| A lack of winter persistence in alfalfa (Medicago sativa L.) can limit economic returns. Soluble carbohydrates, including sucrose, are believed to play a cryoprotective role; freezing tolerance may be enhanced by raising the concentration of sucrose in the plant. Sucrose-phosphate synthase (SPS) diverts carbohydrate from starch production towards sucrose accumulation. The purpose of this study was to test whether expression of a constitutively active form of SPS would result in an increase in the sucrose:starch ratio, and whether altered carbohydrate concentrations would produce improved winter hardiness. This is the first study to investigate whether transgenic plants with increased soluble sugar concentrations are more cold-tolerant. The role of SPS and sugars in cold acclimation and the potential of SPS overexpression to improve winter persistence was investigated. A gene and a cDNA encoding alfalfa SPS were isolated. Both contained an identical coding region. SPS existed as a single copy gene. A Ser150 to Arg150 substitution abolished downregulation of the enzyme by phosphorylation. Northern and western dot blots revealed that SPS expression increased within three hours of exposure to 4°C, and rose to levels three to five times higher than seen in control plants within twelve hours. SPS expression was also upregulated in response to low temperatures in the field. Raffinose and stachyose accumulated after the first frost, and reached higher concentrations in more dormant cultivars. A similar response was seen under controlled conditions. Transgenic plants expressing the constitutively active mutant of SPS were generated, but did not consistently display increased sucrose concentrations or altered sucrose:starch partitioning relative to the controls. Upon exposure to 4°C, some lines overexpressing SPS had greater sucrose:starch ratios than the controls. As well, some transgenic lines overexpressing SPS accumulated higher concentrations of raffinose family oligosaccharides after exposure to 4°C than seen in other lines. |
