Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
Botany & Plant Pathology
Non Technical Summary
Arsenic is a naturally occurring metalloid that is toxic to most organisms. It is a contaminant of soils and ground water in many regions of the world, including the US (reviewed in [11]. Arsenic exists in two main forms in the environment, as arsenate (As(V)) or arsenite (As(III)), depending on the redox potential of the surrounding environment [12]. Arsenate (AsO4-3), an analog of phosphate, is the predominant form of arsenic in aerobic soils and aquifers, although numerous other organic arsenic forms exist in the biosphere. In humans, chronic exposure to arsenic causes developmental defects, cancers, cardiovascular disease and mortality in young adults (reviewed in [13]). In addition to being an environmental problem, for which Pteris vittata can be used to reduce arsenic levels in soils by 6-13% [14-16], arsenic is used in the treatment of leukemia and parasitic diseases. Arsenic trioxide, for example, has recently become a drug of choice in treating acute leukemia (reviewed in [17]). Given its unique and remarkable ability to tolerate and accumulate very high levels of arsenic in its tissues, P. vittata is a exceptional system for understanding natural mechanisms responsible for arsenic metabolism, toxicity, and resistance in a multicellular organism, which is the long-term goal of our research. Using this knowledge to reduce arsenic contamination in our food chain is a long term application of the proposed research?.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
We have successfully identified and characterized two genes that play important roles in arsenic tolerance in the fern P. vittata. One gene (PvACR2) encodes an arsenic-specific arsenate reductase, and another gene (PvACR3) encodes a vacuolar arsenic transporter. More recently, we have identified a small number of genes whose expression is up-regulated after treatment with arsenate. The three P. vittata genes we will focus on during the duration of the project include: glyceraldehyde-3-phosphate dehydrogenase (PvGAPDH), glutathione S-transferase (PvGST) and organic cation/carnitine transporter (PvOCT). All three are up-regulated at least 50 fold by arsenate in P. vittata. The goal of the project is to understand their role in arsenic tolerance and accumulation in P. vittata.The first objective tests the hypotheses that PvGAPDH, PvGST and PvOCT are necessary for arsenic tolerance and accumulation in P. vittata. To test this, each gene will be knocked-down in P. vittata gametophyte by RNAi. Arsenic accumulation in the same gametophytes will be assessed using scanning electron microscopy combined with energy dispersive X-rayspectroscopy (SEM-EDS). To test whether GAPDH, GST and OCT are sufficient for arsenic tolerance and accumulation, each gene will be ectopically expressed in Arabidopsis and the growth and arsenic levels in transgenic plants measured. The second objective addresses the biochemical functions of the proteins encoded by PvGAPDH and PvOCT using a variety of physiological and biochemical approaches.
Project Methods
Objective 1. Are GAPDH, GST and OCT necessary for arsenic tolerance and accumulation in P. vittata?An RNA-Seq approach was used to identify 20 genes that are up-regulated by arsenate treatment. Among them were GAPDH (up-regulated 370-fold), GST (up-regulated 234-fold) and OCT1 (up-regulated 50-fold). Included this list of up-regulated was PvACR3, known from previous studies to be up-regulated 20-fold. Their high abundance in arsenate treated gametophytes suggests that they play important roles in either arsenic tolerance or accumulation. To test the hypothesis that they are necessary for this trait, we will knock down the expression of each gene by RNAi using methods developed in the Banks lab [1]. Six-day old gametophytes will be biolistically bombarded with gold that has been coated with two plasmids: one a fluorescent marker for transformation (35S::DsRed) and the other a plastid containing a hairpin forming sequence specific to each of the targeting genes. Two days following bomabardment, red fluorescing gametophytes will be transferred to media lacking arsenate for two days, then transferred again to media with or without 5mM arsenate. The circumference of all transferred gametophytes will be measured seven days later. If a gene is necessary for arsenic tolerance, it will not grow (or will not grow as well) on media containing arsenate. The amount of arsenic in the RNAi gametophytes will be measured by scanning electron microscopy combined with energy dispersive X-rayspectroscopy (SEM-EDS), which is located in the Life Science Microscopy Facility at Purdue. Dr. Christopher Gilpin, an expert in electron microscopy and director of the facility, will collaborate with us on this aspect of the project.To see if the same genes are sufficient for arsenic tolerance and accumulation, each gene will be expressed in the fern Ceratopteris richardii (a transformation system does not exist for P. vittata), which is in the same family (Pteridaceae) as P. vittata. Each gene will be driven by the 35S promoter; stably transformed C. richardii plants will be generated using a recently published method [2]. Following transformation, the spores will be harvested from the transformed parent plants then grown on hygromycin (to eliminate non-transformed individuals) plus varying concentrations of arsenate. Because C. richardii is not tolerant to arsenic, varying the amount of arsenate will be necessary for assessing arsenic tolerance. The amount of arsenic in hygromycin resistant gametophytes also grown on arsenate will be determined by SEM-EDS.Objective 2. What are the molecular functions of the proteins encoded by PvGAPDH, PvGST and PvOCT?In P. vittata, arsenate is taken up and quickly reduced to arsenite, which accumulates in the vacuoles of gametophytes and sporophyte shoots [3, 4]. Arsenate (AsV) and arsenite (AsIII) both disrupt plant metabolism but though different mechanisms (reviewed in [5]). AsV, a phosphate analog, replaces phosphate in biological molecules, disrupting processes central to metabolism, information storage and retrieval, and cellular signaling. AsIII can bind to thiol-containing proteins and affect their proper folding. AsIII also induces the production of reactive oxygen species (ROS), including superoxide, the hydroxyl radical and H2O2, which in turn compromise cellular functions (oxidative stress). Clearly, P. vittata is uniquely adept at counteracting the toxic effects of AsV and AsIII. We hypothesize that PvGAPDH, PvGST and PvOCT play important roles in avoiding arsenic toxicity in P. vittata based upon our observation that they are highly up-regulated by arsenate in gametophytes. We will first address whether these genes are induced by arsenic (both AsV and AsII), and/or are induced by oxidative stress in response to arsenic exposure. In these experiments, the expression of each gene will be assessed by qRT-PCR. Gametophytes will be grown in the presence of absence of varying concentrations of AsV, AsIII and the ROS-generating chemicals paraquat and H2O2 (with and without stabilizers, available from Sigma) for varying periods of time prior to RNA isolation. Oxidative stress will also be measured in similarly treated gametophytes using commercially available oxidative stress sensors (e.g., CellROX reagents, ThermoFisher Scientific). What would of interest in these experiments is to determine whether the ROS-generating chemicals are able to induce the expression of one or more of these genes in the absence of arsenic. If their expression is not affected by the ROS-generating chemicals, it would suggest that the up-regulation of these genes is a specific response to arsenic and not the downstream effects of arsenic exposure.As shown in the following figure, the protein encoded by PvOCT is predicted to be a membrane protein with 12 membrane spanning domains. It is 40% identical to the Arabidopsis organic cation/carnitine transporter4 (OCT4) protein (e value=4e-119). Unfortunately, little is known about the function of any of the OCT proteins in plants (there are six in Arabidopsis) with the exception of one study [6] showing that a mutation of OCT1 in Arabidopsis affects root branching. In yeast, the protein most similar to the OCT family of proteins in plants regulates the transport of polyamines. In plants, polyamines can provide protection from environmental stress as free-radical scavengers [7]. Because polyamine levels have not been reported in P. vittata, polyamine levels will be assessed in gametophytes treated with and without arsenate using methods described in [8]. If their levels are affected by arsenic, future experiments will be designed to test the possibility that the OCT1 protein is involved in the localization and cellular trafficking of polyamines. In the meantime (during the current funding period), the cellular localization of OCT1 will be determined by generating a 35S::OCT1::GFP protein fusion construct, introducing the construct into Ceratopteris richardii cells, and determining it's localization by confocal microscopy.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is well known for its role in glycolysis. It also has other "moonlighting" functions, including arsenate reduction in human red blood cells [9]. To test the possibility that PvGAPDH is an arsenate reductase in vitro, it will be over-produced in E. coli and purified using the same methodology employed in the characterization of ACR2 in the PI's lab [10]. Arsenate reductase activity of the purified protein will assayed using the method developed for ACR2 [10].Altogether, the proposed studies should help clarify the roles of three genes whose function in arsenic tolerance and/or hyperaccumulation are completely unknown at this time.