Background

Cyanogenic glycosides (CNglcs) are secondary plant metabolites arisen from amino acids and found in over 3,000 plant species, including economically important crops as Prunus spp., Manihot spp., Malus spp., or Sorghum spp. (Gleadow and Møller, 2014). CNglcs play crucial roles in different physiological processes such as germination, transport of essential nutrients, turnover processes, oxidative stress regulation, or defense responses (Møller, 2010; Kadow et al., 2012; Flematti et al., 2013; Neilson et al., 2013). The metabolism of CNglcs is complex and derives in cyanohydrin production, which in turn can be converted into hydrogen cyanide and ketones or aldehydes in the presence of α-hydroxynitrile lyase enzymes (HNLs). Plant cyanohydrins have been demonstrated to act as direct defense molecules, with a highly toxic action against herbivores and pathogens (Park et al., 2002; Beran et al., 2019). In addition, they can also work as signaling molecules, triggering the defense response (Bernal-Vicente et al., 2020). However, some herbivores, mainly lepidopteran insects, have developed the ability to counteract or take advantage of these cyanogenic molecules, and alternatively may degrade or use these compounds for their benefit (Zagrobelny and Møller, 2011; Zagrobelny et al., 2014).

The focus of this protocol is one of these CNglcs derivatives, mandelonitrile. Mandelonitrile is a cyanohydrin derived from CNglc metabolism, as prunasin or amygdalin among others (Gleadow and Møller, 2014), that occurs naturally in cyanogenic plants. It is characterized by having hydroxyl and cyano groups attached to the same carbon atom (Figure 1). Some works have reported the quantification of mandelonitrile in different cyanogenic plant species such as Prunus cerasus L. and Aronia melanocarpa Ell using high-performance liquid chromatography (HPLC) and gas chromatography–mass spectrometry (GC/MS) techniques, respectively (Hirvi and Honkanen, 1985; Chandra and Nair, 1993).

Chassagne et al. (1996) detected amygdalin and its corresponding parental ion, benzaldehyde cyanohydrin (or mandelonitrile), in Passiflora fruits using GC/MS. More recently, mandelonitrile has been quantified in peach micro-propagated shoots by ultra-performance liquid chromatography (UPLC) coupled to a mass spectrophotometer (Bernal-Vicente et al., 2020). However, as far as we know, mandelonitrile levels haven’t been determined in the model plant A. thaliana until now, maybe because it is considered a non-cyanogenic plant (CNglcs have not been detected in A. thaliana wild-type plants). Despite this, an alternative pathway to cyanohydrin production has recently been described—the 4-hydroxy-indole-3-carbonylnitrile (4-OH-ICN) route—as a rare metabolic re-invention leading to alternative cyanogenic compounds, to expand A. thaliana defenses and also contribute to the production of cyanohydrins (Rajniak et al., 2015; Pastorczyk et al., 2020). In addition, an HNL has also been found in A. thaliana, and its encoding gene has been cloned and the recombinant protein has been purified and crystalized (Andexer et al., 2007). Biochemical studies revealed that Arabidopsis HNL recognized a broad range of substrates, was enantioselective, and transformed aliphatic and aromatic aldehydes and/or ketones into the corresponding R-cyanohydrins, including mandelonitrile (Andexer et al., 2007).

Therefore, the study of the relationship between mandelonitrile levels and the role of HNL in Arabidopsis has allowed the establishment of their defense role against the two-spotted spider mite Tetranychus urticae (Arnaiz et al., 2022). Consequently, mandelonitrile can be considered a target molecule for the study of Arabidopsis–spider mite interaction in the plant defense process. This is why the development of a protocol to determine the levels of mandelonitrile in A. thaliana plants under controlled conditions and after spider mite infestation is of crucial interest. In addition, this protocol was established since it was not possible to adapt previous protocols already described for other plant species. In fact, no results were obtained using HPLC techniques and with the available quantity of infested A. thaliana sample as starting plant material, suggesting the need to develop a specific quantification procedure for such a non-cyanogenic species. GC/MS was selected for mandelonitrile detection as it allowed to detect the target molecule in standard samples. Moreover, the spectrum information related to mandelonitrile-derivatized found in the databases helped us to confirm our obtained results. Additionally, the derivatization process of mandelonitrile makes it a very volatile molecule and for that reason, it was better to use GC/MS than HPLC for analysis. Finally, it is likely that this protocol will work in other plant species by applying some modifications, e.g., quantity of starting material, solvent volume, or incubation and sonication times.