Supplemental calcium improves freezing tolerance of spinach (Spinacia oleracea L.) by mitigating membrane and photosynthetic damage, and bolstering anti-oxidant and cell-wall status

•Ca2+-supplementation improves in situ freeze-tolerance of spinach seedlings by.•Membrane stabilization, i.e. reducing ion-leakage, specifically K+, Mg2+-leakage.•Improving oxidative stress-tolerance, i.e. bolstering antioxidant enzymes activity.•Improving PSII efficiency as evidenced by chlorophyll fluorescence.•Quantitative and qualitative augmentation of the cell wall. Supplemental calcium (Ca2+) fertilization has been reported to enhance plant tolerance against various abiotic stresses, however, its effect on freezing tolerance has not been well studied. We investigated the effect of Ca2+ supplementation on freezing tolerance of spinach (Spinacia oleracea L.) at whole-plant as well as excised-leaf level. Seedlings supplemented with 20 and 25 mM Ca2+ through sub-irrigation efficiently assimilated Ca2+, evident by higher leaf-Ca2+content. Supplemental Ca2+ did not impede leaf-growth (leaf-area) but slightly increased dry weight/fresh weight ratio compared to the control. Supplemental Ca2+ significantly improved freezing tolerance as evident by reducing K+/Mg2+/total ion-leakage (i.e. indicative of less membrane damage), alleviating oxidative stress (less accumulation of O2 and H2O2), and enhancing PSII potential quantum yield /energy trapping efficiency (Fv/Fm) following a relatively moderate or severe freeze-thaw stress, i.e. −5.5 and −6.5 ˚C. Improved freezing tolerance by Ca2+ supplementation was paralleled by enhanced activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX). Our data further suggests that, besides a bolstered antioxidant status, improved freezing tolerance of Ca2+-fed seedlings may also be associated with reduced loss of Ca2+ from plasma membrane (due to lower abundance of leaked K+ in extracellular spaces) as well as due to increased mechanical strength of cell wall as evident by cell wall augmentation.