The Roles of β-Oxidation and Cofactor Homeostasis in Peroxisome Distribution and Function in Arabidopsis thaliana

Abstract

Key steps of essential metabolic pathways are housed in plant peroxisomes. We conducted a microscopy-based screen for anomalous distribution of peroxisomally targeted fluorescence in Arabidopsis thaliana This screen uncovered 34 novel alleles in 15 genes affecting oil body mobilization, fatty acid β-oxidation, the glyoxylate cycle, peroxisome fission, and pexophagy. Partial loss-of-function of lipid-mobilization enzymes conferred peroxisomes clustered around retained oil bodies without other notable defects, suggesting that this microscopy-based approach was sensitive to minor perturbations, and that fatty acid β-oxidation rates in wild type are higher than required for normal growth. We recovered three mutants defective in PECTIN METHYLESTERASE31, revealing an unanticipated role in lipid mobilization for this cytosolic enzyme. Whereas mutations reducing fatty acid import had peroxisomes of wild-type size, mutations impairing fatty acid β-oxidation displayed enlarged peroxisomes, possibly caused by excess fatty acid β-oxidation intermediates in the peroxisome. Several fatty acid β-oxidation mutants also displayed defects in peroxisomal matrix protein import. Impairing fatty acid import reduced the large size of peroxisomes in a mutant defective in the PEROXISOMAL NAD TRANSPORTER (PXN), supporting the hypothesis that fatty acid accumulation causes pxn peroxisome enlargement. The diverse mutants isolated in this screen will aid future investigations of the roles of β-oxidation and peroxisomal cofactor homeostasis in plant development.

The PEX1 ATPase Stabilizes PEX6 and Plays Essential Roles in Peroxisome Biology

“…We isolated pex1-3 in a screen for seedlings displaying a pattern of peroxisome-targeted GFP (GFP-PTS1) distribution that differed from that of the wild type (Rinaldi et al, 2016). The pex1-3 mutant displayed second AAA ATPase domain of PEX1 was aligned to that of human (Hs) PEX1, mouse (Mm) p97, human PEX6, and Arabidopsis PEX6 using ClustalW on Lasergene MegAlign (DNAStar).…”

“…Peroxisomespecific autophagy (pexophagy) was discovered recently in plants (for review, see Young and Bartel, 2016); autophagy-defective seedlings have increased numbers of peroxisomes (Kim et al, 2013;Shibata et al, 2013). We isolated PEX1/pex1-3 from a microscopybased screen for mutants with altered GFP-PTS1 patterning that were visible under relatively low magnification (Rinaldi et al, 2016). Although this screen yielded mutations in 15 genes that conferred aberrantly large or clustered peroxisomes (Rinaldi et al, 2016), PEX1/pex1-3 was the only peroxin mutant recovered.…”

“…Such mutants often are also resistant to the synthetic IBA analog 2,4-dichlorophenoxybutyric acid (2,4-DB), which is similarly b-oxidized to the auxinic herbicide 2,4-dichlorophenoxyacetic acid (Wain and Wightman, 1954;Hayashi et al, 1998). At a cellular level, defects in peroxisomal size, positioning, and matrix protein import can be visualized by tagging GFP with a PTS, which also has formed the basis of screens for mutants with peroxisome defects (Mano et al, 2006;Burkhart et al, 2013;Rinaldi et al, 2016). Because PTS2 cleavage occurs after import, mutations in peroxin genes often impair PTS2 processing (for review, see Bartel et al, 2014).…”

Peroxisome Function, Biogenesis, and Dynamics in Plants

“…Various mutants defective in b-oxidation, the glyoxylate cycle, or NADH reduction display peroxisomes clustered near oil bodies after oil bodies are depleted in wild type (Germain et al, 2001;Eastmond, 2007;Pracharoenwattana et al, 2007;Cassin-Ross and Hu, 2014;Rinaldi et al, 2016). Moreover, diphenyl methylphosphonate confers triacylglycerol retention (Brown et al, 2013) similar to sdp1 and pxa1 mutants (Eastmond, 2006;Slocombe et al, 2009;Kelly et al, 2013).…”

“…MFP2 and AIM1 require NAD + , which is imported by the PXN peroxisomal NAD + carrier and recycled by peroxisomal NAD + -malate dehydrogenase (PMDH) and monodehydroascorbate reductase (MDAR4). Consequently, pmdh, mdar4, and pxn mutants also display b-oxidation defects (Eastmond, 2007;Pracharoenwattana et al, 2007;Bernhardt et al, 2012;Rinaldi et al, 2016;van Roermund et al, 2016).…”