Abstract
Horseradish allyl isothiocyanate (AITC, a volatile oil) and cyanobacterial microcystin-LR (MCY-LR, a cyclic heptapeptide) affect eukaryotic cell cycle. MCY-LR inhibits protein phosphatases PP1 and PP2A. We aimed to reveal the mechanisms of their cellular effects in a model eukaryote, Vicia faba. We have shown for the first time that AITC had minor effects on PP1 and PP2A activities in vitro, but it inhibited significantly PP1 in vivo. The combination of 10 μM AITC with 10 μM MCY-LR induced metaphase arrest after short-term (12 h) treatments. 10 μM AITC, 0.2–10 μM MCY-LR and their combinations induced histone H3 hyperphosphorylation, associated with the regulation of metaphase-anaphase transition. This hyperphosphorylation event occurred at any treatment which led to the inhibition of PP1 activity. 10 μM AITC + 10 μM MCY-LR increased the frequency of metaphase spindle anomalies, associated with metaphase arrest. We provide new insights into the mechanisms of metaphase-anaphase transition. Metaphase arrest is induced at the concomitant hyperphosphorylation of histone H3, alteration of metaphase spindle assembly and strong inhibition of PP1 + PP2A activity. Near-complete blocking of metaphase-anaphase transition by rapid protein phosphatase inhibition is shown here for the first time in plants, confirming a crucial role of serine-threonine phosphatases in this checkpoint of cell cycle regulation. Tissue-dependent differences in PP1 and PP2A activities induced by AITC and MCY-LR suggest that mainly regulatory subunits are affected. AITC is a potential tool for the study of protein phosphatase function and regulation. We raise the possibility that one of the biochemical events occurring during AITC release upon wounding is the modulation of protein phosphatase dependent signal transduction pathways during the plant defense response.
Highlights
Allyl-isothiocyanate (AITC) with a chemical formula of CH2 = CH-CH2-N = C = S is a main component of the volatile oil of several Brassicaceae species
We aimed to study (i) the cytological effects of the main compound of horseradish volatile oil, AITC alone or in combination with MCY-LR in order to see whether it can be used as a tool for studying the regulation of eukaryotic subcellular events; (ii) to investigate whether PP1 and PP2A are modulated during the subcellular changes detected and to answer the question: is a certain type of phosphatases involved in the regulation of certain mitotic events, e.g., metaphaseanaphase transition and mitotic spindle organization? In this study, we provide novel contributions for the understanding of the mechanisms of metaphase-anaphase transition in plants and in eukaryotes in general
LC-MS analysis did not reveal any significant reaction product or adduct between AITC and MCY-LR for short-term (12 h) that was used in our experiments (Supplementary Figure S1)
Summary
Allyl-isothiocyanate (AITC) with a chemical formula of CH2 = CH-CH2-N = C = S is a main component of the volatile oil of several Brassicaceae species. The main AITC source is the root system of horseradish (Armoracia sp.). It is a simple lipophylic molecule, produced from degradation by myrosinase of sinigrin, a glucosinolate after wounding of plant tissues (Zhang, 2010; Nguyen et al, 2013). There are multiple biochemical and cellular/ physiological effects of isothiocyanates and among them, AITC in eukaryotes. Its effects were observed mainly in mammalian tumor cell lines (see the review of Nguyen et al, 2013) and here we list those related to alterations of cell cycle and cell viability:
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