Targeted quantitative profiling of metabolites and gene transcripts associated with 4-aminobutyrate (GABA) in apple fruit stored under multiple abiotic stresses

Abstract

4-Aminobutyrate accumulates in plants under abiotic stress. Here, targeted quantitative profiling of metabolites and transcripts was conducted to monitor glutamate- and polyamine-derived 4-aminobutyrate production and its subsequent catabolism to succinate or 4-hydroxybutyrate in apple ( Malus x domestica Borkh.) fruit stored at 0 °C with 2.5 kPa O₂ and 0.03 or 5 kPa CO₂ for 16 weeks. Low-temperature-induced protein hydrolysis appeared to be responsible for the enhanced availability of amino acids during early storage, and the resulting higher glutamate level stimulated 4-aminobutyrate levels more than polyamines. Elevated CO₂ increased the levels of polyamines, as well as succinate and 4-hydroxybutyrate, during early storage, and 4-aminobutyrate and 4-hydroxybutyrate over the longer term. Expression of all of the genes likely involved in 4-aminobutyrate metabolism from glutamate/polyamines to succinate/4-hydroxybutyrate was induced in a co-ordinated manner. CO₂-regulated expression of apple GLUTAMATE DECARBOXYLASE 2, AMINE OXIDASE 1, ALDEHYDE DEHYDROGENASE 10A8 and POLYAMINE OXIDASE 2 was evident with longer term storage. Evidence suggested that respiratory activities were restricted by the elevated CO₂/O₂ environment, and that decreasing NAD⁺ availability and increasing NADPH and NADPH/NADP⁺, respectively, played key roles in the regulation of succinate and 4-hydroxybutyate accumulation. Together, these findings suggest that both transcriptional and biochemical mechanisms are associated with 4-aminobutyrate and 4-hydroxybutyrate metabolism in apple fruit stored under multiple abiotic stresses.

References

[1] Shelp, B. J., Bown, A. W. & Zarei, A. 4-Aminobutyrate (GABA): metabolite and signal with practical significance. Botany 95, 1015-1032 (2017).

[2] Brikis, C. J. et al. Ancient plant glyoxylate/succinic semialdehyde reductases: GLYR1s are cytosolic, whereas GLYR2s are localized to both mitochondria and plastids. Front. Plant Sci. 8, 601 (2017).

[3] Zarei, A. et al. Plant glyoxylate/succinic semialdehyde reductases: comparative biochemical properties, function during chilling stress, and subcellular localization. Front. Plant Sci. 8, 1399 (2017).

[4] Tiburcio, A. F., Altabella, T., Bitrián, M. & Alcázar, R. The roles of polyamines during the lifespan of plants: from development to stress. Planta 240, 1-18 (2014).

[5] Zarei, A. et al. Apple fruit copper amine oxidase isoforms: peroxisomal MdAO1 prefers diamines as substrates, whereas extracellular MdAO2 exclusively utilizes monoamines. Plant Cell Physiol. 56, 137-147 (2015).

[6] Signorelli, S., Dans, P. D., Coitiño, E. L., Borsani, O. & Monza, J. Connecting proline and γ-aminobutyric acid in stressed plants through non-enzymatic reactions. PLoS ONE 10, 1-14 (2015).

[7] Zarei, A., Trobacher, C. P. & Shelp, B. J. NAD⁺-aminoaldehyde dehydrogenase candidates for 4-aminobutyrate (GABA) and β-alanine production during terminal oxidation of polyamines in apple fruit . FEBS Lett. 589, 2695-2700 (2015).

[8] Zarei, A., Trobacher, C. P. & Shelp, B. J. Arabidopsis aldehyde dehydrogenase 10 family members confer salt tolerance through putrescine-derived 4-aminobutyrate (GABA). Sci. Rep. 6, 35115 (2016).

[9] Deyman, K. L. et al. Effects of elevated CO₂ and 1-methylcyclopropene on storage-related disorders of Ontario-grown ‘Empire’ apples . Can. J. Plant Sci. 94, 857-865 (2014).

[10] Johnson, F. T. & Zhu, Y. Transcriptome changes in apple peel tissues during CO₂ injury symptom development under controlled atmosphere regimens . Hortic. Res. 2, 15061 (2015).

[11] Shelp, B. J. et al. Strategies and tools for studying the metabolism and function of γ-aminobutyrate in plants. II. Integrated analysis. Botany 90, 781-793 (2012).

[12] Chiu, G. Z., Shelp, B. J., Bowley, S. R., DeEll, J. R. & Bozzo, G. G. Controlled atmosphere related injury in ‘Honeycrisp’ apples is associated with γ-aminobutyrate accumulation. Can. J. Plant Sci. 95, 879-886 (2015).

[13] Lum, G. B. et al. Pre-storage conditioning ameliorates the negative impact of 1-methylcyclopropene on physiological injury and modifies the response of antioxidants and γ-aminobutyrate in ‘Honeycrisp’ apples exposed to controlled-atmosphere conditions. Postharvest Biol. Technol. 116, 115-128 (2016).

[14] Deyman, K. L., Brikis, C. J., Bozzo, G. G. & Shelp, B. J. Impact of 1-methylcyclopropene and controlled atmosphere storage on polyamine and γ-aminobutyrate levels in ‘Empire’ apple fruit. Front. Plant Sci. 5, 144 (2014).

[15] Trobacher, C. P. et al. Catabolism of GABA in apple fruit: Subcellular localization and biochemical characterization of two γ-aminobutyrate transaminases. Postharvest Biol. Technol. 75, 106-113 (2013).

[16] Ho, Q. T. et al. A three-dimensional multiscale model for gas exchange in fruit. Plant Physiol. 155, 1158-1168 (2011).

[17] Ho, Q. T., Verboven, P., Verlinden, B. E., Schenk, A. & Nicolaï, B. M. Controlled atmosphere storage may lead to local ATP deficiency in apple. Postharvest Biol. Technol. 78, 103-112 (2013).

[18] Allan, W. L., Simpson, J. P., Clark, S. M. & Shelp, B. J. γ-Hydroxybutyrate accumulation in Arabidopsis and tobacco plants is a general response to abiotic stress: putative regulation by redox balance and glyoxylate reductase isoforms. J. Exp. Bot. 59, 2555-2564 (2008).

[19] Allan, W. L. et al. Glyoxylate reductase and NAD kinase mutants subjected to submergence stress. Botany 90, 51-61 (2012).

[20] Lum, G. B., Shelp, B. J., DeEll, J. R. & Bozzo, G. G. Oxidative metabolism is associated with physiological disorders in fruits stored under multiple environmental stresses. Plant Sci. 245, 143-152 (2016).

[21] Chehab, E. W., Eich, E. & Braam, J. Thigmomorphogenesis: a complex plant response to mechano-stimulation. J. Exp. Bot. 60, 43-56 (2009).

[22] Benikhlef, L. et al. Perception of soft mechanical stress in Arabidopsis leaves activates disease resistance. BMC Plant Biol. 13, 133-145 (2013).

[23] Allan, W. L. & Shelp, B. J. Fluctuations of γ-aminobutyrate, γ-hydroxybutyrate, and related amino acids in Arabidopsis leaves as a function of the light, dark cycle, leaf age, and N stress. Can. J. Bot. 84, 1339-1346 (2006).

[24] Trobacher, C. P. et al. Calmodulin-dependent and calmodulin-independent glutamate decarboxylases in apple fruit. BMC Plant Biol. 13, 144 (2013).

[25] Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25, 402-408 (2001).

[26] Poupard, P., Parisi, L., Campion, C., Ziadi, S. & Simoneau, P. A wound- and ethephon-inducible PR-10 gene subclass from apple is differentially expressed during infection with a compatible and an incompatible race of Venturia inaequalis. Physiol. Mol. Plant Pathol. 62, 3-12 (2003).

[27] Czechowski, T., Stitt, M., Altmann, T. & Udvardi, M. K. Genome-wide identification and testing of superior reference genes for transcript normalization. Plant Physiol. 139, 5-17 (2005).

[28] SAS Institute Inc. Base SAS® 9.3 Procedures Guide. (SAS Institute Inc., Cary, NC, 2011).

[29] R Core Team . R: a language and environment for statistical computing (R Foundation for Statistical Computing, Vienna, Austria. 2016) http://www.Rproject.org/.

[30] Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. Roy. Stat. Soc. Ser. B 57, 289-300 (1995).

[31] Good, A. G. & Crosby, W. L. Anaerobic induction of alanine aminotransferase in barley root tissue. Plant Physiol. 90, 1305-1309 (1989).

[32] Moschou, P. N. et al. Bridging the gap between plant and mammalian polyamine catabolism: a novel peroxisomal polyamine oxidase responsible for a full back-conversion pathway in Arabidopsis. Plant Physiol. 147, 1845-1857 (2008).

[33] Queval, G. & Noctor, G. A plate reader method for the measurement of NAD, NADP, glutathione, and ascorbate in tissue extracts: application to redox profiling during Arabidopsis rosette development. Anal. Biochem. 363, 58-69 (2007).

[34] Adyanthaya, I., Kwon, Y., Apostolidis, E. & Shetty, K. Apple postharvest preservation is linked to phenolic content and superoxide dismutase activity. J. Food Biochem. 33, 535-556 (2009).

[35] Magné, C., Bonenfant-Magné, M. & Audran, J. Nitrogenous indicators of postharvest ripening and senescence in apple fruit (Malus domestica Borkh. Cv. Granny Smith). Intern. J. Plant Sci. 158, 811-817 (1997).

[36] Feng, F., Li, M., Ma, F. & Cheng, L. Effects of location within the tree canopy on carbohydrates, organic acids, amino acids and phenolic compounds in the fruit peel and flesh from three apple (Malus x domestica) cultivars. Hortic. Res. 1, 14019 (2014).

[37] Shi, Y., Jiang, L., Zhang, L., Kang, R. & Yu, Z. Dynamic changes in proteins during apple (Malus x domestica) fruit ripening and storage. Hortic. Res. 1, 6 (2014).

[38] Scott-Taggart, C. P., Van Cauwenberghe, O. R., McLean, M. D. & Shelp, B. J. Regulation of γ-aminobutyric acid synthesis in situ by glutamate availability. Physiol. Plant. 106, 363-369 (1999).

[39] Winter, G., Todd, C. D., Trovato, M., Forlani, G. & Funck, D. Physiological implications of arginine metabolism in plants. Front. Plant Sci. 6, 534 (2015).

[40] Fait, A., Fromm, H., Walter, D., Galili, G. & Fernie, A. R. Highway or byway: the metabolic role of the GABA shunt in plants. Trends Plant. Sci. 13, 14-19 (2008).

[41] Zarei, A., Chiu, G. Z., Yu, G., Trobacher, C. P. & Shelp, B. J. Salinity-regulated expression of genes involved in GABA metabolism and signaling. Botany 95, 621-627 (2017).

[42] Deewatthanawong, R., Rowell, P. & Watkins, C. B. γ-Aminobutyric acid (GABA) metabolism in CO₂ treated tomatoes . Postharvest Biol. Technol. 57, 97-105 (2010).

[43] Kinnersley, A. M. & Turano, F. J. γ-Aminobutyric acid (GABA) and plant responses to stress. Crc. Crit. Rev. Plant. Sci. 19, 479-509 (2000).

[44] Shelp, B. J., Bozzo, G. G., Trobacher, C. P., Chiu, G. & Bajwa, V. S. Strategies and tools for studying the metabolism and function of γ-aminobutyrate in plants. I. Pathway structure. Botany 90, 651-668 (2012).

[45] Juda, G. A., Shepard, E. M., Elmore, B. O. & Dooley, D. M. A comparative study of the binding and inhibition of four copper-containing amine oxidases by azide: Implications for the role of copper during the oxidative half-reaction. Biochemistry 45, 8788-8800 (2006).

[46] Mukherjee, A. et al. An inner-sphere mechanism for molecular oxygen reduction catalyzed by copper amine oxidases. J. Am. Chem. Soc. 23, 9459-9473 (2008).

[47] Forde, B. G. & Lea, P. J. Glutamate in plants: metabolism, regulation and signalling. J. Exp. Bot. 58, 2339-2358 (2007).

[48] Mathooko, F. M. Regulation of respiratory metabolism in fruits and vegetables by carbon dioxide. Postharvest Biol. Technol. 9, 247-264 (1996).

[49] Tcherkez, G. et al. Respiratory metabolism of illuminated leaves depends on CO₂ and O₂ conditions . Proc. Natl Acad. Sci. USA 105, 797-802 (2008).

[50] Tokumura, A., Tanaka, T., Yotsumoto, T. & Tsukatani, H. Identification of sn-2-ω-hydroxycarboxylate-containing phospholipids in a lipid extract from bovine brain. Biochem. Biophys. Res. Commun. 177, 466-473 (1991).

[51] Tanaka, T., Minamino, H., Unezaki, S., Tsukatani, H. & Tokumura, A. Formation of platelet-activating factor-like phospholipids by Fe²⁺/ascorbate/EDTA-induced lipid peroxidation . Biochim. Biophys. Acta 1166, 264-274 (1993).

[52] Mamelak, M. & Kuksis, A. Gammahydroxybutyrate and lipid peroxidation. Int. News Fat., Oils, Relat. Mater. 17, 607-610 (2006).

[53] Mamelak, M. Alzheimer’s disease, oxidative stress and gammahydroxybutyrate. Neurobiol. Aging 28, 1340-1360 (2007).

[54] Renault, H. et al. γ-Aminobutyric acid transaminase deficiency impairs central carbon metabolism and leads to cell wall defects during salt stress in Arabidopsis roots. Plant Cell Environ. 36, 1009-1018 (2013).