In silico and in vivo studies of an Arabidopsis thaliana gene, ACR2, putatively involved in arsenic accumulation in plants

Abstract

Previously, our in silico analyses identified four candidate genes that might be involved in uptake and/or accumulation of arsenics in plants: arsenate reductase 2 (ACR2), phytochelatin synthase 1 (PCS1) and two multi-drug resistant proteins (MRP1 and MRP2) [Lund et al. (2010) J Biol Syst 18:223-224]. We also postulated that one of these four genes, ACR2, seems to play a central role in this process. To investigate further, we have constructed a 3D structure of the Arabidopsis thaliana ACR2 protein using the iterative implementation of the threading assembly refinement (I-TASSER) server. These analyses revealed that, for catalytic metabolism of arsenate, the arsenate binding-loop (AB-loop) and residues Phe-53, Phe-54, Cys-134, Cys-136, Cys-141, Cys-145, and Lys-135 are essential for reducing arsenate to arsenic intermediates (arsenylated enzyme-substrate intermediates) and arsenite in plants. Thus, functional predictions suggest that the ACR2 protein is involved in the conversion of arsenate to arsenite in plant cells. To validate the in silico results, we exposed a transfer-DNA (T-DNA)-tagged mutant of A. thaliana (mutation in the ACR2 gene) to various amounts of arsenic. Reverse transcriptase PCR revealed that the mutant exhibits significantly reduced expression of the ACR2 gene. Spectrophotometric analyses revealed that the amount of accumulated arsenic compounds in this mutant was approximately six times higher than that observed in control plants. The results obtained from in silico analyses are in complete agreement with those obtained in laboratory experiments.

OsHAC1;1 and OsHAC1;2 Function as Arsenate Reductases and Regulate Arsenic Accumulation

“…Because AtACR2 and AtHAC1 share sequence identity within the region used by Dhankher et al (2006) to knock down expression of AtACR2 by RNA interference, this sequence may also have suppressed AtHAC1 expression in their RNAi lines, thus resulting in decreased As(V) tolerance and As hyperaccumulation in the shoots (Chao et al, 2014). Nahar et al (2012) reported increased As(V) sensitivity and As accumulation in the shoots of a single T-DNA line (SALK_005882C) with a T-DNA insertion to the neighboring gene (At5g03452) of AtACR2 (At5g03455), which appeared to knockdown the expression of AtACR2.…”

Functional studies of AtACR2 gene putatively involved in accumulation, reduction and/or sequestration of arsenic species in plants

“…), which has important implications for how As is redistributed within the plants (Zhao et al 2009). Previously, by employing the in silico and in vivo studies on A. thaliana, we have postulated that the AtACR2 gene is involved in reduction of arsenate (AsV) to arsenite (AsIII) in the plant cells (Nahar et al 2012). One advantage of this explanation is that arsenite (but not arsenate) can be sequestered in the vacuoles of the plant cells although it is more toxic than AsV (Cobbett 2000;Mukhopadhyay et al 2000;Dhankher et al 2006;Blum et al 2007).…”

“…PCR tubes containing the reaction mixture were then placed in the thermocycler (Flexcycler, model: Block assembly 96G). The RT-PCR cycle setups for the first strand cDNA synthesis and the second strand DNA synthesis were carried out as described by Nahar at al. (2012).…”

“…The primers were specific to the first and the third exons of ACR2 gene. The transcription level of A. thaliana actin gene was analysed similarly as described by Nahar et al (2012) and used as a control. For visualization and separation of the target cDNA fragment, 10 µL of the amplified product were then loaded onto agarose gel for electrophoresis.…”

“…This enzyme, called alternatively AtACR2, AtASR and AtCDC25, has been identified and characterised as an arsenate reductase both in microorganisms and in plants (Bleeker et al 2006;Dhanker et al 2006;Nahar et al 2012). Disruption of arsenate reductase gene leads to arsenate hypersensitivity in all Escherichia coli, Saccharomyces cerevisiae and A. thaliana (Duan et al 2007).…”