Melatonin enhances salt tolerance by promoting CcCAD10-mediated lignin biosynthesis in pigeon pea

doi:10.21203/rs.3.rs-4439371/v1

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Research Article

Melatonin enhances salt tolerance by promoting CcCAD10-mediated lignin biosynthesis in pigeon pea

https://doi.org/10.21203/rs.3.rs-4439371/v1

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Melatonin plays an important role in improving plant resistance to salt stress by regulating biosynthesis of specialized metabolites. Phenylpropanoids, especially lignin, contribute to all aspects of plant responses towards biotic and abiotic stresses. However, the crosstalk between melatonin and lignin is largely unknown in pigeon pea under salt stress. In this study, the cinnamyl alcohol dehydrogenase CcCAD10 was identified to be involved in melatonin treatment and salt stress. The content of lignin was increased substantially in CcCAD10 over-expression (OE) lines, the activities of antioxidant enzyme was increased, indicating enhanced salt resistance. As a parallel branch of the lignin synthesis pathway, the content of flavonoids was further determined. The accumulations of luteolin, genistin, genistein, biochain A, apigenin and isovitexin were down-regulated in CcCAD10-OE hairy root. The results indicate that CcCAD10-OE mediated carbon flow from the phenylalanine pathway is redirected to the lignin pathway at the expense of less carbon flow in the flavonoid pathway, enhancing the salt-tolerance. Furthermore, we found the exogenous melatonin stimulated endogenous melatonin production mainly by upregulating the expression of CcASMT2 gene. This study found a new way for melatonin to improve the salt tolerance of pigeon pea, which laid a foundation for exploring the molecular mechanism of melatonin in salt stress response.

Pigeon pea

Lignin

Cinnamyl alcohol dehydrogenase

Luteolin

Salt tolerance

It is estimated that soil salinization affects more than 20% of all cultivated land and approximately half of all irrigated land worldwide [1]. Soil salinization has a significant toxic impact on plant growth. The presence of large amounts of Na⁺ and Cl⁻ can block the efficient transportation of key nutrients such as nitrogen, phosphorus, and potassium [2]. In recent years, the role of melatonin, an emerging indole plant hormone, in regulating plant growth and development has been increasingly explored [3]. Melatonin is ubiquitous and present in all plant that it regulates growth and development, seed germination, root growth. In addition, it also plays a significant role in plants response to salt stress [4] Current studies have shown that melatonin enhances plant resistance to salt stress mainly in two ways.: One is through direct pathways, such as direct scavenging of reactive oxygen species (ROS); the other is through indirect pathways, such as increasing antioxidant enzyme activity, secondary metabolite content, photosynthetic efficiency, and regulating the expression of stress-related genes [5–7]. Exogenous melatonin improves Zea mays resistance to salt stress by increasing the activity of antioxidant enzymes and reducing membrane conductivity and membrane damage [8]. Exogenous melatonin can enhance the salt stress resistance of upland Gossypium hirsutum by increasing the flavonoid content [9].

Lignin is a type of secondary metabolite found in plants. It is an aromatic phenolic compound that is widely present in the cell walls of vascular plants and accounts for approximately 18–35% of the total plant biomass [10]. Lignin, a polymer composed of phenylacetone units, is one of the main components of the secondary cell wall of plant and is involved in coping with various biotic and abiotic stresses [11–13]. Studies have shown that increased lignification strengthens plant structure and facilitates plant adaptation to salt stress, such as Malus pumila [14], Cucumis melo [15], Arabidopsis thaliana [16], under salt stress, the increased lignin content was accompanied by the up-regulation of a number of key enzyme genes of the lignin synthesis pathway, such as HCT, COMT and CAD [17]. Cinnamyl alcohol dehydrogenase (CAD), is an enzyme at the end of the lignin monomer synthesis pathway [18]. The role of CAD is to convert cinnamaldehyde (coniferaldehyde, sinapaldehyde, and coumaraldehyde) into the corresponding alcohols (coniferol, sinapol, and coumarol), controlling the lignin content [19, 20]. Since the first CAD gene was successfully identified, the CAD gene family have been successfully identified in other species [21], such as Arabidopsis thaliana [22], Oryza sativa [23], Populus tremula [24]. The function of CAD in regulating lignin synthesis to increase plant resistance to stress has been confirmed. Overexpression of GsCAD1, in Wild Soybean increased resistance to Glycine max Mosaic Virus [19]. However, the function of the CAD gene in pigeon pea remains to be investigated.

Pigeon pea (Cajanus cajan) is a widely cultivated perennial woody plant in the Leguminosae family, that is found in tropical and subtropical areas [25]. Pigeon pea have a well-developed root system and strong nitrogen fixation ability, making them an ideal tree species for vegetation restoration and soil improvement. In addition, pigeon pea also have high medicinal value, as they can clear heat and detoxify the body, treat diseases such as chickenpox, malaria, and femoral head necrosis [26]. However, the yield of pigeon pea has declined significantly over the years due to environmental factors [27]. Salt stress is a major constraint limiting productivity of pigeon pea. Due to the antagonistic effect of salt stress on plant growth, physiology and metabolism, plant biomass and economic yield are significantly reduced [28]. Therefore, the breeding of salt-tolerant pigeon pea is of great significance to improve the aboveground biomass and increase the yield of pigeon pea.

In the present study, the application of exogenous melatonin reduced the damage of salt stress to pigeon pea. Notably, both exogenous melatonin and salt stress treatments promoted the accumulation of lignin in pigeon pea. Further qRT-PCR analysis showed that CAD genes were significantly differentially expressed under salt stress and exogenous melatonin treatment. We conducted a comprehensive identification and bioinformatics analysis of the CAD gene family of pigeon pea. Finally, CcCAD10 was selected for further analysis. Overexpression of CcCAD10 in hairy root and transient transgenic pigeon pea revealed that CcCAD10 enhanced the tolerance of pigeon pea to salt stress by promoting lignin synthesis and antioxidant enzyme activities. Luteolin were reduced in CcCAD10 overexpressing pigeon pea, but exogenous melatonin treatment significantly promoted the accumulation of luteolin. In order to reveal whether the effect of exogenous melatonin depends on endogenous melatonin, the content of melatonin in pigeon pea was further determined. The results showed that exogenous melatonin treatment could increase the accumulation of endogenous melatonin in pigeon pea. Meanwhile, the genes in the melatonin synthesis pathway were differentially expressed under exogenous melatonin treatment, especially CcASMT2 was significantly up-regulated. Taken together, we revealed that exogenous melatonin treatment plays an important role in improving the salt stress resistance of pigeon pea..

2.1 Plant growth and treatment

Pigeon pea seeds (ICPL87119) were preserved in Northeast Forestry University. The pigeon pea seeds were soaked in water overnight to fully absorb water and transfer them to a seedling pot the next day. The pots were incubated at 37°C for 2–3 days until the seeds germinated. For hydroponic cultivation, 1/5 Hoagland solution was used. When the pigeon pea seedlings grew to 5–6 cm, the pigeon pea seedlings were transferred to a mixed nutrient soil culture pot, and kept at a constant temperature of 24 ℃, under 16 hours of light/8 hours of darkness, with a light intensity of 5000 lx. After 30 days of cultivation, pigeon pea seedlings with uniform growth were selected for the experiments. The plants in the melatonin treatment group were watered once a week with 50 mM melatonin, 500 ml each time, Abbreviation M. The salt stress group was watered once a week with 150 mM NaCl, 500 ml each time, to simulate salt stress, Abbreviation N. The mixed treatment group was treated simultaneously with melatonin and NaCl, Abbreviation MN, Blank control group treated with equal volume of distilled water, Abbreviation WT. The culture conditions of pigeon pea, NaCl and melatonin treatment concentrations were referred to Song et al. [29]. After 0, 3, 6, 12, and 24 h of different stress treatments, each collected material was stored in a -80℃ refrigerator for subsequent experiments.

2.2 Determination of physiological and biochemical indexes

The lignin content was determined using the acetyl bromide method [30]. Determination of SOD activity by riboffavin-NBT method, determination of POD activity by peroxidase guaiacol method, determination of CAT activity by Beer-Lambert Law method [31]. MDA accumulation was measured using the thiobarbituric acid-based method [32]. The endogenous melatonin content was measured using plant melatonin enzyme-linked immunosorbent assay ELISA kit (China), and the specific operating steps were carried out according to the instructions of the kit.

2.3 Identification and bioinformatics analysis of melatonin synthesis pathway gene and lignin synthesis gene CcCAD

The tryptophan decarboxylase (TDC) gene ,tryptamine 5-hydroxylase (T5H) gene, serotonin N-acetyl transferase (SNAT) gene, and acetyl-serotonin methyltransferase (ASMT) gene were identified following the methods of Zhao et al.[4]. Caffeic acid o-methyltransferase (COMT) gene identification followed the experimental method of Liang et al [33]. The identification of the cinnamyl alcohol dehydrogenase (CAD) gene family was performed according to the methods of Yang et al [34]. CcCAD protein physiochemical properties were analyzed through the ExPASy website (https://web.expasy.org/compute_pi/) [35]. In addition, the subcellular localization was evaluated by the WoLF-PSORT (https://wolfpsort.hgc.jp/) [36] The analysis results of physical and chemical properties of proteins are shown in Table.S1. The target sequences were compared with known functional protein sequences by CLUSTALW (https://www.genome.jp/ tools-bin/clustalw). A Phylogenetic tree was constructed by MEGA7 with the maximum likelihood method, the bootstrap of the phylogenetic tree was set to 1000. The conserved domains was were predicted using the NCBI Batch CD-Search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). The MEME tool software was used to analyze conserved motifs [37].

2.4 RNA extraction and quantitative real-time RT-PCR

In our experiment, the whole pigeon pea seedlings were collected and processed for qRT-PCR. A Plant RNA Kit (USA) was used to draw the total RNA from the whole seedlings, according to the manufacturer’s protocol. The concentration and quality of total RNAs were determined by a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies). Finally, we synthesized the first-strand cDNA using the PrimeScriptTM·1st Strand cDNA Synthesis Kit (JAPAN), following the manufacturer’s instructions. To perform qRT-PCR assays, we designed primers with Primer Premier 5.0 software (Premier Biosoft International), and the details are listed in Table S1. The reactions were carried out as previously described [38]. The relative expression levels were normalized to the Actin gene (Accession No. XM_020358530.2) and calculated using the 2^−ΔΔCt method.

2.5 Construction of the CcCAD10 overexpression vector and pigeon pea transformation

The CDS sequence of the CcCAD10 gene was cloned and inserted into the vector pCAMBIA1303. An overexpression vector of CcCAD10 (CcCAD10-OE) was constructed and transformed into Agrobacterium tumefaciem EAH105 and Agrobacterium rhizogenes LBA9402 for immediate transformation of pigeon pea and induction of transgenic in hairy roots. Transient transformation using Agrobacterium tumefaciem EAH105, 30-days-old pigeon pea seedlings were immersed in Agrobacterium tumefaciem EAH105 solution (OD600 = 0.8-1.0) and vacuumed in a vacuum container of -50 kPa for 5 minutes. After vacuum treatment, the seedlings were washed three times with sterile water and cultured in Hoagland nutrient solution for subsequent experiments [37]. Transgenic hairy roots were grown using Agrobacterium rhizogenes LBA9402 (OD600 = 0.8 ~ 1.0), soaked for 5 min with shaking, washed with distilled water and incubated for 2 days, then decontaminated with 200µg/ml ceftriaxone, and the transgenic hairy roots could be grown after 7 days [39].

2.6 Salt stress treatment of transgenic pigeon pea

A total of 108 pigeon pea seedlings underwent transient transformation and were then exposed to salt stress. The seedlings were divided into two groups pCAMBIA1303 and CcCAD10-OE and were subjected to Hoagland's solution supplemented with 150 mM NaCl. Moreover, the control group received Hoagland's solution without any treatment. After 24 hours and 7 days of salt stress, the entire pigeon pea seedlings were collected rapidly frozen in liquid nitrogen, and then stored at -80 ℃ until analysis. Transgenic hairy roots were grown in 1/2MS liquid medium for one month and then transferred into 1/2MS liquid medium containing 150 mm NaCl. Samples were collected after 24 hours and 7 days of salt stress. Quickly frozen in liquid nitrogen and stored at -80°C for analysis. For the physiological experiments and quantification analysis of stress-related genes, pCAMBIA1303 and CcCAD10-OE transgenic plants subjected to salt stress for 24 hours were used. Samples were collected after 7 days of salt stress treatment for lignin content determination and quantification of lignin synthesis genes.

2.7 Determination of flavonoid levels

The hairy root of pigeon peas underwent desiccation until reaching a state of constant mass within a freeze-drying apparatus. Subsequently, flavonoids were extracted utilizing a 70% ethanol aqueous solution, employing a 1:3 ratio relative to the initial volume of the substrate. This extraction was accompanied by ultrasonication at 80 W and 25°C for a duration of 20 minutes. Following centrifugation at 10,000×g for 5 minutes, the resultant supernatant was meticulously collected.

Quantification of total flavonoid content was conducted as follows: Initially, 0.2 mL of the hairy root flavonoid extract was introduced to 0.3 mL of a 5% sodium nitrite solution, followed by agitation and subsequent incubation for 6 minutes. Subsequently, 0.3 mL of a 10% aluminum nitrate solution was added, and the mixture was subjected to further agitation and static incubation for 6 minutes. Finally, 2 mL of a 4% NaOH solution was introduced, followed by another round of agitation and static incubation for 10 minutes. After adjusting the volume to 10 mL, the absorbance value was measured at 510 nm.

The UPLC-MS/MS analysis was conducted using the Agilent 1290 Ultra-High Performance Liquid Chromatograph, interconnected with the Agilent 6460 Triple Quadrupole Mass Spectrometer, which was equipped with an electrospray ionization (ESI) source to scrutinize the designated compounds. Analyte separation transpired on an Agilent ZORBAX Eclipse Plus C18 column, measuring 50 mm in length with a 2.1 mm internal diameter and 1.8 µm particle size, maintained at a steady temperature of 30°C. The binary solvent system consisted of acetonitrile (Component A) and water (Component B), employed in a gradient elution protocol at a flow rate of 0.4 mL/min. The elution gradient was as follows: from 0 to 2 minutes, a shift from 25–35% A; between 2 and 3.5 minutes, increasing to 90% A; held at 90% A from 3.5 to 5 minutes; a rapid decrease to 25% A between 5 and 5.1 minutes, and maintained at 25% A until 6 minutes. The injected sample volume was 1 µL, with the post-chromatographic eluate directed into the mass spectrometer for analysis. The mass spectrometry was conducted in Multiple Reaction Monitoring (MRM) mode, with key parameters set as follows: a capillary voltage of 4 KV for ESI + and 3.5 KV for ESI-, a gas temperature of 350°C, a gas flow rate of 10 L/min, a nebulizer pressure of 50 psi, and an accelerator voltage of 4V for the quadrupole [39].

2.8 Statistical analysis

SPSS 20.0 was used for the statistical significance of the data in this study, which were analyzed by one-way ANOVA followed by a post hoc Tukey's test. Significant differences were marked: *P < 0.05 **P < 0.01 ***P < .0.0001.

3.1 Effect of melatonin on the salt tolerance of pigeon pea

To study the effect of melatonin on pigeon pea under salt stress, one-month-old soil-cultured pigeon peas were watered with 50 µM melatonin (the mock group was watered with distilled water). As shown in Fig. 1a. Leaves turned yellow and wilting after 7 days of salt stress in the N group, but the pigeon peas in the MN group treated with melatonin were greener than those in N group. Meanwhile, the growth status of pigeon peas in M group was similar to that of W group. Analysis of antioxidant enzyme activities showed that pigeon peas had high antioxidant enzyme activities in MN group (Fig. 1b,c,d). Compared with pigeon peas in the W, M and N group, the lignin content in MN groups was significantly increased (Fig. 1e). The results showed that melatonin pretreatment significantly alleviated the damage of salt stress to pigeon pea.

3.2 Expression pattern analysis of CcCAD10 gene under salt stress and melatonin treatment

Since CAD proteins were key enzyme in lignin biosynthesis, we conducted a comprehensive identification and bioinformatics analysis of the CAD gene family of pigeon pea. A total of 12 CAD proteins were identified in pigeon pea. The physical and chemical properties of CcCAD are analysed in Table 1.The length of CcCAD proteins ranged from 203 aa (CcCAD2) to 378 aa (CcCAD12). The mean of pI and molecular weight was 6.55 and 35315.77 Da, respectively. 12 CcCAD proteins were found to be located in the cytoplasm, peroxisome, cytoskeleton. Phylogenetic tree was created to study the evolutionary relationship of the CAD gene family in pigeon pea (Fig. 2a). The phylogenetic tree included 12 members of the CAD gene family in pigeon pea, 9 members of the CAD gene family in Arabidopsis thaliana, and several genes with known functions from Populus tremula and Medicago sativa (Table S2). The CcCAD gene family of pigeon pea is mainly divided into three branches. The genes in the same branch have similar gene structure and Motif composition (Fig. S1). Notably, pigeon pea CcCAD1 and CcCAD10 are closely related to populus CAD1 of known function located in the same branch [40]. To check the sequence similarity of CcCAD1, CcCAD10 and PtrCAD1, multiple sequences were aligned.. The conserved Zinc-containing ADH_N domain [GHE(X)₂G(X)₅G(X)₂V] (68–82 aa), and AlaDh_PNT_C domain [V(X)G(X)GG(X)G] (186–193 aa) were found in these genes [41]. However, the sequence similarity between CcCAD10 and PtrCAD1 is 80%, which is slightly higher than the 79.38% sequence similarity between CcCAD1 and PtrCAD1 (Fig. 2b). qRT-PCR analysis showed that CcCAD10 was significantly up-regulated under salt stress and melatonin treatment, where CcCAD10 was up-regulated 12.84-fold at 24h of melatonin treatment and 4.17-fold at 24h of salt stress treatment (Fig. 2c,d). Therefore, we speculated that CcCAD10 may be a key gene involved in lignin biosynthesis, improving salt-tolerance of pigeon pea.

Table 1

The physiochemical properties of CcCAD proteins
Name	Protein length (aa)	Molecular weight (Da)	pI	location
CcCAD1	354	38496.25	6.75	cyto
CcCAD2	204	21784.36	6.97	cyto
CcCAD3	360	39049.91	6.39	pero
CcCAD4	297	31819.68	5.54	extr
CcCAD5	160	17764.03	5.59	chlo
CcCAD6	359	39154.23	5.73	cysk
CcCAD7	361	39070.2	8.26	cyto
CcCAD8	365	38810.71	6.49	cysk
CcCAD9	362	38918.04	6.26	cyto
CcCAD10	356	38457	5.94	cyto
CcCAD11	367	39774.96	8.41	cyto
CcCAD12	378	40689.85	6.31	cyto
Average	326.92	35315.77	6.55	-

3.3 CcCAD10-OE transient transformation increases the salt stress resistance of pigeon pea

To further investigate the biological function of CcCAD10 in pigeon pea, the CcCAD10 overexpression vector (CcCAD10-OE) were constructed (Fig. 3a). After 24 h of recovery, the transformed pigeon pea seedlings were further incubated in Hoagland nutrient solution supplemented with 150 mM for 7 days. No significant phenotype difference existed between the transiently transformed pCAMBIA1303 and WT under salt conditions. However, after 7 days of salt treatment, the leaves of the control plants withered and curled, while the leaves of the CcCAD10-OE line grew well (Fig. 3b). Compared to the WT and empty-vector control (pCAMBIA1303), the CcCAD10-OE transiently transformed plants showed a 9.7-fold increase in CcCAD10 expression, and transgenic hairy root showed a 68-fold increase in CcCAD10 expression (Fig. 3c). As shown in Fig. 3d, the MDA content in the transiently transformed CcCAD10-OE group was lower than that in the WT. These findings indicate that overexpression of CcCAD10 gene reduced the oxidative damage of cell membrane induced by salt stress. The enzyme activities of POD, SOD, and CAT were measured, and it was found that the salt resistance of the transiently transformed CcCAD10-OE group was higher than that of the pCMBIA1303 and WT group (Fig. 3e-g). At the same time, the lignin content was also found to be higher in the transiently transformed CcCAD10-OE group than in the WT group (Fig. 3h).

3.4 Overexpression of CcCAD10 increases salt stress resistance in pigeon pea hairy roots

To further investigate the function of CcCAD10, we generated hairy roots of CcCAD10-OE using Agrobacterium rhizogenes. The generated hairy root should be further verified by PCR detection of some reference genes (rolB, rolC, aux1, and virD). RolB, rolC, and aux1 genes located at pRiT-DNA. From Fig. 4a, there is no gene insertion in the sterile seedlings, and the above four genes are present on Agrobacterium rhizogenes LBA9402, while there is no vir-D gene in the hairy roots, indicating that the hairy roots were successfully obtained [39]. After 7 days of salt treatment, the hairy roots of CcCAD10-OE group were more robust and the growth state was better than that of the plants in the WT group, green fluorescence can be observed in transgenic hairy roots (Fig. 4b). Compared to the WT and empty-vector control (pCAMBIA1303), the CcCAD10-OE transgenic hairy root showed a 68-fold increase in CcCAD10 expression (Fig. 4c).As shown in Fig. 4d, the MDA content in the transgenic hairy roots CcCAD10-OE group was lower than that in the WT. The POD CAT SOD activity of the CcCAD10-OE transgenic hairy root group was higher than that of the pCMBIA1303 and WT groups (Fig. 4e-g). The CcCAD10-OE transgenic hairy root group was higher than that of the pCMBIA1303 and WT groups. These results indicate that CcCAD10 improves salt stress resistance of pigeon pea by increasing antioxidant ability capacity.

3.5 The impact of CcCAD10 overexpression on flavonoids and lignin levels

We utilized transgenic hairy roots of pigeon pea as the foundation of our experimental framework. Our investigation focused on elucidating the influence of CcCAD10 on the synthesis and accumulation of flavonoids, pivotal compounds within the pigeon pea. Eleven flavonoids with important functions in pigeonpea were determined. The findings of the study unveiled a marked augmentation in lignin content within the CcCAD10-OE specimens, juxtaposed with a notable decrease in the concentrations of six flavonoid compounds, genistin, genistein, biochain A, apigenin, isovitexin, and luteolin (Fig. 5).unveiled a notable downregulation in the levels of six flavonoids within the CcCAD10-OE samples, specifically genistin, genistein, biochain A, apigenin, isovitexin, and luteolin (Fig. 5). Furthermore, we meticulously evaluated the total flavonoid content in the transgenic hairy roots, revealing that the augmentation of CcCAD10 had no significant impact on the overall flavonoid levels (Fig. S2). Research conducted by Song et al has shown that melatonin treatment significantly increases the content of luteolin in the roots of pigeon pea. The luteolin is the main compound that enhances the salt tolerance of pigeon pea [29]. To investigate the effect of CcCAD10 overexpression on luteolin synthesis. In the study, the hairy roots of CcCAD10-OE and WT were treated with melatonin and then the luteolin content was measured. As shown in Fig. S3, the luteolin content in the CcCAD10-OE plants was lower than that in the WT. However, after the hairy roots were treated with exogenous melatonin, the luteolin content increased in the CcCAD10-OE and WT groups.

3.6 CcCAD10 increases the lignin levels

To further explore the molecular mechanism of CcCAD10 in improving salt stress tolerance in pigeon pea, the content of lignin was quantitatively analyzed. The lignin content was also found to be higher in the transgenic hairy roots CcCAD10-OE group than in the WT group (Fig. 5). Lignin biosynthesis genes were significantly up-regulated in CcCAD10-OE lines under salt conditions (Fig. 6). The expression levels of lignin biosynthesis genes were selected for quantitative analysis (Table. S3). A total of 12 genes were significantly upregulated. Specifically, Cc4CL1, Cc4CL7, CcCCoAOMT4, CcCCR11, CcCAD1, CcCAD8, along with members of the LAC gene family CcLAC1, CcLAC2, CcLAC5, CcLAC6, CcLAC9, and CcLAC18, exhibited pronounced elevation in their expression within CcCAD10-OE lines under conditions of salt stress. Collectively, these results indicate that CcCAD10 improves salt stress resistance of pigeon pea by increasing lignin content.

3.7 Effect of exogenous melatonin on the endogenous melatonin content

In this study, the levels of endogenous melatonin were measured in both control group and treatment group of pigeon peas, and it was discovered that exogenous melatonin can enhance the synthesis of endogenous melatonin. It was found that salt stress could also increase the content of endogenous melatonin in pigeon pea (Fig. S4). We identified key enzyme genes in the melatonin synthesis pathway, and the phylogenetic tree of key enzyme genes of melatonin synthesis pathway is shown in Fig. S5, the genes are shown in Table. S4.The phylogenetic trees of the key enzyme genes of the melatonin synthesis pathway in Arabidopsis thaliana, Sorghum bicolor, Oryza sativa and pigeon pea were constructed and found that the five gene families were divided into three branches, AtTDC1 and AtTDC3 with known functions of CcTDC1 have a close relationship (Fig. S5a). CcSNAT2 has a close relationship with AtSNAT1 with known functions (Fig. S5c). CcCOMT1 has a close relationship with AtCMOT1 (Fig. S5d). Differentially expressed genes in the melatonin synthesis pathway were found by transcriptome analysis of melatonin treatment, as shown in Fig. S6. The expression of the genes CcTDC2, CcSNAT1, CcSANT2, CcCOMT3, CcCOMT4 and CcASMT2 were up-regulated.

As a plant hormone, melatonin plays an important role in plant growth, development, synthesis of specialized metabolites and resistance to stress [42]. The activity of antioxidant enzymes (SOD, CAT and POD) in plants is closely related to plant salt tolerance. A large number of studies have confirmed that there is a positive correlation between the activity of antioxidant enzymes and plant resistance [43]. Melatonin can improve plant resistance to salt stress by increasing the activity of POD, SOD, CAT and other antioxidant enzymes [44]. Our findings showned that when melatonin is added externally, can boost the activities of these antioxidant enzymeslead to an increase in the salt-tolerance of pigeon pea (Fig. 1b,c,d).Research discovery that lignin has many functions such as anti-oxidation and anti-bacteria. The most important function of lignin in plants is to increase the mechanical support and resistance to stress of plants as an important part of secondary cell wall [17]. Lignin can improve the stem strength and dehydration tolerance of Arabidopsis to protect itself from salt stress [45]. Research finding that treating plants with melatonin increased the expression of several key enzyme encodinge genes involved in lignin synthesis promotes the increase of lignin content [46, 47]. After conducting research, we determined that the lignin content in pigeon pea significantly increased after 7 days of melatonin treatment. This suggests that melatonin can affect the synthesis of lignin in pigeon pea (Fig. 1e). Therefore, we believe that treatment with melatonin can increase lignin synthesis and improve the self-protection of pigeon pea against salt stress.

The CAD gene, which encodes a crucial enzyme in the lignin synthesis pathway, has the ability to regulate lignin synthesis in response to environmental conditions such as high salt levels. This can help increase the plant's tolerance to stress. Additionally, research has shown that the activity of cinnamyl alcohol dehydrogenase increases when plants are exposed to high saltlevels concentrations [49]. The results show that CmCAD2 may be the main gene in response to lignin deposition and can also resist abiotic stress [15]. Using phosphorylated proteomics, the study reported that CAD1 phosphorylation was detected in resistant tomato Hawaii 7996 [50]. Silencing of the CAD gene enhances the susceptibility of leaf tissues to the fungal pathogen Blumeria graminis f. sp. Tritici, causing the powdery mildew disease [51]. Zhao et al.found that when the function of MsCAD gene is lost, Medicago truncatula will show the characteristics of reduced lignin content, dwarf plant and reduced tolerance to high temperature environment [18]. We found that CcCAD10 was also involved in salt stress response while responding to melatonin treatment (Fig. 2c,d).Therefore functional validation of CcCAD10 by transient transformation and transgenic hairy roots revealed that antioxidant enzyme activity of salt tolerance in transgenic plant materials were higher than those in the WT group, however, the MDA content of transgenic plants was lower than that of WT group (Fig. 3, Fig. 4). The possible reason is that the increase of lignin content and the enhanced protective effect of secondary cell wall on cells reduce the oxidative damage of membrane system and reduce the content of MDA [52]. Therefore, as an important gene regulating lignin synthesis, CcCAD10 plays an important role in the process of melatonin increasing the resistance of pigeon peas to salt stress.

The metabolic balance between the flavonoid and lignin pathways, which are also downstream of the phenylpropanoid pathway, has been discussed by researchers [53], overexpression of CsHCT increased lignin content and decreased flavonol content [48], The levels of rosmarinic acid and salvianolic acid B decreased, while the lignin content increased in plants and hairy roots overexpressing SmLAC25 [54]. Previous studies have demonstrated that the production of luteolin, a flavonoid compound, is a crucial process regulated by melatonin to enhance the salt tolerance of pigeon pea [29]. To investigate the impact of CAD gene overexpression on these pathways, researchers explored the metabolic relationship between them. We determined the luteolin content in CcCAD10-OE hairy roots and found that it was lower than that in the blank control group. This suggests that CcCAD10-OE can alter the carbon flow of the phenylpropane metabolic pathway and promote lignin synthesis. In the CCoAOMT gene mutant of alfalfa, there was a discernible reduction in lignin content, concomitant with heightened levels of isoflavones and their pathway intermediates compared to the wild-type cohort. Notably, compounds such as medicarpin and 7,4'-dihydroxyflavone exhibited notably elevated accumulation in the mutant line [55]. Melatonin promotes the synthesis of lignin and also promotes the synthesis of luteolin (Fig. S3). Yin et al. found that exogenous melatonin can increase the synthesis of Glycine max isoflavones under salt stress [56]. Yang et al. found that the catechin content of Vitis vinifera almost doubled under exogenous melatonin treatment [57]. Exogenous melatonin also plays an important role in increasing the content of flavonoids in Brassica campestris and anthocyanins in Malus spectabilis leaves [58, 59]. There is a noticeable difference in melatonin levels under varying growth conditions due to its sensitivity to the environment and rhythm as a plant hormone [60]. After receiving salt stress, we observed that endogenous melatonin levels increased to varying degrees (Fig. S4). The content of endogenous melatonin in Vitis vinifera also increased under salt stress [61]. By analyzing the transcriptome of Song, we found that the expressions of CcTDC, CcASMT, CcCOMT genes were significantly up-regulated (Fig. S6). Sharafi's results showed that exogenous melatonin treatment increased the expression of TDC, T5H, SNATand ASMT genes, thereby promoting endogenous melatonin synthesis [6]. Samanta et al. found that exogenous melatonin can regulate the expression of TDC, SNAT and ASMT genes and promote the synthesis of endogenous melatonin in Oryza sativa [62]. We can explore the balance between exogenous and endogenous melatonin and determine the optimal cycle of exogenous melatonin use to maximise the influence on endogenous melatonin synthesis and improve the efficiency of exogenous melatonin use while conserving exogenous melatonin.

Taken together, Our data indicate that exogenous melatonin improves the salt tolerance of pigeonpea by increasing the expression of the last step enzyme CcCAD10 in lignin biosynthesis. At the same time, luteolin and endogenous melatonin are regulated by exogenous melatonin, and there is a dynamic balance between luteolin and lignin in content. They interact to affect the salt tolerance of pigeon pea (Fig. 7).

To improve the resistance of pigeon pea to salt stress, the exogenous melatonin was imposed to explore its effects on the physiology, biochemistry, metabolism and expression of related genes in pigeon pea. The results showed that exogenous melatonin can significantly improve the antioxidant capacity, lignin content, and endogenous melatonin content of pigeon pea. Transcriptome analysis revealed significant differences in the expression of CAD gene family members under melatonin treatment. Further qRT-PCR analysis showed that CcCAD10 responds to salt stress and melatonin treatment. We verified through transient transformation and hairy root genetic transformation that CcCAD10 overexpression can increased the pigeon pea salt-resistant capacity by upregulating the expression of laccase and other relevant genes, thereby elevating the lignin content within the pigeon pea. In addition, the content of luteolin in pigeon pea was further determined. The findings indicated that the overexpression of CcCAD10 led to a reduction in the concentration of luteolin and other flavonoid compounds, while exogenous melatonin application promoted the accumulation of luteolin. We also measured the content of melatonin, the results showed that exogenous melatonin application and salt stress could induce the content of endogenous melatonin. Transcriptome data analysis unraveled that the application of exogenous melatonin up-regulated the expression of melatonin synthesis pathway genes, especially CcASMT2. Take together, Our results suggest that the application of exogenous melatonin may improve the salt stress resistance of pigeon pea by mediating lignin and flavonoid biosynthesis through endogenous melatonin signaling. Our findings provide new insights into the function of melatonin in salt stress and a crucial reference for the genetic improvement of pigeon pea resistant to salt stress.

Authors’ contribution

Feng Pan and Hongquan Li conceived the work and designed the experiments. Feng pan performed all experiments, analyzed the data, and wrote the original draft. Hongquan Li reviewed and edited the manuscript. Xiaoli An collected the experimental samples and attended to the curation of RNA-Seq data. Ming Qu contributed to the statistical analysis of the data. Jie Yang and Yujie Fu provided revisions, editing, supervision, and project administration. All authors have read and agreed to the published version of the manuscript.

Acknowledgements

This work was supported by the National Key R&D Program of China (2022YFD2200602), the National Key Natural Science Foundation of China (31930076), the National Natural Science Foundation of China (32271805), the National XA Science and Technology innovation project (2022XACX1100), the 111 Project (B20088).

Declaration of competing interest

The authors declare that they have no conflict of interests.

Data availability statement

The data that support the findings of this study are available within the manuscript and its supporting information files. Datasets generated and analyzed in the current study are available from the corresponding author on reasonable request.

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Melatonin enhances salt tolerance by promoting CcCAD10-mediated lignin biosynthesis in pigeon pea

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Plant growth and treatment

2.2 Determination of physiological and biochemical indexes

2.3 Identification and bioinformatics analysis of melatonin synthesis pathway gene and lignin synthesis gene CcCAD

2.4 RNA extraction and quantitative real-time RT-PCR

2.5 Construction of the CcCAD10 overexpression vector and pigeon pea transformation

2.6 Salt stress treatment of transgenic pigeon pea

2.7 Determination of flavonoid levels

2.8 Statistical analysis

3. Results

3.1 Effect of melatonin on the salt tolerance of pigeon pea

3.2 Expression pattern analysis of CcCAD10 gene under salt stress and melatonin treatment

3.3 CcCAD10-OE transient transformation increases the salt stress resistance of pigeon pea

3.4 Overexpression of CcCAD10 increases salt stress resistance in pigeon pea hairy roots

3.5 The impact of CcCAD10 overexpression on flavonoids and lignin levels

3.6 CcCAD10 increases the lignin levels

3.7 Effect of exogenous melatonin on the endogenous melatonin content

4. Discussion

Conclusion

Declarations

References

Supplementary Files

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