A Cloned Gene HuBADH from Hylocereus undatus Enhanced Salt Stress Tolerance in Transgenic Arabidopsis thaliana Plants

Background : Betaine aldehyde dehydrogenase (BADH) catalyzes the synthesis of glycine betaine and is considered to be a type of osmoregulator, so it can play a role in plants’ responses to abiotic stresses. Methods : In this study, a novel HuBADH gene from Hylocereus undatus (pitaya) was cloned, identified


Introduction
Abiotic stresses, such as salinity, extremely low or high temperatures, and drought, influence plant growth and development, so they are a major challenge for sustainable agricultural development because they can reduce crop yield [1,2].Soil-based salinity has become a worldwide problem because of poor irrigation systems, salt infiltration, water pollution, reduced rainfall, and other environmental factors, so the areas affected by saline stress are likely to increase [3][4][5].Salinization has affected an estimated 400 million ha of land around the world, or about 3% of the globe's arable land [6].Salt stress interferes with osmotic balance and ion homeostasis in plants, decreasing photosynthetic activity, inducing metabolic dysfunction, and finally resulting in decreased crop production, so many agricultural lands and crops suffer from the secondary effects of salinization [7].Plants employ a range of mechanisms to respond to salt stress, including minimizing the amount of salt absorbed via roots and the partitioning of salt at cellular and tissue levels to prevent its accumulation in the cytosol of physiologically functional leaves [8].The accumulation of compatible solutes is one such important mecha-nism.Comptible solutes, including amino acids, sugar alcohols, quaternary ammonium compounds, and tertiary sulphonium compounds vary depending on the plant species [9,10].Glycine betaine (GB) is an important compatible solute for achieving salinity tolerance in many plants [11].In plants, GB is abundant in the Graminaceae, Asteraceae, Malvaceae, and Amaranthaceae [9].GB can induce plant stress tolerance by increasing the levels of expression of stress resistance genes, stress signal transduction, enhancing the activities of antioxidant enzymes, protecting cell osmotic pressure, maintaining cell membrane integrity, as well as protecting the photosystem II (PSII) complex [12].GB achieves this by maintaining a high Na + :K + ratio by regulating osmotic balance and reducing the toxic effects of ions on the cell's structures [13].GB can decrease adverse effects of drought and salinity stresses and increase photosynthetic efficiency under stress [14].The exogenous application of GB increased the activity of antioxidant enzymes in Axonopus compressus [14].
Betaine aldehyde dehydrogenase (BADH) is a key enzyme related to the biosynthetic pathway of GB because it oversees the second step in the GB biosynthetic pathway, and the introduction of its gene through transgenetics has fortified the tolerance of various plant species to abiotic stresses [12].BADH is coded by multifunctional genes that can enhance stress tolerance an improve the productivity and longevity of plants under stress by protecting their photosynthetic apparatus [12].As one example, LrAMADH1 in Lycium ruthenicum increased GB content under salt stress [15].
Salt stress also induces the accumulation of reactive oxygen species (ROS), a high level of which may cause molecular damage, including to proteins, DNA and lipids, or ultimately result in cell death [16][17][18].ROS can also increase the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), thereby decreasing oxidative stress [19].By revealing the molecular mechanisms underlying salinity tolerance in plants, it may be possible to improve the tolerance of crops to salinity [20].
Hylocereus undatus (pitaya) belongs to the Cactaceae family [31,32].Three pitaya varieties with red-skinned fruit and white flesh, all native to Central and South America [33], have been industrialized [34].These are now widely cultivated in tropical and subtropical areas of the world, particularly in Asian countries including Vietnam, the Philippines, Malaysia, Thailand and China [35].At present, the main regions of pitaya cultivation in China include Guangxi, Hainan, Yunnan, Guangdong, Fujian and Taiwan [36].Pitaya can tolerate different abiotic stresses, including cold, heat, drought, nutritionally poor soil [37,38], and salt [39,40].Pitaya is thus an outstanding plant species to mine genes related to drought and salt tolerance.Some research on pitaya has focused on the biosynthesis of betaine and the formation of pigments [41,42], as well as on antioxidant and radical-scavenging capacity [43].A transcriptomic analysis identified several key genes in the betaine biosynthetic pathway [44].
Pitaya plants typically display high salt tolerance [45].To investigate the function of the HuBADH gene in salt stress, we cloned the HuBADH gene from pitaya and transformed it into A. thaliana for the first time.We found that transgenic A. thaliana plants harboring the overexpressed HuBADH gene showed significantly higher salt tolerance than wild type (WT) plants.Our results will be useful for investigating the detailed function of the HuBADH gene in salt tolerance and for exploring a new method to develop abiotic stress-resistant pitaya.

Plant Material and Culture Conditions
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 µmol 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 µmol m −2 s −1 ).Adult pitaya plants were grown in a greenhouse of South China Botanical Garden (Guangzhou, Guangdong, China) under ambient conditions.

Vector Construction and Genetic Transformation
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].HuBADHoverexpressing (OE) transgenic A. thaliana lines were cultured on MS medium with 50 µg 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 µg 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.

RNA Extraction and qRT-PCR
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 µg of DNA-free RNA product was syntheszed from firststrand cDNA by the GoScript TM  Relative gene expression was quantified using the 2 −∆∆Ct method [52].

Semi-qRT-PCR
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.

Salt Stress Treatment of Pitaya Seedlings and Transgenic A. thaliana Lines
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 HuBADHoverexpressing (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.

Assessment of CAT and SOD Content
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 man-ufacturer's intructions.CAT activity was determined at 240 nm [53] while SOD activity was determined at 560 nm [54].

Assessment of Proline and MDA Content
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.

Assessment of GB Content
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.

3,3'-Diaminnobenzidine (DAB) Staining
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].

Bioinformatics Analysis of the HuBADH Protein
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 1), which depicts dehydrogenation, and several residues are denoted as being related to catalytic and NAD + -binding sites.

Expression Patterns of the HuBADH Gene under NaCl Stress
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).

Overexpression of HuBADH in Transgenic A. thaliana Enhanced Tolerance to Salt Stress
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 µg/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.
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 ± 1.29% seed germination whereas transgenic A. thaliana OE lines OE1, OE2 and OE3 displayed 89.25 ± 2.21%, 90.25 ± 1.26% and 90.75 ± 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).
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 ± 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.

HuBADH-Overexpression Lines Eliminated Reactive Oxygen Species
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).
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 pro-line 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).
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.
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).

Discussion
A previous study profiled the metabolites of two species of pitaya (H.undatus and H. polyrhizus) to compare their antioxidant activities and betalain biosynthesis [62].Plants must efficiently adapt their growth and development to stressful conditions.Salt stress, a globally impactful abiotic stress [63], negatively impacts plant growth and development, although some plants have developed regulatory mechanisms permitting them to adapt to adverse environments [64,65].To date, only a few salt stress-related genes have been identified in pitaya, such as HuCAT3 [66], miR396b-GRF [67] and HuERF1 [51].The exact functions of BADH proteins in pitaya remain unkown, but they have been found to play a major function in modulating the response of plants to different abiotic stresses, including drought in Nicotiana tabacum [68], heat in Hylocereus polyrhizus [69], cold in Hordeum vulgare [70] and salt in Leymus chinensis [71].
In our study, a new gene named HuBADH was cloned from pitaya for the first time.Sequence alignment and phylogenetic analysis of HuBADH and related proteins in other plants indicated that HuBADH is highly homologous to AhBADH from Amaranthus hypochondriacus (Figs.1,2).Bioinformatics analysis showed that the BADH protein had 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).Therefore, we deduced that the BADH motif was highly conserved, not only in terms of its amino acid sequence, but also biological and biochemical roles.
The overexpression of stress-related genes endowing plants with salt resistance is a popular method to enhance the salt stress tolerance of crops.The expression of HuBADH was stimulated by salt treatment (Fig. 3A).Therefore, HuBADH plays a role in salt tolerance.HuBADH was strongly expressed in the petals and calyx of pitaya plants, but was weakly expressed in the squama (Fig. 3B).Also in pitaya, HuERF1 was strongly expressed in the roots [51] but miR396-GRF was weakly expressed in the roots, relative to stems [67].In summary, the genes involved in salt stress have different expression patterns in different tissues in pitaya.
Semi-qRT-PCR and qRT-PCR analyses showed that HuBADH expression levels were significantly higher in transgenic A. thaliana OE lines than in WT plants (Fig. 4), suggesting that HuBADH is a candidate gene to improve salt stress tolerance in pitaya.
The level of HuBADH expression increased under salt stress (Fig. 3A).Molecular, morphological and physiological analyses revealed a relationship between HuBADH overexpression and salt stress tolerance (Fig. 5A-G).At the germination stage, transgenic seeds overexpressing HuBADH had significantly higher germination rates on MS medium containing NaCl than WT seeds (Fig. 5A,D).At the seedling stage, the salt stress tolerance of transgenic A. thaliana was enhanced (Fig. 5B,E,F).At the flowering stage, transgenic A. thaliana plants showed enhanced tolerance to salt stress, as demonstrated by significantly higher survival relative to WT plants (Fig. 5C,G).These findings indicate that HuBADH plays a modulatory role in pitaya salt stress resistance and/or tolerance.
When ROS overaccumulates, there is interference with cellular homeostasis, and this induces oxidative stress in mitochondria, leading to their dysfunction [71].ROS can be reduced in the ROS-scavenging pathway [72].H 2 O 2 is one form of ROS, so improving the expression of genes related to the ROS-scavenging pathway is one way to prevent the accumulation of H 2 O 2 [73].In our study, the accumulation of ROS was significantly lower in transgenic A. thaliana lines harboring the HuBADH gene than in WT plants exposed to salt stress (Fig. 6).In addition, four genes involved in the ROS-scavenging pathway were upregulated in transgenic A. thaliana plants after exposure to salt stress (Fig. 8).This result indicates that HuBADH enhanced salt stress tolerance by regulating the expression of at least these four stress-responsive marker genes.
HuBADH-OE transgenic plants had higher tolerance to salt stress than WT plants under saline conditions.The levels of GB were higher in transgenic lines than WT lines in plants growing in control or salt-stressed conditions (Fig. 9).The level of NaCl in plants could be determined in future research.

Conclusions
BADH is a central enzyme in the biosynthetic pathway of GB.Overexpression of the BADH has been shown to increase tolerance to different abiotic stresses in a range of plants.The HuBADH gene from pitaya was cloned.Its fulllength cDNA had an ORF of 1512 bp that encodes a 54.17 KDa protein with 503 amino acids.When the HuBADH gene was genetically transformed into A. thaliana, transgenic HuBADH-OE lines displayed tolerance to salt (300 mM NaCl).Transgenic A. thaliana accumulated less ROS than WT plants and showed higher activities of two antioxidant enzymes (SOD, CAT) in the NaCl treatment.Four oxidation-related stress-responsive marker genes (FSD1, CSD1, CAT1, and APX2) were significantly upregulated in WT and transgenic A. thaliana plants under salt stress.The level of GB was 32-36% higher in transgenic A. thaliana lines than in WT in the control, and 70-80% higher in NaCl stress.Our research indicates that HuBADH in pitaya plays a positive role in modulating the negative impact of salt stress, so it may be an ideal candidate to increase salt tolerance in pitaya breeding programs.
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-Iα 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.

2. 9
Assessment of H 2 O 2 and O 2 − Content 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.

Fig. 3 .
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-α 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.

Fig. 4 .
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.

Fig. 5 .
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.

Fig. 6 .
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.

Fig. 7 .
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) O2 − content.(D) H2O2 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.

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 saltstressed (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.