title: An inhibitor of oil body mobilization in Arabidopsis.
abstract: Fatty acid β-oxidation is an essential process in many aspects of plant development, and storage oil in the form of triacylglycerol (TAG) is an important food source for humans and animals, for biofuel and for industrial feedstocks. In this study we characterize the effects of a small molecule, diphenyl methylphosphonate, on oil mobilization in Arabidopsis thaliana. Confocal laser scanning microscopy, transmission electron microscopy and quantitative lipid profiling were used to examine the effects of diphenyl methylphosphonate treatment on seedlings. Diphenyl methylphosphonate causes peroxisome clustering around oil bodies but does not affect morphology of other cellular organelles. We show that this molecule blocks the breakdown of pre-existing oil bodies resulting in retention of TAG and accumulation of acyl CoAs. The biochemical and phenotypic effects are consistent with a block in the early part of the β-oxidation pathway. Diphenyl methylphosphonate appears to be a fairly specific inhibitor of TAG mobilization in plants and whilst further work is required to identify the molecular target of the compound it should prove a useful tool to interrogate and manipulate these pathways in a controlled and reproducible manner.
authors.affiliation: Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Laura-Anne
authors.initials: LA
authors.lastname: Brown
authors.affiliation: Centre for Novel Agricultural Products, Department of Biology, University of York, Wentworth Way, Heslington, York, YO10 5DD, UK.
authors.forename: Tony R
authors.initials: TR
authors.lastname: Larson
authors.affiliation: Centre for Novel Agricultural Products, Department of Biology, University of York, Wentworth Way, Heslington, York, YO10 5DD, UK.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Graham
authors.affiliation: Department of Biological and Medical Sciences, Oxford Brookes University, Gipsy Lane, Oxford, OX3 0BP, UK.
authors.forename: Chris
authors.initials: C
authors.lastname: Hawes
authors.affiliation: Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Rupesh
authors.initials: R
authors.lastname: Paudyal
authors.affiliation: School of Chemistry, Faculty of Mathematics and Physical Sciences, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Stuart L
authors.initials: SL
authors.lastname: Warriner
authors.affiliation: Centre for Plant Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Alison
authors.initials: A
authors.lastname: Baker
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D010084:Oxidation-Reduction; D020675:Peroxisomes / Q000378:metabolism*; D010938:Plant Oils / Q000378:metabolism*; D036226:Seedlings; D014280:Triglycerides / Q000378:metabolism*
chemical_list: D000214:Acyl Coenzyme A; D029681:Arabidopsis Proteins; D005227:Fatty Acids; D009943:Organophosphorus Compounds; D010938:Plant Oils; D014280:Triglycerides; C032627:methylphosphonic acid
keywords: Arabidiopsis; inhibitor; lipid metabolism; oil body; peroxisome; triacylglycerol; β-oxidation
references.citation: Afitlhile MM, Fukushige H, Nishimura M, Hildebrand DF. 2005. A defect in glyoxysomal fatty acid β-oxidation reduces jasmonic acid accumulation in Arabidopsis. Plant Physiology and Biochemistry 43: 603-609.
references.citation: Agrimi G, Russo A, Pierri C, Palmieri F. 2012. The peroxisomal NAD+ carrier of Arabidopsis thaliana; transports coenzyme A and its derivatives. Journal of Bioenergetics and Biomembranes 44: 333-340.
references.citation: Arai Y, Hayashi M, Nishimura M. 2008. Proteomic identification and characterization of a novel peroxisomal adenine nucleotide transporter supplying ATP for fatty acid beta-oxidation in soybean and Arabidopsis. Plant Cell 20: 3227-3240.
references.citation: Baker A, Graham I, Holdsworth M, Smith S, Theodoulou F. 2006. Chewing the fat: β-oxidation in signalling and development. Trends in Plant Science 11: 124-132.
references.citation: Batoko H, Zheng H-Q, Hawes C, Moore I. 2000. A Rab1 GTPase is required for transport between the endoplasmic reticulum and Golgi apparatus and for normal Golgi movement in plants. Plant Cell 12: 2201-2218.
references.citation: Bernhardt K, Wilkinson S, Weber APM, Linka N. 2012. A peroxisomal carrier delivers NAD+ and contributes to optimal fatty acid degradation during storage oil mobilization. Plant Journal 69: 1-13.
references.citation: Brown L-A, O'Leary-Steele C, Brookes P, Armitage L, Kepinski S, Warriner SL, Baker A. 2011. A small molecule with differential effects on the PTS1 and PTS2 peroxisome matrix import pathways. Plant Journal 65: 980-990.
references.citation: Burgal J, Shockey J, Lu C, Dyer J, Larson T, Graham I, Browse J. 2008. Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil. Plant Biotechnology Journal 6: 819-831.
references.citation: Cutler SR, Ehrhardt DW, Griffitts JS, Somerville CR. 2000. Random GFP: cDNA fusions enable visualization of subcellular structures in cells of Arabidopsis at a high frequency. Proceedings of the National Academy of Sciences, USA 97: 3718-3723.
references.citation: De Marcos Lousa C, van Roermund CWT, Postis VLG, Dietrich D, Kerr ID, Wanders RJA, Baldwin SA, Baker A, Theodoulou FL. 2013. Intrinsic acyl-CoA thioesterase activity of a peroxisomal ATP binding cassette transporter is required for transport and metabolism of fatty acids. Proceedings of the National Academy of Sciences, USA 110: 1279-1284.
references.citation: Dietrich D, Schmuths H, De Marcos Lousa C, Baldwin JM, Baldwin SA, Baker A, Theodoulou FL, Holdsworth MJ. 2009. Mutations in the Arabidopsis peroxisomal ABC transporter COMATOSE allow differentiation between multiple functions in planta: insights from an allelic series. Molecular Biology of the Cell 20: 530-543.
references.citation: Eastmond PJ. 2006. SUGAR-DEPENDENT1 encodes a patatin domain triacylglycerol lipase that initiates storage oil breakdown in germinating Arabidopsis seeds. Plant Cell 18: 665-675.
references.citation: Eastmond PJ. 2007. MONODEHYROASCORBATE REDUCTASE4 is required for seed storage oil hydrolysis and postgerminative growth in Arabidopsis. Plant Cell 19: 1376-1387.
references.citation: Faso C, Chen Y-N, Tamura K, Held M, Zemelis S, Marti L, Saravanan R-S, Hummel E, Kung L, Miller E et al. 2009. A missense mutation in the Arabidopsis COPII coat protein Sec24A induces the formation of clusters of the endoplasmic reticulum and Golgi apparatus. Plant Cell 21: 3655-3671.
references.citation: Footitt S, Dietrich D, Fait A, Fernie AR, Holdsworth MJ, Baker A, Theodoulou FL. 2007. The COMATOSE ATP-binding cassette transporter is required for full fertility in arabidopsis. Plant Physiology 144: 1467-1480.
references.citation: Footitt S, Slocombe SP, Larner V, Kurup S, Wu YS, Larson T, Graham I, Baker A, Holdsworth M. 2002. Control of germination and lipid mobilization by COMATOSE, the Arabidopsis homologue of human ALDP. EMBO Journal 21: 2912-2922.
references.citation: Fulda M, Schnurr J, Abbadi A, Heinz E, Browse J. 2004. Peroxisomal acyl-CoA synthetase activity is essential for seedling development in Arabidopsis thaliana. Plant Cell 16: 394-405.
references.citation: Germain V, Rylott EL, Larson TR, Sherson SM, Bechtold N, Carde JP, Bryce JH, Graham IA, Smith SM. 2001. Requirement for 3-ketoacyl-CoA thiolase-2 in peroxisome development, fatty acid beta-oxidation and breakdown of triacylglycerol in lipid bodies of Arabidopsis seedlings. Plant Journal 28: 1-12.
references.citation: Goepfert S, Poirier Y. 2007. beta-oxidation in fatty acid degradation and beyond. Current Opinion in Plant Biology 10: 245-251.
references.citation: Graham IA. 2008. Seed storage oil mobilization. Annual Review of Plant Biology 59: 115-142.
references.citation: Hayashi M, Toriyama K, Kondo M, Nishimura M. 1998. 2,4-dichlorophenoxybutyric acid-resistant mutants of Arabidopsis have defects in glyoxysomal fatty acid beta- oxidation. Plant Cell 10: 183-195.
references.citation: Hernandez ML, Whitehead L, He Z, Gazda V, Gilday AD, Kozhevnikova E, Vaistij FE, Larson TR, Graham IA. 2012. A cytosolic acyl transferase contributes to triacylglycerol synthesis in sucrose-rescued Arabidopsis seed oil catabolism mutants. Plant Physiology 160: 215-225.
references.citation: Hu J, Baker A, Bartel B, Linka N, Mullen RT, Reumann S, Zolman BK. 2012. Plant peroxisomes: biogenesis and function. Plant Cell 24: 2279-2303.
references.citation: Kelly AA, Quettier A-L, Shaw E, Eastmond PJ. 2011. Seed storage oil mobilization is important but not essential for germination or seedling establishment in Arabidopsis. Plant Physiology 157: 866-875.
references.citation: Kunze M, Pracharoenwattana I, Smith SM, Hartig A. 2006. A central role for the peroxisomal membrane in glyoxylate cycle function. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1763: 1441.
references.citation: Larson TR, Edgell T, Byrne J, Dehesh K, Graham IA. 2002. Acyl CoA profiles of transgenic plants that accumulate medium- chain fatty acids indicate inefficient storage lipid synthesis in developing oilseeds. Plant Journal 32: 519-527.
references.citation: Larson TR, Graham IA. 2001. A novel technique for the sensitive quantification of acyl CoA esters from plant tissues. Plant Journal 25: 115-125.
references.citation: Linka N, Theodoulou FL, Haslam RP, Linka M, Napier JA, Neuhaus HE, Weber APM. 2008. Peroxisomal ATP import is essential for seedling development in Arabidopsis thaliana. Plant Cell 20: 3241-3257.
references.citation: Logan DC, Leaver CJ. 2000. Mitochondria-targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells. Journal of Experimental Botany 51: 865-871.
references.citation: Murphy DJ. 2012. The dynamic roles of intracellular lipid droplets: from archaea to mammals. Protoplasma 249: 541-585.
references.citation: Oskolkova OV, Hermetter A. 2002. Fluorescent inhibitors reveal solvent-dependent micropolarity in the lipid binding sites of lipases. Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology 1597: 60-66.
references.citation: Pinfield-Wells H, Rylott EL, Gilday AD, Graham S, Job K, Larson TR, Graham IA. 2005. Sucrose rescues seedling establishment but not germination of Arabidopsis mutants disrupted in peroxisomal fatty acid catabolism. Plant Journal 43: 861-872.
references.citation: Pracharoenwattana I, Cornah JE, Smith SM. 2005. Arabidopsis peroxisomal citrate synthase is required for fatty acid respiration and seed germination. Plant Cell 17: 2037-2048.
references.citation: Richmond TA, Bleecker AB. 1999. A defect in β-oxidation causes abnormal inflorescence development in Arabidopsis. Plant Cell 11: 1911-1923.
references.citation: Rubio S, Larson TR, Gonzalez-Guzman M, Alejandro S, Graham IA, Serrano R, Rodriguez PL. 2006. An Arabidopsis mutant impaired in CoA biosynthesis is sugar-dependent for seedling establishment. Plant Physiology 140: 830-843.
references.citation: Rylott EL, Eastmond PJ, Gilday AD, Slocombe SP, Larson TR, Baker A, Graham IA. 2006. The Arabidopsis thaliana multifunctional protein gene (MFP2) of peroxisomal beta-oxidation is essential for seedling establishment. Plant Journal 45: 930-941.
references.citation: Saint-Jore CM, Evins J, Batoko H, Brandizzi F, Moore I, Hawes C. 2002. Redistribution of membrane proteins between the Golgi apparatus and endoplasmic reticulum in plants is reversible and not dependent on cytoskeletal networks. Plant Journal 29: 661-678.
references.citation: Salisbury CM, Ellman JA. 2006. Rapid identification of potent nonpeptidic serine protease inhibitors. ChemBioChem 7: 1034-1037.
references.citation: Schulz H. 1983. Metabolism of 4-pentanoic acid and inhibition of thiolase by metabolites of 4-pentanoic acid. Biochemistry 22: 1827-1832.
references.citation: Schulz H. 1987. Inhibitors of fatty acid oxidation. Life Sciences 40: 1443-1449.
references.citation: Sheahan MB, Staiger CJ, Rose RJ, McCurdy DW. 2004. A green fluorescent protein fusion to actin-binding domain 2 of Arabidopsis fimbrin highlights new features of a dynamic actin cytoskeleton in live plant cells. Plant Physiology 136: 3968-3978.
references.citation: Slocombe SP, Cornah J, Pinfield-Wells H, Soady K, Zhang Q, Gilday A, Dyer JM, Graham IA. 2009. Oil accumulation in leaves directed by modification of fatty acid breakdown and lipid synthesis pathways. Plant Biotechnology Journal 7: 694-703.
references.citation: Surpin M, Rojas-Pierce M, Carter C, Hicks GR, Vasquez J, Raikhel NV. 2005. The power of chemical genomics to study the link between endomembrane system components and the gravitropic response. Proceedings of the National Academy of Sciences, USA 102: 4902-4907.
references.citation: Theodoulou FL, Job K, Slocombe SP, Footitt S, Holdsworth M, Baker A, Larson TR, Graham IA. 2005. Jasmonic acid levels are reduced in COMATOSE ATP-binding cassette transporter mutants. Implications for transport of jasmonate precursors into peroxisomes. Plant Physiology 137: 835-840.
references.citation: Tilton GB, Shockey JM, Browse J. 2004. Biochemical and molecular characterization of ACH2, an acyl-CoA thioesterase from Arabidopsis thaliana. Journal of Biological Chemistry 279: 7487-7494.
references.citation: Wahlroos T, Soukka J, Denesyuk A, Wahlroos R, Korpela T, Kilby NJ. 2003. Oleosin expression and trafficking during oil body biogenesis in tobacco leaf cells. Genesis 35: 125-132.
references.citation: Wiszniewski AAG, Zhou W, Smith SM, Bussell JD. 2009. Identification of two Arabidopsis genes encoding a peroxisomal oxidoreductase-like protein and an acyl-CoA synthetase-like protein that are required for responses to pro-auxins. Plant Molecular Biology 69: 503-515.
references.citation: Youssef J, Abdel-aleem S, Badr M. 1994. Enoximone inhibits hepatic mitochondrial long-chain acyl-CoA synthetase. Toxicology Letters 74: 15-21.
references.citation: Zolman BK, Monroe-Augustus M, Thompson B, Hawes JW, Krukenberg KA, Matsuda SPT, Bartel B. 2001a. chy1, an Arabidopsis mutant with impaired beta-oxidation, is defective in a peroxisomal beta-hydroxyisobutyryl-CoA hydrolase. Journal of Biological Chemistry 276: 31037-31046.
references.citation: Zolman BK, Silva ID, Bartel B. 2001b. The Arabidopsis pxa1 mutant is defective in an ATP-binding cassette transporter-like protein required for peroxisomal fatty acid beta-oxidation. Plant Physiology 127: 1266-1278.
references.citation: Zolman BK, Yoder A, Bartel B. 2000. Genetic analysis of indole-3-butyric acid responses in Arabidopsis thaliana reveals four mutant classes. Genetics 156: 1323-1337.