No abstract

Carotenoids have essential roles in light-harvesting processes and protecting the photosynthetic machinery from photo-oxidative damage. Phytoene synthase (PSY) and Orange (Or) are key plant proteins for carotenoid biosynthesis and accumulation. We previously isolated the sweetpotato (Ipomoea batatas) Or gene (IbOr), which is involved in carotenoid accumulation and salt stress tolerance. The molecular mechanism underlying IbOr regulation of carotenoid accumulation was unknown. Here, we show that IbOr has an essential role in regulating IbPSY stability via its holdase chaperone activity both in vitro and in vivo. This protection results in carotenoid accumulation and abiotic stress tolerance. IbOr transcript levels increase in sweetpotato stem, root, and calli after exposure to heat stress. IbOr is localized in the nucleus and chloroplasts, but interacts with IbPSY only in chloroplasts. After exposure to heat stress, IbOr predominantly localizes in chloroplasts. IbOr overexpression in transgenic sweetpotato and Arabidopsis conferred enhanced tolerance to heat and oxidative stress. These results indicate that IbOr holdase chaperone activity protects IbPSY stability, which leads to carotenoid accumulation, and confers enhanced heat and oxidative stress tolerance in plants. This study provides evidence that IbOr functions as a molecular chaperone, and suggests a novel mechanism regulating carotenoid accumulation and stress tolerance in plants.

The effect of simultaneous expression of genes encoding three antioxidant enzymes, copper zinc superoxide dismutase (CuZnSOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), and dehydroascorbate (DHA) reductase (DHAR, EC 1.8.5.1), in the chloroplasts of tobacco plants was investigated under oxidative stress conditions. In previous studies, transgenic tobacco plants expressing both CuZnSOD and APX in chloroplast (CA plants), or DHAR in chloroplast showed enhanced tolerance to oxidative stresses, such as paraquat and salt. In this study, in order to develop transgenic plants that were more resistant to oxidative stress, we introduced the gene encoding DHAR into CA transgenic plants. Mature leaves of transgenic plants expressing all three antioxidant genes (CAD plants) had approximately 1.6-2.1 times higher DHAR activity, and higher ratios of reduced ascorbate (AsA) to DHA, and oxidized glutathione (GSSG) to reduced glutathione (GSH) compared to CA plants. CAD plants were more resistant to paraquat-induced stress, exhibiting only 18.1% reduction in membrane damage relative to CA plants. In addition, seedlings of CAD plants had enhanced tolerance to NaCI (100 mM) compared to CA plants. These results indicate that the simultaneous expression of multiple antioxidant enzymes, such as CuZnSOD, APX, and DHAR, in chloroplasts is more effective than single or double expression for developing transgenic plants with enhanced tolerance to multiple environmental stresses.

Oxidative stress is a major damaging factor for plants exposed to environmental stresses. In order to develop transgenic potato plants with enhanced tolerance to environmental stress, the genes of both Cu/Zn superoxide dismutase and ascorbate peroxidase were expressed in chloroplasts under the control of an oxidative stress-inducible SWPA2 promoter (referred to as SSA plants). SSA plants showed enhanced tolerance to 250 microM methyl viologen, and visible damage in SSA plants was one-fourth that of non-transgenic (NT) plants that were almost destroyed. In addition, when SSA plants were treated with a high temperature of 42 degrees C for 20 h, the photosynthetic activity of SSA plants decreased by only 6%, whereas that of NT plants decreased by 29%. These results suggest that the manipulation of the antioxidative mechanism of the chloroplasts may be applied in the development of industrial transgenic crop plants with increased tolerance to multiple environmental stresses.