The model plant A. thaliana (ecotype Col-0) was used for ectopic gene expression. Seeds were surface sterilized in 1 mL of 70% (v/v) ethanol for 15 min and washed three times with autoclaved water. A. thaliana seeds were placed on agar-solidified Murashige and Skoog (MS) medium [46], and kept at 4 °C in the dark for 3 d. Plates were then transferred to a climate-controlled growth chamber where plants were grown for 4 or 5 d at 22 °C in a 16-h photoperiod. WT and transgenic A. thaliana seedlings were transferred to pots with nutrient soil for 2 weeks. Pots were placed in a growth chamber at 22 °C and grown in light (100 $\mu$mol m${}^{-2}$ s${}^{-1}$; 16-h photoperiod). Pitaya seedlings were cultured in a growth chamber at 25 °C in a 16-h photoperiod, and at 70–80% relative humidity (light intensity: 100 $\mu$mol m${}^{-2}$ s${}^{-1}$). Adult pitaya plants were grown in a greenhouse of South China Botanical Garden (Guangzhou, Guangdong, China) under ambient conditions.

DNAMAN 7.0 software was used to align the sequences of HuBADH and BADH proteins from other plants (Lynnon Biosoft Corp., San Ramon, CA, USA) [47]. Protein sequences of proteins homologous to H. undatus BADH (MK160492), Amaranthus hypochondriacus BADH (AAB70010), Sesuvium portulacastrum BADH (AEK98521), Spinacia oleracea BADH (XP_021837164), Tamarix hispida BADH (AIL24123), and A. thaliana BADH (AAG51938), were retrieved from GenBank (https://www.ncbi.nlm.nih.gov/). A phylogenetic tree was built by MEGA v7.0.14 software (https://www.megasoftware.net/) [48] using the neighbor-joining (NJ) method [49]. Bootstrap values were assessed as 1000 replicates. The conserved domain of the HuBADH protein was analyzed according to the conserved domains within the protein coding nucleotide sequence (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The sequences for protein HuBADH was submitted to ExPASy (https://web.expasy.org/protparam/) to determine MWs and theoretical pIs. All BADH proteins similarity were further identified by BlastP in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

To obtain a plasmid construct of the HuBADH gene, its full-length ORF was amplified by Real-time reverse transcription PCR (RT-PCR) from pitaya seedlings using specific primers (Supplementary Table 1). The polymerase chain reaction (PCR) product of the HuBADH gene was inserted into the pCAMBIA1302 vector to obtain 35Spro::HuBADH recombinant plasmids and sequence. The constructed vector was mobilized to Agrobacterium tumefaciens EHA105 by a 42 °C heat shock in a water bath (ZX-S24, Southeast Yicheng Laboratory Equipment Co., Ltd., Beijing, China), then used to transform A. thaliana using the floral dip method [50]. HuBADH-overexpressing (OE) transgenic A. thaliana lines were cultured on MS medium with 50 $\mu$g mL${}^{-1}$ kanamycin (1162GR005 Yibaishun Technology Co., Ltd., Guangdong, China) to select resistant lines [51]. After 5 d, A. thaliana seedlings (T0 generation) with true green leaves were selected as transformants and transplanted to pots containing soil (WT-NTT-5, Witte, Fuller, Germany). Seeds of T1 and T2 generations were screened on MS medium with 50 $\mu$g mL${}^{-1}$ kanamycin (1162GR005). Positive transgenic lines were selected when the resistant : sensitive segregation ratio was 3:1. Positive transgenic (T0, T1, T2) and WT lines were identified using PCR with the following procedure: stage 1 (94 °C for 3 min); stage 2 (30 cycles of 94 °C for 10 s, 55 °C for 30 s, 72 °C for 2 min); stage 3 (72 °C for 5 min). PCR products were detected on 1% agarose under ultraviolet light. DL2000 is used as control (MYDEER, Guangzhou Anbang biotechnology Co. Ltd. Guangzhou, China) PCR primers are listed in Supplementary Table 1.

To detect the expression level of HuBADH, total RNA was isolated from the leaves of A. thaliana plants, and from the roots, stems, petals, calyces, and squamas of adult pitaya plants with the Eastep® Super Total RNA Extraction Kit (Promega, Beijing, China). RNase-free DNase I (Promega) was used to degrade residual genomic DNA, and 1 $\mu$g of DNA-free RNA product was syntheszed from first-strand cDNA by the GoScript${}^{\text{TM}}$ Reverse Transcription Mix (Promega). Products were diluted five-fold for quantitative real-time reverse transcription PCR (qRT-PCR). The Eastep qPCR Master Mix Kit (Promega) was used to perform qRT-PCR. qRT-PCR was conducted with Eastep qPCR Master Mix Kit (Promega) on a Roche Light Cycler 480 Real-time PCR System (Roche, Basel, Switzerland). Thermal cycling was 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. Melting curve analysis was achieved with the following program after 40 PCR cycles: 95 °C for 15 s, 72 °C for 5 min. A. thaliana Actin2 and pitaya HuEF-I$\alpha$were used as internal controls. Dissociation kinetic curves were established at the end of each qRT-PCR run. All reactions were executed in triplicate for three biological replicates. Relative gene expression was quantified using the 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method [52].

Total RNA from the leaves of 14-d-old WT and three OE transgenic lines were extracted and purified as described above. Semi-qRT-PCR was performed with the following protocol: stage 1 (95 °C for 5 min); stage 2 (35 cycles of 94 °C for 10 s, 57 °C for 30 s, 72 °C for 2 min); stage 3 (72 °C for 5 min). AtUBQ10 (At4g05320) from A. thaliana was used as the internal control. PCR products were detected on a 1% agrose gel under ultraviolet light. The primers designed for semi-qRT-PCR are listed in Supplementary Table 1.

Pitaya seedlings (14-d-old) were treated with 300 mM NaCl (S805277-500 g; Macklin, Beijing Ruizhi Hanxing, Beijing, China) for different periods of time (0, 3, 6, 9, 12, and 24 h). HuBADH expression levels were then analyzed. To assess the percentage of seed germination, more than 200 seeds of homozygous transgenic A. thaliana HuBADH-overexpressing (OE) lines and WT were sown onto MS medium containining 0 or 120 mM NaCl for 3 d. Seeds were also sown onto MS medium with 300 mM NaCl, but they did not germinate (data not shown), so this treatment was not used in further assays. Germination percentage was assessed after growth in the presence of 120 mM NaCl for 3 d. For the in vitro assay, 5-d-old transgenic A. thaliana seedlings were transplanted to MS medium containining 0 or 120 mM NaCl for 15 d. Root length and fresh weight was investigated after culture for 15 d. In the salt stress assay, 5-d-old transgenic and WT A. thaliana seedlings were transplanted to pots containing sterilized soil (WT-NTT-5) for 14 d under normal conditions (no salt stress). Seedlings were irrigated with 300 mM NaCl every 3 d. Treatments lasted for a total of 21 d to maintain long-term salt stress. Survival percentage was investigated after 21 d.

To assess biochemical traits under salt stress, 5-d-old A. thaliana seedlings, including the WT and transgenic lines, were transplanted to pots with nutrient soil for 14 d under normal conditions (no salt stress). Both WT and transgenic lines were watered with 0 or 300 mM NaCl, respectively for 24 h. The spectrophotometer used for all biochemical measurements (AS11D-H, Asone, Merrill Biochemical Technology Co., Ltd. Shanghai, China).Twenty leaves of 10 A. thaliana lines were sampled for both 0 or 300 mM NaCl treatments. CAT and SOD assay kits (CAT: BC0205; SOD: BC0175. Solarbio Science and Technology Co. Ltd., Beijing, China) were used to measure of CAT (E.C. 1.11.1.6) and SOD (E.C. 1.15.1.1) activities, respectively. Each assay used 0.1 g of fresh leaves per 1 mL of extract and activity was determined by following the manufacturer’s intructions. CAT activity was determined at 240 nm [53] while SOD activity was determined at 560 nm [54].

Proline content was measured according to the Zhang et al. [55] method. Briefly, fresh leaves (0.1 g) were homogenized in 1 mL of extraction buffer of a Solarbio Science and Technology Co. Ltd. kit (BC0295), and proline content was determined at 520 nm by following the manufacturer’s intructions.

Malondialdehyde (MDA) content were measured using the Liu et al. [56] method. Briefly, fresh leaves (0.1 g) were homogenized in 1 mL of extraction buffer of a Solarbio Science and Technology Co. Ltd. kit (BC0025) and MDA content was determined at 532 and 600 nm by following the manufacturer’s intructions.

Hydrogen peroxide (H${}_2$O${}_2$) content was measured using the Tiryaki et al. [57] method. Briefly, fresh leaves (0.1 g) were homogenized in 1 mL of extraction buffer of a Solarbio Science and Technology Co. Ltd. kit (BC3590), and H${}_2$O${}_2$ content was determined at 415 nm by following the manufacturer’s intructions.

Superoxide radical (O${}_2$${}^{-}$) content was measured using the Cai et al. method [58]. Briefly, fresh leaves (0.1 g) were homogenized in 1 mL of extraction buffer of a Solarbio Science and Technology Co. Ltd. kit (BC1295), and Superoxide radical (O${}_2$${}^{-}$) content was determined at 530 nm by following the manufacturer’s intructions.

GB content was measured by the Wang et al. method [59]. Briefly, fresh leaves (0.1 g) were homogenized in 1 mL of extraction buffer of a Solarbio Science and Technology Co. Ltd. kit (BC3135), and GB content was determined at 525 nm by following the manufacturer’s intructions.

To detect O${}_2$${}^{-}$$$and H${}_2$O${}_2$ in situ, 30 fresh terminal leaves for each of 35-d-old WT and transgenic A. thaliana OE lines growing in control and salt stress (300 mM NaCl) treatments were soaked in a solution of 1 mg/mL 3,3’-diaminnobenzidine (DAB) (1024, Nanjing Jiancheng, China) for 10 h then washed in 95% ethanol, as suggested by a previous protocol [60].

All data were plotted in Sigmaplot 12.5 (Systat Software Inc., San Jose, CA, USA) [61]. Data were analyzed by Duncan’s multiple range test (p 0.05) in SPSS version 20.0 software (IBM Corp., Armonk, NY, USA).

Among several BADH homologs that were identified in a previous pitaya transcriptomic analysis, HuBADH was shown to be upregulated in a salt stress treatment [45]. The BADH open reading frame (ORF) was 1512 bp long, it encoded 503 amino acids (aa), had a molecular weight of 54.792 KDa, and a theoretical pI of 5.75. BlastP results indicated that HuBADH had 89.2% similarity with BADH of Sesuvium portulacastrum, 92.0% similarity with BADH of Amaranthus hypochondriacus, 88.3% similarity with BADH of Spinacia oleracea, 88.3% similarity with BADH of Tamarix hispida, and 79.2% similarity with BADH of A. thaliana (Fig. 1).

Fig. 1.

Multiple sequence alignment analysis of the BADH protein amino acids. Multiple alignment of betaine aldehyde dehydrogenase (BADH) amino acid sequences performed in DNAMAN 7.0. Protein sequences of BADH homologues (in order of listing) from Hylocereus undatus (MK160492), Amaranthus hypochondriacus (AAB70010), Tamarix hispida (AI L24123), Sesuvium portulacastrum (AEK98521), Spinacia oleracea (XP_021837164), and Arabidopsis thaliana (AAG51938).

Using MEGA7.0 software, a NJ-based phylogenetic tree of these BADH sequences was constructed to evaluate the phylogenetic relationships among Hylocereus undatus (MK160492), Amaranthus hypochondriacus (AAB70010), Tamarix hispida (AIL24123), Sesuvium portulacastrum (AEK98521), Spinacia oleracea (XP_021837164), and A. thaliana (AAG51938) (Fig. 2). The results indicate that HuBADH is likely a member of the BADH family.

Fig. 2.

Phylogenetic tree analysis comparing HuBADH and BADH proteins from other plants. The phylogenetic tree of BADH subfamily proteins was developed in MEGA7.0 software. These BADH proteins include Hylocereus undatus (MK160492), Amaranthus hypochondriacus (AAB70010), Sesuvium portulacastrum (AEK98521), Spinacia oleracea (XP_021837164), Arabidopsis thaliana (AAG51938), and Tamarix hispida (AI L24123). GenBank accession numbers of these proteins are indicated in parentheses.

Since BADH catalyzes the last biosynthetic step, i.e., the transfer of betaine aldehyde into betaine, we evaluated the HuBADH protein in pitaya. The BADH protein sequences from other plants were aligned. The HuBADH protein contains a fairly conserved domain F-G-C-F-W-T-N-G-Q-I-C-S-A-T-S-R-L-L-V-H-E (Fig. 1), which depicts dehydrogenation, and several residues are denoted as being related to catalytic and NAD${}^{+}$-binding sites.

To appreciate the expression levels of the HuBADH gene in response to 300 mM NaCl, qRT-PCR was performed using total RNA from 14-d-old pitaya seedlings as the template. In the 300 mM NaCl treatment, the level of HuBADH transcript was initially upregulated, peaking at 9 h, and declining thereafter (Fig. 3A). This result indicates that HuBADH responds to salt stress. HuBADH gene expression levels, which were determined in different tissues of pitaya at the flowering stage, were highest in petals and the calyx, and lowest in the squama (Fig. 3B).

Fig. 3.

Expression analysis of HuBADH of 14-d-old pitaya seedlings treated with 300 mM NaCl. (A) HuBADH expression levels at 0, 3, 6, 9, 12, and 24 h after salt treatment. The value at 0 h served as the control. (B) HuBADH expression levels in different tissues. HuEF1-$\alpha$ was used as the internal standard. Different lower-case letters above error bars indicate significant differences at p 0.05 (ANOVA followed by Duncan’s multiple range test). Data are shown as the means (20 seedlings for each experiment) and SD of three biological replicates.

The overexpression of HuBADH in A. thaliana was achieved by transforming this gene into A. thaliana lines using an Agrobacterium-based floral dip method [50], driven by the CaMV-35S promoter. Five positively transgenic A. thaliana lines were selected on MS medium with 50 $\mu$g/mL kanamycin (Supplementary Fig. 1) using PCR and a kanamycin-resistance assay [51]. Homozygous lines (T2-T3) were examined. The expression levels of three HuBADH OE transgenic A. thaliana lines with a higher expression level were identified by semi-qRT-PCR (Fig. 4A). There were no differences in the phenotype (size, flowering time, color, leaves number) between WT and the three OE transgenic lines (Fig. 4B). A. thaliana OE lines showed relatively higher expression levels of HuBADH than WT A. thaliana in normal (non-salt stressed) conditions (Fig. 4C). These results indicate that HuBADH was successfully transformed into the A. thaliana genome and expressed normally.

Fig. 4.

Expression levels of three HuBADH-overexpressing (OE) transgenic A. thaliana lines identified by semi-qRT-PCR. (A) Semi-qRT-PCR analysis of the HuBADH gene in wild-type (WT) and three 35S::HuBADH transgenic (OE) A. thaliana lines. AtUBQ10 served as the internal control. (B) 15-d-old A. thaliana seedlings of WT and HuBADH OE lines. (C) qRT-PCR analysis of HuBADH in three transgenic A. thaliana OE lines and WT. In (C), different lower-case letters above error bars indicate significant differences at p 0.05 (ANOVA followed by Duncan’s multiple range test). Data shown as the means (20 seedlings for each experiment) and SD of three biological replicates. Bar in (B) = 2 cm.

To appreciate the function of HuBADH in seed germination under salt stress, all seeds of transgenic A. thaliana OE and WT plants were sown on MS medium supplemented with 0 or 120 mM NaCl for 3 d. Under salt stress, WT plants showed 74.5 $\pm$ 1.29% seed germination whereas transgenic A. thaliana OE lines OE1, OE2 and OE3 displayed 89.25 $\pm$ 2.21%, 90.25 $\pm$ 1.26% and 90.75 $\pm$ 2.22% seed germination, respectively. Seed germination of transgenic A. thaliana was at least 16% higher than that of WT seeds exposed to 120 mM NaCl, although no significant differences were found between WT and OE lines in the control (0 mM NaCl) treatment after 3 d (Fig. 5A,D).

Fig. 5.

Overexpressing HuBADH in transgenic A. thaliana improved tolerance to salt stress and enhanced seed germination. (A) Seed germination of HuBADH-overexpression (OE) transgenic A. thaliana lines on MS medium with 0 (above) and 120 mM NaCl (below) for 3 d, when germination rates were measured (D). (B) Four-d-old transgenic A. thaliana and WT seedlings were grown on MS medium with 0 (above) and 120 mM NaCl (below) for 15 d. Root length (E) and fresh weight (F) were measured. (C) Five-d-old HuBADH OE transgenic A. thaliana and WT seedlings were transplanted in plastic pots for 14 d, then treated with 300 mM NaCl for 21 d, when survival was measured (G). (D–G) Different lower-case letters above error bars indicate significant differences at p 0.05 (ANOVA followed by Duncan’s multiple range test). Mean values ($>$200 seeds per line for each experiment) and SD of three biological replicates are shown. Bars (A–C) = 1 cm.

The root length and fresh weight of transgenic A. thaliana OE and WT seedlings were assessed after they were transplanted for 15 d on MS medium supplemented with 0 and 120 mM NaCl. Transgenic A. thaliana seedlings displayed significantly longer roots and higher fresh weight than WT seedlings under salt stress but no differences were observed between WT and transgenic OE lines in the control (no salt stress) (Fig. 5B,E,F). These results indicate that HuBADH OE transgenic A. thaliana lines experienced an obvious increase in salt tolerance during seed germination and at the seedling stage.

To further assess whether transgenic A. thaliana OE lines could enhance salt stress tolerance at a later stage, in the flowering period, 5-d-old WT and transgenic A. thaliana seedlings were cultivated in salt-free conditions for 14 d. Thereafter, WT and OE lines were watered with a 300 mM NaCl solution for 21 d. Transgenic A. thaliana OE lines showed significantly higher tolerance to salt stress than WT (Fig. 5C,G). While survival of transgenic A. thaliana OE1, OE2 and OE3 lines was 100%, that of WT plants was only 66.67 $\pm$ 9.07%. Under normal (non-salt stressed) growth conditions, in contrast, there were no significant differences in survival percentage between transgenic A. thaliana OE lines and WT plants (Fig. 5C,G). These results indicate that transgenic A. thaliana OE lines showed greater salt tolerance than WT plants under salt stress, even during the flowering period.

To study the function of HuBADH in oxidative stress, we assayed the level of O${}_2$${}^{-}$ and H${}_2$O${}_2$ accumulation in the leaves of HuBADH OE transgenic A. thaliana plants (Fig. 6A,B). Transgenic A. thaliana OE1, OE2, and OE3 lines accumulated less O${}_2$${}^{-}$ and H${}_2$O${}_2$ than WT in the 300 mM NaCl treatment (Fig. 6B) whereas there were no significant differences in the levels of O${}_2$${}^{-}$ and H${}_2$O${}_2$ between WT and OE plants in the control (0 mM NaCl) treatment (Fig. 6A).

Fig. 6.

Detection of ROS accumulation in the leaves of HuBADH-overexpressing A. thaliana and wild-type (WT) seedlings under salt stress. (A) ROS detected by DAB staining in 35-d-old WT and transgenic seedlings under control conditions. (B) ROS detected under salt stress. For the salt stress treatment, plants grown in pots were irrigated with 300 mM NaCl for 24 h. Bars = 0.5 cm.

To further verify the possible physiological mechanisms mediated by HuBADH, several physiological indices, such as the content of H${}_2$O${}_2$, proline, O${}_2$${}^{-}$, the activities of antioxidant enzymes, and the content of MDA were measured in WT and transgenic A. thaliana plants in the 0 and 300 mM NaCl treatments (Fig. 7A–F). Compared to WT, H${}_2$O${}_2$, O${}_2$${}^{-}$ and MDA contents were significantly less in transgenic A. thaliana plants exposed to salt stress, but not significant in the control (0 mM NaCl) (Fig. 7A,C,D). Proline content and activities of antioxidant enzymes (SOD; CAT) were significantly higher in transgenic A. thaliana plants than in WT in the 300 mM NaCl treatment (Fig. 7E,F), but there were no differences in proline content or in the activities of both antioxidant enzymes between WT and OE1, OE2, and OE3 lines in the no-stress (0 mM NaCl) treatment (Fig. 7). This indicates that the overexpression of HuBADH in transgenic A. thaliana reduced the accumulation of ROS by decreasing MDA, H${}_2$O${}_2$ and O${}_2$${}^{-}$ contents and by increasing the activities of two antioxidant enzymes and proline content in transgenic A. thaliana lines under salt stress, suggesting that the transgenic A. thaliana OE lines are salt tolerant, unlike WT plants (Fig. 7B).

Fig. 7.

Activities of two antioxidant enzymes and different physiological parameters in the salt stress treatment (300 mM NaCl) in 35-d-old transgenic A. thaliana (HuBADH-overexpressing; OE) and wild-type (WT) plants. (A) MDA content. (B) Proline content. (C) O${}_2$${}^{-}$ content. (D) H${}_2$O${}_2$ content. (E) CAT activity. (F) SOD activity after treatment for 24 h. Different lower-case letters above error bars indicate significant differences at p 0.05 (ANOVA followed by Duncan’s multiple range test). Mean values (10 seedlings for each experiment) and SD of three biological replicates are shown.

To further clarify the possible functional molecular mechanisms of the HuBADH gene under salt stress, four oxidation-related stress-responsive marker genes (FSD1, CSD1, CAT1, and APX2) were analyzed by qRT-PCR in WT and transgenic A. thaliana lines exposed to 300 mM NaCl. All four marker genes were significantly upregulated in WT and transgenic A. thaliana plants under salt stress (Fig. 8A–D). This result indicates that HuBADH induced the expression of oxidative stress-responsive genes, leading to improved salt stress tolerance of transgenic plants.

Fig. 8.

Oxidation-related genes were analyzed by qRT-PCR in 15-d-old transgenic A. thaliana (HuBADH-overexpressing; OE) and wild-type (WT) plants under salt stress (300 mM NaCl) for 24 h. (A) FSD1. (B) CSD1. (C) CAT1. (D) APX2. Different lower-case letters above error bars indicate significant differences at p 0.05 (ANOVA followed by Duncan’s multiple range test). Mean values (10 seedlings for each experiment) and SD of three biological replicates are shown.

GB content in WT and transgenic lines was determined in control (0 mM NaCl) or salt stress (300 mM NaCl) treatments. In the control, transgenic lines OE1, OE2 and OE3 showed a 32.20, 34.96 and 36% increase, respectively in GB content relative to WT. After 24 h of exposure to 300 mM NaCl, GB content in OE1, OE2 and OE3 increased 70.00, 73.87 and 80.97% compared to WT, respectively (Fig. 9).

Fig. 9.

Glycine betaine content in HuBADH-overexpressing transgenic Arabidopsis thaliana. Five-d-old HuBADH OE transgenic A. thaliana and WT seedlings were transplanted in plastic pots for 14 d, then treated with 300 mM NaCl for 24 h. Glycine betaine content was measured in control (0 mM NaCl) and salt-stressed (300 mM NaCl) conditions. Different lower-case letters above error bars indicate significant differences at p 0.05 (ANOVA followed by Duncan’s multiple range test). Mean values (10 seedlings for each experiment) and SD of three biological replicates are shown.