Hydrogen Peroxide and Nitric Oxide Crosstalk Mediates Brassinosteroids Induced Cold Stress Tolerance in Medicago truncatula.

Brassinosteroids (BRs) play pivotal roles in modulating plant growth, development, and stress responses. In this study, a Medicago truncatula plant pretreated with brassinolide (BL, the most active BR), enhanced cold stress tolerance by regulating the expression of several cold-related genes and antioxidant enzymes activities. Previous studies reported that hydrogen peroxide (H2O2) and nitric oxide (NO) are involved during environmental stress conditions. However, how these two signaling molecules interact with each other in BRs-induced abiotic stress tolerance remain largely unclear. BL-pretreatment induced, while brassinazole (BRZ, a specific inhibitor of BRs biosynthesis) reduced H2O2 and NO production. Further, application of dimethylthiourea (DMTU, a H2O2 and OH- scavenger) blocked BRs-induced NO production, but BRs-induced H2O2 generation was not sensitive to 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO, a scavenger of NO). Moreover, pretreatment with DMTU and PTIO decreased BL-induced mitochondrial alternative oxidase (AOX) and the photosystem capacity. However, pretreatment with PTIO was found to be more effective than DMTU in reducing BRs-induced increases in Valt, Vt, and MtAOX1 gene expression. Similarly, BRs-induced photosystem II efficiency was found in NO dependent manner than H2O2. Finally, we conclude that H2O2 was involved in NO generation, whereas NO was found to be crucial in BRs-induced AOX capacity, which further contributed to the protection of the photosystem under cold stress conditions in Medicago truncatula.

Nitric oxide as a signaling molecule in brassinosteroid-mediated virus resistance to Cucumber mosaic virus in Arabidopsis thaliana.

Brassinosteroids (BRs) are growth-promoting plant hormones that play a crucial role in biotic stress responses. Here, we found that BR treatment increased nitric oxide (NO) accumulation, and a significant reduction of virus accumulation in Arabidopsis thaliana. However, the plants pre-treated with NO scavenger [2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-1-oxyl-3-oxide (PTIO)] or nitrate reductase (NR) inhibitor (tungstate) hardly had any NO generation and appeared to have the highest viral replication and suffer more damages. Furthermore, the antioxidant system and photosystem parameters were up-regulated in brassinolide (BL)-treated plants but down regulated in PTIO- or tungstate-treated plants, suggesting NO may be involved in BRs-induced virus resistance in Arabidopsis. Further evidence showed that NIA1 pathway was responsible for BR-induced NO accumulation in Arabidopsis. These results indicated that NO participated in the BRs-induced systemic resistance in Arabidopsis. As BL treatment could not increase NO levels in nia1 plants in comparison to nia2 plants. And nia1 mutant exhibited decreased virus resistance relative to Col-0 or nia2 plants after BL treatment. Taken together, our study addressed that NIA1-mediated NO biosynthesis is involved in BRs-mediated virus resistance in A. thaliana.

Orchestration of hydrogen peroxide and nitric oxide in brassinosteroid-mediated systemic virus resistance in Nicotiana benthamiana.

Brassinosteroids (BRs) play essential roles in modulating plant growth, development and stress responses. Here, involvement of BRs in plant systemic resistance to virus was studied. Treatment of local leaves in Nicotiana benthamiana with BRs induced virus resistance in upper untreated leaves, accompanied by accumulations of H2O2 and NO. Scavenging of H2O2 or NO in upper leaves blocked BR-induced systemic virus resistance. BR-induced systemic H2O2 accumulation was blocked by local pharmacological inhibition of NADPH oxidase or silencing of respiratory burst oxidase homolog gene NbRBOHB, but not by systemic NADPH oxidase inhibition or NbRBOHA silencing. Silencing of the nitrite-dependent nitrate reductase gene NbNR or systemic pharmacological inhibition of NR compromised BR-triggered systemic NO accumulation, while local inhibition of NR, silencing of NbNOA1 and inhibition of NOS had little effect. Moreover, we provide evidence that BR-activated H2O2 is required for NO synthesis. Pharmacological scavenging or genetic inhibiting of H2O2 generation blocked BR-induced systemic NO production, but BR-induced H2O2 production was not sensitive to NO scavengers or silencing of NbNR. Systemically applied sodium nitroprusside rescued BR-induced systemic virus defense in NbRBOHB-silenced plants, but H2O2 did not reverse the effect of NbNR silencing on BR-induced systemic virus resistance. Finally, we demonstrate that the receptor kinase BRI1(BR insensitive 1) is an upstream component in BR-mediated systemic defense signaling, as silencing of NbBRI1 compromised the BR-induced H2O2 and NO production associated with systemic virus resistance. Together, our pharmacological and genetic data suggest the existence of a signaling pathway leading to BR-mediated systemic virus resistance that involves local Respiratory Burst Oxidase Homolog B (RBOHB)-dependent H2O2 production and subsequent systemic NR-dependent NO generation.

Role of Transcription Factor HAT1 in Modulating Arabidopsis thaliana Response to Cucumber mosaic virus.

Arabidopsis thaliana homeodomain-leucine zipper protein 1 (HAT1) belongs to the homeodomain-leucine zipper (HD-Zip) family class II that plays important roles in plant growth and development as a transcription factor. To elucidate further the role of HD-Zip II transcription factors in plant defense, the A. thaliana hat1, hat1hat3 and hat1hat2hat3 mutants and HAT1 overexpression plants (HAT1OX) were challenged with Cucumber mosaic virus (CMV). HAT1OX displayed more susceptibility, while loss-of-function mutants of HAT1 exhibited less susceptibility to CMV infection. HAT1 and its close homologs HAT2 and HAT3 function redundantly, as the triple mutant hat1hat2hat3 displayed increased virus resistance compared with the hat1 and hat1hat3 mutants. Furthermore, the induction of the antioxidant system (the activities and expression of enzymatic antioxidants) and the expression of defense-associated genes were down-regulated in HAT1OX but up-regulated in hat1hat2hat3 when compared with Col-0 after CMV infection. Further evidence showed that the involvement of HAT1 in the anti-CMV defense response might be dependent on salicylic acid (SA) but not jasmonic acid (JA). The SA level or expression of SA synthesis-related genes was decreased in HAT1OX but increased in hat1hat2hat3 compared with Col-0 after CMV infection, but there were little difference in JA level or JA synthesis-related gene expression among HAT1OX or defective plants. In addition, HAT1 expression is dependent on SA accumulation. Taken together, our study indicated that HAT1 negatively regulates plant defense responses to CMV.

GOLDEN2-LIKE transcription factors coordinate the tolerance to Cucumber mosaic virus in Arabidopsis.

Arabidopsis thaliana GOLDEN2-LIKE (GLKs) transcription factors play important roles in regulation of photosynthesis-associated nuclear genes, as well as participate in chloroplast development. However, the involvement of GLKs in plants resistance to virus remains largely unknown. Here, the relationship between GLKs and Cucumber mosaic virus (CMV) stress response was investigated. Our results showed that the Arabidopsis glk1glk2 double-mutant was more susceptible to CMV infection and suffered more serious damages (such as higher oxidative damages, more compromised in PSII photochemistry and more reactive oxygen species accumulation) when compared with the wild-type plants. Interestingly, there was little difference between single mutant (glk1 or glk2) and wild-type plants in response to CMV infection, suggesting GLK1 and GLK2 might function redundant in virus resistance in Arabidopsis. Furthermore, the induction of antioxidant system and defense-associated genes expression in the double mutant were inhibited when compared with single mutant or wild-type plants after CMV infection. Further evidences showed that salicylic acid (SA) and jasmonic acid (JA) might be involved in GLKs-mediated virus resistance, as SA or JA level and synthesis-related genes transcription were impaired in glk1glk2 mutant. Taken together, our results indicated that GLKs played a positively role in virus resistance in Arabidopsis.

[Induction on callus culture and regeneration of Orostachyis fimbriatae].

OBJECTIVE: To explore the effects of different hormonal combinations on induction, proliferation and differentiation of Orostachyis fimbriatae callus culture. METHODS: Aseptic seedling leaves were used as explants,the different concentrations of IAA,NAA, 6-BA and KT on induction proliferation of callus were optimized by orthogonal test to explore the optimum medium for differentiation of callus by tissue culture techniques. RESULTS: The best medium for induction was MS + IAA 1.0 mg/L + NAA 0.5 mg/L + KT 1.0 mg/L, and the best hormonal combination for proliferation was MS + IAA 0.5 mg/L + 6-BA 0.5 mg/I. + KT 1.0 mg/L. The best medium for differentiation was MS + IAA 0.1 mg/L + KT 2.0 mg/L, and 1/2MS + IAA 0.2 mg/L was the optimum medium for rooting culture. CONCLUSION: The system of regeneration of Orostachyis fimbriatae is establishd by tissue culture techniques in this study.

[Karyotype analysis of three Lonicera species growth in Sichuan].

OBJECTIVE: To provide the genetic reference for development and application of Lonicera species growth in Sichuan. METHODS: Shoot apices cells from Lonicera japonica, Lonicera japonica var. chinensis and Lonicera similis (cultivars) were used to make the chromosomal preparation. Their karyotypes were analyzed and the relevant parameters of chromosomes were measured by improved chromosome preparation technique. RESULTS: The chromosome numbers of three Lonicera species were 2n = 2X = 18; their chromosome length was 30.747, 33.231 and 36.948 microm; Their karyotype formula was 2n = 2X = 18 = 2m + 7sm, 2n = 2X = 18 = 8m + 8sm + 2st and 2n = 2X = 18 = 8sm + 10m; Their As. k was 64.013%, 64.380% and 61.949%; And their karyotypes belonged to 2B, 2B and 2A type, respectively. CONCLUSION: These three Lonicera species can be of the germplasm resources for widely cultivating since they have high degree genetic evolution.

[Callus induction and plant regeneration of Sambucus chinensis].

OBJECTIVE: To optimize the medium for callus and multiple shoot induction and cultural plantlets rooting of Sambucus chinensis, and to provide a basis for the development of the seedling of mass production and germplasm conservation. METHODS: The plantlet leaves, petioles, stems and shoot tips were used as explants for the study of influences of different explants and different hormones on callus and multiple shoot induction, and cultural plantlets rooting. RESULTS: The optimal medium for callus induction of leaves was MS +2, 4-D 1.5 mg/L; and optimum mediums for petiole were MS + NAA 0.2 mg/L + KT 1.0 mg/L and MS +2, 4-D 0. 2 mg/L + KT 1.0 mg/L, MS + NAA 0.2 mg/L + KT 3.0 mg/L + GA, 2.0 mg/L medium was best for multiple shoot induction. And the best medium for tissue culture rooting was 1/2 MS + NAA 0. 3 mg/L. On the optimized medium, the roots were well-developed and the plants were healthier. CONCLUSION: Both the leaves and petioles can be well induced to callus on suitable medium, the stems and shoot tips are the best materials for multiple shoot induction and plant regeneration.

ß-Elemene radiosensitizes lung cancer A549 cells by enhancing DNA damage and inhibiting DNA repair.

ß-Elemene is a broad-spectrum antitumor agent. In China, several studies have indicated that ß-elemene enhances the cytotoxic effect of radiation in vitro and in vivo. In this study, the alkaline comet assay and neutral comet assay were used to measure both DNA strand breaks and DNA repair activity in A549 cells exposed to ß-elemene, irradiation or combination treatment. The overall object of the study was to test whether ß-elemene radiosensitization is associated with an enhancement in radiation-induced DNA damage or with a decrease in the repair of radiation-induced damage. The results revealed high levels of DNA single strand breaks (SSB) and double strand breaks (DSB) in A549 cells after exposure to the combination of ß-elemene and irradiation. To assess SSB and DSB repair, alkaline comet assay and neutral comet assay were performed at 24 h postirradiation. The damage induced by the combination of ß-elemene and irradiation was repaired at a slower rate. These findings suggest that ß-elemene can enhance A549 cell radiosensitivity through the enhancement of DNA damage and the suppression of DNA repair.

Proteomic changes in Arthrobotrys oligospora conidia in response to benzaldehyde-induced fungistatic stress.

Soil fungistasis limits the effect of fungal agents designed to control plant-parasitic nematodes. Benzaldehyde is a fungistatic factor produced by soil microorganisms that can suppress conidial germination, but the molecular mechanism of this suppression is unknown. In this study, three conidial proteomes of Arthrobotrys oligospora ATCC24927, a nematode-trapping fungus, were obtained, quantified, and compared. Under benzaldehyde fungistatic stress, conidial protein expression profile changed significantly. Screening with a twofold selection criterion revealed 164 up-regulated and 110 down-regulated proteins. 17 proteins related to protein translation were down-regulated and gene transcription analysis suggested that the repression of proteins translation might be one mechanism by which benzaldehyde inhibites conidial germination. Benzaldehyde also resulted in the down-regulation of respiratory chain proteins and mitochondrial processes, as well as the repression of conidial DNA synthesis. In addition, the conidia up-regulated several proteins that enable it to resist benzaldehyde-induced fungistatis, and this was confirmed by a functional assessment of two knockout mutants. This study reveals putative mechanisms by which benzaldehyde causes fungistasis as well as the proteomic response of conidia to benzaldehyde. SIGNIFICANCE: Soil fungistasis limits the effect of fungal agents designed to control plant-parasitic nematodes. Benzaldehyde is one of fungistatic factors produced by soil microorganisms that can suppress conidial germination. In this study, we found that conidial protein expression profile changed significantly under benzaldehyde fungistatic stress. This research revealed new mechanistic data that describe how benzaldehyde is responsible for fungiststis by inhibiting conidial germination. Moreover, we also found that conidia can resist benzaldehyde by up-regulating proteins such as benzaldehyde dehydrogenase and heat shock proteins. This study also showed that proteomics methods play important roles in addressing soil fungistatic mechanisms.

[Tissue culture and rapid propagation of Phytolacca americana].

OBJECTIVE: To explore the technique of rapid propagation for Phytolacca americana. METHODS: Aseptic seedling were used as explants. RESULTS: The best explants were the stems from strong aseptic seedling. The optimal culture media were MS + NAA (0.2 mg/L) +6-BA (1.0 mg/L) for primarily culture, MS + NAA (0.2 mg/L) +6-BA (2.0 mg/L) for the induction of clustered shoots 1/2MS with NAA 0.4 mg/L for rooting. CONCLUSION: The propagating coefficient of Phytolacca americana can be improved by inducing the clustered shoots from aseptic seedling.

Quantitative proteomics revealed partial fungistatic mechanism of ammonia against conidial germination of nematode-trapping fungus Arthrobotrys oligospora ATCC24927.

Ammonia is one of the fungistatic factors in soil that can suppress conidial germination, but the molecular mechanism underlying the suppression is unknown. In this study, the proteomes of fungistatic conidia, fresh conidia and germinated conidia of Arthrobotrys oligospora ATCC24927 were determined and quantified. The protein expression profile of fungistatic conidia was significantly different from those in the other two conditions. 281 proteins were down expressed in fungistatic conidia and characterized by GO annotation. Gene transcription analysis and inhibition of puromycin (a protein translation inhibitor) on conidial germination suggested that down expression of 33 protein translation related proteins might well result in repression of protein synthesis and inhibition of conidial germination. In addition, 16 down-expressed proteins were mapped to the Ras/mitogen-activated protein (Ras/MAP) regulatory networks which regulate conidial DNA synthesis. The conidial DNA synthesis was found to be definitely inhibited under by ammonia, and function studies of two Ras/MAP proteins by using knock-out strains provided partial evidence that Ras/MAP pathway regulate the conidial germination. These results suggested that down-expression of Ras/MAP related proteins might result in inhibition of DNA synthesis and finally result in inhibition conidial germination. This study revealed partial fungistatic mechanism of ammonia against conidial germination.

Medicago truncatula genotypes Jemalong A17 and R108 show contrasting variations under drought stress.

Drought is one of the most significant abiotic stresses that restrict crop productivity. Medicago truncatula is a model legume species with a wide genetic diversity. We compared the differential physiological and molecular changes of two genotypes of M. truncatula (Jemalong A17 and R108) in response to progressive drought stress and rewatering. The MtNCED and MtZEP activation and higher abscisic acid (ABA) content was observed in Jemalong A17 plants under normal conditions. Additionally, a greater increase in ABA content and expression of MtNCED and MtZEP in Jemalong A17 plants than that of R108 plants were observed under drought conditions. A more ABA-sensitive stomatal closure and a slower water loss was found in excised leaves of Jemalong A17 plants. Meanwhile, Jemalong A17 plants alleviated leaf wilting and maintained higher relative water content under drought conditions. Exposed to drought stress, Jemalong A17 plants exhibited milder oxidative damage which has less H2O2 and MDA accumulation, lower electrolyte leakage and higher chlorophyll content and PSII activity. Furthermore, Jemalong A17 plants enhanced expression of stress-upregulated genes under drought conditions. These results suggest that genotypes Jemalong A17 and R108 differed in their response and adaptation to drought stress. Given the relationship between ABA and these physiological responses, the MtNCED and MtZEP activation under normal conditions may play an important role in regulation of greater tolerance of Jemalong A17 plants to drought stress. The activation of MtNCED and MtZEP may lead to the increase of ABA content which may activate expression of drought-stress-regulated genes and cause a series of physiological resistant responses.

Lysosomal membrane permeabilization contributes to elemene emulsion-induced apoptosis in A549 cells.

Elemene is a broad-spectrum antitumor agent. In the present study, lysosomal membrane permeabilization (LMP) was detected after short elemene emulsion--exposure (12 h) that preceded a decrease of the mitochondrial membrane potential and DNA damage (24 h) in A549 cells. At later time points (36 h) elemene emulsion caused the appearance of A549 cells with apoptotic features, including apoptotic morphology, phosphatidylserine exposure, and caspase-3 activation. A significant increase in protein expression for cathepsin D was also observed utilizing Western blot analysis after exposure to elemene emulsion for 12 h. The present study showed that elemene emulsion induced the increased levels of reactive oxygen species (ROS) and depletion of glutathione (GSH) in A549 cells. Cells treated with pepstatin A, an inhibitor for cathepsin D, showed a significant inhibition in DNA damage, mitochondrial membrane permeabilization, caspase-3 activation, and phosphatidylserine exposure. These results demonstrated that apoptosis induced by elemene emulsion in A549 cells is mediated in part through LMP and lysosomal protease cathepsin D.

[Chronic administration of CRF makes depression like changes in rats].

For studying the role of CRF in the process of depression, chronic intra-cerebral ventricular administration of CRF in normal rats for 21 days were performed and compared with the depression model of chronic unpredictable miled stress (CUMS) in rats. The Open-field Test and Morris water Maze were employed to test the ability of locomotion and learning and memory. HPLC-UV, RT-PCR were employed to analyze the level of blood serum cortisol and the expression of CRF as well as its receptors (R1 and R2) of CUMS models. After chronic stress, the locomotion activity and spatial learning and memory ability decreased obviously, while the level of serum cortisol increased evidently, and the CRF and its receptor-1 mRNA levels were higher compared with those in normal rats. The rats with chronic administration of CRF also consistently decreased the weight gain, locomotion activity and the ability of spatial learning and memory as the CUMS model. This work demonstrates that CRF plays a very important role in the depression genesis and development, sustained elevation of CRF induced by stress may be the chief factor for depression.

[The expression of galanin and galanin receptor-2 in the brain of chronic stress model of depression].

The expression of galanin and galanin receptor-2 in hippocampus and dorsal raphe nucleus of depression model was studied. The chronic stress rat model was adopted as the modal of depression. Open-field test was used to observe the transformation of their behavior and HPLC-EC was employed to analysis the level of blood serum cortisol. The method of in situ hybridization was used for testing the expression of Galanin and galanin receptor-2 in hippocampus and dorsal raphe nucleus and the method of RT-PCR was used to further analysis of the expression. The results showed that the locomotion activity decreased extremely after chronic stress, but the level of serum cortisol increased evidently. The expression of Galanin and its receptor-2 in hippocampus and dorsal raphe nucleus increased obviously. The higher expression for galanin and galanin receptor-2 in some brain area suggested that galanin probably takes part in the modulation of the function of neurons during the stress process.

[Expression of aurora-A kinase in human lung cancer cell lines PG, A549, and NCI-H460].

BACKGROUND & OBJECTIVE: Recent researches found that Aurora-A overexpresses in various malignancies. This study was to detect the expression of Aurora-A in lung cancer cell lines PG (highly-metastatic giant cell lung cancer), A549 (lung adenocarcinoma), and NCI-H460 (large cell lung cancer) and explore its correlation to DNA content, provide a theoretical basis for screening tumor marker and molecular therapeutic target of lung cancer. METHODS: mRNA and protein levels of Aurora-A in PG, A549, and NCI-H460 cells were detected by reverse transcription-polymerase chain reaction(RT-PCR) and Western blot. Flow cytometry was used to analyze DNA contents in cell cycles of PG, A549, and NCI-H460 cells. RESULTS: mRNA level of Aurora-A was 1.14 in PG cells, 1.16 in A549 cells, and 0.84 in NCI-H460 cells, respectively; protein level of Aurora-A was 8.96 in PG cells, 21.13 in A549 cells, and 6.43 in NCI-H460 cells, respectively. The proportion of cells with tetraploid DNA was 19.88% in PG cells, 14.97% in A549 cells, and 10.6% in NCI-H460 cells, respectively (P<0.01); the proportion of cells with polyploid DNA was 2.66% in PG cells, 3.59% in A549 cells, and 2.30% in NCI-H460 cells, respectively. CONCLUSION: Aurora-A is overexpressed in the 3 lung cancer cell lines, but the mRNA levels are different.