title: ThePhyscomitrella patensChloroplast Proteome Changes in Response to Protoplastation.
abstract: Plant protoplasts are widely used for genetic manipulation and functional studies in transient expression systems. However, little is known about the molecular pathways involved in a cell response to the combined stress factors resulted from protoplast generation. Plants often face more than one type of stress at a time, and how plants respond to combined stress factors is therefore of great interest. Here, we used protoplasts of the mossPhyscomitrella patensas a model to study the effects of short-term stress on the chloroplast proteome. Using label-free comparative quantitative proteomic analysis (SWATH-MS), we quantified 479 chloroplast proteins, 219 of which showed a more than 1.4-fold change in abundance in protoplasts. We additionally quantified 1451 chloroplast proteins using emPAI. We observed degradation of a significant portion of the chloroplast proteome following the first hour of stress imposed by the protoplast isolation process. Electron-transport chain (ETC) components underwent the heaviest degradation, resulting in the decline of photosynthetic activity. We also compared the proteome changes to those in the transcriptional level of nuclear-encoded chloroplast genes. Globally, the levels of the quantified proteins and their corresponding mRNAs showed limited correlation. Genes involved in the biosynthesis of chlorophyll and components of the outer chloroplast membrane showed decreases in both transcript and protein abundance. However, proteins like dehydroascorbate reductase 1 and 2-cys peroxiredoxin B responsible for ROS detoxification increased in abundance. Further, genes such as thylakoid ascorbate peroxidase were induced at the transcriptional level but down-regulated at the proteomic level. Together, our results demonstrate that the initial chloroplast reaction to stress is due changes at the proteomic level.
authors.affiliation: Laboratory of Proteomics, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences Moscow, Russia.
authors.forename: Igor
authors.initials: I
authors.lastname: Fesenko
authors.affiliation: Laboratory of Proteomics, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences Moscow, Russia.
authors.forename: Anna
authors.initials: A
authors.lastname: Seredina
authors.affiliation: Laboratory of Proteomics, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences Moscow, Russia.
authors.forename: Georgij
authors.initials: G
authors.lastname: Arapidi
authors.affiliation: Department of Bioenergetics, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State UniversityMoscow, Russia; Department of Biocatalysis, Emanuel Institute of Biochemical Physics, Russian Academy of SciencesMoscow, Russia.
authors.forename: Vasily
authors.initials: V
authors.lastname: Ptushenko
authors.affiliation: Laboratory of Proteomics, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences Moscow, Russia.
authors.forename: Anatoly
authors.initials: A
authors.lastname: Urban
authors.affiliation: Laboratory of the Proteomic Analysis, Research Institute for Physico-Chemical Medicine Moscow, Russia.
authors.forename: Ivan
authors.initials: I
authors.lastname: Butenko
authors.affiliation: Laboratory of Proteomics, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences Moscow, Russia.
authors.forename: Sergey
authors.initials: S
authors.lastname: Kovalchuk
authors.affiliation: Laboratory of Proteomics, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences Moscow, Russia.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Babalyan
authors.affiliation: Laboratory of Proteomics, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences Moscow, Russia.
authors.forename: Andrey
authors.initials: A
authors.lastname: Knyazev
authors.affiliation: Laboratory of Proteomics, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences Moscow, Russia.
authors.forename: Regina
authors.initials: R
authors.lastname: Khazigaleeva
authors.affiliation: Laboratory of Proteomics, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences Moscow, Russia.
authors.forename: Elena
authors.initials: E
authors.lastname: Pushkova
authors.affiliation: Laboratory of Proteomics, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences Moscow, Russia.
authors.forename: Nikolai
authors.initials: N
authors.lastname: Anikanov
authors.affiliation: Laboratory of Proteomics, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences Moscow, Russia.
authors.forename: Vadim
authors.initials: V
authors.lastname: Ivanov
authors.affiliation: Laboratory of Proteomics, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of SciencesMoscow, Russia; Laboratory of the Proteomic Analysis, Research Institute for Physico-Chemical MedicineMoscow, Russia.
authors.forename: Vadim M
authors.initials: VM
authors.lastname: Govorun
keywords: MRM-MS; SWATH-MS; chloroplast proteome; label-free quantification; moss Physcomitrella patens; stress conditions