High AN1 variability and interaction with basic helix-loop-helix co-factors related to anthocyanin biosynthesis in potato leaves.

AN1 is a regulatory gene that promotes anthocyanin biosynthesis in potato tubers and encodes a R2R3 MYB transcription factor. However, no clear evidence implicates AN1 in anthocyanin production in leaves, where these pigments might enhance environmental stress tolerance. In our study we found that AN1 displays intraspecific sequence variability in both coding/non-coding regions and in the promoter, and that its expression is associated with high anthocyanin content in leaves of commercial potatoes. Expression analysis provided evidence that leaf pigmentation is associated to AN1 expression and that StJAF13 acts as putative AN1 co-regulator for anthocyanin gene expression in leaves of the red leaf variety 'Magenta Love,' while a concomitant expression of StbHLH1 may contribute to anthocyanin accumulation in leaves of 'Double Fun.' Yeast two-hybrid experiments confirmed that AN1 interacts with StbHLH1 and StJAF13 and the latter interaction was verified and localized in the cell nucleus by bimolecular fluorescence complementation assays. In addition, transgenic tobacco (Nicotiana tabacum) overexpressing a combination of either AN1 with StJAF13 or AN1 with StbHLH1 showed deeper purple pigmentation with respect to AN1 alone. This further confirmed AN1/StJAF13 and AN1/StbHLH1 interactions. Our findings demonstrate that the classical loci identified for potato leaf anthocyanin accumulation correspond to AN1 and may represent an important step to expand our knowledge on the molecular mechanisms underlying anthocyanin biosynthesis in different plant tissues.

The Integrated System of Phytodepuration of Sile River Natural Park.

The water conservation topic is likely to become increasingly important and alternative water resources employment should be considered as one possible response to the challenges of fresh water demand and environmental protection; among alternative water sources, municipal wastewaters represent one of the most profitable source but in order to reuse them they need adequate and advanced depuration techniques, such as the use of Integrated System of Phytodepuration (ISP). Across a 3-year sampling period, the performances of an ISP within the Natural Park of the Sile River in the Northern Italy were evaluated, analyzing raw wastewater and final effluent characteristics according to the recommendations of European and Italian legislation. The investigated ISP represents one of the first attempts designed in Italy to improve the efficiency of an existing wastewater treatment plant, able to serve 8000 equivalent inhabitants. The results obtained during the 3 years of analysis show that the designed ISP is characterized by a general efficiency value higher than 87% for TSS removal, 79% for TN, 91% for BOD5 and 86% for COD; moreover the ISP final effluent is characterized by a quality not only suited for release into surface waters but also for irrigation.

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.

Transgenic tobacco plants expressing a fungal laccase are able to reduce phenol content from olive mill wastewaters.

A biotechnological approach was applied to reduce phenol content in olive mill wastewaters by transgenic tobacco plants. The cDNA laccase of poxC gene from Pleurotus ostreatus, carrying its own signal peptide for extracellular secretion, was transferred into the Nicotiana tabacum genome. Transgenic tobacco plants were obtained and the recombinant enzyme was secreted into the rhizosphere by the plant root apparatus, confirming the ability of the plant machinery to recognize the fungal POXC peptide signal leader appropriately as secretory tag. Total laccase activity assayed by ABTS in transgenic lines increased sharply compared to control plants. Moreover, plants cultivated in a hydroponic solution with the addition of olive mill wastewaters were able to reduce the total phenol content up to 70%.

Engineered tobacco and microalgae secreting the fungal laccase POXA1b reduce phenol content in olive oil mill wastewater.

Olive oil mill wastewaters (OMWs) are characterised by low pH and a high content of mono- and polyaromatic compounds that exert microbial and phytotoxic activity. The laccase cDNA of the poxA1b gene from Pleurotus ostreatus, carrying a signal peptide sequence for enzyme secretion and driven by the CaMV 35S promoter, was cloned into a plant expression vector. Nuclear genetic transformation was carried out by co-cultivation of Agrobacterium tumefaciens with tobacco cv Samsun NN leaves and cells of five different microalgae accessions belonging to the genera Chlamydomonas, Chlorella and Ankistrodesmus. Transgenic plants and microalgae were able to express and secrete the recombinant laccase in the root exudates and the culture medium, respectively. In comparison to untransformed controls, the ability to reduce phenol content in OMW solution was enhanced up to 2.8-fold in transgenic tobacco lines and by up to about 40% in two microalgae accessions. The present work provides new evidence for metabolic improvement of green organisms through the transgenic approach to remediation.