Germanium as a tool to dissect boron toxicity effects in barley and wheat Julie E. Hayes A,C , Margaret Pallotta A , Ute Baumann A , Bettina Berger B , Peter Langridge A and Tim Sutton A A Australian Centre for Plant Functional Genomics, School of Agriculture, Food and Wine, The University of Adelaide, Urrbrae, SA 5064, Australia. B The Plant Accelerator, The University of Adelaide, Waite Campus, Urrbrae, SA 5064, Australia. C Corresponding author. Email: julie.hayes@acpfg.com.au Abstract. Tolerance to boron (B) toxicity in barley (Hordeum vulgare L.) is partially attributable to HvNIP2;1, an aquaporin with permeability to B, as well as to silicon, arsenic and germanium (Ge). In this study, we mapped leaf symptoms of Ge toxicity in a doubled-haploid barley population (Clipper  Sahara 3771). Two quantitative trait loci (QTL) associated with Ge toxicity symptoms were identified, located on Chromosomes 6H and 2H. These QTL co-located with two of four B toxicity tolerance loci previously mapped in the same population. The B toxicity tolerance gene underlying the 6H locus encodes HvNIP2;1, whereas the gene(s) and mechanisms underlying the 2H locus are as yet unknown. We provide examples of the application of Ge in studying specific aspects of B toxicity tolerance in plants, including screening of wheat (Triticum aestivum L.) and barley populations for altered function of HvNIP2;1 and related proteins. In particular, Ge may facilitate elucidation of the mechanism and gene(s) underlying the barley Chromosome 2H B tolerance locus. Additional keywords: aquaporins, HvNIP2;1, LemnaTec digital imaging and analysis. Received 2 November 2012, accepted 12 February 2013, published online 2 April 2013 Introduction The elements boron (B), silicon (Si), arsenic (As) and germanium (Ge) are classified in the periodic table as metalloids and they share several similar properties. All form weak acids in solution with high dissociation constants (pK a values of 8.6–9.8), so that in physiological pH ranges, they occur predominantly as free acid molecules. The free acids of each metalloid are small molecules (atomic radii of 3.43–4.48 Å), and all have recently been identified as substrates for transport through certain members of the nodulin-26-like intrinsic protein (NIP) family of aquaporins (water channel proteins; Takano et al. 2006; Bienert et al. 2008; reviewed in Bhattacharjee et al. 2008). The chemical similarities between Si and Ge, and the implications of this for plant biology have been recognised for some time. For example, Takahashi et al.(1976) found that the uptake of Ge and Si appeared to be similar, and that Si-accumulating plant species were particularly sensitive to Ge toxicity. This enabled Ge toxicity screens to be used to identify rice (Oryza sativa L.) mutants defective in Si accumulation (Ma et al. 2002), and was also useful in later identifying and characterising the genes underlying the mutations (Ma et al. 2006; Ma et al. 2007). Ge competitively inhibits the uptake of Si in wheat (Triticum aestivum L.) (Rains et al. 2006) and, recently, the radioactive 68 Ge isotope was validated as a suitable tracer for use in studies of Si transport in higher plants (Nikolic et al. 2007). There have also been a small number of studies looking at the ability of Ge to substitute for B in plant metabolism (Loomis and Durst 1991; Cakmak et al. 1995; Ishii et al. 2002). These studies focussed on the ability of Ge to overcome symptoms of B deficiency, and it was found that Ge is unable to substitute directly for B in its primary role in crosslinking cell wall pectins (Ishii et al. 2002). The relationship between Ge and B toxicities in plants has not been investigated, although barley (Hordeum vulgare L.) is reportedly much more sensitive to Ge (toxicity symptoms observed in the mM range; Halperin et al. 1995) compared with B (1–5 mM; Reid et al. 2004) when grown in nutrient solution. B toxicity has been recognised as a significant problem affecting cereal crop production for nearly 30 years (Cartwright et al. 1984). Considerable intraspecific variation for B toxicity tolerance exists, however, and wheat and barley breeding populations have been developed and exploited to identify the chromosomal regions associated with tolerance (Paull et al. 1995; Jefferies et al. 1999, 2000; Schnurbusch et al. 2007). In barley, four quantitative trait loci (QTL) associated with B toxicity tolerance were identified, using a doubled-haploid (DH) population developed from a cross between the malt variety Clipper and an Algerian landrace Sahara 3771 (Jefferies et al. 1999; Karakousis et al. 2003). A major QTL on Chromosome 4H is associated with shoot B concentration, leaf symptom expression, root growth CSIRO PUBLISHING Functional Plant Biology, 2013, 40, 618–627 http://dx.doi.org/10.1071/FP12329 Journal compilation Ó CSIRO 2013 www.publish.csiro.au/journals/fpb