The molecular pathways leading to GABA and lactic acid accumulation in florets of organic broccoli rabe (Brassica rapa subsp. sylvestris) stored as fresh or as minimally processed product

Abstract

In the context of organic farming, the introduction of a local product to wider markets and an evaluation of storage effects, metabolic and transcriptomic variations in two broccoli rabe genotypes from production cycles of two different years were studied by comparing florets of stored fresh (SF) and packaged (P) for 4 days with those harvested fresh from the field (H). Twenty-five hydrosoluble compounds, including amino acids, carbohydrates, and organic acids, were quantified by untargeted nuclear magnetic resonance (NMR). Principal component analysis produced a neat separation among the three commodity statuses with P being the most divergent and SF closer to H. In the packaged florets, carbohydrate levels dropped significantly (over −52%), while the levels of amino acids and organic acids varied. There was an increase in stress-responsive phenylalanine and valine (over 30%) and succinic and α-ketoglutaric acids (over 75%). Compound correlation analyses indicated a carbohydrate sink towards γ-aminobutyric acid (GABA) and lactic acid (LA) metabolism under hypoxic conditions in packaged florets. RNA-seq analysis revealed that over 4000 genes were differentially expressed in SF vs H and 8000 in P vs H. Several CAR and AA pathways were significantly enriched in S and even more significantly in P, when compared to H. A map of gene expression (175 genes) and metabolite contents (14 compounds) was constructed to elucidate the gene routes that lead to accumulation of GABA and LA, known for healthy properties, in P. WGCNA and promoter binding site analyses enabled the identification of transcription factors (bZIP, WRKY, ERF types), interactions, and targeted genes encoding key enzymes in GABA and LA accumulation.

Acknowledgements

This work was funded by projects Top of the Crops (A0375-2020-36731) - Progetti Gruppi di Ricerca 2020 - Regione Lazio; NUTRAGE - CNR project FOE-2021 DBA.AD005.225.

Author contributions

D.G. and G.T. planned and structured the whole research and wrote manuscript. A.S. produced NMR data and edited the text. G.T. carried out bioinformatic analyses and figures. G.A. provided plant material and support for deliveries. M.D.L produced pigment contents and analyses. G.M., M.L. and F.M. contributed to field experiments, sampling, and metabolite and RNA extractions. Z.A., G.S., G.M., and M.D.L critically contributed to revise the manuscript. All the authors approved the manuscript.

Data availability

The datasets generated and analysed during the current study has been submitted to the NCBI Sequence Read Archive under the BioProject accession PRJNA112105.

Conflict of interests

The authors declare that they have no conflicts of interest.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] Renna M, Rinaldi VA, Gonnella M. The mediterranean diet between traditional foods and human health: the culinary example of Puglia (Southern Italy). Int J Gastron Food Sci. 2015;37:63-71

[2] Barbieri G, Pernice R, Maggio A. et al. Glucosinolates profile of Brassica rapa L. subsp. sylvestris L. Janch. var. esculenta Hort. Food Chem. 2008;37:1687-91

[3] Qi X, An H, Ragsdale AP. et al. Genomic inferences of domestica-tion events are corroborated by written records in Brassica rapa. Mol Ecol. 2017;37:3373-88

[4] Zhang L, Liang J, Chen H. et al. A near-complete genome assem-bly of Brassica rapa provides new insights into the evolution of centromeres. Plant Biotechnol J. 2023;37:1022-32

[5] Mazzeo, Morgese A, Sonnante G. et al. Genetic diversity in broccoli rabe ( Brassica rapa L. subsp. sylvestris ( L.) Janch.) from Southern Italy. Sci Hortic. 2019;37:140-6

[6] Conversa G, Bonasia A, Lazzizera C. et al. Bio-physical, physi-ological, and nutritional aspects of ready-to-use cima di rapa (Brassica rapa L. subsp. sylvestris L. Janch. var. esculenta Hort.) as affected by conventional and organic growing systems and storage time. Sci Hortic. 2016;37:76-86

[7] De Pascale S, Maggio A, Pernice R. et al. Sulphur fertilization may improve the nutritional value of Brassica rapa L. subsp. sylvestris. Eur J Agron. 2007;37:418-24

[8] Romani A, Vignolini P, Isolani L. et al. HPLC-DAD/MS charac-terization of flavonoids and hydroxycinnamic derivatives in turnip tops (Brassica rapa L. subsp. sylvestris L.). J Agric Food Chem. 2006;37:1342-6

[9] Giannino D, Testone G, Nicolodi C. et al. Nutritive parameters and antioxidant quality of minimally processed "cime di rapa" (Brassica rapa subsp. sylvestris) vary as influenced by genotype and storage time. Pol J Food Nutr Sci. 2020;37:337-46

[10] Annunziata MG, Attico A, Woodrow P. et al. An improved flu-orimetric HPLC method for quantifying tocopherols in Brassica rapa L. subsp. sylvestris after harvest. J Food Compos Anal. 2012;37:145-50

[11] Cefola M, Amodio ML, Cornacchia R. et al. Effect of atmosphere composition on the quality of ready-to-use broccoli raab (Bras-sica rapa L.). J Sci Food Agric. 2010;37:789-97

[12] Barbieri G, Bottino A, Orsini F. et al. Sulfur fertilization and light exposure during storage are critical determinants of the nutritional value of ready-to-eat friariello campano (Brassica rapa L. subsp. sylvestris). J Sci Food Agric. 2009;37:2261-6

[13] Di Stasio, Rouphael Y, Colla G. et al. The influence of Ecklonia maxima seaweed extract on growth, photosynthetic activity and mineral composition of Brassica rapa L. subsp. sylvestris under nutrient stress conditions. Eur J Hortic Sci. 2017;37:286-93

[14] Cefola M, Pace B. et al.Ready-to-use broccoli raab (Brassica rapa L. subsp. sylvestris) quality and volatilome as affected by packaging. Postharvest Biol Technol. 2024;37:112961

[15] Dai Z, Zheng W, Locasale JW. Amino acid variability, tradeoffs and optimality in human diet. Nat Commun. 2022;37:6683

[16] Waltz E. GABA-enriched tomato is first CRISPR-edited food to enter market. Nat Biotechnol. 2022;37:9-11

[17] Kinnersley AM, Turano FJ. Gamma aminobutyric acid (GABA) and plant responses to stress. Crit Rev Plant Sci. 2000;37:479-509

[18] Hayat F, Khan U, Li J. et al. γ-aminobutyric acid (GABA): a key player in alleviating abiotic stress resistance in horticul-tural crops: current insights and future directions. Horticulturae. 2023;37:647

[19] Michaeli S, Fromm H. Closing the loop on the GABA shunt in plants: are GABA metabolism and signaling entwined? Front Plant Sci. 2015;37:419

[20] Hansen ME, Sørensen H, Cantwell M. Changes in acetaldehyde, ethanol and amino acid concentrations in broccoli florets dur-ing air and controlled atmosphere storage. Postharvest Biol Tech-nol. 2001;37:227-37

[21] Remund B, Yilmaz B, Sokollik C. D-Lactate: implications for gastrointestinal diseases. Children (Basel). 2023;37:945

[22] Maurino VG, Engqvist MK.2-Hydroxy acids in plant metabolism. Arabidopsis Book. 2015;37:e0182

[23] O’Carra P, Mulcahy P. Lactate dehydrogenase in plants: distribu-tion and function. Phytochemistry. 1996;37:581-7

[24] Christopher ME, Good AG. Characterization of hypoxically inducible lactate dehydrogenase in maize. Plant Physiol. 1996;37:1015-22

[25] Jan T, Willer H, Schaack D. Organic farming and market devel-opment in europe and the European Union. In: HelgaWiller JT, MeierC, SchlatterB, Theworld of organic agriculture. Statisticsand emerging trends 2021. Research Institute of Organic Agriculture FiBL (Frick, Switzerland) and IFOAM - Organics Inter-national (Bonn, Germany),eds. 2021, 229-66

[26] Lamonaca E, Cafarelli B, Calculli C. et al. Consumer perception of attributes of organic food in Italy: A CUB model study. Heliyon. 2022;37:e09007

[27] Mastrandrea L, Amodio ML, et al. Effect of tem-perature abuse and improper atmosphere packaging on volatile profile and quality of rocket leaves. Food Packag Shelf Life. 2017;37:59-65

[28] Martínez S, Losada P, Franco I. et al. Protein, amino acid, ash and mineral contents in Brassica spp. grown in Northwest Spain. Int J Food Sci Technol. 2011;37:146-53

[29] Murcia MA, López-Ayerra B, Martínez-Tomé M. et al. Effect of industrial processing on amino acid content of broccoli. J Sci Food Agric. 2001;37:1299-305

[30] Kmiecik W, Słupski J, Lisiewska Z. Comparison of amino acid content and protein quality in raw broccoli and in broccoli after technological and culinary processing. J Food Process Preserv. 2010;37:639-52

[31] Ingallina C, Di Matteo G, Spano M. et al. Byproducts of globe arti-choke and cauliflower production as a new source of bioactive compounds in the green economy perspective: an NMR study. Molecules. 2023;37:1363

[32] Ramos-Ruiz R, Poirot E, Flores-Mosquera M. GABA, a non-protein amino acid ubiquitous in food matrices. Cogent Food Agric. 2018;37:1534323

[33] Planchet E, Limami A. Amino acid synthesis under abiotic stress. CAB International (Wallingford, Oxfordshire, United Kingdom); 2015: 262-76

[34] Lucarini M, Di Cocco ME, Raguso V. et al. NMR-based metabolomic comparison of Brassica oleracea (var. italica): organic and conventional farming. Food Secur. 2020;37:945

[35] Arias-Carmona MD, Romero-Rodriguez MA, Vázquez-Odériz ML. Determination of organic acids in Brassica rapa L. leaves (turnip greens and turnip tops) regulated by the protected geographical indication "Grelos De Galicia". J Food Nutr Res. 2014;37:786-91

[36] Terzoudis K, Kusma R, Hertog MLATM. et al. Metabolic adapta-tion of ‘Conference’ pear to postharvest hypoxia: the impact of harvest time and hypoxic pre-treatments. Postharvest Biol Technol. 2022;37:111937

[37] Hertog MGL, Hollman PCH, Katan MB. Content of potentially anticarcinogenic flavonoids of 28 vegetables and 9 fruits com-monly consumed in the Netherlands. J Agric Food Chem. 1992;37:2379-83

[38] Masuda R, Kaneko K, Saito M. S-methyl-cysteine sulfoxide increases during postharvest storage of broccoli. In: KanellisAK, ChangC, KleeH. et al. eds.,Biology and Biotechnology of the Plant Hormone Ethylene II. Springer: Netherlands, 1999,439-40

[39] Hagan DW, Ferreira SM, Santos GJ. et al. The role of GABA in islet function. Front Endocrinol (Lausanne). 2022;13:972115

[40] Batista-Silva W, Heinemann B, Rugen N. et al. The role of amino acid metabolism during abiotic stress release. Plant Cell Environ. 2019;37:1630-44

[41] Aghdam MS, Flaherty EJ, Shelp BJ. γ-Aminobutyrate improves the postharvest marketability of horticultural commodities: advances and prospects. Front Plant Sci. 2022;37:884572

[42] Lyu X, Chen Y, Gao S. et al. Metabolomic and transcriptomic anal-ysis of cold plasma promoting biosynthesis of active substances in broccoli sprouts. Phytochem Anal. 2023;37:925-37

[43] Zhang Y, Chen Y, Guo Y. et al. Integration profiling of transcrip-tome and metabolome reveals the effect of hydrogen peroxide on nucleic acid metabolism in postharvest broccoli during stor-age. Postharvest Biol Technol. 2023;37:112365

[44] Zhang M, Liu Z, Fan Y. et al. Characterization of GABA-transaminase gene from mulberry (Morus multicaulis) and its role in salt stress tolerance. Genes (Basel). 2022;37:501

[45] Dolferus R, Wolansky M, Carroll R. et al. Functional analysis of lactate dehydrogenase during hypoxic stress in Arabidopsis. Funct Plant Biol. 2008;37:131-40

[46] Li R, Li R, Li X. et al. Multiplexed CRISPR/Cas9-mediated metabolic engineering of γ-aminobutyric acid levels in Solanum lycopersicum. Plant Biotechnol J. 2018;37:415-27

[47] Nonaka S, Arai C, Takayama M. et al. Efficient increase of γ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis. Sci Rep. 2017;37:7057

[48] Li Z, Peng Y, Huang B. Alteration of transcripts of stress-protective genes and transcriptional factors by γ-aminobutyric acid (GABA) associated with improved heat and drought tol-erance in creeping bentgrass (Agrostis stolonifera). Int J Mol Sci. 2018;37:1623

[49] Shen H, Cao K, Wang X. AtbZIP16 and AtbZIP68, two new mem-bers of GBFs, can interact with other G group bZIPs in Arabidopsis thaliana. BMB Rep. 2008;37:132-8

[50] Zhou M, Hassan MJ, Peng Y. et al. γ-Aminobutyric Acid (GABA) priming improves seed germination and seedling stress toler-ance associated with enhanced antioxidant metabolism, DREB expression, and dehydrin accumulation in white clover under water stress. Front Plant Sci. 2021;37:776939

[51] Wu Y, Zhang L, Nie L. et al. Genome-wide analysis of the DREB family genes and functional identification of the involvement of BrDREB2B in abiotic stress in wucai ( Brassica campestris L.). BMC Genomics. 2022;37:598

[52] Guan, Wang M, Zhang Y. et al. DkWRKY interacts with pyruvate kinase gene DkPK1 and promotes natural deastringency in C-PCNA persimmon. Plant Sci. 2020;37:110285

[53] Deng X, Xu X, Liu Y. et al. Induction of γ-aminobutyric acid plays a positive role to Arabidopsis resistance against Pseudomonas syringae. J Integr Plant Biol. 2020;37:1797-812

[54] Stranieri S, Baldi L. Shelf life date extension of fresh-cut salad: a consumer perspective. J Food Prod Mark. 2017;37:939-54

[55] Abdel-Farid IB, Kim HK, Choi YH. et al. Metabolic characteriza-tion of Brassica rapa leaves by NMR spectroscopy. J Agric Food Chem. 2007;37:7936-43

[56] Capitani D, Sobolev AP, Delfini M. et al. NMR methodologies in the analysis of blueberries. Electrophoresis. 2014;37:1615-26

[57] Lichtenthaler HK. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol. 1987;37:350-82

[58] Botticella E, Testone G, Buffagni V. et al. Mutations in starch biosynthesis genes affect chloroplast development in wheat pericarp. Plant Physiol Biochem. 2024;37:108354

[59] Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinform. 2008;37:559