Abstract
Organic acids (OAs) play an essential role in several cellular biochemical pathways. Released by plant roots, OAs can help them tolerate phytotoxic forms of aluminum (Al) present in acidic soils. Some studies use high-performance liquid chromatography (HPLC), ion chromatography and, more rarely, gas chromatography-mass spectrometry (GC–MS) to analyze exuded OAs, but they are limited to hours or 1–2 days. The main limitation is that OAs are measured only in CaCl2 and AlCl3 solutions, and not in “complete” nutrient solutions, only enabling short-term collection of secreted OAs. Here, we aimed to test a method to quantify citric, malic, oxalic and succinic acids in “complete” nutrient solution with 0, 740 and 1480 μM Al, using a GC–MS. First, a calibration curve was established through a chromatogram that was generated using six concentrations of OAs in methanol. Thus, samples containing OAs only were derived by methylation. Then, analytical curves were set up using seven OAs concentrations added in the three nutrient solutions. The OAs were concentrated by drying the solutions. After re-suspension, samples were derived by methylation. Instrumental precision and the method repeatability were also checked. Chromatograms showed adequate resolution, with distinct peak heights between OAs concentrations and their respective retention times. Both calibration and analytical curves indicated consistent linearity (R > 0.99), evidencing the method and equipment parameters were able to provide results directly proportional to OAs concentrations. This low-cost method is recommended for evaluation of Al-induced OA secretion by roots of whole plants in nutrient solutions for several days or weeks.
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References
Baetz U, Martinoia E (2014) Root exudates: the hidden part of plant defense. Trends Plant Sci 19:90–98
Banhos OFAA, Carvalho BMO, Veiga EB, Bressan ACG, Tanaka FAO, Habermann G (2016a) Aluminum-induced decrease in CO2 assimilation in ‘Rangpur’ lime is associated with low stomatal conductance rather than low photochemical performances. Sci Hortic 205:133–140
Banhos OFAA, Souza MC, Habermann G (2016b) High aluminum availability may affect Styrax camporum, an Al non-accumulating species from the Brazilian savanna. Theor Exp Plant Physiol 28:321–332
Bansal S, DeStefano A (2007) Key elements of bioanalytical method validation for small molecules. AAPS J 9(01):E109–E114
Bittencourt BMOC, Silva CMS, Filho SZ, Habermann G (2019) Aluminum (Al)-induced organic acid exudation in an Al-accumulating species from the Brazilian savanna. Trees 34:155–162
Brazilian National Agency of Sanitary Surveillance (ANVISA) (2003) Resolution RE nº 899, 05/29/2003
Brazilian National Institute of Metrology, Standardization and Industrial Quality (INMETRO) (2003) Orientações sobre Validação de Métodos de Ensaios Químicos, DOQ-CGCRE-008 (in Portuguese)
Bressan ACG, Coan AI, Habermann G (2016) X-ray spectra in SEM and staining with chrome azurol S show Al deposits in leaf tissues of Al-accumulating and non-accumulating plants from the cerrado. Plant Soil 404:293–306
Brunner I, Sperisen C (2013) Aluminum exclusion and aluminum tolerance in woody plants. Front Plant Sci 4:1–12
Cavalheiro MF, Gavassi MA, Silva GS, Nogueira MA, Silva CMS, Domingues DS, Habermann G (2020) Low root PIP1-1 and PIP2 aquaporins expression could be related to reduced hydration in ‘Rangpur’ lime plants exposed to aluminum. Funct Plant Biol 47:112–121
Chen ZC, Liao H (2016) Organic acid anions: An effective defensive weapon for plants against aluminum toxicity and phosphorus deficiency in acidic soils. J Genet Genom 43:631–638
Clark RB (1975) Characterization of phosphatase of intact maize roots. J Agric Food Chem 23:458–460
Delhaize E, Hebb DM, Ryan PR (2001) Expression of a Pseudomonas aeruginosa citrate synthase gene in tobacco is not associated with either enhanced citrate accumulation or efflux. Plant Physiol 125:2059–2067
Delhaize E, Ryan PR, Randall PJ (1993) Aluminum tolerance in wheat (Triticum aestivum L.) II. Aluminum stimulated excretion of malic acid from root apices. Plant Physiol 103:695–702
Deng W, Luo K, Li Z, Yang Y, Hu N, Wu Y (2009) Overexpression of Citrus junos mitochondrial citrate synthase gene in Nicotiana benthamiana confers aluminum tolerance. Planta 230:355–365
Fischer E, Speier A (1895) Darstellung der Ester. Chem Ber 28:3252–3258
Green JM (1996) Peer reviewed: a practical guide to analytical method validation. Anal Chem 68(9):305A–309A
Guo P, Qi YP, Yang LT, Lai NW, Ye X, Yang Y, Chen LS (2017) Root Adaptive responses to aluminum-treatment revealed by RNA-seq in two citrus species with different aluminum-tolerance. Front Plant Sci 8:330
Huber L (1998) Validation of analytical methods: review and strategy. LC/GC Int 11:96–105
Igamberdiev AU, Eprintsev AT (2016) Organic acids: the pools of fixed carbon involved in redox regulation and energy balance in higher plants. Front Plant Sci 7:1042
Jenke DR (1998) Chromatographic method validation: a review of current practices and procedures. Part II. Guidelines for primary validation parameters. Instrum Sci Technol 26(1):19–35
Kaur R, Kaur R, Sharma A, Kumar V, Sharma M, Bhardwaj R, Thukral AK (2018) Microbial production of dicarboxylic acids from edible plants and milk using GC-MS. J Anal Sci Technol 9:21
Kidd PS, Llugany M, Poschenrieder C, Gunsé B, Barceló J (2001) The role of root exudates in aluminium resistance and silicon-induced amelioration of aluminium toxicity in three varieties of maize (Zea mays L.). J Exp Bot 52(359):1339–1352
Kochian LV, Hoekenga OA, Piñeros MA (2004) How do crop plants tolerate acid soils? Mechanisms of aluminum tolerance and phosphorous efficiency. Annu Rev Plant Biol 55:459–493
Kopittke PM, Moore KL, Lombi E, Gianoncelli A, Ferguson BJ, Blamey FPC, Menzies NW, Nicholson TM, McKenna BA, Wang P, Gresshoff PM, Kourousias G, Webb RI, Green K, Tollenaere A (2015) Identification of the primary lesion of toxic aluminum in plant roots. Plant Physiol 167:1402–1411
Kumar V, Sharma A, Bhardwaj R, Thukral AK (2017) Analysis of organic acids of tricarboxylic acid cycle in plants using GC-MS, and system modeling. J Anal Sci Technol 8:20
Li XF, Ma JF, Matsumoto H (2000) Pattern of Al-induced secretion of organic acids differ between rye and wheat. Plant Physiol 123:1537–1543
Lopez-Bucio J, Nieto-Jacobo MF, Ramırez-Rodrıguez V, Herrera-Estrella L (2000) Organic acid metabolism in plants: from adaptive physiology to transgenic varieties for cultivation in extreme soils. Plant Sci 160:1–13
Ma JF (2000) Role of organic acids in detoxification of aluminum in higher plants. Plant Cell Physiol 41:383–390
Ma JF (2005) Physiological mechanisms of Al resistance in higher plants. Soil Sci Plant Nutr 51:609–612
Ma JF, Ryan PR, Delhaize E (2001) Aluminium tolerance in plants and the complexing role of organic acids. Trends Plant Sci 6:273–278
Ma JF, Zheng SJ, Hiradate S, Matsumoto H (1997a) Detoxifying aluminum with buckwheat. Nature 390:569–570
Ma JF, Zheng SJ, Hiradate S, Matsumoto H (1997b) Specific secretion of citric acid induced by Al stress in Cassia tora L. Plant Cell Physiol 38:1019–1025
Ryan PR, Delhaize E, Jones DL (2001) Function and mechanism of organic anion exudation from plants. Annu Rev Plant Physiol Plant Mol Biol 52:527–560
Sade H, Meriga B, Surapu V, Gadi J, Sunita M, Suravajhala P, Kishor PK (2016) Toxicity and tolerance of aluminum in plants: tailoring plants to suit to acid soils. Biometals 29:187–210
Shabir GA (2003) Validation of high-performance liquid chromatography methods for pharmaceutical analysis: understanding the differences and similarities between validation requirements of the US Food and Drug Administration, the US Pharmacopeia and the International Conference on Harmonization. J Chromatogr A 987:57–66
Sharma A, Thakur S, Kumar V, Kanwar MK, Kesavan AK, Thukral AK, Bhardwaj R, Alam P, Ahmad P (2016) Pre-sowing seed treatment with 24-epibrassinolide ameliorates pesticide stress in Brassica juncea L. through the modulation of stress markers. Front Plant Sci. https://doi.org/10.3389/fpls.2016.01569
Silva CMS, Cavalheiro MF, Bressan ACG, Carvalho BMO, Banhos OFAA, Purgatto E, Harakava R, Tanaka FAO, Habermann G (2019) Aluminum-induced high IAA concentration may explain the Al susceptibility in Citrus limonia. Plant Growth Regul 87:123–137
Silva CMS, Zhang C, Habermann G, Delhaize E, Ryan PR (2018) Does the major aluminium-resistance gene in wheat, TaALMT1, also confer tolerance to alkaline soils? Plant Soil 424:451–462
Silva IR, Novais RF, Jham GN, Barros NF, Gebrim FO, Nunes FN, Neves JCL, Leite FP (2004) Responses of eucalypt species to aluminum: the possible involvement of low molecular weight organic acids in the Al tolerance mechanism. Tree Physiol 24:1267–1277
Souza MC, Franco A, Haridasan M, Rossatto DR, de Araújo JF, Morellato LPC, Habermann G (2015) The length of the dry season may be associated with leaf scleromorphism in cerrado plants. Ann Braz Acad Sci 87:1691–1699
Wang H, Chen RF, Iwashita T, Shen RF, Ma JF (2015) Physiological characterization of aluminum tolerance and accumulation in tartary and wild buckwheat. New Phytol 205:273–279
Yang JL (2005) Aluminium resistance requires resistance to acid stress: a case study with spinach that exudes oxalate rapidly when exposed to Al stress. J Exp Bot 56:1197–1203
Yang JL, Zhang L, Li YY, You JF, Wu P, Zheng SJ (2006) Citrate transporters play a critical role in aluminium-stimulated citrate efflux in rice bean (Vigna umbellata) roots. Ann Bot 97:579–584
Yang LT, Jiang HX, Tang N, Chen LS (2011) Mechanisms of aluminum-tolerance in two species of citrus: secretion of organic acid anions and immobilization of aluminum by phosphorus in roots. Plant Sci 180:521–530
Yang LT, Qi YP, Jiang HX, Chen LS (2013) Roles of organic acid anion secretion in aluminium tolerance of higher plants. Biomed Res Int 2013:173682
Zheng SJ, Ma JF, Matsumoto H (1998) High aluminum resistance in buckwheat. I. Al-induced specific secretion of oxalic acid from root tips. Plant Physiol 117:745–751
Acknowledgements
BMOC Bittencourt acknowledges Grant #2016/14216-3, São Paulo Research Foundation (FAPESP) and Coordination for the Improvement of Higher Education Personnel (CAPES), for a Msc. scholarship. We acknowledge the Brazilian National Council for Scientific and Technological Development (CNPq) for financial support (474169/2013-8 Grant to GH) and for a productivity fellowship (309149/2017-7 Grant to GH).
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de Oliveira Carvalho Bittencourt, B.M., Filho, S.Z. & Habermann, G. Method to quantify aluminum-induced organic acids secretion by roots of plants in nutrient solution using GC–MS. Theor. Exp. Plant Physiol. 32, 121–131 (2020). https://doi.org/10.1007/s40626-020-00171-0
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DOI: https://doi.org/10.1007/s40626-020-00171-0