Cadmium-induced responses in duckweed <Emphasis Type="Italic">Lemna minor</Emphasis> L.

Although duckweed Lemna minor L. is a known accumulator of cadmium, detailed studies on its physiological and/or defense responses to this metal are still lacking. In this study, the effects of 10 μM CdCl₂ on Lemna minor were monitored after 6 and 12 days of treatment, while growth was estimated every 2 days. Cadmium treatment resulted in progressive accumulation of the metal in the plants and led to a decrease in the growth rate to 54% of the control value. The metal also considerably impaired chloroplast ultrastructure and caused a significant reduction in pigment content, i.e., at day 12, by 30 and 34% for chlorophylls a and b, and by 25% for carotenoids. During cadmium treatment, the contents of malondialdehyde and endogenous H₂O₂ progressively increased (rising 77 and 46% above the controls by day 12), indicating that cadmium induced considerable oxidative stress. On the other hand, higher activities of pyrogallol peroxidase (PPX), ascorbate peroxidase (APX) and catalase (CAT), as well as the induction of a new APX isoform, in cadmium-treated plants, clearly showed activation of an antioxidative response. At day 6, only PPX activity was significantly above the controls (15%), while, at day 12, PPX, APX and CAT activities were increased (74, 78 and 63%). Cadmium also led to accumulation of the heat shock protein 70 (HSP70) and induced an additional isoform of this protein. The obtained results suggest that cadmium (10 μM) is phytotoxic to Lemna minor, inducing oxidative stress, and that antioxidative enzymes and HSP70 play important roles in the defense against cadmium toxicity. M. Tkalec and T. Prebeg contributed equally to this work     

Influence of long-term exposure to copper on the lichen photobiont <Emphasis Type="Italic">Trebouxia erici</Emphasis> and the free-living algae <Emphasis Type="Italic">Scenedesmus quadricauda</Emphasis>

In the present work, the long-term effect (14 days) of copper on the levels of intracellular and total copper accumulation, growth, assimilation pigment composition, chlorophyll a fluorescence, soluble protein content and oxidative status (production of hydrogen peroxide and superoxide) in two algal species (Scenedesmus quadricauda and Trebouxia erici) was assessed. Scenedesmus quadricauda is a free-living alga while Trebouxia erici is the photobiont of a lichen. The presence of copper negatively affected growth, assimilation pigments, chlorophyll a fluorescence, soluble protein content and oxidative status in both the algae. However, Scenedesmus was much more sensitive compared to Trebouxia.     

Drought-induced oxidative damage and antioxidant responses in peanut (<Emphasis Type="Italic">Arachis</Emphasis><Emphasis Type="Italic">hypogaea</Emphasis> L.) seedlings

Two cultivars of peanut (Arachis hypogaea L.) which were designated as resistant (Florispan) and sensitive (Gazipasa) according to their growth retardation under drought stress conditions were compared for their oxidative damage and antioxidant responses. Sixteen days-old peanut seedlings were subjected to PEG-6000 solutions of two different osmotic potentials; −0.4 and −0.8 MPa, and various growth parameters, photosystem II activity, changes in malondialdehyde (MDA), hydrogen peroxide (H₂O₂) and proline levels, activities of ascorbate peroxidase (APX), catalase (CAT), peroxidase (POX) and gluthatione reductase (GR) enzymes were determined. Both cultivars exhibited water deficit at −0.8 MPa osmotic potential of PEG-6000 and H₂O₂ levels significantly increased during exposure to −0.4 MPa osmotic potential. However, H₂O₂ levels were under control in both cultivars at exposure to −0.8 MPa osmotic potential. Significant proline accumulation was observed in the tissues of cv. Florispan at −0.8 MPa osmotic potential, whereas proline accumulation did not appear to be an essential part of the protection mechanism against drought in cv. Gazipasa. No significant variation in chlorophyll fluorescence values were detected in neither of the cultivars. Enzyme activity measurements revealed that Gazipasa copes well with lesser magnitudes of drought stress by increasing the activity of mainly APX, and during harsh stress conditions, only APX maintains its activity in the tissues. In cultivar Florispan, GR activity appears to take role in lesser magnitudes of drought stress, whereas CAT and APX activities appear to be very crucial antioxidative defenses during intense stress conditions. The results indicate that, the level of proline and activities of the enzymes CAT and APX are important mechanisms for the maintenance of drought tolerance in peanut plants.     

Expression analysis and functional characterization of a novel cold-responsive gene <Emphasis Type="Italic">CbCOR15a</Emphasis> from <Emphasis Type="Italic">Capsella bursa</Emphasis>-<Emphasis Type="Italic">pastoris</Emphasis>

The cold-responsive (COR) genes involved in C-repeat binding factor signaling pathway function essentially in cold acclimation of higher plants. A novel COR gene CbCOR15a from shepherd’s purse (Capsella bursa-pastoris) was predicted to be a homolog of COR15 in Arabidopsis. The analysis of tissue specific expression pattern as well as characterization of the CbCOR15a promoter revealed that the expression of CbCOR15a was induced by coldness not only in leaves and stem but also in roots. Sequence analysis showed that a 909 bp promoter region of CbCOR15a contained two CRT/DRE elements, two ABRE elements, one auxin-responsive TGA-element and one MeJA-responsive CGTCA-motif. In young seedlings the expression of CbCOR15a could be apparently increased by SA, ABA, MeJA and IAA, and transiently increased by GA₃ accompanied by obvious feedback suppression. According to the altered physiological index values in tobacco under cold treatments, the overexpression of CbCOR15a significantly increased the cold tolerance of transgenic tobacco plants. It can be suggested that CbCOR15a was involved in cold response of Capsella bursa-pastoris associated with SA, ABA, MeJA, IAA and GA₃ regulation and confers enhanced cold acclimation in transgenic plants.     

Disruption of <Emphasis Type="Italic">RCI2A</Emphasis> leads to over-accumulation of Na<Superscript>+</Superscript> and increased salt sensitivity in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

For plant salt tolerance, it is important to regulate the uptake and accumulation of Na⁺ ions. The yeast pmp3 mutant which lacks PMP3 gene accumulates excess Na⁺ ions in the cell and shows increased Na⁺ sensitivity. Although the function of PMP3 is not fully understood, it is proposed that PMP3 contributes to the restriction of Na⁺ uptake and consequently salt tolerance in yeasts. In this paper, we have investigated whether the lack of RCI2A gene, homologous to PMP3 gene, causes a salt sensitive phenotype in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants; and to thereby indicate the physiological role of RCI2A in higher plants. Two T-DNA insertional mutants of RCI2A were identified. Although the growth of rci2a mutants was comparable with that of wild type under normal conditions, high NaCl treatment caused increased accumulation of Na⁺ and more reduction of the growth of roots and shoots of rci2a mutants than that of wild type. Undifferentiated callus cultures regenerated from rci2a mutants also accumulated more Na⁺ than that from wild type under high NaCl treatment. Furthermore, when wild-type and rci2a plants were treated with NaCl, NaNO₃, Na₂SO₄, KCl, KNO₃, K₂SO₄ or LiCl, the rci2a mutants showed more reduction of shoot growth than wild type. Under treatments of tetramethylammonium chloride, CaCl₂, MgCl₂, mannitol or sorbitol, the growth reduction was comparable between wild-type and rci2a plants. These results suggested that RCI2A plays a role directly or indirectly for avoiding over-accumulation of excess Na⁺ and K⁺ ions in plants, and contributes to salt tolerance.     

<Emphasis Type="Italic">Pm37</Emphasis>, a new broadly effective powdery mildew resistance gene from <Emphasis Type="Italic">Triticum </Emphasis><Emphasis Type="Italic">timopheevii</Emphasis>

Powdery mildew is an important foliar disease in wheat, especially in areas with a cool or maritime climate. A dominant powdery mildew resistance gene transferred to the hexaploid germplasm line NC99BGTAG11 from T. timopheevii subsp. armeniacum was mapped distally on the long arm of chromosome 7A. Differential reactions were observed between the resistance gene in NC99BGTAG11 and the alleles of the Pm1 locus that is also located on chromosome arm 7AL. Observed segregation in F_2:3 lines from the cross NC99BGTAG11 × Axminster (Pm1a) demonstrate that germplasm line NC99BGTAG11 carries a novel powdery mildew resistance gene, which is now designated as Pm37. This new gene is highly effective against all powdery mildew isolates tested so far. Analyses of the population with molecular markers indicate that Pm37 is located 16 cM proximal to the Pm1 complex. Simple sequence repeat (SSR) markers Xgwm332 and Xwmc790 were located 0.5 cM proximal and distal, respectively, to Pm37. In order to identify new markers in the region, wheat expressed sequence tags (ESTs) located in the distal 10% of 7AL that were orthologous to sequences from chromosome 6 of rice were targeted. The two new EST-derived STS markers were located distal to Pm37 and one marker was closely linked to the Pm1a region. These new markers can be used in marker-assisted selection schemes to develop wheat cultivars with pyramids of powdery mildew resistance genes, including combinations of Pm37 in coupling linkage with alleles of the Pm1 locus.     

Selection <Emphasis Type="Italic">in vitro</Emphasis> and accumulation of phytochelatins in cadmium tolerant cell line of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis>)

Cucumber (Cucumis sativus L.) cells from suspension culture were selected for their ability to grow and divide rapidly in toxic concentration of cadmium. As a result of selection a cell suspension tolerant to 100 M cadmium chloride (CdCl₂) was initiated. The selected tolerant line exhibited stable and repeatable increase in fresh and dry weight of cells in the presence of cadmium. The accumulated level of phytochelatins in cadmium sensitive (unselected) and tolerant cell line was measured by high performance liquid chromatography (HPLC) after 3, 24 h and 5 days of cadmium treatment. It was shown that in both cell lines Cd induced accumulation of phytochelatins and simultaneous glutathione depletion occurred. No distinct changes were found after 3 and 24 h of cadmium treatment whereas after 5 days of exposure to the metal, the level of phytochelatins was two times higher in the sensitive cell line as compared to the tolerant one. The accumulation of phytochelatins was correlated with cadmium concentration that increased in both cell lines during the course of cell exposure to metal. However, the level of cadmium was always lower in the tolerant cell line. The results showed no direct correlation between the tolerance of cucumber cells to Cd and the accumulated level of phytochelatins. Other mechanisms responsible for the increased tolerance of cucumber cells exposed to Cd are discussed.     

Chloroplast-targeted BrMT1 (<Emphasis Type="Italic">Brassica rapa</Emphasis> type-1 metallothionein) enhances resistance to cadmium and ros in transgenic<Emphasis Type="Italic">atabidopsis</Emphasis> plants

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins that bind to heavy metals. Type-1 MTs function under various abiotic stresses, including exposure to the cadmium ion. We have now isolated theBrassica rapa type-1 metallothioneirt gene (BrMT1)using yeast systems, and have found that it confers resistance to Cd in otherwise Cd-sensitive yeast. Using a constitutive CaMV35S promoter and an RbsS transit peptide, we successfully targeted BrMT1 to the chloroplastsof Arabidopsis. Overexpression in either the chloroplasts or the cytosol effectively detoxified cadmium and H₂O₂ stresses in transgenicArabidopsis. in particular, the chloropfast-targeted BrMTl was associated with a significant reduction in paraquat-induced chlorosis and the accumulation of H₂O₂. This is the first report regarding the effects of type-1 MT1 targeted to chloroplasts. Our results suggest that this may be applicable to the development of plants with enhanced tolerance against environmental stresses.     

Characterization of the cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from marine green alga, <Emphasis Type="Italic">Bryopsis corticulans</Emphasis>

A pure, active cytochrome b ₆ f was isolated from the chloroplasts of the marine green alga, Bryopsis corticulans. To investigate and characterize this cytochrome b ₆ f complex, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), absorption spectra measurement and HPLC were employed. It was shown that this purified complex contained four large subunits with apparent molecular masses of 34.8, 24, 18.7 and 16.7 kD. The ratio of Cyt b ₆ to Cytf was 2.01 : 1. The cytochromeb ₆ f was shown to catalyze the transfer of 73 electrons from decylplastoquinol to plastocyanin–ferricyanide per Cyt f per second. α-Carotene, one kind of carotenoid that has not been found to present in cytochrome b ₆ f complex, was discovered in this preparation by reversed phase HPLC. It was different from β-carotene usually found in cytochrome b ₆ f complex. The configuration of the major α-carotene component was assigned to be 9-cis by resonance Raman spectroscopy. Different from the previous reports, the configuration of this α-carotene in dissociated state was determined to be all-trans. Besides this carotene, chlorophyll a was also found in this complex. It was shown that the molecular ratios of chlorophylla, cis and all-trans-α-carotene to Cyt f in this complex were 1.2, 0.7 and 0.2, respectively.     

<Emphasis Type="Italic">Chenopodium</Emphasis>: a prospective plant for phytoextraction

Leaf samples were collected from 40 accessions of Chenopodium spp. and assessed for six heavy metals (Fe, Zn, Cu, Ni, Cr and Cd) accumulation to explore the use of Chenopodium for phytoextraction of heavy metals. The results suggest that Chenopodium spp. have the ability to accumulate large quantities of heavy metals in the leaf tissues even when they are present in low concentrations in the soil. C. quinoa is a better accumulator of Ni, Cr and Cd than the rest of the species, while C. album accessions are good copper accumulators. Bioconcentration factor for chromium ranged from 0.36 (C. album “Chandanbathua”) to 6.57 (C. quinoa Ames 13719) with 13 accessions of C. quinoa scoring above the mean value. High heritability coupled with high genetic advance was recorded for Ni, Cr and Cd, which indicated a major role of additive gene action in the inheritance of these characters. Zinc showed significant positive association with iron (0.351**), nickel (0.659**), chromium (0.743**) and cadmium (0.288**). Nickel was significantly and negatively associated with copper (−0.663**), while it was positively and significantly correlated with chromium (0.682**) and cadmium (0.461**). Considering the accumulation efficiency of Chenopodium spp. with respect to heavy metals, this genus should be further explored for decontamination of metal polluted soils, with plant breeding playing an important role in evolving new plant types with higher capacity of heavy metal accumulation.     

Improving Toxicity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain Contains the <Emphasis Type="Italic">cry8Ca</Emphasis> Gene Specific to <Emphasis Type="Italic">Anomala corpulenta</Emphasis> Larvae

The cry8C-type gene designated cry8Ca2, which was cloned and sequenced from a Bacillus thuringiensis isolate HBF-1 in China, consisted of an open reading frame of 3483 bp encoding a protein of 1160 amino-acid residues. Sequence analysis showed that the Cry8Ca2 protoxin of 130.5 kDa had 99.9% sequence homology with the previously reported Cry8Ca1 protein, with one mismatch between the two amino-acid sequences. When the Cry8Ca2 toxin was expressed in a crystal-negative strain of B. thuringiensis (HD-73⁻), elliptical crystals were produced. Cell extracts from this recombinant strain showed insecticidal activity against Anomala corpulenta larva. Mutant cry8Ca2 genes, produced by polymerase chain reaction amplification with Taq DNA polymerase, were used to develop recombinant B. thuringiensis strains. Mutants producing higher levels of insecticidal activity were identified by bioassay. Thirty-five mutants forming crystals were characterized, and two of them showed significantly increased insecticidal activity against A. corpulenta larva. The 50% lethality concentrations (LC₅₀) of the two mutants were 0.2334 × 10⁸ and 0.2591 × 10⁸ colony-forming units g⁻¹, considerably lower than the LC₅₀ of the wild-type strain HBF-1 (0.9583 × 10⁸ CFU g⁻¹) and that of B. thuringiensis serovar japonensis strain Buibui (1.0752 × 10⁸ CFU g⁻¹).     

Overexpression of the pepper antimicrobial protein <Emphasis Type="Italic">CaAMP1</Emphasis> gene regulates the oxidative stress- and disease-related proteome in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Proteomics facilitates our understanding of cellular processes and network functions in the plant defense response during abiotic and biotic stresses. Here, we demonstrate that the ectopic expression of the Capsicum annuum antimicrobial protein CaAMP1 gene in Arabidopsis thaliana confers enhanced tolerance to methyl viologen (MV)-induced oxidative stress, which is accompanied by lower levels of lipid peroxidation. Quantitative comparative proteome analyses using two-dimensional gel electrophoresis coupled with mass spectrometry identified some of the oxidative stress- and disease-related proteins that are differentially regulated by CaAMP1 overexpression in Arabidopsis leaves. Antioxidant- and defense-related proteins, such as 2-cys peroxiredoxin, l-ascorbate peroxidase, peroxiredoxin, glutathione S-transferase and copper homeostasis factor, were up-regulated in the CaAMP1 transgenic leaf tissues. In contrast, GSH-dependent dehydroascorbate reductase and WD-40 repeat family protein were down-regulated by CaAMP1 overexpression. In addition, CaAMP1 overexpression enhanced resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 infection and also H₂O₂ accumulation in Arabidopsis. The identified antioxidant- and defense-related genes were differentially expressed during MV-induced oxidative stress and Pst DC3000 infection. Taken together, we conclude that CaAMP1 overexpression can regulate the differential expression of defense-related proteins in response to environmental stresses to maintain reactive oxygen species (ROS) homeostasis.     

Cadmium biosorption by <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic"> circulans</Emphasis> strain EB1

Summary A heavy metal resistant bacterium, Bacillus circulans strain EB1 showed a high cadmium biosorption capacity coupled with a high tolerance to this metal when grown in its presence. Bacillus circulans EB1 cells grown in the presence of 28.1 mg cadmium/l were capable of removing cadmium with a specific biosorption capacity of 5.8 mg Cd/g dry wt biomass in the first 8 h. When the cells were pre-conditioned with low concentrations of cadmium in pre-grown medium, the uptake was increased to 6.7 mg Cd/g dry wt biomass. The maximum uptake of␣cadmium was during mid-logarithmic phase of growth. The resting cells (both wet and dry) of EB1 were also able to biosorb cadmium. Specific biosorption capacities of wet and dry biomass were 9.8 and 26.5 mg Cd/g dry wt biomass, respectively. Maximum cadmium removals by both wet and dry cells were at pH 7.0. The results showed that the cadmium removal capacity of resting cells was markedly higher than that of growing cells. Since both growing and resting cells had a high biosorption capacity for cadmium, EB1 cells could serve as an excellent biosorbent for removal of cadmium from natural environments.