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The nutraceutical interest in quinoa ( Chenopodium quinoa Willd.) seeds is associated with the presence of macronutrients, micronutrients, minerals, vitamins, and polyphenols. In particular, polyphenols contribute to the health-promoting effects of this food crop, and their levels are influenced by environmental conditions. Production of quinoa is recently being explored in temperate climate areas, including Italy. The aim of this research was to assess the profile of bioactive compounds in seeds of two quinoa varieties, Regalona-Baer and Titicaca, grown in northern Italy, compared to that of seeds of those varieties grown in Chile and Denmark, respectively. High-performance liquid chromatography-diode array detector (HPLC-DAD) analysis of phenolic acid and flavonoid profiles, both in their free and soluble conjugated forms, showed that the main differences between Regalona grown in Chile and Italy were for the free vanillic acid and daidzein contents, while the two Titicaca samples mainly differed in quercetin derivative levels. The total phenolic index was comparable in Titicaca and Regalona, and only a slight decrease in this parameter was found in seeds of the two varieties grown in Italy. The in vitro antioxidant activity of seed extracts, evaluated by means of three different assays, indicated that it correlated with flavonol (quercetin derivative) levels. In conclusion, the results indicate that, although environmental conditions alter the polyphenolic profile and biological activities, it is possible to grow good-quality quinoa in northern Italy.

Many areas of the world are affected simultaneously by salinity and heavy metal pollution. Halophytes are considered as useful candidates in remediation of such soils due to their ability to withstand both osmotic stress and ion toxicity deriving from high salt concentrations. Quinoa (Chenopodium quinoa Willd) is a halophyte with a high resistance to abiotic stresses (drought, salinity, frost), but its capacity to cope with heavy metals has not yet been fully investigated. In this pot experiment, we investigated phytoextraction capacity, effects on nutrient levels (P and Fe), and changes in gene expression in response to application of Cr(III) in quinoa plants grown on saline or non-saline soil. Plants were exposed for three weeks to 500 mg kg -1 soil of Cr(NO 3 ) 3 ·9H 2 O either in the presence or absence of 150 mM NaCl. Results show that plants were able tolerate this soil concentration of Cr(III); the metal was mainly accumulated in roots where it reached the highest concentration (ca. 2.6 mg g -1 DW) in the presence of NaCl. On saline soil, foliar Na concentration was significantly reduced by Cr(III). Phosphorus translocation to leaves was reduced in the presence of Cr(III), while Fe accumulation was enhanced by treatment with NaCl alone. A real-time RT-qPCR analysis was conducted on genes encoding for sulfate, iron, and phosphate transporters, a phytochelatin, a metallothionein, glutathione synthetase, a dehydrin, Hsp70, and enzymes responsible for the biosynthesis of proline (P5CS), glycine betaine (BADH), tocopherols (TAT), and phenolic compounds (PAL). Cr(III), and especially Cr(III)+NaCl, affected transcript levels of most of the investigated genes, indicating that tolerance to Cr is associated with changes in phosphorus and sulfur allocation, and activation of stress-protective molecules. Moderately saline conditions, in most cases, enhanced this response, suggesting that the halophytism of quinoa could contribute to prime the plants to respond to chromium stress., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Quinoa ( Chenopodium quinoa Willd.), a model halophytic crop species, was used to shed light on salt tolerance mechanisms at the transcriptomic level. An RNA-sequencing analysis of genotype R49 at an early vegetative stage was performed by Illumina paired-ends method comparing high salinity and control conditions in a time-course pot experiment. Genome-wide transcriptional salt-induced changes and expression profiling of relevant salt-responsive genes in plants treated or not with 300 mM NaCl were analyzed after 1 h and 5 days. We obtained up to 49 million pairs of short reads with an average length of 101 bp, identifying a total of 2416 differentially expressed genes (DEGs) based on the treatment and time of sampling. In salt-treated vs. control plants, the total number of up-regulated and down-regulated genes was 945 and 1471, respectively. The number of DEGs was higher at 5 days than at 1 h after salt treatment, as reflected in the number of transcription factors, which increased with time. We report a strong transcriptional reprogramming of genes involved in biological processes like oxidation-reduction, response to stress and response to abscisic acid (ABA), and cell wall organization. Transcript analyses by real-time RT- qPCR supported the RNA-seq results and shed light on the contribution of roots and shoots to the overall transcriptional response. In addition, it revealed a time-dependent response in the expression of the analyzed DEGs, including a quick (within 1 h) response for some genes, suggesting a "stress-anticipatory preparedness" in this highly salt-tolerant genotype.

Biostimulants improve yield, quality, and stress acclimation in crops. In this work, we tested the possibility of using phenolics-rich extracts from spelt (Triticum dicoccum L.) husks to attenuate the effects of salt stress (100-200 mM NaCl) in maize. Two methanolic extracts were prepared from the soluble-conjugated (SC), and the insoluble-bound (IB) phenolic acid fractions of the spelt husk, and their effects were investigated on several stress-associated biochemical parameters, such as proline, lipid peroxidation, H 2 O 2 , GSH levels, and ion content. Results show that SC and IB fractions of husk extracts behaved very differently, no doubt due to their greatly divergent chemical composition, as revealed by both GC-MS and HPLC analyses. The efficacy of treatments in mitigating salt stress was also dose- and timing-dependent. IB, even at the lower concentration tested, was able to recover the performance of stressed plants in terms of growth, photosynthetic pigments content, and levels of salt stress markers. Recovery of shoot growth to control levels and reduction of stress-induced proline accumulation occurred regardless of whether plants were pre-treated or post-treated with IB, whereas only pre-treatment with the higher dose of IB was effective in mitigating oxidative stress. Although in some cases SC and even methanol alone exerted some positive effects, they could also be deleterious whereas IB never was. Overall, results indicate that a polyphenol-containing extract obtained from spelt by-products can behave as biostimulant in maize plants and can mitigate their response to salt stress, by acting on different biochemical targets., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Quinoa (Chenopodium quinoa Willd), an ancient Andean seed crop, exhibits exceptional nutritional properties and resistance to abiotic stress. The species' tolerance to heavy metals has, however, not yet been investigated nor its ability to take up and translocate chromium (Cr). This study aimed to investigate the metabolic adjustments occurring upon exposure of quinoa to several concentrations (0.01-5mM) of CrCl3. Young hydroponically grown plants were used to evaluate Cr uptake, growth, oxidative stress, and other biochemical parameters three and/or seven days after treatment. Leaves accumulated the lowest amounts of Cr, while roots and stems accumulated the most at low and at high metal concentrations, respectively. Fresh weight and photosynthetic pigments were reduced only by the higher Cr(III) doses. Substantially increased lipid peroxidation, hydrogen peroxide, and proline levels were observed only with 5mM Cr(III). Except for a significant decrease at day 7 with 5mM Cr(III), total polyphenols and flavonoids maintained control levels in Cr(III)-treated plants, whereas antioxidant activity increased in a dose-dependent manner. Maximum polyamine accumulation was observed in 1mM CrCl3-treated plants. Even though α- and γ-tocopherols also showed enhanced levels only with the 1mM concentration, tyrosine aminotransferase (TAT, EC 2.6.1.5) activity increased under Cr(III) treatment in a dose- and time-dependent manner. Taken together, results suggest that polyamines, tocopherols, and TAT activity could contribute to tolerance to 1mM Cr(III), but not to the highest concentration that, instead, generated oxidative stress., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Quinoa (Chenopodium quinoa Willd) is an ancient Andean seed-producing crop well known for its exceptional nutritional properties and resistance to adverse environmental conditions, such as salinity and drought. Seed storage proteins, amino acid composition, and bioactive compounds play a crucial role in determining the nutritional value of quinoa. Seeds harvested from three Chilean landraces of quinoa, one belonging to the salares ecotype (R49) and two to the coastal-lowlands ecotype, VI-1 and Villarrica (VR), exposed to two levels of salinity (100 and 300 mM NaCl) were used to conduct a sequential extraction of storage proteins in order to obtain fractions enriched in albumins/globulins, 11S globulin and in prolamin-like proteins. The composition of the resulting protein fractions was analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Results confirmed a high polymorphism in seed storage proteins; the two most representative genotype-specific bands of the albumin/globulin fraction were the 30- and 32-kDa bands, while the 11S globulin showed genotype-specific polymorphism for the 40- and 42-kDa bands. Spot analysis by mass spectrometry followed by in silico analyses were conducted to identify the proteins whose expression changed most significantly in response to salinity in VR. Proteins belonging to several functional categories (i.e., stress protein, metabolism, and storage) were affected by salinity. Other nutritional and functional properties, namely amino acid profiles, total polyphenol (TPC) and flavonoid (TFC) contents, and antioxidant activity (AA) of protein extracts were also analyzed. With the exception of Ala and Met in R49, all amino acids derived from protein hydrolysis were diminished in seeds from salt-treated plants, especially in landrace VI-1. By contrast, several free amino acids were unchanged or increased by salinity in R49 as compared with VR and VI-1, suggesting a greater tolerance in the salares landrace. VR had the highest TPC and AA under non-saline conditions. Salinity increased TPC in all three landraces, with the strongest increase occurring in R49, and enhanced radical scavenging capacity in R49 and VR. Overall, results show that salinity deeply altered the seed proteome and amino acid profiles and, in general, increased the concentration of bioactive molecules and AA of protein extracts in a genotype-dependent manner.

Quinoa (Chenopodium quinoa Willd.) is a highly salt-tolerant species subdivided into five ecotypes and exhibiting broad intra-specific differences in tolerance levels. In a greenhouse study, Chilean landraces belonging either to the salares (R49) or coastal lowlands (VI-1, Villarrica) ecotype with contrasting agro-ecological origins were investigated for their responses to high salinity. The effects of two levels of salinity, 100 (T1) and 300 (T2) mM NaCl, on plant growth and on some physiological parameters were measured. Leaf and root Na(+) accumulation differed among landraces. T2 reduced growth and seed yield in all landraces with maximum inhibition relative to controls in R49. Salinity negatively affected chlorophyll and total polyphenol content (TPC) in VI-1 and Villarrica but not R49. Germination on saline or control media of seeds harvested from plants treated or not with NaCl was sometimes different; the best performing landrace was R49 insofar as 45-65% of seeds germinated on 500 mM NaCl-containing medium. In all landraces, average seedling root length declined strongly with increasing NaCl concentration, but roots of R49 were significantly longer than those of VI-1 and Villarrica up to 300 mM NaCl. Salt caused increases in seed TPC relative to controls, but radical scavenging capacity was higher only in seeds from T2 plants of R49. Total SDS-extractable seed proteins were resolved into distinct bands (10-70 kDa) with some evident differences between landraces. Salt-induced changes in protein patterns were landrace-specific. The responses to salinity of the salares landrace are discussed in relation to its better adaptation to an extreme environment., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Neochloris oleoabundans (Chlorophyta) is widely considered one of the most promising microalgae for biotechnological applications. However, the large-scale production of microalgae requires large amounts of water. In this perspective, the possibility of using exhausted growth media for the re-cultivation of N. oleoabundans was investigated in order to simultaneously make the cultivation more economically feasible and environmentally sustainable. Experiments were performed by testing the following media: autotrophic exhausted medium (E+) and mixotrophic exhausted medium after cultivation with glucose (EG+) of N. oleoabundans cells grown in a 20-L photobioreactor (PBR). Both exhausted media were replenished with the same amounts of nitrate and phosphate as the control brackish medium (C). Growth kinetics, nitrate and phosphate consumption, photosynthetic pigments content, photosynthetic efficiency, cell morphology, and lipid production were evaluated. Moreover, the free fatty acid (FFA) composition of exhausted media and the polyamine (PA) concentrations of both algae and media were analyzed in order to test if some molecules, released into the medium, could influence algal growth and metabolism. Results showed that N. oleoabundans can efficiently grow in both exhausted media, if appropriately replenished with the main nutrients (E+ and EG+), especially in E+ and to the same extent as in C medium. Growth promotion of N. oleoabundans was attributed to PAs and alteration of the photosynthetic apparatus to FFAs. Taken together, results show that recycling growth medium is a suitable solution to obtain good N. oleoabundans biomass concentrations, while providing a more sustainable ecological impact on water resources.

It was previously shown that arbuscular mycorrhizal fungi (AMF) exert a significant improvement of growth in a tolerant white poplar (Populus alba L.) clone (AL35) grown on Cu- and Zn-polluted soil via foliar alterations in the levels of defence/stress-related transcripts and molecules. However, nothing is known about the epigenetic changes which occur during tolerance acquisition in response to heavy metals (HMs) in the same mycorrhizal vs. non-mycorrhizal poplar plants. In order to analyse the epigenome in leaves of AL35 plants inoculated or not with AMF and grown in a greenhouse on multimetal polluted or unpolluted soil, the Methylation Sensitive Amplification Polymorphism (MSAP) approach was adopted to detect cytosine DNA methylation. Modest changes in cytosine methylation patterns were detected at first sampling (4 months from planting), whereas extensive alterations (hypomethylation) occurred at second sampling (after 6 months) in mycorrhizal plants grown in the presence of HMs. The sequencing of MSAP fragments led to the identification of genes belonging to several Gene Ontology categories. Seven MSAP fragments, selected on the basis of DNA methylation status in treated vs control AL35 leaves at the end of the experiment, were analysed for their transcript levels by means of qRT-PCR. Gene expression varied in treated samples relative to controls in response to HMs and/or AMF inoculation; in particular, transcripts of genes involved in RNA processing, cell wall and amino acid metabolism were upregulated in the presence of AMF with or without HMs.

Phytoremediation is a cost-effective and environment friendly in situ technique for the reclamation of heavy metal-polluted soils. The efficacy of this technique, which relies on tolerant plant species, can be improved by the use of chelating agents. A pot experiment was carried out to evaluate the phytoextraction and phytostabilisation capacities of a white poplar (Populus alba L.) clone named AL35 previously selected for its marked tolerance to copper (Cu) and zinc (Zn). Cuttings were grown on agricultural soil highly contaminated with Cu and Zn, in the presence or not (controls) of a chelant mixture (EDTA/EDDS) known to enhance metal bioavailability and, hence, uptake by plant roots, or the not yet investigated synthetic, highly biodegradable polyaspartic acid (PASP). Both chelant treatments improved the phytostabilisation of Cu and Zn in AL35 plants, whilst the phytoextraction capacity was enhanced only in the case of Cu. Considering that the effectiveness of PASP as phytostabilizer was comparable or better than that of EDTA/EDDS, the low cost of its large-scale chemical synthesis and its biodegradability makes it a good candidate for chelant-enhanced metal phytoextraction from soil while avoiding the toxic side-effects previously described for both EDTA and EDDS., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Chenopodium quinoa (Willd.) is an Andean plant showing a remarkable tolerance to abiotic stresses. In Chile, quinoa populations display a high degree of genetic distancing, and variable tolerance to salinity. To investigate which tolerance mechanisms might account for these differences, four genotypes from coastal central and southern regions were compared for their growth, physiological, and molecular responses to NaCl at seedling stage. Seeds were sown on agar plates supplemented with 0, 150 or 300mM NaCl. Germination was significantly reduced by NaCl only in accession BO78. Shoot length was reduced by 150mM NaCl in three out of four genotypes, and by over 60% at 300mM (except BO78 which remained more similar to controls). Root length was hardly affected or even enhanced at 150mM in all four genotypes, but inhibited, especially in BO78, by 300mM NaCl. Thus, the root/shoot ratio was differentially affected by salt, with the highest values in PRJ, and the lowest in BO78. Biomass was also less affected in PRJ than in the other accessions, the genotype with the highest increment in proline concentration upon salt treatment. Free putrescine declined dramatically in all genotypes under 300mM NaCl; however (spermidine+spermine)/putrescine ratios were higher in PRJ than BO78. Quantitative RT-PCR analyses of two sodium transporter genes, CqSOS1 and CqNHX, revealed that their expression was differentially induced at the shoot and root level, and between genotypes, by 300mM NaCl. Expression data are discussed in relation to the degree of salt tolerance in the different accessions., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Chenopodium quinoa Willd. (quinoa) is a halophyte for which some parameters linked to salt tolerance have been investigated separately in different genotypes and under different growth conditions. In this study, several morphological and metabolic responses were analysed in parallel after exposure to salinity. In vitro seed germination was initially delayed by a 150mM NaCl treatment but eventually reached the same level as the control (0mM NaCl), whereas seedling root growth was enhanced; both parameters were moderately inhibited (~35-50%) by 300mM NaCl. In pot grown plants, plant size was reduced by increasing salinity (0-750mM NaCl). Transpiration and stomatal conductance were decreased at the highest salinity levels tested, consistent with reduced stomatal density and size. The density of epidermal bladder cells (EBCs) on the leaf surface remained unaffected up to 600mM NaCl. Tissue contents of Na+ and Cl- increased dramatically with salt treatment, but resulted in only a 50% increase in Na+ from 150 to 750mM NaCl. Internal K+ was unaffected up to 450mM NaCl but increased at the highest salinity levels tested. Excretion through sequestration into EBCs was limited (generally ≤20%) for all ions. A modest dose-dependent proline accumulation, and concomitant reduction in total polyamines and putrescine efflux occurred in NaCl-treated plants. Results confirm the importance of inorganic ions for osmotic adjustment, the plant's ability to maintain K+ levels and the involvement of putrescine efflux in maintaining ionic balance under high salinity conditions. Conversely, ion excretion and proline appear to play a minor role. Taken together these results indicate which parameters could be used for future comparison among different genotypes.

The phytopathogenic actinomycete Rhodococcus fascians drives its host to form a nutrient-rich niche by secreting a mixture of cytokinins that triggers plant cell division and shoot formation. The discrepancy between the relatively low amount of secreted cytokinins and the severe impact of R. fascians infection on plant development has puzzled researchers for a long time. Polyamine and transcript profiling of wild-type and cytokinin receptor mutant plants revealed that the bacterial cytokinins directly stimulated the biosynthesis of plant putrescine by activating arginine decarboxylase expression. Pharmacological experiments showed that the increased levels of putrescine contributed to the severity of the symptoms. Thus, putrescine functions as a secondary signal that impinges on the cytokinin-activated pathway, amplifying the hormone-induced changes that lead to the formation of a leafy gall. Exogenous putrescine and treatment with polyamine biosynthesis inhibitors combined with transcript and polyamine analyses of wild-type and mutant plants indicated that the direct target of both the bacterial cytokinins and plant putrescine was the expression of D3-type cyclins. Hence, the activated d-type cyclin/retinoblastoma/E2F transcription factor pathway integrates both external and internal hormonal signals, stimulating mitotic cell divisions and inducing pathological plant organogenesis.

In the present study, the effects of epibrassinolide (EBL) on indole-3-acetic acid (IAA), abscisic acid (ABA) and polyamine (PA) tissue concentrations and antioxidant potential of 7-day-old Raphanus sativus L. cv. 'Pusa chetki' seedlings grown under Cu stress were investigated. EBL treatment alone or in combination with Cu enhanced free and bound IAA titers when compared with the metal alone. Modest increases in free and bound ABA contents were observed for EBL treatment alone. However, the combination of EBL with Cu caused major increases in both forms of ABA, over Cu alone. Among the PAs analyzed, only putrescine and cadaverine concentrations were enhanced by EBL treatment alone. By contrast, a significant decline in putrescine and spermine contents was found in seedlings treated with EBL plus Cu. EBL treatments alone or in combination with Cu enhanced activities of guaiacol peroxidase (EC1.11.1.7), catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) and glutathione reductase (EC 1.6.4.2) and protein contents in comparison with metal and control treatments. A major decrease in malondialdehyde content was also recorded for EBL treatments with or without Cu. An increase in phytochelatin content was also observed in seedlings treated with EBL alone or in combination with Cu. Major improvement in radical scavenging activities, as attested by the antioxidant activity assay using DPPH (1,1-diphenylpicrylhydrazyl), and elevated deoxyribose and reducing powers, along with increased contents of ascorbic acid, total phenols and proline, also suggest a major influence of EBL application in mitigating copper-induced oxidative stress in radish seedlings., (Copyright © Physiologia Plantarum 2010.)

Background and Aims: It is increasingly evident that plant tolerance to stress is improved by mycorrhiza. Thus, suitable plant-fungus combinations may also contribute to the success of phytoremediation of heavy metal (HM)-polluted soil. Metallothioneins (MTs) and polyamines (PAs) are implicated in the response to HM stress in several plant species, but whether the response is modulated by arbuscular mycorrhizal fungi (AMF) remains to be clarified. The aim of the present study was to check whether colonization by AMF could modify growth, metal uptake/translocation, and MT and PA gene expression levels in white poplar cuttings grown on HM-contaminated soil, and to compare this with plants grown on non-contaminated soil., Methods: In this greenhouse study, plants of a Populus alba clone were pre-inoculated, or not, with either Glomus mosseae or G. intraradices and then grown in pots containing either soil collected from a multimetal- (Cu and Zn) polluted site or non-polluted soil. The expression of MT and PA biosynthetic genes was analysed in leaves using quantitative reverse transcription-PCR. Free and conjugated foliar PA concentrations were determined in parallel., Results: On polluted soil, AMF restored plant biomass despite higher Cu and Zn accumulation in plant organs, especially roots. Inoculation with the AMF caused an overall induction of PaMT1, PaMT2, PaMT3, PaSPDS1, PaSPDS2 and PaADC gene expression, together with increased free and conjugated PA levels, in plants grown on polluted soil, but not in those grown on non-polluted soil., Conclusions: Mycorrhizal plants of P. alba clone AL35 exhibit increased capacity for stabilization of soil HMs, together with improved growth. Their enhanced stress tolerance may derive from the transcriptional upregulation of several stress-related genes, and the protective role of PAs.

The phytopathogenic actinomycete Rhodococcus fascians D188 relies mainly on the linear plasmid-encoded fas operon for its virulence. The bacteria secrete six cytokinin bases that synergistically redirect the developmental program of the plant to stimulate proliferation of young shoot tissue, thus establishing a leafy gall as a niche. A yeast-based cytokinin bioassay combined with cytokinin profiling of bacterial mutants revealed that the fas operon is essential for the enhanced production of isopentenyladenine, trans-zeatin, cis-zeatin, and the 2-methylthio derivatives of the zeatins. Cytokinin metabolite data and the demonstration of the enzymatic activities of FasD (isopentenyltransferase), FasE (cytokinin oxidase/dehydrogenase), and FasF (phosphoribohydrolase) led us to propose a pathway for the production of the cytokinin spectrum. Further evaluation of the pathogenicity of different fas mutants and of fas gene expression and cytokinin signal transduction upon infection implied that the secretion of the cytokinin mix is a highly dynamic process, with the consecutive production of a tom initiation wave followed by a maintenance flow.

In this work, we have analyzed the changes in the protein expression profile elicited by chromium (Cr) exposure in the freshwater green alga Pseudokirchneriella subcapitata, a well known bio-indicator of water pollution. We tested two experimental conditions, namely 0.2 and 1ppm of potassium dichromate; this concentration range includes the environmentally-relevant concentrations. Results show that neither concentration of potassium dichromate tested inhibited algal growth. However, the proteomic approach allowed the identification of relevant modifications in protein expression. In fact, among 800 protein spots detected by two-dimensional electrophoresis, 16 Cr-regulated proteins, including predicted and novel ones, were identified using tandem mass spectromic protein analysis. The results demonstrate a Cr-specific action in altering several photosynthetic proteins, such as ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), RuBisCO activase, Light Harvesting Chla/b protein complex, and stress related Chla/b binding protein1. Although Cr toxicity with respect to photosynthesis has been already documented, here we have identified, for the first time, the target proteins of this toxicity. Cr also induced a modulation of some proteins involved in the metabolism of the amino acids glutamine, arginine and methionine. These data are supported by changes in cellular polyamine (PA) accumulation. Present findings provide new insight into the molecular mechanisms underlying Cr toxicity in P. subcapitata.

Marker-free transgenic white poplar (Populus alba L., cv 'Villafranca') plants, expressing the PsMT (A1) gene from Pisum sativum for a metallothionein-like protein, were produced by Agrobacterium tumefaciens-mediated transformation. The 35SCaMV-PsMT (A1)-NosT cassette was inserted into the ipt-type vector pMAT22. The occurrence of the abnormal ipt-shooty phenotype allowed the visual selection of transformants, while the yeast site-specific recombination R/RS system was responsible for the excision of the undesired vector sequences with the consequent recovery of normal marker-free transgenic plants. Molecular analyses confirmed the presence of the 35SCaMV-PsMT (A1)-NosT cassette and transgene expression. Five selected lines were further characterized, revealing the ability to withstand heavy metal toxicity. They survived 0.1 mM CuCl(2), a concentration which strongly affected the nontransgenic plants. Moreover, root development was only slightly affected by the ectopic expression of the transgene. Reactive oxygen species were accumulated to a lower extent in leaf tissues of multi-auto-transformation (MAT)-PsMT(A1) plants exposed to copper and zinc, compared to control plants. Tolerance to photo-oxidative stress induced by paraquat was another distinctive feature of the MAT-PsMT(A1) lines. Finally, low levels of DNA damage were detected by quantifying the amounts of 8-hydroxy-2'-deoxyguanosine in leaf tissues of the transgenic plants exposed to copper.

The effects of a high concentration of zinc on two registered clones of poplar (Populus alba Villafranca and Populus nigra Jean Pourtet), inoculated or not with two arbuscular mycorrhizal fungi (Glomus mosseae or Glomus intraradices) before transplanting them into polluted soil, were investigated, with special regard to the extent of root colonization by the fungi, plant growth, metal accumulation in the different plant organs, and leaf polyamine concentration. Zinc accumulation was lower in Jean Pourtet than in Villafranca poplars, and it was mainly translocated to the leaves; the metal inhibited mycorrhizal colonization, compromised plant growth, and, in Villafranca, altered the putrescine profile in the leaves. Most of these effects were reversed or reduced in plants pre-inoculated with G. mosseae. Results indicate that poplars are suitable for phytoremediation purposes, confirming that mycorrhizal fungi can be useful for phytoremediation, and underscore the importance of appropriate combinations of plant genotypes and fungal symbionts.

Long-sized oligogalacturonides (OGs) are cell wall fragments that induce defence and developmental responses. The Ca(2+)-dependent "egg-box" conformation is required for their activity, and polyamines may prevent them from adopting this conformation. Although OGs are known to inhibit auxin-induced growth processes, their effect on cytokinin-induced ones requires investigation. In the present work OGs were shown to promote cytokinin (benzyladenine, BA)-induced vegetative shoot formation from tobacco leaf explants, independent of the presence of CaCl(2) in the medium and of auxin (indoleacetic acid, IAA) supply. The effect of polyamines, putrescine (PU) and spermidine (SD) supplied with/without their biosynthetic inhibitors (DFMO, CHA) was also investigated, and showed that spermidine enhanced adventitious vegetative shoot formation, but only on medium containing Ca(2+) and IAA. Treatments with inhibitors blocked this promotive effect. OGs did not alter free polyamine concentrations, but caused a moderate increase of conjugated ones, and exhibited an early inhibitory effect on polyamine biosynthetic gene expression. OGs, but not SD, caused long-term changes in calcium-associated epifluorescent signals in the cell walls, and, later, inside the cells of specific tissues. Electron microscopy analysis (ESI system) demonstrated that calcium accumulated in the cell walls and vacuoles of OG-cultured explants. The relationship between OGs, cytokinin, calcium, and polyamines in adventitious vegetative shoot formation is discussed.

Peach (Prunus persica L. Batsch) was chosen as a model to shed light on the physiological role of jasmonates (JAs) during fruit ripening. To this aim, the effects of methyl jasmonate (MJ, 0.40 mM) and propyl dihydrojasmonate (PDJ, 0.22 mM), applied in planta at different fruit developmental stages, on the time-course of ethylene production and fruit quality traits were evaluated. MJ-induced changes in fruit transcriptome at harvest and the expression profiling of relevant JA-responsive genes were analysed in control and JA-treated fruit. Exogenously applied JAs affected the onset of ripening depending upon the fruit developmental stage, with PDJ being more active than MJ. Both compounds enhanced the transcription of allene oxide synthase (PpAOS1), the first specific enzyme in the biosynthesis of jasmonic acid, and altered the pattern of jasmonic acid accumulation. Microarray transcriptome profiling showed that MJ down-regulated some ripening-related genes, such as 1-aminocyclopropane-1-carboxylic acid oxidase (PpACO1) and polygalacturonase (PG), and the transcriptional modulator IAA7. MJ also altered the expression of cell wall-related genes, namely pectate lyase (PL) and expansins (EXPs), and up-regulated several stress-related genes, including some of those involved in JA biosynthesis. Time-course expression profiles of PpACO1, PL, PG, PpExp1, and the transcription factor LIM confirmed the array results. Thus, in peach fruit, exogenous JAs led to a ripening delay due to an interference with ripening- and stress/defence-related genes, as reflected in the transcriptome of treated fruit at harvest.

Callus cultures from several species of Passiflora were initiated in vitro, and their capacity to produce four glycosyl flavonoids (orientin, isoorientin, vitexin, isovitexin) was analysed. The aim of the present work was to examine the possible role of UV-B irradiation and elicitation with methyl jasmonate (MJ) on the production of these compounds in callus cultures. All the species tested (P. incarnata, P. quadrangularis, P. edulis) formed friable callus from leaf explants after 4 weeks on medium supplemented with kinetin and 2,4-dichlorophenoxyacetic acid. Among them, P. quadrangularis turned out to have a faster growth rate and a more friable texture, and was therefore chosen for experiments with elicitors. In callus cultures only small amounts of isoorientin were found, while the concentration of the other flavonoids was below the detection limit. UV-B irradiation of calluses was able to increase the production of all four glycosyl flavonoids. After a 7-day exposure of cultures to UV-B light, the production of isoorientin reached concentrations similar to those found in fresh leaves from glasshouse-grown plants. Elicitation with methyl jasmonate also enhanced orientin, vitexin and isovitexin concentrations, even though the stimulation was about 6-fold weaker for orientin and vitexin and about 40-fold for isovitexin, than that exerted by UV-B treatment. Callus cultures treated with the UV-B dose which most enhanced flavonoid production showed a higher antioxidant activity compared to untreated calluses, with an increase ranging from 28% to 76%. Results show that the secondary metabolite biosynthetic capacity of Passiflora tissue cultures can be enhanced by appropriate forms of elicitation.

Poplar is a good candidate for phytoremediation purposes because of its rapid growth, extensive root system, and ease of propagation and transformation; however its tolerance to heavy metals has not been fully investigated yet. In the present work, an in vitro model system with shoot cultures was used to investigate the tolerance to high concentrations of zinc (Zn) of a commercial clone (Villafranca) of Populus alba. Based on chlorophyll content (leaf chlorosis) and the rate of adventitious root formation from shoot cuttings as parameters of damage, 0.5-4mM zinc concentrations were all toxic albeit to different extents. Northern blot and reverse transcriptase (RT)-PCR analyses were used to examine the expression profiles of types 1, 2 and 3 PaMT genes in stems, leaves and roots of plants exposed to Zn treatments. In leaves, MT1 and MT3 mRNA levels were enhanced by Zn, while MT2 transcripts were not affected. The PaMT expression profiles were differentially affected by Zn in an organ-specific manner, and the relationship with Zn concentration and exposure time was rarely linear. The developmental and molecular data reveal that the in vitro model is a sensitive and reliable system to study heavy metal stress responses.

The time course of ethylene biosynthesis and perception was investigated in ripening peach fruit (Prunus persica) following treatments with the polyamines putrescine (Pu) and spermidine (Sd), and with aminoethoxyvinylglycine (AVG). Fruit treatments were performed in planta. Ethylene production was measured by gas chromatography, and polyamine content by high-performance liquid chromatography; expression analyses were performed by Northern blot or real-time polymerase chain reaction. Differential increases in the endogenous polyamine pool in the epicarp and mesocarp were induced by treatments; in both cases, ethylene production, fruit softening and abscission were greatly inhibited. The rise in 1-aminocyclopropane-1-carboxylate oxidase (PpACO1) mRNA was counteracted and delayed in polyamine-treated fruit, whereas transcript abundance of ethylene receptors PpETR1 (ethylene receptor 1) and PpERS1 (ethylene sensor 1) was enhanced at harvest. Transcript abundance of arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC) was transiently reduced in both the epicarp and mesocarp. AVG, here taken as a positive control, exerted highly comparable effects to those of Pu and Sd. Thus, in peach fruit, increasing the endogenous polyamine pool in the epicarp or in the mesocarp strongly interfered, both at a biochemical and at a biomolecular level, with the temporal evolution of the ripening syndrome.

S-adenosylmethionine decarboxylase activity (SAMDC; EC 4.1.1.21) leads to spermidine and spermine synthesis through specific synthases which use putrescine, spermidine and decarboxylated S-adenosylmethionine as substrates. In order to better understand the regulation of polyamine (PA), namely spermidine and spermine, biosynthesis, a SAMDC cDNA of Datura stramonium was introduced in tobacco (Nicotiana tabacum L. cv. Xanthi) in antisense orientation under the CaMV 35S promoter, by means of Agrobacterium tumefaciens and leaf disc transformation. The effect of the genetic manipulation on PA metabolism, ethylene production and plant morphology was analysed in primary transformants (R0), and in the transgenic progeny (second generation, R1) of self-fertilised primary transformants, relative to empty vector-transformed (pBin19) and wild-type (WT) controls. All were maintained in vitro by micropropagation. Primary transformants, which were confirmed by Southern and northern analyses, efficiently transcribed the antisense SAMDC gene, but SAMDC activity and PA titres did not change. By contrast, in most transgenic R1 shoots, SAMDC activity was remarkably lower than in controls, and the putrescine-to-spermidine ratio was altered, mainly due to increased putrescine, even though putrescine oxidising activity (diamine oxidase, EC 1.4.3.6) did not change relative to controls. Despite the reduction in SAMDC activity, the production of ethylene, which shares with PAs the common precursor SAM, was not influenced by the foreign gene. Some plants were transferred to pots and acclimatised in a growth chamber. In these in vivo-grown second generation transgenic plants, at the vegetative stage, SAMDC activity was scarcely reduced, and PA titres did not change. Finally, the rhizogenic potential of in vitro-cultured leaf explants excised from antisense plants was significantly diminished as compared with WT ones, and the response to methyl jasmonate, a stress-mimicking compound, in terms of PA conjugation, was higher and differentially affected in transgenic leaf discs relative to WT ones. The effects of SAMDC manipulation are discussed in relation to plant generation, culture conditions and response to stress.

To test the feasibility of altering polyamine levels by influencing their catabolic pathway, we obtained transgenic tobacco (Nicotiana tabacum) plants constitutively expressing either maize (Zea mays) polyamine oxidase (MPAO) or pea (Pisum sativum) copper amine oxidase (PCuAO), two extracellular and H(2)O(2)-producing enzymes. Despite the high expression levels of the transgenes in the extracellular space, the amount of free polyamines in the homozygous transgenic plants was similar to that in the wild-type ones, suggesting either a tight regulation of polyamine levels or a different compartmentalization of the two recombinant proteins and the bulk amount of endogenous polyamines. Furthermore, no change in lignification levels and plant morphology was observed in the transgenic plants compared to untransformed plants, while a small but significant change in reactive oxygen species-scavenging capacity was verified. Both the MPAO and the PCuAO tobacco transgenic plants produced high amounts of H(2)O(2) only in the presence of exogenously added enzyme substrates. These observations provided evidence for the limiting amount of freely available polyamines in the extracellular space in tobacco plants under physiological conditions, which was further confirmed for untransformed maize and pea plants. The amount of H(2)O(2) produced by exogenously added polyamines in cell suspensions from the MPAO transgenic plants was sufficient to induce programmed cell death, which was sensitive to catalase treatment and required gene expression and caspase-like activity. The MPAO and PCuAO transgenic plants represent excellent tools to study polyamine secretion and conjugation in the extracellular space, as well as to determine when and how polyamine catabolism actually intervenes both in cell wall development and in response to stress.

Transcript levels and activities of the polyamine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19), ornithine decarboxylase (ODC, EC 4.1.1.17) and S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), as well as free polyamine titres, were analysed throughout the four growth stages S1-S4 leading up to ripening in the mesocarp from peach fruit (Prunus persica L. Batsch cv. Redhaven) grown under field conditions. SAMDC mRNA, which was northern analysed by using a PCR-generated homologous SAMDC probe, and ADC mRNA levels appeared quite stable during fruit development, while ODC transcript accumulation showed a discontinuous trend. The pattern of transcript levels during growth did not correlate with that of the relative enzyme activity, which instead correlated well with free polyamine levels. Both exhibited maximum levels in S1 and a smaller peak in S3. The behaviour of the polyamine biosynthetic machinery is discussed in relation to the different cell growth rates occurring during fruit development.

Despite the tremendous importance of secondary metabolites for humans as for the plant itself, plant secondary metabolism remains poorly characterized. Here, we present an experimental approach, based on functional genomics, to facilitate gene discovery in plant secondary metabolism. Targeted metabolite analysis was combined with cDNA-amplified fragment length polymorphism-based transcript profiling of jasmonate-elicited tobacco Bright yellow 2 cells. Transcriptome analysis suggested an extensive jasmonate-mediated genetic reprogramming of metabolism, which correlated well with the observed shifts in the biosynthesis of the metabolites investigated. This method, which in addition to transcriptome data also generates gene tags, in the future might lead to the creation of novel tools for metabolic engineering of medicinal plant systems in general.

The effect of various concentrations of aminoethoxyvinylglycine (AVG; 0.32 and 1.28 mM), an ethylene biosynthesis inhibitor, and of the polyamines putrescine (10 mM), spermidine (0.1, 1 and 5 mM) and spermine (2 mM) on peach (Prunus persica L. Batsch cv. Redhaven) fruit ripening was evaluated under field conditions. Treatments were performed 19 (polyamines) and 8 (AVG) days before harvest. Fruit growth (diameter, fresh and dry weight), flesh firmness, soluble solids content and ethylene emission were determined on treated and untreated (controls) fruits. Moreover, endogenous polyamine content and S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21) activity were determined to check for a possible competition between polyamines and ethylene for their common precursor S-adenosylmethionine (SAM). Both treatments strongly inhibited ethylene emission and delayed flesh softening. On a biochemical level, AVG and exogenous polyamines both reduced the free-to-conjugate ratio of endogenous polyamines, and transiently altered SAMDC activity. The possible use of these compounds to control fruit ripening is discussed also in the light of their rejuvenating effect on peach fruits.

•  Change is reported in the biosynthetic and oxidative activity of hypersensitive (NN) and susceptible (nn) tobacco (Nicotiana tabacum) plants in response to tobacco mosaic virus (TMV). •  Mature leaves of nn and NN tobacco were collected over 0-72 h as uninoculated controls or after inoculation with TMV or phosphate buffer (mock-inoculation). The polyamine response to inoculation was analysed by measuring activity and gene expression of S-adenosylmethionine decarboxylase (SAMDC), arginine-(ADC) and ornithine decarboxylases (ODC); incorporation of labelled putrescine; and activity of diamine oxidase (DAO). •  In NN leaves SAMDC activity and transcript levels, and DAO activity increased in the TMV-inoculated plants but not in the other treatments; a two-fold increase in DAO activity was seen after 72 h. Both ADC and ODC activity increased in NN leaves at 72 h in TMV-inoculated plants; ADC mRNA increased with activity. The increase in SAMDC mRNA (24 h) preceded the rise in activity (72 h). [ 3 H]putrescine added to NN leaves led to enhanced label recovery and incorporation into spermidine and spermine in TMV-inoculated plants. No significant changes in biosynthetic or oxidative activity occurred in nn plants. •  After TMV inoculation, NN, unlike nn, tobacco plants upgrade polyamine synthesis and oxidation; this leads to changes in cellular components which might induce programmed cell death.