Dataset Information

Quantitative tandem affinity purification, an effective tool to investigate protein complex composition in plant hormone signaling: strigolactones in the spotlight

ABSTRACT: Phytohormones tightly regulate plant growth by integrating changing environmental and developmental cues. Although the key players have been identified in many plant hormonal pathways, the molecular mechanisms and mode of action of perception and signaling remain incompletely resolved. Characterization of protein partners of known signaling components provides insight into the formed protein complexes, but, unless quantification is involved, does not deliver much, if any, information about the dynamics of the induced or disrupted protein complexes. Therefore, in proteomics research, the discovery of what actually triggers, regulates or interrupts the composition of protein complexes is gaining importance. Here, tandem affinity purification coupled to mass spectrometry (TAP-MS) is combined with label-free quantification (LFQ) to a highly valuable tool to detect physiologically relevant, dynamic protein-protein interactions in Arabidopsis thaliana cell cultures. To demonstrate its potential, we focus on the signaling pathway of one of the most recently discovered phytohormones, strigolactones.

INSTRUMENT(S): LTQ Orbitrap Velos

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

TISSUE(S): Cell Suspension Culture, Cell Culture

SUBMITTER: Sylwia Struk

LAB HEAD: Sofie Goormachtig

PROVIDER: PXD009083 | Pride | 2018-04-26

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 20

Publications

Quantitative Tandem Affinity Purification, an Effective Tool to Investigate Protein Complex Composition in Plant Hormone Signaling: Strigolactones in the Spotlight.

Struk Sylwia S Braem Lukas L Walton Alan A De Keyser Annick A Boyer François-Didier FD Persiau Geert G De Jaeger Geert G Gevaert Kris K Goormachtig Sofie S

Frontiers in plant science 20180426

Phytohormones tightly regulate plant growth by integrating changing environmental and developmental cues. Although the key players have been identified in many plant hormonal pathways, the molecular mechanisms and mode of action of perception and signaling remain incompletely resolved. Characterization of protein partners of known signaling components provides insight into the formed protein complexes, but, unless quantification is involved, does not deliver much, if any, information about the d ...[more]

PMID: 29755490

Similar Datasets

Project description:The fungal pathogen Sclerotinia sclerotiorum infects a broad range of dicotyledonous plant species and is the causative agent of stem rot in Brassica napus. To elucidate the mechanisms underlying the defense response, we studied the patterns of gene expression in a partially resistant variety of ZhongYou 821 (ZY821) and a susceptible line from Westar over five time points, 6, 12, 24, 48, and 72 hours post-inoculation (hpi) using a B. napus oligonucleotide microarray. Maximum differential gene expression was observed at 48 hpi in both genotypes with ZY821 responding more quickly than Westar. Specific sets of genes exhibited higher levels of expression at the earlier stages of the infection (6-12 hpi), including genes encoding defense-associated proteins such as chitinases, glucanases, osmotins and lectins and genes encoding transcription factors belonging to the zinc finger, WRKY, AP2, and MYB classes. Genes encoding enzymes involved in JA, ethylene, and auxin synthesis were induced in both genotypes, as were those for gibberellin degradation. Changes in metabolic pathways affecting carbohydrate and energy metabolism appeared to be directed toward shuttling carbon reserves to the TCA cycle. Genes involved in glucosinolate and phenylpropanoid biosynthesis were highly up-regulated suggesting that secondary metabolites are also important components of the response to S. sclerotiorum in B. napus. Keywords: Time course, infected vs mock infected Time course experiment (6hr, 12 hr, 24 hr, 48 hr, 72 hrs), sclerotinia infected vs mock-infected. Biological replicates: 3, independently harvested. Technical replicates: 2, dye swapped within each time for each biological replicate (total 6 slides per time point) time after innoculation: 6 hours: zy 6h inf vs control rep1, zy 6h control vs inf rep1, zy 6h inf vs control rep2, zy 6h control vs inf rep2, zy 6h inf vs control rep3, zy 6h control vs inf rep3 time after innoculation: 12 hours: zy 12h inf vs control rep1, zy 12h control vs inf rep1, zy 12h inf vs control rep2, zy 12h control vs inf rep2, zy 12h inf vs control rep3, zy 12h control vs inf rep3 time after innoculation: 24 hours: zy 24h inf vs control rep1, zy 24h control vs inf rep1, zy 24h inf vs control rep2, zy 24h control vs inf rep2, zy 24h inf vs control rep3, zy 24h control vs inf rep3 time after innoculation: 48 hours: zy 48h inf vs control rep1, zy 48h control vs inf rep1, zy 48h inf vs control rep2, zy 48h control vs inf rep2, zy 48h inf vs control rep3, zy 48h control vs inf rep3 time after innoculation: 72 hours: zy 72h inf vs control rep1, zy 72h control vs inf rep1, zy 72h inf vs control rep2, zy 72h control vs inf rep2, zy 72h inf vs control rep3, zy 72h control vs inf rep3 biological replicate: zy 6h inf vs control rep1, zy 6h inf vs control rep2, zy 6h inf vs control rep3 biological replicate: zy 6h control vs inf rep1, zy 6h control vs inf rep2, zy 6h control vs inf rep3 technical replicate - labeled-extract: zy 6h inf vs control rep1, zy 6h control vs inf rep1 technical replicate - labeled-extract: zy 6h inf vs control rep2, zy 6h control vs inf rep2 technical replicate - labeled-extract: zy 6h inf vs control rep3, zy 6h control vs inf rep3 biological replicate: zy 12h inf vs control rep1, zy 12h inf vs control rep2, zy 12h inf vs control rep3 biological replicate: zy 12h control vs inf rep1, zy 12h control vs inf rep2, zy 12h control vs inf rep3 technical replicate - labeled-extract: zy 12h inf vs control rep1, zy 12h control vs inf rep1 technical replicate - labeled-extract: zy 12h inf vs control rep2, zy 12h control vs inf rep2 technical replicate - labeled-extract: zy 12h inf vs control rep3, zy 12h control vs inf rep3 biological replicate: zy 24h inf vs control rep1, zy 24h inf vs control rep2, zy 24h inf vs control rep3 biological replicate: zy 24h control vs inf rep1, zy 24h control vs inf rep2, zy 24h control vs inf rep3 technical replicate - labeled-extract: zy 24h inf vs control rep1, zy 24h control vs inf rep1 technical replicate - labeled-extract: zy 24h inf vs control rep2, zy 24h control vs inf rep2 technical replicate - labeled-extract: zy 24h inf vs control rep3, zy 24h control vs inf rep3 biological replicate: zy 48h inf vs control rep1, zy 48h inf vs control rep2, zy 48h inf vs control rep3 biological replicate: zy 48h control vs inf rep1, zy 48h control vs inf rep2, zy 48h control vs inf rep3 technical replicate - labeled-extract: zy 48h inf vs control rep1, zy 48h control vs inf rep1 technical replicate - labeled-extract: zy 48h inf vs control rep2, zy 48h control vs inf rep2 technical replicate - labeled-extract: zy 48h inf vs control rep3, zy 48h control vs inf rep3 biological replicate: zy 72h inf vs control rep1, zy 72h inf vs control rep2, zy 72h inf vs control rep3 biological replicate: zy 72h control vs inf rep1, zy 72h control vs inf rep2, zy 72h control vs inf rep3 technical replicate - labeled-extract: zy 72h inf vs control rep1, zy 72h control vs inf rep1 technical replicate - labeled-extract: zy 72h inf vs control rep2, zy 72h control vs inf rep2 technical replicate - labeled-extract: zy 72h inf vs control rep3, zy 72h control vs inf rep3

Project description:The fungal pathogen Sclerotinia sclerotiorum infects a broad range of dicotyledonous plant species and is the causative agent of stem rot in Brassica napus. To elucidate the mechanisms underlying the defense response, we studied the patterns of gene expression in a partially resistant variety of ZhongYou 821 (ZY821) and a susceptible line from Westar over five time points, 6, 12, 24, 48, and 72 hours post-inoculation (hpi) using a B. napus oligonucleotide microarray. Maximum differential gene expression was observed at 48 hpi in both genotypes with ZY821 responding more quickly than Westar. Specific sets of genes exhibited higher levels of expression at the earlier stages of the infection (6-12 hpi), including genes encoding defense-associated proteins such as chitinases, glucanases, osmotins and lectins and genes encoding transcription factors belonging to the zinc finger, WRKY, AP2, and MYB classes. Genes encoding enzymes involved in JA, ethylene, and auxin synthesis were induced in both genotypes, as were those for gibberellin degradation. Changes in metabolic pathways affecting carbohydrate and energy metabolism appeared to be directed toward shuttling carbon reserves to the TCA cycle. Genes involved in glucosinolate and phenylpropanoid biosynthesis were highly up-regulated suggesting that secondary metabolites are also important components of the response to S. sclerotiorum in B. napus. Keywords: Time course, and infected vs mock infected Time course experiment (6hr, 12 hr, 24 hr, 48 hr, 72 hrs), sclerotinia infected vs mock-infected. Biological replicates: 3, independently harvested. Technical replicates: 2, dye swapped within each time for each biological replicate (total 6 slides per time point) time after innoculation: 6 hours: westar 6h inf vs control rep1, westar 6h control vs inf rep1, westar 6h inf vs control rep2, westar 6h control vs inf rep2, westar 6h inf vs control rep3, westar 6h control vs inf rep3 time after innoculation: 12 hours: westar 12h inf vs control rep1, westar 12h control vs inf rep1, westar 12h inf vs control rep2, westar 12h control vs inf rep2, westar 12h inf vs control rep3, westar 12h control vs inf rep3 time after innoculation: 24 hours: westar 24h inf vs control rep1, westar 24h control vs inf rep1, westar 24h inf vs control rep2, westar 24h control vs inf rep2, westar 24h inf vs control rep3, westar 24h control vs inf rep3 time after innoculation: 48 hours: westar 48h inf vs control rep1, westar 48h control vs inf rep1, westar 48h inf vs control rep2, westar 48h control vs inf rep2, westar 48h inf vs control rep3, westar 48h control vs inf rep3 time after innoculation: 72 hours: westar 72h inf vs control rep1, westar 72h control vs inf rep1, westar 72h inf vs control rep2, westar 72h control vs inf rep2, westar 72h inf vs control rep3, westar 72h control vs inf rep3 biological replicate: westar 6h inf vs control rep1, westar 6h inf vs control rep2, westar 6h inf vs control rep3 biological replicate: westar 6h control vs inf rep1, westar 6h control vs inf rep2, westar 6h control vs inf rep3 technical replicate - labeled-extract: westar 6h inf vs control rep1, westar 6h control vs inf rep1 technical replicate - labeled-extract: westar 6h inf vs control rep2, westar 6h control vs inf rep2 technical replicate - labeled-extract: westar 6h inf vs control rep3, westar 6h control vs inf rep3 biological replicate: westar 12h inf vs control rep1, westar 12h inf vs control rep2, westar 12h inf vs control rep3 biological replicate: westar 12h control vs inf rep1, westar 12h control vs inf rep2, westar 12h control vs inf rep3 technical replicate - labeled-extract: westar 12h inf vs control rep1, westar 12h control vs inf rep1 technical replicate - labeled-extract: westar 12h inf vs control rep2, westar 12h control vs inf rep2 technical replicate - labeled-extract: westar 12h inf vs control rep3, westar 12h control vs inf rep3 biological replicate: westar 24h inf vs control rep1, westar 24h inf vs control rep2, westar 24h inf vs control rep3 biological replicate: westar 24h control vs inf rep1, westar 24h control vs inf rep2, westar 24h control vs inf rep3 technical replicate - labeled-extract: westar 24h inf vs control rep1, westar 24h control vs inf rep1 technical replicate - labeled-extract: westar 24h inf vs control rep2, westar 24h control vs inf rep2 technical replicate - labeled-extract: westar 24h inf vs control rep3, westar 24h control vs inf rep3 biological replicate: westar 48h inf vs control rep1, westar 48h inf vs control rep2, westar 48h inf vs control rep3 biological replicate: westar 48h control vs inf rep1, westar 48h control vs inf rep2, westar 48h control vs inf rep3 technical replicate - labeled-extract: westar 48h inf vs control rep1, westar 48h control vs inf rep1 technical replicate - labeled-extract: westar 48h inf vs control rep2, westar 48h control vs inf rep2 technical replicate - labeled-extract: westar 48h inf vs control rep3, westar 48h control vs inf rep3 biological replicate: westar 72h inf vs control rep1, westar 72h inf vs control rep2, westar 72h inf vs control rep3 biological replicate: westar 72h control vs inf rep1, westar 72h control vs inf rep2, westar 72h control vs inf rep3 technical replicate - labeled-extract: westar 72h inf vs control rep1, westar 72h control vs inf rep1 technical replicate - labeled-extract: westar 72h inf vs control rep2, westar 72h control vs inf rep2 technical replicate - labeled-extract: westar 72h inf vs control rep3, westar 72h control vs inf rep3

Project description:Plant inflorescence meristems and floral meristems possess specific boundary domains that result in floral organ separation, and in proper numbers of floral organs. HANABA TARANU (HAN) encodes a boundary-expressing GATA type zinc finger transcription factor that regulates shoot apical meristem organization, cell division and flower development in Arabidopsis, but the underlying mechanism remains unclear. Through time-course whole genome oligonucleotide microarray analyses following transient overexpression of HAN, we find that HAN represses hundreds of genes, especially genes involved in hormone responses and floral organ regulation. Transient overexpression of HAN also causes the repression of HAN itself and three other HAN family genes: HANL2 (HAN-LIKE2), GNC (GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM-INVOLVED) and GNL (GNC LIKE), forming a negative regulatory feedback loop. Double- and triple-mutant strains of han with hanl2, gnc and gnl show synergistic effects on sepal fusion, petal number, and silique length, and embryo development, as well as carpelloid stamens. Transcripts of HANL2, GNC and GNL have similar accumulation patterns, specifically in petals, stamens, carpels and inflorescence meristems, which are partially overlapping with the expression pattern of HAN, suggesting that HAN and HAN family genes share redundant functions during flower development. We further show by yeast two hybrid assays that HAN can homodimerize as well as heterodimerize with other HAN family proteins. Chromatin-immunoprecipitation analyses indicate that HAN directly binds to its own promoter and the promoter of GNC in vivo. These findings, together with the fact that constitutive overexpression of HAN has an even stronger phenotype than a loss of function mutation, support the hypothesis that HAN may function as a key repressor that regulates floral development via intricate regulatory networks involving genes in the GATA3 family, hormone actions and floral organ specification. Time course induction experiment. Arabidopsis inflorescences containing flower buds from stages 1-9 were collected for microarray experiment 0h, 4h, 12h and 72h after 10M-BM-5M Dex treatment. RNA samples from mock- and Dex-treated plants at each time point were co-hybridized, and labeling dyes were swapped between replicates to reduce dye-related bias. Four biological replicates were used for the microarray hybridization.

			Action	DRS
	PEAK-SMXL7.zip	Other
	PEAKdeltaSMXL7.zip	Other
	SEARCH-SMXL7.zip	Other
	SEARCHdeltaSMXL7.zip	Other
	SMXL7_GR24_1.raw	Raw