The Physcomitrella patens Chloroplast 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 moss Physcomitrella patens as 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.

Distinct types of short open reading frames are translated in plant cells

“…Endogenous peptide extraction was conducted as described previously (Fesenko et al 2015). Proteins were extracted as described previously (Fesenko et al 2016). Additional details are described in the Supplemental Methods.…”

Comparison and optimization of protein extraction and two-dimensional gel electrophoresis protocols for liverworts

“…In the past two decades, few bryophytes, namely, Physcomitrella patens [8] and Marchantia polymorpha [9], have been developed as model organisms, which promoted plant development and genomic studies. Unfortunately, very few proteomic studies have been conducted for bryophytes [10][11][12].…”

An Arabidopsis protoplast isolation method reduces cytosolic acidification and activation of the chloroplast stress sensor SENSITIVE TO FREEZING 2

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