Cucumber metal tolerance protein 7 (CsMTP7) is involved in the accumulation of Fe in mitochondria under Fe excess.

The plant metal tolerance protein family (MTP) includes 12 members that have been classified into three phylogenetically different subgroups - Zn-cation diffusion facilitator (CDF), Fe/Zn-CDF and Mn-CDF - based on their putative metal specificity. To date, only members belonging to the Zn-CDF or Mn-CDF group have been characterized functionally. The plant Fe/Zn-CDF subgroup includes two proteins, MTP6 and MTP7, but their function and metal specificity have not been confirmed. In this study we showed that cucumber CsMTP7 is a highly specific mitochondrial Fe importer that is able to confer yeast tolerance to Fe excess through increased accumulation of Fe in the mitochondria. We also demonstrated that CsMTP7 contributes to the increased accumulation of Fe in the mitochondria of Arabidopsis thaliana protoplasts. The transcripts and mitochondrial levels of CsMTP7 and ferritin - the iron-storing protein - are significantly increased in cucumber roots in response to Fe excess. This finding suggests that CsMTP7 and ferritin work in concert to accumulate Fe in plant mitochondria. As genes that encode orthologous proteins have been identified in phylogenetically distant organisms, including Archaea, cyanobacteria, humans and plants, but not in yeast, we concluded that the MTP7-mediated mitochondrial Fe accumulation may be conserved in the species, and express mitochondrial ferritin for mitochondrial Fe storage.

Metal tolerance protein MTP6 affects mitochondrial iron and manganese homeostasis in cucumber.

Members of the cation diffusion facilitator (CDF) family have been identified in all kingdoms of life. They have been divided into three subgroups, namely Zn-CDF, Fe/Zn-CDF, and Mn-CDF, based on their putative specificity to transported metal ions. The plant metal tolerance protein 6 (MTP6) proteins fall into the Fe/Zn-CDF subgroup; however, their function in iron/zinc transport has not yet been confirmed. Here, we characterized the MTP6 protein from cucumber, Cucumis sativus. When expressed in yeast and in protoplasts isolated from Arabidopsis cells, CsMTP6 localized in mitochondria and contributed to the efflux of Fe and Mn from these organelles. Immunolocalization of CsMTP6 in cucumber membranes confirmed this association with mitochondria. Root expression and protein levels of CsMTP6 were significantly up-regulated in conditions of Fe deficiency and excess, but were not affected by Mn availability. These results indicate that MTP6 proteins contribute to the distribution of Fe and Mn between the cytosol and mitochondria of plant cells, and are regulated by Fe to maintain mitochondrial and cytosolic iron homeostasis under varying conditions of Fe availability.

Functional and Biochemical Characterization of Cucumber Genes Encoding Two Copper ATPases CsHMA5.1 and CsHMA5.2.

Plant copper P1B-type ATPases appear to be crucial for maintaining copper homeostasis within plant cells, but until now they have been studied mostly in model plant systems. Here, we present the molecular and biochemical characterization of two cucumber copper ATPases, CsHMA5.1 and CsHMA5.2, indicating a different function for HMA5-like proteins in different plants. When expressed in yeast, CsHMA5.1 and CsHMA5.2 localize to the vacuolar membrane and are activated by monovalent copper or silver ions and cysteine, showing different affinities to Cu(+) (Km ∼1 or 0.5 µM, respectively) and similar affinity to Ag(+) (Km ∼2.5 µM). Both proteins restore the growth of yeast mutants sensitive to copper excess and silver through intracellular copper sequestration, indicating that they contribute to copper and silver detoxification. Immunoblotting with specific antibodies revealed the presence of CsHMA5.1 and CsHMA5.2 in the tonoplast of cucumber cells. Interestingly, the root-specific CsHMA5.1 was not affected by copper stress, whereas the widely expressed CsHMA5.2 was up-regulated or down-regulated in roots upon copper excess or deficiency, respectively. The copper-induced increase in tonoplast CsHMA5.2 is consistent with the increased activity of ATP-dependent copper transport into tonoplast vesicles isolated from roots of plants grown under copper excess. These data identify CsHMA5.1 and CsHMA5.2 as high affinity Cu(+) transporters and suggest that CsHMA5.2 is responsible for the increased sequestration of copper in vacuoles of cucumber root cells under copper excess.

Cucumber metal tolerance protein CsMTP9 is a plasma membrane H⁺-coupled antiporter involved in the Mn²âº and Cd²âº efflux from root cells.

Members of the plant metal tolerance protein (MTP) family have been classified into three major groups - Zn-CDF, Mn-CDF and Zn/Fe-CDF - however, the selectivity of most of the MTPs has not been confirmed yet. Cucumber gene CsMTP9 encoding a putative CDF transporter homologous to members of the Mn-CDF cluster is expressed exclusively in roots. The relative abundance of CsMTP9 transcript and protein in roots is significantly increased under Mn excess and Cd. Immunolocalization with specific antibodies revealed that CsMTP9 is a plasma membrane transporter that localizes to the inner PM domain of root endodermal cells. The plasma membrane localization of CsMTP9 was confirmed by the expression of the fusion proteins of GFP (green fluorescent protein) and CsMTP9 in yeast and protoplasts prepared from Arabidopsis cells. In yeast, CsMTP9 transports Mn(2+) and Cd(2+) via a proton-antiport mechanism with an apparent Km values of approximately 10 µm and 2.5 µm for Mn(2+) and Cd(2+) , respectively. In addition, CsMTP9 expression in yeast rescues the Mn- and Cd-hypersensitive phenotypes through the enhanced efflux of Mn(2+) and Cd(2+) from yeast cells. Similarly, the overexpression of CsMTP9 in A. thaliana confers increased resistance of plants to Mn excess and Cd but not to other heavy metals and leads to the enhanced translocation of manganese and cadmium from roots to shoots. These findings indicate that CsMTP9 is a plasma membrane H(+) -coupled Mn(2+) and Cd(2+) antiporter involved in the efflux of manganese and cadmium from cucumber root cells by the transport of both metals from endodermis into vascular cylinder.

Molecular and biochemical properties of two P1B2-ATPases, CsHMA3 and CsHMA4, from cucumber.

P1B-ATPases (heavy metal ATPases, HMAs) constitute a multigenic subfamily of P-ATPases involved in the transport of monovalent and divalent heavy metals in plant cells. Here, we present the organization of genes encoding the HMA family in the cucumber genome and report the function and biochemical properties of two cucumber proteins homologous to the HMA2-4-like plant HMAs. Eight genes encoding putative P1B -ATPases were identified in the cucumber genome. Among them, CsHMA3 was predominantly expressed in roots and up-regulated by Pb, Zn and Cd excess, whereas the CsHMA4 transcript was most abundant in roots and flowers of cucumber plants, and elevated under Pb and Zn excess. Expression of CsHMA3 in Saccharomyces cerevisiae enhanced yeast tolerance to Cd and Pb, whereas CsHMA4 conferred increased resistance of yeast cells to Cd and Zn. Immunostaining with specific antibodies raised against cucumber proteins revealed tonoplast localization of CsHMA3 and plasma membrane localization of CsHMA4 in cucumber root cells. Kinetic studies of CsHMA3 and CsHMA4 in yeast membranes indicated differing heavy metal cation affinities of these two proteins. Altogether, the results suggest an important role of CsHMA3 and CsHMA4 in Cd and Pb detoxification and Zn homeostasis in cucumber cells.

Two metal-tolerance proteins, MTP1 and MTP4, are involved in Zn homeostasis and Cd sequestration in cucumber cells.

Metal-tolerance proteins (MTPs) are divalent cation transporters that have been shown to be essential for metal homeostasis and tolerance in model plants and hyperaccumulators. Due to the lack of genomic resources, studies on MTPs in cultivated crops are lacking. Here, we present the first functional characterization of genes encoding cucumber proteins homologous to MTP1 and MTP4 transporters. CsMTP1 expression was ubiquitous in cucumber plants, whereas CsMTP4 mRNA was less abundant and was not detected in the generative parts of the flowers. When expressed in yeast, CsMTP1 and CsMTP4 were able to complement the hypersensitivity of mutant strains to Zn and Cd through the increased sequestration of metals within vacuoles using the transmembrane electrochemical gradient. Both proteins formed oligomers at the vacuolar membranes of yeast and cucumber cells and localized in Arabidopsis protoplasts, consistent with their function in vacuolar Zn and Cd sequestration. Changes in the abundance of CsMTP1 and CsMTP4 transcripts and proteins in response to elevated Zn and Cd, or to Zn deprivation, suggested metal-induced transcriptional, translational, and post-translational modifications of protein activities. The differences in the organ expression and affinity of both proteins to Zn and Cd suggested that CsMTP1 and CsMTP4 may not be functionally redundant in cucumber cells.

Cucumber metal transport protein MTP8 confers increased tolerance to manganese when expressed in yeast and Arabidopsis thaliana.

Cation diffusion facilitator (CDF) proteins are ubiquitous divalent cation transporters that have been proved to be essential for metal homeostasis and tolerance in Archaebacteria, Bacteria, and Eukaryota. In plants, CDFs are designated as metal tolerance proteins (MTPs). Due to the lack of genomic resources, studies on MTPs in other plants, including cultivated crops, are lacking. Here, the identification and organization of genes encoding members of the MTP family in cucumber are described. The first functional characterization of a cucumber gene encoding a member of the Mn-CDF subgroup of CDF proteins, designated as CsMTP8 based on the highest homology to plant MTP8, is also presented. The expression of CsMTP8 in Saccharomyces cerevisiae led to increased Mn accumulation in yeast cells and fully restored the growth of mutants hypersensitive to Mn in Mn excess. Similarly, the overexpression of CsMTP8 in Arabidopsis thaliana enhanced plant tolerance to high Mn in nutrition media as well as the accumulation of Mn in plant tissues. When fused to green fluorescent protein (GFP), CsMTP8 localized to the vacuolar membranes in yeast cells and to Arabidopsis protoplasts. In cucumber, CsMTP8 was expressed almost exclusively in roots, and the level of gene transcript was markedly up-regulated or reduced under elevated Mn or Mn deficiency, respectively. Taken together, the results suggest that CsMTP8 is an Mn transporter localized in the vacuolar membrane, which participates in the maintenance of Mn homeostasis in cucumber root cells.

Characterization of Hspb8 in Zebrafish.

Hspb8 is a member of the small heat shock protein (sHSP) family. Its expression is known to be upregulated under heat shock. This protein interacts with different partners and can, therefore, be involved in various processes relevant to tissue integrity and functioning. In humans, mutations in the gene encoding Hspb8 can lead to the development of various diseases such as myopathies and neuropathies. In our study, we aimed to perform an in-depth characterization of zebrafish Hspb8 during zebrafish development. We applied techniques such as RT-qPCR, Western blot, immunofluorescence, co-immunoprecipitation, LC-MS, and morpholino-mediated knockdown. We broadened the knowledge regarding zebrafish hspb8 expression during development under normal and heat shock conditions as well as its tissue- and subcellular-specific localization. A co-IP analysis allowed us to conclude that zebrafish Hspb8 can interact with proteins such as Bag3 and Hsc70, which are crucial for formation of an autophagy-inducing complex. We also demonstrated that hspb8 morpholino-mediated knockdown has an impact on zebrafish embryos' morphology, muscle ultrastructure, and motility behavior. Our research provides a valuable resource for the potential use of the zebrafish as a model for studying pathological conditions associated with hspb8 disorders.

Cucumber Golgi protein CsMTP5 forms a Zn-transporting heterodimer with high molecular mass protein CsMTP12.

Heterodimeric complexes formed by members of the cation facilitator (CDF) family catalyse the import of Zn into the secretory pathway of yeast and vertebrate cells. Orthologous proteins AtMTP5 and AtMTP12 from Arabidopsis have also been shown to form a heterodimeric complex at the Golgi compartment of plant cells that possibly transport Zn. In this study we show that cucumber proteins CsMTP5 and CsMTP12 form a functional heterodimer that is involved in the loading of Zn into the ER lumen under low Zn, and not in the detoxification of yeast from Zn excess through vesicle-mediated exocytosis. Using specific antibodies, we demonstrate that CsMTP5 is localized at the Golgi compartment of cucumber cells and is markedly up-regulated upon Zn deficiency. The level of CsMTP5 transcript in cucumber is also significantly elevated in Zn-limiting conditions, whereas the expression of CsMTP12 is independent of the availability of Zn. Therefore we propose that the cucumber heterodimeric complex CsMTP5-CsMTP12 functions to deliver Zn to Zn-dependent proteins of the Golgi compartment and is regulated by zinc at the level of CsMTP5 transcription.

Does the grass snake (Natrix natrix) (Squamata: Serpentes: Natricinae) fit the amniotes-specific model of myogenesis?

In the grass snake (Natrix natrix), the newly developed somites form vesicles that are located on both sides of the neural tube. The walls of the vesicles are composed of tightly connected epithelial cells surrounding the cavity (the somitocoel). Also, in the newly formed somites, the Pax3 protein can be observed in the somite wall cells. Subsequently, the somite splits into three compartments: the sclerotome, dermomyotome (with the dorsomedial [DM] and the ventrolateral [VL] lips) and the myotome. At this stage, the Pax3 protein is detected in both the DM and VL lips of the dermomyotome and in the mononucleated cells of the myotome, whereas the Pax7 protein is observed in the medial part of the dermomyotome and in some of the mononucleated cells of the myotome. The mononucleated cells then become elongated and form myotubes. As myogenesis proceeds, the myotome is filled with multinucleated myotubes accompanied by mononucleated, Pax7-positive cells (satellite cells) that are involved in muscle growth. The Pax3-positive progenitor muscle cells are no longer observed. Moreover, we have observed unique features in the differentiation of the muscles in these snakes. Specifically, our studies have revealed the presence of two classes of muscles in the myotomes. The first class is characterised by fast muscle fibres, with myofibrils equally distributed throughout the sarcoplasm. In the second class, composed of slow muscle fibres, the sarcoplasm is filled with lipid droplets. We assume that their storage could play a crucial role during hibernation in the adult snakes. We suggest that the model of myotomal myogenesis in reptiles, birds and mammals shows the same morphological and molecular character. We therefore believe that the grass snake, in spite of the unique features of its myogenesis, fits into the amniotes-specific model of trunk muscle development.