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Investigation of the transcriptional regulation of the antioxidant network in bundle sheath cells of Arabidopsis using a novel immunocapture technique for bundle sheath specific polysomes

Final Activity Report Summary - ANTIOXARAB (Investigation of the transcriptional regulation of the antioxidant network in bundle sheath cells of Arabidopsis using a novel immunocapture technique)

The aim of this project was to investigate the regulation of genes encoding the antioxidant network, which is central to plants’ ability to meet environmental challenges. Many genes can be controlled by the so-called transcriptional factors (TFs) and the antioxidant network of the leaf must be controlled by such proteins, although we are currently aware of only a few. However, the leaf consists of different tissues and each tissue may contain different versions of the antioxidant network.

This project was about the development of a technique that might address this issue focussing on one specific cell type, namely the bundle sheath of the arabidopsis leaf. Based on previous data concerning the bundle sheath cell-specific stress responsive expression of a gene called APX2, it was postulated that the expression of the antioxidant defence gene network and its controlling TFs in bundle sheath cells of the leaf subject to different types of stress were constituted differently from those in cells of the leaf lamina. Therefore, it was postulated that a regulatory network model and, by implication, its description of cellular reactive oxygen species (ROS) metabolism, would have different properties to those derived from the entire leaf. This hypothesis was approached in an interdisciplinary way.

We firstly attempted to establish a novel technique based on epitope tagging of poly-ribosomes (polysomes) in a particular cell type only. This would allow us to immunopurify polysomes and extract ribonucleic acid (RNA) from that specific cell type. With this aim, we used APX2 expression sequences, called promoters, to obtain arabidopsis transgenic plants that specifically expressed a ribosome protein, namely HF-RPL18, tagged with a recognition peptide, called FLAG, in bundle sheath and mesophyll cells respectively. We also fused HF-RPL18 to a constitutive promoter, CaMV35S, to be used as a control. Antibodies against FLAG were used to detect the transgenic protein HF-RPL18 and to show that it was at a too low level in most transgenic lines, excluding CaMV35S:HF-RPL18, in which it was very abundant. Then, we decided to concentrate on the APX2:HF-RPL18 transgenic plants, since expression was confined to bundle sheath cells, and on CaMV35S:HF-RPL18. CaMV35S:HF-RPL18 lines were used to demonstrate that the method which was applied to specifically inmunopurify the FLAG tagged ribosomes and recover the RNA associated with them worked. Subsequently, immunocapture of polysomes was attempted from bundle sheath cells specifically using the APX2:HF-RPL18 transgenic lines. Using heat stressed material, since heat stress was shown to induce the expression of the fusion gene by 20-fold, we were able to prepare a very low amount of RNA, even though the method was not reproducible enough, probably due to the impossibility of maintaining the expression of the APX2:HF-RPL18 for a long period. Therefore, very little of the tagged HPL18 found its way into polysomes.

A second approach was more recently initiated, based on crossing the highest heat-inducible APX2:HF-RPL18 transgenic line with CaMV35S:HSF3, a line over-expressing the heat TF HSF3, which showed basal expression of APX2 circa 200 times higher than wild type plants. The strategy was that the hybrid double transgenic line would increase the yield of bundle sheath cell-specific polysomes enough to allow for their inmunocapture and purification. It was anticipated that that the level of expression of the chimeric protein HF-RPL18 would be higher than that in the line APX2:HF-RPL18 and that it could be used to inmunopurify specific bundle sheath polysomes. The intention was then to examine the expression of antioxidant genes and TF genes known to influence their function and to use this in order to provide feedback to developing computational models that were used in the field, including the host laboratory.