Enhanced Tolerance to Multiple Abiotic Stresses in Transgenic Alfalfa Accumulating Trehalose

Abstract

Alfalfa (Medicago sativa L.) is an important forage crop in many countries with high biomass production and the third‐most‐cultivated crop in the United States, with high protein content and potential use as biofuel. Here we describe the use of a chimeric translational fusion of yeast trehalose‐6‐phosphate synthase (EC 2.4.1.15) and trehalose‐6‐phosphate phosphatase (EC 3.1.3.12) genes to engineer stress tolerance in alfalfa. The gene fusion was overexpressed using either the constitutive 35S promoter or the stress‐regulated rd29A promoter. Transgene insertion in the genome was checked by polymerase chain reaction and transcript expression by reverse transcription polymerase chain reaction. Several independent transgenic plants were selected in the presence of kanamycin and further analyzed. Yeast gene encoding trehalose‐6‐phosphate synthase (TPS1)–yeast gene encoding trehalose‐6‐phosphate phosphatase (TPS2) gene expression under the 35S promoter led to plants with stunted growth and less biomass, whereas TPS1‐TPS2 expression driven by the rd29A promoter improved growth and provoked a significant increase in plant biomass at the foliage level in the different transgenic lines. Trehalose accumulated in all the different lines at similar levels under stress conditions. Transgenic plants displayed a significant increase in drought, freezing, salt, and heat tolerance. This is the first time that genetic engineering of trehalose metabolism is reported in alfalfa and demonstrates that it can improve multiple stress protection in this crop. Thus, the yeast TPS‐TPP fusion protein represents a great potential for generating stress‐tolerant crop plants for agriculture.

Salicornia europaea L. Na+/H+ antiporter gene improves salt tolerance in transgenic alfalfa (Medicago sativa L.)

“…Alfalfa, a perennial and outcrossing species, is one of the most valuable forage crops with high-protein content and biomass production and has been widely cultivated as an economic crop worldwide. However, it is moderately tolerant to salinity and can only be cultivated in moderate salt-alkaline soils, which limit the growth range of alfalfa (Suárez et al, 2009;Li and Brummer, 2012;Zhang et al, 2012). The expression of foreign genes, such as GmDREB1, SsNHX1, AhBADH, and TaNHX2, increases salt tolerance in transgenic alfalfa plants compared with that of wild-type plants (Jin et al, 2010;Liu et al, 2011;Zhang et al, 2012).…”

Salt Stress in Higher Plants: Mechanisms of Toxicity and Defensive Responses

“…In a different approach, the gene encoding the trehalose-6-phosphate synthase from yeast was expressed in rice, tomato (Cortina and Culiáñez-Maci a 2005), Arabidopsis (Miranda et al 2007), and alfalfa (Suárez et al 2009), imparting enhanced salt tolerance to these transgenic plants.…”

Beyond osmolytes and transcription factors: drought tolerance in plants via protective proteins and aquaporins

“…Therefore, the widely adopted strategy till now is to engineer or overexpress certain osmolytes which serves as potential route to breed stress tolerant crops. This strategy has resulted in several successful studies where different osmoprotectants have been overexpressed, like proline (Mani et al 2002, Simon-Sarkadi et al 2005, Yamada et al 2005, Gubis et al 2007, Vendruscolo et al 2007, Kumar et al 2010, glycine betaine (Park et al 2004, Quan et al 2004, Lv et al 2007, Park et al 2007, Ahmad et al 2008 sugars and sugar alcohols (Chen and Murata 2002, Cortina and Culianez-Macia 2005, Karim et al 2007, Miranda et al 2007, Paul et al 2008, Stiller et al 2008, Suzuki et al 2008, Livingston III et al 2009, Suárez et al 2009.…”