Genome-Wide Analysis of the ZRT,IRT-Like Protein (ZIP) Family and Their Responses to Metal Stress in <Emphasis Type="Italic">Populus trichocarpa</Emphasis>

The ZRT, IRT-like protein (ZIP) family plays an important role in the transport of zinc (Zn) and iron (Fe) across the cell membrane in many different species. However, studies on ZIP family are mainly limited in herbaceous species; hence, we investigated functional divergence of ZIP family in Populus trichocarpa. We identified 21 ZIP genes in P. trichocarpa and classified them into four groups based on phylogenetic analysis. Structural analyses revealed that most of the PtrZIP transporters have eight transmembrane domains (TMDs). PtrZIP members were unequally positioned in 19 P. trichocarpa linkage groups (LGs), with six tandem duplications and four segmental duplications. The promoter regions of PtrZIP genes contain Zn, Fe, copper (Cu), and other metal stress-related cis-elements. Additionally, tissue-specific expression of PtrZIP genes showed that most of them had relatively high expression levels in the root. Quantitative real-time RT-PCR (qRT-PCR) analysis revealed that the expression of PtrZIP genes were induced not only under deficiency or excess condition of Zn, Fe, Cu and manganese (Mn) but also under excess condition of cadmium (Cd) and lead (Pb) stress. These findings indicated that PtrZIP genes may have played potential roles in metal transporters. Genome-wide analysis of PtrZIP genes in P. trichocarpa provided more comprehensive insights on the structure and function of this gene family.     

The crucial role of roots in increased cadmium-tolerance and Cd-accumulation in the pea mutant <Emphasis Type="Italic">SGECd</Emphasis><Superscript><Emphasis Type="Italic">t</Emphasis></Superscript>

Elucidation of mechanisms underlying plant tolerance to cadmium, a widespread toxic soil pollutant, and accumulation of Cd in plants are urgent tasks. For this purposes, the pea (Pisum sativum L.) mutant SGECd^t (obtained by treatment of the laboratory pea line SGE with ethylmethane sulfonate) was reciprocally grafted with the parental line SGE, and four scion/rootstock combinations were obtained: SGE/SGE, SGECd^t/SGECd^t, SGE/SGECd^t, and SGECd^t/SGE. They were grown in hydroponics in the presence of 1 μM CdCl₂ for 30 d. The SGE and SGECd^t scions on the SGECd^t rootstock had a higher root and shoot biomass and an elevated root and shoot Cd content compared with the grafts having SGE rootstock. Only the grafts with the SGE rootstock showed chlorosis and roots demonstrating symptoms of Cd toxicity. The content of nutrient elements in roots (Fe, K, Mg, Mn, Na, P, and Zn) was higher in the grafts having the SGECd^t rootstock, and three elements, namely Ca, Fe, and Mn, were efficiently transported by the SGECd^t root to the shoot of these grafts. The content of other measured elements (K, Mg, Na, P, and Zn) was similar in the root and shoot in all the grafts. Then, the non-grafted plants were grown in the presence of Cd and subjected to deficit or excess concentrations of Ca, Fe, or Mn. Exclusion of these elements from the nutrient solution retained or increased differences between SGE and SGECd^t in growth response to Cd toxicity, whereas excess of Ca, Fe, or Mn decreased or eliminated such differences. The obtained results assign a principal role of roots to realizing the increased Cd-tolerance and Cdaccumulation in the SGECd^t mutant. Efficient translocation of Ca, Fe, and Mn from roots to shoots appeared to counteract Cd toxicity, although Cd was actively taken up by roots and accumulated in shoots.     

The biogeochemical background and trace metal accumulation by brown algae of the genus <Emphasis Type="Italic">Fucus</Emphasis> in coastal waters of the Sea of Japan,the Sea of Okhotsk,and the White Sea

The ranges of background concentrations of Fe, Mn, Zn, Cu, and Cd in Fucus algae from the Sea of Japan, the Sea of Okhotsk, and the White Sea were determined. The lower background threshold of element concentration was calculated as Me₁₅–2MAD₁₅, the upper background threshold was determined as Me+2MAD. The upper background threshold of the metal contents in Fucus algae can be used as the maximum permissible regional concentration in assessing pollution levels of marine habitats. Comparison of the Me₁₅–2MAD₁₅ values showed that Fucus algae of the White Sea had increased Mn and decreased Cd concentrations compared to those in Pacific algae. The concentrations of Zn were higher in Fucus algae of the Sea of Japan than in macrophytes from the White Sea and the Sea of Okhotsk. The background concentration range of Fe in Fucus algae of the Sea of Japan was much narrower compared to that in their counterparts from the White Sea and the Sea of Okhotsk. The background metal concentrations in Fucus algae are specific to a sea region; their variations are connected not only with the trace element contents in abiotic components of the environment, but also with the functioning of coastal ecosystems.     

Overexpression of a <Emphasis Type="Italic">Miscanthus sacchariflorus</Emphasis> yellow stripe-like transporter <Emphasis Type="Italic">MsYSL1</Emphasis> enhances resistance of <Emphasis Type="Italic">Arabidopsis</Emphasis> to cadmium by mediating metal ion reallocation

The yellow stripe-like (YSL) family of transporters mediates the uptake, translocation, and distribution of various mineral elements in vivo by transferring metal ions chelated with phytosiderophore or nicotianamine (NA). However, little is known about the roles of the YSL genes against cadmium in planta. In this study, we first cloned and characterized a vital member of the YSL gene family, MsYSL1, from the bioenergy plant Miscanthus sacchariflorus. MsYSL1 localized in the plasma membrane and was widely expressed throughout the whole seedling with the highest expression level in the stem. In addition, its expression in the root was stimulated by excess manganese (Mn), cadmium (Cd), and lead, and a shortage of iron (Fe), zinc (Zn), and copper. Functional complementation in yeast indicated that MsYSL1 showed transport activity for Fe(II)–NA and Zn–NA, but not for Cd–NA. Although they exhibited no significant differences versus the wild type under normal cultivation conditions, MsYSL1-overexpressing Arabidopsis lines displayed a higher resistance to Cd accompanied by longer root lengths, lower Cd, Zn, and Mn levels in roots, and higher Cd, Fe, and Mn translocation ratios under Cd stress. Moreover, genes related to NA synthesis, metal translocation, long-distance transport, and Cd exclusion were highly induced in transgenic lines under Cd stress. Thus, MsYSL1 may be an essential transporter for diverse metal–NAs to participate in the Cd detoxification by mediating the reallocation of other metal ions.     

Analysis of compound heterozygotes reveals that the mouse floxed <Emphasis Type="Italic">Pax6</Emphasis><Superscript><Emphasis Type="Italic">tm1Ued</Emphasis></Superscript> allele produces abnormal eye phenotypes

Analysis of abnormal phenotypes produced by different types of mutations has been crucial for our understanding of gene function. Some floxed alleles that retain a neomycin-resistance selection cassette (neo cassette) are not equivalent to wild-type alleles and provide useful experimental resources. Pax6 is an important developmental gene and the aim of this study was to determine whether the floxed Pax6 ^tm1Ued (Pax6 ^fl ) allele, which has a retained neo cassette, produced any abnormal eye phenotypes that would imply that it differs from the wild-type allele. Homozygous Pax6 ^fl/fl and heterozygous Pax6 ^fl/+ mice had no overt qualitative eye abnormalities but morphometric analysis showed that Pax6 ^fl/fl corneas tended be thicker and smaller in diameter. To aid identification of weak effects, we produced compound heterozygotes with the Pax6 ^Sey-Neu (Pax6 ^?) null allele. Pax6 ^fl/? compound heterozygotes had more severe eye abnormalities than Pax6 ^+/? heterozygotes, implying that Pax6 ^fl differs from the wild-type Pax6 ⁺ allele. Immunohistochemistry showed that the Pax6 ^fl/? corneal epithelium was positive for keratin 19 and negative for keratin 12, indicating that it was abnormally differentiated. This Pax6 ^fl allele provides a useful addition to the existing Pax6 allelic series and this study demonstrates the utility of using compound heterozygotes with null alleles to unmask cryptic effects of floxed alleles.     

Gene <Emphasis Type="Italic">angora</Emphasis> as a modifier of the <Emphasis Type="Italic">hairless</Emphasis> gene in mouse

The interaction between mouse angora-Y (Fgf5 ^go-Y) and hairless (hr) genes have been studied. Homozygous mutant gene Fgf5 ^go-Y increases length of all hair types, while homozygotes for the h gene lose hair completely starting on day 14 after birth. We obtained mice with genotypes +/+ hr/hr F₂, +/Fgf5 ^go-Y hr/hr and Fgf5 ^go-Y/Fgf5 ^go-Y hr/hr. Both +/Fgf5 ^go-Y hr/hr and +/+ hr/hr mice began to loose hair from their heads on day 14. This further extended on the whole body. On day 21 the mice were completely deprived of hair. Therefore a single dose of gene Fgf5 ^go-Y does not modify alopecia in mice homozygous for hr. However in double homozygotes Fgf5 ^go-Y/Fgf5 ^go-Y hr/hr alopecia started 4 days later, namely on day 18. It usually finished 10–12 days after detection of first bald patches. On days 28–30 double homozygotes lose coat completely. Hair loss in double homozygous mice was 1.5-fold slower than in +/+ hr/hr mice. This resulted from a significant extension of anagen phase induced by a mutant homozygous gene Fgf5 ^go-Y in morphogenesis of the hair follicle. The hr gene was expressed at the transmission phase from anagen to catagen. Our data shows that the angora gene is a modifier of the hairless gene and this results in a strong repression of alopecia progression in double homozygous mice compared to +/+ hr/hr animals.     

Expression of iron-regulators in the bone tissue of rats with and without iron overload

Recently, more and more studies indicate that iron overload would cause osteopenia or osteoporosis. However, the molecular mechanism of it remains unclear. Moreover, very little is known about the iron metabolism in bone tissue at present. Therefore, the mRNA expression of iron-regulators, transferrin receptor1 (Tfr1), divalent metal transporter1 (Dmt1?+?IRE and Dmt1???IRE), ferritin (FtH and FtL), and ferroportin1 (Ireg1), and the localization of ferroportin1 protein were examined in the bone tissue of rats. In addition, the mRNA expression of each gene was compared between groups of rats with and without iron overload. The results showed that ferroportin1 protein was localized in the cytoplasm of osteoblast, osteocyte, chondrocyte and osteoclast of rats’ femur. The six iron-regulatory genes, Tfr1, ferritin (FtH and FtL), (Dmt1?+?IRE and Dmt1???IRE) and ferroportin1 (Ireg1), were found in femurs of rats. In addition, significantly up-regulated expression of FtH and FtL mRNA, and markedly down-regulated expression of Tfr1, Dmt1?+?IRE and Ireg1 mRNA, were observed in the iron overload group compared with the control group. The result indicates that ferroportin1 protein is localized in the cytoplasm of bone cells of rats. Tfr1, Dmt1, ferritin and ferroportin1 exist in bone tissue of rats, and they may be involved in the pathological process of iron overload-induced bone lesion.     

Metabolism-mediated induction of zinc tolerance in <Emphasis Type="Italic">Brassica rapa</Emphasis> by <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> CS2-1

Brassica rapa (Chinese cabbage) is an essential component of traditional Korean food. However, the crop is often subject to zinc (Zn⁺) toxicity from contaminated irrigation water, which, as a result, compromises plant growth and production, as well as the health of human consumers. The present study investigated the bioaccumulation of Zn⁺ by Burkholderia cepacia CS2-1 and its effect on the heavy metal tolerance of Chinese cabbage. Strain CS2-1 was identified and characterized on the basis of 16S rRNA sequences and phylogenetic analysis. The strain actively produced indole-3-acetic acid (3.08 ± 0.21 μg/ml) and was also able to produce siderophore, solubilize minerals, and tolerate various concentrations of Zn⁺. The heavy metal tolerance of B. rapa plants was enhanced by CS2-1 inoculation, as indicated by growth attributes, Zn⁺ uptake, amino acid synthesis, antioxidant levels, and endogenous hormone (ABA and SA) synthesis. Without inoculation, the application of Zn⁺ negatively affected the growth and physiology of B. rapa plants. However, CS2-1 inoculation improved plant growth, lowered Zn⁺ uptake, altered both amino acid regulation and levels of flavonoids and phenolics, and significantly decreased levels of superoxide dismutase, endogenous abscisic acid, and salicylic acid. These findings indicate that B. cepacia CS2-1 is suitable for bioremediation against Zn⁺-induced oxidative stress.     

Molecular characterization and expression analysis of the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger gene family in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

One important mechanism plants use to cope with salinity is keeping the cytosolic Na⁺ concentration low by sequestering Na⁺ in vacuoles, a process facilitated by Na⁺/H⁺ exchangers (NHX). There are eight NHX genes (NHX1 through NHX8) identified and characterized in Arabidopsis thaliana. Bioinformatics analyses of the known Arabidopsis genes enabled us to identify six Medicago truncatula NHX genes (MtNHX1, MtNHX2, MtNHX3, MtNHX4, MtNHX6, and MtNHX7). Twelve transmembrane domains and an amiloride binding site were conserved in five out of six MtNHX proteins. Phylogenetic analysis involving A. thaliana, Glycine max, Phaseolus vulgaris, and M. truncatula revealed that each individual MtNHX class (class I: MtNHX1 through 4; class II: MtNHX6; class III: MtNHX7) falls under a separate clade. In a salinity-stress experiment, M. truncatula exhibited ~?20% reduction in biomass. In the salinity treatment, sodium contents increased by 178 and 75% in leaves and roots, respectively, and Cl^? contents increased by 152 and 162%, respectively. Na⁺ exclusion may be responsible for the relatively smaller increase in Na⁺ concentration in roots under salt stress as compared to Cl^?. Decline in tissue K⁺ concentration under salinity was not surprising as some antiporters play an important role in transporting both Na⁺ and K ⁺ . MtNHX1, MtNHX6, and MtNHX7 display high expression in roots and leaves. MtNHX3, MtNHX6, and MtNHX7 were induced in roots under salinity stress. Expression analysis results indicate that sequestering Na⁺ into vacuoles may not be the principal component trait of the salt tolerance mechanism in M. truncatula and other component traits may be pivotal.     

The <Emphasis Type="Italic">angora</Emphasis> gene weakens the effect of interaction of the mutant genes <Emphasis Type="Italic">wellhaarig</Emphasis> and <Emphasis Type="Italic">waved alopecia</Emphasis> in mice

The interaction of the mutant genes wellhaarig (we) and waved alopecia (wal) in mice was earlier demonstrated in our laboratory. The we gene significantly accelerates the appearance of alopecia in double we/wewal/wal homozygotes as compared to that in single +/+wal/wal homozygotes. It has been found in this work that the mutant gene angora-Y (Fgf5 ^go-Y ) weakens the effect of interaction of the we and wal genes. The first signs of alopecia appear in mice of the we/wewal/wal genotype at the age of 14 days, in triple Fgf5 ^go-Y /Fgf5 ^go-Y we/wewal/wal homozygotes alopecia is observed seven days later, i. e., in 21-day-old animals. The progression of alopecia in triple homozygotes is expressed to a lesser degree than in double +/+we/wewal/wal homozygotes. A single dose of the Fgf5 ^go-Y gene also decreases the effect of interaction of the we and wal genes, but less than a double dose of this gene. The first signs of alopecia in mice of the +/Fgf5 ^go-Y we/wewal/wal genotype appear only three days later than in double +/+we/wewal/wal homozygotes. The data obtained demonstrate that the Fgf5 ^go-Y gene is a powerful modifier of mutant genes determining the process of alopecia.     

Concentration of some metals in soil and plant organs and their biochemical profiles in <Emphasis Type="Italic">Tulipa luanica</Emphasis>, <Emphasis Type="Italic">T. kosovarica</Emphasis> and <Emphasis Type="Italic">T. albanica</Emphasis> native plant species

The purpose of this study was to determine the concentration of some metals (Al, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Zn, Ca and Mg) in soil of serpentine and limestone sites, their bioaccumulation and impact on some biochemical parameters in T. luanica, T. kosovarica and T. albanica plants. T. kosovarica and T. albanica grows in serpentine soil, while T. luanica grow in limestone soil. The research showed that concentrations of Cd, Co, Cr, Fe, Mn and Ni were significantly higher at serpentine soil sites in comparison with limestone sites, while concentrations of Pb, Cd, Co and Cr in bulbs, leaves and seeds were under the limit of detection. The concentration of Ni in plant samples of T. kosovarica was significantly higher in comparison with its concentration in T. albanica, but it was under the limit of detection in T. luanica. Moreover, concentrations of Al and Fe in leaves of T. kosovarica and T. albanica were higher in comparison with T. luanica. The concentration of Mg was significantly higher in T. kosovarica and T. albanica than in T. luanica. The δ-aminolevulinic acid dehydratase activity, malondialdehyde and glutathione contents in leaves of T. luanica were higher in comparison with T. kosovarica and T. albanica. In addition, the amounts of total chlorophyll and δ-aminolevulinic acid (ALA) in leaves of T. albanica were higher in comparison with T. kosovarica and T. luanica. Our findings show that target organs of metal accumulation in three Tulip species appears to be leaves?>?seeds?>?bulbs, while the biochemical parameters show that limestone sites represent a less stressful habitat for growing these plant species in comparison with serpentine sites.     

Phosphites attenuate <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>-induced physiological impairments in common bean

Phosphites, marketed as foliar fertilizers and resistance activators, have been shown to be useful for the control of diseases in many profitable crops. Despite the importance of white mold, caused by Sclerotinia sclerotiorum, to reduce common bean yield, knowledge of the phosphites´ effect on disease control at the physiological level is still missing. In this study, the leaf gas exchange and chlorophyll a fluorescence parameters variable-to-maximum chlorophyll a fluorescence ratio (F_v/F_m), photochemical yield [Y(II)], yield for dissipation by down-regulation [Y(NPQ)], yield for non-regulated dissipation [Y(NO)], and electron transport rate (ETR) as well as the concentrations of photosynthetic pigments in common bean plants that were sprayed with zinc (Zn) or copper (Cu) phosphites and challenged or not with S. sclerotiorum were determined. Based on the in vitro assays, Zn and Cu phosphites inhibited fungal mycelial growth in a dose-dependent manner, but the Cu phosphite showed to be more fungitoxic. Lesion area and white mold severity were reduced by Zn and Cu phosphites, but the Zn phosphite was more effective. Fungal infection dramatically decreased the values of net carbon assimilation rate, stomatal conductance to water vapor and transpiration rate on non-sprayed plants. Increases in internal CO₂ concentration indicated that fungal-induced photosynthetic impairments were chiefly governed by biochemical limitations, but these impairments were greatly abrogated in the Zn and Cu phosphite-sprayed plants. Similarly, the photochemical dysfunctions stemmed from S. sclerotiorum infection were limited in the Zn and Cu phosphite-sprayed plants. Concentrations of chlorophyll a?+?b and carotenoids decreased on inoculated plants, but lower reductions were recorded on Zn and Cu phosphites-sprayed plants. In conclusion, the potential of Zn and Cu phosphites in attenuate the S. sclerotiorum-induced physiological impairments in common bean leaflets was demonstrated and may be an effective mean for managing this disease under field conditions.     

A nodule endophytic plant growth-promoting <Emphasis Type="Italic">Pseudomonas</Emphasis> and its effects on growth,nodulation and metal uptake in <Emphasis Type="Italic">Medicago lupulina</Emphasis> under copper stress

The aim of this study was to determine the plant growth-promoting potential of the nodule endophytic Pseudomonas brassicacearum strain Zy-2-1 when used as a co-inoculant of Medicago lupulina with Sinorhizobium meliloti under copper (Cu) stress conditions. Strain Zy-2-1 was capable of producing ACC deaminase activity, IAA and siderophores, and was able to grow in the presence of Cu²⁺ up to 2.0 mmol/L. Co-inoculation of S. meliloti with Zy-2-1 enhanced M. lupulina root fresh weight, total plant dry weight, number of nodules, nodule fresh weight and nitrogen content in the presence of 100 or 300 mg/kg Cu²⁺. In the presence of 500 mg/kg Cu²⁺, co-inoculation with S. meliloti and strain Zy-2-1 increased plant height, number of nodules, nodule fresh weight and nitrogen content in comparison to S. meliloti inoculation alone. Furthermore, a higher amount of Cu accumulation in both shoots and roots and a higher level of Cu translocation to shoots were observed in co-inoculated plants. These results demonstrate that co-inoculation of M. lupulina with S. meliloti and P. brassicacearum Zy-2-1 improves plant growth, nitrogen nutrition and metal extraction potential. This can be of practical importance in the remediation of heavy metal-contaminated soils.