cDNA-AFLP analysis of salicylic acid- and calcium chloride-induced transcript derived fragments under drought in tomato (Solanum lycopersicum)

Abstract

Drought seriously threatens tomato production worldwide. Despite much research on exogenous salicylic acid (SA) and Ca²⁺ improving plant resistance to biotic and abiotic stress, the molecular mechanisms of exogenous SA- and Ca²⁺-mediated drought resistance response in tomato remain unclear. In this study, we analyzed SA- and Ca²⁺ -induced differentially expressed transcripts under drought in tomato plants using cDNA-amplified fragment length polymorphism (cDNA-AFLP). In total, 34 transcript derived fragments (TDFs) were differentially expressed. The functions identified through NCBI BLAST alignment mainly involved signal transduction, amino acid metabolism, transcription factors, transfer transport and stress response. The quantitative real-time polymerase chain reaction results of 12 TDFs associated with drought response were consistent with the patterns of changes observed with cDNA-AFLP analysis. These differentially expressed transcripts may be used for functional verification, transgenic research and breeding of drought-resistant tomato varieties.

Keywords:

• cDNA-AFLP
• differentially expressed transcripts
• drought stress
• exogenous salicylic acid and calcium chloride
• tomato

Introduction

Plants are vulnerable to stresses from the external environment, mainly including drought, heat, cold, salinity and toxic metals [1]. In particular, crop yields and agricultural economies worldwide have suffered enormous losses due to these abiotic stresses [2]. However, drought stress has become a top priority in plant research due to global climate change [3]. Tomato is one of the most important horticultural crops and the second most important vegetable worldwide [4]. Almost all varieties of cultivated tomato are sensitive to drought to varying degrees during the entire growth period, especially during the germination and seedling stages. Therefore, drought resistance is a target trait in tomato breeding programs.

Plant hormones play crucial roles in the resistance response against abiotic and biotic stresses. Naturally occurring salicylic acid (SA) regulates plant growth and participates in plant signal transduction in response to biotic and abiotic stresses [5,6]. Exogenous application of SA can affect many physiological processes, such as seed germination, photosynthesis, reactive oxygen species metabolism, osmotic adjustment, plant growth and fruit yield. Several studies have investigated whether SA can mitigate the adverse effects of water stress on barley, rice and safflower and have assessed its role in the adaptation of cell processes associated with plant tolerance [7–9].

Ca²⁺ participates in plant signaling networks as an important secondary messenger [10]. At present, several calcium signaling elements have been identified, including calcium signaling receptors such as calmodulin (CaM) and calcium-dependent protein kinases (CDPKs), intracellular calcium transport channels and calcium extravasation channels. Calcium can increase the thickness of the cell wall, promote the synthesis of defensive hormones, and alter oxygen activity. Exogenous applications of calcium induce resistance to drought, cold, heat, and salt stresses as well as modulate stress-induced reactive oxygen species (ROS) metabolism in plants, such as tobacco, bermudagrass and soybean [11–14].

Screening for differentially expressed genes (DEGs) may uncover the relevant molecular mechanism. cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis is a quick and effective method to identify both known and unknown transcripts [15]. This technique ligates adaptor molecules to digest double-stranded cDNA to greatly reduce the number of false positives. The cDNA-AFLP method has been used to confirm drought-related genes and to analyze differentially expressed transcripts in many plant species [16–18]. Here, we analyzed SA- and Ca²⁺-induced differentially expressed transcripts under drought in tomato using cDNA-AFLP analysis. Our findings provide an insight into genes associated with the response to drought stress. Our study may provide a basis for cloning drought-related genes, which will be useful for understanding the response mechanism of exogenous SA and Ca²⁺ to enhance drought resistance in tomato and for breeding drought-resistant tomato varieties.

Materials and methods

Plant materials and drought treatment

The tomato cultivar Dongnong 11537 (provided by the Tomato Institute, Northeast Agricultural University) was used in this study. Tomato seeds were sown in pots filled with soil. Based on our previous research on the optimal spray concentration [19], 0.3 mmol·L⁻¹ SA and 10 mmol·L⁻¹ CaCl₂ were used in this study. Seeds were divided into the following six groups: CK1 (control group with normal watering), CK2 (control group with drought treatment only), CKS (control group with exogenous SA only), CKC (control group with exogenous CaCl₂ only), S0.3 (exogenous SA group with drought) and C10 (exogenous CaCl₂ group with drought). At the four-five leaf stage, the seedlings were sprayed for three consecutive days according to the corresponding conditions. Subsequently, the S0.3, C10 and CK2 groups were deprived of water continuously, and the CK1, CKS and CKC groups were maintained with full irrigation. Leaf samples were collected from each group at 0 h, 12 h, 24 h, 36 h, 48 h, 3 d and 5 d from the beginning of drought stress. Each group had 60 plants, and the experiments were repeated three times.

cDNA-AFLP analysis

Total RNA was extracted for each group using the TRIzol method [20]. Double strand cDNA was synthesized using a PrimeScript^TM Double Strand cDNA Synthesis Kit (TaKaRa, Dalian). EcoR I and Mse I restriction enzymes were used to completely digest the double-stranded cDNA, and then the adaptors were used to ligate the fragments for subsequent amplification (Supplementary information Table S1). Preamplification polymerase chain reaction (PCR) was performed using the following program: 94 °C for 5 min; 24 cycles of 94 °C or 30 s, 56 °C for 1 min, and 72 °C for 1 min; and 72 °C for 10 min (ThermoFisher, USA). Subsequently, further selective PCR amplification was performed using the following program: 94 °C for 3 min; 12 cycles of 94 °C for 30 s, 65 °C for 30 s (−0.7 °C per cycle), and 72 °C for 1 min; and 25 cycles of 94 °C for 30 s, 56 °C for 30 s and 72 °C for 1 min. The PCR products were separated by electrophoresis using 6% polyacrylamide gel [21].

Sequence analysis and functional annotation

The target bands were selected from the differential bands in the cDNA-AFLP reaction, excised from the polyacrylamide gel and used as templates for re-amplification. The PCR products were purified with a PCR purification kit (Tiangen, China), cloned into the pMD18-T vector (Takara, Dalian) and sequenced. The homologous genes of each transcript derived fragment (TDF) sequence were searched in National Center for Biotechnology Information (NCBI) and SOL Genomics Network (SGN) databases after removing the vector sequence.

Validation of identified TDFs by quantitative real-time PCR (qRT-PCR)

Twelve TDFs were validated using qRT-PCR to verify the expression profiles obtained by cDNA-AFLP and to obtain a more detailed description of the differential expression patterns of the target TDFs. qRT-PCR was performed using AceQ® qPCR SYBR^® Green Master Mix (Vazyme, USA) on a qTOWER³G Detection System (Analytik Jena, Germany). Each sample was replicated three times, and data were analyzed using the 2^−△△CT method [22]. The following PCR protocol was used: 95 °C for 10 min; and 40 cycles of 95 °C for 5 s, 59 °C for 15 s and 72 °C for 30 s. Subsequently, melting curve analysis was performed to detect primer dimerization and other artifacts of amplification. The actin gene (NM_001321306.1) was used as a reference control for normalization (Supplementary Information Table S2).

Results and discussion

Phenotypic changes of tomato under drought

To visually determine the optimal exogenous chemical concentration, we photographed plants in each group on the third day of drought stress. As shown in Figure 1, there was no difference among plants in the CK1, S0.3 and C10 groups, which showed normal growth. However, CK2 plants showed wilting and leaf curling. Thus, these findings indicated that appropriate concentrations of SA and CaCl₂ effectively improve the drought resistance of tomato by exogenous application.

Figure 1. Plant phenotype on the third day of drought stress. CK1, control group with normal watering; CK2, control group with drought only; S0.3, exogenous SA group with drought; and C10, exogenous CaCl₂ group with drought.

cDNA-AFLP analysis

To identify DEGs induced by drought stress mediated by exogenous SA and CaCl₂ in tomato, we analyzed the six groups via cDNA-AFLP analysis with 256 primer combinations. In total, 6650 clear bands were obtained. Of the 6650 TDFs, 1436 (approximately 21.6%) were differentially expressed and were involved in seven expression patterns (Figure 2). Finally, 34 of these TDFs were sequenced successfully (Table 1). The sequenced TDFs were annotated by blasting against the SGN and NCBI databases.

Figure 2. Seven differential expression patterns were used to analyze the drought resistance response induced by exogenous SA and CaCl₂ in tomato. (A) Expressed in all groups; (B) induced by combination of drought and CaCl₂ (expressed in C10 only); (C) induced by combination of drought and SA (expressed in S0.3 only); (D) repressed by drought stress (expressed in CK1, CKC and CKS); (E) repressed by combination of drought and exogenous substances (expressed in CK1, CK2, CKC and CKS); (F) repressed by drought or exogenous substances (expressed in CK1 only); and (G) induced by drought stress (expressed in CK2, C10 and S0.3).

Table 1. Homology analysis of SA- and Ca²⁺-induced differentially expressed transcripts under drought in tomato.

TDF	Accession number	Homology to sequences in the NCBI database (Accession)	E-value	Expression pattern	Identity (%)
TDF3	JZ948559	Von Willebrand factor A domain-containing protein, transcript variant X3 (XM_010320173.1)	3e − 73	E	95
TDF7	JZ948562	B2 protein (LOC101243655), transcript variant X4 (XM_004231734.2)	1e − 82	E	97
TDF10	JZ948565	PREDICTED: titin (LOC101259599), transcript variant X10 (XM_010318008.1)	2e − 51	C	99
TDF11	JZ948566	PREDICTED: 4-hydroxyphenylpyruvate dioxygenase-like (LOC101257377) (XM_004240123.2)	2e − 126	G	99
TDF15	JZ948569	plasma membrane H+-ATPase (LHA2) (NM_001247548.1)	9e − 119	F	96
TDF16	JZ948570	PREDICTED: probable transcription factor PosF21 (LOC101250636) (XM_004230853.2)	5e − 116	C	97
TDF20	JZ948572	PREDICTED: Solanum lycopersicum, nucleolin (LOC101258799), transcript variant X5, mRNA (XM_004248939.2)	0.0	E	99
TDF22	JZ948576	PREDICTED: Solanum lycopersicum titin (LOC101259599), transcript variant X10, mRNA (XM_010318008.1)	2e − 114	C	98
TDF24	JZ948576	voucher H7072 cytochrome oxidase subunit I (COI) gene, partial cds; mitochondrial (EU827430.1)	8e − 12	B	85
TDF29	JZ948581	ARGOS (LOC100316893) (NM_001247750.2)	7e − 17	G	95
TDF34	JZ948586	Solanum lycopersicum transmembrane amino acid transporter family protein (LOC101261384), mRNA	1e − 04	B	100
TDF44	JZ948594	PREDICTED: microsomal glutathione S-transferase 3 (LOC101263072) (XM_004232613.2)	5e − 37	F	82
TDF50	JZ948600	PREDICTED: lisH domain and HEAT repeat-containing protein KIAA1468 homolog (LOC101253751) (XM_004233660.2)	2e − 41	C	96
TDF53	JZ948603	PREDICTED: calcium-dependent protein kinase 11 (LOC101251099), transcript variant X2 (XM_010324456.1)	3e − 26	D	89
TDF54	JZ948604	PREDICTED: chitin elicitor receptor kinase 1-like (LOC102595953), transcript variant X2 (XM_006361830.2)	0.14	D	77
TDF55	JZ948605	PREDICTED: calcium-dependent protein kinase 11 (XP_015077134.1)	0.013	D	71
TDF57	JZ948607	PREDICTED: UDP-D-apiose/UDP-D-xylose synthase 2 (LOC101247451) (XM_004228410.2)	3e − 102	F	99
TDF59	JZ948609	PREDICTED: elongation factor G, chloroplastic (LOC101245683) (XM_004245684.2)	4e − 87	E	96
TDF62	JZ948612	phytochromobilin synthase (AUREA) (NM_001247204.3)	3e − 88	C	98
TDF63	JZ948613	PREDICTED: Solanum lycopersicum transcription factor WER-like (LOC101245233), mRNA (XM_004235149.2)	2e − 20	F	76
TDF64	JZ948614	PREDICTED: heat shock protein 83-like (LOC101260143) (XM_004240669.2)	2e − 39	C	89
TDF65	JZ948615	PREDICTED: proteinase inhibitor 1-like (LOC101266562) (XM_004247647.2)	4e − 147	F	99
TDF67	JZ948617	PREDICTED: coatomer subunit beta'-3-like (LOC107007027), transcript variant X4 (XR_001455002.1)	1e − 06	B	100
TDF73	JZ948620	PREDICTED: Ciona intestinalis mitogen-activated protein kinase kinase kinase 4 (LOC778975), mRNA (XM_009863224.1)	0.24	F	91
TDF74	JZ948621	PREDICTED: ABC transporter G family member 11-like (LOC102600699) (XM_006351457.2)	0.083	B	70
TDF75	JZ948622	PREDICTED: Solanum pennellii elongation factor G, chloroplastic (LOC107028360), mRNA (XM_015229387.1)	2E − 70	G	93
TDF78	JZ948625	PREDICTED: protein ECERIFERUM 26-like (LOC101245886) (XM_004247830.2)	2e − 74	E	99
TDF81	JZ948628	PREDICTED: proteinase inhibitor 1-like (LOC101266562) (XM_004247647.2)	7e − 109	D	98
TDF82	JZ948629	PREDICTED: elongation factor G, chloroplastic (LOC107028360) (XM_015229387.1)	2e − 59	D	97
TDF87	JZ948634	PREDICTED: probable trans-2-enoyl-CoA reductase, mitochondrial (LOC101247598) (XM_004252983.2)	1e − 95	F	99
TDF93	JZ948638	PREDICTED: protein transport protein SEC13 homolog B (LOC107011415) (XM_015210912.1)	1e − 14	F	89
TDF98	JZ948641	PREDICTED: RINT1-like protein MAG2 (LOC101250005) (XM_004236156.2)	4e − 111	B	99
TDF99	JZ948642	PREDICTED: abscisic acid 8′-hydroxylase 1 (LOC101249565) (XM_004244388.2)	1e − 20	F	89
TDF100	JZ948643	PREDICTED: LRR receptor-like serine/threonine-protein kinase FLS2 (LOC101259509) (XM_004233819.2)	6e − 87	C	92

Note: Expression pattern B represents genes induced by combination of drought and CaCl₂; Expression pattern C represents genes induced by combination of drought and SA; Expression pattern D represents genes repressed by drought stress; Expression pattern E represents genes repressed by combination of drought and exogenous substances; Expression pattern F represents genes repressed by drought or exogenous substances; Expression pattern G represents genes induced by drought stress.

Validation of expression patterns by qRT-PCR analysis

To verify the results of cDNA-AFLP analysis, 12 sequenced TDFs were selected for qRT-PCR using 3 biological replicates. These TDFs were selected based on significantly different expression patterns identified by cDNA-AFLP analysis and homology to genes known to have a role in stress response, signal transduction and photosynthesis. The expression patterns of the 12 TDFs are shown in Table 1. The same expression patterns were found for each TDF with qRT-PCR analysis and cDNA-AFLP analysis. As shown in Figure 3, the expression of TDFs induced by CaCl₂ and drought (TDF34 and TDF74) peaked at different time points with increases of 11.2-fold at 24 h and 16.2-fold at 5 d, respectively. This result suggested that TDF34 may be induced at the early stage of drought stress and that TDF74 may play an important role at the late stage of drought stress. TDF62, TDF64 and TDF100 were all induced by SA and drought, and their expression levels were significantly increased, with maximum increases of 36.9-fold, 76.9-fold and 5.8-fold, respectively. Among them, TDF62 and TDF64 had higher expression levels at 3 and 5 d. These results were highly consistent with the cDNA-AFLP analysis, indicating the reliability of the cDNA-AFLP results. Moreover, these TDFs may play crucial roles in the drought resistance response induced by exogenous SA and CaCl₂ in tomato.

Figure 3. qRT-PCR analysis of 12 differentially expressed transcripts induced by exogenous SA and CaCl₂ under drought stress in tomato. Note: Mean values (n = 3) with standard deviation (±SD).

Sequence analysis and functional annotation

According to the annotation results, the functional classification of 34 TDFs mainly involved stress response, signal transmission and transportation (Figure 4). In particular, the number of TDFs involved in stress response accounted for 26.47%. These TDFs may play an important role in the drought resistance mechanism of tomato.

Figure 4. Functional annotation distribution of 34 differentially expressed fragments.

The basic-leucine zipper (bZIP) family of transcription factors (TFs) is known to play a role in abiotic stress responses [23]. TDF16 was identified as transcription factor POSF21 belonging to the bZIP class. Moreover, previous studies have suggested that POSF21 plays a positive role in the ABA signaling pathway and that POSF21 expression is positively modulated by ABA and cold stress treatment [24]. TDF16 was induced by SA under drought stress, and the relationship and interaction among POSF21, SA and ABA under abiotic stresses deserves further study. Heat shock proteins (HSPs) are the key components that act as molecular chaperones responsible for protein translocation and degradation under stress conditions and in many normal cellular processes [25,26]. In the present study, TDF64 (HSP83-like) was induced by SA under drought stress, and the expression level peaked at 3 d under drought stress (Figure 3h). The molecular chaperone protein, HSP83, which is a homologue of HSP90, protects organisms and cells against thermal damage [27,28]. In the present study, however, transcriptional reprogramming analysis revealed that HSP also plays a critical role in the defense mechanism induced by exogenous SA during drought stress in tomato. In addition, the FLS2 gene (TDF100), encoding pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), was induced in response to drought stress (Figure 3l). Similarly, previous studies have shown that FLS2 is upregulated in grapevines under drought stress [29]. However, the results of the present study help us to understand the response mechanism by which exogenous SA enhances drought resistance in tomato and suggest defense-related genes against drought stress for tomato breeding programs in the future.

Amino acids can improve plant resistance by regulating osmotic pressure, participating in ion transport and removing oxidative damage under various stresses. The transmembrane transport of these amino acids is critically linked to the specific amino acid transporter family protein [30,31]. In the present study, TDF34 was identified as a transmembrane amino acid transporter family protein, and the expression level of TDF34 was the highest at 24 h under drought stress (Figure 3d). Similar results have been observed in Populus simonii, in which amino acid transporter genes may contribute to drought stress tolerance [32]. These results suggest that transmembrane amino acid transporter family proteins may play a critical role in the drought resistance response induced by exogenous Ca²⁺. In the present study, TDF74 was identified as the ATP-binding cassette (ABC) transporter G family member 11-like, and its expression level peaked on 5 d under drought stress (Figure 3i). ABC transporters are important in plant growth, development and environmental stress responses [33,34]. Matsuda et al. [35] and Li et al. [36] reported similar findings wherein the ABC transporter responded to drought and salt stress, respectively. MAG2 provides tolerance to common osmotic stresses [37]. In this study, TDF98 was identified as the RINT1-like protein, MAG2, and was induced by exogenous Ca²⁺ under drought stress. However, these results suggest that MAG2 involves the response to drought resistance induced by exogenous Ca²⁺.

In this study, TDF11 was identified as 4-hydroxyphenylpyruvate dioxygenase-like (HPPD) and was induced under drought stress. In particular, the expression level of HPPD under exogenous SA or Ca²⁺ and drought stress treatment was significantly higher than that under drought stress alone (Figure 3b). The increase in HPPD expression might improve the resistance to drought in the plant. HPPD is associated with the biosynthesis of plastoquinones and tocopherols, and it participates in tyrosine catabolism [38]. Similarly, Xu et al. [39] have reported that HPPD is detected only under drought in Hippophae rhamnoides L. Previous studies have suggested that HPPD is important against different stresses as transcriptional regulators of tocopherol production [40]. These results indicate that the expression of HPPD may play a critical role in drought defense. In the present study, TDF29 was identified as Auxin Regulated Gene involved in Organ Size (ARGOS) and was induced by drought, especially by drought and exogenous SA or Ca²⁺, and ARGOS was indicated to be involved in the ethylene signaling pathway, leading to enhanced drought tolerance. Moreover, ARGOS has been reported to improve stress resistance in tobacco, maize, wheat and Arabidopsis [41–44].

Conclusions

In this study, we successfully analyzed the gene expression patterns associated with the exogenous SA- and Ca²⁺-induced defense response against drought stress in tomato. Several DEGs, including HSP, FLS2, transcription factor POSF21 and amino acid transporters, were determined to be related to drought resistance. These results facilitate our understanding of the potential mechanism by which exogenous SA and Ca²⁺ enhance drought resistance in tomato. More importantly, our study can provide a basis for cloning drought-related genes and breeding drought-resistant tomato varieties in the future.

Acknowledgements

The authors thank the Key Laboratory of Cold Biology in Heilongjiang University, Key Laboratory of Hereditary Improvement and Facility for Cultivation of Horticultural Crops in the North of Heilongjiang Province.

Disclosure statement

The authors report no conflict of interest.

Additional information

Funding

This study was funded by the China Agriculture Research System (CARS-23-A-16), the National Key R&D Program of China (2017YFD0101900), the Natural Science Foundation of Heilongjiang Province (C2017024) and the Breeding of New Staple Vegetable Varieties with High Quality and Disease Resistance (2019ZX16B02).