Guangxiao Yang

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.

A Wheat WRKY Transcription Factor TaWRKY10 Confers Tolerance to Multiple Abiotic Stresses in Transgenic Tobacco

WRKY transcription factors are reported to be involved in defense regulation, stress response and plant growth and development. However, the precise role of WRKY transcription factors in abiotic stress tolerance is not completely understood, especially in crops. In this study, we identified and cloned 10 WRKY genes from genome of wheat (Triticum aestivum L.). TaWRKY10, a gene induced by multiple stresses, was selected for further investigation. TaWRKY10 was upregulated by treatment with polyethylene glycol, NaCl, cold and H2O2. Result of Southern blot indicates that the wheat genome contains three copies of TaWRKY10. The TaWRKY10 protein is localized in the nucleus and functions as a transcriptional activator. Overexpression of TaWRKY10 in tobacco (Nicotiana tabacum L.) resulted in enhanced drought and salt stress tolerance, mainly demonstrated by the transgenic plants exhibiting of increased germination rate, root length, survival rate, and relative water content under these stress conditions. Further investigation showed that transgenic plants also retained higher proline and soluble sugar contents, and lower reactive oxygen species and malonaldehyde contents. Moreover, overexpression of the TaWRKY10 regulated the expression of a series of stress related genes. Taken together, our results indicate that TaWRKY10 functions as a positive factor under drought and salt stresses by regulating the osmotic balance, ROS scavenging and transcription of stress related genes.

TaASR1, a transcription factor gene in wheat, confers drought stress tolerance in transgenic tobacco

Abscisic acid (ABA)-, stress-, and ripening-induced (ASR) proteins are reported to be involved in abiotic stresses. However, it is not known whether ASR genes confer drought stress tolerance by utilizing the antioxidant system. In this study, a wheat ASR gene, TaASR1, was cloned and characterized. TaASR1 transcripts increased after treatments with PEG6000, ABA and H(2)O(2). Overexpression of TaASR1 in tobacco resulted in increased drought/osmotic tolerance, which was demonstrated that transgenic lines had lesser malondialdehyde (MDA), ion leakage (IL) and reactive oxygen species (ROS), but higher relative water content (RWC) and superoxide dismutase (SOD) and catalase (CAT) activities than wild type (WT) under drought stress. Overexpression of TaASR1 in tobacco also enhanced the expression of ROS-related and stress-responsive genes under osmotic stress. In addition, transgenic lines exhibited improved tolerance to oxidative stress by retaining more effective antioxidant system. Finally, TaASR1 was localized in the cell nucleus and functioned as a transcriptional activator. Taken together, our results showed that TaASR1 functions as a positive factor under drought/osmotic stress, involved in the regulation of ROS homeostasis by activating antioxidant system and transcription of stress-associated genes.

Overexpression of the Wheat Aquaporin Gene, TaAQP7, Enhances Drought 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 stress caused by drought. However, the precise role of AQPs in drought stress response is not completely understood in plants. In this study, a PIP2 subgroup gene AQP, designated as TaAQP7, was cloned and characterized from wheat. Expression of TaAQP7-GFP fusion protein revealed its localization in the plasma membrane. TaAQP7 exhibited high water channel activity in Xenopus laevis oocytes and TaAQP7 transcript was induced by dehydration, and treatments with polyethylene glycol (PEG), abscisic acid (ABA) and H2O2. Further, TaAQP7 was upregulated after PEG treatment and was blocked by inhibitors of ABA biosynthesis, implying that ABA signaling was involved in the upregulation of TaAQP7 after PEG treatment. Overexpression of TaAQP7 increased drought tolerance in tobacco. The transgenic tobacco lines had lower levels of malondialdehyde (MDA) and H2O2, and less ion leakage (IL), but higher relative water content (RWC) and superoxide dismutase (SOD) and catalase (CAT) activities when compared with the wild type (WT) under drought stress. Taken together, our results show that TaAQP7 confers drought stress tolerance in transgenic tobacco by increasing the ability to retain water, reduce ROS accumulation and membrane damage, and enhance the activities of antioxidants.

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

BackgroundIn 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.ResultsWe 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.ConclusionsOur results demonstrated that OsCPK9 is a positive regulator of abiotic stress tolerance, spikelet fertility, and ABA sensitivity.

Proteomic analysis of soybean [Glycine max (L.) Meer.] seeds during imbibition at chilling temperature

Many of the world’s important crops such as soybean are now widely distributed, beyond their original zones of natural selection and chilling temperature is one of the major abiotic environmental factors which limit their growth and yield. Various molecular mechanisms, underlying chilling stress and plant responses are yet to be discovered. Chilling temperatures impart maximum damage to the soybean seeds, sown in wet soils, during imbibition, causing poor germination rate, reduced seedling emergence, decreased seedling vigor, and ultimately severe loss in yield. Here we report the changes in proteome of soybean seeds exposed to low temperature (4°C) during imbibition in a known chilling-resistant soybean cultivar Z22, using two-dimensional gel electrophoresis (2-DE) and Matrix-Assisted Laser Ionization Time of Flight Mass Spectrometry (MALDI-TOF/MS). Total 40 protein spots were differentially expressed in response to low temperature, in which, 25 protein spots were up-regulated and fifteen protein spots were down-regulated. According to the search in primary databases, these proteins were a part of many metabolic pathways including, cell defense, energy, protein synthesis, cell growth/division, storage, transcription and transport. To investigate the change in mRNA levels during imbibition at 4°C, quantitative RT-PCR of nine up-regulated protein genes was carried out, which clearly demonstrated an increase in the transcript levels of five genes, whereas no change was observed for other four genes. Results of this study provide some invaluable insights about the response of soybean seeds to low temperature imbibition which include; (1) alcohol dehydrogenase I and RAB21 might contribute in decreasing the effect of anoxia, resulting from water up-take during imbibition, (2) stress-related proteins such as LEA and GST24 probably played a pivotal role in confronting low temperature stress and (3) expression of some crucial enzymes (malate dehydrogenase and phosphoenolpyruvate carboxylase) involved in TCA cycle was enhanced, which might be beneficial for seeds, in stress conditions during germination.

Genome-Wide Identification and Analysis of MAPK and MAPKK Gene Families in Brachypodium distachyon

MAPK cascades are universal signal transduction modules and play important roles in plant growth, development and in response to a variety of biotic and abiotic stresses. Although MAPKs and MAPKKs have been systematically investigated in several plant species including Arabidopsis, rice and poplar, no systematic analysis has been conducted in the emerging monocot model plant Brachypodium distachyon. In the present study, a total of 16 MAPK genes and 12 MAPKK genes were identified from B. distachyon. An analysis of the genomic evolution showed that both tandem and segment duplications contributed significantly to the expansion of MAPK and MAPKK families. Evolutionary relationships within subfamilies were supported by exon-intron organizations and the architectures of conserved protein motifs. Synteny analysis between B. distachyon and the other two plant species of rice and Arabidopsis showed that only one homolog of B. distachyon MAPKs was found in the corresponding syntenic blocks of Arabidopsis, while 13 homologs of B. distachyon MAPKs and MAPKKs were found in that of rice, which was consistent with the speciation process of the three species. In addition, several interactive protein pairs between the two families in B. distachyon were found through yeast two hybrid assay, whereas their orthologs of a pair in Arabidopsis and other plant species were not found to interact with each other. Finally, expression studies of closely related family members among B. distachyon, Arabidopsis and rice showed that even recently duplicated representatives may fulfill different functions and be involved in different signal pathways. Taken together, our data would provide a foundation for evolutionary and functional characterization of MAPK and MAPKK gene families in B. distachyon and other plant species to unravel their biological roles.

On the Mechanism of Plasma Inducing Cell Apoptosis

In recent years, atmospheric pressure nonequilibrium plasmas (APNPs) have been used in many biomedical applications such as bacteria, yeasts, fungi and algae inactivation, surface treatment for cell sheet engineering to controlled cell cultivation, promote blood coagulation and root canal of teeth sterilization. The APNPs can operate at room temperature and APNPs can form numerous short-lived but highly active chemical particles, such as reactive oxygen species, hydroxyl radical (OH), and other excited species. These active chemical particles are essential for various biological processes in cells and human body, reactive oxygen species are involved in initiation, progression and metastasis of cancers and nitric oxide (NO) can induce apoptosis, necrosis or protect cells from death, depending on the cell type and the amount, duration, and site of NO production. Therefore, it is important to study the impact of plasma on cancer cells.

Atmospheric Pressure Room Temperature Plasma Jets Facilitate Oxidative and Nitrative Stress and Lead to Endoplasmic Reticulum Stress Dependent Apoptosis in HepG2 Cells

Atmospheric pressure room temperature plasma jets (APRTP-Js) that can emit a mixture of different active species have recently found entry in various medical applications. Apoptosis is a key event in APRTP-Js-induced cellular toxicity, but the exact biological mechanisms underlying remain elusive. Here, we explored the role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in APRTP-Js-induced apoptosis using in vitro model of HepG2 cells. We found that APRTP-Js facilitated the accumulation of ROS and RNS in cells, which resulted in the compromised cellular antioxidant defense system, as evidenced by the inactivation of cellular antioxidants including glutathione (GSH), superoxide dismutase (SOD) and catalase. Nitrotyrosine and protein carbonyl content analysis indicated that APRTP-Js treatment caused nitrative and oxidative injury of cells. Meanwhile, intracellular calcium homeostasis was disturbed along with the alteration in the expressions of GRP78, CHOP and pro-caspase12. These effects accumulated and eventually culminated into the cellular dysfunction and endoplasmic reticulum stress (ER stress)-mediated apoptosis. The apoptosis could be markedly attenuated by N-acetylcysteine (NAC, a free radical scavenger), which confirmed the involvement of oxidative and nitrative stress in the process leading to HepG2 cell apoptosis by APRTP-Js treatment.

TaNAC29, a NAC transcription factor from wheat, enhances salt and drought tolerance in transgenic Arabidopsis

BackgroundNAC (NAM, ATAF, and CUC) transcription factors play important roles in plant biological processes, including phytohormone homeostasis, plant development, and in responses to various environmental stresses.MethodsTaNAC29 was introduced into Arabidopsis using the Agrobacterium tumefaciens-mediated floral dipping method. TaNAC29-overexpression plants were subjected to salt and drought stresses for examining gene functions. To investigate tolerant mechanisms involved in the salt and drought responses, expression of related marker genes analyses were conducted, and related physiological indices were also measured. Expressions of genes were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR).ResultsA novel NAC transcription factor gene, designated TaNAC29, was isolated from bread wheat (Triticum aestivum). Sequence alignment suggested that TaNAC29 might be located on chromosome 2BS. TaNAC29 was localized to the nucleus in wheat protoplasts, and proved to have transcriptional activation activities in yeast. TaNAC29 was expressed at a higher level in the leaves, and expression levels were much higher in senescent leaves, indicating that TaNAC29 might be involved in the senescence process. TaNAC29 transcripts were increased following treatments with salt, PEG6000, H2O2, and abscisic acid (ABA). To examine TaNAC29 function, transgenic Arabidopsis plants overexpressing TaNAC29 were generated. Germination and root length assays of transgenic plants demonstrated that TaNAC29 overexpression plants had enhanced tolerances to high salinity and dehydration, and exhibited an ABA-hypersensitive response. When grown in the greenhouse, TaNAC29-overexpression plants showed the same tolerance response to salt and drought stresses at both the vegetative and reproductive period, and had delayed bolting and flowering in the reproductive period. Moreover, TaNAC29 overexpression plants accumulated lesser malondialdehyde (MDA), H2O2, while had higher superoxide dismutase (SOD) and catalase (CAT) activities under high salinity and/or dehydration stress.ConclusionsOur results demonstrate that TaNAC29 plays important roles in the senescence process and response to salt and drought stresses. ABA signal pathway and antioxidant enzyme systems are involved in TaNAC29-mediated stress tolerance mechanisms.