1. Introduction
Polyamines (PAs) are a kind of important physiologically bioactive substances in plants, which can indirectly affect the stress tolerance of plants through metabolism [
1,
2]. Polyamine oxidation is the main catabolic pathway of PAs, and polyamine oxidase (PAO) is responsible for catalyzing the oxidation of higher PAs whose amount of amino groups were more than two, such as Spermidine (Spd) or Spermine (Spm), which play a critical role in plant growth, development and stress responses. Kim et al. [
3] found that AtPAO5 regulates Arabidopsis growth through thermospermine oxidase activity, and
AtPAO5 participates in auxin and cytokinin pathways to regulate xylem differentiation [
4]. PAO is involved in the regulation of growth and development by light [
5], whose levels are induced by light and associated with a light-inhibitory role in hypocotyl growth of maize. PAO can also regulate cell wall stiffening and cell growth rate through H
2O
2 produced by PAO function [
6]. In addition, PAO is related to fruit development and ripening [
7]. In one study, the Ca
2+ channel of
AtPAO3 loss-of-function mutants were unable to open, which inhibited pollen tube growth and reduced the number of seeds [
8].
Not only growth of root but also leaf is associated with PAO. Under salt stress, PAO induces cell elongation and helps maintain leaf growth in maize, thereby alleviating the inhibitory effect of stress on growth [
9]. In Arabidopsis, genes encoding PAOs (
AtPAO2-
4) localized in peroxisome are induced by ABA, mechanical damage and salt stress [
10,
11]. The
PAO expression level in
Salinity tolerance 1 (
St1), a salt-tolerant wheat line, was found to be higher than the wild type, indicating that
PAO has an important function in salt tolerance [
12]. Pakdel et al. [
13] compared two maize genotypes with different drought tolerance and found that
PAO expression level, photosynthesis efficiency and antioxidant enzyme activities of relatively tolerant genotype (Karoon) was higher, suggesting that higher activity of polyamine oxidase and antioxidant enzymes might play a role in improving photosynthesis efficiency of this cultivar. Moreover, abiotic stress influenced plants’ responses to pathogen infection, the enhancement of PAO activity in grape induced by osmotic stress mediated expression of pathogen defense genes, which improved resistance to
Botrytis cinerea [
14]. Moreover, PAO function varies among different periods of growth and development. Gemes et al. [
15] discovered that the effect of ZmPAO on salt tolerance of young tobacco plants was strong, while the effect became relatively moderate at the later stage of development. Overexpression of sweet orange
PAO4 in tobacco increased the seed germination rate but inhibited seedlings growth under salt stress [
16].
Genes are transcribed and translated into functional proteins which play physiological roles. However, this process is not independent, and there is always an interaction between proteins. NADPH oxidase and PAO formed a feedback loop to regulate ROS homeostasis in tobacco, which affected stress tolerance [
17]. Toumi et al. [
18] found that under heat stress, silencing
HSP90 genes resulted in an elevated level of polyamine and acetylated polyamine, leading to enhanced
AtPAOs expression, and proposed that HSP90s and PAO cross-talked to influence polyamine acetylation, oxidation and ROS homeostasis. To date, there have been few reports about the interacting proteins of PAO in plants.
Our previous study isolated and characterized PAO-encoding genes (
CsPAO1-
4) in cucumber [
19], from which we could know that
CsPAO2-
4 made obvious responses to NaCl treatment early in both root and leaf. It was found that there was a clear response to the treatment and the expression of
CsPAO2 was higher than the other member genes. Additionally, the
CsPAO2 expression shows apparent tissue-specificity with a much higher level in roots which is impacted primarily by salt. To further investigate the function of
CsPAOs in responses to salt stress, in this study, as our knowledge,
CsPAO2 roles in salt stress were firstly investigated by heterogeneous expression of
CsPAO2 in Arabidopsis, and then screening of yeast two-hybrid (Y2H) cDNA library and virus-induced gene silencing (VIGS) was used to study the interacting functions of CsPAO2 protein and the molecular mechanism underlying the regulation of salt stress tolerance by them was explored.
3. Discussion
Polyamine oxidases (
PAOs) are stress-responsive genes and play an important role in modulating the tolerance of plants to salt stress [
10,
11,
12], which may vary in different members of the gene family. Therefore, our study started with the functional analysis of
CsPAO2 in salt stress resistance by overexpression in Arabidopsis plants. Similar to our previous result of
CsPAO3 [
19],
CsPAO2 was also found to be a candidate gene for enhancing plants’ tolerance to salt, whether seen from the perspective of seed germination rate (
Figure S3), oxidative damage (
Figure 1B–D and
Figure S5) or plant growth (
Figure 1A and
Figure S4). However, the difference between these two genes is that CsPAO2 functioned in the back conversion of Spd and Spm instead of terminal catabolism catalyzed by CsPAO3, due to which the (Spd+Spm)/Put ratio was reduced inevitably in
CsPAO2 overexpression plants (
Figure 1H). A higher (Spd+Spm)/Put ratio would help plants resist abiotic stress [
22,
23], and thus preventing further decrease is vital for
CsPAO2 overexpression lines after salt stress. In our study, compared to control plants, this ratio in transgenic Arabidopsis plants was not decreased further, even increased in OE12 and OE18, indicating that increased CsPAO2 activity could promote the conversion from Put to Spd and Spm to keep the (Spd+Spm)/Put ratio from further decreases, despite the fact that they were in the opposite direction. On the other hand, due to downregulation of
CsPAO2 expression,
CsPAO2-silencing plants were unable to maintain a high ratio of (Spd+Spm)/Put when exposed to salt stress and the ratio decreased dramatically (
Figure 6), which provided further support to the conclusion described above.
Subsequently, CsPSA3 was discovered as a candidate interacting protein of CsPAO2 through yeast-two hybrid library screening and their interaction was confirmed by several methods (
Figure 2), including in vivo and in vitro.
PSA3 has been characterized in several plants such as Arabidopsis, maize, rice and tomato. However, the interesting thing was that there were two different annotations for CsPSA3 in the cucumber genome database: photosystem Ⅰ (PSⅠ) assembly 3 and calcium homeostasis regulator 1, which were identical to maize, rice and Arabidopsis in each database and still remains unexplained [
21]. There have been no studies of PSA in cucumber, but Yang, Liu, Wen and Lu [
20] found that PSⅠ assembly protein was both encoded by both plastid and nuclear genes, and maize PSA3 was encoded by nuclear genes as annotated in NCBI, which was consistent with our observation of subcellular location for CsPSA3. It was localized in chloroplast and nucleus (
Figure 3B,C). The expression profile of
CsPSA3 under salt stress showed that
CsPSA3 made responses differently both in leaf and root, suggesting that CsPSA3 might also play vital roles in root besides participation in assembly of PSⅠ in leaf. Moreover, there was a correlation between expression of
CsPSA3 and
CsPAO2 in response to salt stress when compared with our previous detection [
19], i.e., their expression was both upregulated after 1 h of salt treatment in root, but in leaf,
CsPAO2 expression was enhanced at 6 h and expression level of
CsPSA3 was decreased dramatically at 12 h. So, we speculated that the interaction between these two proteins probably existed in both leaf and root.
In studies, polyamine was shown to bind with photosynthesis compounds and regulate secondary structures of light harvesting complex Ⅱ, PSⅠ and PSⅡ, thus affecting photosynthesis [
24], and antisense-mediated
S-adenosyl-L-methionine decarboxylase silencing which lacked synthesis of Spd and Spm, exhibited significantly reduced photosynthesis rate under heat stress [
25]. In addition, the synthesis and degradation of polyamine was also regulated by light. The activity of ornithine decarboxylase, a key enzyme for Put synthesis, was increased rapidly in dark-treated
Chlamydomonas reinhardtii cells after being transferred to light [
26], and
OsPAO5 expression in rice was upregulated when exposed to light while it was inhibited under dark conditions [
27]. A drought-tolerant maize genotype was previously found to have a higher PAO expression level and photosynthesis efficiency relative to the drought-sensitive genotype [
13]. So, we supposed that CsPAO2 interacted with CsPSA3 to affect photosynthesis, whose change was measured after PAO2 and PSA3 were silenced in cucumber by VIGS. Under the control condition, photosynthesis was impacted because of the relationship between CsPSA3 and PSⅠ assembly, and Pn was reduced (
Figure 4B). However, according to each photosynthetic parameter, photosynthesis of
CsPSA3-silencing cucumber plants suffered significantly less damage than pV190-silecing plants after stress treatment. The increased level of
CsPSA3 expression at 1 h following salt treatment was probably for accelerating PSⅠ accumulation and enhancing its activity, and the decreased expression level at a later stage could induce an increase in Gs, Ci and Tr, which would in turn maintain the normal operation of photosynthesis under salt stress, illustrating that other roles might be played by CsPSA3 in photosynthesis. On the other hand,
CsPAO2 silencing caused more damage to photosynthesis, absorption and utilization of CO
2 was declined and Ci was increased (
Figure 4D).
Oxidative damage could be induced by salt stress, which leads to the increase in MDA content and EL. Similar to photosynthesis in leaf, the root of
CsPAO2-silencing plants was more severely damaged in this study. However, we could not ignore the function of
CsPSA3; EL in the leaf of
CsPSA3-silencing plants was lower than that of the other two gene-silencing plants, while there was no marked difference between EL in the root of
CsPSA3 and pV190-silencing plants (
Figure 5C,D), suggesting that
CsPSA3 played a more important role in leaf than in root and it was involved in the regulation of salt tolerance of leaf by
CsPAO2. Then we found that CsPSA3 could also influence endogenous polyamine content whether under control or salt stress conditions, and notably, conversion from Put to Spd and Spm under salt stress was promoted by
CsPSA3 silencing, which was beneficial for plants to maintain a higher level of (Spd+Spm)/Put ratio and improve salt tolerance.
In summary,
CsPAO2 and
CsPSA3 responded to salt stress in both leaf and root, and they interacted to influence their regulation of salt stress tolerance. This interaction can be summarized as follows (
Figure 7):
CsPAO2 expression was enhanced in leaf after salt stress, on the one hand, oxidative damage was alleviated by the increased activities of antioxidant enzyme induced by CsPAO2, and conversion between polyamines was also promoted; on the other hand, CsPAO2 interacted with CsPSA3 to elevate Gs, Ci and Tr, leading to improved photosynthesis, and the same conversion between polyamines was also accelerated, contributing to the enhanced salt tolerance. However, this regulatory mechanism needs further investigation, and how PSAS affects photosynthetic parameters, such as Gs and Ci, remains to be explored.
4. Materials and Methods
4.1. Plant Materials and Growth Conditions
Uniform seeds of cucumber (Cucumis sativus L.) were sown in quartz sand, and the seedlings were transferred to hydroponic cultivation in 1/2 Hoagland nutrient solution (pH 6.5 ± 0.1, electrical conductivity 2.0~2.2 mS·cm−1) at the second leaves stage. Cucumber seedlings were subjected to salt stress when the third leaves were fully expanded, i.e., the nutrient solution containing 75 mM NaCl. At 0, 1, 2, 6, 12, 24, 48 and 72 h after each treatment, the leaf and root samples were collected.
The floral dip method [
28] was used to perform Arabidopsis (
Arabidopsis thaliana L., cv. Columbia) transformation and homozygous T3 lines (OE7, OE12, OE18) were obtained, whose detailed procedure was described below in “Genetic transformation of Arabidopsis”. After disinfection, WT and transgenic Arabidopsis seeds were sown on a 1/2 Murashige-Skoog (MS) medium for 10 days, then seedlings of similar size were treated as follows: (1) seedlings were transferred to square plate containing 1/2 MS medium with various concentrations of NaCl (0, 100 and 200 mM) and cultivated vertically for 7 days to observe root growth; (2) seedlings were transplanted to vermiculite culture in square flower pots and they were watered with normal water or water supplemented with 200 mM NaCl for 10 days after 3-weeks of growth. All of the samples were frozen instantly in liquid nitrogen and kept at −80 °C for subsequent analysis.
Tobacco (Nicotiana tabacum L.) seeds were randomly sown in square flower plots, and seedlings were transferred to several plots (2 plants per plot) for subsequent agrobacteria infection.
4.2. Genetic Transformation of Arabidopsis
CsPAO2 overexpression vector was constructed by homologous recombination method and introduced into Agrobacteria after sequencing confirmation. Arabidopsis seedlings were infected by this Agrobacteria according to the floral dip method [
28]. Seeds of Arabidopsis plants after infection were collected and plated on a 1/2 MS medium containing kanamycin to screen positive plants growing normally (T1 generation), whose seeds were saved individually (15 plants in total). Seeds of each plant were then sown separately on a 1/2 MS medium supplemented with kanamycin, and lines showing 1:2:1/3:1 segregation ratio were selected (10 lines), among which green plants with relatively better growth were transferred to vermiculite cultivation (10 plants for each line) and seeds of each plant were collected (T2 generation). Subsequently, the seeds of each plant were sown respectively on a 1/2 MS medium containing kanamycin again to choose 5 plants with a relatively high growth uniformity from 10 plants and seeds were saved individually (T3 generation); the plant with higher quality of seeds was used as the representative plant of each line.
4.3. Arabidopsis Seed Germination Rate Analysis
A 1/2 MS medium supplemented with different concentration of NaCl (0, 100 and 200 mM) was used to sow seeds, and the number of geminated seeds was recorded once a day for 7 days. The germination rate was calculated by multiplying the proportion of germinated seeds by the total number of seeds sowed (%).
4.4. Estimation of Malondialdehyde (MDA) Content and Electrolyte Leakage (EL)
Samples were homogenized in trichloroacetic acid (TCA, 5%). After centrifugation at 4000×
g for 10 min, 2 mL of supernatant was incorporated with thiobarbituric acid (TBA, 0.67%) of the equal volume, and the obtained mixture was centrifuged at 3000×
g for 15 min after heated for 30 min using boiling water bath. The absorbance of the supernatant was measured at 450, 532 and 600 nm, which were used to calculate the MDA content according to the method of Dhindsa et al. [
29]. The method described by Xu et al. [
30] was used as a reference for EL detection with slight modifications. The samples were soaked with 20 mL of ddH
2O in 50 mL tube, and kept at room temperature for 8 h and then conductivity was measured (initial conductivity, C
i). After this, the tubes containing samples inside were autoclaved (121 °C, 20 min) and the conductivity (C
max) was measured again. The proportion of C
i to C
max was calculated as EL (%).
4.5. Determination of H2O2 Content
The H
2O
2 content was measured according to the method developed by Alexieva et al. [
31]. After grinding the samples in TCA (0.1%), the homogenates were centrifuged at 12,000×
g for 20 min. 0.2 mL supernatant, 1 mL of 1 M KI solution and 0.25 mL of 0.1 M potassium phosphate buffer (pH 7.8) were mixed together and placed in darkness for 1 h. The absorbance of the mixture was read at 390 nm, and the H
2O
2 concentration was calculated with a standard curve based on serial concentrations gradient of H
2O
2 and the corresponding absorbance.
4.6. Antioxidant Enzyme Activity Assay
The enzymatic extract was obtained by using pre-cooled 0.05 M phosphate buffer (pH 7.8) to grind the samples and centrifuged at 12,000×
g for 20 min, then the supernatant was used to measure enzymes activity. The nitro blue tetrazolium (NBT) photochemical reduction method was used to detect superoxide dismutase (SOD) activity [
32]. One unit of SOD activity was defined as the amount of enzyme required to inhibit 50% of NBT reduction. Peroxidase (POD) and catalase (CAT) activities were measured using the method of Lin and Kao [
33] and Li et al. [
34], respectively. The reaction solution for POD activity assay included 0.2 M phosphate buffer (PBS, pH 6.0), 3.5 M guaiacol, and 30% H
2O
2, while that the solution for CAT assay included 30% H
2O
2 and PBS with different concentration and pH (0.05 M, pH 7.0). One unit of POD or CAT activity was defined as an increase of 0.01 A
470·or a decrease of A
240 per minute. For the measurement of ascorbate peroxidase (APX) activity, the enzyme extract was mixed with reaction solution containing 0.05 M PBS (pH 7.0) containing 0.1 mM EDTA-Na
2, 5 mM ascorbic acid and 20 mM H
2O
2; its activity was then calculated by the method described by Nakano and Asad [
35] using the decrease in A
290 within 1 min.
4.7. Measurement of Polyamines Content
The polyamine content was determined according to the method of Shu et al. [
36]. Leaf sample was ground in 1.6 mL of pre-cooled perchloric acid (PCA, 5%) followed by 20 min centrifugation at 12,000×
g and 4 °C. The supernatant was collected for detecting free and conjugated Pas, while the pellet was used for the assay of bound Pas. 1.4 mL of 2 M NaOH and 15 μL benzoyl chloride was added to 0.7 mL supernatant and then placed at 30 °C for 30 min after 20-s vortex. Next, 2 mL of saturated NaCl solution and cold diethyl ether was mixed with the above solution and centrifuged at 12,000×
g, 4 °C for 5 min, 1 mL of ether phase on the top was placed in new tubes until evaporation to dryness and 1 mL of chromatographically pure methanol was used to redissolve benzoyl Pas. Conjugated and bound Pas were extracted after 18-h hydrolyzation at 110 °C in sealed ampoule bottle. The samples redissolved in methanol were stored at −20 °C and filtered before testing. A UPLC system (Thermo, UltiMate 3000, Waltham, MA, USA) was used for detection the content of Pas.
4.8. Quantitative Real-Time PCR Analysis
RNAsimple Total RNA Kit (TIANGEN, China) was used to extract total RNA according to the manufacturer’s instructions. Then cDNA was synthesized through reverse transcription of total RNA (1 μg) using HiScript
® Ⅲ RT SuperMix for qPCR (Vazyme, Nanjing, China). Beacon Designer 7.9 (Premier Biosoft International, CA, USA) was used to design primers and presented in
Table S1. QuantStudio
TM 6 Real-Time PCR System (Applied Biosystems) with ChamQ SYBR qPCR Master Mix (Vazyme, China) was applied for qRT-PCR. The relative expression of the gene was calculated by the 2
−ΔΔCT method [
37] with the
actin gene of cucumber or Arabidopsis as the internal reference gene.
4.9. Amino Acid Sequence Alignment of PSA3
The amino acid sequence of PSA3 was aligned by ClustalX (version 1.81) and Genedoc (version 2.7) software.
4.10. Observation of PSA3 Localization at Subcellular Level
The coding sequence (CDS) of
PSA3 without stop codon was amplified using primers pAC402-PSA3
F/R (
Supplementary Table S1) and then inserted into the N terminal of the GFP gene in the pAC402 vector. The empty vector and the constructed vector was transformed into Agrobacteria after sequence confirmation, which were then transiently transformed into tobacco leaves through the syringe infiltration method [
38]. After 12 h of exposure to dark treatment, tobacco plants were grown under normal conditions for 2 days, and tobacco leaves were observed and images were obtained using confocal laser scanning microscope (LSM 780, Zeiss, Oberkochen, Germany).
4.11. Determination of Photosynthetic Parameters
The LI-6400 portable photosynthesis system (LI-COR Inc., Lincoln, NE, USA) was used to detect Pn, intercellular CO2 concentration (Ci), stomatal conductance (Gs) and Tr values with light intensity, chamber temperature, relative humidity and CO2 concentration set as 1000 μmol·m−2·s−1, 25 °C, 70% and 400 ± 10 μmol·mol−1, respectively, were maintained.
4.12. Yeast Two-Hybrid cDNA Library Screening and Assay
The CDS of
CsPAO2 was ligated into the bait vector pGBKT7, and the yeast two-hybrid cDNA library of cucumber constructed by Meiwen [
39] was screened with the yeast cotransformation method. The positive clones were sequenced and analyzed by sequence blast. Next, yeast two-hybrid validation of interaction between proteins was carried out. PSA3 were fused to the pGADT7 vector as prey plasmids and co-transformed into the yeast strain Y2H Gold with the bait plasmids, among which yeast cotransformed with pGBKT7-53 and pGADT7-T was used as a positive control, while the one cotransformed with pGBKT7-Lam and pGADT7-T was used as a negative control, and then transformed yeast was plated on SD-Trp/-Leu, SD-Trp/-Leu/-His and SD-Trp/-Leu/-His/-Ade plates.
4.13. Bimolecular Fluorescence Complementation (BiFC) Analysis
The full-length CDSs (without stop codon) of CsPAO2 and PSA3 were inserted separately into the ZYN and ZYC vectors. The methods of vector construction, Agrobacteria transformation and tobacco infection were the same as described above except that Agrobacteria carrying different recombinant plasmids should be mixed in equal volumes according to the protein combination needed to verify the interaction before injection. Confocal laser scanning microscope (LSM 780, Zeiss, Germany) was used to observe YFP fluorescence of tobacco leaves after two-days of growth.
4.14. Luciferase Complementation Assay (LCA)
LCA was conducted by the method of Hou et al. [
40]. In accordance with the method of vector construction and Agrobacteria transformation in subcellular localization of PSA3, the CDSs of CsPAO2 and PSA3 were cloned into the nLUC and cLUC vectors, respectively, and then transformed into agrobacteria. Similar to the BiFC assay, Agrobacteria must be mixed prior to injection, and the difference is that the injection area was a circle rather than the whole leaf. After 2 days of growth, D-Luciferin potassium salt was evenly sprayed on the leaves as substrate and plant living imaging system (Lumazone PLXIS 1024B, Princeton Instruments, Trenton, NJ, USA) was used to observe luminescence.
4.15. GST Pull Down Assay
The method of vector construction mentioned above was used to ligate the CDS of CsPAO2 into pET32a and the CDS of PSA3 into pGEX4T-1. The constructed vector was then transformed into Escherichia coli Rosseta (DE3) to perform prokaryotic expression and extract protein. The solubilities of recombinant proteins and optimum induction time were tested first: incubated overnight grown culture in 20 mL of LB broth at 37 °C until OD600 achieved 0.6–0.8; 500 μL of culture was taken out and the pellets were collected at -80 °C for further analysis after centrifugation at 5000× g, 4 °C for 15 min; IPTG was added to the remanent culture and continued to grow at 25 °C with shaking; 500 μL of the culture was taken out at 2, 6, 8, 10 and 12 h after addition of IPTG, and the pellet was collected and stored at the same time; 100 μL of BugBuster master mix (Novagen) was used to resuspend collected pellet and 20 μL of solution was used as total protein after placed at room temperature for 15 min, then the rest was centrifuged, and the supernatant was used as soluble protein; at last, SDS-PAGE was performed after protein denaturation.
Next, inoculate the culture overnight into the larger volume of LB (400 mL) to extract enough protein according to the optimal induction time learned from the previous step. The pellet was collected after centrifugation and resuspended in 1 × PBS, and high- pressure cell cracker (JNBIO, Guangzhou, China) was used to break cells until the suspension became clear. Then, the supernatant was collected and stored at −80 °C after centrifugation. For pGEX4T-1-PSA3, a recombinant protein with low solubility, its protein was contained in the pellet and needed to be extracted after dialysis treatment.
GST-tag Purification Resin (BeyoGold, Shanghai, China) was used to perform GST pull down with the manufacturer instructions. The samples were separated by SDS-PAGE after denaturation and analyzed by Western blot with anti-His or anti-GST antibodies.
4.16. Co-Immunoprecipitation (Co-IP) Assay
The CDSs of CsPAO2 and PSA3 were ligated into pAC402 carrying GFP tag and pAC330 carrying Myc tag, respectively. The method of vector construction and tobacco injection was the same as described above. A total of 1 g samples of infected tobacco leaf were used for protein extraction: leaves powder was obtained after leaves ground in liquid nitrogen and extraction buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF) was added to produce the homogenate and the supernatant was collected as protein extract after centrifugation. Then Anti-c-Myc magnetic beads (MedChemExpress, South Brunswick, NJ, USA) was used to conduct Co-IP assay following the instructions. Samples were analyzed via Western blot.
4.17. VIGS in Cucumber Plants
The vector pV190 used for VIGS in cucumber was provided by the lab of Gu et al. [
41]. The methods of vector construction and Agrobacteria transformation were the same as mentioned above; meanwhile, as the control, the empty vector and the vector silencing
phytoene desaturase (
PDS) were also transformed into agrobacteria. Germinated-seed vacuum inoculation system was used to inject cucumber. Cucumber seeds were treated with bud forcing and vacuum infiltrated twice in infection solution for 10 min when the length of radicle reached about 5 mm. After injection, seeds were sown in substrate. Then, leaves were sampled for RNA extraction and qRT-PCR analysis of
CsPAO2 and
PSA3 expression until the leaves of cucumber plants infected by agrobacteria carrying
PDS-silencing plasmid showed photobleaching symptoms (
Figure S1), and the plants whose
CsPAO2 or
PSA3 expression level was lower than 0.5 were transferred to a hydroponic culture and treated with 75 mM NaCl. About 120 sprouted seeds were used for the vacuum infiltration of each gene every time, and 96 of which with uniform size were sown after injection. The efficiency of gene silencing was around 30% to 50%.
4.18. Statistical Analysis
There were at least three independently tested biological replicates for each experiment, and the values are the means ± SE of these independent experiments. The bars represent the standard error. All the data were statistically analyzed with the SPSS 17.0 software program (SPSS Inc., Chicago, IL, USA) and differences between treatments were compared using Tukey’s test at the p < 0.05 level of significance.