Expression of Specific Alleles of Zinc-Finger Transcription Factors, HvSAP8 and HvSAP16, and Corresponding SNP Markers, Are Associated with Drought Tolerance in Barley Populations.

Two genes, HvSAP8 and HvSAP16, encoding Zinc-finger proteins, were identified earlier as active in barley plants. Based on bioinformatics and sequencing analysis, six SNPs were found in the promoter regions of HvSAP8 and one in HvSAP16, among parents of two barley segregating populations, Granal × Baisheshek and Natali × Auksiniai-2. ASQ and Amplifluor markers were developed for HvSAP8 and HvSAP16, one SNP in each gene, and in each of two populations, showing simple Mendelian segregation. Plants of F6 selected breeding lines and parents were evaluated in a soil-based drought screen, revealing differential expression of HvSAP8 and HvSAP16 corresponding with the stress. After almost doubling expression during the early stages of stress, HvSAP8 returned to pre-stress level or was strongly down-regulated in plants with Granal or Baisheshek genotypes, respectively. For HvSAP16 under drought conditions, a high expression level was followed by either a return to original levels or strong down-regulation in plants with Natali or Auksiniai-2 genotypes, respectively. Grain yield in the same breeding lines and parents grown under moderate drought was strongly associated with their HvSAP8 and HvSAP16 genotypes. Additionally, Granal and Natali genotypes with specific alleles at HvSAP8 and HvSAP16 were associated with improved performance under drought via higher 1000 grain weight and more shoots per plant, respectively.

Identification, gene expression and genetic polymorphism of zinc finger A20/AN1 stress-associated genes, HvSAP, in salt stressed barley from Kazakhstan.

BACKGROUND: A family of genes designated as the Zinc finger A20/AN1 Transcription factors encoding stress-associated proteins (SAP) are well described in Arabidopsis and rice, and include 14 AtSAP and 18 OsSAP genes that are associated with variable tolerances to multiple abiotic stresses. The SAP gene family displays a great diversity in its structure and across different plant species. The aim of this study was to identify all HvSAP genes in barley (Hordeum vulgare L.), to analyse the expression of selected genes in response to salinity in barley leaves and develop SNP marker for HvSAP12 to evaluate the association between genotypes of barley plants and their grain yield in field trials. RESULTS: In our study, 17 HvSAP genes were identified in barley, which were strongly homologous to rice genes. Five genes, HvSAP5, HvSAP6, HvSAP11, HvSAP12 and HvSAP15, were found to be highly expressed in leaves of barley plants in response to salt stress in hydroponics compared to controls, using both semi-quantitative RT-PCR and qPCR analyses. The Amplifluor-like SNP marker KATU-B30 was developed and used for HvSAP12 genotyping. A strong association (R2 = 0.85) was found between KATU-B30 and grain yield production per plant of 50 F3 breeding lines originating from the cross Granal × Baisheshek in field trials with drought and low to moderate salinity in Northern and Central Kazakhstan. CONCLUSIONS: A group of HvSAP genes, and HvSAP12 in particular, play an important role in the tolerance of barley plants to salinity and drought, and is associated with higher grain yield in field trials. Marker-assisted selection with SNP marker KATU-B30 can be applied in barley breeding to improve grain yield production under conditions of abiotic stress.

Salt-induced expression of intracellular vesicle trafficking genes, CaRab-GTP, and their association with Na+ accumulation in leaves of chickpea (Cicer arietinum L.).

BACKGROUND: Chickpea is an important legume and is moderately tolerant to salinity stress during the growing season. However, the level and mechanisms for salinity tolerance can vary among accessions and cultivars. A large family of CaRab-GTP genes, previously identified in chickpea, is homologous to intracellular vesicle trafficking superfamily genes that play essential roles in response to salinity stress in plants. RESULTS: To determine which of the gene family members are involved in the chickpea salt response, plants from six selected chickpea accessions (Genesis 836, Hattrick, ICC12726, Rupali, Slasher and Yubileiny) were exposed to salinity stress and expression profiles resolved for the major CaRab-GTP gene clades after 5, 9 and 15 days of salt exposure. Gene clade expression profiles (using degenerate primers targeting all members of each clade) were tested for their relationship to salinity tolerance measures, namely plant biomass and Na+ accumulation. Transcripts representing 11 out of the 13 CaRab clades could be detected by RT-PCR, but only six (CaRabA2, -B, -C, -D, -E and -H) could be quantified using qRT-PCR due to low expression levels or poor amplification efficiency of the degenerate primers for clades containing several gene members. Expression profiles of three gene clades, CaRabB, -D and -E, were very similar across all six chickpea accessions, showing a strongly coordinated network. Salt-induced enhancement of CaRabA2 expression at 15 days showed a very strong positive correlation (R2 = 0.905) with Na+ accumulation in leaves. However, salinity tolerance estimated as relative plant biomass production compared to controls, did not correlate with Na+ accumulation in leaves, nor with expression profiles of any of the investigated CaRab-GTP genes. CONCLUSION: A coordinated network of CaRab-GTP genes, which are likely involved in intracellular trafficking, are important for the salinity stress response of chickpea plants.

Energy costs of salt tolerance in crop plants.

Agriculture is expanding into regions that are affected by salinity. This review considers the energetic costs of salinity tolerance in crop plants and provides a framework for a quantitative assessment of costs. Different sources of energy, and modifications of root system architecture that would maximize water vs ion uptake are addressed. Energy requirements for transport of salt (NaCl) to leaf vacuoles for osmotic adjustment could be small if there are no substantial leaks back across plasma membrane and tonoplast in root and leaf. The coupling ratio of the H+ -ATPase also is a critical component. One proposed leak, that of Na+ influx across the plasma membrane through certain aquaporin channels, might be coupled to water flow, thus conserving energy. For the tonoplast, control of two types of cation channels is required for energy efficiency. Transporters controlling the Na+ and Cl- concentrations in mitochondria and chloroplasts are largely unknown and could be a major energy cost. The complexity of the system will require a sophisticated modelling approach to identify critical transporters, apoplastic barriers and root structures. This modelling approach will inform experimentation and allow a quantitative assessment of the energy costs of NaCl tolerance to guide breeding and engineering of molecular components.

AtNDB2 Is the Main External NADH Dehydrogenase in Mitochondria and Is Important for Tolerance to Environmental Stress.

In addition to the classical electron transport pathway coupled to ATP synthesis, plant mitochondria have an alternative pathway that involves type II NAD(P)H dehydrogenases (NDs) and alternative oxidase (AOX). This alternative pathway participates in thermogenesis in select organs of some species and is thought to help prevent cellular damage during exposure to environmental stress. Here, we investigated the function and role of one alternative path component, AtNDB2, using a transgenic approach in Arabidopsis (Arabidopsis thaliana). Disruption of AtNDB2 expression via T-DNA insertion led to a 90% decrease of external NADH oxidation in isolated mitochondria. Overexpression of AtNDB2 led to increased AtNDB2 protein abundance in mitochondria but did not enhance external NADH oxidation significantly unless AtAOX1A was concomitantly overexpressed and activated, demonstrating a functional link between these enzymes. Plants lacking either AtAOX1A or AtNDB2 were more sensitive to combined drought and elevated light treatments, whereas plants overexpressing these components showed increased tolerance and capacity for poststress recovery. We conclude that AtNDB2 is the predominant external NADH dehydrogenase in mitochondria and together with AtAOX1A forms a complete, functional, nonphosphorylating pathway of electron transport, whose operation enhances tolerance to environmental stress. This study demonstrates that at least one of the alternative NDs, as well as AOX, are important for the stress response.

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.

TaDrAp1 and TaDrAp2, Partner Genes of a Transcription Repressor, Coordinate Plant Development and Drought Tolerance in Spelt and Bread Wheat.

Down-regulator associated protein, DrAp1, acts as a negative cofactor (NC2α) in a transcription repressor complex together with another subunit, down-regulator Dr1 (NC2ß). In binding to promotors and regulating the initiation of transcription of various genes, DrAp1 plays a key role in plant transition to flowering and ultimately in seed production. TaDrAp1 and TaDrAp2 genes were identified, and their expression and genetic polymorphism were studied using bioinformatics, qPCR analyses, a 40K Single nucleotide polymorphism (SNP) microarray, and Amplifluor-like SNP genotyping in cultivars of bread wheat (Triticum aestivum L.) and breeding lines developed from a cross between spelt (T. spelta L.) and bread wheat. TaDrAp1 was highly expressed under non-stressed conditions, and at flowering, TaDrAp1 expression was negatively correlated with yield capacity. TaDrAp2 showed a consistently low level of mRNA production. Drought caused changes in the expression of both TaDrAp1 and TaDrAp2 genes in opposite directions, effectively increasing expression in lower yielding cultivars. The microarray 40K SNP assay and Amplifluor-like SNP marker, revealed clear scores and allele discriminations for TaDrAp1 and TaDrAp2 and TaRht-B1 genes. Alleles of two particular homeologs, TaDrAp1-B4 and TaDrAp2-B1, co-segregated with grain yield in nine selected breeding lines. This indicated an important regulatory role for both TaDrAp1 and TaDrAp2 genes in plant growth, ontogenesis, and drought tolerance in bread and spelt wheat.

Genomic structure and expression of alternative oxidase genes in legumes.

Mitochondria isolated from chickpea (Cicer arietinum) possess substantial alternative oxidase (AOX) activity, even in non-stressed plants, and one or two AOX protein bands were detected immunologically, depending on the organ. Four different AOX isoforms were identified in the chickpea genome: CaAOX1 and CaAOX2A, B and D. CaAOX2A was the most highly expressed form and was strongly expressed in photosynthetic tissues, whereas CaAOX2D was found in all organs examined. These results are very similar to those of previous studies with soybean and siratro. Searches of available databases showed that this pattern of AOX genes and their expression was common to at least 16 different legume species. The evolution of the legume AOX gene family is discussed, as is the in vivo impact of an inherently high AOX capacity in legumes on growth and responses to environmental stresses.

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.

A methyltransferase essential for the methoxypyrazine-derived flavour of wine.

Methoxypyrazines are a family of potent volatile compounds of diverse biological significance. They are used by insects and plants in chemical defence, are present in many vegetables and fruit and, in particular, impart herbaceous/green/vegetal sensory attributes to wines of certain varieties, including Cabernet Sauvignon. While pathways for methoxypyrazine biosynthesis have been postulated, none of the steps have been confirmed genetically. We have used the F2 progeny of a cross between a rapid flowering grapevine dwarf mutant, which does not produce 3-isobutyl-2-methoxypyrazine (IBMP), and Cabernet Sauvignon to identify the major locus responsible for accumulation of IBMP in unripe grape berries. Two candidate methyltransferase genes within the locus were identified and one was significantly associated with berry IBMP levels using association mapping. The enzyme encoded by this gene (VvOMT3) has high affinity for hydroxypyrazine precursors of methoxypyrazines. The gene is not expressed in the fruit of Pinot varieties, which lack IBMP, but is expressed in Cabernet Sauvignon at the time of accumulation of IBMP in the fruit. The results suggest that VvOMT3 is responsible for the final step in methoxypyrazine synthesis in grape berries and is the major determinant of IBMP production.

Zinc finger knuckle genes are associated with tolerance to drought and dehydration in chickpea (Cicer arietinum L.).

Chickpea (Cicer arietinum L.) is a very important food legume and needs improved drought tolerance for higher seed production in dry environments. The aim of this study was to determine diversity and genetic polymorphism in zinc finger knuckle genes with CCHC domains and their functional analysis for practical improvement of chickpea breeding. Two CaZF-CCHC genes, Ca04468 and Ca07571, were identified as potentially important candidates associated with plant responses to drought and dehydration. To study these genes, various methods were used including Sanger sequencing, DArT (Diversity array technology) and molecular markers for plant genotyping, gene expression analysis using RT-qPCR, and associations with seed-related traits in chickpea plants grown in field trials. These genes were studied for genetic polymorphism among a set of chickpea accessions, and one SNP was selected for further study from four identified SNPs between the promoter regions of each of the two genes. Molecular markers were developed for the SNP and verified using the ASQ and CAPS methods. Genotyping of parents and selected breeding lines from two hybrid populations, and SNP positions on chromosomes with haplotype identification, were confirmed using DArT microarray analysis. Differential expression profiles were identified in the parents and the hybrid populations under gradual drought and rapid dehydration. The SNP-based genotypes were differentially associated with seed weight per plant but not with 100 seed weight. The two developed and verified SNP molecular markers for both genes, Ca04468 and Ca07571, respectively, could be used for marker-assisted selection in novel chickpea cultivars with improved tolerance to drought and dehydration.

Altering the balance between AOX1A and NDB2 expression affects a common set of transcripts in Arabidopsis.

Stress-responsive components of the mitochondrial alternative electron transport pathway have the capacity to improve tolerance of plants to abiotic stress, particularly the alternative oxidase AOX1A but also external NAD(P)H dehydrogenases such as NDB2, in Arabidopsis. NDB2 and AOX1A can cooperate to entirely circumvent the classical electron transport chain in Arabidopsis mitochondria. Overexpression of AOX1A or NDB2 alone can have slightly negative impacts on plant growth under optimal conditions, while simultaneous overexpression of NDB2 and AOX1A can reverse these phenotypic effects. We have taken a global transcriptomic approach to better understand the molecular shifts that occur due to overexpression of AOX1A alone and with concomitant overexpression of NDB2. Of the transcripts that were significantly up- or down- regulated in the AOX1A overexpression line compared to wild type (410 and 408, respectively), the majority (372 and 337, respectively) reverted to wild type levels in the dual overexpression line. Several mechanisms for the AOX1A overexpression phenotype are proposed based on the functional classification of these 709 genes, which can be used to guide future experiments. Only 28 genes were uniquely up- or down-regulated when NDB2 was overexpressed in the AOX1A overexpression line. On the other hand, many unique genes were deregulated in the NDB2 knockout line. Furthermore, several changes in transcript abundance seen in the NDB2 knockout line were consistent with changes in the AOX1A overexpression line. The results suggest that an imbalance in AOX1A:NDB2 protein levels caused by under- or over-expression of either component, triggers a common set of transcriptional responses that may be important in mitochondrial redox regulation. The most significant changes were transcripts associated with photosynthesis, secondary metabolism and oxidative stress responses.

Height to first pod: A review of genetic and breeding approaches to improve combine harvesting in legume crops.

Height from soil at the base of plant to the first pod (HFP) is an important trait for mechanical harvesting of legume crops. To minimise the loss of pods, the HFP must be higher than that of the blades of most combine harvesters. Here, we review the genetic control, morphology, and variability of HFP in legumes and attempt to unravel the diverse terminology for this trait in the literature. HFP is directly related to node number and internode length but through different mechanisms. The phenotypic diversity and heritability of HFP and their correlations with plant height are very high among studied legumes. Only a few publications describe a QTL analysis where candidate genes for HFP with confirmed gene expression have been mapped. They include major QTLs with eight candidate genes for HFP, which are involved in auxin transport and signal transduction in soybean [Glycine max (L.) Merr.] as well as MADS box gene SOC1 in Medicago trancatula, and BEBT or WD40 genes located nearby in the mapped QTL in common bean (Phaseolus vulgaris L.). There is no information available about simple and efficient markers associated with HFP, which can be used for marker-assisted selection for this trait in practical breeding, which is still required in the nearest future. To our best knowledge, this is the first review to focus on this significant challenge in legume-based cropping systems.

The role of light in the regulation of ascorbate metabolism during berry development in the cultivated grapevine Vitis vinifera L.

BACKGROUND: The accumulation of L-ascorbate (Asc) in fruits is influenced by environmental factors including light quantity. Fruit exposure to ambient light is often reduced by the surrounding leaf canopy, and can be altered by cultivation practices. The influence of reduced sunlight exposure on the accumulation of Asc and its catabolites was investigated in field-grown berries of the cultivated grapevine Vitis vinifera L. cv. Shiraz. RESULTS: Growth under sunlight-eliminated conditions resulted in reduced berry fresh weight, chlorosis and a reduced total L-ascorbate pool size. The concentration of the Asc catabolite L-tartaric acid (TA) was reduced in berries grown without light. Conversely, concentrations of oxalic acid (OA), an alternative catabolite of Asc, and malic acid (MA), were unaffected by shading the berries during development. Brief and significant reductions in transcription of the Asc metabolic genes were observed in shade-grown berries after 4 weeks of dark acclimatisation whilst a key TA biosynthetic gene was not regulated by light. CONCLUSIONS: The results demonstrate that light-regulation of Asc and TA occurs only at brief stages of development and that OA and MA accumulation is light independent. Additionally, the comparable ratios of TA product to Asc precursor under both light regimes suggest that the diversion of Asc to TA is driven by factors that are not responsive to light. These findings suggest that an altered light regime is not the key to manipulating TA or MA levels in the harvested berry.

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

The proposed method is a modified and improved version of the existing "Allele-specific q-PCR" (ASQ) method for genotyping of single nucleotide polymorphism (SNP) based on fluorescence resonance energy transfer (FRET). This method is similar to frequently used techniques like Amplifluor and Kompetitive allele specific PCR (KASP), as well as others employing common universal probes (UPs) for SNP analyses. In the proposed ASQ method, the fluorophores and quencher are located in separate complementary oligonucleotides. The ASQ method is based on the simultaneous presence in PCR of the following two components: an allele-specific mixture (allele-specific and common primers) and a template-independent detector mixture that contains two or more (up to four) universal probes (UP-1 to 4) and a single universal quencher oligonucleotide (Uni-Q). The SNP site is positioned preferably at a penultimate base in each allele-specific primer, which increases the reaction specificity and allele discrimination. The proposed ASQ method is advanced in providing a very clear and effective measurement of the fluorescence emitted, with very low signal background-noise, and simple procedures convenient for customized modifications and adjustments. Importantly, this ASQ method is estimated as two- to ten-fold cheaper than Amplifluor and KASP, and much cheaper than all those methods that rely on dual-labeled probes without universal components, like TaqMan and Molecular Beacons. Results for SNP genotyping in the barley genes HvSAP16 and HvSAP8, in which stress-associated proteins are controlled, are presented as proven and validated examples. This method is suitable for bi-allelic uniplex reactions but it can potentially be used for 3- or 4-allelic variants or different SNPs in a multiplex format in a range of applications including medical, forensic, or others involving SNP genotyping.

Two O-methyltransferases involved in the biosynthesis of methoxypyrazines: grape-derived aroma compounds important to wine flavour.

Methoxypyrazines (MPs) are volatile, grape-derived aroma compounds that contribute to the distinct herbaceous characters of some wines. Although the full pathway leading to MP production has not been elucidated, there is strong evidence that the final step involves the methylation of non-volatile hydroxypyrazine (HP) precursors. Two cDNA encoding O-methyltransferases (OMTs) that have homology to an enzyme previously purified and shown to catalyse the methylation of HPs were isolated from Cabernet Sauvignon. Recombinant protein from the cDNAs (VvOMT1 and VvOMT2) was produced in E. coli and activity assays demonstrated that both encode OMTs able to methylate HPs to produce MPs, however both showed greatest activity against the flavonol quercetin. VvOMT1 has higher catalytic activity against isobutyl hydroxypyrazine compared to isopropyl hydroxypyrazine, whereas the converse is true for VvOMT2. The timing of the expression of VvOMT1 in the skin and the flesh of developing Cabernet Sauvignon grape berries was associated with the period of MP accumulation in these tissues, while VvOMT2 expression was greatest in roots, which were found to contain high levels of MPs. The MP composition of these tissues also reflects the relative levels of expression of these genes and their substrate preference. The identification of genes responsible for MP production in grapevine will help in understanding the effect of different viticultural and environmental factors on MP accumulation.

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.

Alternative NAD(P)H dehydrogenases of plant mitochondria.

Plant mitochondria have a highly branched electron transport chain that provides great flexibility for oxidation of cytosolic and matrix NAD(P)H. In addition to the universal electron transport chain found in many organisms, plants have alternative NAD(P)H dehydrogenases in the first part of the chain and a second oxidase, the alternative oxidase, in the latter part. The alternative activities are nonproton pumping and allow for NAD(P)H oxidation with varying levels of energy conservation. This provides a mechanism for plants to, for example, remove excess reducing power and balance the redox poise of the cell. This review presents our current understanding of the alternative NAD(P)H dehydrogenases present in plant mitochondria.

Analysis of Type II NAD(P)H Dehydrogenases.

Plant mitochondria contain at least four type II NAD(P)H dehydrogenases that link NAD(P)H oxidation to the inner membrane electron transport chain and bypass proton pumping at Complex I, hence ATP synthesis. These activities have been found in mitochondria isolated from all plant species analyzed to date. In this chapter, methods are presented to analyze the expression of genes encoding these dehydrogenases and to detect protein levels in mitochondria isolated from Arabidopsis (Arabidopsis thaliana). In addition, methods and assay conditions are presented to detect the activity of each of these four type II NAD(P)H dehydrogenases in isolated plant mitochondria.