Exogenous salicylic acid improves freezing tolerance of spinach (Spinacia oleracea L.) leaves

Salicylic acid (SA)-treatment has been reported to improve plant tolerance to various abiotic stresses. However, its effect on freezing tolerance has not been well investigated. We investigated the effect of exogenous SA on freezing tolerance of spinach (Spinacia oleracea L.) leaves. We also explored if nitric oxide (NO) and/or hydrogen peroxide (H2O2)-mediation was involved in this response, since these are known as primary signaling molecules involved in many physiological processes. A micro-centrifuge tube-based system used to apply SA to petiolate spinach leaves (0.5 mM over 4-d) was effective, as evident by SA content of leaf tissues. SA-treatment did not hamper leaf growth (fresh and dry weight; equatorial and longitudinal length) and was also not significantly different from 25% Hoagland controls vis-à-vis growth. SA application significantly improved freezing tolerance as evidenced by reduced ion-leakage and alleviated oxidative stress (lower accumulation of O2·- and H2O2) following freeze-thaw stress treatments (−6.5, −7.5, and −8.5 °C). Improved freezing tolerance of SA-treated leaves was paralleled by increased proline and ascorbic acid (AsA) accumulation. A 9-d cold acclimation (CA) treatment also improved leaf freezing tolerance (compared to non-acclimated control) and was accompanied by accumulation of SA and proline. Our results indicate that increased freezing tolerance may be associated with accumulation of compatible solutes (proline) and antioxidants (AsA). Notably, the beneficial effect of SA on freezing tolerance was abolished when either H2O2- or NO-scavenger (1 μM N-acetylneuraminic acid, NANA or 100 μM hemoglobin, HB, respectively) was added to SA as pretreatment. Our data suggest that SA-induced freezing tolerance in spinach may be mediated by NO and H2O2 signaling.