Exploring Influence of Production Area and Harvest Time on Specialized Metabolite Content of Glycyrrhiza glabra Leaves and Evaluation of Antioxidant and Anti-Aging Properties.

Calabrian Glycyrrhiza glabra is one of the most appreciated licorice varieties worldwide, and its leaves are emerging as a valuable source of bioactive compounds. Nevertheless, this biomass is usually discarded, and its valorization could contribute to boost the economic value of the licorice production chain. In this study, the effects of production area and harvest time on the specialized metabolite content of G. glabra leaves (GGL) and also the antioxidant and anti-aging properties are evaluated to explore the potential of this untapped resource and to select the most optimal harvesting practices. GGL exhibited high levels of specialized metabolites (4-30 g/100 g of dry leaf) and the most abundant ones are pinocembrin, prenylated flavanones (licoflavanone and glabranin), and prenylated dihydrostilbenes. Their levels and antioxidant capacity in extracts are influenced by both production area and harvest time, showing a decisive role on specialized metabolites accumulation. Interestingly, GGL extracts strongly attenuate the toxicity of α-synuclein, the intracellular reactive oxygen species (ROS) content, and cellular senescence on Saccharomyces cerevisiae expressing human α-synuclein model, showing great potential to prevent aging and age-related disorders. These results provide insights into the phytochemical dynamics of GGL, identifying the best harvesting site and period to obtain bioactive-rich sources with potential uses in the food, nutraceutical, and pharmaceutical sectors.

Enzyme-Based Biostimulants Influence Physiological and Biochemical Responses of Lactuca sativa L.

Biostimulants (BSs) are natural materials (i.e., organic or inorganic compounds, and/or microorganisms) having beneficial effects on plant growth and productivity, and able to improve resilience/tolerance to biotic and abiotic stresses. Therefore, they represent an innovative alternative to the phyto- and agrochemicals, being environmentally friendly and a valuable tool to cope with extreme climate conditions. The objective of this study was to investigate the effects of several biomolecules (i.e., Xylanase, ß-Glucosidase, Chitinase, and Tramesan), alone or in combinations, on lettuce plant growth and quality. With this aim, the influence of these biomolecules on biomass, pigment content, and antioxidant properties in treated plants were investigated. Our results showed that Xylanase and, to a lesser extent, ß-Glucosidase, have potentially biostimulant activity for lettuce cultivation, positively influencing carotenoids, total polyphenols, and ascorbic acid contents; similar effects were found with respect to antioxidative properties. Furthermore, the effect of the more promising molecules (Xylanase and ß-Glucosidase) was also evaluated in kiwifruit cultured cells to test their putative role as sustainable input for plant cell biofactories. The absence of phytotoxic effects of both molecules at low doses (0.1 and 0.01 µM), and the significantly enhanced cell biomass growth, indicates a positive impact on kiwifruit cells.

Development of a High Oleic Cardoon Cell Culture Platform by SAD Overexpression and RNAi-Mediated FAD2.2 Silencing.

The development of effective tools for the sustainable supply of phyto-ingredients and natural substances with reduced environmental footprints can help mitigate the dramatic scenario of climate change. Plant cell cultures-based biorefineries can be a technological advancement to face this challenge and offer a potentially unlimited availability of natural substances, in a standardized composition and devoid of the seasonal variability of cultivated plants. Monounsaturated (MUFA) fatty acids are attracting considerable attention as supplements for biodegradable plastics, bio-additives for the cosmetic industry, and bio-lubricants. Cardoon (Cynara cardunculus L. var. altilis) callus cultures accumulate fatty acids and polyphenols and are therefore suitable for large-scale production of biochemicals and valuable compounds, as well as biofuel precursors. With the aim of boosting their potential uses, we designed a biotechnological approach to increase oleic acid content through Agrobacterium tumefaciens-mediated metabolic engineering. Bioinformatic data mining in the C. cardunculus transcriptome allowed the selection and molecular characterization of SAD (stearic acid desaturase) and FAD2.2 (fatty acid desaturase) genes, coding for key enzymes in oleic and linoleic acid formation, as targets for metabolic engineering. A total of 22 and 27 fast-growing independent CcSAD overexpressing (OE) and CcFAD2.2 RNAi knocked out (KO) transgenic lines were obtained. Further characterization of five independent transgenic lines for each construct demonstrated that, successfully, SAD overexpression increased linoleic acid content, e.g., to 42.5%, of the relative fatty acid content, in the CcSADOE6 line compared with 30.4% in the wild type (WT), whereas FAD2.2 silencing reduced linoleic acid in favor of the accumulation of its precursor, oleic acid, e.g., to almost 57% of the relative fatty acid content in the CcFAD2.2KO2 line with respect to 17.7% in the WT. Moreover, CcSADOE6 and CcFAD2.2KO2 were also characterized by a significant increase in total polyphenolic content up to about 4.7 and 4.1 mg/g DW as compared with 2.7 mg/g DW in the WT, mainly due to the accumulation of dicaffeoyl quinic and feruloyl quinic acids. These results pose the basis for the effective creation of an engineered cardoon cells-based biorefinery accumulating high levels of valuable compounds from primary and specialized metabolism to meet the industrial demand for renewable and sustainable sources of innovative bioproducts.

Abiotic Stresses Elicitation Potentiates the Productiveness of Cardoon Calli as Bio-Factories for Specialized Metabolites Production.

Cultivated cardoon (Cynara cardunculus L. var altilis) is a Mediterranean traditional food crop. It is adapted to xerothermic conditions and also grows in marginal lands, producing a large biomass rich in phenolic bioactive metabolites and has therefore received attention for pharmaceutical, cosmetic and innovative materials applications. Cardoon cell cultures can be used for the biotechnological production of valuable molecules in accordance with the principles of cellular agriculture. In the current study, we developed an elicitation strategy on leaf-derived cardoon calli for boosting the production of bioactive extracts for cosmetics. We tested elicitation conditions that trigger hyper-accumulation of bioactive phenolic metabolites without compromising calli growth through the application of chilling and salt stresses. We monitored changes in growth, polyphenol accumulation, and antioxidant capability, along with transcriptional variations of key chlorogenic acid and flavonoids biosynthetic genes. At moderate stress intensity and duration (14 days at 50-100 mM NaCl) salt exerted the best eliciting effect by stimulating total phenols and antioxidant power without impairing growth. Hydroalcoholic extracts from elicited cardoon calli with optimal growth and bioactive metabolite accumulation were demonstrated to lack cytotoxicity by MTT assay and were able to stimulate pro-collagen and aquaporin production in dermal cells. In conclusion, we propose a "natural" elicitation system that can be easily and safely employed to boost bioactive metabolite accumulation in cardoon cell cultures and also in pilot-scale cell culture production.

Towards a Cardoon (Cynara cardunculus var. altilis)-Based Biorefinery: A Case Study of Improved Cell Cultures via Genetic Modulation of the Phenylpropanoid Pathway.

Cultivated cardoon (Cynara cardunculus var. altilis L.) is a promising candidate species for the development of plant cell cultures suitable for large-scale biomass production and recovery of nutraceuticals. We set up a protocol for Agrobacterium tumefaciens-mediated transformation, which can be used for the improvement of cardoon cell cultures in a frame of biorefinery. As high lignin content determines lower saccharification yields for the biomass, we opted for a biotechnological approach, with the purpose of reducing lignin content; we generated transgenic lines overexpressing the Arabidopsis thaliana MYB4 transcription factor, a known repressor of lignin/flavonoid biosynthesis. Here, we report a comprehensive characterization, including metabolic and transcriptomic analyses of AtMYB4 overexpression cardoon lines, in comparison to wild type, underlining favorable traits for their use in biorefinery. Among these, the improved accessibility of the lignocellulosic biomass to degrading enzymes due to depletion of lignin content, the unexpected increased growth rates, and the valuable nutraceutical profiles, in particular for hydroxycinnamic/caffeoylquinic and fatty acids profiles.

Specialized Metabolites and Valuable Molecules in Crop and Medicinal Plants: The Evolution of Their Use and Strategies for Their Production.

Plants naturally produce a terrific diversity of molecules, which we exploit for promoting our overall well-being. Plants are also green factories. Indeed, they may be exploited to biosynthesize bioactive molecules, proteins, carbohydrates and biopolymers for sustainable and large-scale production. These molecules are easily converted into commodities such as pharmaceuticals, antioxidants, food, feed and biofuels for multiple industrial processes. Novel plant biotechnological, genetics and metabolic insights ensure and increase the applicability of plant-derived compounds in several industrial sectors. In particular, synergy between disciplines, including apparently distant ones such as plant physiology, pharmacology, 'omics sciences, bioinformatics and nanotechnology paves the path to novel applications of the so-called molecular farming. We present an overview of the novel studies recently published regarding these issues in the hope to have brought out all the interesting aspects of these published studies.

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.

Onion Peel: Turning a Food Waste into a Resource.

Food waste is a serious problem for food processing industries, especially when it represents a loss of a valuable source of nutrients and phytochemicals. Increasing consumer demand for processed food poses the problem of minimizing waste by conversion into useful products. In this regard, onion (Allium cepa) waste consisting mainly of onion skin is rich in bioactive phenolic compounds. Here, we characterized the flavonoid profiles and biological activities of onion skin wastes of two traditional varieties with protected geographical indication (PGI), the red "Rossa di Tropea" and the coppery "Ramata di Montoro", typically cultivated in a niche area in southern Italy. The phytochemical profiles of exhaustive extracts, characterized by ultra-high-performance liquid chromatography coupled with ultraviolet (UV) detection and high-resolution mass spectrometry, revealed that flavonols and anthocyanins were the characteristic metabolite classes of onion skins. Quercetin, quercetin glucosides and their dimer and trimer derivatives, and, among anthocyanins, cyanidin 3-glucoside, were the most abundant bioactive compounds. The potential of onion skins was evaluated by testing several biological activities: ABTS/oxygen radical absorbance capacity (ORAC) and in vitro alpha-glucosidase assays were performed to evaluate the antioxidant and anti-diabetic properties of the extracts and of their main compounds, respectively, and proliferative activity was evaluated by MTT assay on human fibroblasts. In the present study, by observing various biological properties of "Rossa di Tropea" and "Ramata di Montoro" onion-dried skins, we clearly indicated that this agricultural waste can provide bioactive molecules for multiple applications, from industrial to nutraceutical and cosmetical sectors.

Changes in Phenolics and Fatty Acids Composition and Related Gene Expression during the Development from Seed to Leaves of Three Cultivated Cardoon Genotypes.

Cultivated cardoon (Cynara cardunculus var. altilis) has long been used as a food and medicine remedy and nowadays is considered a functional food. Its leaf bioactive compounds are mostly represented by chlorogenic acids and coumaroyl derivatives, known for their nutritional value and bioactivity. Having antioxidant and hepatoprotective properties, these molecules are used for medicinal purposes. Apart from the phenolic compounds in green tissues, cultivated cardoon is also used for the seed oil, having a composition suitable for the human diet, but also valuable as feedstock for the production of biofuel and biodegradable bioplastics. Given the wide spectrum of valuable cardoon molecules and their numerous industrial applications, a detailed characterization of different organs and tissues for their metabolic profiles as well as an extensive transcriptional analysis of associated key biosynthetic genes were performed to provide a deeper insight into metabolites biosynthesis and accumulation sites. This study aimed to provide a comprehensive analysis of the phenylpropanoids profile through UHPLC-Q-Orbitrap HRMS analysis, of fatty acids content through GC-MS analysis, along with quantitative transcriptional analyses by qRT-PCR of hydroxycinnamoyl-quinate transferase (HQT), stearic acid desaturase (SAD), and fatty acid desaturase (FAD) genes in seeds, hypocotyls, cotyledons and leaves of the cardoon genotypes "Spagnolo", "Bianco Avorio", and "Gigante". Both oil yield and total phenols accumulation in all the tissues and organs indicated higher production in "Bianco Avorio" and "Spagnolo" than in "Gigante". Antioxidant activity evaluation by DPPH, ABTS, and FRAP assays mirrored total phenols content. Overall, this study provides a detailed analysis of tissue composition of cardoon, enabling to elucidate value-added product accumulation and distribution during plant development and hence contributing to better address and optimize the sustainable use of this natural resource. Besides, our metabolic and transcriptional screening could be useful to guide the selection of superior genotypes.

Physiological, Biochemical, and Metabolic Responses to Short and Prolonged Saline Stress in Two Cultivated Cardoon Genotypes.

Cultivated cardoon is a multipurpose crop with adaptability to limiting environments. Two genotypes ("Bianco Avorio" and "Spagnolo") were comparatively characterized in response to short and prolonged 100 mM NaCl stress in hydroponics. Salt induced no growth variations between genotypes or symptoms of NaCl toxicity, but boosted ABA accumulation in roots and leaves. Both genotypes had high constitutive phenol content, whose major components were depleted upon 2 days of stress only in "Bianco Avorio". Prolonged stress stimulated accumulation of proline, phenylpropanoids, and related transcripts, and non-enzymatic antioxidant activity. Decreased antioxidant enzymes activities upon short stress did not occur for APX in "Spagnolo", indicating a stronger impairment of enzymatic defenses in "Bianco Avorio". Nonetheless, H2O2 and lipid peroxidation did not increase under short and prolonged stress in both genotypes. Overall, the two genotypes appear to share similar defense mechanisms but, in the short term, "Bianco Avorio" depends mainly on non-enzymatic antioxidant phenylpropanoids for ROS scavenging, while "Spagnolo" maintains a larger arsenal of defenses. Upon prolonged stress, proline could have contributed to protection of metabolic functions in both genotypes. Our results provide cues that can be exploited for cardoon genetic improvement and highlight genotypic differences for breeding salinity tolerant varieties.

Transcriptional, metabolic and DNA methylation changes underpinning the response of Arundo donax ecotypes to NaCl excess.

MAIN CONCLUSION: Arundo donax ecotypes react differently to salinity, partly due to differences in constitutive defences and methylome plasticity. Arundo donax L. is a C3 fast-growing grass that yields high biomass under stress. To elucidate its ability to produce biomass under high salinity, we investigated short/long-term NaCl responses of three ecotypes through transcriptional, metabolic and DNA methylation profiling of leaves and roots. Prolonged salt treatment discriminated the sensitive ecotype 'Cercola' from the tolerant 'Domitiana' and 'Canneto' in terms of biomass. Transcriptional and metabolic responses to NaCl differed between the ecotypes. In roots, constitutive expression of ion transporter and stress-related transcription factors' genes was higher in 'Canneto' and 'Domitiana' than 'Cercola' and 21-day NaCl drove strong up-regulation in all ecotypes. In leaves, unstressed 'Domitiana' confirmed higher expression of the above genes, whose transcription was repressed in 'Domitiana' but induced in 'Cercola' following NaCl treatment. In all ecotypes, salinity increased proline, ABA and leaf antioxidants, paralleled by up-regulation of antioxidant genes in 'Canneto' and 'Cercola' but not in 'Domitiana', which tolerated a higher level of oxidative damage. Changes in DNA methylation patterns highlighted a marked capacity of the tolerant 'Domitiana' ecotype to adjust DNA methylation to salt stress. The reduced salt sensitivity of 'Domitiana' and, to a lesser extent, 'Canneto' appears to rely on a complex set of constitutively activated defences, possibly due to the environmental conditions of the site of origin, and on higher plasticity of the methylome. Our findings provide insights into the mechanisms of adaptability of A. donax ecotypes to salinity, offering new perspectives for the improvement of this species for cultivation in limiting environments.

Transcriptome reprogramming, epigenetic modifications and alternative splicing orchestrate the tomato root response to the beneficial fungus Trichoderma harzianum.

Beneficial interactions of rhizosphere microorganisms are widely exploited for plant biofertilization and mitigation of biotic and abiotic constraints. To provide new insights into the onset of the roots-beneficial microorganisms interplay, we characterised the transcriptomes expressed in tomato roots at 24, 48 and 72 h post inoculation with the beneficial fungus Trichoderma harzianum T22 and analysed the epigenetic and post-trascriptional regulation mechanisms. We detected 1243 tomato transcripts that were differentially expressed between Trichoderma-interacting and control roots and 83 T. harzianum transcripts that were differentially expressed between the three experimental time points. Interaction with Trichoderma triggered a transcriptional response mainly ascribable to signal recognition and transduction, stress response, transcriptional regulation and transport. In tomato roots, salicylic acid, and not jasmonate, appears to have a prominent role in orchestrating the interplay with this beneficial strain. Differential regulation of many nutrient transporter genes indicated a strong effect on plant nutrition processes, which, together with the possible modifications in root architecture triggered by ethylene/indole-3-acetic acid signalling at 72 h post inoculation may concur to the well-described growth-promotion ability of this strain. Alongside, T. harzianum-induced defence priming and stress tolerance may be mediated by the induction of reactive oxygen species, detoxification and defence genes. A deeper insight into gene expression and regulation control provided first evidences for the involvement of cytosine methylation and alternative splicing mechanisms in the plant-Trichoderma interaction. A model is proposed that integrates the plant transcriptomic responses in the roots, where interaction between the plant and beneficial rhizosphere microorganisms occurs.

Metabolic and Molecular Changes of the Phenylpropanoid Pathway in Tomato (Solanum lycopersicum) Lines Carrying Different Solanum pennellii Wild Chromosomal Regions.

Solanum lycopersicum represents an important dietary source of bioactive compounds including the antioxidants flavonoids and phenolic acids. We previously identified two genotypes (IL7-3 and IL12-4) carrying loci from the wild species Solanum pennellii, which increased antioxidants in the fruit. Successively, these lines were crossed and two genotypes carrying both introgressions at the homozygous condition (DHO88 and DHO88-SL) were selected. The amount of total antioxidant compounds was increased in DHOs compared to both ILs and the control genotype M82. In order to understand the genetic mechanisms underlying the positive interaction between the two wild regions pyramided in DHO genotypes, detailed analyses of the metabolites accumulated in the fruit were carried out by colorimetric methods and LC/MS/MS. These analyses evidenced a lower content of flavonoids in DHOs and in ILs, compared to M82. By contrast, in the DHOs the relative content of phenolic acids increased, particularly the fraction of hexoses, thus evidencing a redirection of the phenylpropanoid flux toward the biosynthesis of phenolic acid glycosides in these genotypes. In addition, the line DHO88 exhibited a lower content of free phenolic acids compared to M82. Interestingly, the two DHOs analyzed differ in the size of the wild region on chromosome 12. Genes mapping in the introgression regions were further investigated. Several genes of the phenylpropanoid biosynthetic pathway were identified, such as one 4-coumarate:CoA ligase and two UDP-glycosyltransferases in the region 12-4 and one chalcone isomerase and one UDP-glycosyltransferase in the region 7-3. Transcriptomic analyses demonstrated a different expression of the detected genes in the ILs and in the DHOs compared to M82. These analyses, combined with biochemical analyses, suggested a central role of the 4-coumarate:CoA ligase in redirecting the phenylpropanoid pathways toward the biosynthesis of phenolic acids in the pyramided lines. Moreover, analyses here carried out suggest the presence in the introgression regions of novel regulatory proteins, such as one Myb4 detected on chromosome 7 and one bHLH detected in chromosome 12. Overall our data indicate that structural and regulatory genes identified in this study might have a key role for the manipulation of the phenylpropanoid metabolic pathway in tomato fruit.

Phenylpropanoids Accumulation in Eggplant Fruit: Characterization of Biosynthetic Genes and Regulation by a MYB Transcription Factor.

Phenylpropanoids are major secondary metabolites in eggplant (Solanum melongena) fruits. Chlorogenic acid (CGA) accounts for 70-90% of total phenolics in flesh tissues, while anthocyanins are mainly present in the fruit skin. As a contribution to the understanding of the peculiar accumulation of these health-promoting metabolites in eggplant, we report on metabolite abundance, regulation of CGA and anthocyanin biosynthesis, and characterization of candidate CGA biosynthetic genes in S. melongena. Higher contents of CGA, Delphinidin 3-rutinoside, and rutin were found in eggplant fruits compared to other tissues, associated to an elevated transcript abundance of structural genes such as PAL, HQT, DFR, and ANS, suggesting that active in situ biosynthesis contributes to anthocyanin and CGA accumulation in fruit tissues. Putative orthologs of the two CGA biosynthetic genes PAL and HQT, as well as a variant of a MYB1 transcription factor showing identity with group six MYBs, were isolated from an Occidental S. melongena traditional variety and demonstrated to differ from published sequences from Asiatic varieties. In silico analysis of the isolated SmPAL1, SmHQT1, SmANS, and SmMyb1 promoters revealed the presence of several Myb regulatory elements for the biosynthetic genes and unique elements for the TF, suggesting its involvement in other physiological roles beside phenylpropanoid biosynthesis regulation. Transient overexpression in Nicotiana benthamiana leaves of SmMyb1 and of a C-terminal SmMyb1 truncated form (SmMyb1Δ9) resulted in anthocyanin accumulation only of SmMyb1 agro-infiltrated leaves. A yeast two-hybrid assay confirmed the interaction of both SmMyb1 and SmMyb1Δ9 with an anthocyanin-related potato bHLH1 TF. Interestingly, a doubled amount of CGA was detected in both SmMyb1 and SmMyb1Δ9 agro-infiltrated leaves, thus suggesting that the N-terminal region of SmMyb1 is sufficient to activate its synthesis. These data suggest that a deletion of the C-terminal region of SmMyb1 does not limit its capability to regulate CGA accumulation, but impairs anthocyanin biosynthesis. To our knowledge, this is the first study reporting a functional elucidation of the role of the C-term conserved domain in MYB activator proteins.

Molecular characterization of edestin gene family in Cannabis sativa L.

Globulins are the predominant class of seed storage proteins in a wide variety of plants. In many plant species globulins are present in several isoforms encoded by gene families. The major seed storage protein of Cannabis sativa L. is the globulin edestin, widely known for its nutritional potential. In this work, we report the isolation of seven cDNAs encoding for edestin from the C. sativa variety Carmagnola. Southern blot hybridization is in agreement with the number of identified edestin genes. All seven sequences showed the characteristic globulin features, but they result to be divergent members/forms of two edestin types. According to their sequence similarity four forms named CsEde1A, CsEde1B, CsEde1C, CsEde1D have been assigned to the edestin type 1 and the three forms CsEde2A, CsEde2B, CsEde2C to the edestin type 2. Analysis of the coding sequences revealed a high percentage of similarity (98-99%) among the different forms belonging to the same type, which decreased significantly to approximately 64% between the forms belonging to different types. Quantitative RT-PCR analysis revealed that both edestin types are expressed in developing hemp seeds and the amount of CsEde1 was 4.44 ± 0.10 higher than CsEde2. Both edestin types exhibited a high percentage of arginine (11-12%), but CsEde2 resulted particularly rich in methionine residues (2.36%) respect to CsEde1 (0.82%). The amino acid composition determined in CsEde1 and CsEde2 types suggests that these seed proteins can be used to improve the nutritional quality of plant food-stuffs.

Role of Arabidopsis UV RESISTANCE LOCUS 8 in plant growth reduction under osmotic stress and low levels of UV-B.

In high-light environments, plants are exposed to different types of stresses, such as an excess of UV-B, but also drought stress which triggers a common morphogenic adaptive response resulting in a general reduction of plant growth. Here, we report that the Arabidopsis thaliana UV RESISTANCE LOCUS 8 (UVR8) gene, a known regulator of the UV-B morphogenic response, was able to complement a Saccharomyces cerevisiae osmo-sensitive mutant and its expression was induced after osmotic or salt stress in Arabidopsis plants. Under low levels of UV-B, plants overexpressing UVR8 are dwarfed with a reduced root development and accumulate more flavonoids compared to control plants. The growth defects are mainly due to the inhibition of cell expansion. The growth inhibition triggered by UVR8 overexpression in plants under low levels of UV-B was exacerbated by mannitol-induced osmotic stress, but it was not significantly affected by ionic stress. In contrast, uvr8-6 mutant plants do not differ from wild-type plants under standard conditions, but they show an increased shoot growth under high-salt stress. Our data suggest that UVR8-mediated accumulation of flavonoid and possibly changes in auxin homeostasis are the underlying mechanism of the observed growth phenotypes and that UVR8 might have an important role for integrating plant growth and stress signals.

Early phenylpropanoid biosynthetic steps in Cannabis sativa: link between genes and metabolites.

Phenylalanine ammonia-lyase (PAL), Cinnamic acid 4-hydroxylase (C4H) and 4-Coumarate: CoA ligase (4CL) catalyze the first three steps of the general phenylpropanoid pathway whereas chalcone synthase (CHS) catalyzes the first specific step towards flavonoids production. This class of specialized metabolites has a wide range of biological functions in plant development and defence and a broad spectrum of therapeutic activities for human health. In this study, we report the isolation of hemp PAL and 4CL cDNA and genomic clones. Through in silico analysis of their deduced amino acid sequences, more than an 80% identity with homologues genes of other plants was shown and phylogenetic relationships were highlighted. Quantitative expression analysis of the four above mentioned genes, PAL and 4CL enzymatic activities, lignin content and NMR metabolite fingerprinting in different Cannabis sativa tissues were evaluated. Furthermore, the use of different substrates to assay PAL and 4CL enzymatic activities indicated that different isoforms were active in different tissues. The diversity in secondary metabolites content observed in leaves (mainly flavonoids) and roots (mainly lignin) was discussed in relation to gene expression and enzymatic activities data.

Selection and validation of reference genes for quantitative gene expression studies in Erythroxylum coca.

Real-time quantitative PCR is a powerful technique for the investigation of comparative gene expression, but its accuracy and reliability depend on the reference genes used as internal standards. Only genes that show a high level of expression stability are suitable for use as reference genes, and these must be identified on a case-by-case basis. Erythroxylum coca produces and accumulates high amounts of the pharmacologically active tropane alkaloid cocaine (especially in the leaves), and is an emerging model for the investigation of tropane alkaloid biosynthesis. The identification of stable internal reference genes for this species is important for its development as a model species, and would enable comparative analysis of candidate biosynthetic genes in the different tissues of the coca plant. In this study, we evaluated the expression stability of nine candidate reference genes in E. coca ( Ec6409, Ec10131, Ec11142, Actin, APT2, EF1α, TPB1, Pex4, Pp2aa3). The expression of these genes was measured in seven tissues (flowers, stems, roots and four developmental leaf stages) and the stability of expression was assessed using three algorithms (geNorm, NormFinder and BestKeeper). From our results we conclude that Ec10131 and TPB1 are the most appropriate internal reference genes in leaves (where the majority of cocaine is produced), while Ec10131 and Ec6409 are the most suitable internal reference genes across all of the tissues tested.