Understanding the Molecular Mechanism of Arsenic Tolerance and Accumulation in Pteris vittata

Arsenic is a toxic metalloid that is naturally occurring and widely distributed. The consumption of arsenic-contaminated food and water is threatening the health of millions of people in the world. The fern Pteris vittata is unusual in its ability to tolerate and hyperaccumulate exceptionally high concentrations of arsenic in its fronds. How P. vittata tolerates and accumulates arsenic is fundamentally different from that in angiosperms; however, the molecular mechanism underlying this trait in P. vittata is not well-defined. Because the haploid phase of the fern (the gametophyte) is morphologically simple, easy to grow and also tolerates and hyperaccumulates arsenic, the research described here focuses on the P. vittata gametophyte as a system for study. Combining differential gene expression (DEG) analysis using RNA-seq and knocking down gene expression by RNA interference, three genes (PvGAPC1, PvOCT4 and PvGSTF1) were identified that are necessary for arsenic tolerance in P. vittata gametophytes. Subcellular localization of the proteins encoded by these genes using live-cell imaging and biochemical enzyme kinetic analysis showed that the proteins encoded by all three genes localized in speckled patterns within the cell, and may be involved in sequestering arsenate as it moves from the cytoplasm into the vacuole, where arsenic is sequestered. cDNA libraries generated from P. vittata sporophytes were also sequenced and used to generate a comprehensive, high quality transcriptome of genes expressed in both gametophytes and sporophytes. By identifying genes that are expressed only in either the gametophyte or sporophyte generation, lignin biosynthesis and stomata differentiation genes that are only expressed in the sporophyte generation were discovered. This discovery may explain the lack of lignified cell types and stomata in the gametophyte. New phosphate transporter genes were also discovered, demonstrating that the transcriptome generated from this studyis an excellent resource for the identification of new genes potentially involved in arsenic tolerance and accumulation in P. vittata. Lastly, 3,292 genes that were differentially expressed (DEGs) in shoots or roots from P. vittata sporophytes treated with arsenic were identified from the transcriptome. Gene ontology analysis of these DEGs revealed clusters of genes that are involved in response to phosphate deficiency and transporter genes that are potentially important for arsenic trafficking in the roots and shoots of P. vittata sporophytes