Effects of medium-induced osmotic and nutrient stress on cyanogenic glycosides in flax (Linum usitatissimum)

<title>Abstract</title> Background Cyanogenic glycosides (CGs) are multifunctional plant metabolites involved in defense, nitrogen turnover, and stress responses. Study examined effects of medium-induced osmotic and nutrient stress on cyanogenic glycosides in flax (Linum usitatissimum L.)cv. Linola. Plants were cultivated under mannitol-induced osmotic stress or on media with modified sulfate or nitrate supply, and sampled throughout early development. Transcript levels of genes involved in CGs biosynthesis, degradation, and cyanide detoxification were analyzed by real-time RT-PCR, together with cyanogenic potential, free cyanide content, β-cyanoalanine synthase (β-CAS) activity. Results Drought caused the strongest response, leading to late-stage induction of CG-related genes, increased β-CAS activity, higher cyanogenic potential, and elevated free cyanide under prolonged stress, indicating intensified CGs turnover and a link between cyanide metabolism and drought responses. Sulfur availability exerted a stronger influence on CGs homeostasis than nitrogen, primarily affecting detoxification capacity and overall pathway balance, whereas nitrogen effects were more subtle and reflected modulation of temporal dynamics rather than sustained changes in CGs accumulation. In both cases, transcriptional responses were not consistently mirrored at the enzymatic or metabolite levels, highlighting a partial decoupling across regulatory layers. In particular, β-CAS activity did not closely follow transcript abundance, supporting the concept of substrate-driven regulation of cyanide detoxification, whereby enzyme activity is primarily governed by substrate availability rather than transcriptional control. Conclusions These findings indicate that CGs metabolism in flax is developmentally regulated and highly responsive to environmental and nutritional cues, with its functional output shaped by integrated transcriptional, post-transcriptional, and metabolic buffering mechanisms.