A novel analytical method for in vivo phosphate tracking

Gu, Hongchen; Lalonde, Sylvie; Okumoto, Sakiko; Looger, Loren L.; Scharff-Poulsen, Anne Marie; Grossman, Arthur R.; Koßmann, Jens; Jakobsen, Iver; Frommer, Wolf B.

doi:10.1016/j.febslet.2006.09.048

Cited by 97 publications

(122 citation statements)

References 43 publications

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“…Recently, a system using NMR or positron emission tomography provided very useful information, but would be even more difficult to apply in practice (De Smet et al 2012). Another promising development is in vivo phosphate tracking by fluorescent reporter proteins (Gu et al 2006;Sun et al 2008;review: Frommer et al 2009), but this will also not be easily implemented in a high-throughput screening system.…”

Section: Hydroponics: Growing Plants On Water Solutionsmentioning

confidence: 99%

Improving phosphorus use efficiency in agriculture: opportunities for breeding

Wiel¹,

Linden²,

Scholten³

2015

Euphytica

195

115

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Phosphorus (P) is often an important limiting factor for crop yields, but rock phosphate as fertilizer is a non-renewable resource and expected to become scarce in the future. High P input levels in agriculture have led to environmental problems. One of the ways to tackle these issues simultaneously is improving phosphorus use efficiency (PUE) of the crops through breeding. In this review, we describe plant architectural and physiological traits important for PUE. Subsequently, we discuss efficient methods of screening for PUE traits. We address targeted cultivation methods, including solid and hydroponic systems, as well as testing methods, such as image analysis systems, and biomass and photosynthesis measurements. Genetic variation for PUE traits has been assessed in many crops, and genetics of PUE has been studied by quantitative trait loci (QTL) analyses and genome-wide association study. A number of genes involved in the plant's response to low P have been characterized. These genes include transcription factors, and genes involved in signal transduction, hormonal pathways, sugar signalling, P saving metabolic pathways, and in P scavenging, including transporters and metabolites and/or ATP-ases mobilizing P in the soil. In addition, the role of microorganisms promoting PUE of plants, particularly arbuscular mycorrhizal fungi is discussed. An overview is given of methods for selecting for optimal combinations of plant and fungal genotypes, and their genetics, incl. QTLs and genes involved. In conclusion, significant progress has been made in selecting for traits for PUE, developing systems for the difficult but highly relevant root phenotyping, and in identifying QTLs and genes involved.

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Section: Hydroponics: Growing Plants On Water Solutionsmentioning

confidence: 99%

Improving phosphorus use efficiency in agriculture: opportunities for breeding

Wiel¹,

Linden²,

Scholten³

2015

Euphytica

195

115

View full text Add to dashboard Cite

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“…Flippi 5U was purified as described previously (37). All buffers were made phosphate-free by dialysis with 1 unit of nucleoside phosphorylase (Sigma) and 2 mM inosine (Sigma).…”

Section: Expression and Purification Of His 6 -Wt Kras K104q And Acmentioning

confidence: 99%

“…Single-turnover GTP hydrolysis assays were performed as described previously (36) using the phosphate-binding protein Flippi 5U (Addgene) to detect inorganic phosphate released upon GTP hydrolysis (37). Flippi 5U was purified as described previously (37).…”

Section: Expression and Purification Of His 6 -Wt Kras K104q And Acmentioning

confidence: 99%

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation

Yin

Kistler

George

et al. 2017

Journal of Biological Chemistry

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Edited by Norma AllewellThe KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cellbased properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity.RAS proteins function as molecular switches that cycle between active GTP-and inactive GDP-bound states to regulate signal transduction pathways that modulate cellular growth control. In the unstimulated cell, RAS proteins are populated in their inactive GDP-bound state. However, in response to growth-stimulatory signals, guanine nucleotide exchange factors (GEFs) 2 co-localize and up-regulate RAS by facilitating exchange of GDP for GTP. Inactivation of RAS is achieved through GTPase-activating proteins (GAPs) that bind to GTPbound RAS and promote GTP hydrolysis (1, 2). Several point mutations in RAS have been identified that dysregulate RAS nucleotide exchange or hydrolysis, often leading to hyperactivation and promoting tumorigenesis. The most common RAS mutations identified in cancer occur at residues 12, 13, and 61 and render RAS GAP defective, thereby populating RAS in its active GTP-bound state (3). Constitutive hyperactivation of RAS promotes chronic stimulation of effector-mediated downstream pathways, causing deregulated growth and tumorigenic growth transformation.RAS contains two dynamic regions termed switch I (SWI; residues 30 -37) and switch II (SWII; residues 60 -76 with 66 -74 corresponding to helix 2 (H2)) that populate distinct conformations when the protein is bound to GDP versus GTP. Effectors and GAP proteins recognize specific conformations of the switch regions and bind with preferential affinity to the active GTP-bound state. Activated GTP-bound RAS can interact with multiple effectors (e.g. RAF kinase, RAL exchang...

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“…FRET-based sensors have been used in live plants to assess a variety of analytes, including Glc, maltose, Suc, Gln, calcium, zinc, and pH (Deuschle et al, 2006;Chaudhuri et al, 2008Chaudhuri et al, , 2011Kaper et al, 2008;Rincón-Zachary et al, 2010;Adams et al, 2012;Gjetting et al, 2012Gjetting et al, , 2013Krebs et al, 2012). Gu et al (2006) engineered a FRET-based Pi sensor named fluorescence indicator protein for inorganic phosphate (FLIPPi) that consists of a cyanobacterial inorganic phosphate binding protein (PiBP) fused to enhanced cyan fluorescent protein (eCFP) and enhanced yellow fluorescent protein (eYFP) and showed the use of one of these sensors for monitoring cytosolic Pi in cultured animal cells. In this study, we generated a series of second generation FLIPPi sensors that were modified and optimized for use in live plants.…”

mentioning

confidence: 99%

Live Imaging of Inorganic Phosphate in Plants with Cellular and Subcellular Resolution

et al. 2015

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Despite variable and often scarce supplies of inorganic phosphate (Pi) from soils, plants must distribute appropriate amounts of Pi to each cell and subcellular compartment to sustain essential metabolic activities. The ability to monitor Pi dynamics with subcellular resolution in live plants is, therefore, critical for understanding how this essential nutrient is acquired, mobilized, recycled, and stored. Fluorescence indicator protein for inorganic phosphate (FLIPPi) sensors are genetically encoded fluorescence resonance energy transfer-based sensors that have been used to monitor Pi dynamics in cultured animal cells. Here, we present a series of Pi sensors optimized for use in plants. Substitution of the enhanced yellow fluorescent protein component of a FLIPPi sensor with a circularly permuted version of Venus enhanced sensor dynamic range nearly 2.5-fold. The resulting circularly permuted FLIPPi sensor was subjected to a high-efficiency mutagenesis strategy that relied on statistical coupling analysis to identify regions of the protein likely to influence Pi affinity. A series of affinity mutants was selected with dissociation constant values of 0.08 to 11 mM, which span the range for most plant cell compartments. The sensors were expressed in Arabidopsis (Arabidopsis thaliana), and ratiometric imaging was used to monitor cytosolic Pi dynamics in root cells in response to Pi deprivation and resupply. Moreover, plastid-targeted versions of the sensors expressed in the wild type and a mutant lacking the PHOSPHATE TRANSPORT4;2 plastidic Pi transporter confirmed a physiological role for this transporter in Pi export from root plastids. These circularly permuted FLIPPi sensors, therefore, enable detailed analysis of Pi dynamics with subcellular resolution in live plants.

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A novel analytical method for in vivo phosphate tracking

Cited by 97 publications

References 43 publications

Improving phosphorus use efficiency in agriculture: opportunities for breeding

Improving phosphorus use efficiency in agriculture: opportunities for breeding

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation

Live Imaging of Inorganic Phosphate in Plants with Cellular and Subcellular Resolution

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