staining results showed that the total cell number in apoptosis associated with a dose-dependent up-regulation of Bax and down-regulation of Bcl-2/Bcl-xl was increased. In the meantime, the up-regulation of p53 and reactive oxygen species increase were observed, which suggested that germacrone might be a new potent chemopreventive drug candidate for liver cancer via regulating the expression of proteins related to G2/M cell cycle and apoptosis, and p53 and oxidative damage may play important roles in the inhibition of human hepatoma cells growth by germacrone.

Overexpression of a wheat aquaporin gene, TaAQP8, enhances salt stress tolerance in transgenic tobacco.

Aquaporin (AQP) proteins have been shown to transport water and other small molecules through biological membranes, which is crucial for plants to combat salt stress. However, the precise role of AQP genes in salt stress response is not completely understood in plants. In this study, a PIP1 subgroup AQP gene, designated TaAQP8, was cloned and characterized from wheat. Transient expression of TaAQP8-green fluorescent protein (GFP) fusion protein revealed its localization in the plasma membrane. TaAQP8 exhibited water channel activity in Xenopus laevis oocytes. TaAQP8 transcript was induced by NaCl, ethylene and H(2)O(2). Further investigation showed that up-regulation of TaAQP8 under salt stress involves ethylene and H(2)O(2) signaling, with ethylene causing a positive effect and H(2)O(2) acting as a negative factor. Overexpression of TaAQP8 in tobacco increased root elongation compared with controls under salt stress. The roots of transgenic plants also retained a high K(+)/Na(+) ratio and Ca(2+) content, but reduced H(2)O(2) accumulation by an enhancement of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities under salt stress. Further investigation showed that whole seedlings from transgenic lines displayed higher SOD, CAT and POD activities, increased NtSOD and NtCAT transcript levels, and decreased H(2)O(2) accumulation and membrane injury under salt stress. Taken together, our results demonstrate that TaAQP8 confers salt stress tolerance not only by retaining high a K(+)/Na(+) ratio and Ca(2+) content, but also by reducing H(2)O(2) accumulation and membrane damage by enhancing the antioxidant system.

Characterization of alkaline phosphatase labeled UidA(Gus) probe and its application in testing of transgenic tritordeum.

Hybridization is a very important molecular biology technique to measure the degree of genetic similarity between DNA sequences, and detect the foreign genes in transgenic organisms. To label a DNA or RNA probe plays a key role in hybridization. A method using nonradioactive material alkaline phosphatase to label UidA(Gus) DNA as probe has been studied. On that basis of Renz and our previous work, alkaline phosphatase-labeled DNA was used as a probe to examine the transformation of the foreign UidA(Gus) gene in transgenic tritordeum. Such DNA-enzyme complexes were characterized and examined carefully, the results showed that it was a sensitive, specific, safe and economical probe. For dot hybridization and Southern blot under full-stringency conditions with alkaline phosphatase as the detector and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)-Nitro Blue Tetrazolium (NBT) as the substrate, dot hybridization showed that the UidA(Gus) gene was transformed into the target plants and inherited stable, Southern blot showed that at least two copies of UidA(Gus) gene were inserted into one line of our transgenic tritordeum. Histochemical staining with X-Gluc of transgenic tritordeum also certified that the foreign UidA(Gus) DNA were transformed into the transgenic tritordeum.

cDNA cloning and expression analysis of wheat (Triticum aestivum L.) phytoene and ζ-carotene desaturase genes.

Carotene desaturation, an essential step in the carotenoid biosynthesis pathway, is catalyzed by two carotene desaturases, phytoene desaturase (PDS) and ζ-carotene desaturase (ζ-carotene desaturase, ZDS). Full-length cDNAs designated TaPDS and TaZDS were cloned from wheat (Triticum aestivum cv. Chinese Spring) respectively, using the rapid amplification of cDNA ends (RACE) approach. The cDNA of TaPDS sequence was 2076 bp long, containing a 1731 bp open reading frame (ORF) which deduced protein having 576 amino acid residues with predicted molecular mass of 64.3 kDa and having a putative transit sequence for plastid targeting in the N-terminal region. While the cDNA sequence of TaZDS was 2150 bp long, contained an ORF sequence of 1707 bp, and encoded a putative protein of 568 amino acid residues with an estimated molecular mass of 62.5 kDa. Phylogenetic analysis demonstrated that TaPDS and TaZDS showed high homology with other PDSs and ZDSs in higher plant species, respectively. Moreover, sequences analysis also showed a high degree of conservation among plant PDSs and ZDSs. The deduced TaPDS and TaZDS protein both have the dinucleotide binding domain and conserved regions characteristic of other carotene desaturases. Analysis of the expression pattern of wheat TaPDS and TaZDS in different tissues revealed that the transcripts levels were higher in leaves and flowers petals, followed by in inflorescences, and were nearly absent in the roots and seeds. Southern analysis of genomic DNA indicated that the wheat TaPDS and TaZDS probably belong to a low-copy-number gene family.

Isolation and heterologous transformation analysis of a pollen-specific promoter from wheat (Triticum aestivum L.).

The promoter of a pollen-specific gene TaPSG719 was isolated from wheat (Triticum aestivum L.) by inverse-PCR (IPCR). Sequence analysis revealed that the promoter contains two cis-acting elements (AGAAA and GTGA) known to confer anther/pollen-specific gene expression which suggests that the promoter of TaPSG719 gene is a pollen-specific one. To ascertain the regulatory function of TaPSG719 promoter, two deleted fragments (-1,776 to -1 bp and -1,019 to -1 bp) were fused to the beta-glucuronidase (GUS) gene and transformed into tobacco plants. Similar GUS expression patterns were observed in all transformed plants and its activity was detected exclusively in pollen. No GUS activity in any other floral or vegetative tissue was observed. The results confirm that TaPSG719 promoter is pollen-specific and active during the middle stages of pollen development till anther matured, and it can drive pollen-specific gene expression across the species.

Rice proteomics: a step toward functional analysis of the rice genome.

The technique of proteome analysis with two-dimensional PAGE has the power to monitor global changes that occur in the protein expression of tissues and organisms and/or expression that occurs under stresses. In this study, the catalogues of the rice proteome were constructed, and a functional characterization of some of these proteins was examined. Proteins extracted from tissues of rice and proteins extracted from rice under various kinds of stress were separated by two-dimensional PAGE. An image analyzer was used to reveal a total of 10,589 protein spots on 10 kinds of two-dimensional PAGE gels stained by Coomassie Brilliant Blue. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane, and the N-terminal amino acid sequences of 272 of 905 proteins were determined. The internal amino acid sequences of 633 proteins were determined using a protein sequencer or mass spectrometry after enzyme digestion of the proteins. Finally, a data file of rice proteins that included information on amino acid sequences and sequence homologies was constructed. The major proteins involved in the growth and development of rice can be identified using the proteome approach. Some of these proteins, including a calcium-binding protein that turned out to be calreticulin and a gibberellin-binding protein, which is ribulose-1,5-bisphosphate carboxylase/oxygenase activase in rice, have functions in the signal transduction pathway. The information thus obtained from the rice proteome will be helpful in predicting the function of the unknown proteins and will aid in their molecular cloning.