Frost resistance and biochemical changes during cold acclimation in different annual legumes

In Mediterranean areas of southern Europe growth of forages is seriously limited by the ability of each species to growth during cold winter. Therefore, the objective of this work was to prove the capacity for cold acclimation and frost resistance in four annual legumes native of Mediterranean region under laboratory conditions. Plants from subterranean clover (Trifolium subterraneum L. ssp. brachycalycinum cv. Clare), balansa clover (T. michelianum Savi cv. Giorgia), burr medic (Medicago polymorpha L. cv. Anglona) and barrel medic (M. truncatula Gaertn. cv. Paraggio) were cold acclimated by growing them at 10/5 °C. Non-acclimated plants developed at 20/15 °C. Dry matter partition, root solute content and enzymes for sucrose synthesis were determined during cold acclimation period. Freezing tolerance was determined by measuring the lethal temperature of 50% of the population (LT50) and a thermal analysis was performed to study the dynamics for ice formation in plant tissues. Finally, by measuring forage production after 4 weeks at 20/15 °C it was assessed regrowth capacity. Cold-acclimated plants have increased root solute concentrations coupled with changes in the activity of some enzymes involved in sucrose synthesis in leaves. However, activities of sucrose synthesis pathway are not related with freezing tolerance in a simple way. Lower LT50 was related to lower specific leaf area (SLA), higher root to shoot ratio and higher root solutes. Non-acclimated plants have LT50 similar or higher than ice nucleation temperature indicating that these species cannot tolerate freezing. Cold acclimation decreased LT50 in all species, being lower in Giorgia and Anglona; in Anglona did not change nucleation temperature, resulting values significantly higher than corresponding LT50. This suggests that this cultivar was able to tolerate freezing. By contrast, in Giorgia, nucleation temperature decreased, which suggests that changes occurring during cold-acclimation might contribute to increase their capacity for avoidance by moderate supercooling. Cold-acclimated plants have higher regrowth capacity than non-acclimated ones except in Paraggio, which exhibited a poor regrowth. This might be due to an ineffective cold acclimation process related to impaired SPS activity and low starch reserves observed in this cultivar. On the other hand, Anglona and Giorgia have the better frost tolerance and higher potential for production of forage mass after freezing, indicating that they may be suitable cultivars for sow under cold winter conditions predominant in some Mediterranean zones.