Abstract
Plants use two pathways for the production of the same universal isoprenoid precursors: the mevalonic acid (MVA) pathway and the methylerythritol 4-phosphate (MEP) pathway. Inhibitors of the MVA pathway prevent the activity of the shoot apical meristem and the development of true leaves in seedlings, whereas those inhibiting the MEP pathway show an additional bleaching phenotype. Here, we describe two methods to quantify plant resistance to inhibitors of the MVA pathway or the MEP pathway based on seedling establishment and photosynthetic pigment measurements. Although the methods are presented for Arabidopsis, they are valid for other plant species. These methods can be used as inexpensive and high-throughput alternatives to in vitro assays to estimate the activity of the corresponding target enzymes and to screen for mutants with altered levels or activities of these enzymes.
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References
Pulido P, Perello C, Rodriguez-Concepcion M (2012) New insights into plant isoprenoid metabolism. Mol Plant 5:964–967
Vranova E, Coman D, Gruissem W (2013) Network analysis of the MVA and MEP pathways for isoprenoid synthesis. Annu Rev Plant Biol 64:665–700
Campos N, Boronat A (1995) Targeting and topology in the membrane of plant 3-hydroxy-3-methylglutaryl coenzyme A reductase. Plant Cell 7:2163–2174
Rodríguez-Concepción M (2006) Early steps in isoprenoid biosynthesis: multilevel regulation of the supply of common precursors in plant cells. Phytochem Rev 5:1–15
Pulido P, Toledo-Ortiz G, Phillips MA, Wright LP, Rodriguez-Concepcion M (2013) Arabidopsis J-protein J20 delivers the first enzyme of the plastidial isoprenoid pathway to protein quality control. Plant Cell 25:4183–4194
Rodríguez-Concepción M, Forés O, Martínez-García JF, González V, Phillips MA, Ferrer A, Boronat A (2004) Distinct light-mediated pathways regulate the biosynthesis and exchange of isoprenoid precursors during Arabidopsis seedling development. Plant Cell 16:144–156
Carretero-Paulet L, Cairo A, Botella-Pavia P, Besumbes O, Campos N, Boronat A, Rodriguez-Concepcion M (2006) Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase. Plant Mol Biol 62:683–695
Alberts AW, Chen J, Kuron G, Hunt V, Huff J, Hoffman C, Rothrock J, Lopez M, Joshua H, Harris E, Patchett A, Monaghan R, Currie S, Stapley E, Albers-Schonberg G, Hensens O, Hirshfield J, Hoogsteen K, Liesch J, Springer J (1980) Mevinolin: a highly potent competitive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent. Proc Natl Acad Sci U S A 77:3957–3961
Istvan ES, Deisenhofer J (2001) Structural mechanism for statin inhibition of HMG-CoA reductase. Science 292:1160–1164
Re EB, Jones D, Learned RM (1995) Co-expression of native and introduced genes reveals cryptic regulation of HMG CoA reductase expression in Arabidopsis. Plant J 7:771–784
Kasahara H, Hanada A, Kuzuyama T, Takagi M, Kamiya Y, Yamaguchi S (2002) Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of gibberellins in Arabidopsis. J Biol Chem 277:45188–45194
Zeidler J, Schwender J, Mueller C, Lichtenthaler HK (2000) The non-mevalonate isoprenoid biosynthesis of plants as a test system for drugs against malaria and pathogenic bacteria. Biochem Soc Trans 28:796–798
Matsue Y, Mizuno H, Tomita T, Asami T, Nishiyama M, Kuzuyama T (2010) The herbicide ketoclomazone inhibits 1-deoxy-d-xylulose 5-phosphate synthase in the 2-C-methyl-d-erythritol 4-phosphate pathway and shows antibacterial activity against Haemophilus influenzae. J Antibiot (Tokyo) 63:583–588
Kuzuyama T, Shimizu T, Takahashi S, Seto H (1998) Fosmidomycin, a specific inhibitor of 1-deoxy-d-xylulose 5-phosphate reductoisomerase in the nonmevalonate pathway for terpenoid biosynthesis. Tetrahedron Lett 39:7913–7916
Steinbacher S, Kaiser J, Eisenreich W, Huber R, Bacher A, Rohdich F (2003) Structural basis of fosmidomycin action revealed by the complex with 2-C-methyl-d-erythritol 4-phosphate synthase (IspC). Implications for the catalytic mechanism and anti-malaria drug development. J Biol Chem 278:18401–18407
Flores-Pérez U, Sauret-Güeto S, Gas E, Jarvis P, Rodríguez-Concepción M (2008) A mutant impaired in the production of plastome-encoded proteins uncovers a mechanism for the homeostasis of isoprenoid biosynthetic enzymes in Arabidopsis plastids. Plant Cell 20:1303–1315
Laule O, Furholz A, Chang HS, Zhu T, Wang X, Heifetz PB, Gruissem W, Lange M (2003) Crosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis in Arabidopsis thaliana. Proc Natl Acad Sci U S A 100:6866–6871
Crowell DN, Packard CE, Pierson CA, Giner JL, Downes BP, Chary SN (2003) Identification of an allele of CLA1 associated with variegation in Arabidopsis thaliana. Physiol Plant 118:29–37
Sauret-Güeto S, Botella-Pavia P, Flores-Perez U, Martinez-Garcia JF, San Roman C, Leon P, Boronat A, Rodriguez-Concepcion M (2006) Plastid cues posttranscriptionally regulate the accumulation of key enzymes of the methylerythritol phosphate pathway in Arabidopsis. Plant Physiol 141:75–84
Kobayashi K, Suzuki M, Tang J, Nagata N, Ohyama K, Seki H, Kiuchi R, Kaneko Y, Nakazawa M, Matsui M, Matsumoto S, Yoshida S, Muranaka T (2007) Lovastatin insensitive 1, a Novel pentatricopeptide repeat protein, is a potential regulatory factor of isoprenoid biosynthesis in Arabidopsis. Plant Cell Physiol 48:322–331
Flores-Perez U, Perez-Gil J, Closa M, Wright LP, Botella-Pavia P, Phillips MA, Ferrer A, Gershenzon J, Rodriguez-Concepcion M (2010) Pleiotropic regulatory locus 1 (PRL1) integrates the regulation of sugar responses with isoprenoid metabolism in Arabidopsis. Mol Plant 3:101–112
Leivar P, Antolin-Llovera M, Ferrero S, Closa M, Arro M, Ferrer A, Boronat A, Campos N (2011) Multilevel control of Arabidopsis 3-hydroxy-3-methylglutaryl coenzyme A reductase by protein phosphatase 2A. Plant Cell 23:1494–1511
Rodríguez-Concepción M, Boronat A (2002) Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol 130:1079–1089
Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol 148:351–382
Acknowledgements
Our work is funded by grants from the Catalan AGAUR (2009SGR-26 and XRB), Spanish DGI (BIO2011-23680 and PIM2010IPO-00660), and European Union FP7 (TiMet, contract 245143). We are members of the IBERCAROT network funded by Programa Iberoamericano de Ciencia y Tecnologia para el Desarrollo (CYTED, code 112RT0445). C.P. received a predoctoral fellowship of the JAE-PRE program (CSIC).
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Perelló, C., Rodríguez-Concepción, M., Pulido, P. (2014). Quantification of Plant Resistance to Isoprenoid Biosynthesis Inhibitors. In: Rodríguez-Concepción, M. (eds) Plant Isoprenoids. Methods in Molecular Biology, vol 1153. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0606-2_20
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DOI: https://doi.org/10.1007/978-1-4939-0606-2_20
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