Abstract
The amino acid cysteine plays a major role in plant response to abiotic stress by being the donor of elemental sulfur for the sulfuration of the molybdenum cofactor, otherwise the last step of ABA biosynthesis, the oxidation of abscisic aldehyde, is inactivated. Additionally, cysteine serves as a precursor for the biosynthesis of glutathione, the reactive oxygen species scavenger essential for redox status homeostasis during stress. Cysteine is generated by the sulfate reductive pathway where sulfite oxidase (SO; EC 1.8.3.1) is an important enzyme in the homeostasis of sulfite levels (present either as a toxic intermediate in the pathway or as a toxic air pollutant that has penetrated the plant tissue via the stomata). SO is localized to the peroxisomes and detoxifies excess sulfite by catalyzing its oxidation to sulfate. Here we show a kinetic assay that relies on fuchsin colorimetric detection of sulfite, a substrate of SO activity. This SO assay is highly specific, technically simple, and readily performed in any laboratory.
5′-adenylylsulfate (APS) reductase (APR, E.C. 1.8.4.9) enzyme regulates a crucial step of sulfate assimilation in plants, algae and some human pathogens. The enzyme is upregulated in response to oxidative stress induced by abiotic stresses, such as salinity and hydrogen peroxide, to generate sulfite an intermediate for cysteine generation essential for the biosynthesis of glutathione, the hydrogen peroxide scavenger. Here we present two robust, sensitive, and simple colorimetric methods of APR activity based on sulfite determination by fuchsin.
Sulfite reductase (SiR) is one of the key enzymes in the primary sulfur assimilation pathway. It has been shown that SiR is an important plant enzyme for protection plant against sulfite toxicity and premature senescence. Here we describe two methods for SiR activity determination: a kinetic assay using desalted extract and an in-gel assay using crude extract.
Due to the energetically favorable equilibrium, sulfurtransferase (ST) activity measured as sulfite generation or consumption. Sulfite-generating ST activity is determined by colorimetric detection of SCN− formation at 460 nm as the red Fe(SCN)3 complex from cyanide and thiosulfate using acidic iron reagent. Sulfite-consuming (MST) activity is detected as sulfite disappearance in the presence of thiocyanate (SCN−) or as SCN− disappearance. To abrogate interfering SO activity, total ST activities is detected by inhibiting SO activity with tungstate.
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References
Marschner H (2005) Mineral nutrition of higher plants, 2nd edn. Academeic Press, London
Saito K (2000) Regulation of sulfate transport and synthesis of sulfur-containing amino acids. Curr Opin Plant Biol 3:188–195
Lewandowska M, Sirko A (2008) Recent advances in understanding plant response to sulfur-deficiency stress. Acta Biochim Pol 55(3):457–471
Rausch T, Wachter A (2005) Sulfur metabolism: a versatile platform for launching defence operations. Trends Plant Sci 10(10):503–509
Muttucumaru N, Halford NG, Elmore JS, Dodson AT, Parry M, Shewry PR, Mottram DS (2006) Formation of high levels of acrylamide during the processing of flour derived from sulfate-deprived wheat. J Agric Food Chem 54(23):8951–8955
Leustek T, Martin MN, Bick JA, Davies JP (2000) Pathways and regulation of sulfur metabolism revealed through molecular and genetic studies. Ann Rev Plant Phys Plant Mol Biol 51:141–165
Murray F (1997) Urban air pollution and health effects. In: Brune D, Chapman DV, Gwynne MD, Pacyna JM (eds) The global environment: science, technology and management. Scandinavian Science Publisher, Weinheim, Germany
Herschbach C, Rennenberg H (2001) Significance of phloem-translocated organic sulfur compounds for the regulation of sulfur nutrition. Prog Bot 62:177–192
Van der Kooij TAW, De Kok LJ, Haneklaus S, Schnug E Uptake and metabolism of sulfur dioxide by Arabidopsis thaliana. New Phytol 135:101–107
Noji M, Saito M, Nakamura M, Aono M, Saji H, Saito K (2001) Cysteine synthase overexpression in tobacco confers tolerance to sulfur-containing environmental pollutants. Plant Physiol 126:973–980
Legge AH, Krupa SV (2002) Effects of sulphur dioxide. In: Bell JNB, Treshow M (eds) Air pollution and plant life, 2nd edn. John Wiley, Chichester
Matityahu I, Kachan L, Ilan IB, Amir R (2006) Transgenic tobacco plants overexpressing the Met25 gene of Saccharomyces cerevisiae exhibit enhanced levels of cysteine and glutathione and increased tolerance to oxidative stress. Amino Acids 30:185–194
Brychkova G, Xia Z, Yang G, Yesbergenova Z, Zhang Z, Davydov O, Fluhr R, Sagi M (2007) Sulfite oxidase protects plants against sulfur dioxide toxicity. Plant J 50:696–709
Sanda S, Leustek T, Theisen MJ, Garavito RM, Benning C (2001) Recombinant Arabidopsis SQD1 converts UDP-glucose and sulfite to the sulfolipid head group precursor UDP-sulfoquinovose in vitro. J Biol Chem 276:3941–3946
Eilers T, Schwarz G, Brinkmann H, Witt C, Richter T, Nieder J, Koch B, Hille R, Hänsch R, Mendel RR (2001) Identification and biochemical characterization of Arabidopsis thaliana sulfite oxidase: a new player in plant sulfur metabolism. J Biol Chem 276:46989–46994
Kopriva S (2006) Regulation of sulfate assimilation in Arabidopsis and beyond. Ann Bot 97:479–495
Papenbrock J, Schmidt A (2000) Characterization of two sulfurtransferase isozymes from Arabidopsis thaliana. Eur J Biochem 267:5571–5579
Tsakraklides G, Martin M, Chalam R, Tarczynski MC, Schmidt A, Leustek T (2002) Sulfate reduction is increased in transgenic Arabidopsis thaliana expressing 5′-adenylylsulfate reductase from Pseudomonas aeruginosa. Plant J 32:879–889
Brychkova G, Yarmolinsky D, Sagi M (2012) Kinetic assays for determining in vitro APS reductase activity in plants without the use of radioactive substances. Plant Cell Physiol 53(9):1648–1658
Brychkova G, Yarmolinsky D, Ventura Y, Sagi M (2012) A novel in-gel assay and an improved kinetic assay for determining in vitro sulfite reductase activity in plants. Plant Cell Physiol 53(8):1507–1516
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Brychkova, G. et al. (2017). Determination of Enzymes Associated with Sulfite Toxicity in Plants: Kinetic Assays for SO, APR, SiR, and In-Gel SiR Activity. In: Sunkar, R. (eds) Plant Stress Tolerance. Methods in Molecular Biology, vol 1631. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7136-7_14
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DOI: https://doi.org/10.1007/978-1-4939-7136-7_14
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