Frozen mountain pine needles: The endodermis discriminates between the ice-containing central tissue and the ice-free fully functional mesophyll.

Conifer (Pinaceae) needles are the most frost-hardy leaves. During needle freezing, the exceptional leaf anatomy, where an endodermis separates the mesophyll from the vascular tissue, could have consequences for ice management and photosynthesis. The eco-physiological importance of needle freezing behaviour was evaluated based on the measured natural freezing strain at the alpine treeline. Ice localisation and cellular responses to ice were investigated in mountain pine needles by cryo-microscopic techniques. Their consequences for photosynthetic activity were assessed by gas exchange measurements. The freezing response was related to the microchemistry of cell walls investigated by Raman microscopy. In frozen needles, ice was confined to the central vascular cylinder bordered by the endodermis. The endodermal cell walls were lignified. In the ice-free mesophyll, cells showed no freeze-dehydration and were found photosynthetically active. Mesophyll cells had lignified tangential cell walls, which adds rigidity. Ice barriers in mountain pine needles seem to be realised by a specific lignification patterning of cell walls. This, additionally, impedes freeze-dehydration of mesophyll cells and enables gas exchange of frozen needles. At the treeline, where freezing is a dominant environmental factor, the elaborate needle freezing pattern appears of ecological importance.