Variable expression of cyanide detoxification and tolerance genes in cyanogenic and acyanogenic white clover (Trifolium repens L.)

<p><strong>Premise of the study:</strong> β-cyanoalanine synthase (β-CAS) and alternative oxidase (AOX) play important roles in the ability of plants to detoxify and tolerate hydrogen cyanide (HCN) stress.  These functions are critical for all plants, as HCN is produced at low levels during basic metabolic processes, but are likely to be especially important in cyanogenic species, which release high levels of HCN following tissue damage. However, their expression has not been examined in cyanogenic species, nor has it been compared between cyanogenic and acyanogenic genotypes within a species.</p> <p><strong>Methods:</strong> We used a natural polymorphism for cyanogenesis in white clover to examine β-CAS and Aox gene expression in relation to cyanogenesis-associated HCN exposure.  We identified all β-CAS and Aox gene copies present in the genome, including members of the <em>Aox1, Aox2a</em> and <em>Aox2d </em>subfamilies previously reported in legumes.  Expression levels were compared between cyanogenic and acyanogenic genotypes, and under conditions of leaf tissue damage compared to undamaged tissue.  </p> <p><strong>Key results:</strong> Results indicate that β-CAS and Aox2a expression are differentially elevated in cyanogenic genotypes, and that tissue damage is not required to induce this increased expression.  <em>Aox2d</em>, in contrast, appears to be upregulated as a generalized wounding response.</p> <p><strong>Conclusions:</strong> These findings suggest a heightened constitutive role for both HCN detoxification (via elevated β-CAS expression) and HCN-toxicity mitigation (via elevated <em>Aox2a </em>expression) in plants that are capable of cyanogenesis.  As such, freezing-induced cyanide autotoxicity is unlikely to be the primary selective factor in the evolution of climate-associated cyanogenesis clines.  </p>