The role of melatonin on caspase-3-like activity and expression of the genes involved in programmed cell death (PCD) induced by in vitro salt stress in alfalfa (Medicago sativa L.) roots.

BACKGROUND: Alfalfa (Medicago sativa L.) is the most cultivated forage plant as a model in legumes. Salinity stress due to Na+ toxicity causes severe, oxidative stress as a main reason for program cell death (PCD) in plants. Melatonin application can increase plant productivity in response to diverse stressors via modulating plant antioxidant mechanisms and PCD inhibition in plants. RESULTS: Alfalfa roots were subjected to different concentrations of in vitro salinity supplemented with melatonin (0.1, 10 and 15 µM) for ten days. Application of melatonin under salinity stress reduced ROS, H2O2 and [Formula: see text] content and showed a dramatic impact on TTC reduction and augmented cell viability. Interestingly, melatonin inhibited caspase 3-like protease activity and could decrease DNA fragmentation induced by salinity while increased expression of anti-apoptotic genes BI-1, UCP1-UCP2 involved in PCD pathway. In contrast, in 300 mM salinity, γVPE gene as a proapoptotic of PCD down-regulated significantly. CONCLUSIONS: For the first time, present data showed that, melatonin plays a major function in preventing PCD in alfalfa root meristem cells. We attempted to offer a mechanism for the function of melatonin as an anti-apoptotic agent by demonstrating significant actions of melatonin on mitochondria proteins, such as UCPs, in a manner similar to animal cells.

Rosmarinic acid inhibits programmed cell death in Solanum tuberosum L. calli under high salinity.

Oxidative stress induced by salinity is a prime cause of cell death when Na+ toxicity becomes unbearable. We explored the effect of rosmarinic acid (RA) on the Solanum tuberosum L. cv. Desiree calli against salt-induced programmed cell death (PCD). We showed that PCD events were triggered in calli under 250 mM NaCl by the loss of plasma membrane integrity as measured by the amount of malondialdehyde (MDA) in the cytoplasm, the degree of DNA degradation resulting from the cleavage of nuclear DNA into oligonucleosomal fragments in apoptotic cells, the presence of TUNEL-positive nuclei (90 ± 0.005%) damage in genomic DNA, and activation of caspase 3-like protease. Callus Formation Medium (CFM) supplemented with RA led to the suppression of salt-induced cell death and a dramatic decrease in the MDA level and frequency of TUNEL-positive nuclei under salinity to 4 ± and 7.3 ± % in the presence of 50 and 350 µM RA, respectively. The application of RA also resulted in an increase in GSH content and maintenance of a high GSH/GSSG ratio. Interestingly, these reductions in PCD were accompanied by inhibiting caspase 3-like protease activities due to RA under salinity. Molecular docking predicted high binding energies of RA for binding to subtilisin-like protease (StSCTc-3), which has caspase-3 like activity in Solanum tuberosum, near the active site. This finding supports the notion of a role for RA in PCD protection in plants, which is consistent with earlier reports in animal cells.

Sesquiterpene lactones from shoot culture of Artemisia aucheri with cytotoxicity against prostate and breast cancer cells.

Plant tissue culture is used to grow plant cells, tissues, or organs under sterile and determined conditions on culture media. It is alternative to traditional vegetative propagation, and is applied as an effective technology for the production of valuable secondary metabolites. The Artemisia aucheri (A. aucheri) was obtained from shoot culture grown on MS (Murashige and Skoog 1962) medium. Shade-dried aerial parts of in vitro grown A. aucheri (50 g) were extracted with dichloromethane-acetone (90:10). The extract was submitted for isolation to sephadex gel chromatography and preparative thin layer chromatography, which resulted in identification of one known eudesmanolide named artemin or 2,5-dihydroxy-12, 6-eudesmanolide-4(15)-en for the first time in this plant. In cell cytotoxicity test, artemin showed cytotoxic activity against DU-145,LNCaP prostate cancer, and MCF-7 breast cancer cells with IC50 values of 82.2 ± 5.6, 89.1 ± 6.3 and 111.5 ± 6.7 µM , respectively. Artemin was more active against prostate cancer cells with approximately same cytotoxicity against LNCaP androstane dependent cells and DU 145 which is androstane independent.

Physiological targets of salicylic acid on Artemisia aucheri BOISS as a medicinal and aromatic plant grown under in vitro drought stress.

BACKGROUND: Artemisia aucheri BOISS is a medicinal and aromatic plant, which is endemic to mountainous areas of Iran and surroundings. In this study, we investigated the alleviating effects of salicylic acid (SA) pretreatment (0.01 and 0.1 mM) on A. aucheri under in vitro drought stress induced by 2 and 4% polyethylene glycol (PEG/6000). RESULTS: Plants exposed to PEG stress showed higher levels of H2O2, MDA and electrolyte leakage compared with control. While SA pretreatment decreased these parameters under PEG stress significantly. The activity of CAT, POD, APX, SOD and GR positively changed with PEG and more induction in activity of antioxidant enzymes was observed in SA-pretreated plants under PEG stress. Furthermore, ASA, GSH and their redox ratios (ASC/DHA and GSH/GSSG) enhanced with SA pretreatments. Analysis of our data revealed that MDA, DHA and H2O2 were the best targets for SA under in vitro PEG treatment for A. aucheri plants. CONCLUSIONS: Salicylic acid as a signal molecule mitigated adverse effects of PEG-simulated drought stress on A. aucheri under in vitro condition by improving the activity of antioxidant enzymes. In addition, protective role of SA was also related to promotion of ascorbate-glutathione cycle.

Antioxidant response of Stevia rebaudiana B. to polyethylene glycol and paclobutrazol treatments under in vitro culture.

This investigation was carried out with the aim of determining the effect of paclobutrazol (PBZ) (0 and 2 mg l(-1)) and polyethylene glycol (PEG) (0, 2, 4 and 6 % w/v of PEG 6000) treatments on antioxidant system of Stevia rebaudiana Bertoni under in vitro condition. Analysis of data showed that PEG treatment significantly increased hydrogen peroxide (H2O2) and phenolic contents, while PBZ treatment limited the effect of PEG on them. Our data revealed that PEG treatment significantly increased total antioxidant capacity, catalase (CAT), ascorbate peroxidase (APX), polyphenol oxidase (PPO) and peroxidase (POD) activity, while it inversely decreased glutathione reductase (GR) activity. The superoxide dismutase (SOD) activity was not affected by PEG treatment. PBZ treatment induced significantly higher levels of CAT and GR activity and lower levels of SOD activity in PEG-treated plants. PBZ in combination with PEG resulted in no significant difference on APX activity with PEG treatment alone. PBZ treatment prevented the effect of PEG on the PPO activity. PEG (with or without PBZ) treatment increased the ascorbate pool, whereas total glutathione level was not affected by PEG. Our finding indicated that PBZ reduced the negative effect of PEG treatment by quenching H2O2 accumulation and increasing the CAT activity. Collectively, the antioxidant capacity of S. rebaudiana in PEG treatment condition was associated with active enzymatic and non-enzymatic defence systems which partly could be improved by the PBZ treatment. In addition, a higher accumulation of phenolic compounds leads to a more potent reactive oxygen species scavenging activity in S. rebaudiana.

Proteome analysis of soybean leaves, hypocotyls and roots under salt stress.

BACKGROUND: Salinity is one of the most widespread agricultural problems in arid and semi-arid regions that makes fields unproductive, and soil salinization is a serious problem in the entire world. To determine the effects of salt stress on soybean seedlings, a proteomic technique was used. RESULTS: Soybean plants were exposed to 0, 20, 40, or 80 mM NaCl for one week. The effect of treatment at 20 mM NaCl on plant growth was not severe, at 80 mM NaCl was lethal, and at 40 mM NaCl was significant but not lethal. Based on these results, proteins were extracted from the leaves, hypocotyls and roots of soybean treated with 40 mM NaCl. Nineteen, 22 and 14 proteins out of 340, 330 and 235 proteins in the leaves, hypocotyls and roots, respectively, were up- and down-regulated by NaCl treatment. In leaves, hypocotyls and roots, metabolism related proteins were mainly down-regulated with NaCl treatment. Glyceraldehyde-3-phosphate dehydrogenase was down-regulated in the leaf/hypocotyls, and fructokinase 2 was down-regulated in the hypocotyls/root with NaCl treatment. Stem 31 kDa glycoprotein precursor was up-regulated in all three organs with NaCl treatment. Glyceraldehyde-3-phosphate dehydrogenase was specifically down-regulated at the RNA and protein levels by salt stress. CONCLUSION: These results suggest that metabolism related proteins play a role in each organ in the adaptation to saline conditions.

Proteome analysis of tobacco leaves under salt stress.

The mechanisms responsible for the effects of salt stress on tobacco plants were examined by means of proteomic analysis. Tobacco plants were exposed to 0, 150, 250, 300, or 400 mM NaCl. At 150 mM NaCl or above, the plants showed a reduction in fresh weight and an increase in proline levels. Proteins extracted from the leaves of tobacco plants exposed to 150 mM NaCl were separated by 2-DE. Of 205 protein spots that were detected reproducibly in each gel, 18 were differentially expressed under NaCl treatment. Up-regulated proteins belonged to the photosynthesis category, whereas down-regulated proteins correspond to defense-related functions. Dose- and time-dependent studies showed that a stromal 70-kDa heat shock-related protein was markedly down-regulated by NaCl. Thus, down-regulation of the stromal 70-kDa heat shock protein in response to salt stress is likely the cause of failure to protect cells against salt stress of tobacco plants.

Proteome analysis of potato under salt stress.

Because salt stress is a major abiotic source of stress on potato crops, the molecular mechanism of the response of potato plants to salt stress was examined. On exposure to salt, the salt-sensitive cultivar Concord showed a greater reduction in shoot and root length than did the salt-tolerant cultivar Kennebec. For both cultivars, the reduction in the length of shoots was more severe than that of the roots. Salt exposure increased the content of free proline and total soluble sugars in shoots of Kennebec; these remained unchanged in Concord. Proteins extracted from shoots of both cultivars exposed to 90 mM NaCl were separated by two-dimensional polyacrylamide gel electrophoresis: 322 and 305 proteins were detected in shoots of Kennebec and Concord, respectively. Of these, 47 proteins were differentially expressed under NaCl treatment in shoot of both cultivars. Among the differentially expressed proteins, photosynthesis- and protein-synthesis-related proteins were drastically down-regulated, whereas osmotine-like proteins, TSI-1 protein, heat-shock proteins, protein inhibitors, calreticulin, and five novel proteins were markedly up-regulated. These results suggest that up-regulation of defense-associated proteins may confer relative salt tolerance to potato plants.

Plant regeneration from cell suspension culture of potato (Solanum tuberosum L.).

In the present study, for callus production leaf and stem segments of potato cultivar White Desiree were cultured on MS medium supplemented with 2,4-D, NAA and Kinetin (callus production medium). Calli then were transferred in the same liquid medium for cell suspension production. In the next step cell suspensions were transferred back to the callus production medium. Finally, calli derived from cell suspension were cultured on 6 different shoot initiation media (S1-S6). However, on S6 medium with combination of GA3 and BAP more than 80% of the calli produced shoot buds and shoots. Fully grown shoots then were rooted and produced whole plants. Chromosome and morphological analysis showed no somaclonal variation among regenerated plants.