Characterizations Of Phytase And Response Of Antioxidative System In Pteris Vittata L. Under Arsenic Stressed Conditions

Arsenic is a ubiquitous, priority-controled, and carcinogenic element that is existed in the environment. Chinese brake fern (Pteris vittata L.), a newly found arsenic hyperaccumulator, possessed the capability of removing As from contaminated sites and the utilization of Chinese brake fern to remedy the As-contaminated environment has been a cost-efficient and friendly for environment technology. To better remedy the As-contaminated environment using heavy metal hyperaccumulators, it is important to understand the mechanisms of accumulation, however, to date, the mechanisms of arsenic accumulation in Chinese brake fern are scarce. In this study, we compared the different abilities of four plant species to accumulate and transport arsenic, the traits of phytase and changes in the activities of isoenzymes as well as some antioxidative enzymes. The plants used included two different genotypes of Pteris vittata L. (Chinese brake fern) inhabiting the state of Florida, USA (FU-PVL) and the city of Yi-Chang, HuBei province, China (YCC-PVL), respectively, and two common plant species of Pteris nervosa (PN) and Zea mays L. (ZM). The results were as follows:1. Chinese brake fern, an arsenic hyperaccumulator, possessed a powerful ability to accumulate As. In this study, after being treated with arsenic for 10 days, the highest As concentration record (786.044mg/kg) occurred in YCC-PVL, followed by FU-PVL (678.408 mg/kg), PN (114.715mg/kg) and then ZM (89.116mg/kg). The concentrations of As in the fronds of FU-PVL and YCC-PVL were 5.91 to 8.82 folds higher than that in the fronds of PN and ZM. Consequently, it could be concluded that YCC-PVL and FU-PVL were both considered to the same genotype of As hyperaccumulator.2. Arsenic hyperaccumulating P. vittata possessed greater As-resisited capabilities than non hyperaccumulating PN ZM. In terms of superdismutase (SOD), the addition of As enhanced the frond SOD activities in all selective plants in this study. Remarkably higher activity enhancement (41.61%-average of SOD in two genotypes of Chinese brake fern, as compared with the control) was observed in the fronds of both genotypes of Chinese brake fern, while the lowest activity value of frond SOD (11.87% enhancement) occurred ZM. In addition, the activities of catalase (CAT), ascorbate-peroxidase (AsA-POD), peroxidase (POD) in the fronds of Chinese brake fern were observed to possess 100.18%, 133.33% and 224.51% enhancements compared with theirs controls, respectively. The content of MDA in plants have been considered to be an reliable indicator of plant lipid oxidation extent. The fronds of ZM had the highest increase of MDA content, reaching up to 126.31%, followed by Chinese brake fern (38.06%, mean of MDA contents in two genotypes of Chinese brake fern). MDA in PN was not significantly affected by the addition of As. In respect of non-enzymetic antioxidative substance GSH, 75.88% increase was found in the fronds of Chinese brake fern and however, no significant changes in GSH content were observed in PN and ZM.3. The phytases in the plants of this study were all belonged to acidic-phytase, due to the favorable pH value and temperature being 5.0 and 40℃, respectively. It was worthy to note that the activity of phytase in the fronds of Chinese brake fern still remained 100 % activity upon the exposed temperatures of 40℃-70℃and 73.22% activity upon 80℃, as compared with its control, which suggested that the stability of phytase in the fronds of Chinese brake fern was higher than that of other two species employed in this study. To our knowledge, this is the first time to discover high stability phytase in plants. The optimum concentration of substrate sodium-phytase for Chinese brake fern was 1 mmol/L, whereas 2 mmol/L for PN and ZM, which indicated that Chinese brake fern have more affinity to the substrate than the other two plants in this study.4. The additions of heavy metals (metalloid) Pb, Cd, As(Ⅲ), As(Ⅴ) suppressed significantly the in vitro activities of Pteris vittata L. with the decreasing ratios ranging from 41.20% to 96.10%, in which, the inhibition effect by As( V) were the strongest and by As (Ⅲ) the weakest. The differences existed in the responses of phytase derived from different plants to different heavy metal (metalloid) stresses (Pb, Cd, As(Ⅲ)). In all selective plants, the activities of phytase in the fronds of ZM were most heavily inhibited by Pb, Cd and As(Ⅲ), with 90.58%, 90.65%, 92.19% decreases as compared with theirs controls, respectively, which was followed by PN with 70.28%, 65.27%, 54.99% decreases. The decreasing extents of phytase activities in the fronds of YCC-PVL and FU-PVL were 41.20% to 53.33%. 5. Unexpectedly, the spectrums of phytase isozymes were not obtained from the two genotypes of Chinese brake fern treated with arsenic, but they were achieved in the fronds of PN and ZM. The results indicated that no new spectrums of phytase isozymes were discovered in this study under arsenic exposure, but the intensities of spectrums of phytase isozymes were enhanced by As, which suggested that the addition of As induced more expressions of phytase genes in plants as compared with theirs controls, respectively.