Horticulture is a dynamic sector where a wide variety of crops and their products are continuously innovating, and new market opportunities are considered and explored. It includes the cultivation of fruits, vegetables, nuts, seeds, herbs, sprouts, mushrooms, algae, flowers, seaweeds, and non-food crops such as grass and ornamental trees and plants. However, this sector faces many challenges, from retaining economic competitiveness, achieving full economic and environment sustainability and responding to climate change (Fawad et al., 2021; Taskesenlioglu et al., 2022). In addition, growing population and rise in income level will lead to increase in demand of high-value agriculture (HVA) produce (Ghosh, 2012).

Okra (Abelmoschus esculentus L.) as a vegetable, is now becoming more and more popular in China because of its rich nutritive value (Petropoulos et al., 2018). Generally, okra is extremely vulnerable to mechanical damage and therefore quite difficult for storage after harvest. According to statistics, the loss rate of postharvest okra ranges from 50% to72% (Gajanan et al., 2018). Therefore, it is of great importance to develop its preservation methods and prolong its shelf life.

In recent years, the role of hydrogen against abiotic stress in plants has been widely studied (Wu et al., 2019; Yan et al., 2022; Yu et al., 2023). In addition, hydrogen also plays an important role in maintaining fruit quality and delaying senescence process in several postharvest vegetables and fruit. For instance, 25% hydrogen-rich water (HRW) treatment improved the activities of antioxidant enzymes and inhibited the decay incidence of mushrooms (Chen et al., 2017). HRW treatment also increased fruit quality through regulating antioxidant capacity and energy metabolism in Rosa sterile fruit (Dong et al., 2022).

Plant hormones are chemical compounds present in very low concentration in plant, appearing to be multi-regulatory molecules during fruit development, ripening and senescence (Poveda, 2020). Indolacetic acid (IAA), which exists in almost all plant organs, is the main form of plant auxin (Yue et al., 2019). It was reported that IAA content accumulated during fruit ripening but decreased rapidly during fruit senescence (Trainotti et al., 2007; Chen et al., 2014). Exogenous treatment with IAA could delay the ripening process in harvested kiwifruit (Gan et al., 2021). The protective effects of melatonin as an antioxidant has been frequently investigated (Reiter et al., 2001; Iriti et al., 2006). Its effect on prolonging shelf life and improving fruit quality has been demonstrated in postharvest strawberries and table grapes (Liu et al., 2018; Sun et al., 2020). Abscisic acid (ABA) has a key role in regulating fruit senescence (McAtee et al., 2013) and conferring tolerance to environmental stresses. Previous studies have showed that ABA induced fruit senescence in postharvest sweet cherries (Luo et al., 2014) and enhanced the resistance against chilling in harvested zucchini (Carvajal et al., 2017). A tetracyclic diterpenoid hormone known as gibberellic acid (GA) has been reported to maintain fruit quality and prolong shelf life in postharvest okras (Xiao et al., 2022), Chinese flowering cabbage (Fan et al., 2021), persimmons (Maurer et al., 2019).

In a previous study, postharvest okra softening was delayed and storage life was prolonged by treatment with 0.22 mM HRW (Dong et al., 2023). However, there is no information available on the effect of hydrogen (H₂), as an important signal molecule, on the regulation of metabolism of plant hormones in postharvest fruit or vegetables. Therefore, in order to further understand the mechanism of H₂ in the preservation of okras after harvest, here we determined the levels of several hormones such as melatonin, IAA, ABA and GA3 together with their metabolizing genes in okras treated with HRW to reveal the relationship between H₂ and other signal molecules.

As an important bio-regulator in plants, hydrogen has critical roles in diverse processes in plant growth, development and stress responses (Ren et al., 2017a; Yu et al., 2023). Recently, attention to the role of hydrogen has shifted from plant to postharvest vegetables and fruit. For example, HRW treatment was found to improve antioxidant capacity and extend the shelf life of pakchoi (An et al., 2021). Additionally, kiwifruit and mushrooms treated with HRW showed higher free radical scavenging activity and lower rot rate as compared to the non-treated groups (Chen et al., 2017; Hu et al., 2018). In our previous study, we observed that hydrogen-rich water treatment delayed the softening process and prolonged the storage time by modifying the cell wall metabolism and cell wall composition in postharvest okras (Dong et al., 2023). Similarly, here we demonstrated again that HRW treatment could prolong the shelf life of postharvest okras.

In the last few years, a growing number of studies have revealed how hydrogen interacts with other plant hormone signalling pathways, indicating the key role it plays in protecting plants from stress (Zeng, 2013). H₂ enhanced the drought resistance of tomato seedlings by regulating the expression of genes in ABA biosynthesis and signal transduction (Yan et al., 2022); H₂ was also reported to be involved in auxin-induced lateral root formation (Cao et al., 2017). Melatonin, a well-known animal hormone that the brain produces in response to darkness, was first discovered in plants in 1995 (Dubbels et al., 1995), and since then its role in plants has been discovered and studied widely. Accumulating documents over the past decades demonstrated that melatonin could fortify plants against senescence and biotic stresses such as salt, chilling, drought and heavy metals (Cai et al., 2017; Wang et al., 2017; Yan et al., 2019). In addition to inducing stress resistance in plants, much progress has been made recently in understanding the roles of melatonin in regulating storage time in postharvest fruit. Melatonin application enhanced antioxidant defense and increased tolerance to chilling in cold-stored peaches (Cao et al., 2018). The postharvest treatment with melatonin inhibited decay incidence and delayed in strawberries (Liu et al., 2018). Through our experiments, we found that the melatonin content and its biosynthetic genes such as AeTDC, AeT5H, AeSNAT and AeCOMT were upregulated in okras after HRW treatment, indicating the increased melatonin biosynthesis in postharvest okras treated with HRW contribution to the delay of senescence and prolongation of shelf life. Furthermore, Su et al. (2021) reported that melatonin acted downstream of molecular hydrogen and its combination with molecular hydrogen induced salt tolerance in Arabidopsis. Therefore, it is reasonable to speculate that the crosstalk between molecular hydrogen and melatonin also plays a role in delaying senescence in postharvest fruit and vegetable.

Lately, Wu et al. (2020) revealed that HRW treatment enhanced the growth of mung bean seedlings by stimulating elongation of hypocotyl and root cells via increasing GA and IAA content, suggesting that hydrogen acted upstream of IAA and GA biosynthesis during plant growth. Meanwhile, GA and IAA have been demonstrated their roles in inhibiting the senescence process in postharvest okras and strawberries, respectively (Chen et al., 2014; Xiao et al., 2022). Therefore, to further reveal the crosstalk between hydrogen and GA or IAA, the biosynthetic and signalling pathways of these two phytohormones were investigated in okras after HRW treatment. In our present study, HRW upregulated the anabolic genes such as AeGA20OX, AeKO and AeKAO, but downregulated the catabolic gene AeGA20Xs and AeDELLA the negative regulator of gibberellin signalling, thereby increasing GA content and signalling in postharvest okras. In respect of IAA biosynthesis, the treatment also increased the IAA content along with the enhanced transcripts of biosynthetic genes including AeTAR, AeYUC and AeMES and the early auxin-responsive gene AeSAUR, however, the lower expression of the degradative gene AeDAO was observed in the treated okras. Moro et al. (2017) found that postharvest exogenous IAA treatment postponed fruit ripening by affecting anthocyanin biosynthesis in red raspberries. It has also been reported that the application of exogenous GA inhibited fruit softening and delayed the loss of ascorbic acid and soluble reducing sugar, thereby prolonging the shelf life of banana fruit (Huang et al., 2014). Recently, in carambola fruit, exogenous 2,4-epibrassinolide treatment maintained fruit quality and inhibited fruit ripening due to the increase in levels of IAA and GA in carambola fruit (Zhu et al., 2021). Therefore, taken together, we could conclude that HRW treatment delayed senescence and extended the shelf life of postharvest okras also due to the increase of levels of GA and IAA.

ABA, another plant hormone, regulates numerous aspects of fruit ripening, senescence and stress responses. It not only enhanced the ripening process of climacteric fruit like kiwifruit and mango (Zaharah et al., 2013; Gan et al., 2020), but also promoted the senescence of non-climacteric fruit such as sweet cherries (Luo et al., 2014). Previous studies have showed that the application of alginate oligosaccharide inhibited the accumulation of ABA and reduced the degradation of cell wall components, thereby maintaining fruit quality in strawberries (Bose et al., 2019). Gan et al. (2020) found silencing AeNCED1 the key gene in ABA biosynthesis in kiwifruit could decline ABA content and postpone fruit softening. The inhibition of fruit ripening in carambola fruit treated with 2,4-epibrassinolide was also related to the decrease in ABA content (Zhu et al., 2021). Our experimental results showed that HRW treatment reduced the expression of genes related to ABA synthesis such as AeNCED, AeZEP, and AeAAO but increased the transcripts of degradative gene AeCYP707A, thus finally reducing ABA content, which was associated with the inhibition of senescence in okras with HRW treatment. It has been reported that both PLYs and ABFs play important roles in regulating Arabidopsis leaf senescence triggered by ABA (Gao et al., 2016; Zhao et al., 2016). In our present study, corresponding to the ABA level, AePLY3/9 and AeABF were also downregulate by HRW treatment, suggesting that hydrogen could regulate ABA-associated signals to slow down the senescence process in postharvest okras.

The role of GABA in fruit ripening, senescence and tolerance against stresses was well reported in different kinds of postharvest fruit and vegetables (Sheng et al., 2017; Palma et al., 2019; Asgarian et al., 2022). However, in our present study, there was no difference in GABA content together with the expression of its metabolizing genes between the control and HRW treated okras, suggesting that no interaction between H₂ and GABA existed in postharvest okras.

In conclusion, our data demonstrated that HRW treatment could regulate the contents of several phytohormones and consequently prolong shelf life in postharvest okras. Our results suggested that melatonin, IAA and GA were negatively while ABA was positively associated with fruit senescence in postharvest okras. The increased shelf life and delaying okra senescence by HRW treatment was due to its capability to increase melatonin, IAA and GA contents and inhibit ABA level resulted from its regulation of their metabolizing genes, indicating H₂, as a signaling molecule, could interrelate to these hormones and influenced their biosynthesis and signaling and finally co-adjusted okra senescence after harvest. However, more evidence is needed to determine the underline cross-talk regulation and transduction relationship between H₂ and other phytohormones for further exploration on the role of H₂ in the shelf life prolongation of postharvest products.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

WD: Perform the experiment, write the original manuscript. SC and QZ: Conceived and designed the experiment. SJ, CZ and QL: Formal analysis, Investigation. XL and WC: Conducted the experiments, analyzed the data. LS and ZY: Funding acquisition, supervision and manuscript editing. All authors contributed to the article and approved the submitted version.