Cucumber plants have poor ability to take up water and minerals because of their weak root regeneration and shallow roots system, which seriously reduces yield. The hypocotyl of cucumber seedlings has the ability to generate AR (Qi et al., 2019; Qi et al., 2020), therefore measures that stimulate adventitious root development are crucial for promoting plant growth and development and thus for increasing crop yield and so making better use of scarce agricultural land.

Adventitious roots (ARs) can emerge from a hypocotyl, a stem, a leaf or some other plant organ, with emergence regulated by environmental signals and/or by hormones (Sukumar et al., 2013). Many hormones have been shown to play important roles in the induction of AR, among these auxin is the main one that promotes AR initiation (Li et al., 2009; Da Costa et al., 2013; Pacurar et al., 2014).It is reported that auxin synthesis is necessary for AR formation, with the number of ARs increasing with auxin concentration (Boerjan and W., 1995; Ahkami et al., 2013). Conversely, reduced auxin transport or reduced auxin signaling decreases AR development (Vidoz et al., 2010). In addition to auxin, cytokinin (CK), jasmonic acid (JA), salicylic acid (SA) and ethylene are also involved in regulating AR development, and they interact with one another. Studies have shown that CK inhibits AR formation (Hutchison et al., 2006; Mao et al., 2019), over-expression of cytokinin oxidase (CKX) increases AR formation in transgenic tobacco (Werner et al., 2001). It is reported that SA works synergistically with auxin to induce AR development in cucumber (Dong et al., 2020) and Arabidopsis (Pasternak et al., 2019). Moreover, ethylene is another important hormone that interacts with auxin during root development; with ethylene inducing AR formation by regulating auxin transport (Vidoz et al., 2010). Moreover, the crosstalk between ethylene and JA signaling has been shown to be critical for AR formation in Arabidopsis (Fattorini et al., 2018).

Melatonin is a natural molecule that exists widely in plants (Pelagio-Flores et al., 2012), which is structurally related to indole-3-acetic acid (IAA). Initially, the structural similarity between melatonin and IAA prompted researchers to investigate its function in greater depth (Hernández-Ruiz et al., 2004). In plants, melatonin may act as a growth regulator or new hormone (Arnao and Hernández-Ruiz, 2014; Arnao and Hernandez-Ruiz, 2019). A growing number of studies have reported the possible physiological roles of melatonin and their mechanisms. They have shown that melatonin can perform a number of roles, such as protecting the photosynthetic system and related subcellular structures (Zhao et al., 2016; Altaf et al., 2021), promoting seed germination (Zhang et al., 2014; Hwang and Back, 2020) and providing effective defenses against both biotic and abiotic stresses (Zhang et al., 2017; Li et al., 2018). Moreover, with auxin-like activity, melatonin is able to stimulate root initiation and induce root growth. In recent years, a large number of studies have confirmed that applications of exogenous melatonin can promote root development, especially the development of lateral roots and AR. For example, exogenous melatonin can promote the emergence of lateral roots and AR in lupin (Arnao and Hernández‐Ruiz, 2007) and Arabidopsis (Pelagio-Flores et al., 2012), and can promote AR formation in tomato (Wen et al., 2016) and apple (Mao et al., 2020).

Previous studies have shown that melatonin is an important modulator of gene expression related to hormone production, as well as of the metabolism of auxin, CK, JA, SA and ethylene and other endogenous hormones (Arnao and Hernandez-Ruiz, 2018). For example, melatonin regulates root development by increasing the level of auxin and inducing the expression of genes related to auxin signal transduction, transport and synthesis (Chen et al., 2009; Wen et al., 2016; Liang et al., 2017; Mao et al., 2020). However, applications of exogenous melatonin at high concentration, inhibit root growth by inhibiting auxin transport and synthesis, and is accompanied by a decrease in endogenous IAA (Wang et al., 2016). Under copper stress, melatonin promotes root development in melon by inhibiting the expression of JA synthesis-related genes (Hu et al., 2020). In Arabidopsis, melatonin promotes the expressions of IAA and ethylene synthesis-related genes, whereas it inhibits the expressions of brassinosteroid, JA and CK synthesis-related genes, thereby integrating hormone signals to further mediate the expressions of zinc finger proteins and calmodulin-like proteins, so as to promote lateral roots development (Yang et al., 2021a). Moreover, melatonin also upregulates SA-related genes, leading to an increase in SA, while melatonin synthesis enzyme serotonin N-acetyltransferase mutants exhibited lowered levels of SA (Lee et al., 2015). Though these studies demonstrate a link between melatonin and hormones, the relationship between melatonin and these hormones in the formation of AR in cucumber seedlings is unknown.

To better understand how melatonin induces AR formation in the hypocotyls of cucumber seedlings under shaded conditions, we first analyze the effects of melatonin on AR formation and hormone content in cucumber hypocotyls. Then, combined with transcriptomics analysis, we explore the mechanism of AR formation induced by exogenous melatonin under shaded hypocotyl conditions. This research aimed to explore the effects of melatonin on AR development in cucumber seedlings, and to provide a basis for revealing the mechanism of melatonin in regulating AR formation in cucumber seedlings.

The formation of AR is affected by water, minerals, wounding and hormones. Many studies have shown that hormones contribute greatly to AR formation (Hutchison et al., 2006; Vidoz et al., 2010; Da Costa et al., 2013; Dong et al., 2020). Melatonin treatments could regulate endogenous hormone contents and their related genes (Arnao and Hernandez-Ruiz, 2018) and so stimulate root generation, giving rise to new lateral and adventitious roots (Arnao and Hernández‐Ruiz, 2007; Pelagio-Flores et al., 2012; Mao et al., 2020). Our results show that exogenous melatonin significantly promotes AR formation in cucumber seedings under hypocotyl shading conditions. We measured hormones levels in the hypocotyls among the three treatments and found that melatonin significantly induced the accumulation of the hormones - auxin, CK, SA, JA and ABA. The GO and KEGG analyses showed that DEGs induced by melatonin were significantly enriched in regulation of hormone levels and plant hormone signal transduction, suggesting that hormones and hormone signal transduction may play important roles in cucumber AR formation.

Melatonin and auxin are tryptophan-derived compounds, previous studies have shown that melatonin may increase auxin contents or exhibit auxin-like activity during root development. For example, melatonin regulates root development in apple (Mao et al., 2020), tomato (Wen et al., 2016), rice (Liang et al., 2017) and Arabidopsis (Yang et al., 2021b) by inducing the expression of genes related to auxin synthesis, transport and signaling, as well as increasing auxin contents. However, other studies have shown that melatonin can regulate root development independently of auxin signaling in Arabidopsis (Pelagio-Flores et al., 2012; Zia et al., 2019). In this study, we found that exogenous melatonin induced AR development in cucumber seedlings may be related to auxin. It has been reported that, transport and accumulation of auxin are necessary for root development, PIN proteins are responsible for auxin transport, pin1, pin3 and pin7 mutants show statistically significant reductions in AR formation (Sukumar et al., 2013), while down regulation of the PIN5 promotes lateral root development (Ren et al., 2019). In this study, it was found that compared with EW, the SW treatment induced the expressions of tryptophan decarboxylase (TDC) (CsaV3_1G036910) and PIN7 (CsaV3_3G000190), whereas it inhibited the expressions of genes related to auxin synthesis, such as YUCCA (CsaV3_3G017920 and CsaV3_6G012670) and TAA(CsaV3_1G001250) ( Figure 4A , Supplementary Table S1 ); and decreased the level of IAN, while it increased the levels of IAA and IAA-Asp ( Figure 4B ). Which may be related to the upregulation of the auxin transport gene PIN, resulting in local accumulations of IAA. The melatonin (SM) treatment further activated the expression of TDC (CsaV3_1G036910) and PIN7 (CsaV3_3G000190) ( Figure 4A ) under hypocotyl shading conditions. It also increased the contents of IAN significantly ( Figure 4B ), whereas the content of IAA did not increase significantly in SM. This may be related to the upregulation of DAO (CsaV3_4G000540 and CsaV3_7G032860), which convert IAA to bound state. It is clear that the increased level of IAA induced by hypocotyl shading is required for the formation of AR ( Figure 1 , Figure 4B ), while exogenous melatonin significantly promoted the accumulation of IAN under hypocotyl shading conditions ( Figure 4B ), which may be the factor inducing the significant increase in AR. Auxin signaling plays a vital role in the transition of protocambial cells to AR-initiating cells (Chen et al., 2016), auxin induced primary auxin responsive genes include AUX/IAA, SAUR and GH3. In the absence of auxin, Aux/IAAs protein interacts with ARFs and prevents ARFs from binding to the promoter of auxin-reponsive genes. In the presence of auxin, the Aux/IAA protein is degraded through the ubiquitination pathway, then ARFs are released from inhibition and can regulate the expressions of auxin-responsive genes (Weijers and Jürgens, 2004). SAUR are highly induced in response to auxin, which regulate various aspects of plant development, such as: root initiation, cell division and cell differentiation. Studies show that SAURs may function as positive regulators of cell expansion, which plays vital roles in IAA-mediated adventitious rooting in petunia cuttings (Yang et al., 2019). GH3 controls auxin homeostasis in plants, GH3.3, GH3.5, GH3.6 are required for AR initiation in the Arabidopsis hypocotyl (Gutierrez et al., 2012), GH3.17 is necessary to control meristem size of the root (Di Mambro et al., 2019). The expression of SAUR71 (CsaV3_1G009830) and SAUR36 (CsaV3_6G001030) were upregulated, GH3.17 (CsaV3_4G002130) was downregulated by hypocotyl shading, and the expressions of these genes were upregulated by melatonin under hypocotyl shading conditions ( Figure 4A ). In addition, melatonin inhibited the expression of AUX/IAA26 (CsaV3_3G048460) under hypocotyl shading conditions ( Figure 4A , Supplementary Table S1 ). These results show that hypocotyl shading treatment promoted signal transduction of auxin, and the application of exogenous melatonin under hypocotyl shading conditions further induced auxin signaling, which may promote the formation and elongation of AR. This observation is consistent with the effects of auxin in root induction and elongation (Guan et al., 2019; Qi et al., 2019). From the results, it is clear that the induction effect of melatonin on AR formation in the cucumber seedling hypocotyl may be related to auxin, and there may be interactions between melatonin and auxin. Similar results have been obtained in studies with lupin (Arnao and Hernández‐Ruiz, 2007), tomato (Wen et al., 2016) and apple (Mao et al., 2020).

Cytokinin can regulate cell division, vascular development and root development (Werner et al., 2001; Sakakibara, 2006). It has been reported that, exogenous applications of CK inhibits both lateral (Hutchison et al., 2006; Li et al., 2006) and adventitious root formation (Mahonen et al., 2006) in Arabidopsis. Isopentenyltransferase (IPT) is a key enzyme in CK synthesis, while CK dehydrogenase (CKX) catalyzes the oxidative decomposition of CK (Sakakibara, 2006). Over-expression of IPT can significantly inhibit the development of primary and lateral roots, while over-expressing CKX significantly promotes AR formation in Arabidopsis seedlings (Werner et al., 2001). Transcriptome data analysis found that the expressions of CKX (CsaV3_2G001450 and CsaV3_4G036030) were downregulated by hypocotyls shading ( Supplementary Table S1 ), whereas the content of IP was decreased ( Figure 4D ), which may be related to the upregulation of UGT76C1 (CsaV3_1G035800) and glycosylate free-state IP to the bound state. However, melatonin downregulated the expression of tRNA-IPT (CsaV3_3G013770) and upregulated the expressions of CKX (CsaV3_2G001450 and CsaV3_4G036030) under hypocotyl shading conditions ( Figure 4C , Supplementary Table S1 ); while the level of 2MeScZ was increased significantly in SM ( Figure 4D ). It has been reported that, 2MeS-CKs (unbound methylthiolated CKs) is less cytotoxic and more resistant to CKX than classical CKs, which may be responsible for continuous tissue proliferation during plant-pathogen interactions (Gibb et al., 2020). In this study, the level of 2MeScZ in SM was significantly increased relative to that in SW, which may be related to the stronger resistance of 2MeScZ to CKX and the reduction of decomposition, resulting in local accumulations of 2MeScZ. In addition, KEGG enrichment analysis found the plant-pathogen interaction pathway was significantly enriched in the SW and SM comparison groups ( Figure 3D ). This may be related to pathogenic bacterial infection during AR formation. The accumulation of 2MeScZ in hypocotyls, can mediate plant defenses, promote tissue proliferation around the site of infection and ease deleterious effects on AR development, thereby facilitating the formation of AR. AHPs plays a vital role in CK signal transduction, most AHPs are positive regulators of CK signal transduction (Hutchison et al., 2006), whereas AHP6 is a negative regulator of CK signal transduction (Mahonen et al., 2006). In Arabidopsis, reduced AHP function in ahp1,2,3,4,5 mutants resulted in a short primary root but enhanced AR development. Additionally, AHP2, AHP3 and AHP5 are particularly important and AHP1 and AHP4 may play minor roles in these processes (Hutchison et al., 2006). ahp6 mutants grown on media with increasing concentrations of CK, produce fewer ARs in Arabidopsis (Mahonen et al., 2006). In our study, we found that compared with that in EW, the SW treatment induced the expression of AHP6 (CsaV3_4G001620), while SM treatment activated AHP6 expression further ( Figure 4C , Supplementary Table S1 ). This suggests that melatonin inhibited CK signaling, which may enhance AR development.

Salicylic acid affects seed germination, root elongation and AR development, which is also involved in defense against pathogens. Studies have shown that SA and auxin act synergistically to regulate AR development (Pasternak et al., 2019; Dong et al., 2020). Exogenous SA promoted AR formation in wheat under flooding condition (Koramutla et al., 2022), whereas Arabidopsis eds5-1 and eds5-2 mutants lacking SA synthesis produced fewer AR than the wild type. This suggests SA is a positive regulator of AR development (Gutierrez et al., 2012). In Arabidopsis, melatonin upregulated SA-related genes, resulting in an increased level of SA (Lee et al., 2015). In our study, we found that melatonin significantly upregulated the expression of SA synthetase CM (CsaV3_5G038370) and PPA-ATs (CsaV3_4G000360) ( Figure 5A , Supplementary Table S1 ), thereby significantly increasing the level of SA in the hypocotyl ( Figure 5B ). Moreover, UDP-glycosyltransferase UGT74F2 (CsaV3_6G015940) was also upregulated by melatonin, which glycosylated SA to SAG, resulting in a significant increase in SAG in the SM plants. These results indicate that the significant increase in AR induced by exogenous melatonin may be related to increased levels of SA. This is consistent with the effect of SA in cucumber hypocotyl AR induction (Dong et al., 2020). On the other hand, in the process of AR emergence, the young plant is susceptible to affection by soil-borne pathogens. KEGG enrichment analysis found that the plant-pathogen interaction pathway was significantly enriched in the SW and SM comparison groups ( Figure 3D ). This may be related to a pathogenic bacterial infection during AR formation. To ensure plant growth in an unfavorable environment, SA plays a vital role in regulating the growth-defense balance. The expression of pathogenesis-related gene 1(PR1) is considered a marker for activating SA-mediated systemic acquired resistance. It has been reported that, SA inhibited lateral root formation, enhanced adventitious rooting, promoted cell wall lignification and plasmodesmal closure by inducing the expressions of PR1 and PR2, which can directly prevent pathogens from invading roots and so facilitate plants growth under adverse conditions (Bagautdinova et al., 2022). In our study, we found that under hypocotyl shading conditions, exogenous melatonin significantly induced the expressions of PR1 (CsaV3_7G007550 and CsaV3_7G007610) and of transcription factors TGA (CsaV3_3G039040, CsaV3_4G004890 and CsaV3_4G030930) ( Figure 5A , Supplementary Table S1 ). These can mediate systemic acquired resistance, effectively resisting pathogen infection during AR formation and so facilitating AR development.

It is reported that JA serves as a signal to activate rooting, thereby promoting AR formation in tobacco (Fattorini et al., 2009), pea (Rasmussen et al., 2015), petunia (Lischweski et al., 2015) and Arabidopsis (Fattorini et al., 2018). LOX is a key enzyme in JA synthesis. We found that most LOX genes were upregulated by hypocotyl shading ( Figure 5C , Supplementary Table S1 ), thereby significantly increasing the content of Jasmonoyl-L-isoleucine (JA-ILE) ( Figure 5D ). Meanwhile, melatonin significantly further upregulated the expressions of these genes under hypocotyl shading conditions ( Figure 5C , Supplementary Table S1 ), as well as promoting the accumulation of JA-ILE and JA ( Figure 5D ). JAZ proteins are key repressors of JA signaling. MYC2, a bHLH TF, is considered to be a master regulator of the JA signaling (Fernandez-Calvo et al., 2011). In our study, it was found that the expression of JAZ (CsaV3_1G041270) was significantly downregulated by hypocotyl shading, and the expression of MYC2 (CsaV3_6G014370) was significantly upregulated by melatonin under hypocotyl shading conditions ( Figure 5C , Supplementary Table S1 ). These findings indicate that the formation of ARs induced by hypocotyl shading may be related to the accumulation of JA-ILE and enhanced signaling. Meanwhile, the significant accumulation of JA-ILE and JA, accompany the upregulation of MYC2 induced by exogenous melatonin may further activate the expression of JA-responsive genes, thereby significantly promoting AR formation, in accordance with the finding that JA acts as a accelerator of AR formation in tea plants (Fan et al., 2021).

Ethylene interacts with auxin, gibberellin and ABA to regulate AR formation in tomato (Vidoz et al., 2010) and rice (Steffens and Sauter, 2006). ACS and ACO are key synthase genes of ethylene, which are upregulated during AR formation in petunia (Lischweski et al., 2015). In Arabidopsis, ACS and ACO are significantly regulated by melatonin (Yang et al., 2021a). Our Transcriptome data analysis showed that the expressions of ACO (CsaV3_3G012240 and CsaV3_3G023650) were downregulated by hypocotyl shading ( Supplementary Table S1 ), thereby the level of ACC in SW decreased significantly ( Figure 6B ). However, the melatonin upregulated the expressions of ACO (CsaV3_1G005350 and CsaV3_3G012290) and ACS (CsaV3_6G000890), while it downregulated the expression of SAMs (CsaV3_6G039940) under hypocotyl shading conditions ( Figure 6A , Supplementary Table S1 ). Also, the ACC content did not change significantly ( Figure 6B ). Many ethylene response factors (ERFs) are common mediators of root developmental. It has been found that ERFs are upregulated during AR development in cuttings (Druege et al., 2016). In our study, we found melatonin significantly enhanced the expressions of ERF1 (CsaV3_3G022370 and CsaV3_7G027380) under hypocotyl shading conditions ( Figure 6A , Supplementary Table S1 ). The above results show that the significant increase in AR in our cucumber seedlings may well be related to the enhanced expression of ethylene synthesis and response factor genes.

Earlier studies found that ABA can promote AR formation in cucumber (Li et al., 2016) and mung bean seedlings (Li et al., 2014) under stress conditions. ABA 8’-hydroxylase catalyzes the oxidative degradation of ABA, while β-glucosidase (β-Glu) can hydrolyze ABA gluconate into bioactive ABA. Studies on tea plants showed that ABA content was higher in the easy-to-root cultivar, which indicate that ABA is involved in the occurrence of AR (Fan et al., 2021). In our study, the expression of β-Glu (CsaV3_1G042500, CsaV3_1G042530 and CsaV3_1G042540) were significantly upregulated by hypocotyl shading and melatonin further upregulated these genes under hypocotyl shading conditions, while ABA 8′-hydroxylase was inhibited by melatonin ( Figure 6A , Supplementary Table S1 ). Thereby the level of ABA in SM increased significantly ( Figure 6D ), which will contribute to the occurrence of AR. Additionally, ABA regulates stomatal transpiration and also root hydrophilicity. One prominent response of roots to ABA sensitivity is their ability to perceive differences in water potential and translate this into growth towards water, which depends on ABA signaling kinase SnRK2.2 activity (Dietrich et al., 2017). Calcium dependent protein kinase (CDPK) is a positive effector of ABA signaling transduction and plays a key role between ABA signal perception and response. In our study, we found that exogenous melatonin significantly induced the expressions of CDPK (CsaV3_3G038220 and CsaV3_6G038920) under hypocotyl shading conditions ( Figure 6C , Supplementary Table S1 ). The upregulation of CDPK could phosphorylate downstream target proteins to respond to ABA signals, and so improve the absorptive capacity for water. This too will facilitate the production of AR. These findings indicate that ABA may be involved in melatonin-induced AR development in our cucumber seedlings.

Cellulase-like synthases (CSL) are the main load-bearing components of cell walls. AtCSLD3 is very important for the synthesis of primary cell wall in the root hair region and plays vital roles in root hair growth. In AtCSLD3 deficient plants, the growth of root hairs is affected (Xuan et al., 2001). Peroxidase (POD) induces cell wall loosening and cell elongation by regulating the elongation and cross-linking of cell wall components (Passardi et al., 2004). From our analyses, we found that melatonin significantly induced POD expression under hypocotyl shading conditions ( Supplementary Figure S3 , Supplementary Table S1 ). In melon, the expression of POD was also upregulated by melatonin during lateral root development (Hu et al., 2020). The expansin can restore the expansion of heat-inactivated cell walls under acidic conditions, which loosen the cell wall, thereby affecting the extensibility of the cell wall (McQueen-Mason and S., 1992). In transgenic tobacco plants over-expressing TaEXPB23, the number of lateral roots was significantly increased (Han et al., 2014). In our study, we found exogenous melatonin significantly induced the expression of expansin under hypocotyl shading conditions ( Supplementary Figure S3 , Supplementary Table S1 ). The upregulation of expansin will lead to the loosening of the cell wall where the new lateral roots appear, which will reduce the mechanical resistance of the stem tissue, thereby aiding the emergence of new ARs. Therefore, exogenous melatonin regulates the formation of cell walls by upregulating the expression of CSL, POD and expansin under hypocotyl shading conditions, thereby significantly inducing AR formation in our cucumber seedlings hypocotyl.

Hypocotyl shading was necessary to induce AR formation, while exogenous melatonin application significantly promoted AR formation in cucumber seedlings under hypocotyl shading conditions. To gain a deeper understanding of the underlying regulatory mechanisms, RNA-seq and hormone analyses were carried out. Our results show that exogenous melatonin induced the expressions of auxin, SA, JA, ethylene and ABA synthesis and signaling related genes. It also inhibited the expressions of CK signaling genes, as well as significantly increasing the auxin, CK, JA, SA and ABA contents ( Figure 7 ). In the process of AR formation, melatonin also regulated the expressions of genes related to cell wall formation and induced the loosening of cell walls, thereby facilitating the emergence of AR. Finally, melatonin promoted the formation of a large numbers of AR in the cucumber seedling hypocotyls.

The original contributions presented in the study are publicly available. This data can be found here: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA717318.

YW, ZZ and LZ carried out experimental work. LL, XH, YZ and LH performed the data analyses. YW wrote the manuscript. HZ and ML designed the experiments and assisted with writing the manuscript. All authors contributed to the article and approved the submitted version.

This research was supported by Shanxi Province Key R&D Plan (202102140601013), the National Key R&D Program of China (2019YFD1001900), Shanxi Province Applied basic Research Program (201801D221304), Science and Technology Innovation Fund of Shanxi Agricultural University (2017YJ44, 2018YJ05).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.