Strategies to Modulate Specialized Metabolism in Mediterranean Crops: From Molecular Aspects to Field.

Plant specialized metabolites (SMs) play an important role in the interaction with the environment and are part of the plant defense response. These natural products are volatile, semi-volatile and non-volatile compounds produced from common building blocks deriving from primary metabolic pathways and rapidly evolved to allow a better adaptation of plants to environmental cues. Specialized metabolites include terpenes, flavonoids, alkaloids, glucosinolates, tannins, resins, etc. that can be used as phytochemicals, food additives, flavoring agents and pharmaceutical compounds. This review will be focused on Mediterranean crop plants as a source of SMs, with a special attention on the strategies that can be used to modulate their production, including abiotic stresses, interaction with beneficial soil microorganisms and novel genetic approaches.

Supplementary Light Differently Influences Physico-Chemical Parameters and Antioxidant Compounds of Tomato Fruits Hybrids.

One of the challenges for agriculture in the coming years will be producing more food avoiding reducing the nutritional values of fruits and vegetables, sources of nutraceutical compounds. It has been demonstrated that light-emitting diodes (LEDs) used as a supplementary light (SL) technology improve tomato yield in Mediterranean greenhouses, but few data have been reported about SL effects on fruit physio-chemical parameters. In this study, three tomato hybrid (F1) cultivars were grown for year-round production in a commercial semi-closed glasshouse in Southern Italy: red cherry type ("Sorentyno"), red plum type ("Solarino"), and yellow plum type ("Maggino"). From 120 to 243 days after transplant (DAT), Red/White/Blue LEDs were used as SL. The fruits harvested 180 DAT were analyzed and those obtained under LEDs had 3% more dry weight, 15% more total soluble solids, and 16% higher titratable acidity than fruits grown only under natural light. Generally, the antioxidant activity and the mineral profile of the fruits were not negatively influenced by SL. Lycopene content was unchanged and vitamin C content of "Sorentyno" even increased by 15% under LEDs. Overall, LEDs used as SL technology could be one of the tools used by agriculture in Mediterranean basin to produce more food maintaining high quality production.

Genome-Wide Identification of WRKY Genes in Artemisia annua: Characterization of a Putative Ortholog of AtWRKY40.

Artemisia annua L. is well-known as the plant source of artemisinin, a sesquiterpene lactone with effective antimalarial activity. Here, a putative ortholog of the Arabidopsis thaliana WRKY40 transcription factor (TF) was isolated via reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends in A. annua and named AaWRKY40. A putative nuclear localization domain was identified in silico and experimentally confirmed by using protoplasts of A. annua transiently transformed with AaWRKY40-GFP. A genome-wide analysis identified 122 WRKY genes in A. annua, and a manually curated database was obtained. The deduced proteins were categorized into the major WRKY groups, with group IIa containing eight WRKY members including AaWRKY40. Protein motifs, gene structure, and promoter regions of group IIa WRKY TFs of A. annua were characterized. The promoter region of AaWRKY group IIa genes contained several abiotic stress cis-acting regulatory elements, among which a highly conserved W-box motif was identified. Expression analysis of AaWRKY40 compared to AaWRKY1 in A. annua cell cultures treated with methyl jasmonate known to enhance artemisinin production, suggested a possible involvement of AaWRKY40 in terpenoid metabolism. Further investigation is necessary to study the role of AaWRKY40 and possible interactions with other TFs in A. annua.

Cyclic AMP mediates heat stress response by the control of redox homeostasis and ubiquitin-proteasome system.

Heat stress (HS), causing impairment in several physiological processes, is one of the most damaging environmental cues for plants. To counteract the harmful effects of high temperatures, plants activate complex signalling networks, indicated as HS response (HSR). Expression of heat shock proteins (HSPs) and adjustment of redox homeostasis are crucial events of HSR, required for thermotolerance. By pharmacological approaches, the involvement of cAMP in triggering plant HSR has been recently proposed. In this study, to investigate the role of cAMP in HSR signalling, tobacco BY-2 cells overexpressing the 'cAMP-sponge', a genetic tool that reduces intracellular cAMP levels, have been used. in vivo cAMP dampening increased HS susceptibility in a HSPs-independent way. The failure in cAMP elevation during HS caused a high accumulation of reactive oxygen species, due to increased levels of respiratory burst oxidase homolog D, decreased activities of catalase and ascorbate peroxidase, as well as down-accumulation of proteins involved in the control of redox homeostasis. In addition, cAMP deficiency impaired proteasome activity and prevented the accumulation of many proteins of ubiquitin-proteasome system (UPS). By a large-scale proteomic approach together with in silico analyses, these UPS proteins were identified in a specific cAMP-dependent network of HSR.

Salycilic Acid Induces Exudation of Crocin and Phenolics in Saffron Suspension-Cultured Cells.

The production of crocin, an uncommon and valuable apocarotenoid with strong biological activity, was obtained in a cell suspension culture of saffron (Crocus sativus L.) established from style-derived calli to obtain an in-vitro system for metabolite production. Salycilic acid (SA) was used at different concentrations to elicit metabolite production, and its effect was analyzed after a 4 days of treatment. HPLC-DAD analysis was used for total crocin quantification while the Folin-Ciocâlteu method was applied for phenolic compounds (PC) content. Interestingly, despite cell growth inhibition, a considerable exudation was observed when the highest SA concentration was applied, leading to a 7-fold enhanced production of crocin and a 4-fold increase of phenolics compared to mock cells. The maximum antioxidant activity of cell extracts was evidenced after SA 0.1 mM elicitation. Water-soluble extracts of saffron cells at concentrations of 1, 0.5, and 0.1 µg mL-1 showed significant inhibitory effects on MDA-MB-231 cancer cell viability. The heterologous vacuolar markers RFP-SYP51, GFPgl133Chi, and AleuRFP, were transiently expressed in protoplasts derived from the saffron cell suspensions, revealing that SA application caused a rapid stress effect, leading to cell death. Cell suspension elicitation with SA on the 7th day of the cell growth cycle and 24 h harvest time was optimized to exploit these cells for the highest increase of metabolite production in saffron cells.

Plant Cellular and Molecular Biotechnology: Following Mariotti's Steps.

This review is dedicated to the memory of Prof. Domenico Mariotti, who significantly contributed to establishing the Italian research community in Agricultural Genetics and carried out the first experiments of Agrobacterium-mediated plant genetic transformation and regeneration in Italy during the 1980s. Following his scientific interests as guiding principles, this review summarizes the recent advances obtained in plant biotechnology and fundamental research aiming to: (i) Exploit in vitro plant cell and tissue cultures to induce genetic variability and to produce useful metabolites; (ii) gain new insights into the biochemical function of Agrobacterium rhizogenes rol genes and their application to metabolite production, fruit tree transformation, and reverse genetics; (iii) improve genetic transformation in legume species, most of them recalcitrant to regeneration; (iv) untangle the potential of KNOTTED1-like homeobox (KNOX) transcription factors in plant morphogenesis as key regulators of hormonal homeostasis; and (v) elucidate the molecular mechanisms of the transition from juvenility to the adult phase in Prunus tree species.

Signal transduction in artichoke [Cynara cardunculus L. subsp. scolymus (L.) Hayek] callus and cell suspension cultures under nutritional stress.

Stimulated production of secondary phenolic metabolites and proline was studied by using cell cultures of artichoke [Cynara cardunculus L. subsp. scolymus (L.) Hayek] submitted to nutritional stress. Artichoke cell cultures accumulated phenolic secondary metabolites in a pattern similar to that seen in artichoke leaves and heads (capitula). This paper shows that both callus and cell suspension cultures under nutritional stress accumulated phenolic compounds and proline, at the same time their biomass production was negatively affected by nutrient deficiency. The results obtained strongly suggest that plant tissues respond to nutrient deprivation by a defensive costly mechanism, which determines the establishment of a mechanism of trade-off between growth and adaptive response. Furthermore, the results of this research suggest that perception of abiotic stress and increased phenolic metabolites are linked by a sequence of biochemical processes that also involves the intracellular free proline and the oxidative pentose phosphate pathway. The main conclusion of this paper is that, once calli and cell suspension cultures respond to nutrient deficiency, in acclimated cells the establishment of a negative correlation between primary metabolism (growth) and secondary metabolism (defence compounds) is observed.

ROS Production and Scavenging under Anoxia and Re-Oxygenation in Arabidopsis Cells: A Balance between Redox Signaling and Impairment.

Plants can frequently experience low oxygen concentrations due to environmental factors such as flooding or waterlogging. It has been reported that both anoxia and the transition from anoxia to re-oxygenation determine a strong imbalance in the cellular redox state involving the production of reactive oxygen species (ROS) and nitric oxide (NO). Plant cell cultures can be a suitable system to study the response to oxygen deprivation stress since a close control of physicochemical parameters is available when using bioreactors. For this purpose, Arabidopsis cell suspension cultures grown in a stirred bioreactor were subjected to a severe anoxic stress and analyzed during anoxia and re-oxygenation for alteration in ROS and NO as well as in antioxidant enzymes and metabolites. The results obtained by confocal microscopy showed the dramatic increase of ROS, H2O2, and NO during the anoxic shock. All the ascorbate-glutathione related parameters were altered during anoxia but restored during re-oxygenation. Anoxia also induced a slight but significant increase of α-tocopherol levels measured at the end of the treatment. Overall, the evaluation of cell defenses during anoxia and re-oxygenation in Arabidopsis cell cultures revealed that the immediate response involving the overproduction of reactive species activated the antioxidant machinery including ascorbate-glutathione system, α-tocopherol and the ROS-scavenging enzymes ascorbate peroxidase, catalase, and peroxidase making cells able to counteract the stress toward cell survival.

Carbon Fluxes between Primary Metabolism and Phenolic Pathway in Plant Tissues under Stress.

Higher plants synthesize an amazing diversity of phenolic secondary metabolites. Phenolics are defined secondary metabolites or natural products because, originally, they were considered not essential for plant growth and development. Plant phenolics, like other natural compounds, provide the plant with specific adaptations to changing environmental conditions and, therefore, they are essential for plant defense mechanisms. Plant defensive traits are costly for plants due to the energy drain from growth toward defensive metabolite production. Being limited with environmental resources, plants have to decide how allocate these resources to various competing functions. This decision brings about trade-offs, i.e., promoting some functions by neglecting others as an inverse relationship. Many studies have been carried out in order to link an evaluation of plant performance (in terms of growth rate) with levels of defense-related metabolites. Available results suggest that environmental stresses and stress-induced phenolics could be linked by a transduction pathway that involves: (i) the proline redox cycle; (ii) the stimulated oxidative pentose phosphate pathway; and, in turn, (iii) the reduced growth of plant tissues.

Wheat Bran Phenolic Acids: Bioavailability and Stability in Whole Wheat-Based Foods.

Wheat bran is generally considered a byproduct of the flour milling industry, but it is a great source of fibers, minerals, and antioxidants that are important for human health. Phenolic acids are a specific class of wheat bran components that may act as antioxidants to prevent heart disease and to lower the incidence of colon cancer. Moreover, phenolic acids have anti-inflammatory properties that are potentially significant for the promotion of gastrointestinal health. Evidence on the beneficial effects of phenolic acids as well as of other wheat bran components is encouraging the use of wheat bran as an ingredient of functional foods. After an overview of the chemistry, function, and bioavailability of wheat phenolic acids, the discussion will focus on how technologies can allow the formulation of new, functional whole wheat products with enhanced health-promoting value and safety without renouncing the good-tasting standards that are required by consumers. Finally, this review summarizes the latest studies about the stability of phenolic acids in wheat foods fortified by the addition of wheat bran, pearled fractions, or wheat bran extracts.

Phytochemical composition and anti-inflammatory activity of extracts from the whole-meal flour of Italian durum wheat cultivars.

In this study, the quali-quantitative composition of hydrophilic (phenolic acids) and lipophilic (isoprenoids) extracts from whole-meal flour of five elite Italian durum wheat cultivars was determined. Significant differences in the content of bioactive compounds were observed among the wheat extracts, in particular concerning the content of bound phenolic acids, lutein and ß-tocotrienols. The cultivars Duilio and Svevo showed the highest amount of phenolic acids and isoprenoids, respectively. Extracts were evaluated for their anti-inflammatory activity on HT-29 human colon cells by measuring the levels of interleukin 8 (IL-8) and transforming growth factor ß1 (TGF-ß1). Durum wheat extracts significantly inhibited the secretion of the pro-inflammatory IL-8 mediator at 66 µg/mL of phenolic acids and at 0.2 µg/mL of isoprenoids. Conversely, the secretion of the anti-inflammatory mediator TGF-ß1 was not modified by neither hydrophilic nor lipophilic extracts. These results provide further insight into the potential of durum wheat on human health suggesting the significance of varieties with elevated contents of bioactive components.

Subcellular compartmentalization in protoplasts from Artemisia annua cell cultures: engineering attempts using a modified SNARE protein.

Plants are ideal bioreactors for the production of macromolecules but transport mechanisms are not fully understood and cannot be easily manipulated. Several attempts to overproduce recombinant proteins or secondary metabolites failed. Because of an independent regulation of the storage compartment, the product may be rapidly degraded or cause self-intoxication. The case of the anti-malarial compound artemisinin produced by Artemisia annua plants is emblematic. The accumulation of artemisinin naturally occurs in the apoplast of glandular trichomes probably involving autophagy and unconventional secretion thus its production by undifferentiated tissues such as cell suspension cultures can be challenging. Here we characterize the subcellular compartmentalization of several known fluorescent markers in protoplasts derived from Artemisia suspension cultures and explore the possibility to modify compartmentalization using a modified SNARE protein as molecular tool to be used in future biotechnological applications. We focused on the observation of the vacuolar organization in vivo and the truncated form of AtSYP51, 51H3, was used to induce a compartment generated by the contribution of membrane from endocytosis and from endoplasmic reticulum to vacuole trafficking. The artificial compartment crossing exocytosis and endocytosis may trap artemisinin stabilizing it until extraction; indeed, it is able to increase total enzymatic activity of a vacuolar marker (RGUSChi), probably increasing its stability. Exploring the 51H3-induced compartment we gained new insights on the function of the SNARE SYP51, recently shown to be an interfering-SNARE, and new hints to engineer eukaryote endomembranes for future biotechnological applications.

Enhanced production of bioactive isoprenoid compounds from cell suspension cultures of Artemisia annua L. using ß-cyclodextrins.

Plant cell cultures as valuable tools for the production of specific metabolites can be greatly improved by the application of elicitors including cyclodextrins (CDs) for enhancing the yields of the desired plant compounds. Here the effects of 2,6-dimethyl-ß-cyclodextrins (DIMEB) on the production of carotenoids and quinones from Artemisia annua L. cell suspension cultures were investigated. The addition of 50 mM DIMEB induced an early increase of intracellular carotenoid and quinone contents, which could be observed to a higher extent for lutein (10-fold), Q9 (3-fold) and Q10 (2.5-fold). Real Time PCR analysis revealed that the expression of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) gene in DIMEB treated cell cultures after three days was 2.5-fold higher than in untreated samples, thus suggesting that the DIMEB induced increase of carotenoids and quinones could be due to the induction of the plastidial isoprenoid biosynthetic route. In addition, the DIMEB treatment induced an enhanced release of carotenoids and quinones into the culture medium of A. annua cell suspension cultures possibly due to the ability of CDs to form inclusion complexes with hydrophobic molecules.

Effects of sodium alginate bead encapsulation on the storage stability of durum wheat (Triticum durum Desf.) bran oil extracted by supercritical CO2.

The aim of this study was to investigate the influence of encapsulation on the storage stability of oil extracted by supercritical carbon dioxide from a micronized durum wheat bran fraction. Wheat bran oil was encapsulated in 2% (w/v) sodium alginate beads. Encapsulated and unencapsulated oil samples were stored at 4 or 25 °C, in daylight or darkness, over 90 days, and, at defined time points, subjected to stability evaluation based on fatty acid hydroperoxide production and tocopherol (α, ß, and γ forms), tocotrienol (α, ß, and γ forms) and carotenoid (lutein, zeaxanthin, and ß-carotene) degradation. The encapsulation of the oil into alginate beads significantly increased stability, optimally when stored at 4 °C, maintaining high levels of isoprenoids and low content of fatty acid hydroperoxides over 30 days of storage.

ß-Cyclodextrins enhance artemisinin production in Artemisia annua suspension cell cultures.

Artemisinin is a sesquiterpene antimalarial compound produced, though at low levels (0.1-1% dry weight), in Artemisia annua in which it accumulates in the glandular trichomes of the plant. Due to its antimalarial properties and short supply, efforts are being made to improve our understanding of artemisinin biosynthesis and its production. Native ß-cyclodextrins, as well as the chemically modified heptakis(2,6-di-O-methyl)-ß-cyclodextrin (DIMEB) and 2-hydroxypropyl-ß-cyclodextrins, were added to the culture medium of A. annua suspension cultures, and their effects on artemisinin production were analysed. The effects of a joint cyclodextrin and methyl jasmonate treatment were also investigated. Fifty millimolar DIMEB, as well as a combination of 50 mM DIMEB and 100 µM methyl jasmonate, was highly effective in increasing the artemisinin levels in the culture medium. The observed artemisinin level (27 µmol g(-1) dry weight) was about 300-fold higher than that observed in untreated suspensions. The influence of ß-cyclodextrins and methyl jasmonate on the expression of artemisinin biosynthetic genes was also investigated.

Tocopherol production in plant cell cultures.

Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, essential dietary components for mammals and exclusively synthesized by photosynthetic organisms. Of the four forms (alpha, beta, gamma and delta), alpha-tocopherol is the major vitamin E form present in green plant tissues, and has the highest vitamin E activity. Synthetic alpha-tocopherol, being a racemic mixture of eight different stereoisomers, always results less effective than the natural form (R,R,R) alpha-tocopherol. This raises interest in obtaining this molecule from natural sources, such as plant cell cultures. Plant cell and tissue cultures are able to produce and accumulate valuable metabolites that can be used as food additives, nutraceuticals and pharmaceuticals. Sunflower cell cultures, growing under heterotrophic conditions, were exploited to establish a suitable in vitro production system of natural alpha-tocopherol. Optimization of culture conditions, precursor feeding and elicitor application were used to improve the tocopherol yields of these cultures. Furthermore, these cell cultures were useful to investigate the relationship between alpha-tocopherol biosynthesis and photomixotrophic culture conditions, revealing the possibility to enhance tocopherol production by favouring sunflower cell photosynthetic properties. The modulation of alpha-tocopherol levels in plant cell cultures can provide useful hints for a regulatory impact on tocopherol metabolism.

Tocopherol biosynthesis is enhanced in photomixotrophic sunflower cell cultures.

Alpha-tocopherol is the most biologically active component of vitamin E and is synthesized only by photosynthetic organisms. Two heterotrophic cell lines of sunflower (Helianthus annuus L.) of differing alpha-tocopherol biosynthetic capability, three-fold higher in the high synthesizing cell line, HT, than in the low synthesizing one, LT, were previously identified. To investigate the relationship between alpha-tocopherol biosynthesis and photomixotrophic culture conditions, a new photomixotrophic sunflower cell line HS3 was established by selecting HT cells able to grow in the presence of a ten-fold reduced sucrose concentration in the culture medium. The photosynthetic properties of HS3 cells were characterized in comparison with HT and LT cells, revealing an increase in chlorophyll content, chloroplast number, and level of the photosynthesis related enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Furthermore, an enhanced expression of the gene encoding for the tocopherol biosynthetic enzyme geranyl-geranylpyrophosphate synthase (GGPPS) was observed in HS3 cells. HS3 cells also revealed a 25% and a more than three-fold higher tocopherol level than HT and LT, respectively, indicating a positive correlation between alpha-tocopherol biosynthesis of sunflower cell cultures and their photosynthetic properties. These findings can be useful for improving the tocopherol yields of the sunflower in vitro production system.

Improving alpha-tocopherol production in plant cell cultures.

Suspension cell cultures of Helianthus annuus L. were previously established for the production of the most active component of vitamin E, alpha-tocopherol, by optimizing medium composition and culture conditions. In the present work, the possibility of enhancing alpha-tocopherol production by the addition of jasmonic acid to the culture medium was investigated both in sunflower and Arabidopsis cell cultures. A considerable increase (49% and 66%, respectively) of alpha-tocopherol production was obtained in both, after a 72-h treatment with 5 microM jasmonic acid. The modulation of alpha-tocopherol levels in plant cell cultures can provide useful hints for a regulatory impact on tocopherol metabolism.

Enhancement of vitamin E production in sunflower cell cultures.

The most biologically active component of vitamin E, alpha-tocopherol, is synthesized in its most effective stereoisomeric form only by photosynthetic organisms. Using sunflower cell cultures, a suitable in vitro production system of natural alpha-tocopherol was established. The most efficient medium was found to be MS basal medium with naphthaleneacetic acid and 6-benzylaminopurine with the addition of casaminoacids and myo-inositol. Culture feeding experiments using biosynthetic precursors showed that alpha-tocopherol production improved by 30% when homogentisic acid was used. Interestingly, time-course experiments with sunflower suspension cultures showed a possible increase of 78% in alpha-tocopherol production when using cultures of longer subculture intervals. Compared to the starting plant tissue, an overall 100% increase of alpha-tocopherol was reached by these sunflower cell cultures.