Plant survival in response to freezing stress depends on the efficient activation of tolerance mechanisms. Fritillaria imperialis exposure to freezing stress enhanced signalling molecules Ca2+ and H2O2 along with overexpression of Ca2+ signalling proteins (Ca2+ dependent protein kinases, CPK), followed by upregulation of NHX1 (Na+/H+ antiporter), LEA (late embryogenesis abundant proteins) and P5CS (1-pyrroline-5-carboxylate synthetase). Overexpression of OsCNGC6 was responsible for high accumulation Ca2+, Na+ and K+. The NHX1 gene product transported Na+ to vacuoles and increased cytosolic K+ content to re-establish ionic homeostasis under stress conditions. The reduced water potential of leaves was due to high accumulation of osmolytes and ions. No changes were observed in relative water content of leaves, which might be correlated with overexpression of the LEA gene, which protects against dehydration. High accumulation of H2O2 under freezing stress was responsible for activation of antioxidant systems involving SOD, phenols, anthocyanins, catalase and ascorbate peroxidase. Photosynthesis, suppressed in freezing-stressed plants, returned to normal levels after termination of freezing stress. Taken together, our findings suggest that Fritillaria efficiently tolerated freezing stress through induction of signalling mechanisms and overexpression of cold stress-responsive genes, and prevention of cold-induced water stress, oxidative stress and photosynthetic damage.

CIRSEC Centre for Climatic Change Impact University of Pisa Via del Borghetto 80 1 56124 Pisa Italy

Department of Agriculture Food and Environment University of Pisa Pisa Italy

Department of Botany and Plant Physiology Faculty of Agrobiology Food and Natural Resources Czech University of Life Sciences 16500 Prague Czech Republic

Department of Plant Physiology Faculty of Agrobiology and Food Resources Slovak University of Agriculture 94976 Nitra Slovakia

Interdepartmental Research Center Nutrafood Nutraceuticals and Food for Health University of Pisa Pisa Italy

Plant Biology Department Faculty of Science Behbahan Khatam Alanbia University of Technology Khuzestan Iran

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