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Proteome-wide cellular thermal shift assay reveals novel crosstalk between brassinosteroid and auxin signaling

ABSTRACT: We demonstrated that bikinin changed the thermal stability of some of its target proteins as well as several known or putative interacting proteins of Arabidopsis GSK3s. By combining the thermal- and the phospho-proteomes of bikinin we uncovered the auxin carrier PIN-FORMED1 (PIN1) as a novel substrate of the Arabidopsis GSK3s. We showed that inhibition the kinase activity of GSK3s by brassinosteroids or the specific chemical inhibitor bikinin led to the loss of PIN1 polarity, revealing a novel crosstalk between brassinosteroid and auxin signaling. Hence, our study demonstrates the applicability of CETSA MS in plant intact cells for identification of small-molecule targets and for discovery of novel protein-protein interactions.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Xiangyu Xu

LAB HEAD: Ive DeSmet

PROVIDER: PXD029936 | Pride | 2022-03-08

REPOSITORIES: Pride

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			Action	DRS
	E22632_C2_u_2p_PSB-254_1.raw	Raw
	E22634_C2_u_2p_PSB-254_2.raw	Raw
	E22636_C2_u_2p_PSB-254_3.raw	Raw
	E22640_C2_u_2p_PSB-254_5.raw	Raw
	E22642_C2_u_2p_PSB-254_6.raw	Raw

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Similar Datasets

Proteome-wide cellular thermal shift assay reveals novel crosstalk between brassinosteroid and auxin signaling

Project description:Despite the growing interest in using chemical genetics in plant research, small-molecule target identification remains a major challenge. The cellular thermal shift assay coupled with high-resolution massspectrometry (CETSA MS) that monitors changes in the thermal stability of proteins caused by their interactions with small molecules, other proteins, or post-translational modifications allows the identification of drug targets, or the study of protein-metabolite and protein-protein interactions mainly in mammalian cells. To showcase the applicability of this method in plants, we applied CETSA MS to intact Arabidopsis thaliana cells and identified the thermal proteome of the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, bikinin. A comparison between the thermal- and the phospho-proteomes of bikinin revealed the auxin efflux carrier PIN-FORMED1 (PIN1) as a novel substrate of the Arabidopsis GSK3s that negatively regulate the brassinosteroid signaling. We established that PIN1 phosphorylation by the GSK3s is essential for maintaining its intracellular polarity that is required for auxin-mediated regulation of vascular patterning in the leaf thus, revealing a novel crosstalk between brassinosteroid and auxin signaling.

2022-04-04 | PXD030309 | Pride

Proteome-wide cellular thermal shift assay reveals novel crosstalk between brassinosteroid and auxin signaling

Project description:We demonstrated that bikinin changed the thermal stability of some of its target proteins as well as several known or putative interacting proteins of Arabidopsis GSK3s. By combining the thermal- and the phospho-proteomes of bikinin we uncovered the auxin carrier PIN-FORMED1 (PIN1) as a novel substrate of the Arabidopsis GSK3s. We showed that inhibition the kinase activity of GSK3s by brassinosteroids or the specific chemical inhibitor bikinin led to the loss of PIN1 polarity, revealing a novel crosstalk between brassinosteroid and auxin signaling. Hence, our study demonstrates the applicability of CETSA MS in plant intact cells for identification of small-molecule targets and for discovery of novel protein-protein interactions.

2021-12-08 | MSV000088545 | MassIVE

Flavonol-mediated stabilization of efflux complexes regulates polar auxin transport

Project description:The transport of auxin controls the rate, direction and localization of plant growth and development. The course of auxin transport is defined by the polar subcellular localization of the PIN proteins, a family of auxin efflux transporters. Using blue native PAGE and quantitative mass spectrometry, we identify native PIN core transport units as homo- and heteromers assembled from PIN1, 2, 3, 4 and 7 subunits only. To identify the protein composition of the larger, low stringency-solubilized PIN assembly we affinity-purified GFP-fused PIN proteins from Arabidopsis roots after expression from their native promoters and after the induction of discrete lateral roots, and analyzed the captured proteins by label-free quantitative mass spectrometry (LC-MS/MS).

2020-10-08 | PXD021543 | Pride

Histidine Kinase Inhibitors Impair Shoot Regeneration in Arabidopsis thaliana via Cytokinin Signalling and SAM Patterning Determinants

Project description:Reversible protein phosphorylation is a post-translational modification involved in virtually all plant processes, as it mediates protein activity and signal transduction. Here, we probe dynamic protein phosphorylation during de novo shoot organogenesis in Arabidopsis thaliana. We find that application of three kinase inhibitors in various time intervals has different effects on root explants. We furthermore show that short exposures to the putative His kinase inhibitor TCSA during the initial days on shoot induction medium (SIM) are detrimental for regeneration in seven natural accessions. Investigation of ahk and ahp mutants, as well as reporter lines for shoot markers and hormone responses suggests that TCSA at least partially works by impeding cytokinin signal transduction via AHK3, AHK4, AHP2, AHP3, and AHP5. A mass spectrometry-based phosphoproteome analysis further reveals profound deregulation of Ser/Thr/Tyr phosphoproteins related to protein modification, transcriptional regulation, vesicle trafficking, organ morphogenesis, and cation transport. Among TCSA-responsive factors are prior candidates with a role in shoot apical meristem patterning, such as AGO1, BAM1, PLL5, FIP37, TOP1ALPHA, and RBR1, but also proteins involved in polar auxin transport (e.g., PIN1) and brassinosteroid signalling (e.g., BIN2). Potentially novel regeneration determinants regulated by TCSA include RD2, AT1G52780, PVA11, and AVT1C, while NAIP2, OPS, ARR1, QKY, and aquaporins exhibit differential phospholevels on control SIM.

2022-09-28 | PXD030754 | Pride

Spatiotemporal brassinosteroid signaling and antagonism with auxin control Arabidopsis root growth.

Project description:Analysis of brassinosteroid (BR) and auxin effects on gene expression in Arabidopsis roots. Our genomic results indicate that BR and auxin induce largely opposite gene expression responses in primary roots. RNA-Seq for 7-day-old Arabidopsis Col-0, dwf4, bri1-116, and bri1-116;bzr1-1D roots grown on regular medium and treated with brassinolide, auxin or mock solution for 4 hr.

2015-04-30 | E-GEOD-52966 | biostudies-arrayexpress

Auxin stimulates brassinosteroid biosynthesis in Arabidopsis roots

Project description:We found that auxin stimulates gene expression of DWF4, which encodes a rate-dertermining step in brassinosteroid biosynthesis pathways. This increased gene expressioin subsequently led to elevation of the biosynthetic flux in Arabidopsis roots. To determine the list of genes that are regulated by auxin-synthesizing brassinosteroids, we challenged Arabidopsis seedlings with either auxin only or auxin plus brassinosteroid biosynthetic inhibitor brassinazole. Keywords: Hormone treatment

2011-02-15 | GSE12964 | GEO

Spatiotemporal brassinosteroid signaling and antagonism with auxin control Arabidopsis root growth.

2015-04-30 | GSE52966 | GEO

Interactions of ARF6 with PIF4, BZR1, and RGA integrate auxin signaling with environmental and other hormonal signals in Arabidopsis [RNA-Seq]

Project description:Auxin is a major plant hormone for both development and environmental adaptation. Auxin responses are context dependent and highly modulated by light, temperature, the circadian clock, brassinosteroid, and gibberellin, but the underlying mechanisms remain unclear. Here, we show that auxin signaling integrates with other signals through direct interactions of AUXIN RESPONSE FACTOR6 (ARF6) with PHYTOCHROME INTERACTING FACTOR4 (PIF4), the brassinosteroid-signaling transcription factor BZR1, and the gibberellin-signaling repressor RGA. ChIP-Seq and RNA-Seq experiments show that ARF6, PIF4, and BZR1 bind to largely overlapping targets in the genome and synergistically activate gene expression. In vitro and in vivo assays show that ARF6-promoter binding is enhanced by PIF4 and BZR1 but blocked by RGA. Furthermore, a tripartite HLH/bHLH module feedback regulates PIF activity and thus modulates auxin sensitivity according to additional developmental and environmental cues. Our results demonstrate a central growth-regulation transcriptional network that coordinates hormonal, environmental, and developmental control of cell elongation and plant growth. Seedlings (Col-0 and iaa3) were grown on medium containing 2 µM propiconazole (PPZ) in the dark for 5 days and treated with mock or 100 nM BL for 4 hr before harvesting for total RNA extraction.

2014-06-03 | E-GEOD-51772 | biostudies-arrayexpress

Suppression of pid nulls by mutations in PIN-FORMED1 (PIN1) and PIN1-GFP fusion

Project description:Disruption of either the auxin transporter PIN-FORMED 1 (PIN1) or the protein kinase PINOID (PID) leads to the development of pin-like inflorescences. Previous studies suggested that PID phosphorylates and activates PIN1. Here we report unexpected findings about the genetic interactions between the two genes. We deleted the first 2/3 of the PIN1 coding sequence using CRISPR/Cas9 and the resulting pin1 mutant (pin1-27) was a strong allele. Surprisingly, heterozygous pin1-27 suppressed three independent pid null mutants whereas homozygous pin1-27 enhanced the phenotypes of the pid mutants during embryogenesis. Furthermore, we show that deletion of either the hydrophilic loop or the second half of PIN1 also abolished PIN1 function, yet those heterozygous pin1 mutants were also capable of rescuing pid nulls. Moreover, we inserted GFP into the hydrophilic loop of PIN1 through CRISPR-mediated homology-directed repair (HDR). The GFP signal and pattern in the PIN1-GFP HDR line are similar to those in the previously reported PIN1-GFP transgenic lines. Interestingly, the PIN1-GFP HDR line also rescued various pid null mutants in a semi-dominant fashion. In addition, the previously reported key phosphorylation sites in PIN1 were still phosphorylated in PIN1-GFP pid plants. We conclude that PID is not directly required for phosphorylation and activation of PIN1.

2023-07-07 | MSV000092373 | MassIVE

Auxin-triggered changes in the Arabidopsis root tip (phospho)proteome reveal novel root growth regulators

Project description:Canonical auxin signalling starts with auxin binding to the receptor complex, followed by modulation of gene transcription and protein abundance (Tan et al., 2007; Chapman and Estelle, 2009; Slade et al., 2017). However, recent studies also showed an alternative mechanism in roots involving intra-cellular auxin perception, but not transcriptional reprogramming (Fendrych et al., 2018). Despite knowledge on effects of auxin on Arabidopsis root growth at the protein and phosphorylation level is increasing (Zhang et al., 2013; Mattei et al., 2013; Slade et al., 2017), it still remains incomplete. To address this gap in our knowledge, we explored the impact of auxin on the root tip proteome and phosphoproteome.

2021-09-09 | PXD021267 | Pride

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