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

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

Modified "Allele-Specific qPCR" Method for SNP Genotyping Based on FRET

Peer reviewe

Alternative Respiratory Pathway Component Genes (AOX and ND) in Rice and Barley and Their Response to Stress

Plants have a non-energy conserving bypass of the classical mitochondrial cytochrome c pathway, known as the alternative respiratory pathway (AP). This involves type II NAD(P)H dehydrogenases (NDs) on both sides of the mitochondrial inner membrane, ubiquinone, and the alternative oxidase (AOX). The AP components have been widely characterised from Arabidopsis, but little is known for monocot species. We have identified all the genes encoding components of the AP in rice and barley and found the key genes which respond to oxidative stress conditions. In both species, AOX is encoded by four genes; in rice OsAOX1a, 1c, 1d and 1e representing four clades, and in barley, HvAOX1a, 1c, 1d1 and 1d2, but no 1e. All three subfamilies of plant ND genes, NDA, NDB and NDC are present in both rice and barley, but there are fewer NDB genes compared to Arabidopsis. Cyanide treatment of both species, along with salt treatment of rice and drought treatment of barley led to enhanced expression of various AP components; there was a high level of co-expression of AOX1a and AOX1d, along with NDB3 during the stress treatments, reminiscent of the co-expression that has been well characterised in Arabidopsis for AtAOX1a and AtNDB2

Identification of Alternative Mitochondrial Electron Transport Pathway Components in Chickpea Indicates a Differential Response to Salinity Stress between Cultivars

All plants contain an alternative electron transport pathway (AP) in their mitochondria, consisting of the alternative oxidase (AOX) and type 2 NAD(P)H dehydrogenase (ND) families, that are thought to play a role in controlling oxidative stress responses at the cellular level. These alternative electron transport components have been extensively studied in plants like Arabidopsis and stress inducible isoforms identified, but we know very little about them in the important crop plant chickpea. Here we identify AP components in chickpea (Cicer arietinum) and explore their response to stress at the transcript level. Based on sequence similarity with the functionally characterized proteins of Arabidopsis thaliana, five putative internal (matrix)-facing NAD(P)H dehydrogenases (CaNDA1-4 and CaNDC1) and four putative external (inter-membrane space)-facing NAD(P)H dehydrogenases (CaNDB1-4) were identified in chickpea. The corresponding activities were demonstrated for the first time in purified mitochondria of chickpea leaves and roots. Oxidation of matrix NADH generated from malate or glycine in the presence of the Complex I inhibitor rotenone was high compared to other plant species, as was oxidation of exogenous NAD(P)H. In leaf mitochondria, external NADH oxidation was stimulated by exogenous calcium and external NADPH oxidation was essentially calcium dependent. However, in roots these activities were low and largely calcium independent. A salinity experiment with six chickpea cultivars was used to identify salt-responsive alternative oxidase and NAD(P)H dehydrogenase gene transcripts in leaves from a three-point time series. An analysis of the Na:K ratio and Na content separated these cultivars into high and low Na accumulators. In the high Na accumulators, there was a significant up-regulation of CaAOX1, CaNDB2, CaNDB4, CaNDA3 and CaNDC1 in leaf tissue under long term stress, suggesting the formation of a stress-modified form of the mitochondrial electron transport chain (mETC) in leaves of these cultivars. In particular, stress-induced expression of the CaNDB2 gene showed a striking positive correlation with that of CaAOX1 across all genotypes and time points. The coordinated salinity-induced up-regulation of CaAOX1 and CaNDB2 suggests that the mitochondrial alternative pathway of respiration is an important facet of the stress response in chickpea, in high Na accumulators in particular, despite high capacities for both of these activities in leaf mitochondria of non-stressed chickpeas

Identification of AtNDI1, an Internal Non-Phosphorylating NAD(P)H Dehydrogenase in Arabidopsis Mitochondria

Plant mitochondria contain non-phosphorylating NAD(P)H dehydrogenases (DHs) that are not found in animal mitochondria. The physiological function, substrate specificity, and location of enzymes within this family have yet to be conclusively determined. We have linked genome sequence information to protein and biochemical data to identify that At1g07180 (SwissProt Q8GWA1) from the Arabidopsis Genome Initiative database encodes AtNDI1, an internal NAD(P)H DH in Arabidopsis mitochondria. Three lines of evidence are presented: (a) The predicted protein sequence of AtNDI1 has high homology with other designated NAD(P)H DHs from microorganisms, (b) the capacity for matrix NAD(P)H oxidation via the rotenone-insensitive pathway is significantly reduced in the Atndi1 mutant plant line, and (c) the in vitro translation product of AtNDI1 is imported into isolated mitochondria and located on the inside of the inner membrane