Drought-induced ABA, H2O2 and JA positively regulate CmCAD genes and lignin synthesis in melon stems

Cinnamyl alcohol dehydrogenase (CAD) is an important enzyme functions at the last step in lignin monomer synthesis pathway. Our previous work found that drought induced the expressions of CmCAD genes and promoted lignin biosynthesis in melon stems. Here we studied the effects of abscisic acid (ABA), hydrogen peroxide (H2O2) and jasmonic acid (JA) to CmCADs under drought stress. Results discovered that drought-induced ABA, H2O2 and MeJA were prevented efficiently from increasing in melon stems pretreated with fluridone (Flu, ABA inhibitor), imidazole (Imi, H2O2 scavenger) and ibuprofen (Ibu, JA inhibitor). ABA and H2O2 are involved in the positive regulations to CmCAD1, 2, 3, and 5, and JA is involved in the positive regulations to CmCAD2, 3, and 5. According to the expression profiles of lignin biosynthesis genes, ABA, H2O2 and MeJA all showed positive regulations to CmPAL2-like, CmPOD1-like, CmPOD2-like and CmLAC4-like. In addition, positive regulations were also observed with ABA to CmPAL1-like, CmC4H and CmCOMT, with H2O2 to CmPAL1-like, CmC4H, CmCCR and CmLAC17-like, and with JA to CmCCR, CmCOMT, CmLAC11-like and CmLAC17-like. As expected, the signal molecules positively regulated CAD activity and lignin biosynthesis under drought stress. Promoter::GUS assays not only further confirmed the regulations of the signal molecules to CmCAD1~3, but also revealed the important role of CmCAD3 in lignin synthesis due to the strongest staining of CmCAD3 promoter::GUS. CmCADs but CmCAD4 are positively regulated by ABA, H2O2 and JA under drought stress and participate in lignin synthesis.


Background
Lignin is the second rich substance in plants [1] and plays important roles in sap transport and water barrier in addition to the basal function of support [2]. In lignin monomer synthesis pathway, cinnamyl alcohol dehydrogenase (CAD) is an important enzyme which functions in the last step responsible for the transformation between cinnamyl aldehydes and cinnamyl alcohols. Lignin synthesis is not only complied with regular growth, but also can be regulated by biotic and abiotic stresses. As one frequently happened abiotic stress, drought negatively affects plant growth but promotes lignification process. Some research showed that lignifying enzymes (cinnamoyl CoA reductase (CCR), CAD, kinds of peroxidases) were induced and lignin synthesis was promoted in shoots under drought stress [3][4][5][6]. While in roots, lignin biosynthesis was suppressed at the beginning and induced in the late period when suffering drought stress [7][8][9]. The positive promotion of drought on lignification may confer to plants drought tolerance in aspects of water transport [10] and water loss [11].
As an important enzyme in lignin monomer synthesis pathway, activity of CAD enzyme and expression levels of CAD genes were promoted by drought and signal molecules, resulting in lignin deposition [5,16,23]. CAD genes usually present as a gene family in different species and each member may function distinctly or redundantly from each other [24,25]. Our team discovered five CmCAD genes in melon genome database (http:// melonomics.cragenomica.es/) [26] and found they can be significantly up-regulated by drought stress in melon seedlings [27]. However, it remains unclear which signal molecule is responsible for regulating CmCADs under drought stress.
In this study, we investigated the regulations of ABA, H 2 O 2 and JA to the five CmCAD genes, as well as some lignin biosynthesis genes, and lignin deposition through using their corresponding inhibitor or scavenger. Then promoter::GUS assays were performed to investigate the regulations of the signal molecules to CmCAD1, 2, and 3, which members were chosen according to the phylogenetic analysis [26] and the expression profiles under drought stress [27], as well as the expression patterns in melon seedlings (unpublished data). The results revealed important roles of ABA, H 2 O 2 and JA in regulating CmCAD1~3, 5 and lignin biosynthesis genes, thus in regulating lignin biosynthesis under drought stress.

Results
Drought-induced ABA, H 2 O 2 and MeJA could be inhibited by the corresponding inhibitor or scavenger ABA and H 2 O 2 are frequently reported as important signal molecules in regulating downstream gene expression and metabolism under drought stress [6,28]. While, it remains controversial of the role of JA under drought stress [29]. To investigate the changes of ABA, H 2 O 2 and JA in melon stems under drought stress, contents of ABA, H 2 O 2 and JA were determined and found the signal molecules were all induced by PEG treatment (Fig. 1a-c). ABA and H 2 O 2 responded to drought fast and reached peak value at 2 h (1.6 fold) and 3 h (2.26 fold), respectively, while JA reached peak value at 7 h (1.54 fold) after PEG treatment. Though decreases were observed after reaching peak values, contents of the signal molecules still maintained higher levels than those in control.
Fluridone (Flu), imidazole (Imi) and ibuprofen (Ibu) are used as inhibitor or scavenger in investigating the functions of ABA, H 2 O 2 and JA in plants [30,31]. Here, we evaluated the inhibitory efficiency of Flu, Imi and Ibu to ABA, H 2 O 2 and JA, respectively, in melon stems under drought stress. As shown in Fig. 1d-f, the signal molecules slightly decreased when treated with Flu, Imi or Ibu alone, showing 9.5, 7.5 and 11% decreases compared to those in control, respectively, but were significantly restricted from increasing in PEG-treated melon stems pretreated with Flu, Imi and Ibu, showing 26.4, 36.1 and 22.7% decreases compared to those in PEGtreated samples, respectively. Thus, they can also be used as effective inhibitor or scavenger in melon seedlings for further study. , and Imi pretreatment severely inhibited the up-regulations of them in PEG-treated melon seedlings (2.5, 2.6, 5.3, 2 fold, respectively). MeJA treatment significantly up-regulated the expressions of CmCAD2 (6.8 fold), 3 (7.6 fold), and 5 (4.6 fold), and Ibu pretreatment strongly inhibited the upregulations of them in PEG-treated melon seedlings (2.8, 4.2, 1.9 fold, respectively). Notably, MeJA exhibited stronger regulations to CmCAD2 and 3 than ABA and H 2 O 2 did. CmCAD4 was slightly regulated by the signal molecules. Treated with each inhibitor or scavenger alone had no or little effect on CmCADs expressions.
The lignin biosynthesis genes were all highly upregulated in expressions under drought stress. Among the genes, CmPAL1-like, CmPAL2-like, CmC4H, CmCOMT, CmPOD1-like, CmPOD2-like and CmLAC4-like were induced by ABA treatment and inhibited from increases under PEG+Flu treatment, indicating ABA signalling involves in the regulation of these genes. Similarly, CmPAL1like, CmPAL2-like, CmC4H, CmCCR, CmPOD1-like, CmPOD2-like, CmLAC4-like and CmLAC17-like were upregulated by H 2 O 2 treatment and restricted from inductions under PEG+Imi treatment, suggesting the positive regulation of H 2 O 2 to these genes. The lignin biosynthesis genes except for CmPAL1-like, CmC4H and Cm4CL were induced by MeJA treatment and inhibited by PEG+Ibu treatment, implying JA signalling involves in the regulation of these genes. Cm4CL studied here was only slightly regulated by H 2 O 2 .

CAD activity was positively regulated by ABA, H 2 O 2 and MeJA under drought
Since ABA, H 2 O 2 and MeJA play positive roles in regulating the CmCADs under drought stress, it is necessary to investigate the effects of the signal molecules on CAD activity. Under various treatments, CAD activity was actively induced under PEG treatment and not affected by each inhibitor or scavenger treatment. Signal molecule treatments promoted CAD activity from increasing in melon stems and inhibitor or scavenger pretreatments inhibited CAD activity from increasing in PEG-treated melon stems (Fig. 4). Among the signal molecules, ABA induced CAD activity to significant levels (increased 6.4-15.2%) (Fig. 4a), and MeJA promoted CAD activity from increasing about 4.9-17.9% (Fig. 4c), while H 2 O 2 showed slight induction to CAD activity which only reached significant level (7%) at 3 day after treatment (Fig. 4b). Pretreatments of inhibitor or scavenger all strongly suppressed the up-regulation of CAD activity in PEG-treated melon stems. These results suggest that the Further, assays of lignin content and histochemical staining were performed. Results as shown in Fig. 5, lignin biosynthesis in stems was significantly promoted by PEG treatment and inhibited by inhibitor or scavenger pretreatments. Though not to significant levels, the signal molecule treatments showed slight promotions on lignin biosynthesis and deposition in similar patterns (Fig. 5a, c, e). Histochemical staining with phloroglucinol-HCl is a method commonly used for direct lignin observation.
Consistent with lignin content detection, xylem tissues in vascular bundles exhibited the strongest staining under PEG treatment, the modest staining under treatments of the signal molecules or pretreatments of inhibitor or scavenger, and the control-like staining under treatments of inhibitor or scavenger (Fig. 5b, d, f). These results suggest that the signal molecules play positive roles in lignin biosynthesis under drought stress.
Promoters of CmCAD1, 2, and 3 were positively induced by ABA, H 2 O 2 and MeJA To further study the response of CmCADs to the three signal molecules, we constructed promoter::GUS vectors of CmCAD1 pro::GUS, CmCAD2 pro::GUS and CmCAD3 pro::GUS and carried out GUS assays in tobacco leaves treated with ABA (100 μM), H 2 O 2 (10 mM) or MeJA (100 μM). As Fig. 6a revealed, positive control showed the strongest GUS staining driven by 35S promoter and negative control exhibited no GUS staining without promoter. Under control, CAD1 pro::GUS had the slightest GUS staining, CAD2 pro::GUS the modest, and CAD3 pro::GUS the strongest which is consistent with its highest expression pattern in melon seedlings. Under signal molecule treatments, CmCAD1 pro::GUS was slightly induced by MeJA, but was strongly induced by ABA and H 2 O 2 . While CmCAD2 pro::GUS and CmCAD3 pro::GUS exhibited similar staining patterns, they were both slightly induced by ABA and strongly induced by H 2 O 2 and MeJA. GUS activity assay, Fig. 6b, obtained similar results with those of histochemical staining. GUS activity driven by CmCAD1 promoter increased 9.7, 15.9 and 4.6 fold in response to ABA, H 2 O 2 and MeJA, respectively. GUS activity driven by CmCAD2 promoter observed 1.5, 1.9 and 2.2 fold increases in response to ABA, H 2 O 2 and MeJA, respectively. And 1.2, 1.6 and 1.7 fold increases of GUS activity were observed driven by CmCAD3 promoter in response to ABA, H 2 O 2 and MeJA, respectively. These results demonstrate that the signal molecules play important roles in regulating CmCAD1, 2, and 3.  [36][37][38]. Drought, as a frequently encountered abiotic stress, usually induce ABA, H 2 O 2 and JA from highly increasing [6,39], however, these signal molecules regulate lignification diversely in different species [14,17,20,40]. Thus it becomes necessary to reveal the regulating patterns of these signal molecules to CmCAD genes as well as lignification in melon stems under drought stress for adding an understanding.

Discussion
First of all, we confirmed that ABA, H 2 O 2 and JA all could be induced significantly by drought stress and reached peak values differentially after PEG treatment, and then they were efficiently restricted from reaching each peak value pretreated with inhibitor or scavenger, implying their signalling to downstream can be weakened efficiently. While, treated with inhibitor or scavenger alone had little effect on the signal molecules as well as thereafter indexes, which is similar with the results observed by Hu et al. (2013), Kojo et al. (2006) and Shan and Liang (2010) [30,41,42]. The inhibitor or scavenger exhibited little effect on the signal molecules when treated with each of them alone, but strongly inhibited the singal molecules from increasing under PEG+inhibitor or +scavenger treatments, which can be explained that they do nothing with the existed signal molecules but strongly inhibit the synthesis and accumulation of the signal molecules induced by PEG treatment.
Application of inhibitor or scavenger revealed that ABA and H 2 O 2 exhibited similar positive regulations to CmCAD1, 2, 3, and 5. While, JA showed more direct regulations to CmCAD2 and 3, as well as CmCAD5, and exhibited slight regulation to CmCAD1. CmCAD4 was slightly affected by the signal molecules. This is consistent with previous CmCADs promoter analysis [26]. In the 1.5 kb promoter sequences of CmCADs, promoters of CmCAD1, 2, and 5 contain TC-rich repeats (cis-acting element involved in defense and stress responsiveness) which may drive the genes responding to H 2 O 2 . Promoters of CmCAD1, 2, 3, and 5 contain ABRE element (cis-acting element involved in the abscisic acid responsiveness), and we demonstrated these CmCADs can be regulated by ABA. CmCAD3 promoter contains TGACG-motif and CCAAT-box and CmCAD5 promoter contains CGTCA-motif, all these three elements act as cis-acting regulatory elements involved in the MeJA-responsiveness, which were also confirmed in this study. In addition, some other stress-and signal-  Table S1) may be a reason why certain increases were still observed in PEG-treated melon seedlings pretreated with inhibitor or scavenger. The positive regulating patterns of the signal molecules to CmCADs are consistent with previous reports that one CAD gene can be regulated by several signal molecules [19,43] and different CAD members in a family may respond to signal molecules and stresses differentially due to the distinct function each member fulfilled [24,44]. Expression analysis of lignin biosynthesis genes further confirmed the positive regulations of ABA, H 2 O 2 and JA to lignin biosynthesis under drought stress. Taken together, these results once again demonstrated that drought mediates gene expression depending on various signal molecules [8,9,45].
CAD activity and lignin content was drastically induced by drought stress and slightly induced by signal molecule and PEG+inhibitor or +scavenger treatments. Slight inductions to CAD activity and lignin content by exogenous applications of signal molecules may due to the absorption efficiency as well as time-based effects of exogenous applied signal molecules. While the certain increases of CAD activity and lignin content under PEG+inhibitor or +scavenger treatments may due to the inhibitory efficiency of the inhibitors and scavenger, or exist bypass signalling pathways. These suggest a positive correlation between the signal molecules and CAD activity, resulting in lignin accumulation, which is consistent with previous reports [6]. Among the signal molecules, ABA and H 2 O 2 showed similar regulations to CmCAD genes, but ABA and JA showed similar regulations to CAD activity and lignin deposition. However, Mohr and Cahill (2007) [17] presented an inverse result declaring a negative correlation between ABA and lignin, this may due to the reason that JA and SA are more likely responsible for lignin biosynthesis under biotic stresses [46,47]. Antagonism and cooperation were both observed between ABA and JA under biotic stress [48,49], which mechanism between ABA and JA interactions is considered existing both dependent and independent ways [38].
JA is also an important drought-response signal [42,50] and functions in regulating lignin biosynthesis [21, Our results proved JA functions actively in promoting lignin biosynthesis in drought-stressed melon stems, which is similarly observed in Arabidopsis [40]. Previous studies on H 2 O 2 also reported positive regulations to lignification showing that increased H 2 O 2 content corresponded with higher lignin deposition [52] and decreased H 2 O 2 content correlated with lower lignin deposition [16]. Consistently, our study also observed positive regulations of H 2 O 2 to CmCAD genes and CAD activity, as well as lignification, in a slight degree which may due to the short lifespan of exogenous applied H 2 O 2 .
Earlier, Jin et al. (2014) [26] analyzed and discovered stress and signal response elements in the promoters of CmCAD genes, and later, Liu et al. (2018) [27] revealed that drought could induce CmCADs expressions and promote lignin deposition. As the signal molecule and inhibitor or scavenger treatments revealed in this study, CmCADs are positively regulated by ABA, H 2 O 2 and JA. To further demonstrate the regulating patterns, promoter::GUS constructs were constructed by replacing 35S promoter with promoters of CmCAD1, 2, and 3, respectively, in pCAM-BIA1381Z vector. These three genes were chosen according to their positive responses to drought stress [27], phylogenetic analysis [26] and their major functions in lignin synthesis [53]. GUS staining of tabacco leaves after signal molecule treatments obtained similar regulating patterns of the signal molecules to the three CmCAD genes consistent with expression assay, demonstrating CmCAD genes can be regulated by the stress-induced signal molecules similar as Kim et al. (2010) [43] reported. However, CmCAD1 pro::GUS showed the lightest GUS staining and activity either without or with signal molecule treatments, while CmCAD3 pro::GUS exhibited the strongest staining and activity under all conditions and CmCAD2 pro:: GUS the modest. These GUS assays further confirmed the regulations of ABA, H 2 O 2 and JA to CmCAD1, 2, and 3 on the one hand and demonstrated that CmCAD3 is probably the major lignification member in oriental melon seedlings on the other hand.

Conclusions
The three signal molecules responded to drought stress strongly, while ABA and H 2 O 2 in a fast way. Among the signal molecules, ABA and H 2 O 2 showed similar positive regulation patterns to CmCAD1, 2, 3, and 5, and JA positively regulated CmCAD2, 3, and 5. In addition, these signal molecules also exhibited positive regulations to most of the lignin biosynthesis genes. CAD activity and lignin content are under regulated by the signal molecules, showing positive correlations between lignification and the signal molecules under drought stress. The thereafter promoter::GUS assay provides a further demonstration that the signal molecules function positively in regulating CmCAD1, 2, and 3.

Plant material and treatments
Oriental melon (Cucumis melo var. makuwa Makino) cultivar 'CaiHong7' (purchased from Qiqihar Vegetable Research Institution, Qiqihar, Heilongjiang Province, China) was taken as experimental material for lignification related analysis and tobacco (Nicotiana benthamiana) preserved in our lab (the key laboratory of Key Laboratory of Protected Horticulture of Education Ministry and Liaoning Province, Shenyang Agricultural University, Shenyang, China.) was used for promoter analysis. Seeds were sterilized before sowing. Melon seedlings were cultivated using Yamasaki melon nutrient solution (half strength) [54] which was refreshed every 2 days and tobacco seedlings were cultivated in pots (soil: peat: compost = 1:1:1), both seedlings were cultivated in a growth chamber (25±2°C, 14 h/10 h light/dark cycle) in our lab.
Melon seedlings with four fully expended leaves were used for treatments. For drought treatment, PEG-6000 was added to the nutrient solution to the final concentration of 8% (w/v); for fluridone (Flu, ABA inhibitor, 25 μM), imidazol (Imi, H 2 O 2 scavenger, 10 mM) [30], or ibuprofen (Ibu, JA inhibitor, 1 mM) [31]  Six-week-old tobacco seedlings were used for agroinfiltration. The inoculated tobacco seedlings were firstly cultivated at room temperature under dark for 24 h, and then at room temperature under light for another 24 h. After preliminary 48 h cultivation, the tobacco seedlings were then treated with ABA (100 μM), H 2 O 2 (10 mM) and MeJA (100 μM), respectively. Leaf discs for GUS staining and samples for GUS activity analysis were then collected after 24 h cultivation at 24°C under 16/8 light/ dark condition after treatments in October 2018 for the first batch and January 2020 for the second batch (voucher No. Cm-20,181,013-001 and Cm-20,200,128-001).
Assays of each index contained three biological replicates and each biological replicate included three analytical replicates. The second and third internodes (counted from base to growth point) of treated oriental melon seedlings were collected and used for assays either with fresh or frozen samples. Tobacco leaves for GUS activity assay were collected as frozen samples. Frozen samples were frozen in liquid nitrogen immediately after collection and stored at − 80°C.

Measurements of ABA, JA and H 2 O 2 contents
Contents of ABA and JA were determined using 0.5 g frozen samples according to Li et al. (2011) [55] using ELISA assay kit (made by the China Agricultural University). Specific monoclonal antibodies were used for ABA and JA analysis and standard curve was built for each hormone according to the manufacturer instruction. Contents of ABA and JA were expressed as ng g − 1 fresh weight.
Content of H 2 O 2 was determined using 0.1 g frozen sample following the manufacturer instruction of H 2 O 2 content detection kit (Cat#A064, Nanjing Jiancheng Bioengineering Istitute, Nanjing, China). H 2 O 2 content was expressed as mmol g − 1 fresh weight (FW) calculated by the absorbance at 405 nm.

Measurement of lignin content
Lignin contents were measured according to Zhang et al. (2010) [56] as well as we described previously [27,53]. Extracted lignin solution was measured at 280 nm. Lignin contents were calculated according to a standard curve and expressed as mg g − 1 DW.

Histochemical lignin staining
The phloroglucinol-HCl staining was the same as described in our previous study [27]. While, 10 μm thickness sections were applied for observation.

Assay of CAD activity
CAD activity is performed the same as we described previously [27]. Coniferyl alcohol was used as substrate and CAD activity is presented as U mg − 1 protein. Protein concentration was measured by using BioRad Protein Assay Kit (Code No.T9310A, TaKaRa, Japan) based on the method described by Bradford (1976) [57].

Construction of GUS reporter gene vectors and expression in tobacco
The DNA, used for promoter clone, was extracted from untreated melon leaves using Hi-DNAsecure Plant Kit (Cat#DP350-02, Tiangen, Beijing, China). PrimeSTAR® HS (Premix) (Code No.R040A, TaKaRa, Japan) was used for sequence clone. Promoter sequences of CmCAD1, CmCAD2 and CmCAD3 were firstly cloned using the primers without restriction enzyme cutting site, then added poly-A tails (Cat#RT124, Tiangen, Beijing, China) and ligated into T-vector (pMDTM19(simple), Code No.3271, TaKaRa, Japan) using T4 DNA ligase kit (Cat#RT406, Tiangen, Beijing, China) for sequencing. Correct sequences, which are the same with the sequences downloaded from Melonomics (https://www. melonomics.net/), were secondly cloned using the primers with restriction enzyme cutting sites and ligated into linearized pCAMBIA1381Z by using Double Digest Protocol with Acc I and Hind III restriction enzymes. TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0 (Code No.9761, TaKaRa, Japan) was used for cloned sequence purification when necessary. The constructed vectors were introduced into Agrobacterium tumefaciens (strain GV3101) competent respectively using freeze-thaw protocol, and the virus was screened and inoculated with LB medium containing 25 mg L − 1 rifampicin and 50 mg L − 1 kanamycin. After PCR and gel electrophoresis detection, correct bacterial plaques were inoculated in 50 ml LB liquid medium in the presence of rifampicin and kanamycin in erlenmeyer flasks for 14-16 h. Then, bacterial solutions were centrifuged at 4°C 5000 rpm for 10 min. The harvested Agrobacterium pellets were resuspended to the density of 1.0 at 600 nm in infiltration buffer (10 mM MgCl 2 , 10 mM MES, 0.1 mM acetosyringone). After incubation at room temperature under dark condition for 3 h, infiltration cultures were pressure-injected into fully expended leaves of tobacco plants using a 1 ml needleless syringe avoiding veins. Primers used here are listed in Table S2.

GUS activity
GUS activity was referred to the procedures described by Jefferson et al. (1987) [58] and Wang et al. (2019) [59]. Inoculated tobacco leaf (0.2 g each biological replicate sample) was used for GUS activity detection which is calculated as μmol of substrates converted to products per minute and expressed as U mg − 1 protein. BioRad Protein Assay Kit was used to determine the protein concentration of enzyme extracts based on the method of coomassie brilliant blue G-250 described by Bradford (1976) [57].

Statistical analysis
Data were organized using Excel 2013 and analyzed through SPSS 18.0 using Duncan method with P = 0.05 to test the significance. Then Origin 8.0 and Photoshop CS4 were both used for graph generation and beautification. methyltransferase; PAL: Phenylalanine ammonia lyase; POD: Peroxidase; LAC: Laccase; F5H: Ferulate 5-hydroxylase