Proceedings of the 54th Italian Society of Agricultural Genetics Annual Congress
Matera, Italy – 27/30 September, 2010
ISBN 978-88-904570-0-5
Poster Communication Abstract – 4.33
TRANSGENIC TOBACCO PLANTS EXPRESSING POXA1B GENE ARE
ABLE TO REDUCE OLIVE OIL MILL WASTEWATER PHENOLS
CONTENT
TATINO F., PALOMBA F., CHIAIESE P., FILIPPONE E.
Department of Soil, Plant, Environmental and Animal Production Sciences, School of
Biotechnology, University of Naples “Federico II”, Via Università 100, 80055 Portici (Italy)
Laccase, Nicotiana tabacum, phytoremediation, poxA1b, olive mill wastewater
Olive oil mill wastewaters (OMW) are one of major environmental issue in the Mediterranean
area due to their high content of phytotoxic and low biodegradability of aromatic compounds such
as phenols. Lignin, phenol and other aromatic compounds are degraded by white rot fungi that
release in the environment oxidative enzymes, such as laccases. Our aim is to evaluate the ability of
transgenic plants to reduce OMW phenol content by releasing a heterologous laccase enzyme. In
silico analysis of poxA1b gene, encoding for a multicopper oxidase protein isolated from Pleurotus
ostreatus, shown that signal fungi peptide is recognised in tobacco plants and therefore laccase
protein might be release by root system. Transgenic tobacco plants were obtained by co-cultivation
of leaf explants with Agrobacterium tumefaciens bearing the plant expression vector pGreen
0029::35S poxA1b. Putative transgenic shoots were screened by PCR, western blot and zymogram.
The quantification of laccase activity was performed by 2,2'-azino-bis(3-ethylbenzthiazoline-6sulphonic acid (ABTS) and 2,6 dimethoxyphenol (DMP) coupled assays on young leaves and roots
exudates collected from eight tobacco plants growing in vivo in hydroponic culture. In leaf explants
from transgenic lines the enzymatic activity was detected at an appreciable level and such activity
was 2 times higher in transgenic lines 5 and 8 than in control plants. Roots exudates from the
control plants did not showed any activities whereas in four transgenic plants a significant
enzymatic activity was found. The ability of transgenic plants to reduce OMW-phenol content was
carried out in hydroponic condition. Plants were left to grow into an OMW solution having a phenol
content of 4.2 g l-1 . Two transgenic lines were able to reduce the phenol content to 0.08 g FW-1,
after only 10 days of growth in OMW solution. This figure was 3.5 times lower than the one
observed in treatment with control plants.
Morphological variations were detected in five out eight transgenic plants. These alterations
were observed for size, thickness, colour of leafs and plants inflorescences abortion before
flowering.