Peroxisomes compartmentalize a dynamic suite of biochemical reactions and play a central role in plant metabolism, such as the degradation of hydrogen peroxide, metabolism of fatty acids, photorespiration, and the bio...Peroxisomes compartmentalize a dynamic suite of biochemical reactions and play a central role in plant metabolism, such as the degradation of hydrogen peroxide, metabolism of fatty acids, photorespiration, and the biosyn- thesis of plant hormones. Plant peroxisomes have been traditionally classified into three major subtypes, and in-depth mass spectrometry （MS）-based proteomics has been per- formed to explore the proteome of the two major subtypes present in green leaves and etiolated seedlings. Here, we carried out a comprehensive proteome analysis of perox- isomes from Arabidopsis leaves given a 48-h dark treatment. Our goal was to determine the proteome of the third major subtype of plant peroxisomes from senescent leaves, and further catalog the plant peroxisomal proteome. We identified a total of 111 peroxisomal proteins and verified the peroxisomal localization for six new proteins with potential roles in fatty acid metabolism and stress response by in vivo targeting analysis. Metabolic pathways compartmentalized in the three major subtypes of peroxisomes were also compared, which revealed a higher number of proteins involved in the detoxification of reactive oxygen species in peroxisomes from senescent leaves. Our study takes an important step towards mapping the full function of plant peroxisomes.展开更多
Peroxisome research has been revolutionized by proteome studies combined with in vivo subcellular targeting analyses. Yellow and cyan fluorescent protein(YFP and CFP) are the classical fluorophores of plant peroxisome...Peroxisome research has been revolutionized by proteome studies combined with in vivo subcellular targeting analyses. Yellow and cyan fluorescent protein(YFP and CFP) are the classical fluorophores of plant peroxisome research. In the new transient expression system of Arabidopsis seedlings co-cultivated with Agrobacterium we detected the YFP fusion of one candidate protein in peroxisomes, but only upon co-transformation with the peroxisome marker, CFP-PTS1. The data suggested that the YFP fusion was directed to peroxisomes due to its weak heterodimerization ability with CFP-PTS1,allowing piggy-back import into peroxisomes. Indeed, if co-expressed with monomeric Cerulean-PTS1(mCer-PTS1),the YFP fusion was no longer matrix localized. We systematically investigated the occurrence and extent of dimerization-based piggy-back import for different fluorophore combinations in five major transient plant expression systems. In Arabidopsis seedlings and tobacco leaves both untagged YFP and monomeric Venus were imported into peroxisomes if co-expressed with CFP-PTS1 but not with mCer-PTS1. By contrast, piggy-back import of cytosolic proteins was not observed in Arabidopsis and tobacco protoplasts or in onion epidermal cells for any fluorophore combination at any time point. Based on these important results we formulate new guidelines for fluorophore usage and experimental design to guarantee reliable identification of novel plant peroxisomal proteins.展开更多
基金supported by grants from the National Science Foundation to J.H.(MCB 0618335MCB 1330441)and L.J.O.(MCB 0618279)
文摘Peroxisomes compartmentalize a dynamic suite of biochemical reactions and play a central role in plant metabolism, such as the degradation of hydrogen peroxide, metabolism of fatty acids, photorespiration, and the biosyn- thesis of plant hormones. Plant peroxisomes have been traditionally classified into three major subtypes, and in-depth mass spectrometry （MS）-based proteomics has been per- formed to explore the proteome of the two major subtypes present in green leaves and etiolated seedlings. Here, we carried out a comprehensive proteome analysis of perox- isomes from Arabidopsis leaves given a 48-h dark treatment. Our goal was to determine the proteome of the third major subtype of plant peroxisomes from senescent leaves, and further catalog the plant peroxisomal proteome. We identified a total of 111 peroxisomal proteins and verified the peroxisomal localization for six new proteins with potential roles in fatty acid metabolism and stress response by in vivo targeting analysis. Metabolic pathways compartmentalized in the three major subtypes of peroxisomes were also compared, which revealed a higher number of proteins involved in the detoxification of reactive oxygen species in peroxisomes from senescent leaves. Our study takes an important step towards mapping the full function of plant peroxisomes.
基金financed by start-up funding provided by the University of Hamburg
文摘Peroxisome research has been revolutionized by proteome studies combined with in vivo subcellular targeting analyses. Yellow and cyan fluorescent protein(YFP and CFP) are the classical fluorophores of plant peroxisome research. In the new transient expression system of Arabidopsis seedlings co-cultivated with Agrobacterium we detected the YFP fusion of one candidate protein in peroxisomes, but only upon co-transformation with the peroxisome marker, CFP-PTS1. The data suggested that the YFP fusion was directed to peroxisomes due to its weak heterodimerization ability with CFP-PTS1,allowing piggy-back import into peroxisomes. Indeed, if co-expressed with monomeric Cerulean-PTS1(mCer-PTS1),the YFP fusion was no longer matrix localized. We systematically investigated the occurrence and extent of dimerization-based piggy-back import for different fluorophore combinations in five major transient plant expression systems. In Arabidopsis seedlings and tobacco leaves both untagged YFP and monomeric Venus were imported into peroxisomes if co-expressed with CFP-PTS1 but not with mCer-PTS1. By contrast, piggy-back import of cytosolic proteins was not observed in Arabidopsis and tobacco protoplasts or in onion epidermal cells for any fluorophore combination at any time point. Based on these important results we formulate new guidelines for fluorophore usage and experimental design to guarantee reliable identification of novel plant peroxisomal proteins.
