Faculty Opinions recommendation of An Arabidopsis thaliana knock-out mutant of the chloroplast triose phosphate/phosphate translocator is severely compromised only when starch synthesis, but not starch mobilisation is abolished.

Stress granules (SGs) are dynamic RNA–protein complexes localized in the cytoplasm that rapidly form under stress conditions and disperse when normal conditions are restored. The formation of SGs depends on the Ras-GAP SH3 domain-binding protein (G3BP). Formations, interactions and functions of plant and human SGs are strikingly similar, suggesting a conserved mechanism. However, functional analyses of plant G3BPs are missing. Thus, members of the Arabidopsis thaliana G3BP (AtG3BP) protein family were investigated in a complementation assay in a human G3BP knock-out cell line. It was shown that two out of seven AtG3BPs were able to complement the function of their human homolog. GFP-AtG3BP fusion proteins co-localized with human SG marker proteins Caprin-1 and eIF4G1 and restored SG formation in G3BP double KO cells. Interaction between AtG3BP-1 and -7 and known human G3BP interaction partners such as Caprin-1 and USP10 was also demonstrated by co-immunoprecipitation. In addition, an RG/RGG domain exchange from Arabidopsis G3BP into the human G3BP background showed the ability for complementation. In summary, our results support a conserved mechanism of SG function over the kingdoms, which will help to further elucidate the biological function of the Arabidopsis G3BP protein family.

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Ca2+-Dependent Protein Kinase 6 Enhances KAT2 Shaker Channel Activity in Arabidopsis thaliana

International Journal of Molecular Sciences ◽

10.3390/ijms22041596 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1596

Author(s):

Elsa Ronzier ◽

Claire Corratgé-Faillie ◽

Frédéric Sanchez ◽

Christian Brière ◽

Tou Cheu Xiong

Keyword(s):

Arabidopsis Thaliana ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Physical Interaction ◽

Fluorescence Lifetime Imaging ◽

Dependent Manner ◽

In Planta ◽

Knock Out ◽

Calcium Dependent ◽

Dependent Protein

Post-translational regulations of Shaker-like voltage-gated K+ channels were reported to be essential for rapid responses to environmental stresses in plants. In particular, it has been shown that calcium-dependent protein kinases (CPKs) regulate Shaker channels in plants. Here, the focus was on KAT2, a Shaker channel cloned in the model plant Arabidopsis thaliana, where is it expressed namely in the vascular tissues of leaves. After co-expression of KAT2 with AtCPK6 in Xenopuslaevis oocytes, voltage-clamp recordings demonstrated that AtCPK6 stimulates the activity of KAT2 in a calcium-dependent manner. A physical interaction between these two proteins has also been shown by Förster resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM). Peptide array assays support that AtCPK6 phosphorylates KAT2 at several positions, also in a calcium-dependent manner. Finally, K+ fluorescence imaging in planta suggests that K+ distribution is impaired in kat2 knock-out mutant leaves. We propose that the AtCPK6/KAT2 couple plays a role in the homeostasis of K+ distribution in leaves.

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Free energy landscape for the entire transport cycle of triose-phosphate/phosphate translocator

10.1101/206912 ◽

2017 ◽

Author(s):

Mizuki Takemoto ◽

Yongchan Lee ◽

Ryuichiro Ishitani ◽

Osamu Nureki

Keyword(s):

Free Energy ◽

Conformational Changes ◽

Conformational Transition ◽

Substrate Binding ◽

Energy Landscape ◽

Umbrella Sampling ◽

Free Energy Landscape ◽

Coupling Mechanism ◽

Triose Phosphate ◽

Phosphate Translocator

AbstractSecondary active transporters translocate their substrates using the electrochemical potentials of other chemicals, undergoing large-scale conformational changes. Despite extensive structural studies, the atomic details of the transport mechanism still remain elusive. Here we performed a series of all-atom molecular dynamics simulations of the triose-phosphate/phosphate translocator (TPT), which exports organic phosphates in the chloroplast stroma in strict counter exchange with inorganic phosphate (Pi). Biased sampling methods, including string method and umbrella sampling, successfully reproduced the conformational changes between the inward– and outward-facing states, along with the substrate binding. The free energy landscape of this entire TPT transition pathway demonstrated the alternating access and substrate translocation mechanisms, which revealed Pi is relayed by positively charged residues along the transition pathway. Furthermore, the conserved Glu207 functions as a “molecular switch”, linking the local substrate binding and the global conformational transition. Our results provide atomic-detailed insights into the energy coupling mechanism of antiporter.

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Triple null mutations in starch synthase SSIIa gene homoeologs lead to high amylose and resistant starch in hexaploid wheat

10.21203/rs.3.rs-36596/v2 ◽

2020 ◽

Author(s):

Adam Schoen ◽

Anupama Joshi ◽

Vijay K Tiwari ◽

Bikram S. Gill ◽

Nidhi Rawat

Keyword(s):

Resistant Starch ◽

Amylose Content ◽

Starch Content ◽

Wheat Variety ◽

Starch Synthesis ◽

Starch Synthase ◽

Single Individual ◽

Individual Genome ◽

Mutant Genotype ◽

Knock Out

Abstract Background: Lack of nutritionally appropriate foods is one of the leading causes of obesity in the US and worldwide. Wheat (Triticum aestivum) provides 20% of the calories consumed daily across the globe. The nutrients in the wheat grain come primarily from the starch composed of amylose and amylopectin. Resistant starch content, which is known to have significant human health benefits, can be increased by modifying starch synthesis pathways. Starch synthase enzyme SSIIa, also known as starch granule protein isoform-1 (SGP-1), is integral to the biosynthesis of the branched and readily digestible glucose polymer amylopectin. The goal of this work was to develop a triple null mutant genotype for SSIIa locus in the elite hard red winter wheat variety ‘Jagger’ and evaluate the effect of the knock-out mutations on resistant starch content in grains with respect to wild type. Results: Knock-out mutations in SSIIa in the three genomes of wheat variety ‘Jagger’ were identified using TILLING. Subsequently, these loss-of function mutations on A, B, and D genomes were combined by crossing to generate a triple knockout mutant genotype Jag-ssiia-∆ABD. The Jag-ssiia-∆ABD had an amylose content of 35.70% compared to 31.15% in Jagger, leading to ~118% increase in resistant starch in the Jag-ssiia-∆ABD genotype of Jagger wheat. The single individual genome mutations also had various effects on starch composition. Conclusions: Our full null Jag-ssiia-∆ABD mutant showed a significant increase in RS without the shriveled grain phenotype seen in other ssiia knockouts in elite wheat cultivars. Moreover, this study shows the potential for developing nutritionally improved foods in a non-GM approach. Since all the mutants have been developed in an elite wheat cultivar, their adoption in production and supply will be feasible in future.

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Molecular Cloning and Spatial Expression of an ApL1 cDNA for the Large Subunit of ADP-Glucose Pyrophosphorylase from Arabidopsis thaliana

Zeitschrift für Naturforschung C ◽

10.1515/znc-1999-5-610 ◽

1999 ◽

Vol 54 (5-6) ◽

pp. 353-358 ◽

Cited By ~ 3

Author(s):

Leszek A. Kleszkowski ◽

Lubomir N. Sokolov ◽

Cheng Luo ◽

Per Villand

Keyword(s):

Arabidopsis Thaliana ◽

Large Subunit ◽

Critical Role ◽

Starch Synthesis ◽

Transit Peptide ◽

Homologous Proteins ◽

Peptide Cleavage ◽

Reading Frame ◽

Calculated Molecular Weight ◽

Adp Glucose Pyrophosphorylase

Abstract A cDNA, A p L 1a , corresponding to a homologue of the large subunit of ADP-glucose pyrophosphorylase (AG Pase), has been isolated/characterised by screening a cDNA library prepared from leaves of Arabidopsis thaliana, followed by rapid amplification of cDNA 3′-ends (3′-RACE). Within the 1685 nucleotide-long sequence (excluding polyA tail), an open reading frame encodes a protein of 522 amino acids (aa), with a calculated molecular weight of 57.7 kDa. The derived aa sequence does not contain any discernible transit peptide cleavage site motif, similarly to two other recently sequenced full-length Arabidopsis homo-logues for AGPase, and shows ca. 58–78 % identity to homologous proteins from other plants/tissues. The corresponding gene was found (rosette and stem leaves, stems, flowers and fruits), consistent with its critical role in starch synthesis in

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Photosynthetic efficiency and mesophyll conductance are unaffected in Arabidopsis thaliana aquaporin knock-out lines

Journal of Experimental Botany ◽

10.1093/jxb/erz442 ◽

2019 ◽

Cited By ~ 1

Author(s):

Johannes Kromdijk ◽

Katarzyna Głowacka ◽

Stephen P Long

Keyword(s):

Arabidopsis Thaliana ◽

Photosynthetic Efficiency ◽

Mesophyll Conductance ◽

Knock Out ◽

Extensive Evaluation

Extensive evaluation of gm and photosynthetic efficiency of Arabidopsis aquaporin knock-out lines using a range of methods and measurement conditions failed to establish significant deviations from the Col-0 background.

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