Heterologous expression of two Medicago truncatula putative ERF transcription factor genes, WXP1 and WXP2, in Arabidopsis led to increased leaf wax accumulation and improved drought tolerance, but differential response in freezing tolerance.

Cuticular waxes are the major components of plant cuticle and play an important role in protecting aerial organs from damage caused by biotic and abiotic stresses. Here we report the functional characterization of two putative ERF transcription factor genes WXP1 and its paralog WXP2 from Medicago truncatula. Transgenic expression of WXP1 and WXP2 in Arabidopsis (ecotype Columbia) led to significantly increased cuticular wax deposition on leaves of 4-week-old and 6-week-old transgenic plants, assessed based on fresh weight or based on surface area. Differences in the accumulation of various wax components as well as their chain length distributions were found in the WXP1 and WXP2 plants. The major wax component in Arabidopsis, n-alkanes, increased substantially in both WXP1 and WXP2 transgenics, however, another wax component, primary alcohols, increased in WXP1 plants but decreased in WXP2 plants. Cuticle properties of the transgenic leaves were analyzed by chlorophyll leaching assay; while the WXP1 plants had no change, the WXP2 plants showed more chlorophyll leaching. Analysis of fresh weight loss from detached leaves revealed that the transgenic leaves tend to retain more water than the control. Both WXP1 and WXP2 transgenic plants showed significantly enhanced whole plant drought tolerance. Analysis of freezing tolerance at the whole plant level and measurement of electrolyte leakage from detached leaves revealed that the WXP1 plants had increased freezing tolerance while the WXP2 plants were more sensitive to low temperature when compared to the control. Transgenic expression of WXP1 had no obvious effects on plant growth and development, however, the expression of WXP2 led to slower plant growth. These results indicate that WXP1 is a useful candidate gene for improving plant drought and freezing tolerance by genetic transformation.