Volume 60, Issue 3-4

The plants used for phytoremediation pose special biological risks, whether transgenic or not, as most of the species: (a) are semi-domesticated; (b) are introduced from other habitats; (c) can become established in the contaminated site; (d) can spread and displace native species, and/or; (e) may introgress transgenes into related species. The addition of transgenes can reduce the risks, e.g. to sterilize or render the species and hybrid offspring hypersensitive to environmental effects (heat, cold), or to a chemical that will cull the species. Various measures can contain transgenes used in phytoremediation species to prevent gene flow, but most containment technologies are both uni-directional (prevent either outflow or influx), and are inherently leaky, even a concept specifically utilizable for phytoremediation Ð grafting non-transgenic scions on bioremediating transgenic rootstocks. Containment mechanisms should be either stacked with each other or with “mitigator” genes. Transgenic mitigation (TM) has mitigator genes added in tandem to the desired primary transgene (genetically linked) and the mitigator genes confer traits that are positive or neutral to the desired species but are deleterious to hybrids, keeping them at very low frequencies. The concept was demonstrated in tobacco and oilseed rape with a dwarfing mitigator gene that enhanced the reproductive productivity (harvest index) when cultured alone, but eliminated it from mixed populations. Besides the mitigator genes previously proposed for crop species (sterility, no seed shattering, dwarfing, no secondary dormancy) there are genes especially appropriate for phytoremediation, e.g. overexpression of cytokinin oxidase (reduces cytokinin levels) conferring reduced shoot systems (unfitness to compete) with a more extensive root system that is better for extracting toxic wastes as well as no-flowering for vegetatively propagated species. Thus, biotechnology can be harnessed to reduce risks from both non-transgenic and transgenic phytoremediation species.

Glutathione S-transferases (GSTs) are ubiquitous enzymes and abundant in plants. They are intimately involved in plant metabolism and stress defense related to reactive oxygen species. Our project assigned particular reactions including novel ones to certain GST-isoforms. Transformed E. coli was used to express recombinant GST-isoforms from maize. An N-terminal His tag allowed their purification by affinity chromatography. Three GST-monomers had a molecular weight of 26, 27, 29 kDa, and aggregated to dimers when assayed for their enzymic properties. Four dimeric isoforms were used to study how they interact with tetrapyrroles (of the chlorophyll biosynthesis pathway). It was found that protoporphyrin IX (Proto IX), Mg-protoporphyrin and other tetrapyrroles are bound non-covalently (“liganded”) to GSTs but not conjugated with reduced glutathione. This binding is non-covalent, and results in inhibition of conjugation activity, the degree depends on type of the porphyrin and GST-isoform. I 50 -values between 1-10 μᴍ were measured for Proto IX, the inhibition by mesoporphyrin and Mg-protoporphyrin was 2- to 5-fold less. The ligand binding is noncompetitive for the substrate 1-chloro-2,4-dinitrobenzene and competitive for glutathione. The dimer GST 26/26 prevents the (non-enzymic) autoxidation of protoporphyrinogen to Proto IX, which produces phytotoxic reactive oxygen species in the light. GST 27/27 protects hemin against degradation. Protoporphyrinogen is formed in the plastid and then exported into the cytosol. Apparently binding by a suitable GST-isoform ensures that the highly autoxidizable protoporphyrinogen can safely reach the mitochondrium where it is processed to cytochrome.

Phytoremediation of metal contaminated soils requires high-biomass plants exhibiting tolerance to and accumulation of metal contaminants. However, very little is known about the genes controlling these traits. In order to better understand this, Arabidopsis halleri ssp. halleri (L.) O’Kane and Al-Shehbaz, a naturally selected zinc and cadmium tolerant plant species capable of hyperaccumulating both metals, is a suitable model plant. To date, the scarcity of sequence information from A. halleri is still limiting its use as a model organism. Here we report 128 transcript-derived sequence fragments (TDFs) identified in a cDNAAFLP approach aimed at identifying metal-regulated transcripts in roots. In addition we show that in roots of A. halleri, transcript levels of AhPDR11, encoding an ATP-bindingcassette (ABC) transport protein, are slightly induced in response to metal exposure.

Using plants to extract excess nitrate from soil is important in protecting against eutrophication of standing water, hypoxic conditions in lakes and oceans, or elevated nitrate concentrations in domestic water supplies. Global climate change issues have raised new concerns about nitrogen (N) management as it relates to crop production even though there may not be an immediate threat to water quality. Carbon dioxide (CO 2 ) emissions are frequently considered the primary cause of global climate change, but under anaerobic conditions, animals can contribute by expelling methane (CH 4 ) as do soil microbes. In terms of the potential for global climate change, CH 4 is ~ 25 times more harmful than CO 2 . This differential effect is minuscule compared to when nitrous oxide (N 2 O) is released into the atmosphere because it is ~ 300 times more harmful than CO 2 . N 2 O losses from soil have been positively correlated with residual N (nitrate, NO 3 -) concentrations in soil. It stands to reason that phytoremediation via nitrate scavenger crops is one approach to help protect air quality, as well as soil and water quality. Winter wheat was inserted into a seed corn/soybean rotation to utilize soil nitrate and thereby reduce the potential for nitrate leaching and N 2 O emissions. The net effect of the 2001- 2003 relay cropping sequence was to produce three crops in two years, scavenge 130 kg N/ha from the root zone, produce an extra 2 Mg residue/ha, and increase producer profitability by ~ $ 250/ha.

Thlaspi caerulescens is a well-studied metal-hyperaccumulator of zinc, cadmium and nickel, belonging to the Brassicaceae family. Moreover it is one of the few hyperaccumulators that occur on different metalliferous soil types, as well as on nonmetalliferous soils. We are interested in the development of systems to improve phytoremediation of metal contaminated soils through improved metal-accumulation. About 1900 cDNAs isolated from T. caerulescens roots were hybridized with reverse transcribed RNA from zinc-treated T. caerulescens plants of two accessions originating from two different soil types. This comparative transcript profiling of T. caerulescens plants resulted in the identification of genes that are affected by heavy metals. The developed microarray proved to be an appropriate tool for a large scale analysis of gene expression in this metal-accumulator species.

A functional screening in yeast allowed to identify various cDNAs from the Cd/Zn hyperaccumulator Thlaspi caerulescens. TcMT3 displayed high identity with its closest homologue in Arabidopsis thaliana but variation in its Cys residues. Functional analysis in yeast supported a higher binding capacity for Cu, but not for Cd or Zn, of TcMT3 compared to AtMT3. Expression analysis in plants indicated that metallothionein 3 (MT3) like all the other T. caerulescens genes from the screen studied is overexpressed in all studied populations of T. caerulescens compared to A. thaliana. TcMT3 was induced by Cu, but not by Cd. Moreover significant variation in expression within T. caerulescens populations that have contrasting tolerance and accumulation capacities indicated a possible local adaptation of MT3.

The genetic diversity of Cistus ladanifer ssp. ladanifer (Cistaceae) growing on ultramafic and non-ultramafic (basic and schists) soils in the NE of Portugal was studied in order to identify molecular markers that could distinguish the metal-tolerant ecotypes of this species. Random Amplified Polymorphic DNA (RAPD) markers were used in order to estimate genetic variation and differences between populations. The RAPD dataset was analysed by means of a cluster analysis and an analysis of molecular variance (AMOVA). Our results indicate a significant partitioning of molecular variance between ultramafic and non-ultramafic populations of Cistus ladanifer, although the highest percentage of this variance was found at the intra-population level. Mantel’s test showed no relationship between inter-population genetic and geographic distances. A series of RAPD bands that could be related to heavy metal tolerance were observed. The identification of such markers will enable the use of Cistus ladanifer in phytoremediation procedures.

Mercury-binding peptides from roots, stems, and leaves of Hg-treated Chromolaena odorata plants were isolated and partially characterized using RP-HPLC and ESI-MS. Upon exposure of C. odorata plants to high concentrations of 1.0 and 2.0 μᴍ Hg(NO 3 ) 2 treatments from 0-28 days, they accumulated as much as 125 mg/g (dry wt) Hg in the roots, 15.280 mg/g (dry wt) Hg in the stems, and 0.800 mg/g (dry wt) Hg in the leaves indicating that C. odorata has a high potential as a phytoremediation agent of inorganic mercury. The plant’s ability to accumulate and sequester Hg ions was primarily attributed to the production of Hgbinding peptides, which were initially detected through the use of Ellman’s reagent. Isolation techniques using RP-HPLC equipped with a C18 column manifested a single prominent peak consistently appearing at a retention time of 2.6-2.8 min in all the plant samples treated with different Hg concentrations at varying lengths of exposure. Further characterization of this prominent peak using electrospray ionization mass spectrometry revealed the presence of a peptide containing several cysteine residues with the highest peak concentration recorded at 91 mV and 89 mV in roots and stems of plants treated with 2.0 μᴍ Hg(NO 3 ) 2 for 4 wk (P < 0.05) and 85 mV in leaves treated with 1.0 μᴍ Hg(NO 3 ) 2 for 1 wk.

A field experiment with microelement loads was set up on loamy textured, calcareous chernozem soil formed on loess. The ploughed layer contained ca. 5% CaCO 3 and 3% humus. The soil was well supplied with Ca, Mg, Mn and Cu, moderately supplied with N and K, and weakly supplied with P and Zn. The water table is at the depth of 15 m, the water balance of the area is negative, and the site is drought sensitive. Salts of the 13 examined microelements were applied at 4 doses in the spring of 1991. Treatments were arranged in a split-plot design, in a total of 104 plots with two replications. Loading rates were 0, 90, 270 and 810 kg/ha per element in the form of AlCl 3 , NaAsO 2 , BaCl 2 , CdSO 4 , K 2 CrO 4 , CuSO 4 , HgCl 2 , (NH 4 ) 6 Mo 7 O 24 , NiSO 4 , Pb(NO 3 ) 2 , Na 2 SeO 3 , SrSO 4 , ZnSO 4 . Soil profiles of the control and the 810 kg/ha treated plots were sampled in the 3 rd , 6 th and 10 th year of the trial. The mixed samples, consisting of 5 cores/plot were taken in 30 cm steps to 60 cm (year 1993), 90 cm (year 1996), and 290 cm (year 2000). Ammonium acetate + EDTA-soluble element content was determined. The main conclusions of the study are: 1. In soils contaminated with 810 kg/ha load rates As, Hg, Ni, Cu, Pb, Ba and Sr displayed no significant vertical movement. There is little uptake and translocation in plants of these elements: their concentration in the above-ground plant parts usually remains below 5D 10 mg/kg dry mass with the exception of Ba and Sr showing a somewhat higher accumulation. Under our conditions the above elements did not behave as dangerous contaminants to the soil, groundwater, or plants. 2. Moderate leaching of Zn and Cd was detected when applied at higher doses. Their accumulation was also moderate in the above-ground plants parts. Zn is not a dangerous pollutant for soil, plants or groundwater at our site. Cd, however, is a very dangerous element because of its high toxicity to mammals, soil life and crops. 3. Cr, Se, and Mo (in the form of chromate, selenate and molybdenate anions) exhibited great mobility in the soil and partly in the soil-plant system. Cr was hardly detectable in the above-ground parts of plants. Still, its rapid leaching can jeopardize groundwater quality. Se was strongly accumulated in all plant organs and was highly toxic for all crops. Mo, too, showed 2D3 orders of magnitude greater accumulation in plant parts, resulting in products unfit for animal or human consumption. Under our experimental conditions Cr(VI), Se, Mo can be classified as dangerous contaminants, since the anion forms remained stabile for a long time in this well-aerated calcareous environment.

Our previous study showed that approximately one-third of the nitrogen of 15 N-labeled NO 2 taken up into plants was converted to a previously unknown organic nitrogen (hereafter designated UN) that was not recoverable by the Kjeldahl method (Morikawa et al., 2004). In this communication, we discuss metabolic and physiological relevance of the UN based on our newest experimental results. All of the 12 plant species were found to form UN derived from NO 2 (about 10-30% of the total nitrogen derived from NO 2 ). The UN was formed also from nitrate nitrogen in various plant species. Thus, UN is a common metabolite in plants. The amount of UN derived from NO 2 was greatly increased in the transgenic tobacco clone 271 (Vaucheret et al., 1992) where the activity of nitrite reductase is suppressed less than 5% of that of the wild-type plant. On the other hand, the amount of this UN was significantly decreased by the overexpression of S-nitrosoglutathione reductase (GSNOR). These findings strongly suggest that nitrite and other reactive nitrogen species are involved in the formation of the UN, and that the UN-bearing compounds are metabolizable. A metabolic scheme for the formation of UN-bearing compounds was proposed, in which nitric oxide and peroxynitrite derived from NO 2 or endogenous nitrogen oxides are involved for nitrosation and/or nitration of organic compounds in the cells to form nitroso and nitro compounds, including N-nitroso and S-nitroso ones. Participation of non-symbiotic haemoglobin bearing peroxidase-like activity (Sakamoto et al., 2004) and GSNOR (Sakamoto et al., 2002) in the metabolism of the UN was discussed. The UN-bearing compounds identified to date in the extracts of the leaves of Arabidopsis thaliana fumigated with NO 2 include a ⊿ 2 - 1,2,3-thiadiazoline derivative (Miyawaki et al., 2004) and 4-nitro-β-carotene.

Arabidopsis thaliana was transformed with a gene encoding a nitroreductase (NTR, E.C. 1.6.99.7) with activity against a wide range of nitroaromatic compounds. The gene was transferred from Escherichia coli by an Agrobacterium-mediated in planta method. The ob­tained seeds were sowed to produce T1 plants, and they were assayed for the integration of the transgene in the plant genome. Transgenic plants that were positive with the PCR analysis were self-pollinated to produce T2 generation plants. Seven lines obtained were assayed for the NTR activity. While the noil-transformed wild-type plants showed no detectable NTR activity, the enzyme activity of the transgenic plant lines was approx. 20 times higher. Using the line with the highest NTR activity, the phytoremediation characteristics of plants against 2,4,6-trinitrotoluene (TNT) was investigated. While the wild-type plants did not grow in the presence of 0.1 mᴍ TNT, the transgenic plants grew almost normally in this condition. The uptake of TNT by seedlings of transgenic plants increased by 7 to 8 times when they were floated on TNT solution. HPLC analysis showed that the peak due to TNT taken up into plant body was much smaller in the transgenic plants as compared with that of the wild type, and that a number of peaks attributable to the degradation products of TNT, including 4-amino-2,6-dinitrotoluene, were detected in the extract from the transgenic plants. This indi­cates that the expression of bacterial NTR improved the capability of plants to degrade TNT.

In order to investigate nocturnal uptake and assimilation of NO 2 by C3 and crassulacean acid metabolism (CAM) plants, they were fumigated with 4 μl I -115 N-Iabeled nitrogen diox­ide (NO 2 ) for 8 h. The amound of NO 2 and assimilation of NO 2 by plants were determined by mass spectrometry and Kjeldahl-nitrogen based mass spectrometry, respectively. C3 plants such as kenaf (Hibiscus cannabinus), tobacco (Nicotiana tabacum) and ground cherry (Phy- salis alkekengi) showed a high uptake and assimilation during daytime as high as 1100 to 2700 ng N mg -1 dry weight. While tobacco and ground cherry strongly reduced uptake and assimilation of NO 2 during nighttime, kenaf kept high nocturnal uptake and assimilation of NO 2 as high as about 1500 ng N mg -1 dry weight. Stomatal conductance measurements indicated that there were no significant differences to account for the differences in the uptake of NO 2 by tobacco and kenaf during nighttime. CAM plants such as Sedum sp., Kalanchoe blossfeldiana (kalanchoe) and Aloe arborescens exhibited nocturnal uptake and assimilation of NO 2 . However, the values of uptake and assimilation of NO 2 both during daytime and nighttime was very low (at most about 500 ng N mg -1 dry weight) as compared with those of above mentioned C3 plants. The present findings indicate that kenaf is an efficient phytoremediator of NO 2 both during daytime and nighttime.

Wetland grasses and grass-like monocots are very important natural remediators of pollutants. Their genetic improvement is an important task because introduction of key transgenes can dramatically improve their remediation potential. Tissue culture is prerequisite for genetic manipulation, and methods are reported here for in vitro culture and micropropagation of a number of wetland plants of various ecological requirements such as salt marsh, brackish water, riverbanks, and various zones of lakes and ponds, and bogs. The monocots represent numerous genera in various families such as Poaceae, Cyperaceae, Juncaceae, and Typhaceae. The reported species are in various stages of micropropagation and Arundo donax is scaled for mass propagation for selecting elite lines for pytoremediation. Transfer of key genes for mercury phytoremediation into the salt marsh cordgrass (Spartina alterniflora) is also reported here. All but one transgenic lines contained both the organomercurial lyase (merB) and mercuric reductase (merA) sequences showing that co-introduction into Spartina of two genes from separate Agrobacterium strains is possible.

Clone stability and in vitro phytoextraction capacity of vegetative clones of P. x canescens (2n = 4x = 38) including two transgenic clones (ggs11 and lgl6) were studied as in vitro leaf disc cultures. Presence of the gshI-transgene in the transformed clones was detected in PCR reactions using gshI-specific primers. Clone stability was determined by fAFLP (fluorescent amplified DNA fragment length polymorphism) analysis. In total, 682 AFLP fragments were identified generated by twelve selective primer pairs after EcoRIDMseI digestion. Four fragments generated by EcoAGTDMseCCC were different (99.4% genetic similarity) which proves an unexpectedly low bud mutation frequency in P. \ canescens. For the study of phytoextraction capacity leaf discs (8 mm) were exposed to a concentration series of ZnSO 4 (10 -1 to 10 -5 ᴍ) incubated for 21 days on aseptic tissue culture media WPM containing 1 μᴍ Cu. Zn 2+ caused phytotoxicity only at high concentrations (10 -1 to 10 -2 ᴍ). The transgenic poplar cyt-ECS (ggs11) clone, as stimulated by the presence of Zn, showed elevated heavy metal (Cu) uptake as compared to the non-transformed clone. These results suggest that gshI-transgenic poplars may be suitable for phytoremediation of soils contaminated with zinc and copper.

By learning lessons from weed science we have adopted three approaches to make plants more effective in phytoremediation: 1. The application of functional genomics to identify key components involved in the detoxification of, or tolerance to, xenobiotics for use in subsequent genetic engineering/breeding programmes. 2. The rational metabolic engineering of plants through the use of forced evolution of protective enzymes, or alternatively transgenesis of detoxification pathways. 3. The use of chemical treatments which protect plants from herbicide injury. In this paper we examine the regulation of the xenome by herbicide safeners, which are chemicals widely used in crop protection due to their ability to enhance herbicide selectivity in cereals. We demonstrate that these chemicals act to enhance two major groups of phase 2 detoxification enzymes, notably the glutathione transferases and glucosyltransferases, in both cereals and the model plant Arabidopsis thaliana, with the safeners acting in a chemical- and species-specific manner. Our results demonstrate that by choosing the right combination of safener and plant it should be possible to enhance the tolerance of diverse plants to a wide range of xenobiotics including pollutants.

Unintentional loss of herbicides into drainage ditches, shores or other waterbodies may cause large problems in farmland. Therefore strategies for the phytoremediation of agrochemicals and especially herbicides have become a topic of great interest in many agricultural areas. However, in order to establish effective biological pollution control, information on the detoxification capacity of riparian plants and aquatic macrophytes (e.g., Phragmites australis) is important to build up effective buffer stripes. We determined the detoxification capacity of Phragmites australis roots and leaves for the conjugation of agrochemicals to glutathione by assaying the model substrate CDNB as well as the herbicides fenoxaprop-P, propachlor, pethoxamid and terbuthylazine. Specific GST activities were always higher in the rhizomes (6.78 d 0.88 μkat/mg protein for CDNB) than in leaves (1.08 d 0.21 μkat/mg protein). The detoxification capacity is distributed across an array of GST isoforms. In summary, Phragmites australis seems to be efficient in herbicide detoxification and a good candidate for phytoremediation of effluents from agricultural sites.

Root-microbe interactions are considered to be the primary process of polyaromatic hydrocarbon (PAH) phytoremediation, since bacterial degradation has been shown to be the dominant pathway for environmental PAH dissipation. However, the precise mechanisms driving PAH rhizostimulation symbiosis remain largely unresolved. In this study, we assessed PAH degrading bacterial abundance in contaminated soils planted with 18 different native Michigan plant species. Phenanthrene metabolism assays suggested that each plant species differentially influenced the relative abundance of PAH biodegraders, though they generally were observed to increase heterotrophic and biodegradative cell numbers relative to unplanted soils. Further study of > 1800 phenanthrene degrading isolates indicated that most of the tested plant species stimulated biodegradation of a broader range of PAH compounds relative to the unplanted soil bacterial consortia. These observations suggest that a principal contribution of planted systems for PAH bioremediation may be via expanded metabolic range of the rhizosphere bacterial community.

Two bacterial isolates were obtained in axenic culture from the rhizosphere soil of Astragalus bisulcatus, a legume able to hyperaccumulate selenium. Both strains resulted of particular interest for their high resistance to the toxic oxyanion SeO 3 2- (selenite, Se IV ). On the basis of molecular and biochemical analyses, these two isolates were attributed to the species Bacillus mycoides and Stenotrophomonas maltophilia, respectively. Their capability in axenic culture to precipitate the soluble, bioavailable and highly toxic selenium form selenite to insoluble and relatively non-toxic Se 0 (elemental selenium) was evaluated in defined medium added with 0.2 or 0.5 mm Se IV . Both strains showed to completely reduce 0.2 mᴍ selenite in 120 h, while 0.5 mm Se IV was reduced up to 67% of the initial concentration by B. mycoides and to about 50% by S. maltophilia in 48 h. Together in a dual consortium, B. mycoides and S. maltophilia increased the kinetics of selenite reduction, thus improving the efficiency of the process. A model system for selenium rhizofiltration based on plant-rhizobacteria interactions has been proposed.

The aim of this work was to study the colonization of indigenous arbuscular mycorrhizal fungi (AMF) species in fine-roots of poplar clones. Roots of 7 poplar clones were sampled from a 1-year-old trial established at an industrial site strongly polluted with heavy metals at Balatonfűzfő , Hungary. The poplar clones have shown variable degrees of colonization by AMF, suggesting differential host susceptibility or mycorrhizal dependency. After outplanting the percentage of poplar survival was strongly correlated with the frequency of AMF infection. Two clones that survived at the lowest ratio after outplanting had not been colonized by AMF in contrast to those which survived to a much higher extent.

Vegetation at an abandoned heavy metal bearing mine tailing may have multifunctional roles such as modification of water balance, erosion control and landscape rehabilitation. Research on the vegetation of mine tailings can provide useful information on tolerance, accumulation and translocation properties of species potentially applicable at moderately contaminated sites. Analyses of the relationship between heavy metal content (Pb, Zn and Cu) and vegetation in a mine tailing were carried out. These analyses included: (1) spatial analysis of relationship among heavy metal distribution, pH and vegetation patterns, and (2) analysis of heavy metal accumulation and translocation in some plant species. Presence of vegetation was found to be significantly dependent on pH value, which confirms that phytotoxicity is a function of element concentration in solution, which is primarily controlled by pH value in mine tailings. Among the most abundant plant species, dewberry (Rubus caesius), vipersbugloss (Echium vulgare), scarlet pimpernel (Anagallis arvensis) and narrowleaf plantain (Plantago lanceolata) accumulate significant amounts of Pb, Cu and Zn, while in the case of annual bluegrass (Poa annua) only Pb can be measured in elevated contents. Considering the translocation features, scarlet pimpernel, narrowleaf plantain, and dewberry accumulate heavy metals primarily in their roots, while heavy metal concentration in vipersbugloss and annual bluegrass is higher in the shoots.