Phytoremediation of soils co-contaminated by organic compounds and heavy metals: bioassays with Lupinus luteus L. and associated endophytic bacteria.

In the central part of the Iberian Peninsula there are old sealed landfills containing soils co-contaminated by several heavy metals (Cu, Zn, Pb, Cd, Ni, As, Cr, Fe, Al, Mn) and organic pollutants of different families (hydrocarbons, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, pesticides and other organochlorinated compounds, phenols and volatile compounds), which this work will address. We have focused on phytoremedial plants that are able to deal with this type of complex pollution, not only species that tolerate the joint effect of heavy metals in the soil, but also those that can take advantage of associated bacteria to efficiently break down organic compounds. This study was carried out with Lupinus luteus and its endophytes in two greenhouse experiments: A) growing in a substrate artificially contaminated with benzo(a)pyrene (BaP), and B) using real co-contaminated landfill soils. Endophytes of roots and shoots were isolated in both bioassays. Plant growth-promotion tests and organic pollutant tolerance and degradation tests were conducted on all strains isolated in bioassay A), and on those proving to be pure cultures from bioassay B). The selected landfill is described as are isolation and test procedures. Results indicate that plants did not show toxicity symptoms when exposed to BaP but did when grown in landfill soil. Some endophytes demonstrated plant growth-promotion capacity and tolerance to BaP and other organic compounds (diesel and PCB commercial mixtures). A few strains may even have the capacity to metabolize those organic pollutants. The overall decline in plant growth-promotion capacity in those strains isolated from the landfill soil experiment, compared with those from the bioassay with BaP, may indicate that lupin endophytes are not adapted to metal concentration in roots and shoots and fail to grow. As a result, most isolated root endophytes must have colonized root tissues from the soil. While preliminary degradation tests showed promising results (some strains exhibiting the potential to use organic pollutants as their sole source of carbon), these are not conclusive and further in-depth degradation assays need to be performed.

Integrated approach to assessing the effects of soils polluted with heavy metals on a plant population.

This study addresses the effects of soil polluted with more than one heavy metal in a grass species. A 16-week bioassay with Avena sativa L. was conducted in microcosms using soils from two abandoned mines in central Spain that contained levels above World Health Organization (WHO) reference limits for soils of more than three heavy metals. Pollution effects were examined at cell, tissue, organ, plant and population levels. For this purpose, dry weight, maximum height and number of leaves were determined; leaf tissues were observed by low temperature scanning electron microscopy; the metal contents of roots and shoots were determined by plasma emission spectroscopy and their distribution in different tissues was analyzed by X-ray microanalysis using an environmental scanning electron microscope. The results explain the accumulation and translocation of soil metals by this plant species; their effects in cells, tissues and growth of plants; and allow inference on population effects. The discussion of the methodological approach leads us to propose a valid protocol to assess the effects of a set of heavy metals present in the topsoil of polluted sites on a plant population. We recommend its use for an ecotoxicological diagnosis and risk analysis of similarly polluted sites.

Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis.

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter-beta-glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.

Aspergillus oryzae has two nearly identical Taka-amylase genes, each containing eight introns.

cDNA and genomic DNA for two nearly identical genes, AmyI and AmyII, coding for the enzyme Taka-amylase A (TA-A) of the fungus Aspergillus oryzae have been cloned and characterized. These genes are apparently unlinked, differing by only 3 nucleotides (nt) out of the 2720 nt that span the coding regions. The 617-nt 5'-flanking regions differ only at nt -372 (T or A) from the putative ATG start codon and contain four sets of short, inverted repeats (IR) upstream from the putative TATAAA box at nt -100 and the transcription start point at nt -69. The coding regions consist of 499 codons disrupted by eight intervening sequences. The putative proenzymes differ by only two amino acids (aa) and consist of the 478-aa extracellular enzyme plus a 21-aa hydrophobic leader sequence. Except for the replacement site changes in codons 35 (Arg----Gln) and 151 (Phe----Leu), the identity of the two genes continues downstream for 58 nt past the TGA stop codon before diverging. Exon 9 codes for 94 of the 98 aa of the domain B of mature TA-A. Little conservation of TA-A exons was found when these exons were aligned with those of human amylase. The genes are flanked by at least 6 to 10 kb of unrelated chromosomal nucleotide sequence. The Amy genes are co-expressed, since mRNA (cDNA) specific to the 3'-UTR of both genes was recovered from mycelia grown on starch, a known inducer of TA-A biosynthesis. The 3'-UTRs of cDNAs related to AmyI are shorter (128 nt and 145 nt) than those of AmyII (179 nt and 297 nt). The AmyI specific 3'-UTR is characterized by the absence of IR sequences and the presence of a putative 'AATAAA' polyadenylation signal.