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Thylakoid Protein FPB1 Synergistically Cooperates with PAM68 to Promote CP47 Biogenesis and Photosystem II Assembly

ABSTRACT: LC-MS/MS analysis was used for protein identification of immunoprecipitation. 20 uL eluted samples of immunoprecipitation were separated by 12.5% SDS-PAGE and each gel lane was cut in consecutive gel slices followed by destaining twice using NH4HCO3/ACN. Proteins were digested in-gel with DTT, IAA and trypsin. Digested peptides in the gel slices were then desalted using StageTips with C18 Cartridge (Sigma). Peptides extracts for each gel slice were analyzed by High Performance Liquid Chromatography (HPLC) using EASY column (Fisher Scientific, USA) coupled with a Q-Exactive mass spectrometer (Thermo Finnigan). Resulting MS/MS spectra were searched against the predicted Arabidopsis proteome (35386 entries, 20191018) with maxquant software (version 2.3).

INSTRUMENT(S): High Performance Liquid Chromatography (HPLC)

ORGANISM(S): Arabidopsis Thaliana (ncbitaxon:3702)

SUBMITTER: Lin Zhang

PROVIDER: MSV000094131 | MassIVE | Tue Feb 20 10:42:00 GMT 2024

SECONDARY ACCESSION(S): PXD049968

REPOSITORIES: MassIVE

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Project description:To investigate the developmental gradient of the third maize leaf, the light exposed area of the leaf (corresponding to 18cm of leaf) and 2cm shaded by the sheath were sampled in ten slices. Four replicates were collected, immediately shock frozen in liquid nitrogen and subsequently cut into 2cm slices. At least 10 plants were pooled for each biological replicate. We have systematically analyzed a developmental gradient of the third maize leaf from the point of emergence into the light to the tip in ten continuous leaf slices to study organ development and physiological and biochemical functions. Transcriptome analysis, oxygen sensitivity of photosynthesis, delta-13C values, and photosynthetic rate measurements showed that the maize leaf undergoes a sink to source transition without an intermediate phase of C3 photosynthesis or operation of a photorespiratory carbon pump. Metabolome and transcriptome analysis, chlorophyll and protein measurements, as well as dry weight determination showed continuous gradients for all analyzed items. The absence of binary on-off switches and regulons pointed to a morphogradient along the leaf as the determining factor of developmental stage. Analysis of transcription factors for differential expression along the leaf gradient defined a list of putative regulators orchestrating the sink-to-source transition and establishment of C4 photosynthesis. Finally, transcriptome and metabolome analysis, as well as enzyme activity measurements, and absolute quantification of selected metabolites revised the current model of maize C4 photosynthesis. All datasets are included within the publication to serve as a resource for maize leaf systems biology. For the transcriptional analysis, the goal of the study was to (i) identify whether the leaf contains binary switches for genes involved in photosynthesis, (ii)characterize the patterns of gene expression in the leaf, (iii) provide independent validation of maize leaf expression experiments published in Li et al. (2011) and (iv) determine transcripts co-expressed with key transcripts of C4 photosynthesis. To this end, changed transcripts were determined by ANOVA and characterized by K-means and hierachical clustering. Four replicates were collected for each of the ten consecutive leaf slices resulting in 40 one color arrays. Slice 1 represents the tip of the leaf, slice 10 the lowermost slice which is shaded by the sheath with all slices in between consecutively numbered.

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Thylakoid Protein FPB1 Synergistically Cooperates with PAM68 to Promote CP47 Biogenesis and Photosystem II Assembly

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