In plants, Se have been thought to be beneficial for plant growth and biomass accumulation in low dosages, but can also be toxic leading to retarded growth, reduced biomass, chlorosis and ultimately plant death (Gupta and Gupta, 2017). In this study, different concentrations of selenite were used to treat C. moschata seedlings. Results indicated that the growth of C. moschata seedlings under different concentrations of selenite exhibited obvious differences compared to the control (0 µM selenite treatment). A low concentration (2 µM) of selenite significantly decreased the plant height but had no or slight effect on the other growth parameters that we checked, indicating that the stem growth of C. moschata should be easily affected by selenite. The root vigor of C. moschata seedlings were found to be enhanced under 2 µM selenite treatment. Therefore, we deduced that 2 µM selenite should improve the seedling health index in C. moschata. Moderate concentrations (10–20 µM) of selenite could also elevate the root vigor of C. moschata seedlings but significantly retarded the seedling growth. In addition, S contents in the shoots was found to be enhanced at moderate concentrations of selenite, which was in consistent with previous studies in cucumber (Hawrylak-Nowak et al., 2015) and Arabidopsis (Van Hoewyk et al., 2008). High concentration of selenite (40 µM) seriously decreased the root vigor and inhibited the normal growth and development of C. moschata seedlings, and 80µM selenite even caused symptoms of toxicity, such as leaf chlorosis and root necrosis. The inhibition of root growth under selenite stress might be related to decreased auxin signaling, because all of the 8 DEGs described as auxin transporters in roots of C. moschata were downregulated in response to 40 µM selenite. In cucumber, the toxicity threshold for selenite was determined at the doses of 20 µM (Hawrylak-Nowak et al., 2015), suggesting that C. moschata should have a greater tolerance to selenite compared to cucumber. These results also remind us that the appropriate concentration of Se should be explored and chosen for cucurbitaceae crops in the process of Se biofortification cultivation. both 10µM and 20µM treatments resulted in increased S contents in leaf, but not in the root.
The selenite absorbed in roots can be rapidly converted to organic forms in roots, with limited migration to shoots (Li et al., 2008). Accumulation of Se is mostly found to be in organic forms in rice (Carey et al., 2012). Similarly, the majority of Se were organic forms in both shoots (61.40%-67.44%) and roots (70.06%-72.36%), and most of the Se (more than 91.23–92.44%) were accumulated in the roots under different concentrations of selenite (2 µM -40 µM). The Se contents in both shoots and roots of C. moschata seedlings were obviously higher than control, and they were closely related to the selenite doses, indicating that selenite could be effectively absorbed in C. moschata roots and partially transitioned to shoots. Selenite has been found to be transported across the root cell membrane by phosphate transporters (PHTs) (Zhang and Chu, 2022). In rice, the overexpression and mutant lines of OsPHT2 exhibited noticeably increased and decreased rates of selenite uptake, respectively (Zhang et al., 2014), and Se concentration was elevated in OsPHT8-overexpressing tobacco plants (Nicotiana tabacum) (Song et al., 2017), indicating that PHTs were important for the absorption of selenite in plants. In this study, several PHTs, such as CmoCh12G006790 and CmoCh18G011500, were highly upregulated in response to selenite treatment, suggesting that they should be involved in the uptake of selenite in C. moschata. Se is reported to be chemically similar to S (Zhang and Chu, 2022). Inostroza-Blancheteau et al. (Inostroza-Blancheteau et al., 2013) and Cao et al. (Cao et al., 2018) found that sulfate transporters (SULTRs) can be activated by selenite treatment in plant roots. The low-affinity sulfate transporter SULTR2;1 was reportedly involved in the transport of sulfate and selenate from the roots to the leaves (Zhang and Chu, 2022). In our study, two significantly upregulated sulfate transporters (CmoCh10G006430 and CmoCh11G005950) under selenite treatment were identified in C. moschata, and they were annotated to sulfate transporter 3.5 (SULTR3;5) (Table S3). SULTR3;5 is found to be colocalized with the SULTR2;1 in the root xylem and can strengthen the capacity of SULTR2;1 (Kataoka et al., 2004), meaning that high expressions of SULTR3;5s in C. moschata roots might contribute to the transport of selenium from roots to leaves. Besides, our results also supported the idea that ABC transporters might contribute to Se accumulation in plants (Rao et al., 2021; Ren et al., 2022), because a large of differentially expressed ABC transporters were found in our study and most of them were significantly upregulated under selenite treatment.
Because of the high similarity of Se and S (sulfur) elements, Se usually via S metabolic pathway to form into different forms in plants(Gupta and Gupta, 2017; Zhang and Chu, 2022). Sulfite oxidase (SO) has been reported to catalyze the oxidation of sulfite to sulfate in plants (Kappler and Enemark 2015). In this study, we found that the SO gene CmoCh15G000800 in C. moschata was significantly upregulated in response to selenite treatment. Thus, we inferred that partial selenite might also be oxidized to selenate by SO in C. moschata roots, which could be associated with the upregulated expressions of SULTR3;5s in C. moschata roots under selenite treatment. The conjecture can be further supported by the results that the genes encoding ATP sulfurylase (APS), adenylyl-sulfate kinase (APK), and adenylyl-sulfate reductase (APR), which were involved in the conversion of selenate to selenite in plants (Gupta and Gupta, 2017; Zhang and Chu, 2022), were significantly upregulated in response to selenite treatment. The sulfite reductase (SiR) is thought to catalyze the reduction of selenite to selenide (Van Hoewyk, 2013). However, we found the gene CmoCh11G008630 encoding SiR was downregulated under selenite treatment. Inhibition or knockdown of SiR were likely to not affect the selenite reduction to selenide (Ng and Anderson, 1979; Fisher et al., 2016), meaning that SiR might be not crucial for the selenite reduction in C. moschata. Many studies reported that the reduction of selenite to selenide can also occur non-enzymatically by glutathione in plants and the glutathione reductase (GR) is important in this pathway (Hsieh and Ganther, 1975; Zhang and Chu, 2022). In this study, the expression of CmoCh14G019350 encoding GR greatly increased in response to selenite treatment, suggesting that it should be involved in the selenite reduction in C. moschata. Cysteine synthase (CS) is reported to exhibit stronger affinity for selenide than for sulphide and catalyze the conversion of selenide to selenocystein (SeCys) (Gupta and Gupta, 2017). 2 CS genes were found to be significantly upregulated in our study, implying that they should be important for the synthesis of SeCys in C. moschata.
As a ubiquitous second messenger, Calcium (Ca2+) has been reported to play critical roles in plants response to numerous abiotic stresses (Ren et al., 2023). Cyclic nucleotide-gated channels (CNGCs) belong to calcium-permeable ion channels, and act as “on” component of Ca2 + signaling to mediate stress-induced calcium influx (Ma et al., 2023). Our results revealed that selenite stress significantly increased the expression levels of CNGC-coding genes (especially CmoCh13G011470), meaning that selenite should induce Ca2+ uptake and activate Ca2+ signaling in C. moschata. As expected, a series of genes annotated to calcium sensors, including calmodulins (CaMs), CaM-like proteins (CMLs), calcium-dependent protein kinases (CDPKs), and calcineurin B-like proteins (CBLs) (Kudla et al., 2018), showed changes in expression levels at 24h of selenite treatment, and most of them were upregulated. Rao et al. (Rao et al., 2021) reported that CDPKs were downregulated under selenate treatment in Cardamine violifolia. The reasons for different results should be related to plant species or Se form, concentration and treatment time. Ca2+ signaling can induce plant respiratory burst oxidase homologs (RBOHs)-mediated production of reactive oxygen species (ROS), which in turn promotes calcium influx by activating calcium-permeable ion channels (Dell'Aglio et al., 2019; Ravi et al., 2023). In this study, We identified 2 upregulated and 2 downregulated genes annotated to RBOH, and the former had significantly higher expressions under both control and selenate treatment conditions, meaning that RBOHs should be activated by Ca2+ signaling during selenite treatment in C. moschata, which was consistent with previous report that Se acts as pro-oxidant and generates ROS under high doses in plants (Gupta and Gupta, 2017). For maintaining the Ca2+ homeostasis in cells, plants need to remove the excessive Ca2+ by different Ca2+ efflux. Ca2+-ATPase and Ca2+/H+ exchangers are mainly membrane proteins responsible for Ca2+ efflux under abiotic stresses (Ren et al., 2023; Wang and Luan, 2024). 3 genes annotated to Ca2+-ATPase and 2 genes annotated to Ca2+/H+ exchangers were found to be obviously upregulated in response to selenite treatment in C. moschata. The activity of Ca2+ efflux indirectly reflected that Ca2+ accumulation might be excessive in selenite-treated C. moschata seedlings. Altogether, these DEGs related to Ca2+ influx, sensors and efflux reflected that Ca2+ signaling should be involved in plant responses to selenite treatment.
Se toxicity mainly due to ROS-mediated oxidative stress in plants (Gupta and Gupta, 2017). Antioxidant defense systems including non-enzymatic (GSH, etc) and enzymatic systems (including POD, SOD, etc.) are important for ROS (including O2•–, etc.) scavenging in plants (Pirasteh-Anosheh, Samadi et al. 2023). Our results revealed that there was excess accumulation of O2•– in the leaves of WCO39 seedlings treated with high doses of selenite (above 20 µM). CmoCh10G011710 (CmoCu/ZnSOD) and several POD genes, such as CmoCh15G014290 and CmoCh20G003430 were also significantly and rapidly upregulated under selenite treatment, suggesting that they should contribute to scavenging ROS during Se stress in C. moschata, which could be supported by previous reports that Se participated in increasing the activities of antioxidant enzymes in plant response to abiotic stresses (Sun et al., 2020; Kang et al., 2022). Glutathione (GSH) is an essential non-enzymatic antioxidant used for both detoxification of ROS and transmission of redox signals (Forman et al., 2009; Li et al., 2010) and the genes related to GSH metabolism play vital roles in tolerance of Se stress in plants (Rao et al., 2021; Ren et al., 2022). Se treatment could enhance the levels of GSH and the expressions of GSH genes in tomato (Li et al., 2016). Under Se stress, GSH level was elevated in Se-tolerant plant, and GSH depletion was found to be related to ROS accumulation (Grant et al., 2011), indicating that GSH should be important in plant adaptation to Se stress. In GSH cycle, GSH can be gradually degraded by Gamma-glutamyl trans-peptidase (GGT), Gamma-glutamyl cyclotransferase (GCT) and 5-oxoprolinase (OXP) enzymes (Bachhawat and Yadav, 2018), The products glutamate and L-cysteinylglycine can be further used for the biosynthesis of GSH (Bachhawat and Yadav, 2018). Our study indicated that the expression levels of GGT, GCT and OXP genes obviously increased in response to selenite treatment, implying that GSH metabolism should be reinforced under Se stress in C. moschata. Ascorbate–glutathione (AsA-GSH) pathway has been well documented to be involved in ROS scavenging (Li et al., 2010). In this pathway, we found the genes encoding GR, GPX, DHAR, APX and MDHAR were significantly upregulated under selenite treatment, meaning that the AsA-GSH cycle should contribute to protect cells from oxidative damage under Se stress in C. moschata. Besides, we found all of the 14 DEGs encoding glutathione S − transferase (GST), which can protect plants from oxidative burst by catalyzing the conjugation of GSH to hydrophobic and electrophilic substrates (Kumar and Trivedi, 2018), positively responded to the selenite treatment. The study in tea plant revealed that most of the differentially expressed GSTs were upregulated in the roots in response to selenite treatment (Ren et al., 2022). GSTs has been reported to protect plants from different abiotic stresses (Kumar and Trivedi, 2018). Thus, we think that GST-mediated metabolism should contribute to the survival of C. moschata from high concentration Se stress.
Melatonin is demonstrated to be effective in alleviating ROS-mediated oxidative stress in plants (Colombage et al., 2023). Melatonin biosynthesis in plants occurs via four sequential enzymatic steps involving tryptophan decarboxylase (TDC), tryptophan hydroxylase (TPH), tryptamine 5-hydroxylase (T5H), serotonin N-acetyltransferase (SNAT), N-acetylserotonin methyltransferase (ASMT), and caffeic acid O-methyltransferase (COMT) (Sun et al., 2021). The last step of melatonin biosynthesis is the conversion of N-acetylserotonin to MT catalyzed by COMT in Arabidopsis (Byeon et al., 2014) or ASMT in rice (Kang et al., 2011). Li et al. reported that different forms of Se could significantly induced the expressions of TDC, T5H, SNAT, and ASMT genes, and the induction of TDC is the most significant in tomato (Li et al., 2016). Our results revealed that selenite treatment can significantly induce the expressions of several COMT genes in C. moschata, which might further promote the synthesis of MT. We also found that exogenous application of MT could alleviate the growth-inhibited phenotype caused by selenite toxicity. All of these results suggested that COMT-mediated MT synthesis should be positively involved in Se stress in C. moschata.
The MAPK cascade, mainly consisting of MAP kinase kinase kinase (MAPKKK), MAP kinase kinase kinase (MAPKK), and MAP kinase (MAPK), is a ubiquitous and conserved signal trans-duction pathway in plant and has a dominant role in regulating plant adaptation to various environment stresses (Chen et al., 2021; Mo et al., 2021; Zhang and Zhang, 2022). Ca2+ and ROS signal induced by abiotic stresses can activate the mitogen − activated protein kinas (MAPK) signaling (Chen et al., 2021; Mo et al., 2021). In mice, selenium was found to regulate the ROS-mediated MAPK pathways (Indumathi et al., 2023), while there is little knowledge about the relationship between selenium and MAPK cascade in plants. In this study, we found that selenite treatment obviously promoted the expressions of different MAPK cascade genes, including MAPKs, MPKKs and MAPKKKs in C. moschata. MAPK cascades are also known to participate in the regulation of plant hormone signaling (Jagodzik, Tajdel-Zielinska et al. 2018). In Arabidopsis, MAPKs were found to promote ethylene (ET) biosynthesis by both the phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthases (ACSs, the rate-limiting enzyme of ET biosynthesis) and the upregulated expressions of ACS genes (Liu and Zhang, 2004; Li et al., 2012). Many studies indicated that suppression of MAPKs resulted in decreased expressions of wound-induced jasmonic acid (JA) biosynthesis genes and JA‐dependent defense genes (Haug et al., 2007; Takahashi et al., 2007; Jagodzik et al., 2018). As expected, our results revealed that selenite treatment not only resulted in higher expressions of many MAPK cascade genes, but also induced the expressions of many biosynthesis and signaling genes related to ET and JA, suggesting that MAPK-mediated regulation in the biosynthesis and signaling pathway of ETH and JA might be involved in the response to selenite toxicity in C. moschata. ET and JA are thought as important plant hormones involved in resisting various abiotic stresses (Pérez-Llorca et al., 2023). Hoewyk et al. reported that Arabidopsis mutants defective in ET or JA response pathways exhibited a decreased tolerance to Se (Van Hoewyk et al., 2008). Therefore, we inferred that MAPK cascade (MAPKKK–MAPKK–MAPK), ET and JA should play an important role in Se tolerance in C. moschata.
Various abiotic stresses usually lead to protein disfunction and denaturing in plant cell. As important proteins involved in protecting proteins from mis-folding and maintaining cellular proteostasis, Heat shock proteins (HSPs), such as HSP20s, HSP70s and HSP90s, have been widely reported to be activated by abiotic stresses in plants (Ul Haq et al., 2019; Hu et al., 2022). Studies in C. violifolia showed that the expression of HSP23.6 belonging to HSP20 family was significantly enhanced by selenate treatment (Rao et al., 2021). Our results indicated that selenite treatment could induce the expressions of numerous HSP-encoding genes, including 21 HSP20 family genes, 5 HSP70 family genes and 1 Hsp90 family gene. HSFs were reported to control the expressions of HSPs in plants (Bunch and Calderwood, 2023). Interestingly, we identified 2 heat shock factors (HSFs) showing highly increased expressions in response to selenite stress. In total, our results supported the idea that HSFs and HSPs might participate in the rescue of dysfunctional and misfolding proteins to maintain cellular proteostasis in C. moschata under selenite stress.