1,721,013 research outputs found
THE ROLE OF THE ARABIDOPSIS THALIANA ATYPICAL KINASES ABC1K2 AND ABC1K9 IN STRESS RESPONSES
No full textThe ABC1K protein kinases (activity of bc1 complex kinases) are a large family of proteins with multiple roles in the regulation of respiration and oxidative stress tolerance. In plants they regulate diverse physiological processes in the chloroplasts and mitochondria, but their precise functions are poorly defined. In this work the functional characterization of the chloroplast Arabidopsis thaliana ABC1K2 and ABC1K9 proteins is carried out. To characterize the function of the two proteins, A. thaliana knock-out mutant lines that lack ABC1K2 or ABC1K9 proteins are considered. Under standard growth conditions, abc1k2 and abc1k9 mutant plants do not show morphological or developmental abnormalities if compared to wild type. Pigment analysis reveals a reduced total chlorophyll content and Chla/b ratio in abc1k2 and abc1k9 old leaves. Analysis of ultrathin sections of leaves with transmission electron microscopy does not show any significant difference in the chloroplast ultrastructure between wild type and mutant plants. Interestingly, in abc1k2 and abc1k9 plants plastoglobules with a reduced diameter are associated with thylakoids. Analysis of the photosynthetic performance demonstrates no differences in the major photosynthetic parameters between wild type and mutant plants. Since other ABC1K proteins are involved in plant response to oxidative stress, the effect of oxidative stress on abc1k2 and abc1k9 mutant plants is considered. When plants were grown in the presence of hydrogen peroxide, both abc1k2 and abc1k9 mutants show an increased root length in comparison to wild type. Transcription levels of SOD genes and APX1 are down-regulated in abc1k2 mutant, while they result up-regulated in abc1k9. The level of superoxide anion radical staining is more evident in wild type plants than in abc1k2 and abc1k9 mutants. Because reactive oxygen species take part in ABAmediated processes, the functions of ABC1K2 and ABC1K9 during germination are investigated. Germination is more severely affected by ABA, osmotic stress and salt stress in abc1k2 and abc1k9 mutants. These results suggest that ABC1K2 and ABC1K9 might be involved in the cross-talk between ABA and ROS signallin
The role of ABC1K7 and ABC1K8, two ABC1K kinases of Arabidopsis thaliana
Full text linkThe activity of bc1 complex kinases (ABC1K) belong to a large group of atypical protein kinases found in prokaryotes and eukaryotes. In bacteria and mitochondria, ABC1K kinases are involved in the respiratory pathway, being necessary for the synthesis of the coenzyme Q. In chloroplasts, ABC1K proteins play a role in prenylquinone synthesis and stress responses, but their precise function remains unclear. A functional characterization was carried out for ABC1K7 and ABC1K8, two ABC1K proteins of the Arabidopsis thaliana plastome. The comparison of abc1k7 and abc1k8 mutants, abc1k7/abc1k8 double mutant and wild-type plants revealed a reduction in plastidial ironcontaining proteins of the Cytb6f complex in the mutants. Iron uptake from soil is not hampered in mutant lines, suggesting that ABC1K7 and ABC1K8 affect iron distribution within the chloroplast. Moreover, mutant plants accumulated more ferritin and superoxide, and showed reduced tolerance to reactive oxygen species (ROS). Because ROS take part in abscisic acid (ABA) signaling, we investigated the relation between ABA and ABC1K7 and ABC1K8 and found that both genes were upregulated by ABA treatment, while expression of several ABA-responsive genes resulted affected in mutants. Moreover, analyzing ABA-mediated processes, we determined that germination was more affected by ABA treatment and osmotic and salt stress in the single and double mutants than in wild-type plants. Stomatal aperture was also reduced in the mutants under standard growth conditions and was not further reduced by exogenous ABA application. Furthermore, ABA-induced senescence symptoms were more severe in the leaves of the mutants compared to wild type leaves. Taken together, these data suggest that ABC1K7 and ABC1K8 probably act in signaling pathways that influence responses to ROS production and oxidative stress, such as ABA signaling, probably by influencing the cellular redox state and chloroplast lipid metabolism
5.42 IDENTIFICATION AND ANALYSIS OF GENES INVOLVED IN NICKEL TOLERANCE IN NOCCAEA CAERULESCENS
No full textMetal hyperaccumulator plants are able to accumulate extremely high concentrations of Heavy Metals (HMs) in shoots, in contrast with non-accumulator species. Among them, the european species Noccaea caerulescens, member of the Brassicaeae family, represents an interesting model, because it shows a great variability between different ecotypes in metal tolerance and accumulation, considering, for instance, Zinc (Zn), Nickel (Ni) and Cadmium (Cd), alone or in combination. This work focused on the ecotype Monte Prinzera (MP, Italy) of N. caerulescens, autochthonous in a Natural Reserve in the Tosco-Emilian Appennins (Italy) characterized by serpentine soil; this particular ecotype is able to tolerate and accumulate Ni and Zn, a property that is shared with two other species of the same genus, i.e. N. goesingense and N. japonica. Molecular mechanisms responsible for Ni tolerance and accumulation are still unknown, although different genes seem to play a role in these processes. Metal transporter proteins are essential for metal homeostasis and different studies have demonstrated a fundamental role in HM tolerance and accumulation. In this work, we analysed and compared the response of N. caerulescens MP to different Ni concentrations with that of T. arvense (non-accumulator) and N. caerulescens ecotype Ganges (GA, Zn and Cd hyperaccumulator). Particularly, Real time PCR analysis was performed to evaluate the response of two vacuolar transporters, MTP1 and NRAMP4, and a plasma membrane transporter, ZNT1, to different Ni concentrations. This assay demonstrated that MTP1, which encode for a vacuolar Zn transporter, was highly expressed, suggesting a role in Ni tolerance and/or accumulation. In both GA and MP ecotypes, at least two different forms of MTP1 have been identified, possibly encoding proteins with different metal specificity; the shorter version of MTP1 lacks the highly conserved cytoplasmic His-loop necessary for metal binding specificity. Protoplasts transfection with constructs harbouring the two MTP1 coding sequences fused to a GFP reporter is ongoing to visualize the subcellular localization of both proteins. Tests with yeast mutants will be performed in order to determine whether the genes under investigation have a different role in heavy metal detoxification
IDENTIFICATION AND ANALYSIS OF GENES INVOLVED IN NICKEL TOLERANCE IN NOCCAEA CAERULESCENS
No full textMetal hyperaccumulator plants are able to accumulate extremely high concentrations of Heavy Metals (HMs) in shoots, in contrast with non-accumulator species. Among them, the european species Noccaea caerulescens, member of the Brassicaeae family, represents an interesting model, because it shows a great variability between different ecotypes in metal tolerance and accumulation, considering, for instance, Zinc (Zn), Nickel (Ni) and Cadmium (Cd), alone or in combination. This work focused on the ecotype Monte Prinzera (MP, Italy) of N. caerulescens, autochthonous in a Natural Reserve in the Tosco-Emilian Appennins (Italy) characterized by serpentine soil; this particular ecotype is able to tolerate and accumulate Ni and Zn, a property that is shared with two other species of the same genus, i.e. Noccaea goesingense and Noccaea japonica. Molecular mechanisms responsible for Ni hypertolerance and hyperaccumulation are still unknown, although different genes seem to be involved in these processes. Particularly, several studies suggested that metal transporters, also essential for metal homeostasis, have a fundamental role in HM tolerance and accumulation. Therefore, the expression of vacuolar transporters MTP1 and NRAMP4 and of plasma membrane transporter ZNT1 was compared in N. caerulescens MP exposed to different Ni concentrations with those of Thlaspi arvense (non-accumulator) and N. caerulescens ecotype Ganges (GA, Zn/Cd hyperaccumulator). High expression of MTP1 was observed in the presence of Ni excess in N. caerulescens, GA ecotype and MP ecotype in comparison to the control species T. arvense, suggesting a possible role of MTP1 in Ni hypertolerance. In both N. caerulescens ecotypes, two forms of MTP1 were found, differing for the length of the histidine-rich loop. Co-localization experiments in Nicotiana tabacum leaves confirmed that both MTP1-long and MTP1-short are localized in tonoplast. Complementation assays with yeast mutants showed that the two MTP1 forms may possibly have different metal specificity, and other tests are going to be done in order to confirm this results. The vacuolar transporter NRAMP4 and the plasma membrane transporter ZNT1 have important roles in Fe and Zn homeostasis in plants, although several studies showed their possible involvement in Ni hyperaccumulation/hypertolerance. Upon Ni treatment, ZNT1 was strongly induced in N. caerulescens GA; in the MP ecotype, NRAMP4 showed an high and constitutive expression, whereas ZNT1 was down-regulated in response to Ni treatment. These results point to a possible role of these transporters in Ni hypertolerance/hyperaccumulation. To study their involvement in Ni hyperaccumulation/hypertolerance, plants of Arabidopsis thaliana were transformed with constructs carrying CaMV35S::ZNT1 and CaMV35S::NRAMP4 and the overexpressing lines were crossed to obtain plants overexpressing both gene
THE PROMOTER OF VACUOLAR METAL TRANSPORTER IN ARABIDOPSIS HALLERI: AN EXAMPLE OF EVOLUTION FOR METAL HYPERTOLERANCE/HYPERACCUMULATION
Full text linkIn the metal hyperaccumulator Arabidopsis halleri, the metal vacuolar transporter (VMT) isinvolved in hyperaccumulation and hypertolerance. Three VMT promoter sequences have beenidentified in the genome of A. halleri; expression analysis, conducted by GUS assay, showed thateach promoter sequence of A. halleri induced higher expression than the promoter sequence of theVMT orthologous gene in A. thaliana. Moreover, it was observed that A. halleri VTM members areexpressed in leaf trichomes, whereas VTM of A. thaliana is not present in these organs. In silicoanalysis revealed that the 5’ UTR region of VTM is highly conserved between the two Arabidopsisspecies, although a dimer of MYB-binding motifs harboured by the A. halleri promoters is mutatedin a single nucleotide in the sequence of A. thaliana. Site-specific mutagenesis of these motifs in thepromoter of A. halleri indicates that they are likely involved in trichome-specific expression. Therole of these MYB-binding motifs in the A. halleri VMT promoter was tested in A. thaliana,considering their effect in both metal tolerance and accumulation. Transgenic A. thaliana plantsexpressing VMT under the control of the native A. halleri VMT promoter are more tolerant, in termsof root length, biomass production and chlorophyll content, to high metal concentrations, than theones bearing the same construct with the mutated MYB-binding motifs. Differences in shoots andin roots were also observed regarding metal accumulation. These results underline the divergentevolution of the VMT promoter, conferring higher metal tolerance, accompanied by expression intrichomes, in the hyperaccumulator A. halleri and protection to heavy metal stress in transgenic A.thaliana. Furthermore, a possible biotechnological approach might be applied in phytoremediationor biofortification programs to modulate metal content in plants
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
TOMATO KNOTTIN TCMP-1 PLAYS A ROLE IN CADMIUM TOLERANCE
No full textKnottins or cystine-knot miniproteins (CMPs) are a class of cysteine (Cys)-rich proteins identified in eukaryotes. They are characterized by a very small size and by the presence of a C-terminal knot domain with six conserved Cys forming three disulfide bonds, conferring stability and resistance to high temperatures, proteolysis and chemical chaotropic agents. In plants, members of the knottin family participate in defense against pathogens and herbivores acting as inhibitors of proteases or amylases or displaying antimicrobial and insecticidal properties (Daly & Craik, 2011). Moreover, knottins have been found as associated with nickel (Ni) tolerance in a Ni-hyperaccumulator ecotype of Noccaea caerulescens (Halimaa et al., 2014) and with cadmium (Cd) accumulation in tobacco (Harada et al., 2010). Due to their biological activity, knottins have been considered for their potential application for therapeutical purposes: in particular, two tomato CMPs, TCMP-1 and TCMP-2, are able to inhibit angiogenesis without affecting endothelial cell proliferation and viability, both in vitro and in vivo, and to reduce cell migration (Treggiari et al., 2015). In tomato plants, TCMP-1 and TCMP-2 expression is strictly regulated and associated with flower buds before anthesis and ripe fruits, respectively. This evidence, as well as the earlier fruit setting observed when perturbing TCMP-1 expression, indicate a role in fruit development (Molesini et al., 2018)
FUNCTIONAL ANALYSIS OF THREE ARABIDOPSIS THALIANA ZIP GENES: ZIP4, ZIP6 AND ZIP9
No full textPlants have developed a variety of mechanisms to maintain the cellular concentration of essential metals within physiological limits. A complex homeostatic network controls the uptake, chelation and transport of metal ions to ensure healthy amounts of essential elements, and to avoid excess of non-essential ones (Williams and Salt, 2009). Metal transporters play a key role in metal uptake from the soil, metal homeostasis and metal transport across the cellular membranes. The ZIP (ZRT IRT1-like Proteins: Zinc-regulated transporter Iron-regulated transporter 1-like protein) family of metal transporter is involved in this complex network. Most members of the ZIP family fold into eight transmembrane domains with a variable metal-binding region, rich in histidine residues, between transmembrane domain III and IV (Guerinot, 2000). In Arabidopsis thaliana, 15 members of this family have been identified, and can be clustered in four main groups that share sequence similarity between 38 and 85%. To better understand the role of plant metal transporters, this project focuses on three ZIP proteins: AtZIP4, AtZIP6 and AtZIP9, for which limited information is available. AtZIP4 has been partially studied and the promoter analysis of this gene has been performed (Lin et al., 2016). Wintz et al. have shown that both ZIP4 and ZIP9, that share 77% similarity at the amino acid sequence, are up-regulated in roots and shoots of A. thaliana in Zn-deficiency conditions. On the contrary, AtZIP6, which doesn’t belong to any of the four groups mentioned above, does not show any modulation in expression at varying Zn or Fe concentrations. The promoter analysis for AtZIP6 and AtZIP9 has been performed on transgenic A. thaliana lines carrying the promoter sequence fused to the GUS reporter gene. The expression pattern of AtZIP6 and AtZIP9 showed different tissue localization. AtZIP9 expression is present in stomata in leaves, and localized in the root endodermis, cortex and epidermis, while AtZIP6 is expressed in the vascular tissues of both roots and shoots. Here, single AtZIP4, AtZIP6 and AtZIP9 knock-out mutant, zip4/zip9, zip4/zip6, zip6/zip9 double mutants and zip4/zip6/zip9 triple mutant obtained by crossing single homozygous mutant plants have been studied. The effect on growth of different Zn, Fe and Mn concentrations, and an ionomic analysis at different Zn concentrations, have been tested on these mutants looking for differences in the accumulation of multiple elements. To analyze the effect of the overexpression of ZIP4, ZIP6 and ZIP9, the coding sequences of the three genes have been cloned downstream of the strong promoter CaMV35S. The constructs were introduced into A. thaliana obtaining different over-expressing lines. Furthermore, the subcellular protein localization has been characterized in protoplasts transfected with genes encoding fusion proteins of ZIP4 and ZIP9 with GFP, and GFP observed at both the plasma membrane and tonoplast
Monitoraggio di movimenti verticali di strutture con un livello digitale automatizzato: prime sperimentazioni
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