Mark W. Davey

Catalase is a sink for H2O2 and is indispensable for stress defence in C3 plants

Hydrogen peroxide (H2O2) has been implicated in many stress conditions. Control of H2O2 levels is complex and dissection of mechanisms generating and relieving H2O2 stress is difficult, particularly in intact plants. We have used transgenic tobacco with ∼10% wild‐type catalase activity to study the role of catalase and effects of H2O2 stress in plants. Catalase‐deficient plants showed no visible disorders at low light, but in elevated light rapidly developed white necrotic lesions on the leaves. Lesion formation required photorespiratory activity since damage was prevented under elevated CO2. Accumulation of H2O2 was not detected during leaf necrosis. Alternative H2O2‐scavenging mechanisms may have compensated for reduced catalase activity, as shown by increased ascorbate peroxidase and glutathione peroxidase levels. Leaf necrosis correlated with accumulation of oxidized glutathione and a 4‐fold decrease in ascorbate, indicating that catalase is critical for maintaining the redox balance during oxidative stress. Such control may not be limited to peroxisomal H2O2 production. Catalase functions as a cellular sink for H2O2, as evidenced by complementation of catalase deficiency by exogenous catalase, and comparison of catalase‐deficient and control leaf discs in removing external H2O2. Stress analysis revealed increased susceptibility of catalase‐deficient plants to paraquat, salt and ozone, but not to chilling.

Plant L-ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing

Humans are unable to synthesise L-ascorbic acid (L-AA, ascorbate, vitamin C), and are thus entirely dependent upon dietary sources to meet needs. In both plant and animal metabolism, the biological functions of L-ascorbic acid are centred around the antioxidant properties of this molecule. Considerable evidence has been accruing in the last two decades of the importance of L-AA in protecting not only the plant from oxidative stress, but also mammals from various chronic diseases that have their origins in oxidative stress. Evidence suggests that the plasma levels of L-AA in large sections of the population are sub-optimal for the health protective effects of this vitamin. Until quite recently, little focus has been given to improving the L-AA content of plant foods, either in terms of the amounts present in commercial crop varieties, or in minimising losses prior to ingestion. Further, while L-AA biosynthesis in animals was elucidated in the 1960s, 1 it is only very recently that a distinct biosynthetic route for plants has been proposed. 2 The characterisation of this new pathway will undoubtedly provide the necessary focus and impetus to enable fundamental questions on plant L-AA metabolism to be resolved. This review focuses on the role of L-AA in metabolism and the latest studies regarding its bio- synthesis, tissue compartmentalisation, turnover and catabolism. These inter-relationships are considered in relation to the potential to improve the L-AA content of crops. Methodology for the reliable analysis of L-AA in plant foods is briefly reviewed. The concentrations found in common food sources and the effects of processing, or storage prior to consumption are discussed. Finally the factors that determine the bioavailability of L-AA and how it may be improved are considered, as well as the most important future research needs. # 2000 Society of Chemical Industry

A Mutation of the Mitochondrial ABC Transporter Sta1 Leads to Dwarfism and Chlorosis in the Arabidopsis Mutant starik

A mutation in the Arabidopsis gene STARIK leads to dwarfism and chlorosis of plants with an altered morphology of leaf and cell nuclei. We show that the STARIK gene encodes the mitochondrial ABC transporter Sta1 that belongs to a subfamily of Arabidopsis half-ABC transporters. The severity of the starik phenotype is suppressed by the ectopic expression of the STA2 homolog; thus, Sta1 function is partially redundant. Sta1 supports the maturation of cytosolic Fe/S protein in Δatm1 yeast, substituting for the ABC transporter Atm1p. Similar to Atm1p-deficient yeast, mitochondria of the starik mutant accumulated more nonheme, nonprotein iron than did wild-type organelles. We further show that plant mitochondria contain a putative l-cysteine desulfurase. Taken together, our results suggest that plant mitochondria possess an evolutionarily conserved Fe/S cluster biosynthesis pathway, which is linked to the intracellular iron homeostasis by the function of Atm1p-like ABC transporters.

Genome-wide SNP detection, validation, and development of an 8K SNP array for apple.

As high-throughput genetic marker screening systems are essential for a range of genetics studies and plant breeding applications, the International RosBREED SNP Consortium (IRSC) has utilized the Illumina Infinium® II system to develop a medium- to high-throughput SNP screening tool for genome-wide evaluation of allelic variation in apple (Malus×domestica) breeding germplasm. For genome-wide SNP discovery, 27 apple cultivars were chosen to represent worldwide breeding germplasm and re-sequenced at low coverage with the Illumina Genome Analyzer II. Following alignment of these sequences to the whole genome sequence of ‘Golden Delicious’, SNPs were identified using SoapSNP. A total of 2,113,120 SNPs were detected, corresponding to one SNP to every 288 bp of the genome. The Illumina GoldenGate® assay was then used to validate a subset of 144 SNPs with a range of characteristics, using a set of 160 apple accessions. This validation assay enabled fine-tuning of the final subset of SNPs for the Illumina Infinium® II system. The set of stringent filtering criteria developed allowed choice of a set of SNPs that not only exhibited an even distribution across the apple genome and a range of minor allele frequencies to ensure utility across germplasm, but also were located in putative exonic regions to maximize genotyping success rate. A total of 7867 apple SNPs was established for the IRSC apple 8K SNP array v1, of which 5554 were polymorphic after evaluation in segregating families and a germplasm collection. This publicly available genomics resource will provide an unprecedented resolution of SNP haplotypes, which will enable marker-locus-trait association discovery, description of the genetic architecture of quantitative traits, investigation of genetic variation (neutral and functional), and genomic selection in apple.

Isolation of a cDNA coding for L-galactono-gamma-lactone dehydrogenase, an enzyme involved in the biosynthesis of ascorbic acid in plants - Purification, characterization, cDNA cloning, and expression in yeast

l-Galactono-γ-lactone dehydrogenase (EC 1.3.2.3; GLDase), an enzyme that catalyzes the final step in the biosynthesis of l-ascorbic acid was purified 1693-fold from a mitochondrial extract of cauliflower (Brassica oleracea, var. botrytis) to apparent homogeneity with an overall yield of 1.1%. The purification procedure consisted of anion exchange, hydrophobic interaction, gel filtration, and fast protein liquid chromatography. The enzyme had a molecular mass of 56 kDa estimated by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis and showed a pH optimum for activity between pH 8.0 and 8.5, with an apparent K m of 3.3 mm forl-galactono-γ-lactone. Based on partial peptide sequence information, polymerase chain reaction fragments were isolated and used to screen a cauliflower cDNA library from which a cDNA encoding GLDase was isolated. The deduced mature GLDase contained 509 amino acid residues with a predicted molecular mass of 57,837 Da. Expression of the cDNA in yeast produced a biologically active protein displaying GLDase activity. Furthermore, we identified a substrate for the enzyme in cauliflower extract, which co-eluted withl-galactono-γ-lactone by high-performance liquid chromatography, suggesting that this compound is a naturally occurring precursor of l-ascorbic acid biosynthesis in vivo.

Genetic control of fruit vitamin C contents

An F1 progeny derived from a cross between the apple (Malus x domestica) cultivars Telamon and Braeburn was used to identify quantitative trait loci (QTL) linked to the vitamin C (l-ascorbate [l-AA]) contents of fruit skin and flesh (cortex) tissues. We identified up to three highly significant QTLs for both the mean l-AA and the mean total l-AA contents of fruit flesh on both parental genetic linkage maps, confirming the quantitative nature of these traits. These QTLs account for up to a maximum of 60% of the total population variation observed in the progeny, and with a maximal individual contribution of 31% per QTL. QTLs common to both parents were identified on linkage groups (LGs) 6, 10, and 11 of the Malus reference map, while each parent also had additional unique QTLs on other LGs. Interestingly, one strong QTL on LG-17 of the Telamon linkage map colocalized with a highly significant QTL associated with flesh browning, and a minor QTL for dehydroascorbate content, supporting earlier work that links fruit l-AA contents with the susceptibility of hardfruit to postharvest browning. We also found significant minor QTLs for skin l-AA and total l-AA (l-AA + dehydroascorbate) contents in Telamon. Currently, little is known about the genetic determinants underlying tissue l-AA homeostasis, but the presence of major, highly significant QTL in both these apple genotypes under field conditions suggests the existence of common control mechanisms, allelic heterozygosity, and helps outline strategies and the potential for the molecular breeding of these traits.

γ-Glutamyl Transpeptidase in Transgenic Tobacco Plants. Cellular Localization, Processing, and Biochemical Properties

γ-Glutamyl transpeptidase (γ-GT) is a ubiquitous enzyme that catalyzes the first step of glutathione (GSH) degradation in the γ-glutamyl cycle in mammals. A cDNA encoding an Arabidopsis homolog for γ-GT was overexpressed in tobacco (Nicotiana tabacum) plants. A high level of the membrane-bound γ-GT activity was localized outside the cell in transgenic plants. The overproduced enzyme was characterized by a high affinity to GSH and was cleaved post-translationally in two unequal subunits. Thus, Arabidopsis γ-GT is similar to the mammalian enzymes in enzymatic properties, post-translational processing, and cellular localization, suggesting analogous biological functions as a key enzyme in the catabolism of GSH.

Application of Visible and Near-Infrared Reflectance Spectroscopy (Vis/NIRS) to Determine Carotenoid Contents in Banana (Musa spp.) Fruit Pulp

The analysis of carotenoids is complicated by the tendency of these compounds to react with radical species, leading to oxidative breakdown and isomerization during extraction. Therefore, protocols should be rapid and avoid unnecessary exposure to heat, acids, and so forth. Here, we evaluate the use of visible and near infrared reflectance spectroscopy (Vis/NIRS) to measure carotenoid contents in fruit from 28 Musa (banana and plantain) varieties. Carotenoid contents were first quantified using standardized RP-HPLC protocols, and these results were then used to develop algorithms to predict carotenoid contents from Vis/NIR spectra of the same samples. Cross-validation of the predictive algorithms across a genetically diverse group of varieties demonstrated that correlation coefficients between the HPLC measurements and the Vis/NIRS predictions varied from good for the total carotenoids and beta-carotene fractions (r(2)(cv), 0.84, 0.89) to reasonable for alpha-carotene and cis-carotenes (r(2)(cv), 0.61, 0.66), but there was only a poor correlation (r(2)(cv), 0.30) for the minor lutein component. Nonetheless, since approximately 90% of the Musa carotenoids consist of only alpha- and beta-carotene, results indicate that Vis/NIRS can be used for the high-throughput screening of fruit pulp samples for vitamin A nutritional content on the basis of their total carotenoids content.

Regulation of fruit ascorbic acid concentrations during ripening in high and low vitamin C tomato cultivars

BackgroundTo gain insight into the regulation of fruit ascorbic acid (AsA) pool in tomatoes, a combination of metabolite analyses, non-labelled and radiolabelled substrate feeding experiments, enzyme activity measurements and gene expression studies were carried out in fruits of the ‘low-’ and ‘high-AsA’ tomato cultivars ‘Ailsa Craig’ and ‘Santorini’ respectively.ResultsThe two cultivars exhibited different profiles of total AsA (totAsA, AsA + dehydroascorbate) and AsA accumulation during ripening, but both displayed a characteristic peak in concentrations at the breaker stage. Substrate feeding experiments demonstrated that the L-galactose pathway is the main AsA biosynthetic route in tomato fruits, but that substrates from alternative pathways can increase the AsA pool at specific developmental stages. In addition, we show that young fruits display a higher AsA biosynthetic capacity than mature ones, but this does not lead to higher AsA concentrations due to either enhanced rates of AsA breakdown (‘Ailsa Craig’) or decreased rates of AsA recycling (‘Santorini’), depending on the cultivar. In the later stages of ripening, differences in fruit totAsA-AsA concentrations of the two cultivars can be explained by differences in the rate of AsA recycling activities. Analysis of the expression of AsA metabolic genes showed that only the expression of one orthologue of GDP-L-galactose phosphorylase (SlGGP1), and of two monodehydroascorbate reductases (SlMDHAR1 and SlMDHAR3) correlated with the changes in fruit totAsA-AsA concentrations during fruit ripening in ‘Ailsa Craig’, and that only the expression of SlGGP1 was linked to the high AsA concentrations found in red ripe ‘Santorini’ fruits.ConclusionsResults indicate that ‘Ailsa Craig’ and ‘Santorini’ use complementary mechanisms to maintain the fruit AsA pool. In the low-AsA cultivar (‘Ailsa Craig’), alternative routes of AsA biosynthesis may supplement biosynthesis via L-galactose, while in the high-AsA cultivar (‘Santorini’), enhanced AsA recycling activities appear to be responsible for AsA accumulation in the later stages of ripening. Gene expression studies indicate that expression of SlGGP1 and two orthologues of SlMDHAR are closely correlated with totAsA-AsA concentrations during ripening and are potentially good candidates for marker development for breeding and selection.

Ascorbic acid mapping to study core breakdown development in 'Conference' pears

Abstract Core breakdown is a physiological disorder, characterised by discolouration of the inner core tissue, that can develop during storage of pears under certain controlled atmosphere (CA) conditions. Recent research suggested a relation between this storage disorder and ascorbic acid concentrations. Postharvest changes of ascorbic acid concentrations and spatial distribution have been investigated. Loss of ascorbic acid during delayed CA (cooling period of 3 weeks in air before CA storage) was observed, but further losses during subsequent CA storage were minimal. Browning-inducing CA storage conditions resulted in a more than 4-fold faster decrease in ascorbic acid concentration. A threshold of 0.37 mg 100 g −1 FW ascorbic acid was determined below which the incidence of internal browning was higher than 50%. Ascorbic acid maps show a strong asymmetrical distribution and illustrate that most brown tissue was located in the contour line of 0.4 mg 100 g −1 FW, which supports the 0.37 mg 100 g −1 FW threshold value. The occurrence of sound spots in the brown tissue zone corresponded with higher ascorbic acid concentrations, and could be associated to the protective capability of ascorbic acid.