Improved drought tolerance without undesired side effects in transgenic plants producing trehalose

Abstract

Most organisms naturally accumulating trehalose upon stress produce the sugar in a two-step process by the action of the enzymes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). Transgenic plants overexpressing TPS have shown enhanced drought tolerance in spite of minute accumulation of trehalose, amounts believed to be too small to provide a protective function. However, overproduction of TPS in plants has also been found combined with pleiotropic growth aberrations. This paper describes three successful strategies to circumvent such growth defects without loosing the improved stress tolerance. First, we introduced into tobacco a double construct carrying the genes TPS1 and TPS2 (encoding TPP) from Saccharomyces cerevisiae. Both genes are regulated by an Arabidopsis RuBisCO promoter from gene AtRbcS1A giving constitutive production of both enzymes. The second strategy involved stress-induced expression by fusing the coding region of ScTPS1 downstream of the drought-inducible Arabidopsis AtRAB18 promoter. In transgenic tobacco plants harbouring genetic constructs with either ScTPS1 alone, or with ScTPS1 and ScTPS2 combined, trehalose biosynthesis was turned on only when the plants experienced stress. The third strategy involved the use of AtRbcS1A promoter together with a transit peptide in front of the coding sequence of ScTPS1, which directed the enzyme to the chloroplasts. This paper confirms that the enhanced drought tolerance depends on unknown ameliorated water retention as the initial water status is the same in control and transgenic plants and demonstrates the influence of expression of heterologous trehalose biosynthesis genes on Arabidopsis root development.

Trehalose and Abiotic Stress in Biological Systems

“…In addition, trehalose-6-phosphate has been found to act as an inhibitor of SnRK1, a hexokinase that is an important transcriptional regulator of metabolism, growth and development in plants (Zhang et al, 2009;Paul et al, 2010). Drought tolerant tobacco plants which did not present any growth abnormalities were obtained by targeting the TPS1 gene expression to chloroplast or by using bifunctional fusion yeast trehalose synthesis genes (Karim et al, 2007).…”

“…Since trehalose has been found to participate in increasing tolerance to abiotic stresses in other organisms, many attempts have been made to engineer plants with microbial trehalose biosynthetic genes from OtsA-OtsB pathway in order to create stress tolerant plants: tobacco (Holmstrom et al, 1996;Goddijn et al, 1997;Romero et al, 1997;Pilon-Smits et al, 1998;Lee et al, 2003;Han et al, 2005;Karim et al, 2007), rice (Garg et al, 2002;Jang et al, 2003), tomato (Cortina & Culianez-Macia, 2005), potato (Goddijn et al, 1997) and Arabidopsis (Karim et al, 2007;Miranda et al, 2007). The first trials were partially successful, trehalose accumulated, albeit at a low level, the plants were stress tolerant, however they displayed abnormal phenotype characteristics (Goddijn et al, 1997;Romero et al, 1997;Pilon-Smits et al, 1998;Cortina & Culianez-Macia, 2005).…”

“…The first trials were partially successful, trehalose accumulated, albeit at a low level, the plants were stress tolerant, however they displayed abnormal phenotype characteristics (Goddijn et al, 1997;Romero et al, 1997;Pilon-Smits et al, 1998;Cortina & Culianez-Macia, 2005). Nonetheless, subsequent studies solved the phenotype problem by using fused bacterial trehalose biosynthesis genes, directing the gene constructs to chloroplasts (Garg et al, 2002;Jang et al, 2003;Karim et al, 2007;Miranda et al, 2007), or engineering the plants with alternate trehalose biosynthesis genes, as trehalose phosphorylase (TreP), which circumvented the production of trehalose-6-phoaphate (Han et al, 2005). The symbiotic relationship between rhizobia and legumes has a considerable impact not only on the legumes yields but also on the significant amount of the fixed nitrogen that remains in the soil for future crops use.…”

Overexpression of the Trehalase Gene AtTRE1 Leads to Increased Drought Stress Tolerance in Arabidopsis and Is Involved in Abscisic Acid-Induced Stomatal Closure

“…The undesired abnormalities in these plants were ascribed to altered levels of T6P, perturbing the developmental processes influenced by this important signal metabolite (Eastmond et al, 2003;Schluepmann et al, 2003Schluepmann et al, , 2004. Plants with improved stress tolerance but no obvious morphological defects were obtained by introducing bifunctional TPS-TPP constructs (Garg et al, 2002;Karim et al, 2007;Miranda et al, 2007), by placing the introduced TPS genes under the control of droughtinducible or tissue-specific promoters (Garg et al, 2002;Karim et al, 2007), or by overexpressing the plant endogenous TPS1 in Arabidopsis (Avonce et al, 2004) and rice (Oryza sativa; Li et al, 2011). It seems unlikely that the increased drought tolerance in these transgenic lines could be ascribed to trehalose acting as an osmoprotectant, since the trehalose levels never exceeded 1 mg g 21 fresh weight (Garg et al, 2002;Avonce et al, 2004;Cortina and Culianez-Macia, 2005).…”

Transplastomic plants for innovations in agriculture. A review

“…Several osmoprotectants, e.g., the sugar trehalose (Karim et al 2007;Iordachescu and Imai 2008) and betaines (Giri 2011), are known to confer resistance towards drought, cold, and/or salt stress presumably via macromolecule protection and ROS detoxification in the cell. Therefore, increasing the accumulation of such compounds is an important goal during development of novel crop cultivars tolerant to abiotic stressors.…”