Combined expression of the Arabidopsis metallothionein MT2b and the heavy metal transporting ATPase HMA4 enhances cadmium tolerance and the root to shoot translocation of cadmium and zinc in tobacco

We expressed the AtMt2b and AtHMA4 genes under the 35S cauliflower mosaic virus promoter simultaneously in Nicotiana tabacum (SR1), using leaf disc transformation. A single AtMT2b tobacco T2 line was used for re-transformation with AtHMA4 to obtain the double transformant. Cadmium (Cd) and zinc (Zn) tolerance, uptake and translocation were measured in the double transformant, and compared to untransformed (‘wild type’) tobacco and single gene transformants. The double transformant exhibited enhanced Cd-tolerance, enhanced Cd and Zn root to shoot transport, but unaltered Zn tolerance and Cd and Zn uptake, compared with wild type.The single transformant lines did not show significant phenotypes. Our results suggest that the phenotypes of the double transformant are due to synergistic interaction between the transgenes. Except for Cd tolerance, the phenotypes were moderate for Cd and Zn root to shoot transport, which may be due to use of the 35S promotor, resulting in incorrect tissue-specificity.     

Cadmium Tolerance and Accumulation in Indian Mustard Is Enhanced by Overexpressing γ-Glutamylcysteine Synthetase

To investigate rate-limiting factors for glutathione and phytochelatin (PC) production and the importance of these compounds for heavy metal tolerance, Indian mustard (Brassica juncea) was genetically engineered to overexpress the Escherichia coli gshI gene encoding γ-glutamylcysteine synthetase (γ-ECS), targeted to the plastids. The γ-ECS transgenic seedlings showed increased tolerance to Cd and had higher concentrations of PCs, γ-GluCys, glutathione, and total non-protein thiols compared with wild-type (WT) seedlings. When tested in a hydroponic system, γ-ECS mature plants accumulated more Cd than WT plants: shoot Cd concentrations were 40% to 90% higher. In spite of their higher tissue Cd concentration, the γ-ECS plants grew better in the presence of Cd than WT. We conclude that overexpression of γ-ECS increases biosynthesis of glutathione and PCs, which in turn enhances Cd tolerance and accumulation. Thus, overexpression of γ-ECS appears to be a promising strategy for the production of plants with superior heavy metal phytoremediation capacity.     

Overexpression of Glutathione Synthetase in Indian Mustard Enhances Cadmium Accumulation and Tolerance

An important pathway by which plants detoxify heavy metals is through sequestration with heavy-metal-binding peptides called phytochelatins or their precursor, glutathione. To identify limiting factors for heavy-metal accumulation and tolerance, and to develop transgenic plants with an increased capacity to accumulate and/or tolerate heavy metals, the Escherichia coli gshII gene encoding glutathione synthetase (GS) was overexpressed in the cytosol of Indian mustard (Brassica juncea). The transgenic GS plants accumulated significantly more Cd than the wild type: shoot Cd concentrations were up to 25% higher and total Cd accumulation per shoot was up to 3-fold higher. Moreover, the GS plants showed enhanced tolerance to Cd at both the seedling and mature-plant stages. Cd accumulation and tolerance were correlated with the gshII expression level. Cd-treated GS plants had higher concentrations of glutathione, phytochelatin, thiol, S, and Ca than wild-type plants. We conclude that in the presence of Cd, the GS enzyme is rate limiting for the biosynthesis of glutathione and phytochelatins, and that overexpression of GS offers a promising strategy for the production of plants with superior heavy-metal phytoremediation capacity.     

Enhanced Heavy Metal Tolerance and Accumulation by Transgenic Sugar Beets Expressing Streptococcus thermophilus StGCS-GS in the Presence of Cd,Zn and Cu Alone or in Combination

Phytoremediation is a promising means of ameliorating heavy metal pollution through the use of transgenic plants as artificial hyperaccumulators. A novel Streptococcus thermophilus γ-glutamylcysteine synthetase-glutathione synthetase (StGCS-GS) that synthesizes glutathione (GSH) with limited feedback inhibition was overexpressed in sugar beet (Beta vulgaris L.), yielding three transgenic lines (s2, s4 and s5) with enhanced tolerance to different concentrations of cadmium, zinc and copper, as indicated by their increased biomass, root length and relative growth compared with wild-type plants. Transgenic sugar beets accumulated more Cd, Zn and Cu ions in shoots than wild-type, as well as higher GSH and phytochelatin (PC) levels under different heavy metal stresses. This enhanced heavy metal tolerance and increased accumulation were likely due to the increased expression of StGCS-GS and consequent overproduction of both GSH and PC. Furthermore, when multiple heavy metal ions were present at the same time, transgenic sugar beets overexpressing StGCS-GS resisted two or three of the metal combinations (50 μM Cd-Zn, Cd-Cu, Zn-Cu and Cd-Zn-Cu), with greater absorption in shoots. Additionally, there was no obvious competition between metals. Overall, the results demonstrate the explicit role of StGCS-GS in enhancing Cd, Zn and Cu tolerance and accumulation in transgenic sugar beet, which may represent a highly promising new tool for phytoremediation.     

Co-transformation of canola by chimeric chitinase and <Emphasis Type="Italic">tlp</Emphasis> genes towards improving resistance to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Canola (Brassica napus) plants were co-transformed with two pathogenesis-related protein genes expressing a Trichoderma atroviride chitinase with a chitin-binding domain (chimeric chitinase) and a thaumatin-like protein (tlp) from Oryza sativa conferring resistance to phytopatogenic fungi by Agrobacterium-mediated transformation. The putative transgenic plants were confirmed by PCR. After measuring the specific activity of the chimeric chitinase and glucanase activity for tlp genes, transgenic plants with high specific activity were selected for southern blot analysis to confirm the copy number of the genes. In vitro assays, the antifungal activity of crude extracted protein against Sclerotinia sclerotiorum showed that the inhibition percentage in double transgenic plants was between 55 and 62, whereas the inhibition percentage in single-gene transformants (chimeric chitinase) ranged from 35 to 45 percent. Importantly, in greenhouse conditions, the double transgenic plants showed significant resistance than the single-gene transformant and wild type plants. The results in T₂ generation using the intact leaf inoculation method showed that the average lesion diameters were 10, 14.7 and 29 mm for the double transformant, single-gene transformant and non-transgenic plants, respectively. Combined expression of chimeric chitinase and tlp in transgenic plants showed significantly enhanced resistance against S. sclerotiorum than the one that express single-gene transformant plants. These results suggest that the co-expression of chimeric chitinase and tlp can confer enhanced disease resistance in canola plant.     

Cadmium tolerance and phytochelatin content of Arabidopsis seedlings over-expressing the phytochelatin synthase gene AtPCS1

Previous studies demonstrated that expression of the Arabidopsis phytochelatin (PC) biosynthetic gene AtPCS1 in Nicotiana tabacum plants increases the Cd tolerance in the presence of exogenous glutathione (GSH). In this paper, the Cd tolerance of Arabidopsis plants over-expressing AtPCS1 (AtPCSox lines) has been analysed and the differences between Arabidopsis and tobacco are shown. Based on the analysis of seedling fresh weight, primary root length, and alterations in root anatomy, evidence is provided that, at relatively low Cd concentrations, the Cd tolerance of AtPCSox lines is lower than the wild type, while AtPCS1 over-expressing tobacco is more tolerant to Cd than the wild type. At higher Cd concentrations, Arabidopsis AtPCSox seedlings are more tolerant to Cd than the wild type, while tobacco AtPCS1 seedlings are as sensitive as the wild type. Exogenous GSH, in contrast to what was observed in tobacco, did not increase the Cd tolerance of AtPCSox lines. The PC content in wild-type Arabidopsis at low Cd concentrations is more than three times higher than in tobacco and substantial differences were also found in the PC chain lengths. These data indicate that the differences in Cd tolerance and in its dependence on exogenous GSH between Arabidopsis and tobacco are due to species-specific differences in the endogenous content of PCs and GSH and may be in the relative abundance of PCs of different length.     

Expression of the Arabidopsis metallothionein 2b enhances arsenite sensitivity and root to shoot translocation in tobacco

We expressed the AtMT2b gene under the 35 S cauliflower mosaic virus promoter in Nicotiana tabacum (Sr1), using leaf disc transformation. Arsenite tolerance and uptake, as well as arsenite-induced phytochelatin (PC) accumulation in roots were measured in transgenic lines, and compared to untransformed (‘wild type’) tobacco. Measured after 5 days of exposure, arsenite tolerance was slightly but significantly decreased in the transgenic lines compared to wild type. The highest AtMT2b expressing line exhibited a significantly decreased arsenic accumulation in roots, but an increased accumulation in shoots, while the total amount of arsenic taken up remained unchanged, suggesting that AtMT2b expression enhanced the arsenic root to shoot transport. The same transformant line also exhibited a decreased rate of phytochelatin accumulation in the roots, but the phytochelatin-SH to As molar ratio was higher than in wild type, suggesting that the lower arsenite tolerance in the transformant lines was not due to a potential shortage of cysteine for PC synthesis, imposed by expression of the transgene.     

Differential cadmium stress tolerance in five indian mustard (Brassica juncea L.) cultivars: An evaluation of the role of antioxidant machinery

The presence of Cadmium (Cd) in the agricultural soils affects horticultural cultivars and constrains the crop productivity. A pot experiment was performed using five cultivars of mustard (Brassica juncea L.) to evaluate the difference in their response to Cd toxicity under greenhouse conditions. The pots containing reconstituted soil were supplied with different concentration of CdCl₂ (0, 25, 50, 100 or 150 mg Cd kg⁻¹ soil). Increasing concentration of Cd in the soil resulted in decreased growth, photosynthesis and yield. Maximum significant reduction in growth, photosynthesis and yield were observed with 150 mg Cd kg⁻¹ soil in all the cultivars. Our results indicate that the cultivar Alankar is found to be more tolerant to Cd stress, recording higher plant dry mass, net photosynthesis rate, associated with high antioxidant activity and low Cd content in the plant leaves and thus less oxidative damage. Cultivar RH30 experienced maximum damage in terms of reduction in growth, photosynthesis, yield characteristics and oxidative damage and emerged as sensitive cultivar. The data of tolerance index of Alankar were found to be higher among all tested mustard cultivars which indicate its higher tolerance to Cd. Better coordination of antioxidants protected Alankar from Cd toxicity, whereas lesser antioxidant activity in RH30 resulted in maximum damage. Cultivars of mustard were ranked with respect to their tolerance to Cd: Alankar > Varuna > Pusa Bold > Sakha > RH30, respectively.Key words: antioxidants, cadmium, growth, mustard cultivars, photosynthesis, stress, yield     

Increased Tolerance to Salinity and Drought in Transgenic Indica Rice by Mannitol Accumulation

The osmolytes, including mannitol have been shown to be very important in abiotic stress tolerance. Thus, the present study was undertaken with the aim to enhance abiotic stress tolerance in basmati indica rice by introduction of the E. coli mannitol-1-phospho dehydrogenase (mt/D) gene, which is involved in mannitol synthesis in plants. Several putative transgenic rice plants were generated by Agrobacterium-mediated transformation. The presence of the transgene in the primary transformants was confirmed by PCR using hygromycin phosphotransferase (hpt) and mt/D gene specific primers. Southern hybridization also revealed the integration of the transgene. Transgenic lines exhibited mannitol accumulation, which was correlated to the increased tolerance of the transgenics against salinity and drought stress. The T₁ transgenic seed germination and seedlings growth showed better performance than that of wild type during abiotic stresses under in vitro and in vivo growth conditions.     

Induction of salt tolerance in salicylate-deficient <Emphasis Type="Italic">NahG</Emphasis> Arabidopsis transformants using the nitric oxide donor

The effects of treatment with nitric oxide donor sodium nitroprusside (SNP, 0.5 mM) on salt tolerance of wild type (Col–0) Arabidopsis thaliana plants and Arabidopsis thaliana plants transformed with the bacterial salicylate hydroxylase gene (NahG) were compared. Basic salt tolerance level (200 mM NaCl) was higher in NahG transformants. Under salt stress conditions, these plants showed higher activity levels for antioxidant enzymes as well as higher content of sugars and anthocyanins. The treatment with NO donor induced salt tolerance in the plants of both genotypes, which could be observed as less strong growth inhibition, reduced oxidative damage, and preservation of chlorophyll pool in leaves. After the exposure to salt stress, the activity of both superoxide dismutase and guaiacol peroxidase was higher in SNP-treated wild type plants and NahG transformants than in the nontreated plants. After the imposition of salt stress, proline content in leaves of the wild type plants treated with the nitric oxide donor was lower than in the leaves of the nontreated plants. In contrast, SNP treatment of NahG transformants led to a significant increase in the proline content in leaves under the salt stress conditions. Conclusions have been made that wild type Col-0 plants and NahG transformants differ in how their systems of protection against salt stress are activated and that nitric oxideinduced mobilization of protection systems in A. thaliana may not require the presence of salicylate.     

Overexpression of the glutamine synthetase gene modulates oxidative stress response in rice after exposure to cadmium stress

Key message

Overexpression of OsGS gene modulates oxidative stress response in rice after exposure to cadmium stress. Our results describe the features of transformants with enhanced tolerance to Cd and abiotic stresses.

Abstract

Glutamine synthetase (GS) (EC 6.3.1.2) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine. Exposure of plants to cadmium (Cd) has been reported to decrease GS activity in maize, pea, bean, and rice. To better understand the function of the GS gene under Cd stress in rice, we constructed a recombinant pART vector carrying the GS gene under the control of the CaMV 35S promoter and OCS terminator and transformed using Agrobacterium tumefaciens. We then investigated GS overexpressing rice lines at the physiological and molecular levels under Cd toxicity and abiotic stress conditions. We observed a decrease in GS enzyme activity and mRNA expression among transgenic and wild-type plants subjected to Cd stress. The decrease, however, was significantly lower in the wild type than in the transgenic plants. This was further validated by the high GS mRNA expression and enzyme activity in most of the transgenic lines. Moreover, after 10 days of exposure to Cd stress, increase in the glutamine reductase activity and low or no malondialdehyde contents were observed. These results showed that overexpression of the GS gene in rice modulated the expression of enzymes responsible for membrane peroxidation that may result in plant death.     

Metabolic Engineering of the Carotenoid Biosynthetic Pathway in the Yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a β-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the β-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via β-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3′-4′-didehydro-β-ψ-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.     

Agrobacterium tumefaciens-Mediated Transformation of Aspergillus fumigatus: an Efficient Tool for Insertional Mutagenesis and Targeted Gene Disruption

Agrobacterium tumefaciens was used to transform Aspergillus fumigatus by either random or site-directed integration of transforming DNA (T-DNA). Random mutagenesis via Agrobacterium tumefaciens-mediated transformation (ATMT) was accomplished with T-DNA containing a hygromycin resistance cassette. Cocultivation of A. fumigatus conidia and Agrobacterium (1:10 ratio) for 48 h at 24°C resulted in high frequencies of transformation (>100 transformants/10⁷ conidia). The majority of transformants harbored a randomly integrated single copy of T-DNA and were mitotically stable. We chose alb1, a polyketide synthase gene, as the target gene for homologous integration because of the clear phenotype difference between the white colonies of Δalb1 mutant strains and the bluish-green colonies of wild-type strains. ATMT with a T-DNA-containing alb1 disruption construct resulted in 66% albino transformants. Southern analysis revealed that 19 of the 20 randomly chosen albino transformants (95%) were disrupted by homologous recombination. These results suggest that ATMT is an efficient tool for transformation, random insertional mutagenesis, and gene disruption in A. fumigatus.     

Expression of Tobacco Carbonic Anhydrase in the C4 Dicot Flaveria bidentis Leads to Increased Leakiness of the Bundle Sheath and a Defective CO2-Concentrating Mechanism

Flaveria bidentis (L.) Kuntze, a C₄ dicot, was genetically transformed with a construct encoding the mature form of tobacco (Nicotiana tabacum L.) carbonic anhydrase (CA) under the control of a strong constitutive promoter. Expression of the tobacco CA was detected in transformant whole-leaf and bundle-sheath cell (bsc) extracts by immunoblot analysis. Whole-leaf extracts from two CA-transformed lines demonstrated 10% to 50% more CA activity on a ribulose-1,5-bisphosphate carboxylase/oxygenase-site basis than the extracts from transformed, nonexpressing control plants, whereas 3 to 5 times more activity was measured in CA transformant bsc extracts. This increased CA activity resulted in plants with moderately reduced rates of CO₂ assimilation (A) and an appreciable increase in C isotope discrimination compared with the controls. With increasing O₂ concentrations up to 40% (v/v), a greater inhibition of A was found for transformants than for wild-type plants; however, the quantum yield of photosystem II did not differ appreciably between these two groups over the O₂ levels tested. The quantum yield of photosystem II-to-A ratio suggested that at higher O₂ concentrations, the transformants had increased rates of photorespiration. Thus, the expression of active tobacco CA in the cytosol of F. bidentis bsc and mesophyll cells perturbed the C₄ CO₂-concentrating mechanism by increasing the permeability of the bsc to inorganic C and, thereby, decreasing the availability of CO₂ for photosynthetic assimilation by ribulose-1,5-bisphosphate carboxylase/oxygenase.     

Multiple Heavy Metal Tolerance of Soil Bacterial Communities and Its Measurement by a Thymidine Incorporation Technique

A thymidine incorporation technique was used to determine the tolerance of a soil bacterial community to Cu, Cd, Zn, Ni, and Pb. An agricultural soil was artificially contaminated in our laboratory with individual metals at three different concentrations, and the results were compared with the results obtained by using the plate count technique. Thymidine incorporation was found to be a simple and rapid method for measuring tolerance. Data obtained by this technique were very reproducible. A linear relationship was found between changes in community tolerance levels obtained by the thymidine incorporation and plate count techniques (r = 0.732, P < 0.001). An increase in tolerance to the metal added to soil was observed for the bacterial community obtained from each polluted soil compared with the community obtained from unpolluted soil. The only exception was when Pb was added; no indication of Pb tolerance was found. An increase in the tolerance to metals other than the metal originally added to soil was also observed, indicating that there was multiple heavy metal tolerance at the community level. Thus, Cu pollution, in addition to increasing tolerance to Cu, also induced tolerance to Zn, Cd, and Ni. Zn and Cd pollution increased community tolerance to all five metals. Ni amendment increased tolerance to Ni the most but also increased community tolerance to Zn and, to lesser degrees, increased community tolerance to Pb and Cd. In soils polluted with Pb increased tolerance to other metals was found in the following order: Ni > Cd > Zn > Cu. We found significant positive relationships between changes in Cd, Zn, and Pb tolerance and, to a lesser degree, between changes in Pb and Ni tolerance when all metals and amendment levels were compared. The magnitude of the increase in heavy metal tolerance was found to be linearly related to the logarithm of the metal concentration added to the soil. Threshold tolerance concentrations were estimated from these linear relationships, and changes in tolerance could be detected at levels of soil contamination similar to those reported previously to result in changes in the phospholipid fatty acid pattern (Å. Frostegård, A. Tunlid, and E. Bååth, Appl. Environ. Microbiol. 59: 3605-3617, 1993).     

Comparison of increased expression of wild-type and herbicide-resistant acetolactate synthase genes in transgenic plants, and indication of posttranscriptional limitation on enzyme activity

Genes encoding wild type acetolactate synthase (ALS) and a sulfonylurea herbicide-resistant form of the enzyme, isolated from Arabidopsis thaliana, were expressed in transgenic Nicotiana tabacum plants under the control of their native promoters or of the highly active cauliflower mosaic virus 35S promoter. Expression of the wild type coding region from the 35S promoter resulted in a small, threefold increase in sulfonylurea tolerance above the levels measured in tissue expressing the native wild type gene. A much larger, 300-fold increase in herbicide tolerance was conferred by the mutant gene encoding a herbicide-resistant ALS. An additional 10-fold increase in tolerance was attained by expressing this coding region from the 35S promoter. The increase in both wild type and mutant gene expression directed by the 35S promoter resulted in over 25-fold higher levels of ALS messenger RNA in some transformants as compared with those expressing the native genes. However, ALS specific activity increased at most twofold, indicating that the amount of functional enzyme and messenger RNA are not correlated.     

Isolation and characterization of phi X174 mutants carrying lethal missense mutations in gene G.

A previously constructed Escherichia coli transformant carrying a functional copy of bacteriophage phi X174 gene G on a plasmid, p phi XG, was used to isolate gene G mutants carrying temperature sensitive and lethal missense mutations. Two of the mutations have been characterized by sequencing: one carries a G --> A transition at residue 2821 producing a Gly --> Ser change in codon 143 of the G spike protein; the other carries an A --> G transition at residue 2678 producing Glu --> Gly change in codon 95. Sequencing DNA from 2 other mutants carrying lethal mutations that are rescued with p phi XG did not reveal any changes in the coding sequence. The lesion is believed to be in the intercistronic region between genes F and G. The adsorption kinetics for these mutants appear to be normal. Their burst size is about 25% that of wild type phi X174 on the host carrying p phi XG. These results along with previous results from the senior author's laboratory demonstrate that p phi XG can be used to rescue any gene G mutant of phi X174 regardless of the nature of the mutation involved.     

Engineering Aspergillus oryzae A-4 through the Chromosomal Insertion of Foreign Cellulase Expression Cassette to Improve Conversion of Cellulosic Biomass into Lipids

A genetic modification scheme was designed for Aspergillus oryzae A-4, a natural cellulosic lipids producer, to enhance its lipid production from biomass by putting the spotlight on improving cellulase secretion. Four cellulase genes were separately expressed in A-4 under the control of hlyA promoter, with the help of the successful development of a chromosomal genetic manipulation system. Comparison of cellulase activities of PCR-positive transformants showed that these transformants integrated with celA gene and with celC gene had significantly (p<0.05) higher average FPAase activities than those strains integrated with celB gene and with celD gene. Through the assessment of cellulosic lipids accumulating abilities, celA transformant A2-2 and celC transformant D1-B1 were isolated as promising candidates, which could yield 101%–133% and 35.22%–59.57% higher amount of lipids than the reference strain A-4 (WT) under submerged (SmF) conditions and solid-state (SSF) conditions, respectively. Variability in metabolism associated to the introduction of cellulase gene in A2-2 and D1-B1 was subsequently investigated. It was noted that cellulase expression repressed biomass formation but enhanced lipid accumulation; whereas the inhibitory effect on cell growth would be shielded during cellulosic lipids production owing to the essential role of cellulase in substrate utilization. Different metabolic profiles also existed between A2-2 and D1-B1, which could be attributed to not only different transgene but also biological impacts of different integration. Overall, both simultaneous saccharification and lipid accumulation were enhanced in A2-2 and D1-B1, resulting in efficient conversion of cellulose into lipids. A regulation of cellulase secretion in natural cellulosic lipids producers could be a possible strategy to enhance its lipid production from lignocellulosic biomass.