Constitutively expressed DHAR and MDHAR influence fruit, but not foliar ascorbate levels in tomato

Abstract

Vitamin C (L-ascorbate, AsA) is an essential nutrient required in key metabolic functions in humans and must be obtained from the diet, mainly from fruits and vegetables. Given its importance in human health and plant physiology we sought to examine the role of the ascorbate recycling enzymes monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) in tomato (Solanum lycopersicum), an economically important fruit crop. Cytosolic-targeted tomato genes Mdhar and Dhar were cloned and over-expressed under a constitutive promoter in tomato var. Micro-Tom. Lines with increased protein levels and enzymatic activity were identified and examined. Mature green and red ripe fruit from DHAR over-expressing lines had a 1.6 fold increase in AsA content in plants grown under relatively low light conditions (150 μmol m(-2) s(-1)). Conversely, MDHAR over-expressers had significantly reduced AsA levels in mature green fruits by 0.7 fold. Neither over-expressing line had altered levels of AsA in foliar tissues. These results underscore a complex regulation of the AsA pool size in tomato.

Fig. 1

Western blot analysis of AsA recycling enzymes in leaf total protein extracts from WT and three independent (A) DHAR and (B) MDHAR transgenic lines. Ten µg of total protein was loaded per lane, resolved on a 4–20% gradient SDS-PAGE, transferred to PVDF and probed with respective antibodies.

Fig. 2

In vitro enzymatic activity of (A) DHAR and (B) MDHAR in leaf protein extracts. Activity was normalized for total protein input as determined by Bradford quantification. Data represent the mean ± standard deviation (n=12). Significance at P<0.05 is indicated by asterisks. Diagonal fill=DHAR, dotted fill=MDHAR, solid fill=WT.

Fig. 3

Total ascorbate levels in (A) mature green and (B) red ripe whole fruit extracts. Data represent the mean ± standard deviation (n>9). Significance at P<0.05 is indicated by asterisks. Diagonal fill=DHAR, dotted fill=MDHAR, solid fill=WT. Grey=mature green, black=red ripe.