Abstract
Broccoli (Brassica oleracea L. var. italica Planch) hairy roots can rapidly produce secondary metabolites in massive amounts. However, under prolonged culture conditions, hairy roots undergo browning and aging, and finally die, resulting in the failure to produce secondary metabolites. As an antioxidant, melatonin has been widely used to prevent vegetables and fruits from browning. In this study, we explored the effect of melatonin on the molecular mechanism of browning in broccoli hairy roots. Transcriptomic analysis of broccoli hairy root samples treated with melatonin for 0, 6, 12, 20, and 32 h after 20 days of growth led to identification of 72 genes potentially associated with enzymatic browning. With the increase in melatonin treatment duration, the activities of polyphenol oxidase (PPO), peroxidase (POD), l-phenylalanine ammonia-lyase (PAL), ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (CAT) first increased and then decreased. Compared with the control, the content of phenolic compounds was lower in melatonin-treated samples, whereas that of soluble quinones was higher. Additionally, transcript levels of five out of 72 browning-related genes were verified by qRT-PCR. Finally, a protein–protein interaction network was constructed to identify browning-related proteins and their functional relationship. This study provides a strong theoretical basis for the genetic improvement of broccoli hairy roots and the subsequent development of browning resistant hairy roots lines.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The sequencing raw data has been uploaded to the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) under Bioproject PRJNA796369 and PRJNA764437.
References
Abohatem MA, Bakil Y, Baaziz M (2017) Plant regeneration from somatic embryogenic suspension cultures of date palm. In: Al-Khayri JM, Jain SM, Johnson DV (eds) Date Palm Biotechnology Protocols Volume I. Springer New York, New York, pp 203–214
Afoakwah AN (2020) Anti-browning methods on fresh-cut fruits and fruit juice: A review. Afr J Biol Sci 2:27. https://doi.org/10.33472/AFJBS.2.4.2020.27-32
Alagarsamy K, Shamala LF, Wei S (2018) Protocol: high-efficiency in-planta Agrobacterium-mediated transgenic hairy root induction of Camellia sinensis var. sinensis. Plant Methods 14:17. https://doi.org/10.1186/s13007-018-0285-8
Ali S, Khan AS, Anjum MA, Nawaz A, Naz S, Ejaz S, Hussain S (2020) Effect of postharvest oxalic acid application on enzymatic browning and quality of lotus (Nelumbo nucifera Gaertn.) root slices. Food Chem 312:126051. https://doi.org/10.1016/j.foodchem.2019.126051
Assis JS, Maldonado R, Muñoz T, Escribano MI, Merodio C (2001) Effect of high carbon dioxide concentration on PAL activity and phenolic contents in ripening cherimoya fruit. Postharvest Biol Technol 23:33–39. https://doi.org/10.1016/S0925-5214(01)00100-4
Ban QY, Liu TJ, Ning K, Fan JJ, Cui QX, Guo YL, Zai XM (2021) Effect of calcium treatment on the browning of harvested eggplant fruits and its relation to the metabolisms of reactive oxygen species (ROS) and phenolics. Food Sci Nutr 9:5567–5574. https://doi.org/10.1002/fsn3.2517
Beato VM, Orgaz F, Mansilla F, Montaño A (2011) Changes in phenolic compounds in garlic (Allium sativum L.) owing to the cultivar and location of growth. Plant Foods Hum Nutr 66:218–223. https://doi.org/10.1007/s11130-011-0236-2
Cen HF, Wang TT, Liu HY, Tian DY, Zhang YW (2020) Melatonin application improves salt tolerance of Alfalfa (Medicago sativa L.) by enhancing antioxidant capacity. Plants 9:220. https://doi.org/10.3390/plants9020220
Chen C, Jiang A, Liu CH, Wagstaff C, Zhao QQ, Zhang YH, Hu WZ (2021) Hydrogen sulfide inhibits the browning of fresh-cut apple by regulating the antioxidant, energy and lipid metabolism. Postharvest Biol Technol 175:111487. https://doi.org/10.1016/j.postharvbio.2021.111487
Dan YH, Zhang S, Zhong H, Yi H, Manuel BS (2015) Novel compounds that enhance Agrobacterium-mediated plant transformation by mitigating oxidative stress. Plant Cell Rep 34:291–309. https://doi.org/10.1007/s00299-014-1707-3
Fan JB, Xie Y, Zhang ZC, Chen L (2018) Melatonin: A multifunctional factor in plants. IJMS 19:1528. https://doi.org/10.3390/ijms19051528
Fan J, Du W, Chen QL, Zhang JG, Yang ZP, Hussain SB, Hu HJ (2021) Comparative transcriptomic analyses provide insights into the enzymatic browning mechanism of fresh-cut sand pear fruit. Horticulturae 7:502. https://doi.org/10.3390/horticulturae7110502
Gao H, Lu ZM, Yang Y, Wang D, Yang T, Cao MM, Cao W (2018) Melatonin treatment reduces chilling injury in peach fruit through its regulation of membrane fatty acid contents and phenolic metabolism. Food Chem 245:659–666. https://doi.org/10.1016/j.foodchem.2017.10.008
Gao HS, Huang LZ, Gong ZJ, Wang XT, Qiao XQ, Xiao F, Yang YT, Yu BH, Guo XT, Yu CY, Zhang HX (2022) Exogenous melatonin application improves resistance to high manganese stress through regulating reactive oxygen species scavenging and ion homeostasis in tobacco. Plant Growth Regul. https://doi.org/10.1007/s10725-022-00857-2
Gomes MH, Vieira T, Fundo JF, Almeida DPF (2014) Polyphenoloxidase activity and browning in fresh-cut ‘Rocha’ pear as affected by pH, phenolic substrates, and antibrowning additives. Postharvest Biol Technol 91:32–38. https://doi.org/10.1016/j.postharvbio.2013.12.013
Han M, Gleave AP, Wang T (2010) Efficient transformation of Actinidia arguta by reducing the strength of basal salts in the medium to alleviate callus browning. Plant Biotechnol Reports 4:129–138. https://doi.org/10.1007/s11816-010-0128-1
Hrynets Y, Bhattacherjee A, Betti M (2019) Nonenzymatic browning reactions: Overview. Encyclopedia of Food Chemistry. Elsevier, pp 233–244
Jones AMP, Saxena PK (2013) Inhibition of phenylpropanoid biosynthesis in Artemisia annua L.: A novel approach to reduce oxidative browning in plant tissue culture. PLoS ONE 8:e76802. https://doi.org/10.1371/journal.pone.0076802
Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M (2007) KEGG for linking genomes to life and the environment. Nucleic Acids Res 36:480–484. https://doi.org/10.1093/nar/gkm882
Kim SJ, Park WT, Uddin MR, Kim YB, Nam SY, Jho KH, Park SU (2013) Glucosinolate biosynthesis in hairy root cultures of broccoli (Brassica oleracea var. italica). Nat Prod Commun 8:217–220. https://doi.org/10.1177/1934578X1300800222
Kumar N, Gulati A, Bhattacharya A (2013) L-glutamine and l-glutamic acid facilitate successful Agrobacterium infection of recalcitrant tea cultivars. Appl Biochem Biotechnol 170:1649–1664. https://doi.org/10.1007/s12010-013-0286-z
Laukkanen H, Rautiainen L, Taulavuori E, Hohtola A (2000) Changes in cellular structures and enzymatic activities during browning of Scots pine callus derived from mature buds. Tree Physiol 20:467–475. https://doi.org/10.1093/treephys/20.7.467
Li T, He M, Zeng J, Chen ZS, Qu HX, Duan XW, Jiang YM (2020) Choline chloride alleviates the pericarp browning of harvested litchi fruit by inhibiting energy deficiency mediated programmed cell death. Postharvest Biol Technol 167:111224. https://doi.org/10.1016/j.postharvbio.2020.111224
Liu X, Lu YZ, Yang Q, Yang HY, Li Y, Zhou BY, Li TT, Gao Y, Qiao LP (2018) Cod peptides inhibit browning in fresh-cut potato slices: A potential anti-browning agent of random peptides for regulating food properties. Postharvest Biol Technol 146:36–42. https://doi.org/10.1016/j.postharvbio.2018.08.001
Liu J, Yang J, Jiang C, Zhou JH, Zhao YY, Huang LQ (2021a) Volatile organic compound and endogenous phytohormone characteristics during callus browning in Aquilaria sinensis. Ind Crops Prod 168:113605. https://doi.org/10.1016/j.indcrop.2021.113605
Liu X, Zhang A, Zhao J, Shang J, Zhu ZW, Wu XX, Zha DS (2021b) Transcriptome profiling reveals potential genes involved in browning of fresh-cut eggplant (Solanum melongena L.). Sci Rep 11:16081. https://doi.org/10.1038/s41598-021-94831-z
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Miao H, Zeng W, Zhao M, Wang JS, Wang QM (2020) Effect of melatonin treatment on visual quality and health-promoting properties of broccoli florets under room temperature. Food Chem 319:126498. https://doi.org/10.1016/j.foodchem.2020.126498
Min T, Bao Y, Zhou B, Yi Y, Wang LM (2019) Transcription profiles reveal the regulatory synthesis of phenols during the development of lotus rhizome (Nelumbo nucifera Gaertn). Int J Mol Sci 20:2735. https://doi.org/10.3390/ijms20112735
Nadafzadeh M, Abdanan Mehdizadeh S, Soltanikazemi M (2018) Development of computer vision system to predict peroxidase and polyphenol oxidase enzymes to evaluate the process of banana peel browning using genetic programming modeling. Sci Hort 231:201–209. https://doi.org/10.1016/j.scienta.2017.12.047
Nagai T, Suzuki N (2001) Partial purification of polyphenol oxidase from Chinese cabbage Brassica rapa L. J Agric Food Chem 49:3922–3926. https://doi.org/10.1021/jf000694v
Nguyen T, Ketsa S, Doorn WG (2003) Relationship between browning and the activities of polyphenoloxidase and phenylalanine ammonia lyase in banana peel during low temperature storage. Postharvest Biol Technol 30:187–193. https://doi.org/10.1016/S0925-5214(03)00103-0
Rana M, Han Z, Song D, Liu GF, Li DX (2016) Effect of medium supplements on Agrobacterium rhizogenes mediated hairy root induction from the callus tissues of Camellia sinensis var. sinensis. International Journal of Molecular Sciences 17:1132. https://doi.org/10.3390/ijms17071132
Rayan A, Morsy N (2020) Thermal inactivation kinetics of peroxidase and polyphenol oxidase from pomegranate arils (Punica granatum L. cv. Wonderful). J Food Biochem 44:e13428. https://doi.org/10.1111/jfbc.13428
Ru Z, Lai Y, Xu C, Li L (2013) Polyphenol oxidase (PPO) in early stage of browning of Phalaenopsis leaf explants. J Agric Sci 5:57. https://doi.org/10.5539/jas.v5n9p57
Sharafi Y, Aghdam MS, Luo Z, Jannatizadeh A, Razavi F, Fard ZR, Farmani B (2019) Melatonin treatment promotes endogenous melatonin accumulation and triggers GABA shunt pathway activity in tomato fruits during cold storage. Sci Hort 254:222–227. https://doi.org/10.1016/j.scienta.2019.04.056
Shekari A, Hassani RN, Aghdam MS, Rezaee M, Jannatizadeh A (2021) The effects of melatonin treatment on cap browning and biochemical attributes of Agaricus bisporus during low temperature storage. Food Chem 348:129074. https://doi.org/10.1016/j.foodchem.2021.129074
Sun QQ, Zhang N, Wang JF, Cao YY, Li XS, Zhang HJ, Zhang L, Tan DX, Guo YD (2016) A label-free differential proteomics analysis reveals the effect of melatonin on promoting fruit ripening and anthocyanin accumulation upon postharvest in tomato. J Pineal Res 61:138–153. https://doi.org/10.1111/jpi.12315
Szklarczyk D, Gable AL, Nastou KC, Lyon D, Kirsch R (2021) The STRING database in 2021: customizable protein-protein networks, and functional characterization of user-uploaded gene/measurement sets. Nucleic Acids Res 49:605–612. https://doi.org/10.1093/nar/gkaa1074
Tang TT, Xie XF, Ren X, Wang WJ, Tang XM, Zhang J, Wang ZD (2020) A difference of enzymatic browning unrelated to PPO from physiology, targeted metabolomics and gene expression analysis in Fuji apples. Postharvest Biol Technol 170:111323. https://doi.org/10.1016/j.postharvbio.2020.111323
Teng YX, Murtaza A, Iqbal A, Fu JL, Ali SW, Iqbal MA, Xu XY, Pan SY, Hu WF (2020) Eugenol emulsions affect the browning processes, and microbial and chemical qualities of fresh-cut Chinese water chestnut. Food Bioscience 38:100716. https://doi.org/10.1016/j.fbio.2020.100716
Tian P, Lu X, Bao JY, Zhang XM, Lu YQ, Zhang XL, Wei YC, Yang J, Li S, Ma SY (2021) Transcriptomics analysis of genes induced by melatonin related to glucosinolates synthesis in broccoli hairy roots. Plant Signal Behav 16:1952742. https://doi.org/10.1080/15592324.2021.1952742
Wan SQ, Wang WD, Zhou TS, Zhang YH, Chen JF, Xiao B, Yang YJ, Yu YB (2018) Transcriptomic analysis reveals the molecular mechanisms of Camellia sinensis in response to salt stress. Plant Growth Regul 84:481–492. https://doi.org/10.1007/s10725-017-0354-4
Wang T, Hu MJ, Yuan DB, Yun Z, Gao ZY, Su ZH, Zhang ZK (2020) Melatonin alleviates pericarp browning in litchi fruit by regulating membrane lipid and energy metabolisms. Postharvest Biol Technol 160:111066. https://doi.org/10.1016/j.postharvbio.2019.111066
Wang C, Zhang XL, Gao Y, Han YQ, Wu XZ (2021a) Path analysis of non-enzymatic browning in Dongbei Suancai during storage caused by different fermentation conditions. Food Chem 335:127620. https://doi.org/10.1016/j.foodchem.2020.127620
Wang Q, Wu XY, Liu L, Yao DZ, Li JC, Fang J, Chen XN, Zhu LW, Liu P, Ye ZF, Jia B, Heng W (2021b) Transcriptome and metabolomic analysis to reveal the browning spot formation of ‘Huangguan’ pear. BMC Plant Biol 21:321. https://doi.org/10.1186/s12870-021-03049-8
Wang ZQ, Pu HL, Shan SS, Zhang P, Li JK, Song HM, Xu XB (2021c) Melatonin enhanced chilling tolerance and alleviated peel browning of banana fruit under low temperature storage. Postharvest Biol Technol 179:111571. https://doi.org/10.1016/j.postharvbio.2021.111571
Wang WC, Pang JY, Zhang FH, Sun LP, Yang L, Kadambot HMS (2022) Transcriptomic and metabolomics-based analysis of key biological pathways reveals the role of lipid metabolism in response to salt stress in the root system of Brassica napus. Plant Growth Regul 97:127–141. https://doi.org/10.1007/s10725-021-00788-4
Worarad K, Suzuki T, Norii H, Mochizuki Y, Ishii T, Shinohara K, Miyamoto T, Kuboyama T, Inoue E (2021) Transcriptome profiling for pericarp browning during long-term storage of intact lotus root (Nelumbo nucifera). Plant Growth Regul 95:207–221. https://doi.org/10.1007/s10725-021-00736-2
Young MD, Wakefield MJ, Smyth GK, Oshlack A (2010) Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol 11:R14. http://genomebiology.com/2010/11/2/R14
Yun Z, Gao HJ, Chen X, Chen ZSZ, Zhang ZK, Li TT, Qu HX, Jiang YM (2021) Effects of hydrogen water treatment on antioxidant system of litchi fruit during the pericarp browning. Food Chem 336:127618. https://doi.org/10.1016/j.foodchem.2020.127618
Zhang ZK, Huber DJ, Qu HX, Yun Z, Wang H, Huang ZH, Huang H, Jiang YM (2015) Enzymatic browning and antioxidant activities in harvested litchi fruit as influenced by apple polyphenols. Food Chem 171:191–199. https://doi.org/10.1016/j.foodchem.2014.09.001
Zhang XL, Bao JY, Lu X, Tian P, Yang J, Wei YC, Li S, Ma SY (2022) Transcriptome analysis of melatonin regulating the transformation of glucoraphanin to sulforaphane in broccoli hairy roots. Physiol Mol Biology Plants 28:51–64. https://doi.org/10.1007/s12298-022-01143-1
Zheng HH, Liu W, Liu S, Liu CH, Zheng L (2019) Effects of melatonin treatment on the enzymatic browning and nutritional quality of fresh-cut pear fruit. Food Chem 299:125116. https://doi.org/10.1016/j.foodchem.2019.125116
Zhu H, Liu J, Wen Q, Chen M, Wang B (2017) De novo sequencing and analysis of the transcriptome during the browning of fresh-cut Luffa cylindrica “Fusi-3” fruits. PLoS ONE 12:e0187117. https://doi.org/10.1371/journal.pone.0187117
Acknowledgements
This work was supported by the National Natural Science Foundation of China (31,860,067), Gansu Province Higher Education Innovation Fund Project (2021B-136), Gansu Agricultural University Patent Transformation Project (GSAU-JSZR-2021-001), Longyuan Youth Innovation and Entrepreneurship Project (2016-3-18), Gansu Provincial People’s Livelihood Science and Technology Project (1603FCMG007).
Author information
Authors and Affiliations
Contributions
Jie Yang designed the experiments and wrote the manuscript. Jie Yang, Xiaoling Zhang and Peng Tian performed the experiments. Jie Yang, Jinyu Bao and Xiaotong Shi analyzed the data. Sheng Li, Shaoying Ma, Jinyu Bao and Xu Lu revised the manuscript. All authors read and approved the final manuscript.
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare no conflict of interest.
Additional information
Communicated by Hong-Xia Zhang.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Yang, J., Bao, J., Lu, X. et al. Transcriptomic analysis of the effects of melatonin on genes potentially related to the browning of broccoli (Brassica oleracea L. var. italica Planch) hairy roots. Plant Growth Regul 98, 557–567 (2022). https://doi.org/10.1007/s10725-022-00893-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10725-022-00893-y