Methylated As species were not detected in rice, tomato or red clover tissues exposed to arsenate under axenic conditions, even with nutrient deficiency or symbiosis with root- nodulating bacteria (Figures 3.1; 3.2; 3.5; 3.6). Methylated As was detected however in rice plants exposed to MMA and DMA, demonstrating that plants are able to take these As species up when they are present in the growth medium (Figure 3.3; Table S3.2). MMA and DMA were also detected in rice shoots grown in flooded soil from Bangladesh or the UK, and were also present in the soil pore and standing waters (Figure 3.8).
Furthermore, Arao et al., (2011) reported that addition of the antibiotic chloramphenicol to nutrient solution significantly decreased DMA formation under MMA treatment in hydroponic culture of rice. It is highly likely that microbial methylation explains the presence of methylated As species in plants cultured in non-sterile media reported previously (Nissen & Benson, 1982; Quaghebeur & Rengel, 2003; Raab et al., 2007a). Using GeoChip analysis, Lomax et al. (2012) found that arsM genes were highly abundant in a Bangladeshi paddy soil.
Recently, several studies have reported an increase in MMA and DMA in soils after the addition of fertilisers with high organic matter contents such as dried distillers’ grain (DDG; Jia et al., 2012), biogas slurry (Jia et al., 2013b) and rice straw (Jia et al., 2013a). The addition of organic matter is thought to enhance As mobilisation through competition for sorption sites and by lowering the redox potential (Jia et al., 2013b). Additionally,
increasing the organic matter content of 14 different paddy soils by addition of rice straw was found to increase the abundance of microbial arsM genes by an average of almost 140% (Jia et al., 2013a).
The results also suggest that the methyltransferase containing a UbiE/Coq5 motif found to be upregulated by arsenate exposure (Norton et al., 2008), does not function as an As methyltransferase in planta (Ye et al., 2012). When data from several market basket surveys were combined, the relationship between total grain As and the proportion of inorganic As varied greatly by region (Zhao et al., 2013a). Rice from Asia (including
49 the U.S. displayed a hyperbolic pattern; reaching a maximum around 0.15 mg kg-1. Rice produced in Europe (including Italy, Spain and France) was more variable and displayed an intermediate relationship. Recent studies have found that differences in grain As speciation are due to environmental conditions, rather than genetic differences between cultivars. Norton et al. (2009a) grew 13 rice cultivars at sites in Bangladesh, India and China under normal field conditions and found that site had the biggest influence on grain DMA percentage. There were also significant effects of genotype and site by genotype interaction; however these contributed only slightly to grain As speciation in comparison to site. Therefore there is little evidence to support the classification of rice cultivars to DMA or inorganic type by Zavala et al. (2008).
Although unable to further methylate MMA, rice roots clearly have a capacity to reduce MMA to trivalent MMA (Figure 3.3; Table S3.2). Interestingly, in humans MMA reduction is the rate-limiting step in As methylation (Zakharyan et al., 2001). Recently it was
reported that rice plants are also able to reduce TMAO to volatile TMA gas (Jia et al., 2012). Trivalent DMA is very unstable (Gong et al., 2001) which may explain the lack of detection of this As species in plants, although it has been found in human urine (Le et al., 2000). Rice plants may even be able to demethylate As, as MMA was detected in roots after exposure to DMA (Figure 3.3; Table S3.2). MMA has also been detected in the As hyperaccumulators, P. vittata and Pteris cretica and As-tolerant Boehmeria nivea exposed to DMA in sand culture (Huang et al., 2008), and in radish (Raphanus sativus) grown in soil amended with DMA. De-methylation of DMA (Huang et al., 2007) and MMA (Yoshinaga et al., 2011) mediated by micro-organisms has also been observed in soils, however the significance of this process to the global As biogeocycle is not yet fully understood.
The lack of methylated As species in red clover with nodulation from R. leguminosarum
(Figures 3.6; 3.7) is unsurprising as a BLAST search (tBlastn) failed to find any genes with homology to arsM from Rhodopseudomonas palustris (accession number: NP_948900.1; Qin et al., 2006) in the genomes of Rhizobium species. However, the possibility cannot be excluded that As methylation by other symbiotic micro-organisms may result in
accumulation of methylated As in the host plant. For example, Ultra et al., (2007) found elevated DMA concentrations in soil surrounding sunflower roots colonised with the arbuscular mycorrhizal (AM) fungus Glomus aggregatum. Furthermore, AM fungal
50 hyphae were found to facilitate transport of arsenate in a split-compartment pot
experiment (Meding & Zasoski, 2008), and shoot DMA concentrations of maize (Zea mays) were higher in plants inoculated with the AM fungus G. mosseae compared to the uninoculated control (Yu et al., 2009). However rice plants inoculated with G. intraradices
contained only inorganic As after exposure to arsenate for 10 days in sand culture (Chen
et al., 2013a). Phosphate is transferred from AM fungi to the host in the form of
polyphosphate, however polyarsenate is too unstable to be translocated in the same way (Smith et al., 2010). A further BLAST search (tBlastn) using the protein sequence of arsM
from R. palustris (Qin et al., 2006) did not find any homologous genes in the genomes of members of the phylum Glomeromycota. However, difficulties in culturing AM fungi mean that these species are underrepresented in genetic databases. Further investigation into the contribution of AM fungi to the uptake of As from soils and As methylation is needed.
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