To identify the HpSNAT candidates, the hidden Markov model profile of the SNAT conserved DNA binding domain (PF13508) was used as a query to search the genomic databases of H. perforatum via the PFAM databases.¹ In addition, only those genes with complete GNAT domain can be used as members of the SNAT gene family by using InterProScan² (Zdobnov and Rolf, 2001). The isoelectric point (PI) and molecular weight (MW) of the HpSNAT proteins were predicted by Compute PI/MW tool on the ExPASy³ (Gasteiger, 2005).

Four SNAT genes (rice OsSNAT1 and OsSNAT2, Arabidopsis AtSNAT1 and AtSNAT2) were confirmed to have SNAT activity, identified as bait proteins. A neighbor-joining phylogenetic tree of the candidate HpSNATs and four bait protein sequences was constructed using the software MEGA 9.0 with 1000 replicates bootstrap. The protein sequence of HpSNATs was used to predict the conserved motifs by using the program MEME⁴ (Bailey et al., 2009) with the following parameters: a maximum number of motifs sets at 5, optimum motif width from 70 to 300 bp, and with an e-value less than 1e-10. The amino acid sequence of the conserved GNAT domain of HpSNAT proteins was obtained by SMART,⁵ and then multiple sequence alignment analysis was carried out by using Geneious v10.22. The upstream 2 kb genomic DNA sequences of HpSNAT1/2 were uploaded to the PlantCARE database to search for the cis-acting regulatory elements in the promoter region (Lescot, 2002). The transcriptome data of different tissues (root, stem, leaf, and flower) were used to display the tissue-specific expression profile in the form of a heat map by using Origin v10.5.

Arabidopsis homozygous mutant snat (SALK_033944C) and wild-type Columbia-0 (Col-0) seeds were obtained from the European Arabidopsis Stock Centre (NASC). Three primers (LP, RP, and BP) were used to screen the homozygous mutants (Supplementary Figure 1 and Supplementary Table 1). The seeds of H. perforatum were preserved in our laboratory. The sowing, sterilization, and growth conditions of aseptic tissue culture seedlings of Arabidopsis and H. perforatum can be referred to in the previous detailed description (Zhou et al., 2021). Three-month-old aseptic seedlings of H. perforatum were transferred into liquid Murashige and Skoog (MS; Solarbio) medium containing 250 mM D-Mannitol (Alfa Aesar) and 200 mM NaCl (Solarbio) for drought and high salt stress treatment. One-week-old aseptic seedlings of Arabidopsis were transferred to one-half strength MS agar medium with 200 mM D-mannitol and 150 mM NaCl for 7 days to observe their phenotypes, while 4-week-old seedlings in soil were treated with drought stress without watering for 12 days.

Scaf 151.204 (HpSNAT1) and Scaf 64.495 (HpSNAT2) were cloned from H. perforatum transcriptome cDNA (Zhou et al., 2021) using gene-specific primers (Supplementary Table 1). The full-length cDNAs mentioned above were inserted into PBI221-GFP to produce the 35S::SNAT-GFP constructs as subcellular localization vectors. The HpSNAT genes were cloned with the Gateway™ Recombination Cloning Technology (Invitrogen, Waltham, MA, United States) into the pEarleyGate202 vector to generate final overexpression (OE) vectors pEG202-HpSNAT1 and pEG202-HpSNAT2.

For overexpressing HpSNAT in Arabidopsis, the pEG202-HpSNAT constructs were introduced into Agrobacterium tumefaciens strain GV3101 (self-made) and transformed into Arabidopsis snat plants by the floral dip method (Zhang et al., 2006). According to the resistance of the expression vector to BASTA, the positive transgenic plants were screened out until T3 generation (Supplementary Figure 2).

For overexpressing HpSNAT in H. perforatum, the recombinant constructs were transferred into Agrobacterium rhizogenes strain K599 (Weidi, Shanghai, China). The specific steps follow the product instructions. A culture of A. rhizogenes with pEG202- HpSNAT1 and pEG202-HpSNAT2 was grown for 1 day at 28°C and 120 rpm in TY liquid media (0.5% tryptone, 0.3% yeast extract, 0.5% meat extract, 10 mM CaCl₂, 1.5% agar with 50 μg/ml kanamycin, and 50 μg/ml streptomycin; pH 7.0). The bacterial suspension was centrifuged at 5,000 rpm for 10 min and resuspended in sterile water, then diluted to an OD₆₀₀ of 0.1.

Genomic DNA and RNA were extracted using the FastPure Plant Total RNA/DNA Isolation Kit (Vazyme, Jiangsu, China). The specific extraction steps are by the product instructions. The CaMV35S promoter was amplified from the genomic DNA to detect whether the gene was integrated into the genome of the transgenic plants. PCR amplification was performed as previously described (Wang et al., 2018). Reverse transcription PCR (RT-PCR) was performed to explore the expression patterns of HpSNAT genes in Arabidopsis snat, Col-0, and T3 transgenic plants. An Arabidopsis Actin gene fragment is used as an internal control. Quantitative real-time PCR (qPCR) was used to investigate the expression patterns of HpSNAT genes in H. perforatum plant, and the HpACT2 gene was used as an internal reference. The specific operation steps can be referred to in the previous detailed description (Zhou et al., 2021) and primer sequences used are shown in Supplementary Table 1. The transient transformation of Arabidopsis protoplasts is operated according to Dr. Sheen’s operation manual⁶ (Sheen, 2001). Briefly, the leaves of Arabidopsis plants aged 3–4 weeks were cut into 1 mm wide bands, completely soaked in 10 ml enzymatic hydrolysate and digested in the dark at 55°C water bath for 10 min. Then, digestion was continued at 25°C for about 5 h and gently shaken in the dark. The samples were washed with solution W5 and filtered through a 300-mesh sieve. The obtained protoplasts were resuspended in MMG solution for microscopic examination. After PEG-mediated transfection, the fluorescence was observed by Nikon C2-ER laser confocal microscope.

Two-month-old Arabidopsis OE, Col-0, and snat plants were collected. According to the extraction and detection methods established in our laboratory, melatonin in Arabidopsis leaves was determined by LC/MS (Zhou et al., 2021). The leaves of OE and WT (the line infected by A. rhizogenes, K599 only) lines of H. perforatum were freeze-dried, and a 200 mg dry sample was extracted by ultrasonic treatment in 500 μl 80% methanol (Sigma, St. Louis, MO, United States) (Zuo et al., 2015). The sample preparation and HPLC detection of melatonin and its precursors were performed as previous described (Zhao et al., 2013).

One-week-old OE, Col-0, and snat aseptic seedlings of Arabidopsis under drought and high salt stress were to observe their phenotypes. One-month-old plants without watering to create drought stress for physiological analysis. The concentration of H₂O₂ and Malondialdehyde (MDA) were measured using the Hydroperoxide/Malondialdehyde Fluorometric Assay Kit (Sigma–Aldrich). Reactive oxygen species (ROS) concentration was examined by histochemical staining with CM-H₂DCFDA fluorescent dye (Solarbio, Tongzhou District, Beijing, China) (Oparka et al., 2016). Fluorescence images were taken with a Leica stereomicroscope (Leica, Germany).

All experiments were performed with three biological replicates unless otherwise specified. The data were the average of three technical repetitions expressed as mean ± standard error. ANOVA analysis was used for statistical analysis; the probability value P < 0.05 was considered statistically significant.

A total of 48 HpSNAT members were screened, all of which contained GNAT conserved domain. The MW of the predicted HpSNAT proteins ranged from 14.92 kDa (HpSNAT26) to 132.67 kDa (HpSNAT21), the PIs ranged from 4.98 (HpSNAT23) to 9.97 (HpSNAT1), and protein lengths ranged from 129 (HpSNAT26) to 1,178 (HpSNAT21) amino acids (Supplementary Table 2). Hierarchical clustering of expression profiles of HpSNAT genes in different tissues (root, stem, leaf, and flower) were shown in Supplementary Figure 3. Among them, two full-length HpSNAT showed homology to the four bait proteins, and they were named HpSNAT1 (Scaf 151.204) and HpSNAT2 (Scaf 64.495) according to the phylogenetic analysis (Figure 1A). As shown in Figure 1B, two HpSNATs and four bait proteins have the same motif, showing that the functional proteins have similarities. The qPCR results were consistent with the expression pattern of HpSNAT1 and HpSNAT2 in different tissue transcriptome sequencing results, indicating that the two genes were highly expressed in leaves (Figure 1C). Through the analysis of cis-acting elements in the promoter region of the two genes, it was found that both two genes contain gibberellic acid (GA), methyl jasmonate (MeJA), salicylic acid (SA), auxin (IAA), abscisic acid (ABA), and stress response elements of low temperature, drought, and injury. In addition, HpSNAT1 also contains flavonoids biosynthesis and elements related to plant circadian rhythm regulation, while HpSNAT2 also contains ethylene response and elements related to gene expression regulation in plant meristem.

To determine the subcellular location of HpSNAT1 and HpSNAT2 in vivo, HpSNAT-GFP fusion proteins expression vector driven by the CaMV 35S promoter was constructed, and the empty vector PBI221-GFP was used as a positive control. The fused expression vector was used to perform a transient expression assay in Arabidopsis protoplasts. From Figure 2A, the GFP fluorescence of positive control was almost distributed in the whole cell. In contrast, the GFP fluorescence of HpSNAT1/2 was observed only in the chloroplasts in cytoplasm, which was consistent with the previous subcellular localization of SNAT proteins in other plants (Byeon et al., 2013a; Lee et al., 2015a; Yeong et al., 2016).

Melatonin has strong free radical scavenging and antioxidant ability to prevent cell oxidative damage. To determine the effects of drought and high salt stress on the expression of HpSNAT1 and HpSNAT2, 3-month-old aseptic seedlings of H. perforatum were treated with 250 mM D-Mannitol and 200 mM NaCl for up to 32 h. The expression trend of the HpSNAT1 and HpSNAT2 in H. perforatum at five-time intervals (0, 4, 8, 16, and 32 h) was quantified by qPCR (Figure 2B). Under drought stress, the expression level of HpSNAT1 reached the highest at 8 h, increased by about 2.5 times, while HpSNAT2 had no obvious response to D-mannitol treatment. Similarly, the expression level of HpSNAT1 increased nearly three-fold at 8 h under high salt stress, while HpSNAT2 showed a slight response at 32 h.

By overexpression of HpSNAT1 and HpSNAT2 in the Arabidopsis mutant snat, the OE lines contained the expected 926 bp fragments of the CaMV35S promoter were confirmed by PCR. Five T3 homozygous transgenic lines of OE-HpSNAT1 and OE-HpSNAT2 were obtained for gene expression (Figure 3A) and melatonin detection (Figure 3B). Finally, three OE lines with high HpSNAT1 (OE1, OE3, and OE5) and HpSNAT2 (OE2, OE4, and OE5) gene expression and melatonin content were used for further analysis. The content of melatonin in OE1- HpSNAT1 was 1.86 times higher than that in Col-0 plants, and that in OE5-HpSNAT2 was 1.67 times higher. By analyzing the rosette leaves of different lines (snat, Col-0, OE1- HpSNAT1, and OE5-HpSNAT2) growing to 25 days, we found that the OE lines had more leaves than snat and Col-0. The dry and fresh weight of OE leaves was almost three times that of the snat and twice that of Col-0 (Figure 3C).

In order to explore whether HpSNAT genes can enhance the drought and salt resistance of plants, Arabidopsis root morphology of different lines was observed in one-half-strength MS medium with NaCl and D-mannitol, respectively. Results as shown in Figure 4A, after 10 days of treatment, there was no significant change in the length of the primary root of snat, Col-0, and OE, but the number of lateral roots of OE lines was significantly more than that of Col-0 and snat plants under drought and salt stress. Similarly, the growth of 4-week-old seedlings in soil was better than that of Col-0 and snat after without watering for 12 days (Figure 4B), and the MDA and H₂O₂ contents in OE lines were significantly lower than in Col-0 and snat plants (Figure 4C). From the CM-H₂DCFDA staining, it can be seen that the fluorescence intensity in OE leaves (Figure 4D) is lower than that in snat under drought treatment. The relative staining fluorescence intensities in snat, Col-0, OE-HpSNAT1, and OE-HpSNAT2 leaves were 91.455, 71.502, 63.009, and 60.531, respectively. These results showed that the content of ROS in transgenic leaves was lower than that in Col-0 and snat. In other words, the content of ROS in plants can be greatly reduced by overexpression of HpSNAT genes, so as to improve the drought tolerance of plants.

As shown in Figure 5A, the first candidate transgenic root tips emerged from the wounded areas of the explants about the 2nd week on the selective medium with antibiotics. When growing to the 6th week, 100 mg roots were put into a liquid MS medium containing phytohormones for expanded culture to obtain enough samples for molecular detection. After 10 weeks of growth, the positive lines carrying the HpSNAT gene were verified by PCR. In total, we obtained five positive transgenic HR lines for both constructs that were confirmed in HpSNAT gene expression. The expression levels of the two genes in the OE lines were significantly higher than those in the WT group, especially the OE2, OE4, and OE5 of HpSNAT1 and OE1, OE2, and OE4 of HpSNAT2 detected by qPCR (Figure 5B). Among them, the expression of HpSNAT2 in the OE lines increased by about 1500 times. The above highly expressed HR lines were selected for callus, adventitious bud, and root induction to obtain a complete transgenic plant of H. perforatum.

As shown in Figure 5C, when explants were grown on MS medium containing thidiazuron for 2 months, a large amount of brown callus will be formed with a little green part in it. Then the green callus was separated and transferred to MS medium containing 6-Benzylaminopurine and naphthylacetic acid to induce adventitious buds. About 1 month later, the shoots were cut off and inserted into one-half strength MS medium for rooting induction to obtain complete plants. It began to take root slowly in about 2 weeks, and lots of HRs and branches could be found outside the medium after 1 month. qPCR analysis of HpSNAT expression showed that HpSNAT1 expressed over 20-fold higher than the WT transformed lines, and the expression of HpSNAT2 was 100- to 400-fold higher in adult transgenic H. perforatum lines (Figure 5D). We further evaluate the content of melatonin and its precursors. Three OE and WT lines were used to examine four compounds, such as 5-HT, NAS, 5-MT, and melatonin in adult transgenic H. perforatum. The results showed that the contents of four compounds were significantly up-regulated, and the melatonin increased to 12.28–21.15 μg/g DW in OE lines (Figure 6A). Among them, OE-HpSNAT2-1 accumulated the highest content of melatonin, which was 4.21-folds higher than WT. These results confirmed the positive role of HpSNAT1 and HpSNAT2 in melatonin biosynthesis.