Hydrogen peroxide is involved in hydrogen sulfide-induced carbon assimilation and photoprotection in cucumber seedlings

Highlights

• H₂S alleviates the negative effects of chilling stress on photosynthesis.

• H₂S may trigger endogenous H₂O₂ accumulation.

• H₂O₂ may act as a downstream signaling in H₂S-induced protection of the photosynthetic apparatus under chilling stress.

Abstract

Hydrogen sulfide (H₂S) and Hydrogen peroxide (H₂O₂) are two crucial gaseous signaling molecules that participate in various physiological processes and abiotic stresses. However, how the synergy of H₂S and H₂O₂ regulates photosynthesis have rarely been studied. This study aimed to reveal the mechanism underlying the interaction between H₂S and H₂O₂ in plants response to photosynthesis. Cucumber (Cucumis sativus L., ‘Jinyou 35’) seedlings were used as the material and grown in a climate chamber at 26 °C/18 °C with a 600 μmol m⁻²·s^-1 photon flux density (PFD). Sodium hydrosulfide hydrate (NaHS, an H₂S donor), H₂O₂, and their scavengers or inhibitors were applied as foliar sprayed at the two-leaf seedling stage. The result showed that both NaHS and H₂O₂ increased the CO₂ assimilation, which mainly attributed to an increase in the activity and gene expression of photosynthetic enzymes. NaHS and H₂O₂ also induced photoprotection for both photosystem Ⅱ (PSⅡ) and photosystem Ⅰ (PSⅠ) in cucumber seedlings, by activating the D1 protein repair pathway under chilling stress. Interestingly, 1.0 mM NaHS significantly enhanced the relative gene expression of respiratory burst oxidase homolog (RBOH), which in turn elevated endogenous H₂O₂ accumulation in cucumber seedlings. However, H₂O₂ had little effect on gene expression of L-/D-cysteine desulfhydrase (L-/D-CD) and endogenous H₂S level. The H₂S-induced adaptive response of photosynthesis to chilling stress was suppressed by diphenyleneiodonium (DPI, a H₂O₂ generation inhibitor) or dimethylthiourea (DMTU, a H₂O₂ scavenger). These data suggest that NaHS alleviates the negative effects of chilling stress on photosynthesis by improving photosynthetic carbon assimilation, carbon metabolism, and photoprotection for both PSⅡ and PSⅠ in cucumber seedlings. H₂O₂ may act as a downstream signal in H₂S-induced protection of the photosynthetic apparatus in cucumber seedlings under chilling stress.

Introduction

Cucumber (Cucumissativus L.) is an important thermophilic vegetable that is vulnerable to low temperature. However, cucumber plants commonly encounter chilling stress because they are mainly cultivated through the winter in solar greenhouses in the north of China. Under chilling stress, the photosynthetic capacity of cucumber is immediate impacted, including the inhibition of photosynthetic enzyme activities, degradation of membrane proteins, and the disturbance of carbohydrate metabolism (Allen and Ort, 2001). Therefore, strategies to maintain thylakoid electron transport and carbon assimilation in cucumber under low temperature are currently a pressing issue.

Hydrogen sulfide (H₂S), a novel endogenous gasotransmitter, has potential regulatory functions for the growth and development of plants, including seed germination (Zhang et al., 2008; Chen et al., 2019), root organogenesis (Fang et al., 2014), flower senescence (Zhang et al., 2011a) and photosynthesis (Chen et al., 2011). Exogenous H₂S can enhance photosynthesis by promoting chloroplast biogenesis, gene expression and activity of ribulose-1,5-bisphosphate carboxylase (RuBPCase/Rubisco), as well as proteins involved in thiol redox modifications in Spinacia oleracea seedlings (Chen et al., 2011). H₂S also alleviates various abiotic stresses, such as drought (Shen et al., 2013), salinity (Lai et al., 2014), extreme temperature (Li et al., 2013), and heavy metals (Guan et al., 2018). Related experiments have shown that H₂S effectively decreases the uptake of cadmium (Cd) and alleviates Cd-induced growth inhibition through improvements in photosynthetic performance, nutrients uptake, antioxidants enzyme activities, and ultra-structural changes in Brassica napus(Ali et al., 2014). In response to low temperature conditions, H₂S fumigation can alleviate chilling injury in banana by enhancing antioxidant system and the Δ¹-pyrroline-5-carboxylate synthetase (P5CS) activities, which mainly attributed to an elevation in proline content (Luo et al., 2015). Very recently, it was reported that H₂S had a positive role in inducing cucurbitacin C synthesis as a result of protein modification by S-sulfhydration, finally improving the cold tolerance of plants by regulating the synthesis of secondary metabolites (Liu et al., 2019). Additionally, H₂S could alleviate cucumber seedlings chilling damage, potentially interacting with nitric oxide (NO) signaling (Wu et al., 2016).

Hydrogen peroxide (H₂O₂) is a form of reactive oxygen species (ROS) that is generated as a result of oxidative stresses (Qiao et al., 2014). Oxidative stresses may provoke oxidative damage due to excessive ROS accumulation under various abiotic stress conditions. A high accumulation of ROS considered as an undesirable and harmful event in stress metabolism (Miller et al., 2008). However, several pieces of evidence have suggested that ROS, especially H₂O₂, also acts as a signaling molecule (Choudhury et al., 2013; Baxter et al., 2014), playing an important role in responses to abiotic stress (Wang et al., 2016). H₂O₂ is generated in chloroplasts, mitochondria, and peroxisomes, and at the plasma membrane or cell wall (Quan et al., 2008). H₂O₂ is a non-radical ROS and by-product of oxidative stress metabolism, being the only ROS species that is stable in solution due to no net charge (Sun et al., 2016) and that can retain homeostasis in plant cells via the complex and effective scavenging systems (Carvalho, 2008). Moreover, previous studies have indicated that H₂O₂ acts as a signal that induces a range of molecular, biochemical, and physiological responses within plant cells and mediates cross-talk between signaling pathways (Neill et al., 2002).

A relationship has been reported between H₂S and H₂O₂ in plant resistance to adversity, and exogenous H₂S can resist the oxidative damage caused by various stresses by improving the antioxidant enzyme activities and thereby reducing the level of H₂O₂ (Zhang et al., 2008). Li and He (2015) showed that H₂S-promoted seed germination was eliminated by the H₂O₂ scavenger dimethylthiourea (DTMU), NADPH oxidase inhibitor diphenyleneiodonium (DPI) and free radical scavenger tiron, but the H₂S biosynthesis inhibitor DL-propargylglycine (PAG) and H₂S scavenger hydroxylamine (HT) did not weaken H₂O₂-promoted seed germination. Therefore, they suggested that H₂O₂ might be a downstream signaling molecule of H₂S in seed germination of mung bean. However, another study found that H₂O₂ modulators suppressed the salt stress-induced increases in H₂S levels and L-/D-cysteine desulfhydrase (L-/D-CD) activities, as well as stomatal closure in leaves of Vicia faba. In contrast, there were no effects of H₂S modulators on salt stress-induced H₂O₂ production in guard cells. Thus, the authors suggested that H₂S might function downstream of H₂O₂ in salt stress-induced stomatal movement in Vicia faba (Ma et al., 2019). Based on the above results, we hypothesized that a crosstalk might exist between H₂S and H₂O₂ in photosynthesis under chilling stress. To test this possibility, we investigated the effects of H₂S and H₂O₂ on photosynthesis in cucumber seedlings and the role of H₂O₂ in H₂S-induced regulation of CO₂ assimilation, carbohydrate metabolism and photoprotection in cucumber seedlings in response to chilling stress. The objective was to illustrate the mechanism of the positive effects of H₂S in regulating photosynthesis and its signalling pathways in the chilling stress response of cucumbers.