Cytokinins In Two Pteris Species Under Arsenic Stress:Speciation, Decomposition And Function

Cytokinins play an important role in the process of plant growth. Aiming to study the biosynthesis, decomposition and roles of cytokinins in two Pteris species under arsenic stress. Firstly, we established a method for cytokinin purification, separation and determination. Secondly, we studied some existing forms of endogenous cytokinins in the arsenic hyperaccumulator Pteris cretica var. nervosa and non-hyperaccumulator Pteris ensiformis under As(V) stress, and the correlation between CTKs and the concentrations of arsenic in plant leaves was analyzed. Additionally, the correlation between cytokinin oxidase (CKX) and the content of CTKs under arsenic stress (0-200 mg·kg-1) was also established. Finally, the time-course effect of arsenic accumulation with time extension was examined when different concentrations of kinetin (6-KT) (0,5,20,40 mg·L-1) was exogenously sprayed under the same As (V) (5 mg·L-1) stress, and the ultrastructure of cells in plant leaves was also observed. The main results were listed as follows:1. A high performance liquid chromatographic method was established for the simultaneous determination of conjugates and free cytokinins. The cytokinins were extracted by modified Bieleski’s solution containing a small amount of 2, 6-Di-tert-butyl-4-methylphenol. After filtering, the filtrate was purified by solid phase extraction through PVPP column, DEAE column and Sep-pak C18 column in sequence, and then the cytokinin components were separated by high performance liquid chromatography. The concrete conditions were as follows:Zobax SB-C18 column (5μm×4.6mm×250mm), UV detection detector at 269 nm, a flow rate of 0.6 mL min-1, constant temperature of 45 ℃ and gradient elution with methanol-acetonitrile-1% acetic acid. The cytokinins compounds of iP, iPA, iP7G, tZ, tZR and tZR7G could separated under this condition. The detectable limitation of this method varied from 1.72 to 5.59 ng, with average recovery of 92.9%-104.2%.2. Results from hydroponic experiment showed that the difference in plant growth of P. cretica var. nervosa and P. ensiformis was displayed under different concentrations of arsenic stress (0,2,5,10 mg·L-1). No significance difference in the above-ground fresh weight was observed in P. cretica var. nervosa under different concentrations of As stress, while a significant decrease (23%) of this fresh weight in P. ensiformis was found at 10 mg·L-1 As (V) treatment compared to the control. The endogenous contents of ZR7G, ZR, iP7G, iPA in P. cretica var. nervosa were significantly higher than the control group in 10 mg·L-1 As (V) treatment, while no significant difference was found in Z and iP. The endogenous ZR content in leaves of P. ensiformis was only 36.38% of that in control group in 10 mg·L-1 As (V) treatment, however, no significant difference in Z, iP, iPA, ZR7G and iP7G contents were shown compared to the control. In the 10 mg·L-1 As (V) stress, the contents of conjugated cytokinin in leaves of P. cretica var. nervosa and P. ensiformis were 2.48 and 1.7 times higher than the control group, respectively. Under the low concentration of arsenic stress, the contents of CTK in two plants increased. However, CTKs contents in P. ensiformis were decreased but no significant difference was noted in P. cretica var. nervosa under high concentration of arsenic stress.3. A negative correlation was found between the contents of total CTKs and CKX activity in P. cretica var. nervosa under arsenic stress, but this correlation was not shown in P. ensiformis. In addition, a negative correlation was observed between the content of Z and CKX activity in P. cretica var. nervosa, while a positive correlation was shown between the content of Z and the CKX activity in P. ensiformis.4. The growth of arsenic hyperaccumulator P. cretica var. nervosa and non-hyperaccumulator P. Ensiformis was promoted by exogenously-sprayed 6-KT. The plant biomass and vigor index increased. The translocation of arsenic from root to shoot in P. cretica var. nervosa was significantly increased by 5 mg·L-16-KT, and the chlorophyll a/b values were also increased. Similarly, arsenic uptake of P. ensiformis was increased by 6-KT, but the chlorophyll a/b value were decreased with 6-KT addition. This may result from a lack of efficiency detoxification mechanism in P. ensiformis when high concentration of arsenic entered, and plant photosynthesis is decreased. The storage of starch grains in chloroplast of P. cretica var. nervosa was increased by exogenously-applied 6-KT, so the cell activity was also enhanced.6-KT could repair the ultrastructure of P. ensiformis to some extent. The arsenic accumulation of P. ensiformis was higher in 40 mg·L-1 treatment than that in 5 mg·L-1 treatment, and the cells were complete although some damage was found. Therefore, exogenously-sprayed 6-KT could promote arsenic extraction and growth of plants.