Alteration of proteome in germinating seedlings of piegonpea (Cajanus cajan) after salt stress

Abstract

Pigeonpea (Cajanus cajan) is an important crop in semi-arid regions and a significant source of dietary proteins in India. The plant is sensitive to salinity stress, which adversely affects its productivity. Based on the dosage-dependent influence of salinity stress on the growth and ion contents in the young seedlings of pigeonpea, a comparative proteome analysis of control and salt stressed (150 mM NaCl) plants was conducted using 7 days-old seedlings. Among various amino acids, serine, aspartate and asparagine were the amino acids that showed increment in the root, whereas serine, aspartate and phenylalanine showed an upward trend in shoots under salt stress. Furthermore, a label-free and gel-free comparative Q-Tof, Liquid Chromatography-Mass spectrometry (LC–MS) revealed total of 118 differentially abundant proteins in roots and shoots with and without salt stress conditions. Proteins related to DNA-binding with one finger (Dof) transcription factor family and glycine betaine (GB) biosynthesis were differentially expressed in the shoot and root of the salinity-stressed seedlings. Exogenous application of choline on GB accumulation under salt stress showed the increase of GB pathway in C. cajan. Gene expression analysis for differentially abundant proteins revealed the higher induction of ethanolamine kinase (CcEthKin), choline-phosphate cytidylyltransferase 1-like (CcChoPh), serine hydroxymethyltransferase (CcSHMT) and Dof protein (CcDof29). The results indicate the importance of, choline precursor, serine biosynthetic pathways and glycine betaine synthesis in salinity stress tolerance. The glycine betaine protects plant from cellular damages and acts as osmoticum under stress condition. Protein interaction network (PIN) analysis demonstrated that 61% of the differentially expressed proteins exhibited positive interactions and 10% of them formed the center of the PIN. Further, The PIN analysis also highlighted the potential roles of the cytochrome c oxidases in sensing and signaling cascades governing salinity stress responses in pigeonpea.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-021-01116-w.

Introduction

Pigeonpea (Cajanus cajan (L.) Millspaugh), is a food legume for the subtropical and tropical regions of the world including India, East Africa and South East Asia. India is the major consumer and producer of pigeon pea with the 4.87 million metric tons (mmt) per year under 5.38 million hectares cultivation and an average yield of 904 kg/h (FAOSTAT 2020, http://www.fao.org/faostat/) and dietary energy supplied from pulses is 217 kcal/capita/day for Indians. The relatively low crop yields may be attributed to the various stresses (biotic and abiotic), including salinity, incurred by the plant when grown in diverse agro-climatic conditions.

Abiotic stresses negatively affect plants, thereby limiting their growth and development (Ismail and Horie 2017). It has been predicted that by the middle of the twenty-first century, approximately > 50% of the area will be unfavorably affected by the salinization of soil. Salt stress triggers primary (ionic and osmotic) and secondary (ion inequality, nutrient insufficiency and oxidative) stresses in plants. Ionic stress disturbs the ionic homeostasis of the plant, which can be maintained by various Na⁺ and K⁺ transporters (Ismail and Horie 2017). To combat the osmotic stresses, several plants have the ability to synthesize proline and glycine betaine (Cha-um et al. 2019). Although the proline biosynthesis is conserved in all plants, those for glycine betaine (GB) seems to depend on plant species (Rathinasabapathi et al. 1997; Takabe et al. 2015). Some plants can synthesize GB whereas many plants do not synthesize GB like rice due to nonfunctional choline monooxygenase (CMO) gene and grass species (Shirasawa et al. 2006; Zhang et al. 2020). Sugar beet, spinach, barley and wheat synthesize GB and are known as GB accumulators. GB is synthesized by two step oxidations of choline. Choline is first oxidized to betaine aldehyde by CMO which is further oxidized by betaine aldehyde dehydrogenase (BADH) to GB. The CMO enzyme in GB accumulating dicotyledonous Amaranthaceae (Chenopodiaceae, Beta vulgaris L., spinach, Atriplex etc.) is localized in chloroplast (Rathinasabapathi et al. 1997; Rontein et al. 2002) whereas the peroxisome localization of CMO and BADH in GB accumulating monocot, barley, has been reported (Fujiwara et al. 2008; Mitsuya et al. 2011). Furthermore, CMO like protein could be found in GB non-accumulating plants (Cha-um et al. 2019; Takabe et al. 2015). Arabidopsis is a well-known GB non-accumulator, but CMO homolog gene(s) can be found in Arabidopsis genome (Hibino et al. 2002). The Arabidopsis CMO homolog did not show the choline oxidation activity (Hibino et al. 2002). Hitherto, GB accumulation in pigeonpea has been reported by Kumar et al. (2017) although pathway for glycine betaine synthesis has not yet been studied in pigeonpea.

The proteome profiling is a powerful technique to study the functional complexities under stress. The proteomics studies have been done on various abiotic stresses for soybean (Yin and Komatsu 2017). Various salt stress tolerant mechanisms have been deciphered by using iTRAQ like ionic balance, signal transduction, membrane transport and ROS (Li et al. 2020). Integrated transcriptomics and proteomic analysis revealed regulation mechanism in maize after NaCl stress (Chen et al. 2021). Differentially expressed proteins involved in signal transduction, transcription factors, enzyme modulators and oxidoreductases were identified from the guard cells of Chenopodium quinoa (wild) after 300 mM NaCl stress (Rasouli et al. 2021).

The sequencing of pigeonpea is now available (Mahato et al. 2018; Singh et al. 2012; Varshney et al. 2012). It would be feasible to unravel the molecular mechanisms of salt tolerance in pigeonpea and accelerate breeding using high throughput genomic tools. In this study, we focused on the proteomic analysis of pigeonpea during the seedling stage. Proteome profiling is an attractive paradigm for identifying an array of proteins that are differentially regulated during stress. Amongst legumes, soybean proteome has been documented for flood (Yin et al. 2014; Kamal et al. 2015) and salinity stress (Aghaei et al. 2009). Protein profile of C. cajan are not available. We developed the protocol for protein extraction and 2D-gel electrophoresis from different tissues of pigeonpea (Singh et al. 2015). In the current study, gel-free quadrupole time of flight (Q-ToF) was used for comprehensive proteome profiling and amino acid analysis was done for the root and shoot of the pigeonpea seedlings grown under salt stress. Several proteins were identified that belonged to different functional categories.

Materials and methods

Seed germination

Cajanus cajan var Asha ICPL87119 seeds were given washings with Tween-20 (0.1% (v/v)) for 10 min and seeds were surface sterilized using sodium hypochlorite (4% v/v) for 10 min and rinsed to remove all chemical traces. Ten disinfected seeds each were germinated on germination paper soaked with half-strength Hoagland’s nutrient solution in petri dish (diameter = 9 cm) and maintained at 25 ± 2 °C and 70% relative humidity, under dark conditions, in a growth chamber (Aghaei et al. 2009; Negi et al. 2016). The germination paper was imbibed with 5 mL of Hoagland’s nutrient solution supplemented with six different concentrations of NaCl (0, 50, 100, 150, 200, 300 mM) each following the methods as described earlier (Fercha et al. 2016; Xu et al. 2011). The collected roots and shoots of the seedlings were analyzed for ion composition, morphological traits, proteome profile and gene expression after seven days of germination because the responses of various concentrations of NaCl was clear after one week. Three individual experimental replicates of control and NaCl treated samples were collected and each replicate consisted of collective sample of twenty seedlings. For protein and RNA extraction, the harvested tissues (both shoot and root) were immediately freezed using liquid nitrogen and stored at − 80 °C.

Measurement of ion (Na⁺/K⁺) content

Harvested roots and shoots from the control and salt-stressed seedlings were oven dried and finely powdered for the estimation of Na⁺ and K⁺ contents. The powdered samples (0.5 g) were digested with concentrated nitric acid (15.8 M) in a microwave digestion chamber at 110 °C till the mixture turned colorless and 100 mL of the final volume was adjusted using deionized water. The ion contents in the digested mixture were quantified using flame photometer (Labtronics, Flame Photometer, Model LT-65, India).

Amino acid analysis

Amino acids were extracted from the root and shoot tissue as earlier standardized by (Waditee et al. 2005). The samples were pulverized with nine volumes of pure methanol followed by centrifugation (1500 × g) for 5 min at 4 °C. The supernatant was salvaged and re-extraction of the pellet was done using methanol (90%). Pooled methanol extracts were dried in a vacuum rotary evaporator at 45 °C. The pellet obtained was re-dissolved in MilliQ water (0.5 mL) and mixed with an equal volume of chloroform, followed by quick vortexing and centrifuged (1500 × g) for 5 min at 4 °C. The top most aqueous layer was collected and passed through a membrane filter (0.22 μm) and the filtrate was dried using a vacuum rotary evaporator at 45 °C. The extracts were dissolved in mobile-phase solution (MA) containing 14.1 g of trilithium citrate tetrahydrate, 70 mL of 2-methoxyethanol and 13.3 mL of 60% perchloric acid per liter and injected into an amino acid analyzer with a shim-pack Li column. A Shimadzu Nexera LC 40 X3 with photodiode array and RF detectors were employed for analysis (Shimadzu, Japan).

Analysis of morphological traits

For documenting the morphological features viz. primary root length, shoot length, the number of lateral roots and the total length of lateral roots, the seven-day-old seedlings were transferred to 1% agar (w/v) Petri dishes and roots were spread for scanning. Ten seedlings per experimental replicate (total 30 seedlings) were scanned by using a desktop scanner (GE Image Scanner III) at 600 dpi directly from the Petri dishes. The captured images were analyzed for number and length of roots and shoot by using ImageJ (a Java image-processing program; http://rsb.info.nih.gov/ij).

Statistical analysis

R-package was used for the statistical analysis of the data. The experimental data values were the mean values from three independent sets, each done with ten replicates and the results are presented as mean ± standard error (SE). The statistical significance at P ≤ 0.05 has also been calculated. One-way ANOVA was employed for the statistical analysis of datasets. For significance differences Duncan’s test was done.

Protein extraction and quantification

The protein extraction was done from the root and shoot tissues for both salinity-stressed and control conditions after seven days of germination (Singh et al. 2015). Briefly, the protein was extracted by pulverizing the plant tissue using liquid nitrogen and the fine powder was homogenized with 0.1 M phosphate buffer (pH 7.0). The supernatant was extracted after centrifugation (15,870 × g) for 15 min at 4 °C. The proteins were precipitated using trichloroacetic acid (TCA) buffer containing acetone, 10% (w/v) TCA and 0.07% 2-mercaptoethanol (2-ME) and kept for 1 h at − 20 °C. The protein was pelleted down using centrifugation (15,870 × g) for 15 min at 4 °C. The pellet was washed thrice with wash buffer containing acetone, 0.07% 2-ME, 2 mM ethylenediaminetetraacetic acid (EDTA) and EDTA-free protease inhibitor cocktail tablet (Roche) and a final rinse with pure chilled acetone. The air-dried pellet was kept at − 80 °C overnight and re-suspended in rehydration buffer (7 M urea, 2 M thiourea and 4% (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS)) and was quantified using Bradford method (Bradford 1976).

Sample preparation for LC–MS

An aliquot of 100 μg protein was taken and diluted with ammonium bicarbonate solution to a final concentration of 1 M urea. The sample was treated with 100 mM dithiothreitol (DTT) at 95 °C for one hour, followed by 250 mM iodoacetamide (IAA) at room temperature in the dark for 45 min. The protein was digested using trypsin enzyme (Sigma Aldrich) and incubated at 37 °C overnight. Trifluoro acetic acid (TFA, 0.1%) was added to stop the digestion. The protein digest was dried using vacuum concentrator and dissolved in 50 µL of 0.1% formic acid. The solution was filtered by a syringe filter (0.22 µm) and injected directly into the Ultra Performance Liquid Chromatography (UPLC) system.

LC–MS analysis

UPLC was performed on a Waters ACQUITY UPLC™ system (Waters Corporation, Milford, MA, USA), containing a binary solvent delivery manager and a sample manager. Chromatographic separations were carried on a 75 µm × 150 µm × 1.7 µm Nano Acquity BEH C18 chromatography column (Cat. no:186003543). The extracts and standards were analyzed by UPLC chromatography using a mobile gradient phase consisting of 0.1% formic acid in water as solvent A and acetonitrile (ACN) as solvent B, for 150 min with the flow rate of 0.35 mL/min, 10 µL aliquot was injected into the column and it was maintained at 40 °C. Samples of both roots and shoots of stress and non-stressed conditions were used in triplicates. The nano LC separated peptides were analyzed for MS and MS/MS fragmentation on SYNAPT-G2-Si-High-Definition-Mass-Spectrometry coupled Q-Tof mass spectrometer operating in positive ion electrospray and N₂ was used as the desolvation gas.

Identification of proteins

The UPLC-Q-Tof/MS data of samples were analyzed to identify potential discriminant variables. Peak finding, peak alignment and peak filtering of ES + raw data were carried out with Mass Lynx applications manager version 4.1 (Waters). The raw files were analyzed for protein identification using the WATERS Protein Lynx Global Server (PLGS) v 4.1 against the respective UniProt database with fixed carbamidomethyl modification of cysteine (C) and variable oxidation of methionine (M). The peptide tolerance was set at 20 ppm and for MS/MS the tolerance was kept at 30 ppm. The sequence of proteins being differentially expressed was fetched using the EMBL-EBI dbfetch database and blastn. The functional annotation was done using Blast2Go software to retrieve the Gene Ontology terms. The protein function was deciphered by using the gene index accompanied Uniprot accession number as input for the Uniprot database (http://www.uniprot.org). For understanding the roles and interactions of identified proteins, a protein–protein interaction network (PPI) was predicted using the publicly available program Search Tool for the Retrieval of Interacting Genes/Proteins [(STRING) database version 9.1, http://string-db.org/] with a confidence score greater than 0.7.

Quantification of glycine betaine and choline

For quantification of glycine betaine (GB) and choline, the seeds were sown on germination paper in Petri dishes containing Hoagland’s media (half strength), 150 mM NaCl, 1 mM serine + 150 mM NaCl, 1 mM choline + 150 mM NaCl and 1 mM glycine + 150 mM NaCl. After seven days of germination, shoot and roots of different conditions were washed thrice to remove any contamination and homogenized in 90% methanol and centrifuged at 15,000 × g for 5 min. After discarding supernatant, the pellet was re-extracted using 90% methanol. The methanol extract was phase separated by the addition of methanol/chloroform buffer (10 volumes of methanol: 5 volumes of chloroform: 6 volumes of H₂0). After centrifugation at 15,000 × g, the aqueous phase was taken and concentrated in a rotary vacuum evaporator. The dried pellet was re-suspended in a small volume of H₂O and potassium iodide-Iodine precipitation was done. Glycine betaine and choline was quantified on a Time-of-flight mass spectrometer (AXIMA CFR, Shimadzu/Kratos, Japan) with the internal standards d₁₁-betaine and d₉-choline (Hibino et al. 2002).

RNA extraction and real-time quantitative PCR (qPCR)

RNA extraction was done following the manufacturer’s guidelines using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, USA), from both root and shoot samples of control and stressed plants. The RNA concentration was measured by spectrometry (NanoDrop 1000 Spectrophotometer, Thermo Fisher, France) and purity was checked by agarose gel (0.8% w/v) electrophoresis. Three independent biological samples of each were used in the analysis. The cDNA synthesis was done using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, CA) according to the manufacturer’s protocol.

From the in-house pigeonpea genome sequence database (Singh et al. 2012) the gene sequences corresponding to 20 selected proteins were extracted. The primers (Supplementary Table S1) were designed for different Dof family genes (Dof 5, Dof 11, Dof 19, Dof 22, Dof 23, Dof 24, Dof 25, Dof 27, Dof 29, Dof 38, Dof 5, Dof 7, Dof 34, Dof 36) and their mRNA expression was analyzed under salt stress conditions. IF4 reported to exhibit stable expression in different developmental stages and stresses in pigeonpea, was used as a reference for gene expression normalization. The expression of the selected genes was assessed by using a Stratagene Mx3005P qPCR system (Agilent technologies) with SYBR Green (KAPA SYBR^®FAST (Kapa Biosystems, South Africa), as per manufacturer’s instructions. The PCR was performed at 95 °C for 3 min, followed by 40 cycles at 95 °C for 5 s, 55 °C for 20 s and 72 °C for 20 s. Data were analyzed with MxPro QPCR Software (Agilent technologies).

Results and discussion

Physiological analysis after salinity stress

A dosage-dependent influence of salt stress on the root system architecture like primary root length, lateral root number and the total length of lateral roots, was conducted on a popular pigeon pea variety ‘Asha.’ Various levels of NaCl (0 to 300 mM) were utilized to study the salinity elicited responses on germination of seeds and their growth. In control (0 mM NaCl), 92% seeds were germinated which reduced significantly to 40% in 300 mM NaCl. Germinated seedlings after seven days of growth were scanned and analyzed using ImageJ software for quantifying the root system architecture parameters and the length of shoot (Fig. 1a–f).

Fig. 1.

Dosage-dependent effects of salinity stress on the seedling growth. a Pigeon pea seedlings were grown in a Petri plate lined with germination paper wetted with different concentrations of NaCl (0, 50, 100, 150, 200 and 300 mM) for 7 d. Seedlings were spread on 1% agar (w/v) plate to reveal the morphometric details. Data are presented for b Primary root length, c Number and d Total length of first-and higher-order lateral root, e Total root length and f Shoot length. Values b–f are means ± SE (n = 20) and different letters on the histograms indicate that the values differ significantly (P < 0.05). One-way ANOVA was used for the calculation of significant differences. g Principal component analysis (PCA) of the dosage-dependent salinity stress-mediated modifications in the morphometric traits. Cluster 1, Primary Root Length (PRL); cluster 2, is Shoot area; cluster 3, Number of lateral roots (NLR); cluster 4, Total length of lateral roots (TLLR)

No significant variation in the primary root length at lower doses of salt stress (50 and 100 mM NaCl) was observed, but at higher concentrations (200 and 300 mM NaCl), there was a significant decline in the primary root length (Fig. 1a). Compared to the primary root length (Fig. 1a), lateral root number (Fig. 1b) and lateral root length (Fig. 1c) started to decrease at lower salt (50 mM NaCl) and vanished at 200 and 300 mM NaCl, indicating that the lateral root is more sensitive compared to the principal root. Taken together, with the previous reports on enhanced primary root length with 50 mM NaCl in Arabidopsis (Julkowska et al. 2014; Wang et al. 2009; Zolla et al. 2009), the present results indicate that lower concentrations of salt did not alter the primary root length whereas higher salt leads to the inhibition in growth of both primary root length and lateral roots.

For all other traits (lateral root number, total length of lateral roots, shoot length), there was a linear decline with the increase in salt concentration and at 200 and 300 mM NaCl, the seeds exhibited a little emergence of radical but could not germinate further. To explore the variation and the pattern in all measured parameters, principal component analysis (PCA) was performed. The plot depicts the score of each growth parameters on the first two principal components (Fig. 1g). It was observed that two components demonstrated a sufficient amount of variance. The primary root length and shoot length were close in the space whereas the lateral root number and total length of lateral roots were distantly spaced. For shoot growth, very little observable growth was detected at higher salt concentrations. Similar results were observed for salinity tolerance in ten pigeonpea cultivars with different levels of NaCl (Karajol and Naik 2011). Since 150 mM NaCl significantly affected the growth of seedlings, in the following proteomics study, we compared two types of plants; salt-free (control) and 150 mM NaCl (treatment).

Next, we examined the levels of Na⁺ and K⁺ in seedlings of pigeonpea after exposing them to NaCl stress (Fig. 2). It was observed that Na⁺ increased at escalating concentrations of NaCl in root and shoot tissues up to 200 mM NaCl. However, the K⁺ level showed a similar level till 150 mM NaCl and after that, it started to decrease. Keeping K⁺ at high level and avoiding Na⁺ entry into cells were essential for plants to survive under higher salinity stress conditions (Deinlein et al. 2014). It was shown that the ratio of K⁺/Na⁺ in root and shoot are useful biomarkers for screening of soybean for salt tolerance (Shelke et al. 2017).

Fig. 2.

Dosage-dependent effects of salinity stress on the intracellular concentrations of Na⁺ and K⁺. Seedlings were grown under different concentrations of NaCl (0–200 mM) for 7 d. The roots and shoots were separated and assayed for the intracellular concentrations of Na⁺ and K⁺ by flame photometer. Values are means ± SE (n = 10)

Salt stress induced amino acid profiling

Amino acids were quantified using HPLC in control and 150 mM NaCl stressed roots and shoots of germinated seedlings. It was observed that total amino acid contents increased under salt stress in both root and shoot, showing an increase of 42.8% and 14.6% respectively. Under control conditions, asparagine (Asn) was the major amino acid present in the root, followed by valine (Val), glycine (Gly) and serine (Ser) (Table 1). In the shoot under control conditions, Val was the most abundant amino acid, followed by Ser and threonine (Thr). Under salt stress conditions, the contents of Ser, Asp, Gly and Asn showed a significant increase of 2.22 times, 4.47 times, 1.47 times and 1.76 times, respectively, whereas the contents of Val and Gln decreased by 1.52 times and 5.89 times, respectively in the root. In salt stressed shoots, Phe, Ser and Asp increased by 1.68, 1.66 and 1.52 times, respectively, and, Gly recorded a decline of 0.74-times. The higher levels of Ser in shoot suggested an increase in photorespiration under salt stress (Martino et al. 2003). The increase in amino acid contents was related to protein degradation or inhibition of synthesis of proteins (Dhindsa and Cleland 1975; Macauley et al. 1992).

Table 1.

Free amino acid content in control and salt stressed root and shoot of pigeonpea seedlings after salt stress

Amino acid	Root				Shoot
	Control		NaCl		Control		NaCl
Asp	338.42 ± 40.61		1504.63 ± 180.56		950.12 ± 114.014		1442.50 ± 173.1
Thr	958.89 ± 115.07		1230.78 ± 147.69		1133.20 ± 135.98		1259.44 ± 151.13
Ser	1055.80 ± 126.7		2339.94 ± 280.79		1150.93 ± 138.11		1920.66 ± 230.48
Asn	9852.00 ± 1182.24		17,322.74 ± 2078.73		–(i)		–(i)
Gln	1736.04 ± 208.32		294.50 ± 35.34		730.98 ± 87.72		300.75 ± 36.09
Gly	178.96 ± 21.48		263.35 ± 31.60		332.95 ± 39.95		246.93 ± 29.63
Ala	348.79 ± 41.86		669.11 ± 80.29		751.22 ± 90.15		961.63 ± 115.4
Val	3350.79 ± 402.09		2195.68 ± 263.48		5313.56 ± 637.63		5011.44 ± 601.37
Cys	942.48 ± 113.1		584.74 ± 70.17		707.10 ± 84.85		857.84 ± 102.94
Met	0.00 ± 0.00		21.56 ± 2.59		157.64 ± 18.92		318.12 ± 38.17
Ile	322.04 ± 38.64		416.03 ± 49.92		859.03 ± 103.08		982.74 ± 117.93
Leu	93.59 ± 11.23		216.77 ± 26.01		216.92 ± 26.03		342.35 ± 41.08
Tyr	22.95 ± 2.75		61.04 ± 7.32		53.84 ± 6.46		69.88 ± 8.39
Phe	304.79 ± 36.58		733.68 ± 88.041		833.30 ± 100		1399.39 ± 167.93
Total	19,505.55 ± 2340.67		27,854.55 ± 3342.55		13,190.79 ± 1582.9		15,113.66 ± 1813.64

The concentration of NaCl was 150 mM. The values are means of three replicates with standard error. The values differ significantly (P < 0.05). (i) Not assigned

Expression and classification of salt responsive proteins and genes of pigeonpea

A gel-free comparative proteome profiling coupled with mass spectrometry was done using Waters ACQUITY UPLC™ system as described in Materials and Methods. Total of 105 proteins was significantly (P < 0.05) detected in the shoots and 109 proteins in roots (Fig. 3) of control and stressed (150 mM NaCl) seedlings. Among them, 38 proteins were present only in control and 9 were unique to salt-stressed conditions in the shoot. Similarly, in the root, 28 proteins were observed for salt-stressed conditions but not in control. There were 25 and 39 proteins showed differential expression under salt stress condition in shoots and roots respectively (Supplementary Tables S2-S3). In Medicago sativa, 27 and 36 differentially abundant proteins were identified in shoot and root tissues respectively under salt stress condition which were involved in photosynthesis, ion transport and signal transduction (Xiong et al. 2017). Similarly, in Glycine max 201, differentially abundant proteins were identified which belongs to 20 metabolic pathways after salt stress in the germinated seedlings (Yin et al. 2018).

Fig. 3.

Venn diagram showing the salinity-stress induced differentially expressed proteins in the shoot and root. a Majority of the salinity-stress induced proteins (81%) were detected in both the shoot and root, while 8% and 11% of them were found to be specific to the shoot and root, respectively. b Several salinity-stress induced proteins were identified that were either specific or common in both the shoot and root. Out of the total proteins identified in shoot, 48 proteins are common under control and stress whereas 57 proteins are solely expressed in either stress or control. Similarly, in root 56 proteins are universal under salt and salt free conditions and 53 proteins are unique in either control or stress

The identified salt-responsive proteins were functionally classified to understand the biological processes altered during salinity stress. The function of individual proteins was deduced from the UniProt database. Functional categorization revealed 25% of pigeonpea proteins were actively involved under salt stress environment and the majority of proteins were related to the enzyme activity in the shoot (22%) and root (17%) (Fig. 4a). While the lowest percentage was contributed by photosynthesis activity in both shoot (5%) and root (3%), respectively (Fig. 4a). When biological functions in root and shoot were compared, it was evident that oxidation and reduction process, transcription and translation process, biochemical pathways and DNA methylation were more abundant functional categories in roots, while photosynthesis, respiration, enzyme activity and membrane proteins were more prevalent in shoot tissue. These facts suggest that our proteomic studies reflect the situation well occurring in salt stressed-plant. When different enzyme classes were compared, it was observed that the number of transferases was higher in both shoot and root under salt stress whereas, decreased activity of isomerases was also observed (Fig. 4b). GO enrichment study showed (Fig. 5) a similar trend in root and shoot of pigeonpea after salt stress for cellular component, molecular and biological functions. The cellular component showed that nucleus, integral membrane and chloroplast proteins were abundant in both root and shoot tissues after salt stress. Photorespiration, photosynthesis and mRNA processing were more enriched in shoot as compared with root similar to a recent report in sugar beet (Li et al. 2021).

Fig. 4.

The effects of salinity stress on the tissue-specific distribution of salt-responsive proteins and different enzyme classes. a The biological function of each of the protein expressed in the root and shoot was inferred from the Uniprot database (www.uniprot.org). b Distribution of enzyme category (EC) classes deduced based on their protein sequences retrieved from gene ontology classification: 1—oxidoreductases, 2—transferases, 3—hydrolases, 4—lyases, 5—isomerases

Fig. 5.

Gene Ontology (GO) analysis of differentially expressed proteins in roots for a cellular component, b biological process and c molecular function; and in shoots for d cellular component, e biological process and f molecular function

Transcription-related proteins

Among differentially expressed protein family, one of remarkable proteins is DNA binding one zinc finger protein (Dof). Dofs are related to different biological processes like germination and responses to abiotic and biotic stresses. In pigeonpea genome, there are 38 Dofs, distributed on eight chromosomes and 50% are present in intron-less region (Malviya et al. 2015). In the current study, total of 16 CcDof proteins were observed in shoot and root, 13 proteins in shoot and 14 proteins in root. Among 13 CcDof shoot proteins, 4 CcDof proteins (Dof9, Dof11, Dof24, Dof36) were exclusively found in shoot only under salinity stress conditions, whereas Dof5, Dof22, Dof27 and Dof 29 were highly expressed in both shoot and root under salinity stress conditions (Supplementary Tables S2 and S3). Dof genes were overexpressed in Arabidopsis which showed higher tolerance to drought and salinity stress by inducing many stresses responsive genes like RD10, RD29A and COR15 (Corrales et al. 2014). In sorghum 13 SbDofs showed differential gene expression and SbDof 12, 19 and 24 were highly induced under drought and/or salinity stress (Gupta et al. 2016). Further, tomato Dof22 was shown to be a negative regulator of ascorbic acid (AsA) accumulation in tomato and knockout lines showed higher levels of AsA which also affected the D-mannose/L-galactose pathway genes and recycling of AsA (Cai et al. 2016). Since Dofs were predominantly present TFs, an attempt was made towards the analysis of the diverse set of CcDof family proteins in both shoot and root of pigeonpea in the present study.

The phylogenetic tree derived from the CcDof family of proteins reported from Q-ToF data depicted four different groups (Fig. 6a, b). Interestingly, group I has Dof proteins which were suppressed under salt stress condition i.e., CcDof34, CcDof7and CcDof23 whereas, group II, III and IV have CcDofs which were abundant and/or present only under salt stress condition. Also, majority of salt induced CcDofs were located on chromosome 1 and 11. A MEME 4.10.1 program utility was used for the display of motifs and sequence logos of the Dof proteins (Fig. 7a). All motifs discovered by MEME were annotated by adopting Interpro and 10 conserved motifs were identified and predicted. In all the CcDof proteins there were at least 2 main motifs present across all 10 CcDof proteins. Motif 1 was most abundant in all CcDof proteins except CcDof29 while and motif 2 were present in CcDof11 and CcDof29 (Fig. 7b, c).

Fig. 6.

Bioinformatic analysis of Dof transcription factor family. a Multiple sequence alignment revealed the distribution of Dof family in pigeonpea into five distinct clusters. b Phylogenetic analysis of Dof family protein across Arabidopsis, pigeonpea, rice and soybean categorised them into six discrete groups indicated by different colours

Fig. 7.

Conserved motifs of the members of CcDof family protein. a Ten conserved motifs were identified across 10 members of CcDof family protein of which motif 1 (red) and 2 (cyan) were predominant. b and c Weblogo presentation of the sequences of motif 1 and 2, respectively

Energy metabolism, ROS scavenging and detoxifying enzymes

Analysis depicted that a total of 14 proteins belonging to energy metabolism were expressed only under salt stress condition in root tissues. They were photosystem I, II, cytochrome oxidase, Rubisco and diacylglycerol, glycerol 3 phosphate, 3 phosphatidyl transferase. Photosystem I, II, cytochrome oxidase and ATP synthase complex are thylakoid membrane-bound proteins involved in photosynthesis (Hippler et al. 2001). In an earlier study, it was suggested that the tolerance for cold stress in Thelungiella halophila was due to the regulation of chloroplast functions (Gao et al. 2009). Legumin seed storage protein 1 was highly expressed in salt stress condition while Rubisco, glutathione transferase and alcohol dehydrogenase showed lower expression in the tissue. Legumins belongs to globulin family, a source of antioxidant peptides and its accumulation was reduced in abiotic stress condition (Rerie et al. 1991; Terrasson et al. 2015; Fuentes et al. 2015). In shoot, total 17 proteins were there; out of which, 12 were unique to shoot and 9 of them were present in control and 3 were exclusively found in salt-stressed condition. In salt stressed condition, alcohol dehydrogenase, ascorbate peroxidase and ATP synthase subunit beta were present. Under flooding and drought stress alcohol dehydrogenase enzyme activity was increased in root and leaves of soybean plant (Wang et al. 2017).

Rubisco and lectin were other differentially expressed proteins. The lectin protein which was 60-times higher in salt-stress are the carbohydrate-binding proteins in plants and its expression levels increased under environmental stress conditions including salt stress (Van Damme et al. 2008). Various nucleoplasmic lectins played roles in plant stress signaling mechanism like Nictaba from Nicotiana tobacum (Chen et al. 2002; Van Hove et al. 2015).

There were four proteins present in root tissue for reactive oxygen scavenging and detoxification. Among these, glutathione S-transferase, ascorbate peroxidase and Ras-related proteins showed lower expression in root under salt stress condition (Supplementary Table S2), whereas cold and drought regulatory protein (D3YBH6) was detected in root only after salt stress (Supplementary Table S2). In shoot, once again the cold and drought regulatory protein (D3YBH6), glutathione S-transferase and ascorbate peroxidase were detected under only salt stress conditions, whereas Ras-related proteins were not identified under control and salt stressed conditions (Supplementary Table S3). These data indicate the importance of the cold and drought regulatory protein (D3YBH6) for salt stress. It has been reported that salt stress affects the pivotal role in providing the balance between generation and removal of oxygen free radicals and the excess levels of ROS leads to damage the cell macromolecules like proteins, nucleic acids and lipids (Kapoor et al. 2015; Miller et al. 2010).

Signal transduction network involved in NaCl stress responses

Proteins involved in signal transduction pathways were also identified. A total of 21 and 11 proteins were identified in root and shoot tissue respectively. Among 21 proteins found in root tissues, two proteins were exclusively present in salt-stressed condition which were TIR domain protein and high mobility group B proteins and seven proteins showed lower expression when subjected to salt stress which includes ABC transporters and high mobility group B proteins. High mobility group proteins reduce the growth of plants and make plants sensitive to salt stress (Balasubramanian et al. 2013). The TIR (Toll-interleukin-1 receptor) domain is present in innate immune responsive molecules of plants and animals. The TIR-domain was used by plant resistance proteins during pathogen infection to guide the expression of genes involved in defence responses (Xu et al. 2000). In shoot, among 11 proteins found, one hybrid proline-rich protein was exclusively present in salt stress condition, while three were found only under control condition. There were four highly expressed proteins under salt stress conditions which were high mobility proteins, urease and ABC transporter family proteins. ABC transporter proteins are ATP-dependent ion channels, pump and channel regulators (Theodoulou 2000). They have a pivotal role in protecting plants against the salt and heat stress (Li et al. 2011) by maintaining ionic homeostasis.

Protein processing and amino acid metabolism

We also observed 20 and 15 proteins involved in translation, processing and degradation family, in root and shoot tissues, respectively. Noticeably, expression of 7 proteins was lower under salt stress condition in roots. They were DNA directed RNA polymerase, a protease inhibitor, proteases and alpha elongation factor. In the shoot, among 15 proteins, proline and callose synthase were exclusively found in salt-stressed condition while phosphoethanolamine and RNA polymerases were present in the control condition only. Highly expressed proteins were pentatricopepetide, PRLI interacting factor A and pleiotropic drug resistance protein. Translation elongation factor, a protease inhibitor, LysM domain containing receptor kinase, histone and protein phosphatases showed suppressed expression. Proline is one of the osmolytes which acts in scavenging of ROS and also helps in protein structure stabilisation to protect the damage of the plant cells due to stress (Soda et al. 2018). Callose synthase protects the plant from pathogen infection and also mechanical damage by its accumulation in plasmodesmal channels. Arabidopsis genes for callose synthase (CalS1 and CalS8) were involved in biotic and abiotic stress-induced signalling pathways (Cui and Lee 2016).

Proline was only found for salt-stressed conditions and urease; kinase and protease were highly expressed in salt stress condition. In the earlier studies (Krasensky and Jonak 2012), proline has predominantly featured in the salt stress biochemical pathways. Urease is commonly present in most of the legumes which are a seed protein (Balasubramanian et al. 2013). Proline, late embryogenesis-abundant (LEA) proteins and leucine zipper-like protein are seen to be induced under salt stress condition in roots and mature organs (Hossain et al. 2013). Initial changes in the roots and early developmental stages of soybean have also been studied using proteome and phosphoproteomic approaches to understand the mechanisms governing flooding stress (Yin et al. 2014a, b). Another interesting observation in this study is two ethanolamine kinase proteins (A0A0D6DQI5 and A0A0D6DQJ8) (Supplementary Tables S2–S3). Ethanolamine kinase is an enzyme that catalyses the phosphorylation of ethanolamine using ATP (Lin et al. 2020; Sahashi et al. 2019). Ethanolamine is synthesized from serine and involved in the biosynthesis of choline, phosphatidylcholine and phosphatidylethanolamine (Sahashi et al. 2019; Takabe et al. 2015; Tasseva et al. 2004).

Quantification of glycine betaine and choline

Since ethanolamine is implicated in choline biosynthesis, a precursor for synthesis of glycine betaine (GB), GB and choline were quantified using Time-of-flight mass spectrometer system with NaCl, NaCl + glycine, NaCl + serine and NaCl + choline (Fig. 8a–b). The GB was higher when supplemented with NaCl + choline followed by NaCl + serine. No significant change was observed with NaCl and NaCl + glycine. Choline level was highest in NaCl + choline, but other conditions did not show significant differences. Choline is synthesized by three consecutive methylation of phospho-ethanolamine using adenosyl-methionine which is catalysed by the phospho-ethanolamine N-methyltransferase (PEAMT) (Nuccio et al. 2000). Under salt stress conditions, various osmoprotectants are synthesized, which protects the denaturation of enzymes and helps the membrane stability besides osmotic homeostasis. Biosynthetic pathway of GB in dicot plants has not been studied well (Cha-umet al. 2019; Fujiwara et al. 2008; Mitsuya et al. 2011; Rathinasabapathi et al. 1997). Identification of choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) in pigeonpea is an interesting subject to be clarified.

Fig. 8.

Effect of salt stress on the a glycine betaine, and b choline in control and 150 mM NaCl salt stressed seedlings of C. cajan. Data represented values generated from three independent biological replicates with three technical replicate each. The different letters showed significant difference (P < 0.05) by t-test

Transcriptional analysis of genes corresponding to differentially abundant proteins

Next, the gene expression of Dof family proteins was also analyzed by qRT-PCR. The proteome of pigeon pea has not yet been completely annotated/deciphered. Therefore, using the unpublished pigeonpea database, the primers for differential expression analysis were designed for 24 identified proteins. Out of the 13 Dofs studied, expression of 6 Dofs (Dof11, Dof19, Dof22, Dof23, Dof24 and Dof25) was significantly decreased under salt stress, whereas Dof29 showed an upward trend and rest six did not show any significant change between two samples under consideration (Fig. 9a). This data do not coincide with the proteomic analysis and further study is required to understand the mechanism of Dof gene expression and protein accumulation. Function of Dof proteins in conferring tolerance to stress has been studied in many crops, but available literature in pigeon pea is scanty.

Fig. 9.

Effect of salt stress on the relative expression levels of genes in control and 150 mM NaCl salt stressed seedlings of C. cajan. a mRNA for Dof genes, b mRNA for choline metabolites. EthKin, ethanolamine kinase;GST, glutathione S-transferase; ATP Syn; ATP synthase subunit beta fragment; Lectin, lectin protein; SAMS, S-adenosyl-L-methionine-dependent methyltransferase; ChoPh, choline-phosphate cytidylyltransferase 1-like; SHMT, serine hydroxymethyltransferase; Saroxi, sarcosine oxidase; CMO, choline monooxygenase; BADH, betaine aldehyde dehydrogeanse. Data (n = 9) represented values that were generated from three independent biological replicates with 3 technical replicate each

Genes which are involved in the GB synthesis pathway were also selected for their mRNA expression under salt stress conditions. The genes include choline-phosphate cytidylyltransferase 1-like (ChoPh), serine decarboxylase 1-like (Ser Decar), S-adenosyl-L-methionine-dependent methyltransferase (SAM), probable sarcosine oxidase (Sar oxi), serine hydroxymethyltransferase (SHMT), choline monoxygensae (CMO), betaine aldehyde dehydrogenase (BADH) and lectin protein (Fig. 9b). In Fig. 9b, the ethanolamine kinase was highly expressed and showed a two times increase in salt stressed. Earlier, also it was stated that pathway of ethanolamine to glycine betaine via phosphocholine and betaine aldehyde is triggered in salt stress for mangrove plants which may be due to an augmented expression of genes involved (Suzuki et al. 2003). The exogenous application of ethanolamine in jute, mitigated the damaging effects of salt stress by improving the anti-oxidant machinery and ionic equilibrium (Moussa et al. 2019). The expression of CcCMO and CcBADH also showed an increase of 3 and four times, respectively under salt stress condition (Fig. 9b). These results demonstrated the presence of glycine betaine synthesis pathway in pigeonpea (Fig. 10). The first step of GB synthesis is catalysed by CMO, which is the rate limiting step (Yamada et al. 2015). The metabolism of choline was identified in spinach, which was required for the synthesis of phosphatidylcholine which was a precursor for GB (Summers and Weretilnyk 1993).

Fig. 10.

Simplified scheme of biochemical synthesis of glycine betaine in C. cajan. Compounds are indicated as normal fonts while enzymes are indicated in italics

Protein interaction network

Proteins in living cells tend to function in groups rather than as single entities. We tried to find out mechanism underlying the transmission of salinity stress signals through protein–protein interactions in plants and their effects on cell functions in radical and plumule of pigeon pea after salt stress. Protein interaction analysis revealed that ten proteins formed the center of a protein interaction network (Fig. 11). These proteins may be the master regulator in salt stress conditions, energy metabolism, signal transduction and redox homeostasis. These proteins are important components of oxidative phosphorylation and metabolic pathways. Among the 118 total, 73 proteins showed good interaction while remaining were either least or non-interactive. Protein–protein interaction analysis revealed that many processes tend to function collectively to attain cellular homeostasis under salinity stress condition. A prominent network containing cytochrome oxidase (COX) proteins were identified as having CcCOX1, two and three proteins. COX, a mitochondrial soluble protein located in intermembrane space and contributes in transferring copper to cytochrome c oxidase. In rice, OsCOX6b1 and OsCOX6b2 were cloned and found to be induced by salt stress (Ohtsu et al. 2001; Yan et al. 2005). The activation of COXs has the pivotal role in energy production via respiratory chain under salt stress condition (Yan et al. 2005). Gene silencing of AtCOX17 resulted in the lesser response of salt stress-induced genes under salinity stress condition in Arabidopsis (Garcia et al. 2016) therefore, depicting its role in sensing and signaling pathway. The formation of the close interactive network of CcCOX1, CcCOX2 and CcCOX3 proteins suggested that in C. cajan after salt stress there are many CcCOX genes induced to send signals for other salt stress responsive genes to trigger them in action and function in a network mode.

Fig. 11.

Protein interaction network of salinity-stress responsive proteins commonly expressed in root and shoot. String software (https://string-db.org/) was used for determining the protein–protein interaction network

Conclusions

Pigeonpea is an important crop and it is salt sensitive. A comparative proteome analysis of control and salt stressed (150 mM NaCl) plants was conducted using 7 days-old seedlings. Among various amino acids, serine, aspartate and asparagine were the amino acids that showed increment in the root, whereas serine, aspartate and phenylalanine showed an upward trend in shoots under salt stress. The proteomic profiling exhibited the pivotal role of transcription factors (Dofs) and choline precursors such as ethanolamine kinase and SHMT for conferring salt stress tolerance to C. cajan. The glycine betaine biosynthesis was activated in C. cajan after salt stress thereby playing pivotal role in conferring salt tolerance. The activation of COXs signified its role in signal transduction under salt stress condition.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

NKS and VR envisaged the study, designed experiments, coordinated and helped to draft the manuscript, NJ carried out all the wet lab experiments, SF helped in data analysis and mass spectrometer experiment, RK helped in lab work and figure preparations, NS contributed in bioinformatics data analysis, SK edited the manuscript, TT helped in writing the manuscript. The final manuscript was approved by all the authors.

Funding

The study was funded by ICAR-NPTC 3021 (Central Facility) to NKS, VR and NJ.

Data availability

The data are available in supplementary files.

Declarations

Conflict of interest

There was not any commercial and financial conflict of interest.