Ectopic expression of a fruit phytoene synthase from Citrus paradisi Macf. promotes abiotic stress tolerance in transgenic tobacco

Abstract

Abscisic acid (ABA) is an important regulator of plant responses to environmental stresses and an absolute requirement for stress tolerance. Recently, a third phytoene synthase (PSY3) gene paralog was identified in monocots and demonstrated to play a specialized role in stress-induced ABA formation, thus suggesting that the first committed step in carotenogenesis is a key limiting step in ABA biosynthesis. To examine whether the ectopic expression of PSY, other than PSY3, would similarly affect ABA level and stress tolerance, we have produced transgenic tobacco containing a fruit-specific PSY (CpPSY) of grapefruit (Citrus paradisi Macf.). The transgenic plants contained a single- or double-locus insertion and expressed CpPSY at varying transcript levels. In comparison with the wild-type plants, the CpPSY expressing transgenic plants showed a significant increase on root length and shoot biomass under PEG-, NaCl- and mannitol-induced osmotic stress. The enhanced stress tolerance of transgenic plants was correlated with the increased endogenous ABA level and expression of stress-responsive genes, which in turn was correlated with the CpPSY copy number and expression level in different transgenic lines. Collectively, these results provide further evidence that PSY is a key enzyme regulating ABA biosynthesis and that the altered expression of other PSYs in transgenic plants may provide a similar function to that of the monocot's PSY3 in ABA biosynthesis and stress tolerance. The results also pave the way for further use of CpPSY, as well as other PSYs, as potential candidate genes for engineering tolerance to drought and salt stress in crop plants.

Influence of abiotic stresses on plant proteome and metabolome changes

“…The decreased content of these terpenoid compounds was reported in cotton under drought stress (Parida et al 2007;Massacci et al 2008). Overexpression of key enzymes involved in carotenogenesis was observed in transgenic tobacco under drought and salt stress (Cidade et al 2012). Moreover, the carotenoid content of citrus juice was dependent on the temperature conditions.…”

Effect of overexpression of citrus 9-cis-epoxycarotenoid dioxygenase 3 (CsNCED3) on the physiological response to drought stress in transgenic tobacco

“…Preparation of leafdisk explants of wild-type (WT) tobacco (Nicotiana tabacum cv. Havana), Agrobacterium transformation, shoot regeneration, and rooting were performed as described previously (Cidade et al, 2012). Primary transgenic plants (T 0 generation), representing distinct events of genetic transformation, were micropropagated in vitro and transferred to soil and grown under standardized greenhouse conditions.…”

“…The PCR amplifications were carried out as described previously (Cidade et al, 2012), using the primers 5'-CTATTTCTTTGCCCTCGGACGAG-3' and 5'-ATGAAAAAGCCTGAACTCACCGC-3' for amplification of the hptII gene fragment. The amplified DNA fragments were electrophoresed on 1.0% agarose gel, stained with ethidium bromide (0.5 µg/ml) and visualized under UV light.…”

“…The ABA concentration was determined in a pool of mature leaves of control (irrigated) and drought-stressed (10 days after the suspension of irrigation) WT and transgenic plants, as described previously (Cidade et al, 2012). The extracts were analyzed by HPLC, using a 5-µm C18 reverse-phase column (Shimadzu Shin-pack CLC ODS, Shimadzu, Kyoto, Japan).…”

Ectopic expression of the Lycium barbarum β-carotene hydroxylase gene (chyb) enhances drought and salt stress resistance by increasing xanthophyll cycle pool in tobacco

“…The PCR products were electrophoresed on 0.75 % TAE (Tris-acetate-EDTA)-agarose gels stained with ethidium bromide and visualized under UV light. For Southern blotting analysis, total genomic DNA was digested with BamHI at 37°C for 16 h. Southern blotting was performed according to (Cidade et al 2012). DNA probe was produced using the DIG-High Prime DNA Labeling and Detection Starter Kit I (Roche Diagnostics, Switzerland) in accordance with the manufacturer's instructions.…”