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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.

Effetti di zinco e rame sull’espressione genica delle metallotioneine e sul contenuto di poliammine in Populus alba L. in micropropagazione S. BIONDI1, C. FRANCHIN1, T. FOSSATI2, E. PASQUINI1, V. ZIOSI1, P. TORRIGIANI1, S. CASTIGLIONE2, 1 Dipartimento di Biologia, Università di Bologna, Via Irnerio 42, 40126 Bologna; 2 Dipartimento di Biologia, Università di Milano, via Celoria 26, 20133 Milano Vi sono molte evidenze a favore di un possibile coinvolgimento delle metallotioneine (MT) nelle risposte a stimoli ambientali, in particolare si ipotizza che queste proteine siano strettamente correlate alla capacità di molti organismi di tollerare dosi elevate di metalli pesanti. Nelle piante le MT sono classificate in quattro classi (COBBETT, GOLDSBROUGH, 2002)) e in Arabidopsis, pomodoro e cotone vi sono famiglie geniche di MT (ZHOU & GOLDSBROUGH, 1994; GIRITCH et al., 1998; HUDSPETH et al., 1996). Per chiarire le funzioni delle MT nelle piante, sono stati effettuati numerosi studi sull’espressione di questi geni sia durante lo sviluppo che in risposta a stress biotico e abiotico con risultati ancora contrastanti. Pertanto informazioni certe sull’attività endogena delle MT non sono ancora state raggiunte e il loro ruolo rimane elusivo. Nei vegetali le poliammine (PA) si accumulano in risposta a vari stimoli biotici e abiotici (BOUCHEREAU et al., 1999), compresi gli inquinanti ambientali quali i metalli pesanti (WEINSTEIN et al., 1986; HAUSCHILD, 1993). Esse svolgono probabilmente un ruolo nella resistenza allo stress ossidativo, stabilizzando gli acidi nucleici e le membrane biologiche. Il rapido accrescimento, l’elevata produzione di biomassa, l’apparato radicale esteso, la facile propagazione vegetativa e la possiblità di utilizzo nelle coltivazioni a breve rotazione fanno del pioppo un ottimo candidato come pianta arborea da usare nel fitorisanamento di siti inquinati. Tuttavia, sono molte scarse le informazioni circa le sue capacità di accumulo e di tolleranza nei confronti dei metalli. Inoltre, il pioppo è di recente assurto a “pianta arborea modello” in quanto il suo genoma è stato interamente sequenziato, aprendo la via a studi molecolari di ampio respiro (TUSKAN et al., 2004). Nel presente lavoro, è stata esaminata la risposta di un clone commerciale di Populus alba, la cv Villafranca, allevato in vitro attraverso la micropropagazione ed esposto per tempi diversi, fino a un massimo di 15 giorni, a dosi elevate di ZnSO4 o CuCl2. Sono stati valutati i sintomi visivi di tossicità, i livelli endogeni di poliammine libere e coniugate, e i livelli di trascritto di tre geni codificanti MT (MT1, MT2 e MT3). I risultati hanno evidenziato un’evidente clorosi fogliare già dopo 1 giorno in piante esposte a concentrazioni di Zn di 1, 2 e 4 mM, ma non 0.5 mM. Un forte ingiallimento e senescenza delle foglie si osservava soltanto con le due dosi più alte a partire dal 7° giorno. Tali sintomi di tossicità, associati a un calo significativo nel contenuto di clorofille, si palesavano in piante trattate con Cu anche a concentrazioni molto più basse (0.1 mM). La formazione di radici avventizie sui germogli trattati con Zn era inibita in maniera dose dipendente, arrivando fino al 50%. Al contrario, in presenza di Cu la rizogenesi era totalmente inibita a partire dalla concentrazione 0,1 mM e fortemente ridotta anche a dosi molto più basse (5 mM). Pur essendo già noto l’effetto fortemente inibente del Cu sulla crescita radicale (MURPHY & TAIZ, 1995) i dati mostrano che anche la rizogenesi è molto sensibile alla tossicità di questo metallo. Del resto, il Cu è uno dei metalli più tossici conosciuti. Nel suolo, la sua biodisponibilità è bassa, ma in soluzione acquosa la sua tossicità viene messa in evidenza provocando stress ossidativo (FERNANDES & HENRIQUES, 1991). Come riportato in Figura 1, il contenuto totale di PA, sia libere che coniugate, misurate dopo 15 giorni di esposizione a tre diverse concentrazioni di Zn (0,5, 1 e 2 mM) ...

Saponins (SAPs) are a diverse family of plant secondary metabolites and due to their biological activities, SAPs can be utilised as biopesticides and as therapeutic compounds. Given their widespread industrial use, a search for alternative sources of SAPs is a priority. Quinoa ( Chenopodium quinoa Willd) is a valuable food source that is gaining importance worldwide for its nutritional and nutraceutical properties. SAPs from quinoa seed coats could represent a new sustainable source to obtain these compounds in high quantities due to the increasing production and worldwide expansion of the crop. This research aims to characterise saponins of seed coat waste products from six different quinoa varieties for their potential use as a saponin source. Gas chromatography (GC)- and Liquid chromatography (LC)- with mass spectrometry (MS) were applied for qualitative and relative quantitative analysis of saponins. GC–MS led to the identification of three main aglycones, oleanolic acid (Ole), hederagenin (Hed), and a phytolaccagenic acid (Phy), while LC–MS enabled characterization of 24 SAPs with varying sugar moieties. Hed was the most abundant aglycone, followed by Phy and Oledepending on the genotype. Saponin distribution and relative abundances are discussed in the light of genotype provenance and agronomic features. Improved knowledge on the phytochemicals present in quinoa varieties might help in finding valuable and sustainable uses for quinoa SAPs in agroindustry as biopesticides as well as in the production of food and pharmaceuticals.

To further our understanding of the mechanisms governing salt stress responses and adaptation in halophytes, we explored morphological, metabolic, and gene expression responses to high salinity in quinoa (Chenopodium quinoa Willd). The main objective of this study was to analyze selected responsive genes in a time-course experiment to test for expression kinetics and to compare short-term salt-induced effects at the transcript level between two Chilean landraces belonging to different ecotypes. Quinoa genotypes exhibit a large variability in their responses to salinity, but it is not clear whether this is strictly related to the ecotype to which they belong. We tested this hypothesis by comparing the expression levels of genes involved in growth, ion homeostasis, abscisic acid (ABA) biosynthesis, perception, and conjugate cleavage, polyamine (PA) biosynthesis and oxidation, and proline biosynthesis as well as genes encoding ABA-dependent and −independent transcription factors. Landraces R49 (salares ecotype) and Villarrica (VR, coastal-lowlands ecotype) were analyzed from 0.5 to 120Âh after transfer to saline (300ÂmM NaCl) or non-saline (control) medium. All the genes, except CqSOS1 and CqNHX, were investigated here for the first time in quinoa under salt stress. Transcript levels were determined by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) analysis. Germination, seedling growth, ABA, and PA contents were evaluated in parallel. Even though on saline medium germination was inhibited in VR but not in R49, seedling growth reduction at 120Âh was not substantially different in the two landraces. The ABA biosynthetic enzyme NCED was the most strongly salt-induced gene; ABA content was similarly enhanced (shoots) or unaffected (roots) in both R49 and VR. NaCl treatment also altered transcript levels of some PA metabolic enzymes and the PA profile leading to an enhanced ratio between the higher PAs and putrescine. All other genes also exhibited similar expression profiles in response to salinity in the two landraces especially in roots, while in shoots some differences were observed. Our results provide new information indicating that crucial salt adaptation strategies at the molecular level and in terms of ABA and PA contents are shared by the coastal-lowlands and salares landraces; however, the timing of the onset of transcriptional changes (e.g., NCED, ABF3, and RD22) may reflect genotype-dependent constitutive and/or inducible adaptive strategies.

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.

none 5 no This research was supported by funds from the University of Bologna (RFO2014) to S.B.and from the University of Urbino (RFO 2014) to V.S.. 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. open Scoccianti, Valeria; Bucchini, Anahi E.; Iacobucci, Marta; Ruiz, Karina B.; Biondi, Stefania Scoccianti, Valeria; Bucchini, Anahi E.; Iacobucci, Marta; Ruiz, Karina B.; Biondi, Stefania

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.

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.