Transgenic cotton (Gossypium hirsutum) over-expressing alcohol dehydrogenase shows increased ethanol fermentation but no increase in tolerance to oxygen deficiency

Abstract

Cotton (Gossypium hirsutumL.) was transformed with constructs for the over-expression of two enzymes involved in ethanol fermentation, alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC), with the goal of increasing waterlogging tolerance. Four independent transgenic lines transformed with the cotton Adh2 cDNA driven by the CaMV 35S promoter showed ectopic expression of this isozyme in leaves and up to 20-fold greater in vitro ADH activity in roots. Under conditions of O₂ deficiency, excised roots from these transgenic plants showed up to 80% increase in ethanol evolution compared to untransformed roots. Conversely, one line transformed with a construct containing the Adh2 coding region in the antisense orientation showed a 65% decrease in ADH activity and a 25% decrease in ethanol production from anaerobic roots relative to untransformed cotton. Lines transformed with a rice Pdc1 cDNA driven by the CaMV 35S promoter showed high levels of expression of the transgene-encoded protein in leaves, but only very low levels of protein and no in vitro enzyme activity detectable in the roots of these plants. Roots from plants transformed with the 35S-Pdc construct did not produce more ethanol than roots from untransformed controls. We tested the ability of cotton roots to withstand low O₂ treatments under hydroponic conditions. Neither the ‘ADH’ nor the ‘PDC’ transgenics showed more tolerance than the wild-type on the basis of root growth under a mild stress (5% O₂), a strong stress (0% O₂ with or without a 5% O₂ pretreatment), or in recovery growth following these treatments. Our results show that over-expression of ADH can lead to ethanol over-production (even though the activity of this enzyme by far exceeds that of PDC, its precursor in the pathway), but this is not sufficient to increase waterlogging tolerance in cotton.