Ascorbate metabolism and the developmental demand for tartaric and oxalic acids in ripening grape berries

Abstract

Background: Fresh fruits are well accepted as a good source of the dietary antioxidant ascorbic acid (Asc, Vitamin C). However, fruits such as grapes do not accumulate exceptionally high quantities of Asc. Grapes, unlike most other cultivated fruits do however use Asc as a precursor for the synthesis of both oxalic (OA) and tartaric acids (TA). TA is a commercially important product in the wine industry and due to its acidifying effect on crushed juice it can influence the organoleptic properties of the wine. Despite the interest in Asc accumulation in fruits, little is known about the mechanisms whereby Asc concentration is regulated. The purpose of this study was to gain insights into Asc metabolism in wine grapes (Vitis vinifera c.v. Shiraz.) and thus ascertain whether the developmental demand for TA and OA synthesis influences Asc accumulation in the berry.

Results: We provide evidence for developmentally differentiated up-regulation of Asc biosynthetic pathways and subsequent fluctuations in Asc, TA and OA accumulation. Rapid accumulation of Asc and a low Asc to dehydroascorbate (DHA) ratio in young berries was co-ordinated with up-regulation of three of the primary Asc biosynthetic (Smirnoff-Wheeler) pathway genes. Immature berries synthesised Asc in-situ from the primary pathway precursors D-mannose and L-galactose. Immature berries also accumulated TA in early berry development in co-ordination with up-regulation of a TA biosynthetic gene. In contrast, ripe berries have up-regulated expression of the alternative Asc biosynthetic pathway gene D-galacturonic acid reductase with only residual expression of Smirnoff-Wheeler Asc biosynthetic pathway genes and of the TA biosynthetic gene. The ripening phase was further associated with up-regulation of Asc recycling genes, a secondary phase of increased accumulation of Asc and an increase in the Asc to DHA ratio.

Conclusion: We demonstrate strong developmental regulation of Asc biosynthetic, recycling and catabolic genes in grape berries. Integration of the transcript, radiotracer and metabolite data demonstrates that Asc and TA metabolism are developmentally regulated in grapevines; resulting in low accumulated levels of the biosynthetic intermediate Asc, and high accumulated levels of the metabolic end-product TA.

Figure 1

The proposed pathways of L-ascorbate (Asc) metabolism in plants. Single arrowed lines indicate one enzymatic step whilst dashed lines indicate multiple metabolic steps not shown in detail here. Black arrows represent steps in the primary Smirnoff-Wheeler Asc biosynthetic pathway, green arrows represent steps in the alternative 'carbon salvage' Asc biosynthetic pathway, blue arrows represent steps in Asc recycling and red arrows represent steps in Asc catabolism. Intermediates are represented by circles. Closed circles representing intermediates investigated in this study. The abbreviated names of enzymes catalysing individual steps are displayed in rectangular boxes. Shaded boxes highlight the genes encoding the enzymes investigated in this study. The Smirnoff-Wheeler primary Asc biosynthetic pathway enzymes include GDP-D-mannose-3,5-epimerase (GME), EC 5.1.3.18; GDP-L-galactose phosphorylase (VTC2), EC unassigned; L-galactose-1-phosphate phosphatase (VTC4), EC unassigned; L-galactose dehydrogenase (L-GalDH), EC unassigned; L-galactono-1,4-lactone dehydrogenase (GLDH), EC 1.3.2.3. The alternative Asc biosynthetic pathway enzymes include D-galacturonic acid reductase (GalUR), EC 1.1.1.203 and aldono-lactonase, EC 3.1.1-. Enzyme catalysed steps involved in recycling Asc include monodehydroascorbate reductase (MDAR), EC 1.6.5.4 and L-dehydroascorbate (DHAR), EC 1.8.5.1. C4/C5 cleavage of Asc in Vitaceous plants proceeds via the intermediates 2-keto-L-gulonic acid, L-idonic acid, 5-keto-D-gluconic acid, L-threo-tetruronate and L-tartrate. The only characterised enzyme of this pathway is L-idonate dehydrogenase (L-IdnDH), EC 1.1.1.264. C2/C3 cleavage of Asc or L-dehydroascorbate generates oxalate and L-threonate: this pathway may occur enzymatically or non-enzymatically.

Figure 2

Accumulation of total ascorbate (tAsc) and the ascorbate catabolites tartaric (TA) and oxalic acids (OA). All graphs in the left-hand panel show Vitis vinifera c.v. Shiraz berries grown in 2005-2006 (season 1) where n = 3 and displaying SEM bars. All graphs in the right-hand panel show V. vinifera c.v. Shiraz berries grown in 2007-2008 (season 2) where n = 4 and displaying SEM bars. A. Accumulation of tAsc, B. Accumulation of TA, C. Accumulation of OA. The developmental stage of veraison is indicated by a grey dotted box.

Figure 3

Recovery of ¹⁴C-labeled products in grapevine tissue after infiltration of ¹⁴C-labeled precursors to the excised bunch stem. Two-way ANOVA with Bonferroni Post-test was performed using GraphPad Prism 5.01 (San Diego, California). The mean values with different letters above the SEM bars indicate significant differences between the proportions of radiolabelled substrates recovered in a specific product (P < 0.05). V. vinifera c.v. Shiraz bunches with 3 cm rachis attached were collected at 32 DAF. Data is presented as recovery of each ¹⁴C-labeled form in either the berry or rachis/stem as a percent of that same ¹⁴C-labeled form recovered in all tissues. n = 4, SEM bars. A. Recovery of ¹⁴C-labeled products in the berries and B. Recovery of ¹⁴C-labeled products in the combined rachis and stem tissue.

Figure 4

Transcriptional profiles of selected genes in developing berries, grown in 2007-2008 (season 2). Error bars are standard errors of four biological replicates and three technical (qRT-PCR reaction) replicates. Transcriptional changes of V. vinifera genes: A. GDP-D-mannose-3,5-epimerase (GME), B. GDP-L-galactose phosphorylase (Vtc2). C. L-galactose dehydrogenase (L-GalDH), D. L-galactono-1,4-lactone dehydrogenase (GLDH), E. D-galacturonic acid reductase (GalUR), F. L-idonate dehydrogenase (L-IdnDH), G. monodehydroascorbate reductase (MDAR) and H. dehydroascorbate reductase (DHAR). The developmental stage of veraison is indicated by a grey dotted box.

Figure 5

Accumulation of the redox forms of ascorbate in Shiraz berries across developmental season 2007-2008. The ratio of reduced ascorbate (Asc) to the oxidised form dehydroascorbate (DHA) is presented, n = 4, SEM bars. The graph is fitted with a Lowess curve (medium). The grey horizontal line indicates the developmental stage where the berry tAsc pool is composed of 50% Asc and 50% DHA. The developmental stage of veraison is indicated by a grey dotted box.