scholarly journals Biochemical and functional characterization of a mitochondrial citrate carrier in Arabidopsis thaliana

2020 ◽  
Vol 477 (9) ◽  
pp. 1759-1777 ◽  
Author(s):  
Danielle S. Brito ◽  
Gennaro Agrimi ◽  
Lennart Charton ◽  
Dominik Brilhaus ◽  
Maria Gabriella Bitetto ◽  
...  
Keyword(s):  
Gene Expression Analysis ◽  
Nitrogen Assimilation ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Functional Characterization ◽  
Radicle Growth ◽  
Citrate Carrier ◽  
Green Fluorescent ◽  
Lower Expression

A homolog of the mitochondrial succinate/fumarate carrier from yeast (Sfc1p) has been found in the Arabidopsis genome, named AtSFC1. The AtSFC1 gene was expressed in Escherichia coli, and the gene product was purified and reconstituted in liposomes. Its transport properties and kinetic parameters demonstrated that AtSFC1 transports citrate, isocitrate and aconitate and, to a lesser extent, succinate and fumarate. This carrier catalyzes a fast counter-exchange transport as well as a low uniport of substrates, exhibits a higher transport affinity for tricarboxylates than dicarboxylates, and is inhibited by pyridoxal 5′-phosphate and other inhibitors of mitochondrial carriers to various degrees. Gene expression analysis indicated that the AtSFC1 transcript is mainly present in heterotrophic tissues, and fusion with a green-fluorescent protein localized AtSFC1 to the mitochondria. Furthermore, 35S-AtSFC1 antisense lines were generated and characterized at metabolic and physiological levels in different organs and at various developmental stages. Lower expression of AtSFC1 reduced seed germination and impaired radicle growth, a phenotype that was related to reduced respiration rate. These findings demonstrate that AtSFC1 might be involved in storage oil mobilization at the early stages of seedling growth and in nitrogen assimilation in root tissue by catalyzing citrate/isocitrate or citrate/succinate exchanges.

scholarly journals RNAi-Mediated Knockdown of Imaginal Disc Growth Factors (IDGFs) Genes Causes Developmental Malformation and Mortality in Melon Fly, Zeugodacus cucurbitae

2021 ◽  
Vol 12 ◽  
Author(s):  
Shakil Ahmad ◽  
Momana Jamil ◽  
Muhammad Fahim ◽  
Shujing Zhang ◽  
Farman Ullah ◽  
...  
Keyword(s):  
Growth Factors ◽  
Imaginal Disc ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Small Body ◽  
Functional Characterization ◽  
Oral Feeding ◽  
Developmental Defects ◽  
Melon Fly ◽  
Green Fluorescent

This study reports the first successful use of oral feeding dsRNA technique for functional characterization of imaginal disc growth factors (IDGFs) genes (IDGF1, IDGF3_1, IDGF4_0, IDGF4_1, and IDGF6) in melon fly Zeugodacus cucurbitae. Phylogenetic and domain analysis indicates that these genes had high similarity with other Tephritidae fruit flies homolog and contain only one conserved domain among these five genes, which is glyco-18 domain (glyco-hydro-18 domain). Gene expression analysis at different developmental stages revealed that these genes were expressed at larval, pupal, and adult stages. To understand their role in different developmental stages, larvae were fed dsRNA-corresponding to each of the five IDGFs, in an artificial diet. RNAi-mediated knockdown of IDGF1 shows no phenotypic effects but caused mortality (10.4%), while IDGF4_0 caused malformed pharate at the adult stage where insects failed to shed their old cuticle and remained attached with their body, highest mortality (49.2%) was recorded compared to dsRNA-green fluorescent protein (GFP) or DEPC. Silencing of IDGF3_1 and IDGF4_1 cause lethal phenotype in larvae, (17.2%) and (40%) mortality was indexed in Z. cucurbitae. IDGF6 was mainly expressed in pupae and adult stages, and its silencing caused a malformation in adult wings. The developmental defects such as malformation in wings, larval–larval lethality, pupal–adult malformation, and small body size show that IDGFs are key developmental genes in the melon fly. Our results provide a baseline for the melon fly management and understanding of IDGFs specific functions in Z. cucurbitae.

scholarly journals Functional Characterization of a Drought-Responsive Invertase Inhibitor from Maize (Zea mays L.)

2019 ◽  
Vol 20 (17) ◽  
pp. 4081 ◽  
Author(s):  
Lin Chen ◽  
Xiaohong Liu ◽  
Xiaojia Huang ◽  
Wei Luo ◽  
Yuming Long ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Protein Fusion ◽  
Drought Treatment ◽  
Vacuolar Invertase ◽  
Maize Leaves ◽  
Green Fluorescent ◽  
Proteinaceous Inhibitor

Invertases (INVs) play essential roles in plant growth in response to environmental cues. Previous work showed that plant invertases can be post-translationally regulated by small protein inhibitors (INVINHs). Here, this study characterizes a proteinaceous inhibitor of INVs in maize (Zm-INVINH4). A functional analysis of the recombinant Zm-INVINH4 protein revealed that it inhibited both cell wall and vacuolar invertase activities from maize leaves. A Zm-INVINH4::green fluorescent protein fusion experiment indicated that this protein localized in the apoplast. Transcript analysis showed that Zm-INVINH4 is specifically expressed in maize sink tissues, such as the base part of the leaves and young kernels. Moreover, drought stress perturbation significantly induced Zm-INVINH4 expression, which was accompanied with a decrease of cell wall invertase (CWI) activities and an increase of sucrose accumulation in both base parts of the leaves 2 to 7 days after pollinated kernels. In summary, the results support the hypothesis that INV-related sink growth in response to drought treatment is (partially) caused by a silencing of INV activity via drought-induced induction of Zm-INVINH4 protein.

Four novel splice variants of sulfonylurea receptor 1

2002 ◽  
Vol 283 (2) ◽  
pp. C587-C598 ◽  
Author(s):  
Annette Hambrock ◽  
Regina Preisig-Müller ◽  
Ulrich Russ ◽  
Anke Piehl ◽  
Peter J. Hanley ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Splice Variants ◽  
Functional Characterization ◽  
Pcr Analysis ◽  
Sulfonylurea Receptor ◽  
Transmembrane Segments ◽  
Sulfonylurea Receptor 1 ◽  
Green Fluorescent ◽  
Splice Forms

ATP-sensitive K+ (KATP) channels are composed of pore-forming Kir6.x subunits and regulatory sulfonylurea receptor (SUR) subunits. SURs are ATP-binding cassette proteins with two nucleotide-binding folds (NBFs) and binding sites for sulfonylureas, like glibenclamide, and for channel openers. Here we report the identification and functional characterization of four novel splice forms of guinea pig SUR1. Three splice forms originate from alternative splicing of the region coding for NBF1 and lack exons 17 (SUR1Δ17), 19 (SUR1Δ19), or both (SUR1Δ17Δ19). The fourth (SUR1C) is a COOH-terminal SUR1-fragment formed by exons 31–39 containing the last two transmembrane segments and the COOH terminus of SUR1. RT-PCR analysis showed that these splice forms are expressed in several tissues with strong expression of SUR1C in cardiomyocytes. Confocal microscopy using enhanced green fluorescent protein-tagged SUR or Kir6.x did not provide any evidence for involvement of these splice forms in the mitochondrial KATP channel. Only SUR1 and SUR1Δ17 showed high-affinity binding of glibenclamide ( K d≈ 2 nM in the presence of 1 mM ATP) and formed functional KATPchannels upon coexpression with Kir6.2.

scholarly journals Green fluorescent protein-based assays for high-throughput functional characterization and ligand-binding studies of biotin protein ligase

2016 ◽  
Vol 8 (2) ◽  
pp. 418-424 ◽  
Author(s):  
Samuel P. Askin ◽  
Thomas E. H. Bond ◽  
Patrick M. Schaeffer
Keyword(s):  
Green Fluorescent Protein ◽  
Ligand Binding ◽  
High Throughput ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Binding Studies ◽  
Biotin Protein Ligase ◽  
Green Fluorescent

Rapid functional characterization of GFP-tagged biotin protein ligase (BirA-GFP) with a high-throughput DSF-GTP assay.

scholarly journals Functional characterization of green fluorescent protein-profilin fusion proteins

2000 ◽  
Vol 267 (16) ◽  
pp. 5247-5256 ◽  
Author(s):  
Nina Wittenmayer ◽  
Martin Rothkegel ◽  
Brigitte M. Jockusch ◽  
Kathrin Schlüter
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Green Fluorescent

scholarly journals Functional characterization of the Beet necrotic yellow vein virus RNA-5-encoded p26 protein: evidence for structural pathogenicity determinants

2005 ◽  
Vol 86 (7) ◽  
pp. 2115-2125 ◽  
Author(s):  
Didier Link ◽  
Laure Schmidlin ◽  
Audrey Schirmer ◽  
Elodie Klein ◽  
Mathieu Erhardt ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Chenopodium Quinoa ◽  
Yellow Vein ◽  
Virus Rna ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Necrotic Symptoms

A Beet necrotic yellow vein virus isolate containing a fifth RNA is present in the Pithiviers area of France. A full-length cDNA clone of RNA-5 was obtained and placed under the control of a T7-RNA-pol promoter that allowed the production of infectious transcripts. ‘Pithiviers' isolate-specific necrotic symptoms were obtained on Chenopodium quinoa when RNA-5-encoded p26 was expressed either from RNA-5 or from an RNA-3-derived replicon. By using haemagglutinin- and green fluorescent protein-tagged constructs, virally expressed p26-fusion proteins induced the same necrotic local lesions on host plants and were localized mainly in the nucleus of infected cells. Deletion mutagenesis permitted identification of two domains, responsible respectively for nuclear export and cytoplasmic retention of the p26 mutated proteins. By using a yeast two-hybrid system, Gal4DB–p26 protein self-activated transcription of the His3 reporter gene. The p26 transcription-activation domain was located within its first 55 aa and has been studied by alanine scanning. Resulting p26 mutants were tested for their capability to induce necrotic symptoms and to localize in the nuclear compartment.

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