| Aluminum (Al) is a major factor limiting plant growth in acidic soil. Al toxicity affects the absorption of water and elements by affecting root tip structure and function, which leads to low yield of crop production or even kills plants. It has been known that aluminum regulates the absorption of cation, however, little analysis has been done to investigate the effect of Al toxicity on overall elements absorption that plants need. Moreover, it is less accurate to measure the elements accumulated in the root tip as the total absorption amount, which is common in previous study, than that has been taken into the cells. This is because some elements are specific for apoplast. In this project, we explored Al toxicity response and regulation mechanism in rice bean, which induces the biosynthesis of citric acid in the root tip under Al treatment based on our previous work. Here, we are reporting a fast and accurate method to isolate cell sap and the effect of Al treatment on taking elements into root cells in rice bean. Moreover, we identified the putative membrane transporters for Fe and P and their behavior under Al toxicity. The results are as follows:a. Set up the isolation system for cell sap in rice bean. The optimum conditions to separate root-cell sap in rice bean were determined by studying different centrifugal forces, MDH as the marker enzyme of cytoplasm. The conditions are as follows:Root segments in centrifugal filter units and centrifuged at3000×g for10min to remove apoplastic solution. The roots were then frozen at-80℃overnight. The root-cell sap solution was obtained by thawing the samples at room temperature, and then centrifuging at20600×g for10min. The residual cell wall was washed with70%ethanol for three times.b. Al toxicity limits the absorption of elements differently. Under a serial Al treatment from0to50μM for24h, we found that the concentrations of Ca, Mg and K are decreasing in cells. When the Al concentration is above10μM, they tend to maintain a specific concentration with slowly decrease. It may be the minimum amount of Ca, Mg and K that plant need for metabolism. However, the concentrations of Fe, S and P are increasing with a higher concentration of Al treatment. It indicates that Fe may involve in the ROS response induced by Al toxicity and the biosynthesis of GSH results in the increased concentration of S. For the low-concentration elements or non-Al response ones, we did not study further, for example Zn, Mn, Cu and Co.c. Al regulates the expression of Fe and P membrane transporter proteins, which have a correlation with the aluminum concentration. We got the putative Fe and P membrane transporter protein genes in rice bean database, namely VuIRTl and VuPHT. qPCR results show that the expression level of VuPHT is positively correlated with Al concentration, while under the condition of high Al concentration (>10μM) treatment, the expression level of VuIRTl is increasing with the amount of Al. Both of them are consistent with the absorption of relative element concentrations. However, the expression level of VuIRT1is negatively correlated with the concentration of Al when it is lower than10μM. Besides, because of the whole-genome sequencing of rice bean is not complete yet, we tried to clone the membrane transporters based on the published sequences of their close homologs in other species and carried out functional and structural analysis on these gene fragments preliminarily... |
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