A natural variation in SlSCaBP8 promoter contributes to the loss of saline–alkaline tolerance during tomato improvement

Abstract Saline–alkaline stress is a worldwide problem that threatens the growth and yield of crops. However, how crops adapt to saline–alkaline stress remains less studied. Here we show that saline–alkaline tolerance was compromised during tomato domestication and improvement, and a natural variation in the promoter of SlSCaBP8, an EF-hand Ca2+ binding protein, contributed to the loss of saline–alkaline tolerance during tomato improvement. The biochemical and genetic data showed that SlSCaBP8 is a positive regulator of saline–alkaline tolerance in tomato. The introgression line Pi-75, derived from a cross between wild Solanum pimpinellifolium LA1589 and cultivar E6203, containing the SlSCaBP8LA1589 locus, showed stronger saline–alkaline tolerance than E6203. Pi-75 and LA1589 also showed enhanced saline–alkaline-induced SlSCaBP8 expression than that of E6203. By sequence analysis, a natural variation was found in the promoter of SlSCaBP8 and the accessions with the wild haplotype showed enhanced saline–alkaline tolerance compared with the cultivar haplotype. Our studies clarify the mechanism of saline–alkaline tolerance conferred by SlSCaBP8 and provide an important natural variation in the promoter of SlSCaBP8 for tomato breeding.


Introduction
Saline-alkaline soil is a major abiotic stress affecting crop productivity and quality in the world [1,2].Over 950 million hectares of soils are affected by saline-alkaline stress in the world [3].Salinealkaline soil with high levels of sodium bicarbonate and sodium carbonate is characterized by high salt and high pH (above pH 8.0) [4].Relative to the neutral salts, saline-alkaline salts damage plants much more severely due to the combination of high pH stress, secondary stresses, ion toxicity, and osmotic stress [5][6][7].
To defend against saline-alkaline stress, plants have developed adaptive strategies.Under saline-alkaline conditions, Ca 2+ binding ZmNSA1 (Na + content under saline-alkaline condition) increases the transcription of plasma memrane-H + -ATPases (MHAs) and promotes Na + eff lux mediated by the Na + /H + antiporter SOS1 in maize [8].The Gγ subunit AT1 (Alkaline Tolerance 1) negatively modulates the phosphorylation of PIP2 aquaporins and reduces the H 2 O 2 export activity of PIP2s, leading to the over-accumulation of H 2 O 2 and resulting in alkaline stress sensitivity in sorghum, millet, rice, maize, and wheat [9,10].SCaBP3/CBL7 decodes alkaline-mediated Ca 2+ signaling and releases SCaBP3 inhibition on activities of PM-H + -ATPase AHA2 against alkali stress in Arabidopsis [11].
To deal with ion toxicity, plants activate the Salt-Overly-Sensitive (SOS) pathway.Salt stress promotes calcium accumulation in the cytoplasm.The Ca 2+ signaling is decoded by two EF-hand calcium-binding proteins, SOS3 in roots and SCaBP8 in shoots.SOS3 and SCaBP8 bind and activate the serine/threonine protein kinase SOS2.Then SOS2 phosphorylates and activates SOS1 to extrude the excess Na + from the cytosol to the apoplast [12].To prevent over-activation of SOS signaling, the SCaBP8-SOS2 complex represses the activity of a putative calciumpermeable transporter, AtANN4, by directly interacting and phosphorylating AtANN4, which fine-tunes AtANN4-dependent calcium transients under salt stress [13].
The ancestors of modern crops provide resourceful natural variations for revealing the mechanism of salt tolerance [2,14,15].OsHKT1;1 regulates root Na + content and OsHKT1;1 from indica uptakes Na + with much more efficiency than that from japonica, which contributes to the tolerance difference between indica and japonica in rice [16].Genome-wide association studies (GWAS) revealed that the HAK family Na + -selective transporter Zea mays L. Na + content 2 (ZmNC2), mediating shoot Na + exclusion, confers the natural variation of salt tolerance in maize [17].Another GWAS assay found that variations in salt-tolerance-associated-gene 4 (SAG4) and SAG6 had positive roles in plant salt tolerance [18].
ZmSOS1 and ZmCBL8, the important components of the SOS pathway in maize, confer natural variations in salt tolerance by regulation of Na + exclusion [19].
Tomato domestication history involves two major processes dependent on fruit size: the wild Solanum pimpinellifolium (PIM) was domesticated in south America to Solanum lycopersicum var.cerasiforme (CER), and this process is called domestication; then CER was further improved to Solanum lycopersicum (BIG) in Mesoamerica, which is called improvement [20][21][22].The wild tomato accommodates salt stress, while the cultivar lost tolerance during tomato domestication and improvement [23][24][25].Recent progress has already identified some important variations leading to the lost salt tolerance during tomato domestication.SlHAK20 regulates the homeostasis between Na + and K + , and a variation in SlHAK20 leads to the difference in Na + binding ability in roots, which conferred salt tolerance variations in tomato [25].SlDREB2 could induce the expression of SlSOS1 against salt stress in wild tomato.Natural variations in the SlDREB2-binding cis-element in the promoter of SlSOS1 down-regulates the SlDREB2-induced expression of SlSOS1, which increases salt sensitivity in tomato cultivars [26].A natural variation within a cis-element in the promoter of the SlSOS2 region was associated with the compromised salt tolerance during tomato domestication [27].
Due to the severe damage to plants caused by saline-alkaline stress compared with neutral salt stress, it is necessary to investigate whether saline-alkaline tolerance was selected during tomato domestication and improvement, and, if so, which variations contributed to the lost tolerance.In this study we demonstrated that saline-alkaline tolerance was compromised during tomato domestication and improvement, and a natural variation in the promoter of SlSCaBP8 contributed to the loss of saline-alkaline tolerance during tomato improvement.

Saline-alkaline tolerance was compromised during tomato domestication and improvement
To investigate whether saline-alkaline tolerance was changed during tomato domestication and improvement, we measured chlorophyll contents and survival rates of tomato accessions based on their geographical origins.Our study included 22 wild accessions of S. pimpinellifolium (PIM), 18 domesticated accessions of S. lycopersicum var.cerasiforme (CER), and 21 improved accessions of S. lycopersicum (BIG).We found that the chlorophyll contents and survival rates of the accessions in the BIG group were significantly lower compared with those in the PIM and CER groups after saline-alkaline treatments (Fig. 1, Supplementary Data Table S1).The survival rates but not chlorophyll contents showed significant differences between the PIM and CER groups.These results suggest that saline-alkaline tolerance was compromised in the process of tomato domestication and improvement.

SlSCaBP8 is an important regulator of saline-alkaline tolerance in tomato
The EF-hand Ca 2+ binding protein SCaBP8/CBL10 plays important roles against salt stress in shoots of Arabidopsis [28].Whether SCaBP8/CBL10 is involved in saline-alkaline stress in tomato has not been studied.Based on the phylogenetic tree of CBL proteins, SlSCaBP8/CBL10/Solyc08g065330 and AtSCaBP8/AtCBL10 clustered in the same clade, and this suggests that SCaBP8 is evolutionarily conserved in plants (Fig. 2A, Supplementary Data Table S2).
SCaBP8 decodes the Ca 2+ signal and translates it to SOS2, a serine/threonine protein kinase.We performed yeast twohybrid (Y2H) assays to confirm the interaction of SlSCaBP8 with SlSOS2.Results showed that SlSCaBP8 interacts with SlSOS2 in yeast (Fig. 2B).The interaction was further verified by the firef ly luciferase complementation imaging (LCI) assay.Compared with the negative controls, a strong luminescence signal was observed in Nicotiana benthamiana leaves co-infiltrated with SlSCaBP8-nLUC and SlSOS2-cLUC (Fig. 2C).The co-immunoprecipitation (Co-IP) assay also supported the interaction between SlSCaBP8 and SlSOS2 (Fig. 2D).Together, these results demonstrated that SlSCaBP8 interacts with SlSOS2.
To further clarify whether SlSCaBP8 is involved in salinealkaline stress, we constructed the slscabp8-cr mutant in the cultivar M82 background by CRISPR/Cas9 gene editing technology.All the mutations in slscabp8-cr lead to frame shifts and generation of premature stop codons (Supplementary Data Fig.S1).As expected, slscabp8-cr mutants exhibited compromised tolerance of salinealkaline stress (Fig. 2E-H).The slscabp8-cr mutants maintained remarkably higher malondialdehyde (MDA) contents with salinealkaline stress than M82 (Fig. 2I), which suggested that slscabp8-cr mutants were subjected to more oxidative damage than M82.In line with this, significantly higher levels of H 2 O 2 and O 2 − were found in slscabp8-cr mutants with saline-alkaline treatments compared with that of M82 (Fig. 2J and K).It is known that the maintenance of a proper Na + /K + ratio against saline stress in growing tissues, such as the shoot apex, is essential to maintain plant growth.To demonstrate SlSCaBP8 function in saline-alkaline tolerance, we checked the Na + /K + ratio in shoot apexes, adult leaves, stems, and roots.The ratio was reduced in adult leaves, stems, and roots.Na + was over-accumulated in the shoot apex of slscabp8-cr, which caused a higher Na + /K + ratio compared with that of M82 (Supplementary Data Fig.S2).This might be responsible for the retarded growth of slscabp8-cr in salinealkaline conditions (Fig. 2E).These results suggested that SlSCaBP8 is a positive regulator against saline-alkaline stress, and SlSCaBP8 functions in maintaining the homeostasis of the Na + /K + ratio.

Pi-75 displayed enhanced saline-alkali tolerance
To clarify whether SlSCaBP8 is selected during tomato domestication, we compared the genomic sequence of SlSCaBP8 between LA1589 (PIM) and cultivar E6203.We found plenty of variations in the promoter regions and no variations in protein sequence, which implies that the transcription of SlSCaBP8 in LA1589 differs from that of cultivar E6203 (Supplementary Data Fig.S3).SlSCaBP8 LA1589 is located in Pi-75, one of the well-established introgression-line (IL) populations derived from a cross between LA1589 and E6203 [30].We first examined SlSCaBP8 expression in LA1589, Pi-75, and E6203 under saline-alkaline stress.Without saline-alkaline stress, the basal level of SlSCaBP8 expression was higher in LA1589 and Pi-75 compared with that of E6203.Salinealkaline stress could induce SlSCaBP8 expression in E6203, but the induction in LA1589 and Pi-75 was much stronger than that in E6203 (Fig. 3A and B).Due to the important role of SlSCaBP8 against saline-alkaline stress, we speculate that the variations in promoter regions of SlSCaBP8 could affect saline-alkaline tolerance.Then we compared saline-alkaline-induced shoot growth inhibition among LA1589, Pi-75, and E6203.Saline-alkaline treatment reduced the shoot fresh weight of LA1589, Pi-75, and E6203 by 57.38, 60.80, and 70.85%, respectively, compared with the mock treatment (Fig. 3C and D).LA1589 and Pi-75 also showed higher chlorophyll contents and survival rates against saline-alkaline stress compared with that of E6203 (Fig. 3E and F).Taken together, these results demonstrate that the variations in the SlSCaBP8 promoter region affect its expression, which might lead to the salinealkaline tolerance differences among E6203, LA1589, and Pi-75.

SlSCaBP8 was selected during tomato improvement
To further clarify whether genetic variations in SlSCaBP8 could modulate saline-alkaline tolerance, we first analyzed the sequence variations with the resequencing data of 706 tomato accessions, which consist of 76 wild accessions from PIM, 254 domesticated accessions from CER, and 376 improved accessions from BIG (Supplementary Data Fig.S4) [31].We calculated the nucleotide diversity (π) and the π ratios between PIM and CER and between CER and BIG on chromosome 8.We found that the π value of the SlSCaBP8 locus was significantly lower in BIG compared with CER and PIM, and this locus was associated with an improvement rather than a domestication sweep (Fig. 4A and B, Supplementary Data Table S3).We next analyzed the SNPs (single-nucleotide polymorphisms) and InDels (insertions and deletions) in the promoter and gene body regions of SlSCaBP8 among the 706 accessions [31].Within 3 kb upstream of the ATG start codon of SlScaBP8, eight SNPs (SNP1-8) and one InDel exist (Fig. 4C, Supplementary Data Table S4).No SNP or InDel was found in the coding sequence of SlSCaBP8.The distributions of all these variations were remarkably reduced from PIM to CER and then to BIG (Supplementary Data Table S4).Within these variations, only SNP7 was predicted to be located in a transcription factor binding site (Fig. 4C and D).
To check whether SNP7 could lead to different transcript levels of SlSCaBP8 in LA1589 and E6203, a transactivation assay was carried out with proSlSCaBP8 LA1589 :LUC, proSlSCaBP8 LA1589 mut:LUC, proSlSCaBP8 E6203 :LUC, and proSlSCaBP8 E6203 mut:LUC.The luminescence intensity was strongly enhanced by saline-alkaline treatment with proSlSCaBP8 LA1589 :LUC, and the mutation in SNP7 from G 1903 to A 1903 significantly reduced the luminescence signal of proSlSCaBP8 LA1589 mut:LUC.Saline-alkaline treatment weakly promoted the luminescence intensity with proSlSCaBP8 E6203 :LUC, but the mutation in SNP7 from A 1903 to G 1903 could significantly enhance the luminescence signal of proSlSCaBP8 E6203 mut:LUC.
These results demonstrated that the variation SNP7 could lead to different transcription levels of SlSCaBP8 in LA1589 and E6203 (Supplementary Data Fig.S5).SNP7 potentially impacts the expression of SlSCaBP8 and was selected for further study.
According to the resequencing result of the SlSCaBP8 locus, SNP7 is 1903 bp upstream of the ATG start codon.The 706 accessions were classified into two haplotypes based on SNP7 in the promoter of SlSCaBP8.The wild SlSCaBP8 promoter is representative of Hap (haplotype) 1 (n = 142) with G 1903 , and the cultivated SlSCaBP8 promoter belongs to Hap2 (n = 533) with A 1903 (Fig. 4D, Supplementary Data Table S5).We then analyzed the phenotypes against saline-alkaline stress with the representative accessions and found that Hap1 showed higher survival rates and greater chlorophyll content compared with Hap2 (Fig. 5A-C).The transcription levels of SlSCaBP8 were increased with saline-alkaline treatment with Hap1 varieties, and this induction was significantly reduced in Hap2 varieties (Fig. 5D and E).Consistently, the distribution of these two alleles Hap1 and Hap2 further indicated that the reduced frequency of the wild allele Hap1 is related to the compromised saline-alkaline tolerance during tomato domestication (Fig. 4E).These results show that the variation in the SlSCaBP8 promoter region results in the compromised salinealkaline tolerance during tomato improvement.

Discussion
Compared with the neutral salt stress, saline-alkaline stress damages plants' growth, development, and yield much severely.But how plants adapt to saline-alkaline stress is still not clear.Crop domestication leads to reduced stress resistance and enhanced productivity [32].The common cultivated tomato (BIG) was domesticated from PIM and improved from CER in harsh environments in South America and Mesoamerica [20][21][22].Recent progress showed that salt tolerance was lost during tomato domestication, and the core components in SOS signaling, SlSOS1 and SlSOS2, were selected during this process [25][26][27].Whether saline-alkaline stress tolerance was changed during tomato domestication and improvement, and what is the role of the SOS pathway, are still unknown.In this study, we found that cultivated tomato varieties showed compromised saline-alkaline tolerance compared with their ancestors, which demonstrated that saline-alkaline tolerance was also compromised during tomato domestication and improvement.To explore the molecular mechanism against saline-alkaline stress, we focused on the EF-hand Ca 2+ binding protein SlSCaBP8 in tomato.Y2H, LCI, and Co-IP assays proved the interaction between SlSCaBP8 and SlSOS2, and slscabp8cr showed enhanced sensitivity to saline-alkaline stress.Na + was over-accumulated in the shoot apex of slscabp8-cr and the Na + /K + ratio was enhanced.These results suggested that SlSCaBP8 is a positive regulator against saline-alkaline stress and that SlSCaBP8 functions in maintaining the homeostasis of Na + /K + ratio in tomato.We compared sequence differences of SlSCaBP8 between PIM LA1589 and cultivar E6203 and found some potential variations in promoter regions but no variations in protein sequences, which implies that the expression of SlSCaBP8 in LA1589 differs from that of cultivar E6203.We found that expression of SlSCaBP8 was promoted by saline-alkaline stress, and the induction was much stronger in LA1589 and Pi-75 compared with that in E6203.Saline-alkaline tolerance of LA1589 and Pi-75 was stronger than that of E6203.These lines of data give us some cues that the potential variations in promoter regions of SlSCaBP8 contribute to the saline-alkaline tolerance difference between LA1589 and E6203.We found eight SNPs and one InDel in the promoter region and their distribution remarkably declined from PIM to CER and then to BIG by analyzing the resequencing data, and SNP7 is located in a transcription factor binding motif.Based on SNP7, the wild SlSCaBP8 promoter is representative of Hap1, and the cultivated SlSCaBP8 promoter belongs to Hap2.Accessions with Hap1 showed greater saline-alkaline tolerance than that of Hap2.The distribution of Hap1 and Hap2 in PIM, CER, and BIG of the 706 accessions further suggested that the reduced frequency of the wild allele Hap1 is related to the compromised saline-alkaline tolerance during tomato domestication.The mechanism of SNP7 leading to the expression differences of SlSCaBP8 between the wild and cultivar accessions still need to be further verified.Altogether, these results suggest that the natural

Plant materials and growth conditions
All tomato accessions used in this work were obtained from TGRC (Tomato Genetics Resource Center) and AGIS-CAAS (Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Science).Tomato seeds were germinated at room temperature and then sown in a mixture of vermiculite and Klasmann-Deilmann substrate (1:1 v/v) and watered to saturation with water or 75 mM NaHCO 3 (pH 8.5) solutions.In the greenhouse, seedlings were grown under 16 h of light at 28

Phylogenetic tree construction
The phylogenetic tree was generated using MEGA X software [29] using the neighbor-joining method.The scale represents the branch length, and each node represents the bootstrap value from 1000 replicates.The SCaBP/CBL members in Arabidopsis and tomato are listed in Supplementary Data Table S2.

Hap1:
Hap2: A D E B C

Generation of mutant of slscabp8
Mutants of slscabp8 were made by CRISPR/Cas9 gene editing technology as described in the previous study [33][34][35].A construct containing two sgRNAs targeting SlSCaBP8 was introduced into Agrobacterium tumefaciens strain LBA4404, and then transformed into tomato cultivar M82.Homozygous plants were used for further experiments.Primers are listed in Supplementary Data Table S6.

RNA extraction and RT-qPCR
Total RNA was extracted from tomato seedlings using the TRIzol reagent (Invitrogen).cDNA was made from 2 μg of total RNA with SuperScript III reverse transcriptase (Invitrogen) and quantified on a Bio-Rad CFX96 with the SYBR Green kit (Takara).Tomato EF1a was used as an internal control.Statistical significance was evaluated by Student's t-test.Primers are listed in Supplementary Data Table S6.

Yeast two-hybrid assay
Yeast transformation and growth assays were performed using the Matchmaker Gold Yeast Two-Hybrid System (Clontech).SlSCaBP8 and SlSOS2 were cloned into pGBKT7 and pGADT7 to construct SlSCaBP8-BD and SlSOS2-AD, respectively.Both constructs were co-transformed into yeast strain Y2HGold.SD/−2 medium and SD/−4 medium plus X-α-Gal were used to check protein interaction.Empty pGADT7 or pGBKT7 vectors were co-transformed with SlSCaBP8-BD or SlSOS2-AD in parallel as negative controls.All primers used for Y2H are listed in Supplementary Data Table S6.

Luciferase complementation imaging assay
The LCI assays were performed as described [36].SlSCaBP8 and SlSOS2 were cloned into pCAMBIA1300-nLUC and pCAMBIA1300-cLUC, respectively.Both constructs were separately transformed into A. tumefaciens strain GV3101 for transforming N. benthamiana leaves.Plants were incubated at 22 • C for 72 h before CCD imaging.A CCD camera (NightShade LB 985, Berthold) was used for LUC images and measurement of LUC activity.Primers used for these constructs are listed in Supplementary Data Table S6.

Co-immunoprecipitation assays
The Co-IP assays were performed as described [37,38].Agrobacterium tumefaciens strain GV3101 carrying p35S::SlSOS2-FLAG or p35S::SlSCaBP8-GFP constructs was co-infiltrated into N. benthamiana leaves.Plants were incubated at 22 • C for 72 h and then the cotransformed N. benthamiana leaves were ground to a fine powder and transferred to lysis buffer.FLAG antibody-bound agarose beads were then added to each supernatant for at least 4 h at 4 • C. The precipitated samples were washed at least three times with the lysis buffer and then eluted by boiling the beads in SDS protein loading buffer for 10 min.Immunoblots were detected with anti-FLAG antibody and anti-GFP antibody.Primers used for these constructs are listed in Supplementary Data Table S6.

Measurement of chlorophyll content
For measurement of chlorophyll content, leaf samples were incubated using a mixture of acetone and ethanol (1:1 v/v) in the dark at room temperature for 24 h.Absorbance of chlorophyll was measured at A663 and A645 [39,40].

Measurement of Na + and K + contents
Measurement of contents of Na + and K + of the tomato was as described in the previous study and modified [25,41].To determine Na + and K + contents, 20-day-old seedlings grown under water or 75 mM NaHCO 3 conditions were collected.The collected plant parts were rinsed three times with double-deionized water to remove any contaminants before drying at 80 • C for 24 h and then grinding to powder.Tissue powder (10 mg) was digested by 1 ml of nitric acid for 2 h in a microwave 3000 digestion system (Anton Paar).Germanium was used as the internal standard.Na + and K + concentrations were measured by using inductively coupled plasma mass spectrometry (ICP-MS) (ICAPQ, Thermo Fisher).
Constructs were separately transformed into GV3101 for transforming N. benthamiana leaves.Plants were incubated at 22 • C for 72 h and then used to detect the LUC and REN activity.Primers used for these constructs are listed in Supplementary Data Table S6.

Figure 2 .Figure 3 .
Figure 2. SlSCaBP8 is a homolog of AtSCaBP8 in tomato.A Phylogenetic analysis of SCaBP/CBL proteins in Arabidopsis and tomato.The phylogenetic tree was constructed using MEGA X [29].The scale represents the branch length, and each node represents bootstrap values from 1000 replicates.B Y2H assays showing that SlSCaBP8 interacts with SlSOS2.The full-length coding sequence of SlSCaBP8 was fused with the DNA-binding domain (BD) in pGBKT7, and the full-length coding sequence of SlSOS2 was fused with the activation domain (AD) in pGADT7.Transformed yeast was grown on selective SD/−2 medium or SD/−4 medium plus X-α-Gal to test protein interaction.The empty pGADT7 or pGBKT7 vectors were co-transformed with SlSCaBP8-BD or SlSOS2-AD in parallel as negative controls.C LCI assays showing that SlSCaBP8 interacts with SlSOS2 in N. benthamiana.At least 12 N. benthamiana leaves were infiltrated and analyzed.D Co-IP assays showing that SlSCaBP8 interacts with SlSOS2 in N. benthamiana.SlSOS2-FLAG and SlSCaBP8-GFP vectors were co-transformed into N. benthamiana leaves.E Phenotypes of 21-day-old M82 (wild type) and slscabp8-cr (SlSCaBP8-edited) plants grown under control and saline-alkaline conditions (75 mM NaHCO 3 , pH 8.5).Seedlings were grown in the climate chamber and maintained at 60% relative humidity under 16 h of light at 26 • C and 8 h of dark at 20 • C. F, G Biomass (F) and chlorophyll content (G) of M82 and slscabp8-cr grown in saline-alkaline conditions for 3 weeks.H Survival rates of M82 and slscabp8-cr grown in saline-alkaline conditions for 4 weeks and then recovered for 1 week.I-K MDA content (I), H 2 O 2 content (J), and O 2 − productivity rate (K) of M82 and slscabp8-cr grown under control and saline-alkaline conditions for 3 weeks.The experiments were performed with three biological replicates with similar results.Statistical significance was determined by one-way ANOVA, P < 0.05.Significant differences are indicated by different lowercase letters.Scale bars = 10 cm.

Figure 4 .
Figure 4. SlSCaBP8 was selected during tomato improvement.A Nucleotide diversity (π) of PIM, CER, and BIG groups on chromosome 8.B The π ratios of PIM/CER and CER/BIG on chromosome 8.The dashed horizontal lines indicate the top 5% threshold for entire chromosome 8 (3.07 π PIM /π CER for domestication and 8.21 π CER /π BIG for improvement).The position of SlSCaBP8 indicated by the vertical line is within an improvement sweep.C Schematic of the gene structure and positions of genetic variations in the genomic region of SlSCaBP8 in the tomato accessions.The SlSCaBP8 promoter is designated as 3 kb upstream of the start codon.Eight SNPs and one indel are indicated by vertical lines.REF and ALT represent the reference sequence and the alternative sequence, respectively.D Two haplotypes of SlSCaBP8 in the tomato accession according to SNP7.The red nucleotide represents the nucleotide variation.E Distribution of Hap1 and Hap2 alleles of SlSCaBP8 in PIM, CER, and BIG.

Figure 5 .
Figure 5. Hap1 showed enhanced saline-alkaline tolerance.A Phenotypes of 30 tomato accessions grown under control or saline-alkaline conditions.For the saline-alkaline condition, 30 tomato accessions were grown in saline-alkaline conditions (75 mM NaHCO 3 , pH 8.5) for 4 weeks and then recovered for 1 week.The haplotypes of SlSCaBP8 are shown as Hap1 and Hap2, respectively.The groups of accessions are listed as PIM, CER, and BIG.Seedlings were grown in the climate chamber and maintained at 60% relative humidity under 16 h of light at 26 • C and 8 h of dark at 20 • C. B Chlorophyll content of 30 tomato accessions grown in saline-alkaline conditions for 3 weeks.The chlorophyll content was calculated with six biological replicates with similar results.C Survival rates of 30 tomato accessions grown in saline-alkaline conditions for 5 weeks.Survival rates were obtained from at least seven plants in three repeated experiments.D, E SlSCaBP8 expression of 30 tomato accessions in shoots (D) and roots (E).

Quantification of malondialdehyde content, H 2 O 2 content and O 2 − productivity rate
The MDA content, H 2 O 2 content, and O 2− productivity rate were quantified according to the manufacturer's instructions (Cominbio, catalog number MDA-1-Y for MDA, catalog number H2O2-1-Y for H 2 O 2 , and catalog number SA-1-G for O 2− ).