Down-regulating alpha-galactosidase enhances freezing tolerance in transgenic petunia - PubMed (original) (raw)
Down-regulating alpha-galactosidase enhances freezing tolerance in transgenic petunia
Joyce C Pennycooke et al. Plant Physiol. 2003 Oct.
Abstract
Alpha-galactosidase (alpha-Gal; EC 3.2.1.22) is involved in many aspects of plant metabolism, including hydrolysis of the alpha-1,6 linkage of raffinose oligosaccharides during deacclimation. To examine the relationship between endogenous sugars and freezing stress, the expression of alpha-Gal was modified in transgenic petunia (Petunia x hybrida cv Mitchell). The tomato (Lycopersicon esculentum) Lea-Gal gene under the control of the Figwort Mosaic Virus promoter was introduced into petunia in the sense and antisense orientations using Agrobacterium tumefaciens-mediated transformation. RNA gel blots confirmed that alpha-Gal transcripts were reduced in antisense lines compared with wild type, whereas sense plants had increased accumulation of alpha-Gal mRNAs. alpha-Gal activity followed a similar trend, with reduced activity in antisense lines and increased activity in all sense lines evaluated. Raffinose content of nonacclimated antisense plants increased 12- to 22-fold compared with wild type, and 22- to 53-fold after cold acclimation. Based upon electrolyte leakage tests, freezing tolerance of the antisense lines increased from -4 degrees C for cold-acclimated wild-type plants to -8 degrees C for the most tolerant antisense line. Down-regulating alpha-Gal in petunia results in an increase in freezing tolerance at the whole-plant level in nonacclimated and cold-acclimated plants, whereas overexpression of the alpha-Gal gene caused a decrease in endogenous raffinose and impaired freezing tolerance. These results suggest that engineering raffinose metabolism by transformation with alpha-Gal provides an additional method for improving the freezing tolerance of plants.
Figures
Figure 1.
Composition of sense (A) and antisense (B) constructs used in transformation of petunia. The tomato Lea-Gal gene (1,540 bp) was placed in the antisense orientation relative to the Figwort Mosaic virus promoter (FMV-P), upstream of the E9 3′-terminator sequence (B). LB and RB, Left and right borders; _npt_II, Spc/Str: resistance genes encoding kanamycin and spectomycin/streptomycin; 3′nos, E9 3′, terminator sequences.
Figure 2.
Northern-blot analysis of total RNA isolated from wild-type, sense (S3 and S7), and antisense (150–91) lines (A). Ten micrograms of total RNA was separated by electrophoresis through agarose and was hybridized with an α-32P-labeled tomato Lea-Gal probe. Ribosomal RNA stained with ethidium bromide was used as a loading control (B). α-Gal activity in wild-type, sense, and antisense lines (C).
Figure 3.
Carbohydrate content in wild-type, sense (S3 and S7), and antisense (150–91) lines 2 h into the photoperiod. Sugar content was determined by HPLC-pulsed amperometric detection. Data are from two replicated experiments with three replicates for each genotype. Bars represent the mean ±
sem
; n = 6; nonacclimated (white bars); cold-acclimated (black bars): 15°Cfor7d, 10°Cfor7d, 5°Cfor7d, and 3°C for 3 d. Starch was determined enzymatically and is estimated as micromoles of Glu per gram of dry weight.
Figure 4.
Effect of α-Gal expression and suppression on whole-plant freezing tolerance as indicated by TEL50 values in nonacclimated (A) and cold-acclimated plants (B). Sense lines (S3 and S7); antisense lines (150–91). Cold acclimation consisted of 15°C for 7 d, 10°C for 7 d, 5°C for 7 d, and 3°C for 3 d. Plants were frozen at a rate of 1°C h–1 to –8°C. Three leaves were drawn from the top portions of two separate plants for each genotype at the temperatures indicated above and were tested for electrolyte leakage as described in “Materials and Methods.”
Figure 5.
Phenotype of wild-type, sense (S3 and S7), and antisense (150–91) plants 1 week after freezing stress. A, Nonacclimated; B, cold-acclimated at 15°C for 7 d, 10°C for 7 d, 5°C for 7 d, and 3°C for 3 d. Two plants per genotype were frozen at a rate of 1°C h–1 to –8°C (only one was photographed). After the freezing stress, three leaves were drawn from the top portions of the two separate plants for each genotype at specific temperatures and were tested for electrolyte leakage. Plants were incubated at 4°C overnight and were then placed at 22°C for 1 week. The tips of all plants fell off 2 d after freezing. Full damage is not apparent in B: 149, 105, 147, and 150 due to sampling for electrolyte leakage and abscission due to injury.
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