Detection of Phytochrome Phosphorylation in Plants

Klement, Éva; Gyula, Péter; Viczián, András

doi:10.1007/978-1-4939-9612-4_4

Cited by 3 publications

(5 citation statements)

References 35 publications

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“…Seedlings were collected at the end of the day (EOD) and at the end of the night (EON). The sample preparation and MS analysis are based on Klement et al () and described in detail in the Methods S1.…”

Section: Methodsmentioning

confidence: 99%

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Differential phosphorylation of the N‐terminal extension regulates phytochrome B signaling

et al. 2019

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Summary Phytochrome B (phyB) is an excellent light quality and quantity sensor that can detect subtle changes in the light environment. The relative amounts of the biologically active photoreceptor (phyB Pfr) are determined by the light conditions and light independent thermal relaxation of Pfr into the inactive phyB Pr, termed thermal reversion. Little is known about the regulation of thermal reversion and how it affects plants’ light sensitivity. In this study we identified several serine/threonine residues on the N‐terminal extension (NTE) of Arabidopsis thaliana phyB that are differentially phosphorylated in response to light and temperature, and examined transgenic plants expressing nonphosphorylatable and phosphomimic phyB mutants. The NTE of phyB is essential for thermal stability of the Pfr form, and phosphorylation of S86 particularly enhances the thermal reversion rate of the phyB Pfr–Pr heterodimer in vivo. We demonstrate that S86 phosphorylation is especially critical for phyB signaling compared with phosphorylation of the more N‐terminal residues. Interestingly, S86 phosphorylation is reduced in light, paralleled by a progressive Pfr stabilization under prolonged irradiation. By investigating other phytochromes (phyD and phyE) we provide evidence that acceleration of thermal reversion by phosphorylation represents a general mechanism for attenuating phytochrome signaling.

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Section: Methodsmentioning

confidence: 99%

“…The sample preparation and MS analysis are based on Klement et al (2019) and described in detail in the Methods S1. Seedlings were collected at the end of the day (EOD) and at the end of the night (EON).…”

Section: Plant Sample Collection For Lc-ms/ms Analysis Of Phybmentioning

confidence: 99%

Differential phosphorylation of the N‐terminal extension regulates phytochrome B signaling

et al. 2019

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“…Total protein extracts or recombinant protein samples were immunopurified using ARHGAP25 antibody and protein A‐coupled magnetic beads with an average particle size of 50 nm (MACS® Technology, Bella Vista, New South Wales, Australia, Miltenyi) and digested in column with trypsin 26 . Alternatively, GST‐tagged recombinant ARHGAP25 protein phosphorylated with a cytosolic extract from primary human neutrophils was digested in a solution with trypsin, the resulting tryptic peptide mixture desalted on a C18 ZipTip (Omix C18 100 μl tips, Varian) and subjected to Fe‐NTA phosphopeptide enrichment 27 …”

Section: Methodsmentioning

confidence: 99%

“…26 Alternatively, GST-tagged recombinant ARHGAP25 protein phosphorylated with a cytosolic extract from primary human neutrophils was digested in a solution with trypsin, the resulting tryptic peptide mixture desalted on a C18 ZipTip (Omix C18 100 μl tips, Varian) and subjected to Fe-NTA phosphopeptide enrichment. 27 2.5.2 | Mass spectrometry analysis An aliquot of the tryptic digest (with or without phosphopeptide enrichment) was analyzed by LC-MS/MS using a nanoflow RP-HPLC (Waters, LC program: linear-gradient of 3%-40% B in 30 or 100 min, solvent A: 0.1% formic acid in water, solvent B: 0.1% formic acid in acetonitrile) on-line coupled to an Orbitrap-Fusion™ Lumos (ThermoFisher Scientific, Waltham, MA, USA) mass spectrometer operating in positive ion mode. Data acquisition was carried out in a data-dependent fashion, the 20 most abundant, multiply charged ions were selected from each MS survey for MS/MS analysis using HCD fragmentation (both spectra were acquired in the Orbitrap).…”

Section: Sample Preparationmentioning

confidence: 99%

A novel BRET‐Based GAP assay reveals phosphorylation‐dependent regulation of the RAC‐specific GTPase activating protein ARHGAP25

et al. 2022

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Please remember illegible or unclear comments and corrections may delay publication.Many thanks for your assistance.AUTHOR: Please note that missing content in references have been updated where we have been able to match the missing elements without ambiguity against a standard citation database, to meet the reference style requirements of the journal. It is your responsibility to check and ensure that all listed references are complete and accurate.

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“…The NdeI/BamHI fragments were than exchanged by standard molecular cloning procedures. The full length wild-type and various mutant sequences were PCR amplified by the 5 AtR1 NdeI forward and AtRop1-SalI(lm) Rev reverse pimers to insert at the NdeI and SalI sites of the pGADT7-Dest vector, and by the AtRop1 Fw (SalI + 2ncl) and AtROP1 Rev (Kpnl) primers to clone into the pWEN240 YFP [65] pollen expression vector (for primer sequences see Table S2). The CPK 17 and 34 cDNA sequences were amplified by PCR using appropriate 5 and 3 primers (Table S2) and cloned into NdeI/NotI sites of the pET28a vector for bacterial protein purification.…”

Section: Molecular Cloningmentioning

confidence: 99%

The Arabidopsis Rho of Plants GTPase ROP1 Is a Potential Calcium-Dependent Protein Kinase (CDPK) Substrate

Ménesi

Klement

Ferenc

et al. 2021

Plants

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Plant Rho-type GTPases (ROPs) are versatile molecular switches involved in a number of signal transduction pathways. Although it is well known that they are indirectly linked to protein kinases, our knowledge about their direct functional interaction with upstream or downstream protein kinases is scarce. It is reasonable to suppose that similarly to their animal counterparts, ROPs might also be regulated by phosphorylation. There is only, however, very limited experimental evidence to support this view. Here, we present the analysis of two potential phosphorylation sites of AtROP1 and two types of potential ROP-kinases. The S74 site of AtROP1 has been previously shown to potentially regulate AtROP1 activation dependent on its phosphorylation state. However, the kinase phosphorylating this evolutionarily conserved site could not be identified: we show here that despite of the appropriate phosphorylation site consensus sequences around S74 neither the selected AGC nor CPK kinases phosphorylate S74 of AtROP1 in vitro. However, we identified several phosphorylation sites other than S74 for the CPK17 and 34 kinases in AtROP1. One of these sites, S97, was tested for biological relevance. Although the mutation of S97 to alanine (which cannot be phosphorylated) or glutamic acid (which mimics phosphorylation) somewhat altered the protein interaction strength of AtROP1 in yeast cells, the mutant proteins did not modify pollen tube growth in an in vivo test.

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Detection of Phytochrome Phosphorylation in Plants

Cited by 3 publications

References 35 publications

Differential phosphorylation of the N‐terminal extension regulates phytochrome B signaling

Differential phosphorylation of the N‐terminal extension regulates phytochrome B signaling

A novel BRET‐Based GAP assay reveals phosphorylation‐dependent regulation of the RAC‐specific GTPase activating protein ARHGAP25

The Arabidopsis Rho of Plants GTPase ROP1 Is a Potential Calcium-Dependent Protein Kinase (CDPK) Substrate

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