Premise of the study: 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.

Methods: 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 Aox1, Aox2a and Aox2d 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.  

Key results: 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.  Aox2d, in contrast, appears to be upregulated as a generalized wounding response.

Conclusions: These findings suggest a heightened constitutive role for both HCN detoxification (via elevated β-CAS expression) and HCN-toxicity mitigation (via elevated Aox2a 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.  

All qPCR protocols followed MIQE guidelines, including for numbers of biological and technical replicates and reaction optimization protocols.  Gene expression level was quantified using the CFX96 Real-Time PCR Detection System (BIO-RAD, Hercules, California, USA). For all genes, reactions included 1× iTaq™ Universal SYBR^® Green Supermix, 0.5 µM (0.3 µM for β-CAS) forward primer, 0.5 µM (0.3 µM for β-CAS) reverse primer and 4-10 ng cDNA in a 20 µL reaction volume. Ef1α was used as the reference gene. PCR conditions followed the standard SYBR® Green cycle in CFX96 Real-Time PCR Detection System with slight modifications to maximize reaction efficiency: 95°C for 30 s, then [95°C for 5 s, 60°C (63°C for β-CAS) for 30 s] × 40 cycles. Three technical replicates were applied to each cDNA of the sample. The data were analyzed by the ΔΔC_T method with efficiency calibration in CFX Maestro™ Software (BIO-RAD, Hercules, California, USA) according to the manufacturer’s instructions.