Harvesting at different time-points of day affects glucosinolate metabolism during postharvest storage of broccoli

Highlights

• The moment of the day to harvest influences broccoli glucosinolate metabolism.

• Postharvest storage affects differently aliphatic - indolic glucosinolate metabolisms.

• Expression of genes associated to glucosinolate metabolism are influenced by light.

Abstract

The consumption of broccoli provides a large quantity of compounds with nutraceutical properties to the human diet. Broccoli has a high content of glucosinolates, compounds of the specialized metabolism with anticarcinogenic activity. In a previous work, we found that harvesting different time-points during the day affects the rate of senescence of broccoli heads during postharvest storage. In this work, we tested the same cultural practice to evaluate glucosinolate content and expression of genes involved in glucosinolate metabolism. Broccoli heads were harvested at 8:00, 13:00 and 18:00 h and stored for 5 d at 20 °C in darkness. We found that content and composition of the glucosinolate pool was affected by the time of harvest. Levels of indolic glucosinolates decreased with the time of harvest on the day whereas indolic glucosinolate showed only a moderate decrease. The expression of genes associated to the biosynthesis of aliphatic glucosinolates was variable during the day. In relation to indolic glucosinolates, an increase in the expression of the transcription factor BolMYB51 was detected around 13:00 h, which strongly correlated with the increase in expression of genes associated to their biosynthesis towards the end of the day.

During postharvest, the storage in darkness affected differently the metabolisms of indolic and aliphatic glucosinolates. The content of aliphatics decreased during the postharvest period, as well as the expression of the genes associated with their biosynthesis. In contrast, in the case of indolics, their content remained constant or varied slightly, while the expression of the associated biosynthetic genes decreased only slightly. Finally, the genes related to the degradation of glucosinolates appeared to be strongly regulated by light conditions, since their expression increased during the course of the day and decreased markedly during postharvest storage in darkness.

These results suggest that harvesting of broccolis close to noon would be convenient to maintain higher levels of glucosinolates during postharvest storage.

Introduction

Broccoli is a vegetable with remarkable nutritional properties. It has a low content of calories, lipids and carbohydrates, but it is a rich source of multiple proteins and dietary fibres. It also contains all the essential amino acids, an important amount of vitamins, minerals, dietary folate and antioxidants (ascorbic acid and phenolic compounds) (Jeffery, Brown, Kurilich, Keck, Matusheski, Klein, & Juvik, 2003). However, in the last years, the study of broccoli along with other cruciferous has intensified due to its high concentrations of glucosinolates.

Glucosinolates are secondary metabolites derived from amino acids characteristic of the Brassicaceae family plants. These compounds are thioglycosides chemically stable until they come in contact with the enzyme myrosinase, which is stored in different cell compartments from glucosinolates (Halkier & Gershenzon, 2006). When tissue damage occurs, compartmentalization between glucosinolates and myrosinase is lost. The enzyme catalyses the hydrolysis of the thioglucoside bond resulting in glucose and an unstable aglycone, which rearrange into isothiocyanates, thiocyanates and nitriles. Some of these products have biological activities like antimicrobial, antifungal and insect repellent properties (Halkier and Gershenzon, 2006, Jeffery and Araya, 2009)

Glucosinolates are derived from amino acids through a series of reactions: oxidative decarboxylation, elongation of side chains and secondary modifications (Yan & Chen, 2007). Three types of glucosinolates can be described according to the precursor amino acid: aliphatic (if derived from methionine), aromatic (derived from phenylalanine and tyrosine) and indolic (derived from tryptophan).

Several studies have shown that in broccoli the most abundant glucosinolate is glucoraphanin, an aliphatic glucosinolate (Jones et al., 2006, Mølmann et al., 2015, Yuan et al., 2010). However, other authors suggest that the glucosinolate profile may present variations according to the cultivar studied, with some varieties presenting greater amounts of indolic glucosinolates such as glucobrassicin or neoglucobrassicin (Ávila et al., 2013, Ku et al., 2013). A description of this high variability was also made by Wang, Gu, Yu, Zhao, Sheng, and Zhang (2012), who analyzed up to 143 broccoli lines and found significant variations in the concentration of individual glucosinolates among broccoli populations. The content of glucosinolates in broccoli can be influenced by numerous factors related to the management of the crop (nutrients, fertilization, temperature, photoperiod (Mølmann, Steindal, Bengtsson, Seljåsen, Lea, Skaret, & Johansen, 2015), as well as postharvest storage conditions (Rybarczyk-Plonska et al., 2016, Yuan et al., 2010). Additionally, some authors have found that the accumulation of aliphatic glucosinolates is mainly due to genetic factors, whereas the content of indolic glucosinolates is mainly influenced by environment and genotype interaction (Brown et al., 2002, Farnham et al., 2004).

The biosynthetic pathways of glucosinolates and the enzymes and genes involved in each step are well characterized (Ishida, Hara, Fukino, Kakizaki, & Morimitsu, 2014) and are shown in Supplementary Fig. 1. The degradation of glucosinolates is mediated by myrosinases. The chemical structure of the obtained products depends on the side chain of the glucosinolate, the reaction conditions (pH), and the presence of the epitiospecific protein (ESP), which is a co-factor of myrosinase that favours the formation of nitriles (Ku, Choi, Kim, Kushad, Jeffery, & Juvik, 2013).

In general, the glucosinolate content decreases during broccoli postharvest storage along with the advance of senescence (Jones et al., 2006). Most postharvest treatments carried out in broccoli have focused on the maintenance of colour, given the economic impact that this implies on commercial quality. However, works that determine the content of glucosinolates and simultaneously evaluate their metabolism at the level of gene expression during postharvest storage are scarce. In this sense, it has been determined that treatments with modified atmospheres (Jia, Xu, Wei, Yuan, Yuan, Wang, & Wang, 2009) or 1-MCP (Yuan, Sun, Yuan, & Wang, 2010) can attenuate the decrease in glucosinolates content during postharvest storage. Glucosinolate content can be induced by artificial mechanical damage (Torres-Contreras et al., 2018, Torres-Contreras et al., 2017), or moderate microwave cooking (Lu, Pang, & Yang, 2020). Exogenous application of chemical mediators such as jasmonic acid and ethylene also promotes accumulation and biosynthesis of glucosinolates (Aguilar-Camacho et al., 2019, Torres-Contreras et al., 2018, Villarreal-García et al., 2016).

In a previous work, our group determined that harvesting at different times of the day can affect senescence rate during postharvest storage (Hasperué, Chaves, & Martínez, 2011). Optimizing the harvesting time can help to improve postharvest shelf life of broccoli without using chemicals or technologies that require high economic costs. In this work, we studied the effect of harvesting at different time-points of the day on glucosinolate metabolism during the postharvest storage of broccoli.