Hg-induced And Sa-regulated Oxidative Stressful Response And Isolation And Identification Of Micrornas In Medicago

Mercury has become one of the major sources of toxic heavy metal pollution in agricultural lands. Accumulation of mercury by plants may disrupt many cellular functions. To assess mercury toxicity, we performed the experiment focusing on the responses of alfalfa(Medicago sativa) to Hg+-induced oxidative stress. Alfalfa was treated with mercuric ions in a concentration-and time-dependent manner, and lipid peroxidation was studied biochemically as well as histochemically along with other physiological responses. Histochemical staining with Schiff’s reagent and Evans blue revealed the peroxidation of membrane lipids and loss of plasma membrane integrity in the Hg-treated root apex. The histochemical observations were supported by the quantitative determinations of thiobarbituric acid reactive substances (TB ARS) in the roots and leaves of alfalfa. Analysis of lipoxygenase (LOX) activity by non-denaturing polyacrylamide gel electrophoresis (PAGE) showed that there were two isoforms in the root and three isoforms in the leaves of alfalfa, but they showed different patterns under the Hg exposure. The external concentrations of Hg2+were positively correlated with the generation of O2-in leaves and H2O2in leaves and roots. Treatment with Hg2+increased the activities of NADH oxidase. To understand the biochemical responses under the Hg stress, activities of antioxidant enzymes, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) were assayed. The total activities of SOD, POD, APX, GR and CAT were generally enhanced in leaves after exposure to Hg ions. Activities SOD and POD increased in roots after Hg treatment. Activity of APX was stimulated at relatively high concentration of Hg2+(40μM). In contrast, GR activity was depressed at higher concentrations of Hg+(10-20μM). Analysis of antioxidant enzymes activities in alfalfa by non-denaturing PAGE revealed five and three SOD isoforms, seven and ten POD isoforms, eight and four APX isoforms in leaves and roots, respectively. But they showed different patterns under Hg stress. Only one band of CAT was visualized in leaves. We also measured several antioxidative metabolites such as ascorbate (ASC) and glutathione (GSH). Treatment of seedlings with10-40μM Hg2+decreased the reduced ASC and GSH amounts in the roots but increased in the leaves. These results indicate that the increased levels of O2and H2O2under the Hg stress were closely linked to the changes of the activities of antioxidant enzymes and the content of antioxidative metabolites. The data not only provide the important information for better understanding of the tolerant mechanisms, but as well can be used as a bio-indicator for soil contamination by Hg.Salicylic acid (SA) is a signal molecule mediating many biotic and environmental stress-induced physiological responses in plants. We investigated the role of SA in regulating Hg-induced oxidative stress in the roots of alfalfa. Plants pretreated with0.2mM SA for12h and subsequently exposed to10μM Hg2+for24h displayed attenuated toxicity to the roots. The SA-promoted root elongation was correlated with decreased lipid peroxidation in root cells. The ameliorating effect of SA was confirmed by the histochemical staining for the detection of loss of membrane integrity in Hg-treated roots. We show that treatment with0.2mM SA increased the activity of NADH oxidase, APX and POD in the roots exposed Hg. However, a slightly decreased SOD activity was observed in SA+Hg-treated roots when compared to those of Hg treatment alone. We also measured accumulation of ASC, GSH and proline in the roots of alfalfa and found that roots treated with S A in the presence of Hg accumulated more ASC, GSH and proline than those treated with Hg alone.microRNAs (miRNAs) are a novel class of short, endogenous non-coding small RNAs that regulate gene post-transcriptional expression in bot plants and animals. Thus far, a growing number of miRNAs have been isolated and characterized from a variety of plant species by bioinformatical and experimental methods and deposited in the major miRNAs database (http://www.sanger.ac.uk/). Here we present a bioinformatic approach for ESTs-and GSS-wide prediction of novel miRNAs as well as their targets in Medicago truncatula. We initiated the prediction by using previously known miRNA sequences from Arabidopsis, rice and other plant species to blast the databases of M. truncatula ESTs and GSS to search for the potential miRNAs. We used the enlarged data set to analyze parameters of the plant precursors including secondary structure, stem length and conservation of miRNAs. A total of38potential miRNAs were detected following a variety of filtering criteria. After removal of the12overlapping miRNAs deposited in miRNA Registry (Release9.1),26miRNAs representing15families are found to be new. Using the newly identified miRNA sequences, we were able to further blast the M. truncatula mRNA database and detected16potential targets of miRNAs in M. truncatula. It was found that many miRNA targeted genes were predicted to encode transcription factors that regulate cell growth and development, signaling, and metabolism. To validate the predicted miRNAs in M. truncatula, we performed a RT-PCR based assay of mature miRNA expression. Eight miRNAs were identified in roots, stems, leaves and flowers of M. truncatula. Expression of the eight miRNAs was also performed on samples from the leaves subjected to mercury.Although a number of plant miRNAs have been isolated by computational approach, many of other miRNAs, particularly those in response to abiotic stresses and non-conserved miRNAs appear to be found only by cloning approach. To identify novel abiotic stress-regulated miRNAs, we constructed a library of sRNAs from M. truncatula that were exposed to20μM HgCl2. Sequencing of the clones and subsequent analysis revealed10new miRNAs representing7families except one miRNA previously reported in M. truncatula miRNAs. Four of these were conserved in plant species, one of which was detected by bioinformatic approach above. Five new miRNAs of these from the library have no sequence conservation in any other plant species. The regulatory function of these cloned new miRNAs in M. truncatula under stress will be idetified further.