Integrated transcriptomic and proteomic analysis of Tritipyrum provides insights into the molecular basis of salt tolerance

Background Soil salinity is a major environmental stress that restricts crop growth and yield. Methods Here, crucial proteins and biological pathways were investigated under salt-stress and recovery conditions in Tritipyrum ‘Y1805’ using the data-independent acquisition proteomics techniques to explore its salt-tolerance mechanism. Results In total, 44 and 102 differentially expressed proteins (DEPs) were identified in ‘Y1805’ under salt-stress and recovery conditions, respectively. A proteome-transcriptome-associated analysis revealed that the expression patterns of 13 and 25 DEPs were the same under salt-stress and recovery conditions, respectively. ‘Response to stimulus’, ‘antioxidant activity’, ‘carbohydrate metabolism’, ‘amino acid metabolism’, ‘signal transduction’, ‘transport and catabolism’ and ‘biosynthesis of other secondary metabolites’ were present under both conditions in ‘Y1805’. In addition, ‘energy metabolism’ and ‘lipid metabolism’ were recovery-specific pathways, while ‘antioxidant activity’, and ‘molecular function regulator’ under salt-stress conditions, and ‘virion’ and ‘virion part’ during recovery, were ‘Y1805’-specific compared with the salt-sensitive wheat ‘Chinese Spring’. ‘Y1805’ contained eight specific DEPs related to salt-stress responses. The strong salt tolerance of ‘Y1805’ could be attributed to the strengthened cell walls, reactive oxygen species scavenging, osmoregulation, phytohormone regulation, transient growth arrest, enhanced respiration, transcriptional regulation and error information processing. These data will facilitate an understanding of the molecular mechanisms of salt tolerance and aid in the breeding of salt-tolerant wheat.


INTRODUCTION
Wheat (Triticum aestivum) is the most important food crop in the world. Its total area, total output and total trade volume all rank first among food crops, and it provides 19% of available calories for humans (Yan et al., 2020). It is predicted that by the middle of the 21 st century, increasing salinization will lead to the loss of 50% of the world's arable land quantitative characteristics (accuracy, sensitivity and selectivity) achieved in targeted proteomics but on the scale of thousands of proteins. The E genome species (e.g., halophile wheatgrass Thinopyrum elongatum) of the Triticeae are invaluable sources of salt tolerance (Jauhar, 1990;Munns & Tester, 2008;Margiotta et al., 2020). Tritipyrum derived from the wide crosses of Triticum and Thinopyrum show salt tolerance (King et al., 1997;Yuan & Tomita, 2015). Currently, there are limited reports on the molecular mechanisms of Tritipyrum salt tolerance; therefore, they remain largely unknown. Here, protein markers and pathways were identified using DIA proteomic and transcriptomic profiling, and these will aid in elucidating the molecular mechanisms involved in the salt tolerance of Tritipyrum. The results provide valuable information for the molecular breeding of improved salt tolerance in wheat and other crops.

Plant materials
'Y1805' is a stable progeny from the wide cross between Triticum aestivum and Thinopyrum elongtum. Salt-tolerant Tritipyrum ('Y1805') and salt-sensitive Triticum aestivum ('Chinese Spring', 'CS') were used in this experiment. These genotypes were chosen on the basis of preliminary experiments. 'Y1805' shows plump kernels, mid-maturity and high complex disease-resistance levels, including immunity and high resistance levels to scab, rust and powdery mildew of wheat.

Plant growth conditions and stress treatments
The seeds of 'Y1805' and 'CS' were germinated in a growth chamber (relative humidity of 75% and a 20/15 C light/dark photocycle). The seedlings were sown on a floater board in 1/2 Hoagland's solution with a 16-/8-h light/dark cycle, irradiance of 400 mmol m −2 s −1 and the same temperature and humidity as in the germination chamber. The culture solution was refreshed every 3 days. On the 14 th day (two-leaf stage), salt-stress treatments (1/2 Hoagland's solution supplemented with 250 mM NaCl) were started. A 250-mM NaCl concentration was used as a result of preliminary gradient tests. The pH of the culture solution was 6.0. The first wheat root and shoot samples of a uniform size were selected at 5 h (T1 stage) after exposure to salt stress. The materials were recovered (in 1/2 Hoagland's solution without NaCl) after 24 h of salt stress. The second and third samplings were performed at 1 h (T2 stage) and 24 h (T3 stage) after recovery, respectively. These sampling stages were chosen on the basis of the phenotypic and physiological data. Normal (1/2 Hoagland's solution without NaCl) control cultured materials, CK1, CK2 and CK3, were used as parallel controls for T1, T2 and T3, respectively. The root samples of the T1 and T2 stages were immediately frozen in liquid nitrogen after sampling and stored at −80 C for the transcriptomic analysis, proteomic analysis, and confirmational qRT-PCR. The samples at the T3 stage were only used for growth analysis. Three biological replications and two technical replications were employed, and at least 10 seedlings were mixed per replicate.

Protein analysis
Protein extraction and DIA-MS/MS analysis Total proteins of wheat roots were extracted using the phenol method with slight modifications (Isaacson et al., 2006). Briefly, 0.5 g of wheat roots were ground into a fine powder in a lysis buffer containing 8 M urea, 400 mM ammonium bicarbonate, 10 mM dithiothreitol and a protease inhibitor cocktail (Roche, Basel, Switzerland). Then, 2× volume of phenol saturated with Tris-HCl (pH 7.5) was added, and centrifuged at 25,000 g for 15 min at 4 C. The upper phenolic phase was collected, and then, 5× volume of precooled precipitation solution, containing 0.1 M ammonium acetate in methanol and 10 mM dithiothreitol, was added to the protein mixture. Each sample was maintained for 2 h at −20 C. Then, the samples were centrifuged at 25,000 g for 15 min at 4 C, and the supernatant was removed. The pellets were further washed with precooled acetone with centrifugation at 20,400 g for 40 min. Then, trypsin [protein:trypsin = 40:1 (w/w)] was added for enzymolysis, followed by desalination, vacuum drying, and redissolution. Protein concentrations were measured by using Nanodrop ND-1000 (Thermo Fisher Scientific, San Jose, CA, USA).
The peptide samples were separated using an UltiMate 3000 UHPLC (Thermo Fisher Scientific, San Jose, CA, USA), ionized by a nanoESI source and then put into a Q-Ex Active HF tandem mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) for DIA mode detection. The spectral library was constructed using the DIA of targeted samples. Spectronaut was used to effectively deconvolute, accurately identify and quantitatively analyze the data (Bernhardt et al., 2014).

DEP identification and bioinformatics analysis
The peak area of an ion pair was extracted using Spectronaut. Then, the Msstats software package was used to complete the error correction and normalization steps (Choi et al., 2014). Two conditions, fold change ≥ 2 and false discovery rate < 0.05, were used as the DEP criteria. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, protein-protein interaction (PPI) and subcellular localization analysis were performed for these DEPs.

Proteome-transcriptome-associated analysis
The basis of proteome-transcriptome association analysis is the central principle. Correlation analyses of mRNAs and proteins having the same change trend were carried out, including expression correlation analyses and metabolic pathway map integration analyses.
The quantitative RT-PCR (qRT-PCR) analysis A total of 15 candidate proteins were verified by qRT-PCR (Table S1). Total RNA from the same samples used in the proteome analysis were reverse-transcribed using Power SYBRÒ Green PCR Master Mix (Applied Biosystems, Foster, CA, USA). qRT-PCR amplification was performed on an ABI StepOne Real-Time PCR System. The relative expression levels were calculated in accordance with the 2 −ΔΔCt method, with three biological replications and three technical replications (Depuydt & Hardtke, 2011), and β-actin and 18S RNA were used as the internal control.

Statistical analyses
The data were analyzed using SPSS statistics 19.0 (IBM Corp., Armonk, NY, USA). Statistical differences between means were determined by Duncan's multiple range test at a significance level of p < 0.05 after displaying a significant effect during an ANOVA. Pearson's correlation analysis of binary variables was performed, and two variables were considered significantly correlated at the p < 0.05 level.

Response of plant growth to salt stress and recovery in two wheat varieties
The plant length and dry weight of salt-tolerant Tritipyrum 'Y1805' did not change significantly at any of the three stages compared with the corresponding controls ( Fig. 1). Additionally, 'Y1805' maintained normal growth under salt-stress conditions. The plant length of salt-sensitive 'CS' was significantly (p < 0.05) less than that of the control at the T2 stage. At the T3 stage, the plant length and dry weight of 'CS' decreased significantly (p < 0.05) by 6.37% and 15.69%, respectively, compared with the controls. Thus, 'Y1805' showed a strong salt tolerance under salt-stress conditions and a rapid recovery capability after the salt stress was removed.
In total, 14 DEPs were identified under both salt-stress and recovery conditions in 'Y1805' (Table S4). Therefore, these DEPs might simultaneously participate in the responses to salt stress and recovery. Moreover, these DEPs showed the same expression patterns at the two stages, with 10 DEPs being upregulated and 4 DEPs being downregulated compared with the respective corresponding controls. More specific DEPs were identified after recovery than under salt-stress conditions in 'Y1805'. Thus, salt stress and recovery regulated different types of proteins, allowing plants to adapt to environmental stress or growth restoration, respectively.
15 DEPs closely related to salt stress were selected from 'Y1805' for the qRT-PCR analysis (Table S1). The correlation coefficient of the relative expression levels between DEPs and qRT-PCR was significant (r = 0.802, p = 1.97E −04 ). The up-or downregulation of the proteins, as determined by the proteomics analysis, was corroborated by the qRT-PCR experiment (Fig. 2C). These data demonstrated that the proteomics accurately reflected the responses of Tritipyrum to salt stress and recovery. Plant antioxidant proteins are part of defense systems and play important roles in response to salt stress. 'Antioxidant activity' (GO:0004601) and 'molecular function regulator' (GO:0004867) were two 'Y1805'-specific GO terms (Fig. 3A, Figs. S1, S2). The antioxidative activity was regulated by three peroxidase (POD) superfamily proteins: peroxidase 5 AT1G14550 (TraesCS1B01G096300.1), peroxidase 47 AT4G33420 (TraesCS2B01G616700.1) and peroxidase 50-like AT4G37520 (TraesCS2A01G502100.1) ( Table S5). POD plays pivotal roles in the removal of H 2 O 2 . The molecular functional regulatory protein was subtilisin chymotrypsin inhibitor-2A EMT04243 (TraesCS3A01G093900.1). The subcellular localization of 'Y1805'-specific DEPs under salt-stress conditions revealed that most were cytosolic proteins, followed by chloroplastic and then extracellular (Fig. 3C).
The functional association diagram of 'Y1805' DEPs/DEGs under salt-stress conditions is shown in Fig. 6. Among the associated proteins, galactinol-sucrose galactosyltransferase RFS1 (TraesCS3A01G092800.1) was upregulated in 'galactose metabolism' (ko00052) and is a key protein in raffinose synthesis. PAL1 and AT1G14550, which are involved in the synthesis of cinnamic acid and lignins, respectively, were upregulated in 'phenylpropanoid biosynthesis' (ko00360). In addition, PAL1 is also a key protein in 'phenylalanine metabolism'. ADT6, which takes part in phenylalanine synthesis, was upregulated in 'phenylalanine, tyrosine and tryptophan biosynthesis' (ko00400).
Probable serine acetyltransferase 1 SAT1 (TraesCS3A01G287400.1) and 1-aminocyclopropane-1-carboxylate oxidase 1 EFE (TraesCS5A01G234300.1) were upregulated in 'cysteine and methionine metabolism' (ko00270) during the recovery process in 'Y1805' (Fig. 7). They participate in the synthesis of acetyl-L-serine and ethylene, respectively. Xylanase inhibitor Xip-R1 (TraesCS4A01G173800.1), which is involved in the synthesis of chitobiose and GlcNAc, was upregulated in 'amino sugar and nucleotide sugar metabolism' (ko00520). PAL1, AT1G68850 and berberine bridge enzyme-like 27 AT5G44410 (TraesCS7B01G273600.1) were upregulated in 'phenylpropanoid biosynthesis'. time under stress conditions (Zhu, 2001;Achard et al., 2006). Slower growth may allow plants to redirect resources to overcome or temporarily cope with stress. Here, CHR12 was 'Y1805'-specific under salt-stress conditions, during which the expression level was very high (4.0572), but not after recovery. After recovery, 73 'Y1805'-specific DEPs were identified that should contribute to its fast recovery after removing the salt stress. In total, 14 DEPs were identified under both salt-stress and recovery conditions in 'Y1805'. More specific DEPs were identified after recovery than during the salt stress in 'Y1805'. Therefore, the responses to salt stress and recovery were two independent, but closely related, processes.

Protein responses to salt stress Antioxidant proteins
As members of the defense system, plant antioxidant proteins remove ROS and reduce the damage caused by oxidative stress. The overexpression of class III peroxidase genes enhances the antioxidant capacity of maize under abiotic stress conditions (Wang et al., 2015). POD (B4G0X5) was upregulated in both salt-tolerant and salt-sensitive maize to improve the salt tolerance of the two maize varieties (Chen et al., 2019). Here, the 'antioxidant activity' pathway was enriched, and three peroxide superfamily proteins (AT1G14550, AT4G33420 and AT4G37520) were identified under salt-stress conditions only in 'Y1805'. AT1G14550, which could remove excessive H 2 O 2 , thereby enhancing the antioxidant capacity, was upregulated at both the transcriptional and protein levels. However, no POD was found in 'CS', resulting in excessive ROS accumulation, which led to oxidative damage.

Osmoregulation
Soluble sugars participate in osmotic regulation and also play important roles in maintaining protein stability. Glycosyltransferase (A0a1d6ewj3) was upregulated in both salt-tolerant and -sensitive maize cultivars, and plays an active role in salt tolerance (Chen et al., 2019). RFS1 is involved in the synthesis of cottonseed sugar, which acts as an osmotic protective agent in plant cells (Nishizawa, Yabuta & Shigeoka, 2008). In this work, 'carbohydrate metabolism' was significantly enriched. Trehalose has an important physiological role in osmotic protection, and it improves the tolerance of plants to abiotic stress (Wang et al., 2020). In this experiment, upregulated trehalose-phosphate phosphatase 3 TPP3 (TraesCS2A01G161200.1) was detected only in 'Y1805', and it could remove phosphate from trehalose-6-phosphate to produce free trehalose. Therefore, soluble sugars might contribute to the strong salt tolerance of 'Y1805'.

Phytohormones
ABA plays an important role in regulating water balance, osmotic protection and cell wall component synthesis, and it is also involved in the control of phosphatidyl inositol signaling and chromatin-mediated stress tolerance mechanisms, which alleviate the physiological effects of salt stress and enhance the protective mechanisms against stress (Szypulska, Jankowski & Weidner, 2017). Low doses of ABA (10-100 mM) induce antioxidant defense mechanisms to prevent oxidative damage to membrane lipids and proteins (Jiang & Zhang, 2001;Wang, Kuang & He, 2010). Pathogenic-associated protein 10 is involved in ABA synthesis, which is upregulated in salt-tolerant maize '8723' but stable in salt-sensitive maize 'P138' (Chen et al., 2019). A similar result was obtained in our study. HVA1 was upregulated at both the transcriptional and protein levels under salt-stress conditions only in 'Y1805'. The upregulated expression of HVA1 produces ABA, which is beneficial to maintaining the water balance and preventing oxidative damage.
Ethylene induces the occurrence of adventitious roots and root hairs. The presence of root hairs increases the absorption area of roots, and they secrete a variety of substances, such as organic acids. Moreover, ethylene increases the flavonol content of guard cells. Flavonols act as antioxidants, reduce ROS levels in guard cells and inhibit stomatal closure (Murata, Mori & Munemasa, 2015). An increase in 1-aminocyclopropane-1-carboxylic acid oxidase leads to a large amount of ethylene production (Qin et al., 2007). An ethylene receptor (a negative regulator of ethylene signaling) expression level increased more than nine-fold in salt-sensitive wheat 'Jinan 177' and only two-fold in salt-tolerant 'Shanrong No. 3' in response to salt stress. Therefore, the lower ethylene receptor expression in 'Shanrong No. 3' under salt-stress conditions may be partly responsible for its lower sensitivity to salinity than 'Jinan 177' (O'Malley et al., 2005;Wang, Kuang & He, 2010). Here, upregulated EFE was found only in 'Y1805' under salt-stress conditions and is beneficial for its strong salt tolerance. Under salt-stress conditions, upregulated HVA1 and EFE play vital roles in the salt tolerance of 'Y1805'.

Protein responses to recovery
Cell wall synthesis UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, because its product, UDP-glucuronic acid, is the common precursor for arabinose, xylose, galacturonic acid and apiose residues found in cell walls, which play important roles in primary cell-wall formation (Reboul et al., 2011;Siddique et al., 2012).
Here, an upregulated UDP-glucose 6-dehydrogenase 2, UGD2 (TraesCS4A01G237900.1), which improves the synthesis of cell-wall polymers, was detected during the recovery process only in 'Y1805'. Caffeic acid 3-O-methyltransferase (OMT) catalyzes the multi-step methylation reactions of hydroxylated monomeric lignin precursors, which have important roles in lignin biosynthesis (Ma & Xu, 2008). OMT1 was a 'Y1805'-specific upregulated protein, and it is involved in lignin biosynthesis. With the increase in lignin synthesis, 'Y1805' was able to repair cell walls rapidly during the recovery process, resulting in a rapid return to normal growth. These upregulated UGD2 and OMT1 proteins in 'Y1805' strengthen cell walls and play master roles in the line growth recovery.

Improved respiration
Stress conditions stimulate the expression of GPDHC1, which is related to the availability of oxygen and a key protein affecting mitochondrial respiration (Shen et al., 2006). In this study, GPDHC1 (TraesCS3A01G330600.1) was upregulated only in 'Y1805' during the recovery process, indicating that its respiration was improved, which would provide essential energy and intermediate metabolites for the plant's rapid recovery after being subjected to salt stress.

Phytohormones and signal transduction
During the recovery process, HVA1 was upregulated in both 'Y1805' and 'CS', but the expression level in 'Y1805' was higher than that in 'CS' (Table S6). OMT1 catalyzes the transfer of a methyl group onto N-acetylserotonin, which produces melatonin (N-acetyl-5methoxytryptamine) (Byeon et al., 2015). Melatonin has antioxidative effects in plants. It scavenges ROS mainly by providing electrons. Melatonin also promotes the formation of lateral roots. Indole-3-acetic acid (IAA)-amino acid hydrolase ILR1-like 2 produces free IAA by hydrolyzing IAA-amido (Leclere et al., 2002;Rampey et al., 2004). This enzyme (TraesCS3B01G283900.1) was upregulated only in 'Y1805', resulting in the production of free IAA, which promotes plant growth. Salicylic acid (SA) reduces water loss by inhibiting stomatal opening to alleviate salt stress. EPS1 (a BAHD acyltransferase-family protein) is an essential protein for pathogen-induced SA accumulation (Torrens-Spence et al., 2019). Here, EPS1 (TraesCS3A01G367900.1) was upregulated only in 'Y1805', which resulted in an SA accumulation. Together, these phytohormones had a critical impact on the rapid growth recovery of 'Y1805'.
The responses of plants to abiotic stress are to consolidate their growth and development by regulating complex signaling networks for stress adaptation (Abreu et al., 2013). In plants, Ca 2+ signals play important roles in response to abiotic stresses, such as salinity, drought and temperature. The CML proteins (Ca 2+ sensors) play unique roles in plant Ca 2+ -signaling pathways (Ogunrinde et al., 2017). The Ca 2+ signaling network is closely related to the activation of the salt overly sensitive 'signal transduction' pathway that regulates cellular Na + and K + homeostasis in Arabidopsis (Zhu, 2002;Brini et al., 2007). In this study, the 'signal transduction' pathway was significantly enriched, and CML16 was upregulated only in 'Y1805' after recovery. Therefore, Ca 2+ signaling was involved in the responses to recovery in 'Y1805'.

Transcriptional regulation and error information processing
DExH-box ATP-dependent RNA helicase DExH10 (HEN2) assists the exosome-mediated processing and/or degradation of small nucleolar RNAs and their precursors, microRNA precursors and long non-coding RNAs, as well as a large number of spurious transcripts derived from antisense and non-annotated regions. In addition, HEN2 is involved in the degradation of excised introns and incompletely spliced or otherwise mis-transcribed or mis-processed mRNAs (Lange et al., 2014). DEAD-box ATPdependent RNA helicase 14 (DRH1) is a novel type of ATP/dATP-dependent RNA helicase and polynucleotide-dependent ATPase that is capable of unwinding double-stranded RNA in the presence of ATP or dATP and of hydrolyzing ATP (Okanami, Meshi & Iwabuchi, 1998). In this study, 'transcription regulation activity' pathway was enriched, and upregulated HEN2 (TraesCS2A01G154400.1) and DRH1 (TraesCS3A01G130200.1) were detected only in 'Y1805' during the recovery process, indicating that transcriptional regulation and error information processing performed crucial roles in the line rapid recovery from salt stress.

DEP interactions
Cell wall integrity is an important factor that determines salt-stress tolerance and plant growth (Feng et al., 2018;Zhao et al., 2018). Lignin is produced and the cell wall is hardened through the actions of ADT6 and PAL1 (Huang et al., 2010). CEAS1 is involved in the formation of the primary cell wall. IRX1 participates in the formation of the secondary cell wall and plays an important role in the thickening of the xylem cell wall (Turner & Somerville, 1997). CSLA01 is involved in the synthesis of galactomannan, which is a non-cellulose polysaccharide in plant cell walls. ADT6, PAL1, CEAS1, IRX1 and CSLA01 interacted with each other under salt-stress conditions in 'Y1805', and they were closely related to the synthesis and strength of the cell wall. In addition, IRX1 and CSLA01 were 'Y1805'-specific DEGs. Therefore, the cell wall performed major roles in the salt tolerance of 'Y1805'.
Abiotic stresses, such as salinity, drought, osmotic, cold and freezing temperatures produce cellular water deficits, which lead to the accumulation of highly hydrophilic LEA proteins (Battaglia et al., 2008;Hundertmark & Hincha, 2008). Nine LEA DEGs (Tel3E01G270600, Tel3E01G426400, Tel3E01G628300, Tel4E01G475800, Tel5E01G589800, Tel5E01G665500, Tel6E01G622800, Tel6E01G623000 and Tel7E01G989400) originating from Thinopyrum elongatum were detected among top 30 DEGs under salt-stress conditions in 'Y1805' according to our transcriptome analysis. Histidine is a key component of LEA proteins. APG10 is not only a hydrophilic protein, but also an important protein in histidine biosynthesis. In this study, APG10 was the hub protein during the recovery process in 'Y1805', and it was downregulated. The result revealed that LEA proteins contributed to salt tolerance, and their expression levels fell rapidly during the recovery process. OMT1 is involved in the biosynthesis of lignin, which is used to repair the cell wall rapidly during the recovery process in 'Y1805'. It also produced melatonin, which improved the rapid growth recovery of 'Y1805'. RPL24 is an important protein for photosynthesis and optimal plastid performance, and it plays a key role in the biosynthesis of thylakoid membrane proteins encoded by chloroplast genes (Romani et al., 2012). OMT1 and RPL24 were the second most important hub proteins in the 'Y1805' PPI network. They were both 'Y1805'-specific and upregulated during the recovery process.

CONCLUSION
Forty-four and 102 DEPs were identified in 'Y1805' under salt-stress and recovery conditions, respectively, using the DIA proteomics techniques. In total, 13 and 25 upregulated DEPs/DEGs were found under salt-stress and recovery conditions, respectively, using a proteome-transcriptome-associated analysis. Compared with 'CS', 'Y1805' had specific responses to salt stress and recovery. Scavenging ROS, osmoregulation, phytohormone regulation, transient growth arrest and cell-wall strengthening might be of paramount importance in its strong salt tolerance (Fig. 8). After recovery, 'Y1805' restored growth by cell-wall strengthening, respiration enhancement, phytohormone regulation, signal transduction, and transcriptional regulation and error information processing (Fig. 8). This work provides a new perspective on the molecular mechanisms underlying the adaptation of Tritipyrum to salt stress, and the results may accelerate salt-tolerant crop breeding programs.