Expression of a Pteris vittata glutaredoxin PvGRX5 in transgenic Arabidopsis thaliana increases plant arsenic tolerance and decreases arsenic accumulation in the leaves

Abstract

Chinese brake fern Pteris vittata hyperaccumulates arsenic in its fronds. In a study to identify brake fern cDNAs in arsenic resistance, we implicated a glutaredoxin, PvGRX5, because when expressed in Escherichia coli, it improved arsenic tolerance in recombinant bacteria. Here, we asked whether PvGRX5 transgenic expression would alter plant arsenic tolerance and metabolism. Two lines of Arabidopsis thaliana constitutively expressing PvGrx5 cDNA were compared with vector control and wild-type lines. PvGRX5-expressors were significantly more tolerant to arsenic compared with control lines based on germination, root growth and whole plant growth under imposed arsenic stress. PvGRX5-expressors contained significantly lower total arsenic compared with control lines following treatment with arsenate. Additionally, PvGRX5-expressors were significantly more efficient in their arsenate reduction in vivo. Together, our results indicate that PvGRX5 has a role in arsenic tolerance via improving arsenate reduction and regulating cellular arsenic levels. Paradoxically, our results suggest that PvGRX5 from the arsenic hyperaccumulator fern can be used in a novel biotechnological solution to decrease arsenic in crops.

Stable Transformation of Ferns Using Spores as Targets: Pteris vittata and Ceratopteris thalictroides

“…While transient expression of transgenic constructs does enable research, there is no substitute for stable transformation in functional genomics and plant biology. Researchers have resorted to studying fern gene function using heterologous expression in the angiosperm model plant Arabidopsis (Arabidopsis thaliana; Dhankher et al, 2006;Sundaram et al, 2009;Sundaram and Rathinasabapathi, 2010), which is far from optimal. Overexpression and knockdown analysis of individual genes in a wide variety of fern species would undoubtedly accelerate our ability to learn more about their biology and subsequently develop novel products from ferns.…”

Transgenic expression of fern Pteris vittata glutaredoxin PvGrx5 in Arabidopsis thaliana increases plant tolerance to high temperature stress and reduces oxidative damage to proteins

“…The construction of expression vector and Agrobacteriummediated transformation were done as described previously (Sundaram et al 2009). BrieXy, the open reading frame (726 bp) of the Pv5-6 cDNA clone (Genbank accession number EF052272) was ampliWed and the PCR product, cloned in pCR 2.-1-TOPO vector.…”

“…From the T2 seeds, segregating for the transgene 3:1, several plants were selfed and their progeny analyzed for homozygozity. Homozygous T3 lines identiWed from these analyses were used for evaluations of transgene expression (Sundaram et al 2009) and heat stress tolerance.…”

“…Glutaredoxin activity in leaf protein extracts was determined with a coupled enzyme reaction as described previously (Sundaram et al 2008(Sundaram et al , 2009) using 2-hydroxyethyl disulWde (HED) as the substrate.…”

“…Unlike microbes, plants contain a multitude of glutaredoxins whose functions are not well understood (Rouhier et al 2006). We identiWed a glutaredoxin PvGRX5 from the arsenic hyperaccumulating tropical fern Pteris vittata because ectopic expression of PvGRX5 increased arsenic tolerance in E. coli (Sundaram et al 2008) and in transgenic Arabidopsis thaliana (Sundaram et al 2009). …”