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.
Highlights
In nature, plants are simultaneously exposed to a combination of various stress factors
The mechanisms accounting for plant reactions to complex stress are still poorly understood, especially at the proteomic level, understanding this process is critical for many aspects of plant biology
We found that 577 proteins identified in our study have been previously reported in a quantitative proteomic study of moss chloroplasts (Mueller et al, 2014)
Summary
Plants are simultaneously exposed to a combination of various stress factors. Recent studies have shown that plant responses to two or more stress factors are complex and not merely the result of concurrent, independent reactions to each kind of stress (Atkinson et al, 2013; Rasmussen et al, 2013; Suzuki et al, 2014; Ramegowda and SenthilKumar, 2015). Metabolic and signaling pathways involved in the reaction to complex stresses include specific transcription factors, photosynthetic adjustments, antioxidant protection systems, and biosynthesis of stress hormones (Hirayama and Shinozaki, 2010; Suzuki et al, 2014). Chloroplast interaction with plasma membrane receptor kinases is one of the key mechanisms that modulate plant stress reactions (Trotta et al, 2014)
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