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The presence of anticancer clerodane diterpenoids is a chemotaxonomic marker for the traditional Chinese medicinal plant Scutellaria barbata, although the molecular mechanisms behind clerodane biosynthesis are unknown. Here, we report a high-quality assembly of the 414.98 Mb genome of S. barbata into thirteen pseudochromosomes. Using phylogenomic and biochemical data, we mapped the plastidial metabolism of kaurene (gibberellins), abietane and clerodane diterpenes in three species of the family Lamiaceae (Scutellaria barbata, Scutellaria baicalensis and Salvia splendens), facilitating the identification of genes involved in the biosynthesis of the clerodanes, kolavenol and isokolavenol. We show that clerodane biosynthesis evolved through recruitment and neofunctionalization of genes from gibberellin and abietane metabolism. Despite the assumed monophyletic origin of clerodane biosynthesis which is widespread in species of the Lamiaceae, our data show distinct evolutionary lineages and suggest polyphyletic origins of clerodane biosynthesis in the family Lamiaceae. Our study not only provides significant insights into the evolution of clerodane biosynthetic pathways in the mint family, Lamiaceae, but also will facilitate the elucidation of anticancer clerodanes biosynthesis and future metabolic engineering efforts to increase the production of these high-value chemicals.

A comprehensive collection of 1,254 tomato accessions corresponding to European heirlooms and landraces, together with modern varieties, early domesticates and wild relatives, were analyzed by genotyping by sequencing. A continuous genetic gradient between the vintage and modern varieties was observed. European vintage tomatoes displayed very low genetic diversity, with only 298 loci out of 64,943 variants being polymorphic at the 95% threshold. European vintage tomatoes could be classified in several genetic groups. Two main clusters consisting of Spanish and Italian accessions showed a higher genetic diversity than the rest varieties, suggesting that these regions might be independent secondary centers of diversity and with a different history. Other varieties seem to be the result of a more recent complex pattern of migrations and hybridizations among the European regions. Several polymorphic loci were associated in a GWAS with fruit morphological traits in the European vintage collection, and the corresponding alleles were found to contribute to the distinctive phenotypic characteristic of the genetic varietal groups. The few highly polymorphic loci associated with morphological traits in an otherwise diversity-poor genome suggests a history of balancing selection, in which tomato farmers maintained the morphological variation by applying a high selective pressure within different varietal types.HighlightThe high phenotypic diversity observed among European vintage varieties was created by traditional farmers by combining very few polymorphic loci subjected to balancing selection.

Plants are exposed to numerous abiotic stresses. Drought is probably the most important of them and determines crop distribution around the world. Grapevine is considered to be a drought-resilient species, traditionally covering semiarid areas. Moreover, in the case of grapevine, moderate water deficit is known to improve the quality traits of grape berries and subsequently wine composition. However, against the backdrop of climate change, vines are expected to experience sustained water deficits which could be detrimental to both grape quality and yield. The influence of water deficit on two Greek Vitis vinifera L. cultivars, ‘Agiorgitiko’ and ‘Assyrtiko’, was investigated during the 2019 and 2020 vintages. Vine physiology measurements in irrigated and non-irrigated plants were performed at three time-points throughout berry development (green berry, veraison and harvest). Berry growth and composition were examined during ripening. According to the results, water deficit resulted in reduced berry size and increased levels of soluble sugars, total phenols and anthocyanins. The expression profile of specific genes, known to control grape color, aroma and flavor was altered by water availability during maturation in a cultivar-specific manner. In agreement with the increased concentration of phenolic compounds due to water deficit, genes of the phenylpropanoid pathway in the red-skinned Agiorgitiko exhibited higher expression levels and earlier up-regulation than in the white Assyrtiko. The expression profile of the other genes during maturation or in response to water deficit was depended on the vintage.

Cistus creticus subsp. creticus is a shrubby Mediterranean plant used since ancient times in folk medicine for the treatment of different diseases. C. creticus extracts and resin contain different types of secondary metabolites, such as terpenoids (predominantly labdane type diterpenoids), and phenylpropanoids. Growth conditions seem to influence the content of labdane-type diterpenes and flavan-3-ols in leaves of C. creticus subsp. creticus. Histochemical staining of leaves’ trichomes and comprehensive phytochemical characterization of resin, leaves and their exudates, indicated that long-stalked capitate trichomes of C. creticus subsp. creticus, grown both in vitro (IV) and in greenhouse (GH), are capable of producing bioactive oleoresin-related terpenoids and phenylpropanoids compounds. Bioactivity-guided approach was implemented in search for the major antibacterial compound in C. creticus resin against two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and two Gram-positive bacteria (Bacillus cereus and Micrococcus flavus). Bioautographic assay on TLC plates with separated components of Cistus resin extract, revealed a pronounced zone of microbial growth inhibition, corresponded to a highly active compound with Rf values of 0.45, structurally characterized and identified as ent-3β-acetoxy-13-epi-manoyl oxide. This finding opens the route for focusing on isolation and functional characterization of genes involved in the biosynthesis of ent-3β-acetoxy-13-epi-manoyl oxide and its precursor ent-3β-hydroxy-13-epi-manoyl oxide, with the aim to establish sustainable in vitro biotechnological protocols for its large-scale production in homologous and heterologous plant and microbial hosts.

Ascorbic acid (AsA) is an essential multifaceted phytonutrient for both the human diet and plant growth. Optimum levels of AsA accumulation combined with balanced redox homeostasis are required for normal plant development and defense response to adverse environmental stimuli. Notwithstanding its moderate AsA levels, tomatoes constitute a good source of vitamin C in the human diet. Therefore, the enhancement of AsA levels in tomato fruit attracts considerable attention, not only to improve its nutritional value but also to stimulate stress tolerance. Genetic regulation of AsA concentrations in plants can be achieved through the fine-tuning of biosynthetic, recycling, and transport mechanisms; it is also linked to changes in the whole fruit metabolism. Emerging evidence suggests that tomato synthesizes AsA mainly through the l-galactose pathway, but alternative pathways through d-galacturonate or myo-inositol, or seemingly unrelated transcription and regulatory factors, can be also relevant in certain developmental stages or in response to abiotic factors. Considering the recent advances in our understanding of AsA regulation in model and other non-model species, this review attempts to link the current consensus with novel technologies to provide a comprehensive strategy for AsA enhancement in tomatoes, without any detrimental effect on plant growth or fruit development.

Rosmarinus officinalis L. belongs to the Lamiaceae family and is cultivated worldwide due to its diverse uses as an ornamental evergreen shrub in gardening, as well as a food seasoning and a natural medicine. The present research was conducted to study the morphological traits of seven wild-grown rosemary populations in Greece, as well as the propagation of two selected populations with the most desirable ornamental characteristics, by the use of shoot cuttings and in vitro cultures. From the study of the morphological traits of the seven populations, those with interesting features were grown in the areas of Amaliada and Piperia and, thus, were chosen for studying their propagation. Rooting of the shoot cuttings was carried out during the four seasons of the year, using potassium salt Indole-3-butyric acid (K-IBA) at concentrations of 0.5 and 1 g·L−1, in various substrates under the intermittent mist or fog system. It was found that the shoot cuttings rooted easily in all four seasons, but there were better results for the population of ‘Piperia’ in autumn, with 1 g·L−1 K-IBA (80%), and ‘Amaliada’ in spring, with 0.5 g·L−1 K-IBA (82.5%), while higher rooting percentages were achieved in the fog system, on a substrate consisting of perlite and peat, in a ratio of 2:1 (85%). For the in vitro cultures, shoot tips excised from the two selected populations were successfully disinfested by pre-soaking in an antioxidant solution and then, by sterilizing them in 0.6% (w/v) NaOCl, followed by transferring them onto a Murashige and Skoog (MS) nutrient medium. ‘Amaliada’ cultures proved to be the most productive population (2.1 shoots per explant), with the highest shoot formation frequency (91.6%), when cultivated on the MS nutrient medium without plant growth regulators. For ‘Piperia’ cultures, the highest shoot formation frequency (66.6%) was achieved on the MS nutrient medium supplemented with 0.25 or 0.5 mg·L−1 6-Benzyloaminopurine (BAP) and 0.1 mg·L−1 Indole-3-acetic acid (IAA). Spontaneous root formation frequency was noticed on the MS nutrient medium, containing 0.5 mg·L−1 BAP and 0.1 mg·L−1 IAA, for both ‘Amaliada’ and ‘Piperia’ cultures (50% and 41.6%, respectively) in a single stage, with root lengths of 7.1 and 5.3 cm, respectively. Rosemary plantlets, with roots formed in vitro after transplanting them in soilless substrate, were acclimatized adequately in the greenhouse environment (~70%).

Ascorbate oxidase (AO, EC 1.10.3.3) is a copper-containing enzyme localized at the apoplast, where it catalyzes the oxidation of ascorbic acid (AA) to dehydroascorbic acid (DHA) via monodehydroascorbic acid (MDHA) intermediate. Despite it has been extensively studied, no biological roles have been definitively ascribed. To understand the role of AO in plant metabolism, fruit growth and physiology, we suppressed AO expression in melon (Cucumis melo L.) fruit. Reduction of AO activity increased AA content in melon fruit, which is the result of repression of AA oxidation and simultaneous induction of certain biosynthetic and recycling genes. As a consequence, ascorbate redox state was altered in the apoplast. Interestingly, transgenic melon fruit displayed increased ethylene production rate coincided with elevated levels of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO, EC 1.14.17.4) activity and gene expression, which might contribute to earlier ripening. Moreover, AO suppressed transgenic melon fruit exhibited a dramatic arrest in fruit growth, due to a simultaneous decrease in fruit cell size and in plasmalemma (PM) ATPase activity. All the above, support for the first time, the in vivo AO participation in the rapid fruit growth of Cucurbitaceae and further suggest an alternative route for AA increase in ripening fruit.

Fruits and vegetables are the main source of health promoting phytonutrients, including vitamins, dietary fibers and phytochemicals. Vitamins are key bioactive compounds with strong antioxidant potential including both water-soluble (vitamins B and C) and lipid-soluble (vitamins A, E and K) compounds, and are thought to limit the risk of several chronic diseases such as cancers and cardio-vascular diseases. This chapter review recent advances in the understanding of the multiple roles of vitamins, as well as their major genetic regulatory pathways within plant kingdom. Further, it discusses vitamin occurrence and diversity in different plant tissues, organelles, and horticultural species, as well as throughout fruit development and at postharvest.

Terpenes are a large class of natural products, many of which are used in cosmetics, pharmaceuticals, or biofuels. However, terpene's industrial application is frequently hindered by limited availability of natural sources or low yields of chemical synthesis. In this report, we developed a modular platform based on standardized and exchangeable parts to reproduce and potentially expand the diversity of terpene structures in Saccharomyces cerevisiae. By combining different module-specific parts, we exploited the substrate promiscuity of class I diterpene synthases to produce an array of labdane-type scaffolds. These were subsequently modified by a scaffold decoration module consisting of a mutant library of a promiscuous cytochrome P450 to afford a range of hydroxylated diterpenes. Further P450 protein engineering yielded dedicated and efficient catalysts for specific products. Terpenes produced include precursors of pharmacologically important compounds, molecules that are difficult to obtain from natural sources, or new natural products. The approach described here provides a platform on which additional gene mining, combinatorial biosynthesis, and protein engineering efforts can be integrated to sustainably explore the terpene chemical space.

Ascorbic acid (AsA) and melatonin (Mel) are known molecules participating in stress resistance, however, their combined role in counteracting the impact of salinity in plants is still unknown. In this work the effect of exogenous application of 0.50 mΜ AsA, 1 μΜ Mel and their combination (AsA + Mel) on various stress responses in leaves and roots of Citrus aurantium L. seedlings grown under 100 mΜ NaCl for 30 days was investigated. Application of AsA, Mel or AsA + Mel to saline solution decreased NaCl-induced electrolyte leakage and lipid peroxidation and prevented NaCl-associated toxicity symptoms and pigments degradation. Also, leaves exposed to combined AsA + Mel treatment displayed lower Cl(-) accumulation. Treatments with AsA and/or Mel modulated differently carbohydrates, proline, phenols, glutathione and the total antioxidant power of tissues as well as the activities of SOD, APX, POD, GR and PPO compared to NaCl alone treatment. Exposure of leaves and roots to chemical treatments and especially to combined AsA and Mel application was able to regulate CaMIPS, CaSLAH1 and CaMYB73 expression, indicating that sugar metabolism, ion homeostasis and transcription regulation were triggered by AsA and Mel. These results provide evidence that the activation of the metabolic pathways associated with combined AsA and Mel application are linked with salt adaptation in citrus plants.

Labdane diterpenes (LDs), and especially sclareol, are important feedstocks for the pharmaceutical and cosmetic industries, and therefore several lines of research have led to their heterologous production in non-photosynthetic microbes and higher plants. The potential of microalgae as bioreactors of natural products has been established for a variety of bioactive metabolites, including terpenes. In this work, a codon optimized sequence encoding a key plant labdane-type diterpene (LD) cyclase, copal-8-ol diphosphate synthase from Cistus creticus (CcCLS), was introduced into the chloroplast genome of Chlamydomonas reinhardtii. Of 49 transplastomic algal lines, 12 produced variable amounts of four LD compounds, namely ent-manoyl oxide, sclareol, labda-13-ene-8α,15-diol and ent-13-epi-manoyl oxide. The total LD concentrations measured in the transplastomic lines reached 1.172 ± 0.05 μg/mg cell DW for the highest overall producer, while the highest yield for sclareol was 0.038 ± 0.001 μg/mg cell DW. Thus, transplastomic expression of a key plant labdane diterpene cyclase in the C. reinhardtii chloroplast genome enabled the production of important plant-specific LD compounds.

Epidermal cell differentiation is a paramount and conserved process among plants. In Arabidopsis, a ternary complex formed by MYB, bHLH transcription factors and TTG1 modulates unicellular trichome morphogenesis. The formation of multicellular glandular trichomes of the xerophytic shrub Cistus creticus that accumulate labdane-type diterpenes, has attained much attention renowned for its medicinal properties. Here, we show that C. creticus TTG1 (CcTTG1) interacts with the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPLA/B) proteins, putative homologs of AtSPL4/5 that in turn interact with AtTTG1. These interactions occur between proteins from evolutionarily distant species supporting the conserved function of TTG1-SPL complex. Overexpression of AtSPL4 and AtSPL5 decreased the expression of GLABRA2 (AtGL2), the major regulator of trichome morphogenesis, resulting in trichome reduction on the adaxial surface of cauline leaves, thereby illuminating the significance of TTG1-SPLs interactions in trichome formation control. AtGL2 and AtSPL4 have opposite expression patterns during early stages of leaf development. We postulate an antagonistic effect between SPLs and the heterogeneous MYB-bHLH factors binding to TTG1. Hence, the SPLs potentially rearrange the complex, attenuating its transcriptional activity to control trichome distribution.

Previously, a suppression subtractive hybridization library was constructed to identify differentially expressed genes in peel pitting of navel orange fruit and a cDNA fragment sharing high similarities to cysteine protease genes was identified. In this study, we cloned its full-length cDNA sequence, designated CsCP, using the Rapid amplification of cDNA ends approach. It consists of 1,409 nucleotides and its ORF encodes 361 amino acids predicted to have an N-terminal signal peptide. Phylogenetic analysis revealed that CsCP belonged to the aleurain group in papain family of cysteine proteases. According to quantitative RT-PCR, the expression of CsCP was enhanced during the development of postharvest peel pitting concomitant with senescence, although it was detectable in all tested tissues including root, leaf, flower and peel of fruit. RNA gel blot analysis showed that the CsCP expression was induced by hypoxia (3% O2), but repressed by anoxia (0% O2), wounding, ethylene and high temperature (40°C). Conclusively, the CsCP is a senescence-associated gene and up-regulated during the development of citrus postharvest peel pitting, which provides a basis to understand its role in citrus peel pitting.

In a search for differentially expressed genes in peel pitting of ‘Navel’ orange fruit (Citrus sinensis L. Osbeck), a cDNA subtraction library was constructed and a sequence encoding expansin-like gene was isolated and identified as pitting related gene. Based on sequence information derived from this fragment, a full-length cDNA (CsEXP, GenBank accession no. FJ769424) of 1,083 nucleotides encoding expansin was isolated from ‘Navel’ orange by RACE approaches. CsEXP encoded a protein of 254 amino acid residues with an open reading frame located in the region between 52 and 816 bp. The calculated molecular weight of the mature protein was 27.05 kDa and theoretical isoelectric point was 7.93. The deduced protein contained conserved domains of expansin: the histidine-phenylalanine-aspartate motif in central portion, cysteine residues in N-terminus, and tryptophan residues in C-terminal region. The expression of CsEXP was higher in pitting than the control. Exposure of fruit to stresses, including wounding, anoxia, low temperature (4°C), and treatment with ethylene, increased CsEXP mRNA levels in comparison with the control untreated fruit, whereas high temperature (40°C) decreased its mRNA levels. Since low temperature, low oxygen and wounding were suspected factors inducing peel pitting of citrus fruit. The present results provided us a clue that CsEXP may play a role in response to peel pitting related stresses.

L-Ascorbate (the reduced form of vitamin C) participates in diverse biological processes including pathogen defence mechanisms, and the modulation of plant growth and morphology, and also acts as an enzyme cofactor and redox status indicator. One of its chief biological functions is as an antioxidant. L-Ascorbate intake has been implicated in the prevention/alleviation of varied human ailments and diseases including cancer. To study the regulation of accumulation of this important nutraceutical in fruit, the expression of 24 tomato (Solanum lycopersicon) genes involved in the biosynthesis, oxidation, and recycling of L-ascorbate during the development and ripening of fruit have been characterized. Taken together with L-ascorbate abundance data, the results show distinct changes in the expression profiles for these genes, implicating them in nodal regulatory roles during the process of L-ascorbate accumulation in tomato fruit. The expression of these genes was further studied in the context of abiotic and post-harvest stress, including the effects of heat, cold, wounding, oxygen supply, and ethylene. Important aspects of the hypoxic and post-anoxic response in tomato fruit are discussed. The data suggest that L-galactose-1-phosphate phosphatase could play an important role in regulating ascorbic acid accumulation during tomato fruit development and ripening. ispartof: Journal of Experimental Botany vol:60 issue:2 pages:663-678 ispartof: location:England status: published

Ascorbate is the major water-soluble antioxidant in plants and animals, and it is an essential nutrient for most insect herbivores. Therefore, ascorbate oxidase (AO) has been proposed to function as a plant defense that decreases the availability of ascorbate to insects. This hypothesis was tested by producing transgenic poplar (Populus tremula x Populus alba; Salicaceae) with 14- to 37-fold higher foliar AO activities than control (wild type) leaves and feeding these leaves to Lymantria dispar L. (Lepidoptera: Lymantriidae) caterpillars and Melanoplus sanguinipes (Fabricius) (Orthoptera: Acrididae) grasshoppers. To examine potential mechanisms of activity of AO in these insects, ascorbyl radical and/or ascorbate levels were measured in gut contents. No significant changes in ascorbyl radical or ascorbate levels were found in the midgut contents of L. dispar larvae that ingested the leaves of the AO-overexpressing genotypes compared to the control genotype, and no significant decreases in ascorbate levels were found in the foregut or midgut contents of M. sanguinipes. Treatment of control leaves with commercial AO also produced no changes in the midgut biochemistry of L. dispar larvae, as measured by levels of ascorbyl radicals. Likewise, no increase in oxidative stress was observed in L. dispar that consumed tannin-treated AO-overexpressing leaves compared with tannin-treated control genotype leaves. Performance experiments were carried out on first- and fourth-instar L. dispar larvae on leaf disks and on third instars feeding on intact leaves on trees. In no case was a significant difference found in the contrast between the control and three AO-overexpressing genotypes for relative consumption rate, relative growth rate, or nutritional indices. We conclude that elevated levels of AO in poplar are unlikely to serve as a defense against herbivores such as L. dispar or M. sanguinipes and that the low oxygen levels commonly found in the guts of caterpillars and grasshoppers may limit the activity of ingested AO in these leaf-chewing insects.

Cistus creticus ssp. creticus is an indigenous shrub of the Mediterranean area. The glandular trichomes covering its leaf surfaces secrete a resin called ‘‘ladanum”, which among others contains a number of specific labdane-type diterpenes that exhibit antibacterial and antifungal action as well as in vitro and in vivo cytotoxic and cytostatic activity against human cancer cell lines. In view of the properties and possible future exploitation of these metabolites, it was deemed necessary to study the geranylgeranyl diphosphate synthase enzyme (GGDPS, EC 2.5.1.30), a short chain prenyltransferase responsible for the synthesis of the precursor molecule of all diterpenes. In this work, we present the cloning, functional characterisation and expression profile at the gene and protein levels of two differentially expressed C. creticus full-length cDNAs, CcGGDPS1 and CcGGDPS2. Heterologous yeast cell expression system showed that these cDNAs exhibited GGDPS enzyme activity. Gene and protein expression analyses suggest that this enzyme is developmentally and tissue-regulated showing maximum expression in trichomes and smallest leaves (0.5–1.0 cm). This work is the first attempt to study the terpenoid biosynthesis at the molecular level in C. creticus ssp. creticus. 2008 Elsevier Ltd. All rights reserved.

Rosemary extracts containing the phenolic diterpenes carnosic acid and its derivative carnosol are approved food additives used in an increasingly wide range of products to enhance shelf-life, thanks to their high anti-oxidant activity. We describe here the elucidation of the complete biosynthetic pathway of carnosic acid and its reconstitution in yeast cells. Cytochrome P450 oxygenases (CYP76AH22-24) from Rosmarinus officinalis and Salvia fruticosa already characterized as ferruginol synthases are also able to produce 11-hydroxyferruginol. Modelling-based mutagenesis of three amino acids in the related ferruginol synthase (CYP76AH1) from S. miltiorrhiza is sufficient to convert it to a 11-hydroxyferruginol synthase (HFS). The three sequential C20 oxidations for the conversion of 11-hydroxyferruginol to carnosic acid are catalysed by the related CYP76AK6-8. The availability of the genes for the biosynthesis of carnosic acid opens opportunities for the metabolic engineering of phenolic diterpenes, a class of compounds with potent anti-oxidant, anti-inflammatory and anti-tumour activities., The work was supported in part by the TERPMED project (EC grant number 227448) and by the Leibniz Institute of Plant Biochemistry in Halle, Germany.

The molecular basis for the adaptation of fruit tissues to low oxygen treatments remains largely unknown. RT-PCR differential display (DD) was employed to isolate anoxic and/or hypoxic genes whose expression responded to short, low-oxygen regimes. This approach led to the isolation, cloning, successful sequencing, and bioinformatic analysis of 98 transcripts from Citrus flavedo tissues that were differentially expressed in DD gels in response to 0, 0.5, 3, and 21% O(2) for 24 h. RNA blot analysis of 25 DD clones revealed that 11 genes were induced under hypoxia and/or anoxia, 11 exhibited constitutive expression and three transcripts were suppressed by low oxygen levels. Almost half of the DD cDNAs were either of unknown function or shared no apparent homology to any expressed sequences in the GenBank/EMBL databases. Six DD genes were similar to molecules of the following functions: C-compound and carbohydrate utilization, plant development, amino acid metabolism, and biosynthesis of brasinosteroids. Time-course and stress-related experiments of low O(2)-regulated genes indicated that these genes responded differently in terms of their earliness, band intensity, and their specificity to stresses, showing that some of them can be termed hypoxia- or anoxia-induced genes.

Ascorbate oxidase (AO, EC 1.10.3.3) is a member of the multicopper oxidases family. It catalyzes the oxidation of ascorbic acid (AA) to dehydroascorbic acid (DHA) via monodehydroascorbate (MDHA), with the concomitant reduction of molecular oxygen to water. In melon (Cucumis melo), ascorbate oxidase is encoded by a multigene family comprising at least four genes. Here, we present the detailed characterization of two melon AO genes, CmAO1 and CmAO4. Gene-specific expression studies of the AO gene family in melon revealed that only CmAO1 and CmAO4 are transcriptionally active and differentially regulated dependent on tissue, developmental stage and external stimuli. Transcripts of the CmAO1 gene are present in floral and fruit tissues, whereas CmAO4 mRNA preferentially accumulates in vegetative tissues. CmAO genes were not detected in melon seeds, but CmAO4 expression is activated upon germination. CmAO4 mRNA steady-state levels are also regulated in response to wounding and heat stress, by hormones (abscisic acid, salicylic acid and jasmonates), AA and copper. These findings suggest that AO gene expression is transcriptionally regulated during fruit development and in response to hormonal cues associated with the control of cell growth and the stress response.

Research paper Ascorbate oxidase (AO) is a cell wall-localized enzyme that uses oxygen to catalyse the oxidation of ascorbate (AA) to the unstable radical monodehydroascorbate (MDHA) which rapidly disproportionates to yield dehydroascorbate (DHA) and AA, and thus contributes to the regulation of the AA redox state. Here, it is reported that in vivo lowering of the apoplast AA redox state, through increased AO expression in transgenic tobacco (Nicotiana tabacum L. cv. Xanthi), exerts no effects on the expression levels of genes involved in AA recycling under normal growth conditions, but plants display enhanced sensitivity to various oxidative stress-promoting agents. RNA blot analyses suggest that this response correlates with a general suppression of the plant's antioxidative metabolism as demonstrated by lower expression levels of AA recycling genes. Furthermore, studies using Botrytis cinerea reveal that transgenic plants exhibit increased sensitivity to fungal infection, although the response is not accompanied by a similar suppression of AA recycling gene expression. Our current findings, combined with previous studies which showed the contribution of AO in the regulation of AA redox state, suggest that the reduction in the AA redox state in the leaf apoplast of these transgenic plants results in shifts in their capacity to withstand oxidative stress imposed by agents imposing oxidative stress.

In order to distinguish the interactions between low oxygen and fruit developmental stages, preclimacteric and ripening-initiated with propylene avocado fruit were exposed to different low oxygen levels and the steady-state levels of protein and mRNA accumulation of selected hypoxic and ripening genes were investigated. In addition, the patterns of translatable mRNA were studied by 2D IEF/SDSPAGE analysis in preclimacteric fruit. Analysis of mRNA populations in preclimacteric avocado fruit revealed that low oxygen levels induced new mRNA species possibly implicated in the adaptive mechanism under low oxygen, suppressed de novo synthesized ones, or left unaffected house-keeping and/or pre-existing mRNAs, indicating that the low oxygen response is complex and involves more than a simple adaptation in energy metabolism. The accumulation of cellulase protein and mRNA was irrespective of low oxygen concentrations in preclimacteric fruit and oxygen dependent in ripening-initiated fruit. That is, preclimacteric avocado fruit contained cellulase protein and mRNA which were not suppressed by low oxygen treatment. Low oxygen tensions prevented the accumulation of cellulase protein and mRNA in ripening-initiated with propylene avocado tissues. New ADH isoenzymes were present in preclimacteric and ripening-initiated avocado fruit held in low oxygen atmospheres and correlated with elevated ADH mRNA levels. Low oxygen treatment (0–5%) did not result in increased ADH activity in avocado fruit. Neither the duration of exposure nor the developmental stage of the fruit altered this pattern, suggesting that unknown factor(s) might accumulate which possibly interfere with ADH activity under such conditions.

The last step of ascorbic acid (AA) biosynthesis is catalysed by the enzyme L-galactono-1,4-lactone dehydrogenase (GalLDH, EC 1.3.2.3), located on the inner mitochondrial membrane. The enzyme converts L-galactono-1,4-lactone to ascorbic acid (AA). In this work, the cloning and characterization of a GalLDH full-length cDNA from melon (Cucumis melo L.) are described. Melon genomic DNA Southern analysis indicated that CmGalLDH was encoded by a single gene. CmGalLDH mRNA accumulation was detected in all tissues studied, but differentially expressed during fruit development and seed germination. It is hypothesized that induction of CmGalLDH gene expression in ripening melon fruit contributes to parallel increases in the AA content and so playing a role in the oxidative ripening process. Higher CmGalLDH message abundance in light-grown seedlings compared with those raised in the dark suggests that CmGalLDH expression is regulated by light. Finally, various stresses and growth regulators resulted in no significant change in steady state levels of CmGalLDH mRNA in 20-d-old melon seedlings. To the authors' knowledge, this is the first report of GalLDH transcript induction in seed germination and differential gene expression during fruit ripening.

Dehydrins (DHNs; late embryogenesis abundant D-11) are a family of plant proteins induced in response to environmental stresses such as water stress, salinity and freezing or which occur during the late stages of embryogenesis. Previously, it was reported that citrus contains a small gene family encoding a unique class of dehydrins that differs from most other plant dehydrins in various respects, such as having an unusual K-segment similar to that of gymnosperms. In the present study, we identified by cDNA differential display analysis a 'Navel' orange 202-bp polymerase chain reaction (PCR) fragment, which encoded the typical plant angiosperm-type K-segment consensus sequence, and of which the expression was down-regulated by exposure to low oxygen levels. The full-length cDNA sequence of the orange DHN, designated csDHN (for Citrus sinensis DHN), was further isolated by 5'-and 3'-RACE; it had a total length of 933 bp and encoded a predicted polypeptide of 235 amino acids. In addition, the same 202-bp 'Navel' dehydrin PCR fragment was used to screen a 'Star Ruby' grapefruit flavedo cDNA library, and its full-length grapefruit homologue, designated cpDHN (for C. paradisi DHN) was isolated and found to have a total length of 1024 bp and to encode a predicted polypeptide of 234 amino acids. The defined orange and grapefruit DHN proteins were completely identical in the 196 amino acids of their N-terminus but differed in their C-terminus region. Overall, the csDHN and cpDHN proteins share 84% identity and contain the conserved dehydrin serine cluster (S-segment) and a putative nuclear localization signal, but csDHN has one conserved dehydrin K-segment consensus sequence, whereas cpDHN contains two dehydrin K-segments. Both csDHN and cpDHN represent single copy genes, in 'Navel' orange and 'Star Ruby' grapefruit genomes, respectively. We found that the cpDHN gene was consistently expressed in the fruit peel tissue at harvest, but that its message levels dramatically decreased during storage at either ambient or low temperatures. However, a pre-storage hot water treatment, given to enhance fruit-chilling tolerance, increased cpDHN mRNA levels during the first 3 weeks of cold storage at 2 degrees C, and enabled the message levels to be retained for up to a further 8 weeks of cold storage at 2 degrees C. The hot water treatment by itself had no inductive effect on cpDHN gene expression when the fruits were held at non-chilling temperatures. Other stresses applied to the fruit, such as wounding, UV irradiation, water stress, low oxygen and exposure to the stress hormone ethylene decreased DHN mRNA levels, whereas abscisic acid had no effect at all.

At least 14 ascorbate oxidase (AO) isoenzymes with isoelectric points in the range of pH 3/10 have been identified and their expression profile studied in various Cucurbitaceae tissues and during development and ripening of melon fruit (Cucumis melo L. var. reticulatus). At least four acidic AO isoenzymes were mainly located in the apoplastic fraction, whereas 10 basic AO isoenzymes were found in the soluble fraction. AO isoenzymes actively accumulated in the growing tissues, especially in the apex. Analysis of the AO isoenzyme profile revealed that soluble (basic) isoenzymes were mainly detected in stems and petioles while apoplastic (acidic) isoenzymes were accumulated in the outer mesocarp of melon fruit. As the fruit developed, there was a progressive decrease in the soluble basic AO isoenzymes followed by predominance of the apoplastic acidic AO isoenzymes in mature and ripe melon fruit. Moreover, AO activity and isoenzyme composition was compared in three cucurbit species. All cucurbits exhibited high AO activity in ovaries whereas mature fruit presented lower AO activity levels. In particular, cucumber (Cucumis sativus L.) ovaries showed the highest AO activity. In addition, cucumber and summer squash (Cucurbita pepo L.) exhibited a simpler AO isoenzyme profile compared to the complex profile observed in melon. # 2002 Elsevier Science B.V. All rights reserved.

Background: Salvia diterpenes have been found to have health promoting properties. Among them, carnosic acid and carnosol, tanshinones and sclareol are well known for their cardiovascular, antitumor, antiinflammatory and antioxidant activities. However, many of these compounds are not available at a constant supply and developing biotechnological methods for their production could provide a sustainable alternative. The transcriptome of S. pomifera glandular trichomes was analysed aiming to identify genes that could be used in the engineering of synthetic microbial systems. Results: In the present study, a thorough metabolite analysis of S. pomifera leaves led to the isolation and structure elucidation of carnosic acid-family metabolites including one new natural product. These labdane diterpenes seem to be synthesized through miltiradiene and ferruginol. Transcriptomic analysis of the glandular trichomes from the S. pomifera leaves revealed two genes likely involved in miltiradiene synthesis. Their products were identified and the corresponding enzymes were characterized as copalyl diphosphate synthase (SpCDS) and miltiradiene synthase (SpMilS). In addition, several CYP-encoding transcripts were identified providing a valuable resource for the identification of the biosynthetic mechanism responsible for the production of carnosic acid-family metabolites in S. pomifera. Conclusions: Our work has uncovered the key enzymes involved in miltiradiene biosynthesis in S. pomifera leaf glandular trichomes. The transcriptomic dataset obtained provides a valuable tool for the identification of the CYPs involved in the synthesis of carnosic acid-family metabolites. General Secretariat of Research and Technology (GSRT) {[}09-SYN-23-879]; grant SEE-ERA. NET PLUS {[}ERA 64/01]; grant KRIPIS {[}MIS 448840]

Avocado fruit (Persea americana Mill., cv. Hass), preclimacteric and ripening-initiated, were either hypoxically pretreated (HPT, 3% O2 for 24 h) or exposed directly to 1 and 0.25% O2 for 1–3 days (NHPT) at 20 °C. Low O2 treatments resulted in fruit maintaining higher flesh firmness. Hypoxic acclimation of preclimacteric and ripening-initiated avocado fruit increased their tolerance to subsequent 0.25 and 1% O2 levels, as assessed by visual quality attributes when the fruit returned to air. Hypoxic pre-treatment also produced a stronger expression of the anaerobically induced ADH isoenzymes than in NHPT avocado fruit that were subjected directly to 1 and 0.25% O2 for 1–3 days at 20 °C. Thus, acclimation of avocado fruit to hypoxia resulted in a beneficial increase in tolerance to subsequent ultra-low O2 treatments. © 2001 Elsevier Science B.V. All rights reserved.

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

SummaryPrevious studies (Stavroulakis and Sfakiotakis, 1993) have shown an inhibition of propylene-induced ethylene production in kiwifruit below a critical temperature range of 11–14.8CC. The aim of this research was to identify the biochemical basis of this inhibition in kiwifruit below 11–14.8°C. ‘Hayward’ kiwifruit were treated with increasing propylene concentrations at 10 and 20°C. Ethylene biosynthesis pathways and fruit ripening were investigated. Kiwifruit at 20°C in ah started autocatalysis of ethylene production and ripened after 19 d with a concomitant increase in respiration. Ethylene production and the respiration rise appeared earlier with increased propylene concentrations. Ripening proceeded immediately after propylene treatment, while ethylene autocatalysis needed a lag period of 24#8212;72 h. The latter event was attributed to the delay found in the induction of 1-aminocyclopropane-l-carboxylate synthase (ACC synthase) activity and consequently to the delayed increase of 1-aminocyclopro...

The arginine biosynthetic pathway represents an area of plant biochemistry that has been poorly investigated. Recently, the first enzyme of the arginine pathway, encoded by the N-acetyl-L-glutamate synthase gene (SlNAGS1), was isolated and characterized in tomato, and was found to be structurally similar to other predicted NAGS. SlNAGS1 accumulation patterns suggest a possible role of this gene in hypoxia-induced responses. The 35S::SlNAGS1 Arabidopsis plants accumulated ornithine at high levels and exhibited increased tolerance to salt and drought stresses. Ornithine is the intermediate compound in the arginine biosynthesis where the pathway divaricates to the production of compounds, such as proline and polyamines that are known to serve osmoprotective functions. It is therefore likely that the elevated ornithine accumulation in the SlNAGS1-overexpressing plants be coupled with the production of a pool of osmoprotectants that end up to the improved stress tolerance. The possible implications of ornithine accumulation are discussed.

Terpenes have numerous applications, ranging from pharmaceuticals to fragrances and biofuels. With increasing interest in producing terpenes sustainably and economically, there has been significant progress in recent years in developing methods for their production in microorganisms. In Saccharomyces cerevisiae, production of the 20-carbon diterpenes has so far proven to be significantly less efficient than production of their 15-carbon sesquiterpene counterparts. In this report, we identify the modular structure of geranylgeranyl diphosphate synthesis in yeast to be a major limitation in diterpene yields, and we engineer the yeast farnesyl diphosphate synthase Erg20p to produce geranylgeranyl diphosphate. Using a combination of protein and genetic engineering, we achieve significant improvements in the production of sclareol and several other isoprenoids, including cis-abienol, abietadiene and β-carotene. We also report the development of yeast strains carrying the engineered Erg20p, which support efficient isoprenoid production and can be used as a dedicated chassis for diterpene production or biosynthetic pathway elucidation. The design developed here can be applied to the production of any GGPP-derived isoprenoid and is compatible with other yeast terpene production platforms.

Rosemary (Rosmarinus officinalis) produces the phenolic diterpenes carnosic acid and carnosol, which, in addition to their general antioxidant activities, have recently been suggested as potential ingredients for the prevention and treatment of neurodegenerative diseases. Little is known about the biosynthesis of these diterpenes. Here we show that the biosynthesis of phenolic diterpenes in rosemary predominantly takes place in the glandular trichomes of young leaves, and used this feature to identify the first committed steps. Thus, a copalyl diphosphate synthase (RoCPS1) and two kaurene synthase-like (RoKSL1 and RoKSL2) encoding genes were identified and characterized. Expression in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana demonstrate that RoCPS1 converts geranylgeranyl diphosphate (GGDP) to copalyl diphosphate (CDP) of normal stereochemistry and that both RoKSL1 and RoKSL2 use normal CDP to produce an abietane diterpene. Comparison to the already characterized diterpene synthase from Salvia miltiorrhiza (SmKSL) demonstrates that the product of RoKSL1 and RoKSL2 is miltiradiene. Expression analysis supports a major contributing role for RoKSL2. Like SmKSL and the sclareol synthase from Salvia sclarea, RoKSL1/2 are diterpene synthases of the TPS-e group which have lost the internal gamma-domain. Furthermore, phylogenetic analysis indicates that RoKSL1 and RoKSL2 belong to a distinct group of KSL enzymes involved in specialized metabolism which most likely emerged before the dicot-monocot split., This work was supported by E.U. Grant agreement KBBE-227448 for the TERPMED project (http://www.terpmed.eu). Ric C. H. de Vos acknowledges the Netherlands Metabolomics Centre, which is part of the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research, for additional funding.

A small family of at least four genes encoding melon ascorbate oxidase (AO) has been identified and three members of it have been cloned. Preliminary DNA sequence determination suggested that melon AO genes code for enzymes homologous to ascorbate oxidases from other plants and similar to other multicopper oxidases. We describe detailed molecular studies addressing melon AO expression during organ specific differentiation, fruit development and ripening, and in response to wounding. In particular, AO transcript accumulation was induced in ovaries and the outer mesocarp of mature preclimacteric melon fruits, before the expression of genes encoding the necessary enzymatic activities for ethylene biosynthesis. On the other hand, AO was not expressed in late stages of fruit ripening and was repressed in wounded fruits. The role of ethylene in transcriptional regulation of AO is discussed.

Ascorbate oxidase (AO) is an apoplastic enzyme that uses oxygen to catalyse the oxidation of ascorbate (AA) to dehydroascorbate (DHA) via the unstable radical monodehydroascorbate (MDHA). Here, we report that transgenic tobacco plants ( Nicotiana tabacum L. cv. Xanthi ) with an in vivo lowered apoplastic AA redox state through increased AO expression demonstrate signs of delayed dark-induced senescence compared with wild-type plants, as shown by chlorophyll loss assay. In situ localization of hydrogen peroxide (H 2 O 2 ) suggests that, although transgenic plants have higher constitutive levels of H 2 O 2 under normal growth conditions, imposed dark-induced senescence results in smaller induction levels of H 2 O 2 , an observation which correlates with increased antioxidant enzyme activities and an induction in the expression of AA recycling genes compared with that in wild-type plants. Our current findings, combined with previous studies which showed the contribution of AO in the regulation of AA redox state, suggest that the reduction in AA redox state in the leaf apoplast of these transgenic plants results in an increase in the endogenous levels of H 2 O 2 , which provides a form of ‘acquired tolerance’ to oxidative stress imposed by dark-induced senescence.

Acta Horticulturae,Volume 1012, 15 November 2013, Pages 797-800 Fruit and vegetables constitute a rich source of bioactive compounds with health-promoting properties. Therefore, apart from acceptable qualitative and sensorial attributes, horticultural commodities with proven high nutritional and bioactive content are gaining a significant place in the market. Interestingly, apart from the significant differences in bioactive compounds among different commodities, great variability within cultivars/genotypes of the same species have been monitored, with special reference to ‘forgotten’ and/or indigenous cultivars; such differences may also be attributed to environmental factors and/or to different postharvest treatments. The understanding of bio-fortification enhancement in horticultural crops with health-promoting substances is of prime importance. In the current study, particular attention is given to a special tomato cultivar, grown in the island of Santorini, Greece that has been used together with the model cultivar ‘Ailsa Craig’ for comparative purposes with the aim to further elucidate and simultaneously enhance vitamin C content in tomato fruits. Further, this communication underlines the necessity of the employment of state-of-the-art chromatographic and spectroscopic methodologies in order to define the phytochemical composition of fresh produce. Lastly, the fingerprinting of fruit extracts via frequently applied efficient protocols coupled with system biology approaches may shed further light in the elucidation of such properties with the aim of enhancing their bioactive content.

Background Terpenoids and isoprenoids are an important class of natural products, which includes currently used drugs, high value bioactive and industrial compounds, and fuel candidates. Due to their industrial application, there is increasing interest in the development of S. cerevisiae strains capable of producing high levels of terpenoids. Results Aiming to identify new gene targets which can be manipulated to increase sesquiterpene production, a set of HMG2 positive genetic interactors were assessed as single and digenic heterozygous deletions in the presence or absence of stable HMG2(K6R) overexpression. Upon single allele deletion, most genes examined led to increased sesquiterpene production in yeast cells. Tandem heterozygous deletion of a set of three genes, the ubiquitin ligases ubc7 and ssm4/doa10, and the ER resident protein pho86, led to an 11-fold increase in caryophyllene yields (125 mg/L in shake flasks) compared to cells lacking these modifications. The effect of the heterozygous deletions appears to be due to Hmg1p and Hmg2p stabilization. Conclusion Heterozygous deletions cause significant reductions in protein levels but do not lead to growth impediments frequently seen in haploid strains. By exploiting desirable haploinsufficiencies in yeast, we identified a new set of genes that can be disrupted in tandem and cause significant stabilization of Hmgp and a substantial increase in sesquiterpene production. The approach presented here allows new genetic perturbations to be compiled on yeast cell factory strains without negatively impacting cell growth and viability.

Background To 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. Results The 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. Conclusions Results 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.