Glycine betaine is an osmoprotectant found in many organisms, including bacteria and higher plants. The bacterium Escherichia coli produces glycine betaine by a two-step pathway where choline dehydrogenase (CDH), encoded by betA, oxidizes choline to betaine aldehyde which is further oxidized to glycine betaine by the same enzyme. The second step, conversion of betaine aldehyde into glycine betaine, can also be performed by the second enzyme in the pathway, betaine aldehyde dehydrogenase (BADH), encoded by betB. Transformation of tobacco (Nicotiana tabacum), a species not accumulating glycine betaine, with the E. coli genes for glycine betaine biosynthesis, resulted in transgenic plants accumulating glycine betaine. Plants producing CDH were found to accumulate glycine betaine as did F1 progeny from crosses between CDH- and BADH-producing lines. Plants producing both CDH and BADH generally accumulated higher amounts of glycine betaine than plants producing CDH alone, as determined by 1H NMR analysis. Transgenic tobacco lines accumulating glycine betaine exhibited increased tolerance to salt stress as measured by biomass production of greenhouse-grown intact plants. Furthermore, experiments conducted with leaf discs from glycine betaine-accumulating plants indicated enhanced recovery from photoinhibition caused by high light and salt stress as well as improved tolerance to photoinhibition under low temperature conditions. In conclusion, introduction of glycine betaine production into tobacco is associated with increased stress tolerance probably partly due to improved protection of the photosynthetic apparatus.
Treatments as diverse as exposure to low temperature (LT), exogenous abscisic acid (ABA)
No abstract
SummaryWe isolated a dehydrin-like (DHN-like) gene fragment, PpDHNA, from the moss Physcomitrella patens by PCR amplification using degenerate primers directed against conserved amino acid segments of DHNs of higher plants. The full-length cDNA was found to encode a 59.2-kDa glycine-rich protein, DHNA, with typical characteristics of DHNs, including the presence of several Y repeats and one conserved K segment. DHNA had a high sequence similarity with a protein from Tortula ruralis, Tr288, which is thought to be involved in cellular dehydration tolerance/repair in this moss. Northern and Western analysis showed that PpDHNA is upregulated upon treatment of plants with abscisic acid, NaCl or mannitol, indicating a similar expression pattern to DHNs from higher plants. To analyze the contribution of DHNA to osmotic stress tolerance, we generated a knockout mutant (dhnA) by disruption of the gene using homologous recombination. Growth and stress response studies of the mutant showed that dhnA was severely impaired in its capacity to resume growth after salt and osmotic-stress treatments. We provide direct genetic evidence in any plant species for a DHN exerting a protective role during cellular dehydration allowing recovery when returned to optimal growth conditions.
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.
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