Over-Expression of MYB Transcription Factor Imparts Multi Stress Tolerance in Alfalfa

Alfalfa (Medicago sativa) is a widely used forage crop in the world due to its productivity and high feed value. Improvement for drought and biotic stress tolerance are important breeding objectives in alfalfa. Recently, we identified a MYB96 transcription factor as one of the downregulated gene in Medicago truncatula plants having mutation in Cys(2)His(2) zinc finger transcription factor that led to defects in epicuticular wax accumulation on mutant leaves. We over-expressed Myb96 gene in alfalfa and analyzed the alfalfa Myb96-OE lines for various parameters related to drought tolerance/escape. The Myb96-OE lines show less leaf water loss, low stomatal density, smaller leaf size and high specific leaf weight when compared to wild-type control, indicating a better drought escape mechanism in Myb96-OE lines. The root/shoot ratio in Myb96-OE plants was also high suggesting that these plants can perform better under drought or low soil moisture conditions. Gas chromatography–mass spectrometry (GC-MS) analysis demonstrated more accumulation of leaf epicuticular wax constituents in Myb96-OE leaves than control plant leaves. Wax accumulation was supported by microarray data showing upregulation of most wax biosynthesis and drought responsive genes. Surprisingly, Myb96-OE lines were also tolerant to thrip infestation and had more trichomes on leaf surface compared to control plants. Microarray data also identified upregulation of Lipoxygenase (~80 folds), a jasmonic acid (JA) biosynthetic pathway gene and more accumulation of JA in Myb96-OE lines. Transient expression assay in Nicotiana benthamiana indicated a direct or indirect regulation of Lipoxygenase by MYB96 transcription factor.