Changes in Cell Ca2+ Distribution in Loquat Leaves and Its Effects on Cold Tolerance

Calcium has been associated with improved cold tolerance in many crops. The aim of this study was to investigate the changes in leaf cell $Ca^{2+}$ distribution and cell organelle ultrastructure of loquat (Eriobotrya japonica Lindl.) plants in response to cold stress at $-3^{\circ}C$, using transmission electron microscopy (TEM). Two loquat accessions, Zaozhong 6 (a commercial cultivar) and oakleaf loquat (a wild relative) were used. Cold tolerance, as measured by leaf browning rate, was higher in oakleaf plants, and calcium treatment improved cold tolerance in both species. Cold stress first induced inward transport of $Ca^{2+}$ from the intracellular space. Then, the imported $Ca^{2+}$ was aggregated around the chloroplast membrane, finally entering the chloroplast. This pattern of $Ca^{2+}$ distribution in leaf cells occurred earlier in Zaozhong 6 than in the wild loquat. With increasing time of cold exposure, the chloroplast membranes of Zaozhong 6 leaves were damaged, blurred and even disappeared, while those of wild oakleaf loquat leaves maintained their structure longer. In Zaozhong 6, cold stress induced a clear cavity between poorly structured granal thylakoids and vesicles appearing inside the chloroplast, while in oakleaf leaves cold stress had little effect on the ultrastructure of chloroplasts (although chloroplast membranes looked blurred). Loquat leaves accumulated free calcium ions around chloroplasts in response to cold stress, with earlier calcium accumulation occurring in the cold-sensitive cultivar Zaozhong 6 than in wild oakleaf loquat. These results demonstrate that these two loquat species have differences in both cold tolerance and calcium accumulation dynamics.