Abstract
Genome doubling did not enhance drought tolerance in alfalfa, but may set the stage for long-term adaptation to drought through a novel transcriptional landscape. Whole genome duplication (WGD) has been shown to enhance stress tolerance in plants. Cultivated alfalfa is autotetraploid, but diploid wild relatives are important sources of genetic variation for breeding. Investigating how WGD affects gene expression in stress conditions could provide better understanding for use of diploid genetic resources. In this work, we compared the drought response of neotetraploid plants obtained by bilateral sexual polyploidization with diploid full sibs, by measuring physiological and biochemical traits and RNA-seq. Without drought, 4x plants had lower photosynthetic potential than 2x plants per unit leaf area, but larger leaves allowed them to outperform the per leaf photosynthetic potential of 2x plants. Physiological and biochemical traits were significantly affected by drought in both 2x and 4x plants, but the differences between ploidies were small and nonsignificant. Proline levels were higher in 4x than 2x plants, both in control and drought conditions, indicating that larger cells with higher volume-to-surface ratio of 4x plants require a higher osmolyte concentration. RNA-seq and gene network analyses showed that more genes were affected by drought at 4x than at 2x level, with downregulation of hundreds of genes involved in photosynthesis and stomatal movement at 4x level, suggesting that WGD made the 4x plants more responsive to drought. Genes involved in proline, phytormone and cell wall functions were also transcriptionally affected by drought in 4x plants. We conclude that WGD did not immediately enhance drought tolerance in alfalfa, but may set the stage for long-term adaptation to drought through a novel transcriptional landscape.