55 research outputs found

    Engineering high Zn in tomato shoots through expression of AtHMA4 involves tissue-specific modification of endogenous genes.

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    BACKGROUND: To increase the Zn level in shoots, AtHMA4 was ectopically expressed in tomato under the constitutive CaMV 35S promoter. However, the Zn concentration in the shoots of transgenic plants failed to increase at all tested Zn levels in the medium. Modification of Zn root/shoot distribution in tomato expressing 35S::AtHMA4 depended on the concentration of Zn in the medium, thus indicating involvement of unknown endogenous metal-homeostasis mechanisms. To determine these mechanisms, those metal-homeostasis genes that were expressed differently in transgenic and wild-type plants were identified by microarray and RT-qPCR analysis using laser-assisted microdissected RNA isolated from two root sectors: (epidermis + cortex and stele), and leaf sectors (upper epidermis + palisade parenchyma and lower epidermis + spongy parenchyma). RESULTS: Zn-supply-dependent modification of Zn root/shoot distribution in AtHMA4-tomato (increase at 5 μM Zn, no change at 0.5 μM Zn) involved tissue-specific, distinct from that in the wild type, expression of tomato endogenous genes. First, it is suggested that an ethylene-dependent pathway underlies the detected changes in Zn root/shoot partitioning, as it was induced in transgenic plants in a distinct way depending on Zn exposure. Upon exposure to 5 or 0.5 μM Zn, in the epidermis + cortex of the transgenics' roots the expression of the Strategy I Fe-uptake system (ethylene-dependent LeIRT1 and LeFER) was respectively lower or higher than in the wild type and was accompanied by respectively lower or higher expression of the identified ethylene genes (LeNR, LeACO4, LeACO5) and of LeChln. Second, the contribution of LeNRAMP2 expression in the stele is shown to be distinct for wild-type and transgenic plants at both Zn exposures. Ethylene was also suggested as an important factor in a pathway induced in the leaves of transgenic plants by high Zn in the apoplast, which results in the initiation of loading of the excess Zn into the mesophyll of "Zn accumulating cells". CONCLUSIONS: In transgenic tomato plants, the export activity of ectopically expressed AtHMA4 changes the cellular Zn status, which induces coordinated tissue-specific responses of endogenous ethylene-related genes and metal transporters. These changes constitute an important mechanism involved in the generation of the metal-related phenotype of transgenic tomato expressing AtHMA4

    Functional Analysis of NtZIP4B and Zn Status-Dependent Expression Pattern of Tobacco ZIP Genes

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    Tobacco is frequently considered as a plant useful for phytoremediation of metal-contaminated soil, despite the mechanisms for regulation of uptake and accumulation being largely unknown. Here we cloned and characterized a new tobacco Zn and Cd transporter NtZIP4B from the ZIP family (ZRT-IRT-Like proteins). It complemented the Zn-uptake defective yeast mutant zrt1zrt2, and rendered the wild type DY1457 yeast more sensitive to Cd. Bioinformatic analysis and transient expression of the NtZIP4B-GFP fusion protein in tobacco leaves indicated its localization to the plasma membrane. Real-time q-PCR based analysis showed that it is expressed in all vegetative organs with the highest level in leaves. The Zn status determined transcript abundance; NtZIP4B was upregulated by Zn-deficiency and downregulated by Zn excess. At the tissue level, in roots NtZIP4B is expressed in the vasculature of the middle part of the roots and in surrounding tissues including the root epidermis; in leaves primarily in the vasculature. Bioinformatic analysis identified two copies of ZIP4 in tobacco, NtZIP4A and NtZIP4B with 97.57% homology at the amino acid level, with the same expression pattern for both, indicating a high degree of functional redundancy. Moreover, the present study provides new insights into the coordinated function of NtZIP1, NtZIP2, NtZIP4, NtZIP5, NtZIP8, NtIRT1, and NtIRT1-like in response to low-to-high Zn status. Leaves were the major site of NtZIP4, NtZIP5, and NtZIP8 expression, and roots for NtZIP1, NtZIP2, NtIRT1, and NtIRT1-like. Contrasting expression level in the apical and basal root parts indicates distinct roles in root-specific processes likely contributing to the regulation of Zn root-to-shoot translocation. In summary, new insight into the role of ZIP genes in Zn homeostasis pointing to their overlapping and complementary functions, offers opportunities for strategies to modify Zn and Cd root/shoot partition in tobacco

    Effects of different nitrogen fertilizers on two wheat cultivars: An integrated approach

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    Investigation of cultivated plant physiology grown under low energy input plays an important role to indicate their fitness to the new environmental conditions. The durum‐wheat cultivars Creso and Dylan were tested to evaluate the growth, production, and proteomic and transcriptomic profiles of the crop under different synthetic and organic nitrogen fertilization regimes. In this work, a two‐dimensional gel electrophoresis (2‐DE) approach combined with liquid chromatography–mass spectrometry (LC–MS) was used to investigate the protein changes induced by the use of different nitrogen sources (hydrolysate of proteins 1 and 2, rhizovit, synthesis, leather) on wheat plants. Proteomic studies were integrated with qPCR analysis of genes related to glutamine synthetase/glutamine‐2‐oxoglutarate aminotransferase (GS‐GOGAT) and tricarboxylic acid (TCA) metabolic pathways because most relevant for nitrogen‐dependent plants growth. The proteomic analysis lead to the isolation of 23 spots that were able to distinguish the analyzed samples. These spots yielded the identification of 60 proteins involved in photosynthesis, glycolysis, and nitrogen metabolism. As an example, the quinone oxidoreductase‐like protein and probable glutathione S‐transferase GSTU proteins were identified in two spots that represents the most statistically significant ones in Dylan samples. Transcript analysis indicated that related genes exhibited different expression trends; the heat map also revealed the different behaviors of the hydrolysates of the proteins 1 and 2 nitrogen sources. The effects of nitrogenous fertilizers at the proteomic and agronomic levels revealed that plants fertilized with synthesis or rhizovit gave the best results concerning yield, whereas rhizovit and protein hydrolysates were most effective for proteins content in the grain (% of dry weight). Therefore, all parameters measured in this study indicated that different kinds of nitrogen fertilization used have a relevant impact on plant growth and production

    Differential regulation of alanine aminotransferase homologues by abiotic stresses in wheat (Triticum aestivum L.) seedlings

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    Wheat (Triticum aestivum L.) seedlings contain four alanine aminotransferase (AlaAT) homologues. Two of them encode AlaAT enzymes, whereas two homologues act as glumate:glyoxylate aminotransferase (GGAT). To address the function of the distinct AlaAT homologues a comparative examination of the changes in transcript level together with the enzyme activity and alanine and glutamate content in wheat seedlings subjected to low oxygen availability, nitrogen and light deficiency has been studied. Shoots of wheat seedlings were more tolerant to hypoxia than the roots as judging on the basis of enzyme activity and transcript level. Hypoxia induced AlaAT1 earlier in roots than in shoots, while AlaAT2 and GGAT were unaffected. The increase in AlaAT activity lagged behind the increase in alanine content. Nitrogen deficiency has little effect on the activity of GGAT. In contrast, lower activity of AlaAT and the level of mRNA for AlaAT1 and AlaAT2 in wheat seedlings growing on a nitrogen-free medium seems to indicate that AlaAT is regulated by the availability of nitrogen. Both AlaAT and GGAT activities were present in etiolated wheat seedlings but their activity was half of that observed in light-grown seedlings. Exposure of etiolated seedlings to light caused an increase in enzyme activities and up-regulated GGAT1. It is proposed that hypoxia-induced AlaAT1 and light-induced peroxisomal GGAT1 appears to be crucial for the regulation of energy availability in plants grown under unfavourable environmental conditions

    Reactor control panel, ETS-1 CTO console, drawing 707J888

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    Wykrywanie alfa₁-fetoproteiny metodą immunoelektroforezy przeciwbieżnej

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    Ilościowe oznaczenie frakcji białkowych metodą immunodyfuzji radialnej

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    Immunoelektroforetyczne wykrywanie frakcji białkowych

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