Proteome Analysis of Peroxisomes from Dark-Treated Senescent Arabidopsis Leavesfa
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Journal of Integrative JIPB Plant Biology Proteome analysis of peroxisomes from dark-treated senescent Arabidopsis leavesFA † † Ronghui Pan1 , Sigrun Reumann1,2,3,4 , Piotr Lisik3, Stefanie Tietz1, Laura J. Olsen2 and Jianping Hu1,5* 1. MSU-Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA 2. Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI, USA 3. Center of Organelle Research, University of Stavanger, N-4021 Stavanger, Norway 4. Department of Plant Biochemistry and Infection Biology, Institute of Plant Science and Microbiology, University of Hamburg, D-22609 Hamburg, Germany 5. Plant Biology Department, Michigan State University, East Lansing, MI 48824, USA † These authors contributed equally to this work *Correspondence: Jianping Hu ([email protected]) doi: 10.1111/jipb.12670 High-Impact Article Abstract Peroxisomes compartmentalize a dynamic suite of a total of 111 peroxisomal proteins and verified the peroxisomal biochemical reactions and play a central role in plant localization for six new proteins with potential roles in fatty metabolism, such as the degradation of hydrogen peroxide, acid metabolism and stress response by in vivo targeting metabolism of fatty acids, photorespiration, and the biosyn- analysis. Metabolic pathways compartmentalized in the three thesis of plant hormones. Plant peroxisomes have been major subtypes of peroxisomes were also compared, which traditionally classified into three major subtypes, and in-depth revealed a higher number of proteins involved in the mass spectrometry (MS)-based proteomics has been per- detoxification of reactive oxygen species in peroxisomes formed to explore the proteome of the two major subtypes from senescent leaves. Our study takes an important step present in green leaves and etiolated seedlings. Here, we towards mapping the full function of plant peroxisomes. carried out a comprehensive proteome analysis of perox- Edited by: Li-Xin Zhang, Institute of Botany, the Chinese Academy isomes from Arabidopsis leaves given a 48-h dark treatment. of Sciences, China Our goal was to determine the proteome of the third major Received May 8, 2018; Accepted May 29, 2018; Online on Jun. 7, subtype of plant peroxisomes from senescent leaves, and 2018 further catalog the plant peroxisomal proteome. We identified FA: Free Access, paid by JIPB INTRODUCTION phylloquinone, isoprenoids, biotin and Coenzyme A (CoA) (Kaur et al. 2009; Hu et al. 2012; Pan and Hu 2018). Peroxisomes are ubiquitous and dynamic organelles The peroxisome lacks DNA, and peroxisomal pro- playing vital functions in eukaryotic cells. The metabolic teins are nuclear encoded and imported into the functions of peroxisomes vary significantly among organelle by peroxins (PEX proteins), a majority of kingdoms, and even in different tissues and develop- which are membrane associated. Most peroxisomal Free Access mental stages of the same organism. In plants, matrix proteins carry one of the two major peroxisome peroxisomes have major functions in fatty acid catabo- targeting signals (PTSs): the C-terminal tripeptide PTS1, > > lism, hydrogen peroxide (H2O2) degradation, photores- with a canonical sequence of SKL ( indicates the piration, the glyoxylate cycle, pathogen defense and C-terminal end of the protein); and the nonapeptide the biosynthesis of important plant hormones, such as PTS2, which is located in the N-terminal region and only jasmonic acid (JA), indole-3-acetic acid (IAA) and present in a small number of matrix proteins (Sparkes salicylic acid (SA). Other peroxisomal functions include and Baker 2002; Reumann et al. 2016). the catabolism of polyamines, uric acid, amino acids, To fully understand the physiological roles of plant sulfite and pseudouridine, as well as the biosynthesis of peroxisomes, it is necessary to catalogue the proteome © 2018 Institute of Botany, Chinese Academy of Sciences November 2018 | Volume 60 | Issue 11 | 1028–1050 www.jipb.net Proteome of peroxisomes from senescent leaves 1029 of these organelles. In recent years, experimental peroxisomes from plants undergoing senescence-re- proteomics, based on peroxisome purification and lated processes. Senescence is a genetically-controlled mass spectrometry (MS), have greatly accelerated the global-reprogramming of cellular activities, featured by rate and scale of the identification of novel plant the cessation of photosynthesis, degradation of peroxisomal proteins (Kaur and Hu 2011; Reumann 2011; chlorophyll, disintegration of organelle structures, Pan and Hu 2018). This unbiased and relatively large- and degradation of diverse cellular components, such scale approach has not only successfully identified new as proteins and lipids (Buchanan-Wollaston 1997). Plant auxiliary and regulatory proteins involved in established peroxisomal metabolism during leaf senescence has peroxisomal processes, but also revealed numerous been investigated in pea, from which increased proteins involved in unexpected peroxisomal functions proteolytic activity and more active metabolism of (Kaur and Hu 2011; Reumann 2011; Pan and Hu 2018). reactive oxygen species (ROS), nitric oxide and NADPH Peroxisomes are metabolically and morphologically was discovered (Pastori and del Rio 1997; Corpas et al. dynamic. Environmental factors, including light, are 1999, 2004). However, analysis of the whole peroxi- known to cause changes in peroxisome abundance some proteome undergoing senescence-related pro- (Lopez-Huertas et al. 2000; Koh et al. 2005; Desai and cesses had been lacking. Detached leaves subjected to Hu 2008; Rodrıguez-Serrano et al. 2016; Desai et al. extended (e.g. 48 h) darkness have been widely used as 2017; Fahy et al. 2017). The proteome and primary a system to study plant senescence (Biswal and Biswal functions of plant peroxisomes vary, quantitatively 1984; Oh et al. 1997; Weaver et al. 1998; Weaver and and qualitatively, across developmental stages, and Amasino 2001; Song et al. 2014; Liebsch and Keech distinct names were given to the three major subtypes 2016). To this end, we performed proteome analysis of of plant peroxisomes. Leaf peroxisomes in green peroxisomes isolated from leaves of dark-treated leaves are mainly involved in photorespiration (Tolbert detached Arabidopsis plants. This study identified 111 et al. 1969; Peterhansel et al. 2010), glyoxysomes refer peroxisomal proteins, a higher number compared with to peroxisomes in seeds/fatty tissue, which harbor the previous peroxisome proteomic studies in any organ- b glyoxylate cycle together with fatty acid -oxidation ism, with eight being identified for the first time by MS- (Beevers 1979), and gerontosomes refer to perox- based proteomics of peroxisomes. Our in vivo subcellu- isomes in senescent tissues, which were suggested to lar targeting analysis validated peroxisomal localization resemble glyoxysomes in terms of their metabolic of six new proteins involved in fatty acid metabolism functions (Vicentini and Matile 1993). and stress response. Our study takes an important step To uncover peroxisomal proteins at different towards fully deciphering the peroxisome proteome developmental stages, previous proteomic studies and uncovering novel physiological roles of plant fi analyzed peroxisomes puri ed from various plant peroxisomes. organs and species, including greening and etiolated Arabidopsis cotyledons (Fukao et al. 2002, 2003), Arabidopsis leaves (Reumann et al. 2007, 2009), non- RESULTS green Arabidopsis cell suspension cultures (Eubel et al. 2008), etiolated Arabidopsis seedlings (Quan et al. Mass spectrometry-based interrogation of 2013), etiolated soybean cotyledons (Arai et al. 2008), peroxisomal proteins and spinach leaves (Babujee et al. 2010). These studies Isolation of leaf peroxisomes requires large amounts of revealed common pathways, as well as proteins specific leaves (approximately 60 g FW). Therefore, any abiotic to certain developmental stages, and identified a stress applied to Arabidopsis plants needs to reach all number of novel peroxisome proteins, such as those leaves and preferentially all mesophyll cells relatively involved in methylglyoxal detoxification, phylloquinone uniformly. A comparison of intact and harvested biosynthesis, pseudouridine catabolism, CoA biosynthe- Arabidopsis Col-0 plants subjected to prolonged dark sis, and putative regulatory proteins (Kaur and Hu 2011; treatment demonstrated that only harvested plants Reumann 2011; Pan and Hu 2018). showed uniform progression of leaf senescence in all The goal of this study was to discover additional leaves, consistent with previous reports of chlorophyll peroxisomal proteins and catalog the proteome of degradation (see Introduction). The chlorophyll content www.jipb.net November 2018 | Volume 60 | Issue 11 | 1028–1050 1030 Pan et al. was reduced by approximately 40% after 48 h of dark- identification, 500 mg each of T1 and T2 peroxisome induced senescence, and reached a residual level of proteins were separated on a 1D gel, which was later approximately 15% after 4 d (Figure 1A). By contrast, sliced into eight pieces after electrophoresis. Proteins in each slice were in-gel digested by trypsin, and subjected when intact plants were subjected to dark treatment, to liquid chromatography-electrospray ionization- leaf senescence became visible only slowly and tandem mass spectrometry (LC-ESI-MS/MS). This primarily in the oldest leaves, and as a result, chlorophyll method had been used in our previous studies to content