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At Long Last: Evidence for Pexophagy in Plants

2014; Elsevier BV; Volume: 7; Issue: 8 Linguagem: Inglês

10.1093/mp/ssu029

1674-2052

Tamar Avin‐Wittenberg, Alisdair R. Fernie,

Lipid metabolism and biosynthesis

Peroxisomes are highly dynamic single-membrane-bound eukaryotic organelles displaying great variability in enzymatic content (Platta and Erdmann, 2007Platta H.W. Erdmann R. Peroxisomal dynamics.Trends Cell Biol. 2007; 17: 474-484Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Plant peroxisomes function in a plethora of crucial development and stress ameliorating processes. Among the functions of plant peroxisomes are β-oxidation of fatty acids, phytohormone production, participation in photorespiration, the glyoxyalte cycle, detoxification processes, and signal molecule generation (Hu et al., 2012Hu J. Baker A. Bartel B. Linka N. Mullen R.T. Reumann S. Zolman B.K. Plant peroxisomes: biogenesis and function.Plant Cell. 2012; 24: 2279-2303Crossref PubMed Scopus (315) Google Scholar). Peroxisomes maintain their dynamic character by using a variety of organelle biogenesis and degradation mechanisms. One such mechanism is pexophagy, the selective degradation of peroxisomes via autophagy. Autophagy is a process by which organelles and proteins are transported from the cytosol for degradation in the vacuole (Oku and Sakai, 2010Oku M. Sakai Y. Peroxisomes as dynamic organelles: autophagic degradation.FEBS J. 2010; 277: 3289-3294Crossref PubMed Scopus (68) Google Scholar). Two forms of autophagy have been implicated in pexophagy: macroautophagy and microautophagy. Here we will focus on macroautophagy (hereafter simply referred to as autophagy). During autophagy, portions of the cytoplasm are enclosed by de novo formed vesicles, composed of a double membrane, termed autophagosomes. After transport to the vacuole, the outer membrane of the autophagosome fuses with the tonoplast to release a single-membrane autophagic body which is subsequently degraded in the vacuole. The genes required for the autophagic process have been termed ATG genes (Li and Vierstra, 2012Li F. Vierstra R.D. Autophagy: a multifaceted intracellular system for bulk and selective recycling.Trends Plant Sci. 2012; 17: 526-537Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Autophagy has been characterized in plants, specifically in the model plant Arabidopsis thaliana, where it has been demonstrated to function during senescence as well as under carbon and nitrogen starvation. In addition, autophagy has been shown to facilitate the degradation of chloroplasts, protein aggregates, and oxidized proteins (Li and Vierstra, 2012Li F. Vierstra R.D. Autophagy: a multifaceted intracellular system for bulk and selective recycling.Trends Plant Sci. 2012; 17: 526-537Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Though pexophagy has been described in yeast and mammals, no evidence had accumulated as to its existence in plants (Oku and Sakai, 2010Oku M. Sakai Y. Peroxisomes as dynamic organelles: autophagic degradation.FEBS J. 2010; 277: 3289-3294Crossref PubMed Scopus (68) Google Scholar). However, in the last few months, four independent studies have demonstrated the existence of pexophagy in Arabidopsis (Farmer et al., 2013Farmer L.M. Rinaldi M.A. Young P.G. Danan C.H. Burkhart S.E. Bartel B. Disrupting autophagy restores peroxisome function to an Arabidopsis lon2 mutant and reveals a role for the LON2 protease in peroxisomal matrix protein degradation.Plant Cell. 2013; 25: 4085-4100Crossref PubMed Scopus (101) Google Scholar; Kim et al., 2013Kim J. Lee H. Lee H.N. Kim S.H. Shin K.D. Chung T. Autophagy-related proteins are required for degradation of peroxisomes in Arabidopsis hypocotyls during seedling growth.Plant Cell. 2013; 25: 4956-4966Crossref PubMed Scopus (97) Google Scholar; Shibata et al., 2013Shibata M. Oikawa K. Yoshimoto K. Kondo M. Mano S. Yamada K. Hayashi M. Sakamoto W. Ohsumi Y. Nishimura M. Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis.Plant Cell. 2013; 25: 4967-4983Crossref PubMed Scopus (146) Google Scholar; Yoshimoto et al., 2014Yoshimoto K. Shibata M. Kondo M. Oikawa K. Sato M. Toyooka K. Shirasu K. Nishimura M. Ohsumi Y. Quality control of plant peroxisomes in organ specific manner via autophagy.J. Cell Sci. 2014; 127: 1161-1168Crossref PubMed Scopus (86) Google Scholar). β-oxidation of fatty acids provides the major carbon source in young Arabidopsis seedlings until photosynthesis is established. The product of β-oxidation, acetyl-CoA, is utilized by enzymes of the glyoxylate cycle, located in the peroxisome matrix (Hu et al., 2012Hu J. Baker A. Bartel B. Linka N. Mullen R.T. Reumann S. Zolman B.K. Plant peroxisomes: biogenesis and function.Plant Cell. 2012; 24: 2279-2303Crossref PubMed Scopus (315) Google Scholar). As the seedling grows, degradation of glyoxylate cycle enzymes occurs in parallel to accumulation of photorespiratory enzymes in the peroxisome. Recently, Kim et al., 2013Kim J. Lee H. Lee H.N. Kim S.H. Shin K.D. Chung T. Autophagy-related proteins are required for degradation of peroxisomes in Arabidopsis hypocotyls during seedling growth.Plant Cell. 2013; 25: 4956-4966Crossref PubMed Scopus (97) Google Scholar tested the hypothesis that pexophagy contributes to the degradation of obsolete peroxisomes during seedling growth. They show that autophagy knockout mutants accumulate fluorescent peroxisomal markers in comparison to wild-type (WT) seedlings as well as endogenous glyoxylate cycle enzymes, suggesting that autophagy functions in peroxisomal degradation. In addition, they demonstrate that peroxisomes are targeted to the central vacuole for degradation and that this is facilitated by ATG genes. Surprisingly, peroxisomal function remained intact in autophagy mutants and peroxisomal enzymatic composition did change, albeit later than WT seedlings. The authors suggest that this is due to the function of additional degradation processes occurring in the peroxisomes, such as the existence of endogenous peroxisomal proteases and ubiquitin-mediated transport out of the peroxisome followed by proteasomal degradation (Kim et al., 2013Kim J. Lee H. Lee H.N. Kim S.H. Shin K.D. Chung T. Autophagy-related proteins are required for degradation of peroxisomes in Arabidopsis hypocotyls during seedling growth.Plant Cell. 2013; 25: 4956-4966Crossref PubMed Scopus (97) Google Scholar). The authors postulate that, as the seedling matures, remodeling of the peroxisomal population occurs either by glyoxylate cycle enzymes being degraded simultaneously to photorespiratory enzymes being imported into the peroxisomal matrix or obsolete peroxisomes containing glyoxylate cycle enzymes being degraded by the autophagy machinery (Figure 1A). Remarkably, shortly prior to Kim et al., 2013Kim J. Lee H. Lee H.N. Kim S.H. Shin K.D. Chung T. Autophagy-related proteins are required for degradation of peroxisomes in Arabidopsis hypocotyls during seedling growth.Plant Cell. 2013; 25: 4956-4966Crossref PubMed Scopus (97) Google Scholar, a manuscript was published demonstrating a crosstalk between degradation mechanisms of peroxisomes (Farmer et al., 2013Farmer L.M. Rinaldi M.A. Young P.G. Danan C.H. Burkhart S.E. Bartel B. Disrupting autophagy restores peroxisome function to an Arabidopsis lon2 mutant and reveals a role for the LON2 protease in peroxisomal matrix protein degradation.Plant Cell. 2013; 25: 4085-4100Crossref PubMed Scopus (101) Google Scholar). Farmer et al., 2013Farmer L.M. Rinaldi M.A. Young P.G. Danan C.H. Burkhart S.E. Bartel B. Disrupting autophagy restores peroxisome function to an Arabidopsis lon2 mutant and reveals a role for the LON2 protease in peroxisomal matrix protein degradation.Plant Cell. 2013; 25: 4085-4100Crossref PubMed Scopus (101) Google Scholar investigated the role of LON2, a peroxisomal protease (see Supplemental Text 1 for further information). lon2 mutants are defective in peroxisome metabolism and import but not in matrix protein degradation, displaying enlarged peroxisomes that are less abundant than the WT. A forward genetic screen of lon2 suppressors yielded mutations in several ATG genes. The double mutant possessed peroxisomes that were similar in shape and number to WT peroxisomes, but accumulated peroxisomal matrix proteins. The work of Farmer et al., 2013Farmer L.M. Rinaldi M.A. Young P.G. Danan C.H. Burkhart S.E. Bartel B. Disrupting autophagy restores peroxisome function to an Arabidopsis lon2 mutant and reveals a role for the LON2 protease in peroxisomal matrix protein degradation.Plant Cell. 2013; 25: 4085-4100Crossref PubMed Scopus (101) Google Scholar not only coincides with the model suggested by Kim et al., 2013Kim J. Lee H. Lee H.N. Kim S.H. Shin K.D. Chung T. Autophagy-related proteins are required for degradation of peroxisomes in Arabidopsis hypocotyls during seedling growth.Plant Cell. 2013; 25: 4956-4966Crossref PubMed Scopus (97) Google Scholar, but also implies an additional role for pexophagy as a quality control mechanism for damaged peroxisomes (Figure 1A). Taken together, the two manuscripts demonstrate the existence of pexophagy in plants and its function in seedling development as well as peroxisome quality control. One of the major functions of peroxisomes is the detoxification of reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2) (Platta and Erdmann, 2007Platta H.W. Erdmann R. Peroxisomal dynamics.Trends Cell Biol. 2007; 17: 474-484Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). This function is important in young seedlings during β-oxidation, but also in older plants, as part of photorespiration (Hu et al., 2012Hu J. Baker A. Bartel B. Linka N. Mullen R.T. Reumann S. Zolman B.K. Plant peroxisomes: biogenesis and function.Plant Cell. 2012; 24: 2279-2303Crossref PubMed Scopus (315) Google Scholar). The enzyme facilitating the breakdown of H2O2 is catalase (CAT) (Hu et al., 2012Hu J. Baker A. Bartel B. Linka N. Mullen R.T. Reumann S. Zolman B.K. Plant peroxisomes: biogenesis and function.Plant Cell. 2012; 24: 2279-2303Crossref PubMed Scopus (315) Google Scholar). Yoshimoto et al., 2014Yoshimoto K. Shibata M. Kondo M. Oikawa K. Sato M. Toyooka K. Shirasu K. Nishimura M. Ohsumi Y. Quality control of plant peroxisomes in organ specific manner via autophagy.J. Cell Sci. 2014; 127: 1161-1168Crossref PubMed Scopus (86) Google Scholar have recently demonstrated that autophagy knockout mutants accumulate peroxisomes in comparison to 4-week-old WT plants. In addition, the localization pattern of peroxisomes in the cell was different in autophagy mutants with peroxisomes tending to aggregate (Yoshimoto et al., 2014Yoshimoto K. Shibata M. Kondo M. Oikawa K. Sato M. Toyooka K. Shirasu K. Nishimura M. Ohsumi Y. Quality control of plant peroxisomes in organ specific manner via autophagy.J. Cell Sci. 2014; 127: 1161-1168Crossref PubMed Scopus (86) Google Scholar). A similar observation was reported by Shibata et al., 2013Shibata M. Oikawa K. Yoshimoto K. Kondo M. Mano S. Yamada K. Hayashi M. Sakamoto W. Ohsumi Y. Nishimura M. Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis.Plant Cell. 2013; 25: 4967-4983Crossref PubMed Scopus (146) Google Scholar in a screen of mutants displaying unusual peroxisomal localization. Map-based cloning of the mutants, displaying peroxisomal aggregates and accumulation of peroxisomes, revealed that the mutations occurred in autophagy-related genes (Shibata et al., 2013Shibata M. Oikawa K. Yoshimoto K. Kondo M. Mano S. Yamada K. Hayashi M. Sakamoto W. Ohsumi Y. Nishimura M. Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis.Plant Cell. 2013; 25: 4967-4983Crossref PubMed Scopus (146) Google Scholar). This accumulation was not observed for other organelles, such as the Golgi apparatus, mitochondria, endoplasmic reticulum, and chloroplast (Yoshimoto et al., 2014Yoshimoto K. Shibata M. Kondo M. Oikawa K. Sato M. Toyooka K. Shirasu K. Nishimura M. Ohsumi Y. Quality control of plant peroxisomes in organ specific manner via autophagy.J. Cell Sci. 2014; 127: 1161-1168Crossref PubMed Scopus (86) Google Scholar). Interestingly, both manuscripts report an accumulation of insoluble CAT in the aggregated peroxisomes, also visible as electron-dense bodies within peroxisomes using electron microscopy (EM). This CAT was significantly less active than soluble CAT (Shibata et al., 2013Shibata M. Oikawa K. Yoshimoto K. Kondo M. Mano S. Yamada K. Hayashi M. Sakamoto W. Ohsumi Y. Nishimura M. Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis.Plant Cell. 2013; 25: 4967-4983Crossref PubMed Scopus (146) Google Scholar; Yoshimoto et al., 2014Yoshimoto K. Shibata M. Kondo M. Oikawa K. Sato M. Toyooka K. Shirasu K. Nishimura M. Ohsumi Y. Quality control of plant peroxisomes in organ specific manner via autophagy.J. Cell Sci. 2014; 127: 1161-1168Crossref PubMed Scopus (86) Google Scholar). Shibata et al., 2013Shibata M. Oikawa K. Yoshimoto K. Kondo M. Mano S. Yamada K. Hayashi M. Sakamoto W. Ohsumi Y. Nishimura M. Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis.Plant Cell. 2013; 25: 4967-4983Crossref PubMed Scopus (146) Google Scholar go on to show, using reduction-oxidation-sensitive GFP, that the aggregated peroxisomes in autophagy mutants are more oxidative than the dispersed peroxisomes of the WT, suggesting ROS accumulation in peroxisomes with defective CAT activity. Moreover, the authors show that treatment with H2O2 induces peroxisome aggregation in WT plants and that cat mutants display a slightly higher number for peroxisomal aggregates than WT plants, suggesting that H2O2 indeed causes peroxisomal aggregation (Shibata et al., 2013Shibata M. Oikawa K. Yoshimoto K. Kondo M. Mano S. Yamada K. Hayashi M. Sakamoto W. Ohsumi Y. Nishimura M. Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis.Plant Cell. 2013; 25: 4967-4983Crossref PubMed Scopus (146) Google Scholar). To summarize, both manuscripts postulate that pexophagy is involved in the clearance of malfunctioning peroxisomes in older plants: peroxisomes containing active CAT maintain a low level of H2O2, over time CAT activity is diminished and H2O2 accumulates in the peroxisome, resulting in its degradation via the autophagy machinery. Perturbations such as H2O2 treatment or cat mutation result in accumulation of damaged peroxisomes which, in turn, form aggregates and thus promote autophagy (Figure 1B). This selective degradation of cellular constituents by autophagy is facilitated by receptor proteins that link the targets with ATG proteins. The receptor for pexophagy was initially identified in the methylotrophic yeast Pichia pastoris and termed Atg30. However, homologs of Atg30 were not found in higher eukaryotes (Oku and Sakai, 2010Oku M. Sakai Y. Peroxisomes as dynamic organelles: autophagic degradation.FEBS J. 2010; 277: 3289-3294Crossref PubMed Scopus (68) Google Scholar). Atg8, an autophagosome membrane protein, was shown to play a role in selective cargo recruitment by binding autophagy receptor proteins (Li and Vierstra, 2012Li F. Vierstra R.D. Autophagy: a multifaceted intracellular system for bulk and selective recycling.Trends Plant Sci. 2012; 17: 526-537Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) (see Supplemental Text 1 for further information). Indeed, many of the works described above demonstrate co-localization of Atg8 and peroxisomes, specifically aggregated peroxisomes, suggesting a role for Atg8 in targeting specific peroxisomes for degradation (Figure 1) (Kim et al., 2013Kim J. Lee H. Lee H.N. Kim S.H. Shin K.D. Chung T. Autophagy-related proteins are required for degradation of peroxisomes in Arabidopsis hypocotyls during seedling growth.Plant Cell. 2013; 25: 4956-4966Crossref PubMed Scopus (97) Google Scholar; Shibata et al., 2013Shibata M. Oikawa K. Yoshimoto K. Kondo M. Mano S. Yamada K. Hayashi M. Sakamoto W. Ohsumi Y. Nishimura M. Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis.Plant Cell. 2013; 25: 4967-4983Crossref PubMed Scopus (146) Google Scholar; Yoshimoto et al., 2014Yoshimoto K. Shibata M. Kondo M. Oikawa K. Sato M. Toyooka K. Shirasu K. Nishimura M. Ohsumi Y. Quality control of plant peroxisomes in organ specific manner via autophagy.J. Cell Sci. 2014; 127: 1161-1168Crossref PubMed Scopus (86) Google Scholar). However, the autophagy adaptor protein linking Atg8 to the damaged peroxisomes has not been identified. In mammalian systems, it has been suggested that polyubiquitination of peroxisomal membrane proteins recruits p62 and NBR1, autophagy adaptors which bind Atg8 (Oku and Sakai, 2010Oku M. Sakai Y. Peroxisomes as dynamic organelles: autophagic degradation.FEBS J. 2010; 277: 3289-3294Crossref PubMed Scopus (68) Google Scholar) (see Supplemental Text 1 for further information). A p62/NBR1 homolog exists in Arabidopsis, and nbr1 mutant was shown to be sensitive to oxidative stress, suggesting possible involvement in pexophagy (Zhou et al., 2013Zhou J. Wang J. Cheng Y. Chi Y. Fan B. Yu J. Chen Z. NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses.PLoS Genet. 2013; 9: e1003196Crossref PubMed Scopus (229) Google Scholar). However, immuno-EM analysis using anti-ubiquitin antibodies did not reveal ubiquitination of damaged peroxisomes (Yoshimoto et al., 2014Yoshimoto K. Shibata M. Kondo M. Oikawa K. Sato M. Toyooka K. Shirasu K. Nishimura M. Ohsumi Y. Quality control of plant peroxisomes in organ specific manner via autophagy.J. Cell Sci. 2014; 127: 1161-1168Crossref PubMed Scopus (86) Google Scholar). Additionally, autophagy mutants are suggested to display an early senescence phenotype, stemming from H2O2 accumulation (Yoshimoto et al., 2009Yoshimoto K. Jikumaru Y. Kamiya Y. Kusano M. Consonni C. Panstruga R. Ohsumi Y. Shirasu K. Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis.Plant Cell. 2009; 21: 2914-2927Crossref PubMed Scopus (414) Google Scholar). The nbr1 mutant does not display early senescence, putting the involvement of this protein in pexophagy under question (Zhou et al., 2013Zhou J. Wang J. Cheng Y. Chi Y. Fan B. Yu J. Chen Z. NBR1-mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant stress responses.PLoS Genet. 2013; 9: e1003196Crossref PubMed Scopus (229) Google Scholar). Shibata et al., 2013Shibata M. Oikawa K. Yoshimoto K. Kondo M. Mano S. Yamada K. Hayashi M. Sakamoto W. Ohsumi Y. Nishimura M. Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis.Plant Cell. 2013; 25: 4967-4983Crossref PubMed Scopus (146) Google Scholar directly examined whether CAT was the signal for pexophagy. However, crossing cat mutants with autophagy mutants increased the number of peroxisomal aggregates in the cell compared to cat mutants, suggesting that pexophagy occurs in the absence of CAT. The authors postulate that other damaged proteins and lipids accumulate in the peroxisomes destined for degradation and one of these might be inducing pexophagy (Shibata et al., 2013Shibata M. Oikawa K. Yoshimoto K. Kondo M. Mano S. Yamada K. Hayashi M. Sakamoto W. Ohsumi Y. Nishimura M. Highly oxidized peroxisomes are selectively degraded via autophagy in Arabidopsis.Plant Cell. 2013; 25: 4967-4983Crossref PubMed Scopus (146) Google Scholar). Identification of the autophagy adaptor required for pexophagy will help shed more light on the regulation of this fascinating process. Supplementary Data are available at Molecular Plant Online. Work on autophagy in our laboratory is supported by Minerva, Alexander von Humboldt and EMBO fellowships (T.A.W.), and by the Max-Planck Society (A.R.F.). No conflict of interest declared.