Field plus lab experiments help identify freezing tolerance and associated genes in subtropical evergreen broadleaf trees: A case study of .

The molecular mechanisms of freezing tolerance are unresolved in the perennial trees that can survive under much lower freezing temperatures than annual herbs. Since natural conditions involve many factors and temperature usually cannot be controlled, field experiments alone cannot directly identify the effects of freezing stress. Lab experiments are insufficient for trees to complete cold acclimation and cannot reflect natural freezing-stress responses. In this study, a new method was proposed using field plus lab experiments to identify freezing tolerance and associated genes in subtropical evergreen broadleaf trees using as a case. Cultivated is the dominant woody oil crop in China. Wild at the high-elevation site in Lu Mountain could survive below -30°C, providing a valuable genetic resource for the breeding of freezing tolerance. In the field experiment, air temperature was monitored from autumn to winter on wild at the high-elevation site in Lu Mountain. Leave samples were taken from wild before cold acclimation, during cold acclimation and under freezing temperature. Leaf transcriptome analyses indicated that the gene functions and expression patterns were very different during cold acclimation and under freezing temperature. In the lab experiments, leaves samples from wild after cold acclimation were placed under -10°C in climate chambers. A cultivated variety "Ganwu 1" was used as a control. According to relative conductivity changes of leaves, wild showed more freezing-tolerant than cultivated . Leaf transcriptome analyses showed that the gene expression patterns were very different between wild and cultivated in the lab experiment. Combing transcriptome results in both of the field and lab experiments, the common genes associated with freezing-stress responses were identified. Key genes of the flg22, Ca and gibberellin signal transduction pathways and the lignin biosynthesis pathway may be involved in the freezing-stress responses. Most of the genes had the highest expression levels under freezing temperature in the field experiment and showed higher expression in wild with stronger freezing tolerance in the lab experiment. Our study may help identify freezing tolerance and underlying molecular mechanisms in trees.