Phytoavailability of Heavy Metals and Metalloids in Soils Irrigated with Wastewater,Akaki, Ethiopia: A Greenhouse Study

Irrigation with untreated wastewater from several industrial, commercial, and domestic discharges for decades caused accumulation of various heavy metals and metalloids in soils along the Akaki River in Ethiopia. Assessment of environmental threats and the potential phytoremediation of the soils require understanding of the toxic elements’ uptake and distribution in plant parts. Hence, a greenhouse study was performed to examine the phytoavailability and distribution of Cr, Ni, Co, Cu, Zn, Cd, Pb, Hg, Se, V, and As in forage grasses: Oat (Avena sativa), Rhodes grass (Chloris gayana), Setaria (Setaria sphacelata), and the legumes Alfalfa (Medicago sativa) and Desmodium (Desmodium unicinatum). The average contents of Cr, Ni, Co, Cu, Zn, Pb, Hg, Se, and V in the plants were generally higher than the background levels for forage grasses/legumes, and some of these elements were in the phytotoxic range. Root bioconcentration factor (BCF = root to soil concentration ratio) > 1 was observed for Cu (Oat, Rhodes, Desmodium, and Setaria: Fluvisol), Zn (Setaria: Fluvisol), Cd (Rhodes: Fluvisol; Setaria from both soils) and Hg (Oat and Alfalfa: Fluvisol). Alfalfa and Desmodium displayed translocation factor > 1 (TF = shoot to root concentration ratio) for most heavy metals. Most heavy metals/metalloids may pose a health threat to humans and stock via introduction to the food chain. The plant factors (species and plant part), soil factors (soil type, soil fractions, pH, and CEC), and their interactions significantly (p < 0.05) influenced plant heavy metal and metalloid levels. However, the role of plant part and species emerged as the most important on heavy metal uptake, translocation, sequestration, and ultimately transfer to the food chain. Accordingly, the uptake and distribution of heavy metals/metalloids in the plants reflect the potential environmental and health hazards attributable to the use of fodder grasses, legumes, and cultivation of vegetables in soils with polymetallic and metalloid contamination.     

Potential impact of genetically modified Lepidoptera‐resistant Brassica napus in biodiversity hotspots: Sicily as a theoretical model

The general increase of the cultivation and trade of Bt transgenic plants resistant to Lepidoptera pests raises concerns regarding the conservation of animal and plant biodiversity. Demand for biofuels has increased the cultivation and importation of oilseed rape (Brassica napus L.), including transgenic lines. In environmental risk assessments (ERAs) for its potential future cultivation as well as for food and feed uses, the impact on wild Brassicaeae relatives and on non‐target Lepidoptera should be assessed. Here we consider the potential exposure of butterflies as results of possible cultivation or naturalization of spilled seed in Sicily (Italy). Diurnal Lepidoptera, which are pollinators, can be exposed directly to the insecticidal proteins as larvae (mainly of Pieridae) through the host and through the pollen that can deposit on other host plants. Adults can be exposed via pollen and nectar. The flight periods of butterflies were recorded, and they were found to overlap for about 90% of the flowering period of B. napus for the majority of the species. In addition, B. napus has a high potential to hybridise with endemic taxa belonging to the B. oleracea group. This could lead to an exposure of non‐target Lepidoptera if introgression of the Bt gene into a wild population happens. A rank of the risk for butterflies and wild relatives of oilseed rape is given. We conclude that, in environmental risk assessments, attention should be paid to plant–insect interaction especially in a biodiversity hotspot such as Sicily.     

Enhanced Cd extraction of oilseed rape (Brassica napus) by plant growth-promoting bacteria isolated from Cd hyperaccumulator Sedum alfredii Hance

Four plant growth-promoting bacteria (PGPB) were used as study materials, among them two heavy metal-tolerant rhizosphere strains SrN1 (Arthrobacter sp.) and SrN9 (Bacillus altitudinis) were isolated from rhizosphere soil, while two endophytic strains SaN1 (Bacillus megaterium) and SaMR12 (Sphingomonas) were identified from roots of the cadmium (Cd)/zinc (Zn) hyperaccumulator Sedum alfredii Hance. A pot experiment was carried out to investigate the effects of these PGPB on plant growth and Cd accumulation of oilseed rape (Brassica napus) plants grown on aged Cd-spiked soil. The results showed that the four PGPB significantly boosted oilseed rape shoot biomass production, improved soil and plant analyzer development (SPAD) value, enhanced Cd uptake of plant and Cd translocation to the leaves. By fluorescent in situ hybridization (FISH) and green fluorescent protein (GFP), we demonstrated the studied S. alfredii endophytic bacterium SaMR12 were able to colonize successfully in the B. napus roots. However, all four PGPB could increase seed Cd accumulation. Due to its potential to enhance Cd uptake by the plant and to restrict Cd accumulation in the seeds, SaMR12 was selected as the most promising microbial partner of B. napus when setting up a plant–microbe fortified remediation system.     

Antioxidant defense system in leaves of Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) and rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) under cadmium stress

Plant species capable of hyper-accumulating heavy metals are of considerable interest for phytoremediation, and differ in their ability to accumulate metals from environment. Using two brassica species (Brassica juncea and Brassica napus), nutrient solution experiments were conducted to study variation in tolerance to cadmium (Cd) toxicity based on (1) lipid peroxidation and (2) changes in antioxidative defense system in leaves of both plants (i.e., superoxide dismutase (SOD EC 1.15.1.1), catalase (CAT EC 1.11.1.6), ascorbate peroxidase (APX EC 1.11.1.11), guaiacol peroxidase (GPX EC 1.11.1.7), glutathione reductase (GR EC 1.6.4.2), levels of phytochelatins (PCs), non-protein thiols (NP-SH), and glutathione. Plants were grown in nutrient solution under controlled environmental conditions, and subjected to increasing concentrations of Cd (0, 10, 25 and 50 μM) for 15 days. Results showed marked differences between both species. Brassica napus under Cd stress exhibited increased level of lipid peroxidation, as was evidenced by the increased malondialdehyde (MDA) content in leaves. However, in Brassica juncea treated plants, MDA content remained unchanged. In Brassica napus, with the exception of GPX, activity levels of some antioxidant enzymes involved in detoxification of reactive oxygen species (ROS), including SOD, CAT, GR, and APX, decreased drastically at high Cd concentrations. By contrast, in leaves of Brassica juncea treated plants, there was either only slight or no change in the activities of the antioxidative enzymes. Analysis of the profile of anionic isoenzymes of GPX revealed qualitative changes occurring during Cd exposure for both species. Moreover, levels of NP-SH and PCs, monitored as metal detoxifying responses, were much increased in leaves of Brassica juncea by increasing Cd supply, but did not change in Brassica napus. These results indicate that Brassica juncea plants possess the greater potential for Cd accumulation and tolerance than Brassica napus.     

The isochorismate pathway is negatively regulated by salicylic acid signaling in O<Subscript>3</Subscript>-exposed <Emphasis Type="Italic">Arabidopsis</Emphasis>

Phytochelatins (PCs) are heavy metal binding peptides that play an important role in sequestration and detoxification of heavy metals in plants. In this study, our goal was to develop transgenic plants with increased tolerance for and accumulation of heavy metals from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A 35S promoter fused to a FLAG–tagged AtPCS1 cDNA was expressed in Indian mustard, and transgenic lines, designated pc lines, were evaluated for tolerance to and accumulation of Cd and Zn. Transgenic plants with moderate AtPCS1 expression levels showed significantly higher tolerance to Cd and Zn stress, but accumulated significantly less Cd and Zn than wild type plants in both shoot and root tissues. However, transgenic plants with highest expression of the transgene did not exhibit enhanced Cd and Zn tolerance. Shoots of Cd-treated pc plants had significantly higher levels of phytochelatins and thiols than wild-type plants. Significantly lower concentrations of gluthatione in Cd-treated shoot and root tissues of transgenic plants were observed. Moderate expression levels of phytochelatin synthase improved the ability of Indian mustard to tolerate certain levels of heavy metals, but at the same time did not increase the accumulation potential for Cd and Zn.     

Arbuscular mycorrhiza decreases cadmium phytoextraction by transgenic tobacco with inserted metallothionein

The effect of arbuscular mycorrhiza (AM) on the phytoextraction efficiency of transgenic tobacco with increased ability to tolerate and accumulate cadmium (Cd) was tested in a pot experiment. The tobacco plants bearing the yeast metallothionein CUP1 combined with a polyhistidine cluster were compared to non-transgenic tobacco of the same variety at four Cd concentrations in soil, non-inoculated or inoculated with two isolates of the AM fungus Glomus intraradices. Mycorrhizal inoculation improved the growth of both the transgenic and non-transgenic tobacco and decreased Cd concentrations in shoots and root to shoot translocation. Differences were found between the two AM fungal isolates: one isolate supported more efficient phosphorus uptake and plant growth in the soil without Cd addition, while the other isolate alleviated the inhibitory effect of cadmium on plant growth. The resulting effect of inoculation on Cd accumulation was dependent on Cd level in soil and differed between the more Cd tolerant transgenic plants and the less tolerant non-transgenic plants. Mycorrhiza mostly decreased the phytoextraction efficiency of transgenic plants while increased that of non-transgenic plants at Cd levels in soil inhibitory to tobacco growth. Mechanisms of the observed effects of inoculation on growth and Cd uptake are discussed as well as the possible implications of the results for the exploitation of AM in phytoextraction of heavy metals from contaminated soils.     

Assessment of effects of Bt‐oilseed rape on large white butterfly (Pieris brassicae) in natural habitats

Much effort has been allocated to the definition of risk, relevant for the assessment of genetically modified plants. However, few studies have emphasised the limitations in testing methods. In this study, tests for and effects on non‐target herbivores were exemplified and evaluated for Pieris brassicae (L.) (Lepidoptera: Pieridae) and a genetically modified Brassica napus L. (Brassicaceae) expressing the Bacillus thuringiensis (Bt)‐toxin Cry1Ac. It was established that this herbivore recognises and accepts the transgenic plant as a host. It was found that ovipositing females of P. brassicae preferred the transgenic variety for egg‐laying. Therefore, effects of the transgenic host plant on the herbivore were determined. Larvae feeding on the Bt‐plants experienced 100% mortality for all larval stages. Based on these observations, a population model was established. The model showed that larval survival is increased with amount of food (number of plants) and reduced with the frequency of transgenic specimens, number of host plants needed for completing larval development, and number of egg‐laying butterflies. Such models may both aid the design of further tests for effects and support the assessment whether population effects are likely to occur due to the presence of insect‐resistant plants outside the agricultural area.     

Transgenic Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) plants expressing an <Emphasis Type="Italic">Arabidopsis</Emphasis> phytochelatin synthase (<Emphasis Type="Italic">AtPCS1</Emphasis>) exhibit enhanced As and Cd tolerance

Phytochelatins (PCs) are post-translationally synthesized thiol reactive peptides that play important roles in detoxification of heavy metal and metalloids in plants and other living organisms. The overall goal of this study is to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A FLAG-tagged AtPCS1 gDNA, under its native promoter, is expressed in Indian mustard, and transgenic pcs lines have been compared with wild-type plants for tolerance to and accumulation of cadmium (Cd) and arsenic (As). Compared to wild type plants, transgenic plants exhibit significantly higher tolerance to Cd and As. Shoots of Cd-treated pcs plants have significantly higher concentrations of PCs and thiols than those of wild-type plants. Shoots of wild-type plants accumulated significantly more Cd than those of transgenic plants, while accumulation of As in transgenic plants was similar to that in wild type plants. Although phytochelatin synthase improves the ability of Indian mustard to tolerate higher levels of the heavy metal Cd and the metalloid As, it does not increase the accumulation potential of these metals in the above ground tissues of Indian mustard plants.     

Arrest of embryo development in Brassica napus mediated by modified Pseudomonas aeruginosa exotoxin A

Intracellularly expressed cytotoxins are useful tools both to study the action of plant regulatory sequences in transgenic plants and to modify plant phenotype. We have engineered a low mammalian toxicity derivative of Pseudomonas aeruginosa exotoxin A for intracellular expression in plant cells by fusing the ADP ribosylating domain of the exotoxin gene to plant regulatory sequences. The efficacy of exotoxin A on plant cells was demonstrated by transient expression of the modified exotoxin gene in tobacco protoplasts: the exotoxin gene inhibited the expression of a co-electroporated -glucuronidase gene. An exotoxin with an introduced frameshift mutation was also effective at inhibiting -glucuronidase expression in the transient assay; the activity of the frameshifted gene was presumably a result of frameshifting during translation or initiation of translation at a codon other than AUG. When fused to napin regulatory sequences, the exotoxin gene specifically arrested embryo development in the seeds of transgenic Brassica napus plants concomitant with the onset of napin expression. The napin/exotoxin chimeric gene did not have the same pattern of expression in tobacco as in B. napus; in addition to exhibiting an inhibition of seed development, the transgenic tobacco plants were male-sterile.     

Transgenic banana plants overexpressing a native plasma membrane aquaporin MusaPIP1;2 display high tolerance levels to different abiotic stresses

Water transport across cellular membranes is regulated by a family of water channel proteins known as aquaporins (AQPs). As most abiotic stresses like suboptimal temperatures, drought or salinity result in cellular dehydration, it is imperative to study the cause–effect relationship between AQPs and the cellular consequences of abiotic stress stimuli. Although plant cells have a high isoform diversity of AQPs, the individual and integrated roles of individual AQPs in optimal and suboptimal physiological conditions remain unclear. Herein, we have identified a plasma membrane intrinsic protein gene (MusaPIP1;2) from banana and characterized it by overexpression in transgenic banana plants. Cellular localization assay performed using MusaPIP1;2::GFP fusion protein indicated that MusaPIP1;2 translocated to plasma membrane in transformed banana cells. Transgenic banana plants overexpressing MusaPIP1;2 constitutively displayed better abiotic stress survival characteristics. The transgenic lines had lower malondialdehyde levels, elevated proline and relative water content and higher photosynthetic efficiency as compared to equivalent controls under different abiotic stress conditions. Greenhouse‐maintained hardened transgenic plants showed faster recovery towards normal growth and development after cessation of abiotic stress stimuli, thereby underlining the importance of these plants in actual environmental conditions wherein the stress stimuli is often transient but severe. Further, transgenic plants where the overexpression of MusaPIP1;2 was made conditional by tagging it with a stress‐inducible native dehydrin promoter also showed similar stress tolerance characteristics in in vitro and in vivo assays. Plants developed in this study could potentially enable banana cultivation in areas where adverse environmental conditions hitherto preclude commercial banana cultivation.     

Heavy metal tolerant transgenic Brassica napus L. and Nicotiana tabacum L. plants

Summary A chimeric gene containing a cloned human metallothionein-II (MT-II) processed gene was introduced into Brassica napus and Nicotiana tabacum cells on a disarmed Ti-plasmid of Agrobacterium tumefaciens. Transformants expressed MT protein as a Mendelian trait and in a constitutive manner. Seeds from self-fertilized transgenic plants were germinated on media containing toxic levels of cadmium and scored for tolerance/ susceptibility to this heavy metal. The growth of root and shoot of transformed seedlings was unaffected by up to 100 M CdCl₂, whereas control seedlings showed severe inhibition of root and shoot growth and chlorosis of leaves. The results of these experiments indicate that agriculturally important plants such as B. napus can be genetically engineered for heavy metal tolerance/sequestration and eventually for partitioning of heavy metals in non-consumed plant tissues.     

Co‐expression of NCED and ALO improves vitamin C level and tolerance to drought and chilling in transgenic tobacco and stylo plants

Abscisic acid (ABA) regulates plant adaptive responses to various environmental stresses, while l ‐ascorbic acid (AsA) that is also named vitamin C is an important antioxidant and involves in plant stress tolerance and the immune system in domestic animals. Transgenic tobacco (Nicotiana tabacum L.) and stylo [Stylosanthes guianensis (Aublet) Swartz], a forage legume, plants co‐expressing stylo 9‐cis‐epoxycarotenoid dioxygenase (SgNCED1) and yeast d ‐arabinono‐1,4‐lactone oxidase (ALO) genes were generated in this study, and tolerance to drought and chilling was analysed in comparison with transgenic tobacco overexpressing SgNCED1 or ALO and the wild‐type plants. Compared to the SgNCED1 or ALO transgenic plants, in which only ABA or AsA levels were increased, both ABA and AsA levels were increased in transgenic tobacco and stylo plants co‐expressing SgNCED1 and ALO genes. Compared to the wild type, an enhanced drought tolerance was observed in SgNCED1 transgenic tobacco plants with induced expression of drought‐responsive genes, but not in ALO plants, while an enhanced chilling tolerance was observed in ALO transgenic tobaccos with induced expression of cold‐responsive genes, but not in SgNCED1 plants. Co‐expression of SgNCED1 and ALO genes resulted in elevated tolerance to both drought and chilling in transgenic tobacco and stylo plants with induced expression of both drought and cold‐responsive genes. Our result suggests that co‐expression of SgNCED1 and ALO genes is an effective way for use in forage plant improvement for increased tolerance to drought and chilling and nutrition quality.     

Interaction of silicon and cadmium in <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica napus</Emphasis>

The objective of this study was to determine the effect of silicon (Si) and cadmium (Cd) on root and shoot growth and Cd uptake in two hydroponically cultivated Brassica species (B. juncea (L.) Czern. cv. Vitasso and B. napus L. cv. Atlantic). Both species are potentially usable for phytoextraction. Inhibitory effects of Cd on root elongation were diminished by the impact of Si. Primary roots elongation in the presence of Cd + Si compared with Cd was stronger and the number of lateral roots was lower in B. juncea than in B. napus. Cd content per plant was higher in B. napus roots and shoots compared with B. juncea. Suberin lamellae were formed closer to the root apex in Cd + Si than in Cd treated plants and this effect was stronger in B. napus than in B. juncea. Accelerated maturation of endodermis was associated with reduced Cd uptake. Cd decreased the content of chlorophylls and carotenoids in both species, but Si addition positively influenced the content of photosynthetic pigments which was higher in B. napus than in B. juncea. Si enhanced more substantially translocation of Cd into the shoot of B. napus than of B. juncea. Based on our results B. napus seems to be more suitable for Cd phytoextraction than B. juncea because these plants produce more biomass and accumulate higher amount of Cd. The protective effect of Si on Cd treated Brassica plants could be attributed to more extensive development of suberin lamellae in endodermis.     

Phytochelatin synthase of Thlaspi caerulescens enhanced tolerance and accumulation of heavy metals when expressed in yeast and tobacco

Phytochelatin synthase (PCS) is key enzyme for heavy metal detoxification and accumulation in plant. In this study, we isolated the PCS gene TcPCS1 from the hyperaccumulator Thlaspi caerulescens. Overexpression of TcPCS1 enhanced PC production in tobacco. Cd accumulation in the roots and shoots of TcPCS1 transgenic seedlings was increased compared to the wild type (WT), while Cd translocation from roots to shoots was not affected under Cd treatment. The root length of the TcPCS1 transgenic tobacco seedlings was significantly longer than that of the WT under Cd stress. These data indicate that TcPCS1 expression might increase Cd accumulation and tolerance in transgenic tobacco. In addition, the malondialdehyde content in TcPCS1 plants was below that of the wild type. However, the antioxidant enzyme activities of superoxide dismutase, peroxidase and catalase were found to be significantly higher than those of the WT when the transgenic plant was exposed to Cd stress. This suggests that the increase in PC production might enhance the Cd accumulation and thus increase the oxidative stress induced by the cadmium. The production of PCs could cause a transient decrease in the cytosolic glutathione (GSH) pool, and Cd and lower GSH concentration caused an increase in the oxidative response. We also determined TcPCS1 in Thlaspi caerulescens was regulated after exposure to various concentrations of CdCl₂ over different treatment times. Expression of TcPCS1 leading to increased Cd accumulation and enhanced metal tolerance, but the Cd contents were restrained by adding zinc in Saccharomyces cerevisiae transformants.     

Expression of a Brassica napus Malate Synthase Gene in Transgenic Tomato Plants during the Transition from Late Embryogeny to Germination

To study gene regulation during the transition from late embryogeny to germination, we have analyzed the expression of a gene encoding the glyoxylate cycle enzyme malate synthase in transgenic tomato (Lycopersicon esculentum) plants. We have shown that although there are at least four classes of malate synthase genes in Brassica napus L., one gene is expressed at a high level during both late embryogeny and postgermination. Analyses of transgenic tomato plants containing the expressed B. napus gene along with 4.7 and 1.0 kilobase pairs of 5′ and 3′ flanking sequences, respectively, confirmed that a single gene is expressed at both stages of development. Furthermore, localization studies have shown that mRNA encoded by the B. napus gene is distributed throughout the tissues of a mature embryo but is not detected in the vascular cylinder of a seedling. We conclude that the sequences required to qualitatively regulate the gene correctly over the plant life cycle are present within the transferred gene and/or flanking regions. Moreover, the malate synthase gene is regulated differently during late embryogeny and postgermination in the developing vascular cylinder.