Faculty Opinions recommendation of ATP compartmentation in plastids and cytosol of Arabidopsis thaliana revealed by fluorescent protein sensing.

2018 ◽  
Author(s):  
Xinguang Zhu
Keyword(s):  
Arabidopsis Thaliana ◽  
Fluorescent Protein ◽  
Protein Sensing

scholarly journals ATP compartmentation in plastids and cytosol ofArabidopsis thalianarevealed by fluorescent protein sensing

2018 ◽  
Vol 115 (45) ◽  
pp. E10778-E10787 ◽  
Author(s):  
Chia Pao Voon ◽  
Xiaoqian Guan ◽  
Yuzhe Sun ◽  
Abira Sahu ◽  
May Ngor Chan ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Fluorescent Protein ◽  
Higher Plants ◽  
Electron Flow ◽  
Cyclic Electron Flow ◽  
Linear Electron ◽  
Protein Sensing ◽  
Atp Production ◽  
Respiratory Inhibitors ◽  
Sensor Protein

Matching ATP:NADPH provision and consumption in the chloroplast is a prerequisite for efficient photosynthesis. In terms of ATP:NADPH ratio, the amount of ATP generated from the linear electron flow does not meet the demand of the Calvin–Benson–Bassham (CBB) cycle. Several different mechanisms to increase ATP availability have evolved, including cyclic electron flow in higher plants and the direct import of mitochondrial-derived ATP in diatoms. By imaging a fluorescent ATP sensor protein expressed in livingArabidopsis thalianaseedlings, we found that MgATP2−concentrations were lower in the stroma of mature chloroplasts than in the cytosol, and exogenous ATP was able to enter chloroplasts isolated from 4- and 5-day-old seedlings, but not chloroplasts isolated from 10- or 20-day-old photosynthetic tissues. This observation is in line with the previous finding that the expression of chloroplast nucleotide transporters (NTTs) inArabidopsismesophyll is limited to very young seedlings. Employing a combination of photosynthetic and respiratory inhibitors with compartment-specific imaging of ATP, we corroborate the dependency of stromal ATP production on mitochondrial dissipation of photosynthetic reductant. Our data suggest that, during illumination, the provision and consumption of ATP:NADPH in chloroplasts can be balanced by exporting excess reductants rather than importing ATP from the cytosol.

scholarly journals Colonization of Arabidopsis thaliana with Salmonella enterica and Enterohemorrhagic Escherichia coli O157:H7 and Competition by Enterobacter asburiae

2003 ◽  
Vol 69 (8) ◽  
pp. 4915-4926 ◽  
Author(s):  
Michael B. Cooley ◽  
William G. Miller ◽  
Robert E. Mandrell
Keyword(s):  
Escherichia Coli ◽  
Arabidopsis Thaliana ◽  
Salmonella Enterica ◽  
Fluorescent Protein ◽  
Enterohemorrhagic Escherichia Coli ◽  
Plant Responses ◽  
Human Pathogens ◽  
E Coli ◽  
Green Fluorescent ◽  
Enterobacter Asburiae

ABSTRACT Enteric pathogens, such as Salmonella enterica and Escherichia coli O157:H7, have been shown to contaminate fresh produce. Under appropriate conditions, these bacteria will grow on and invade the plant tissue. We have developed Arabidopsis thaliana (thale cress) as a model system with the intention of studying plant responses to human pathogens. Under sterile conditions and at 100% humidity, S. enterica serovar Newport and E. coli O157:H7 grew to 109 CFU g−1 on A. thaliana roots and to 2 × 107 CFU g−1 on shoots. Furthermore, root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition. Inoculation with green fluorescent protein-labeled S. enterica and E. coli O157:H7 showed invasion of the roots at lateral root junctions. Movement was eliminated and invasion decreased when nonmotile mutants of S. enterica were used. Survival of S. enterica serovar Newport and E. coli O157:H7 on soil-grown plants declined as the plants matured, but both pathogens were detectable for at least 21 days. Survival of the pathogen was reduced in unautoclaved soil and amended soil, suggesting competition from indigenous epiphytes from the soil. Enterobacter asburiae was isolated from soil-grown A. thaliana and shown to be effective at suppressing epiphytic growth of both pathogens under gnotobiotic conditions. Seed and chaff harvested from contaminated plants were occasionally contaminated. The rate of recovery of S. enterica and E. coli O157:H7 from seed varied from undetectable to 19% of the seed pools tested, depending on the method of inoculation. Seed contamination by these pathogens was undetectable in the presence of the competitor, Enterobacter asburiae. Sampling of 74 pools of chaff indicated a strong correlation between contamination of the chaff and seed (P = 0.025). This suggested that contamination of the seed occurred directly from contaminated chaff or by invasion of the flower or silique. However, contaminated seeds were not sanitized by extensive washing and chlorine treatment, indicating that some of the bacteria reside in a protected niche on the seed surface or under the seed coat.

scholarly journals Use of Yellow Fluorescent Protein Fluorescence to Track OPR3 Expression in Arabidopsis Thaliana Responses to Insect Herbivory

2019 ◽  
Vol 10 ◽  
Author(s):  
Mélanie J.A. Body ◽  
Dhruveesh F. Dave ◽  
Clayton M. Coffman ◽  
Taylor Y. Paret ◽  
Abraham J. Koo ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Insect Herbivory ◽  
Protein Fluorescence

scholarly journals Nuclear Dynamics in Arabidopsis thaliana

2000 ◽  
Vol 11 (8) ◽  
pp. 2733-2741 ◽  
Author(s):  
Eva Chytilova ◽  
Jiri Macas ◽  
Elwira Sliwinska ◽  
Susanne M. Rafelski ◽  
Georgina M. Lambert ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Higher Plants ◽  
Chimeric Protein ◽  
Time Lapse ◽  
Confocal Laser ◽  
Long Distance ◽  
General Applicability ◽  
Living Plant

The nucleus is a definitive feature of eukaryotic cells, comprising twin bilamellar membranes, the inner and outer nuclear membranes, which separate the nucleoplasmic and cytoplasmic compartments. Nuclear pores, complex macromolecular assemblies that connect the two membranes, mediate communication between these compartments. To explore the morphology, topology, and dynamics of nuclei within living plant cells, we have developed a novel method of confocal laser scanning fluorescence microscopy under time-lapse conditions. This is used for the examination of the transgenic expression in Arabidopsis thaliana of a chimeric protein, comprising the GFP (Green-Fluorescent Protein of Aequorea victoria) translationally fused to an effective nuclear localization signal (NLS) and to β-glucuronidase (GUS) from E. coli. This large protein is targeted to the nucleus and accumulates exclusively within the nucleoplasm.  This article provides online access to movies that illustrate the remarkable and unusual properties displayed by the nuclei, including polymorphic shape changes and rapid, long-distance, intracellular movement. Movement is mediated by actin but not by tubulin; it therefore appears distinct from mechanisms of nuclear positioning and migration that have been reported for eukaryotes. The GFP-based assay is simple and of general applicability. It will be interesting to establish whether the novel type of dynamic behavior reported here, for higher plants, is observed in other eukaryotic organisms.

scholarly journals Protein hydrolysate-induced cholecystokinin secretion from enteroendocrine cells is indirectly mediated by the intestinal oligopeptide transporter PepT1

2011 ◽  
Vol 300 (5) ◽  
pp. G895-G902 ◽  
Author(s):  
Alice P. Liou ◽  
Diana I. Chavez ◽  
Elvis Espero ◽  
Shuzhen Hao ◽  
Stephen A. Wank ◽  
...  
Keyword(s):  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Protein Hydrolysate ◽  
Cell Activity ◽  
Oligopeptide Transporter ◽  
Enteroendocrine Cell ◽  
Protein Sensing ◽  
Gastric Motor Function ◽  
Green Fluorescent ◽  
I Cells

Dietary protein is a major stimulant for cholecystokinin (CCK) secretion by the intestinal I cell, however, the mechanism by which protein is detected is unknown. Indirect functional evidence suggests that PepT1 may play a role in CCK-mediated changes in gastric motor function. However, it is unclear whether this oligopeptide transporter directly or indirectly activates the I cell. Using both the CCK-expressing enteroendocrine STC-1 cell and acutely isolated native I cells from CCK-enhanced green fluorescent protein (eGFP) mice, we aimed to determine whether PepT1 directly activates the enteroendocrine cell to elicit CCK secretion in response to oligopeptides. Both STC-1 cells and isolated CCK-eGFP cells expressed PepT1 transcripts. STC-1 cells were activated, as measured by ERK1/2 phosphorylation, by both peptone and the PepT1 substrate Cefaclor; however, the PepT1 inhibitor 4-aminomethyl benzoic acid (AMBA) had no effect on STC-1 cell activity. The PepT1-transportable substrate glycyl-sarcosine dose-dependently decreased gastric motility in anesthetized rats but had no affect on activation of STC-1 cells or on CCK secretion by CCK-eGFP cells. CCK secretion was significantly increased in response to peptone but not to Cefaclor, cephalexin, or Phe-Ala in CCK-eGFP cells. Taken together, the data suggest that PepT1 does not directly mediate CCK secretion in response to PepT1 specific substrates. PepT1, instead, may have an indirect role in protein sensing in the intestine.

scholarly journals Water-soluble chlorophyll protein is involved in herbivore resistance activation during greening of Arabidopsis thaliana

2015 ◽  
Vol 112 (23) ◽  
pp. 7303-7308 ◽  
Author(s):  
Edouard Boex-Fontvieille ◽  
Sachin Rustgi ◽  
Diter von Wettstein ◽  
Steffen Reinbothe ◽  
Christiane Reinbothe
Keyword(s):  
Arabidopsis Thaliana ◽  
Stress Responses ◽  
Fluorescent Protein ◽  
Seedling Emergence ◽  
Water Soluble ◽  
Herbivore Resistance ◽  
Porcellio Scaber ◽  
Apical Hook ◽  
Etiolated Plants

Water-soluble chlorophyll proteins (WSCPs) constitute a small family of unusual chlorophyll (Chl)-binding proteins that possess a Kunitz-type protease inhibitor domain. In Arabidopsis thaliana, a WSCP has been identified, named AtWSCP, that forms complexes with Chl and the Chl precursor chlorophyllide (Chlide) in vitro. AtWSCP exhibits a quite unexpected expression pattern for a Chl binding protein and accumulated to high levels in the apical hook of etiolated plants. AtWSCP expression was negatively light-regulated. Transgenic expression of AtWSCP fused to green fluorescent protein (GFP) revealed that AtWSCP is localized to cell walls/apoplastic spaces. Biochemical assays identified AtWSCP as interacting with RD21 (RESPONSIVE TO DESICCATION 21), a granulin domain-containing cysteine protease implicated in stress responses and defense. Reconstitution experiments showed tight interactions between RD21 and WSCP that were relieved upon Chlide binding. Laboratory feeding experiments with two herbivorous isopod crustaceans, Porcellio scaber (woodlouse) and Armadillidium vulgare (pillbug), identified the apical hook as Achilles’ heel of etiolated plants and that this was protected by RD21 during greening. Because Chlide is formed in the apical hook during seedling emergence from the soil, our data suggest an unprecedented mechanism of herbivore resistance activation that is triggered by light and involves AtWSCP.

scholarly journals Interdependence of the Peroxisome-targeting Receptors in Arabidopsis thaliana: PEX7 Facilitates PEX5 Accumulation and Import of PTS1 Cargo into Peroxisomes

2010 ◽  
Vol 21 (7) ◽  
pp. 1263-1271 ◽  
Author(s):  
Naxhiely Martínez Ramón ◽  
Bonnie Bartel
Keyword(s):  
Arabidopsis Thaliana ◽  
Green Fluorescent Protein ◽  
Protein Import ◽  
Fluorescent Protein ◽  
Protein Accumulation ◽  
Animal Development ◽  
Metabolic Reactions ◽  
Targeting Signals ◽  
Green Fluorescent ◽  
Peroxisome Targeting Signals

Peroxisomes compartmentalize certain metabolic reactions critical to plant and animal development. The import of proteins from the cytosol into the organelle matrix depends on more than a dozen peroxin (PEX) proteins, with PEX5 and PEX7 serving as receptors that shuttle proteins bearing one of two peroxisome-targeting signals (PTSs) into the organelle. PEX5 is the PTS1 receptor; PEX7 is the PTS2 receptor. In plants and mammals, PEX7 depends on PEX5 binding to deliver PTS2 cargo into the peroxisome. In this study, we characterized a pex7 missense mutation, pex7-2, that disrupts both PEX7 cargo binding and PEX7-PEX5 interactions in yeast, as well as PEX7 protein accumulation in plants. We examined localization of peroxisomally targeted green fluorescent protein derivatives in light-grown pex7 mutants and observed not only the expected defects in PTS2 protein import but also defects in PTS1 import. These PTS1 import defects were accompanied by reduced PEX5 accumulation in light-grown pex7 seedlings. Our data suggest that PEX5 and PTS1 import depend on the PTS2 receptor PEX7 in Arabidopsis and that the environment may influence this dependence. These data advance our understanding of the biogenesis of these essential organelles and provide a possible rationale for the retention of the PTS2 pathway in some organisms.

A plastidial pantoate transporter with a potential role in pantothenate synthesis

2018 ◽  
Vol 475 (4) ◽  
pp. 813-825 ◽  
Author(s):  
Lili Huang ◽  
Michal Pyc ◽  
Saleh Alseekh ◽  
Donald R. McCarty ◽  
Valérie de Crécy-Lagard ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Potential Role ◽  
Fluorescent Protein ◽  
Comparative Genomic ◽  
Reduction Step ◽  
Knockout Mutants ◽  
Vitamin B5 ◽  
Green Fluorescent ◽  
Inner Envelope ◽  
Pantothenate Biosynthesis

The pantothenate (vitamin B5) synthesis pathway in plants is not fully defined because the subcellular site of its ketopantoate → pantoate reduction step is unclear. However, the pathway is known to be split between cytosol, mitochondria, and potentially plastids, and inferred to involve mitochondrial or plastidial transport of ketopantoate or pantoate. No proteins that mediate these transport steps have been identified. Comparative genomic and transcriptomic analyses identified Arabidopsis thaliana BASS1 (At1g78560) and its maize (Zea mays) ortholog as candidates for such a transport role. BASS1 proteins belong to the bile acid : sodium symporter family and share similarity with the Salmonella enterica PanS pantoate/ketopantoate transporter and with predicted bacterial transporters whose genes cluster on the chromosome with pantothenate synthesis genes. Furthermore, Arabidopsis BASS1 is co-expressed with genes related to metabolism of coenzyme A, the cofactor derived from pantothenate. Expression of Arabidopsis or maize BASS1 promoted the growth of a S. enterica panB panS mutant strain when pantoate, but not ketopantoate, was supplied, and increased the rate of [3H]pantoate uptake. Subcellular localization of green fluorescent protein fusions in Nicotiana tabacum BY-2 cells demonstrated that Arabidopsis BASS1 is targeted solely to the plastid inner envelope. Two independent Arabidopsis BASS1 knockout mutants accumulated pantoate ∼10-fold in leaves and had smaller seeds. Taken together, these data indicate that BASS1 is a physiologically significant plastidial pantoate transporter and that the pantoate reduction step in pantothenate biosynthesis could be at least partly localized in plastids.

scholarly journals Cloning and Expression Analysis of the BocMBF1c Gene Involved in Heat Tolerance in Chinese Kale

2019 ◽  
Vol 20 (22) ◽  
pp. 5637 ◽  
Author(s):  
Lifang Zou ◽  
Bingwei Yu ◽  
Xing-Liang Ma ◽  
Bihao Cao ◽  
Guoju Chen ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Heat Stress ◽  
Green Fluorescent Protein ◽  
Stress Response ◽  
Nicotiana Benthamiana ◽  
Thermal Tolerance ◽  
Fluorescent Protein ◽  
Leaf Tissue ◽  
Chinese Kale ◽  
Green Fluorescent

Chinese kale (Brassica oleracea var. chinensis Lei) is an important vegetable crop in South China, valued for its nutritional content and taste. Nonetheless, the thermal tolerance of Chinese kale still needs improvement. Molecular characterization of Chinese kale’s heat stress response could provide a timely solution for developing a thermally tolerant Chinese kale variety. Here, we report the cloning of multi-protein bridging factor (MBF) 1c from Chinese kale (BocMBF1c), an ortholog to the key heat stress responsive gene MBF1c. Phylogenetic analysis showed that BocMBF1c is highly similar to the stress-response transcriptional coactivator MBF1c from Arabidopsis thaliana (AtMBF1c), and the BocMBF1c coding region conserves MBF1 and helix-turn-helix (HTH) domains. Moreover, the promoter region of BocMBF1c contains three heat shock elements (HSEs) and, thus, is highly responsive to heat treatment. This was verified in Nicotiana benthamiana leaf tissue using a green fluorescent protein (GFP) reporter. In addition, the expression of BocMBF1c can be induced by various abiotic stresses in Chinese kale which indicates the involvement of stress responses. The BocMBF1c-eGFP (enhanced green fluorescent protein) chimeric protein quickly translocated into the nucleus under high temperature treatment in Nicotiana benthamiana leaf tissue. Overexpression of BocMBF1c in Arabidopsis thaliana results in a larger size and enhanced thermal tolerance compared with the wild type. Our results provide valuable insight for the role of BocMBF1c during heat stress in Chinese kale.

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