Proteomics and photosynthetic apparatus response to vermicompost attenuation of salinity stress Vicia faba leaves

Crop production and growth are severely affected by salt stress. Nevertheless, the bio-fertilizer vermicompost (VC) can be participated as a potent inhibitor of salinity on plant growth and crop production by regulating photosynthetic efficiency. We investigated the effect of VC on photosynthetic performance of salt-stressed broad bean (Vicia faba L. Aspani cultivar). Seeds were grown in soil mixture; clay and sand in ratio 1:2 by volume with five different volumetric ratios of VC; 0, 2.5, 5, 10 and 15% irrigated with either water and/or 200 mM NaCl. Leaf area, Na and K contents, chlorophylls, photosystem II efficiency, Rubisco content, soluble sugars, chloroplasts’ organization and proteomics were analyzed. The imposed stress decrease leaf area, chlorophyll contents, maximum quantum efficiency (Fv/Fm), Rubisco content, increase soluble sugars and damage chloroplasts organization. Salinity upregulated glucose-1-phosphate adenylyl transferase, ribulose bisphosphate carboxylase large subunit and chloroplastic peptidyl-prolyl cis–trans isomerase. The increased leaf area, chlorophyll a, b and carotenoids, maximum quantum efficiency of photosystem II, Rubisco content, improving the degeneration of thylakoid lamellae and lessening plastoglobuli number in thylakoid membranes are the major benefits attained with vermicompost treatments under salt stress. Analysis of proteomic revealed that VC upregulated chloroplastic ferredoxin–NADP reductase, plastocyanin, polyphenol oxidase, peptidyl-prolyl cis–trans isomerase, alpha-glucan phosphorylase H isozyme and maturase expression under salt stress. The results suggest that VC controls protein expression at the level of transcriptional and translational which may conserve photosynthetic components and prevent salt-induced harmful effects in broad bean plants.


Introduction
Salinity is a common worldwide problem especially in arid and semi-arid areas; about a third cultivated land has been suffered from salinity by different degrees (Bahmani et al. 2015). Retardation of plant growth under salt stress is particularly caused by osmotic stress and specific ion effects (Khan et al. 2000). Salt stress mostly reduced plant growth and/or causes a significant inhibition in photosynthetic activity (Shu et al. 2012). This inhibition may result from closure of stomata due to osmotic stress (Sudhir and Murthy 2004), decreased efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Shu et al. 2015) and disruption of photosynthetic systems as indicated from chloroplast structure and function or thylakoid membrane disorganization (Demetriou et al. 2007). Salt stress also leads to chloroplasts' aggregation, swelling in the grana, fretting compartments, accumulation of lipid droplets (Hasan et al. 2018), disruption of thylakoid, increased the quantity and extent of pellets of the plastid, and decreased of starch content (Shu et al. 2015).
Proteomics has perceiving power for potential applicant genes that nowadays are used for genetic improve of plant features towards stresses (Barkla et al. 2016). Varies routes in plants are found to be activated in response to stress Communicated by E. Kuzniak-Gebarowska. resulting in a complicated network including transcript components, ROS, antioxidants, hormones, ion balances, kinases formation, and osmolyte biosynthesis (Yin et al. 2015). Analysis of proteome of salt-stressed Arabidopsis revealed upregulation of 200 kinds of protein (Jiang et al. 2007).
Vermicompost is the bio-degradation product for all sources of organic materials through complicated processes between some microorganisms and special type of earthworms; earthworms ingest plant growth-promoting bacteria (PGPB) such as Pseudomonas, Azotobacter, Rhizobium, Bacillus, Azosprillium, etc.; they become active and increased due to the suitable micro-environment of the earthworms gut. Therefore, earthworm activities increase the population of PGPB and facilitate soil enzymatic activities (Sinha et al. 2010). The PGPB promote plant growth by stimulating production of plant growth regulators (Yılmaz and Belliturk 2017), solubilization of nutrients (Satpathy et al. 2020), and enhanced 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity (Correa et al. 2004). It is also reported that PGPB have important impact role as biocontrol agent in controlling plant pathogens (Joshi et al. 2015). Vermicompost is an excellent source of humus nutrients, which enhance soil organic matter status to improve biophysical properties of the soil. The soil became extremely porous, improving good aeration and drainage status, and allowing high-level of water-retention capability (Atiyeh et al. 2000). Vermicompost suggested enhancing plant tolerance to water deficit, stimulate protein synthesis, enhance enzyme activities, and/or improve activity in various plant structures (García et al. 2012). Shilev (2020) reported that Bacillus subtilis and Azotobacter chrooccocum stimulate plant growth in rice, maize, barely, wheat, soybean, and sunflower tolerance to NaCl stress. These microorganisms are also known to inhabit vermicompost supplemented soil and gut of Eisenia foetida earthworm that found to be benefit for the soil structure and the yield of crop plants (Satpathy et al. 2020); This highlights the role of 10% VC cooperated with microorganisms in attenuating the deleterious effect of 200 mM NaCl stress on Vicia faba growth, development, and metabolism (El-Dakak et al. 2021).
Evolving effective, low-cost, easily pliable methods for the salt stress management is a major challenge. There is an increasing concern towards stress management and environmental sustainability. Therefore, the aim of the study was to explore the prospective effects of VC in enhancing broad bean tolerance to salt stress. With this objective, we have evaluated the effects of VC and/or salt stress on broad bean leaf traits including photosynthetic parameters, accumulation protein content of Rubisco subunits, soluble sugars, Na + , K + and Mg 2+ accumulation, ultra-structural modifications in diverse cell organelles, and recent advance on mechanisms of salt tolerance by utilization of high-throughput proteomics under VC application.

Experimental materials
Pure variety of broad beans seeds (Vicia faba L. Aspani cultivar) was obtained from NUBA SEED Company. The seeds were selected for viability and uniformity of shape and size. Seeds were surface sterilized with 0.1% (v/v) mercuric chloride solution for 5 min and then rinsed several times with distilled water.

Vermicompost characterizations
Vermicompost used was obtained from the Agricultural Research Center, Giza, Egypt. It was prepared in vermicomposting bins (100 × 120 × 50 cm), using agricultural residues (rice straw and tree leaves) as materials for VC and fermented natural organic solid plant and animal wastes as worm feeds. The most productive and common type of earth worms "Eisenia foetida" was inoculated (Joshi et al. 2015). Chemical and physical properties of this product are summarized in Table 1.

Experimental design
Stock solution of NaCl (200 mM) was prepared as sub lethal concentration according to Badr (2020). Experiments were conducted in pots (15 cm diameter and 15 cm height) with 1.25 kg soil capacity of homogenous dried soil mixture. A preliminary experiment was carried out by mixing the soil mixture (1clay: 2 washed sand) with different volumes of VC to select the suitable volumetric ratios for the present study; 2.5, 5, 10, 15, and 30% volumetric ratios The selected VC concentrations used here, based on demonstrating the most significant morphological toxicity symptoms. The chemical properties of VC used are given in Table 1. To ensure well and similar distribution of soil mixture components in all replicate pots, volumes of soil mixture and desired volume of VC in each treatment were mixed well in big container, 5 kg capacity, and then distributed in the replicate pots. Six pre-soaked seeds of broad bean were put per pot beneath the soil surface (1 cm depth), and then thinned to three seedlings after emergence. Two sets of experiments with the selected volumetric ratios of VC were performed; in the first set, 15 pots were used including: (1) Control (100% soil mixture); (2) 2.5% vermicompost + 97.5% soil mixture; (3) 5% vermicompost + 95% soil mixture; (4)10% vermicompost + 90% soil mixture and (5) 15% vermicompost + 85% soil mixture. In the second set of experiment, 15 pots were filled as the first set of experiment and irrigated with 200 mM NaCl.
Culture pots conducted under normal light intensity, 10/14 h light/dark cycle, temperature about 20 ± 2 °C, and relative air humidity about 85% in Botanical Garden at the Faculty of Science, Alexandria University, Egypt. On the day of sowing, three pots of each set were irrigated at field capacity with 200 ml demineralized water. All the pots were then regularly irrigated at field capacity with distilled water or 1/4 N Hoagland solution (day after the day) throughout the experimental period. After 10 days of sowing, the first set was irrigated with demineralized water for one time and other time with 1/4 N Hoagland solution to supply plant nutrient requirements (Hoagland and Arnon 1950). The second set was irrigated, once with 200 mM NaCl and other time with 200 mM NaCl in 1/4 N Hoagland solution. After 30 days, broad bean seedlings were carefully harvested, washed with running tap water then with demineralized water, and blotted dry gently between layers of tissue papers. Leaf area for of the second leaf (from the top) is immediately determined by Leaf Area Meter; a Model LI 3000 Portable Area Meter assembled.

Determination of chlorophyll content, carotenoids, fluorescence, and soluble sugars
Chlorophylls and carotenoids contents were determined according to the method of Lichtenthaler (1987). Chlorophyll fluorescence measurements were monitored in fully expanded second leaf with OS-30P pulse modulated chlorophyll fluorimeter (Opti-sciences, Hudson, USA), and the maximum quantum yield of PS II (Fv/Fm) was also calculated (Moustakas et al. 1993). Soluble sugars (SS) were determined according to the method described by Dubois et al. (1956) and calculated as, mg g −1 dry wt.

Protein extraction and relative amount of Rubisco estimation
The second leaf of ten plants was used for protein extraction and subsequent activity and protein amount measurements. Leaves were coarsely ground in a mortar under liquid nitrogen for determination of relative amounts of Rubisco small subunits (SSU) and large subunits (LSU) were measured using HPLC analysis (C 18 column) according to Leitao et al. (2003).

Estimation of Na + , K + , and Mg 2+ contents
Sample preparation, metal analysis, and quality control were carried out according to the standard method of Kimbrough and Wakakuwa (1989).

Transmission electron microscopy (TEM)
The fragments from the second leaf in both control and treated plant are used for ultra-structural evaluation according to Reynolds (1963). The ultrastructure visualization and photographing were carried out using high-resolution TEM (JEOL JEM-2100, Japan).

Protein extraction, digestion, and cleaning of peptides
Leaves from the plants sacrificed at the 4th week were cleaned with distilled water and immediately plunged in liquid nitrogen (LN). The protein extraction was realized according to the phenol method described by Carpentier et al. (2005). Digestion of proteins (20 µg) was determined by addition of dithiotheritol (20 mM) and incubated for 15 min; followed by addition of iodoacetamide (50 mM) and incubation in the dark for 30 min. The samples were diluted three times ammonium bicarbonate (150 mM); finally, trypsin (0.2 µg trypsin per 20 µg protein) was added and incubated overnight at 37 °C; followed by acidification with trifluoroacetic acid to 0.1%.

Peptides separation, identification, and quantification
Samples were desalted using Pierce™ C18 Spin Columns (Thermo Fisher Scientific), speed-vacuum the sample, then re-constitute in 50 μl for elution, and inject the samples on Mass spectrometry. Nano-LC system consisting of Eksigent nano-LC 400 autosampler attached with Ekspert nanoLC425 pump. Injection volume 10 μl containing 1 µg. Needle washes two cycles using 10% isopropanol for 55 min. Sample clean-up using trapping cartridge CHROMXP C18CL 5um (10 × 0.5 mm) pumped at flow rate 10 µl min −1 for 3 min using mobile-phase A DI-H 2 O containing 0.1% formic acid and B acetonitrile containing 0.1% FA. by gradient elution. The acquisition of peptide mass fingerprint data was performed on (MALDI-TOF) system Sciex TripleTOF™ 5600 + . High-resolution TOF MS survey scan followed by product ion scan for the most intense 40 ions. Cycle time is 1.5 s and acquisition time 55 min, MALDI-TOF mass range 400-1250 m/z, (product ion) 170-1500 m/z, ion selection threshold 150 cps and MS calibration are Sciex tuning solution (P/N 4457953).
Protein identification was performed with the MASCOT search engine (Matrix Science, London, UK) searching the analyst TF 1.7.1 is used for data acquisition (Sciex software). Raw MS files from the Triple TOF™ 5600 + were analyzed by peptide shaker (v1.16.26). Database used is Uniprot Vicia faba plant (swiss-prot [58 entery] and TrEMBL [8405 entery] database). Protein identification was performed with the swiss-prot [58 entery] and TrEMBL [8405 entery] database. Search for monoisotopic mass accuracy 20 ppm tolerance, missed cleavages 2, allowed variable modifications: oxidation (Met), carbamidomethylation of C, prolidone from carbamidomethylated C, acetylation of K, acetylation of protein N-terminal, deamidation of N, and deamidation of Q were used as the search parameters.

Statistical analysis
The data obtained were subjected to statistical analysis using analysis of variance (ANOVA) and the mean values were compared using Duncan's Multiple Range Test (DMRT) in SPSS ± standard deviation (SD), following the methods of Sokal and Rohlf (2013).

Vermicompost improves leaf area of Vicia faba plant
To examine the effects of vermicompost on growth performance, we measured leaf area, photosynthetic pigments, and chlorophyll fluorescence under both normal (pots supplemented with VC only) and saline conditions (pots supplemented with VC and 200 mM NaCl). Under normal conditions, VC significantly increased leaf area at 2.5% VC by 20% relative to control plant ( Table 2). As the percentage of VC increased, a gradual decrease in leaf area was recorded. Leaf area reduced by 39% at 200 mM NaCl respective to control. On the other hand, the interaction of 200 mM NaCl and 10% VC showed an enhancement of leaf area by 47%, with respect to salt-stressed plant.

Vermicompost protects photosynthetic apparatus from salt toxicity in Vicia faba plant.
Vermicompost treatments led to an obvious increase in chlorophyll a, b and carotenoid, by 41, 17, and 28% respectively, with respect to the control at 2.5% VC. There was a decrease of chlorophyll a and b by 43 and 74%, respectively, in response to the 200 mM NaCl treatment (Table 2). Fascinatingly, interaction of VC fertilizer and 200 mM NaCl significantly increased the levels of chlorophylls and carotenoid in the leaves of salt-stressed plants. Chla, was increased in broad bean leaves by about 2.9, 5.4, 7.5, and 5.8% with VC levels of 2.5, 5, 10, and 15%, respectively, as compared with salt-stressed leaves.
Vermicompost at 2.5% treatment showed a slight increase in Fv/F 0 and Fv/Fm ratios (Table 3). As the volume of VC increased, these ratios showed slight increase at regular intervals. Under 200 mM NaCl, the values of Fv/F 0 and Fv/ Fm were significantly decreased. These revealed that the PS I1 may be damaged by different degrees under salt stress and the primary reaction of photosynthesis may be inhibited. The decrement for Fv/F 0 and Fv/Fm was obviously different under salt stress and VC treatment; it was ameliorated by about 11 and 4%, respectively. Regarding Rubisco protein, 2nd leaf showed an increase in Rubisco-LSU and Rubisco-SSU contents under vermicompost treatments with respect to the control (Fig. 1). The protein content of LSU represented by 28 and 26% at 2.5 and 10% VC, respectively, compared to control. Simultaneously, it contained average amounts of Rubisco-SSU: about 14 and 10% for 2.5 and 10% VC, respectively. A significant decrease in protein content under saline factor by 10 and 4% was recorded for LSU and SSU, respectively.
The addition of vermicompost by 2.5% significantly increased the content of soluble sugars by 7%. On the other hand, an obvious decrease in SS content was recorded when the volume of VC was increased to 5, 10, or 15%. A significant increase in SS contents of stressed broad bean leaves by about 1.4-fold with respect to nonstressed leaves. Interestingly, a significant reduction of SS content was observed in stressed broad bean tread with 15% vermicompost as compared with its respective control ( Fig. 2).

Vermicompost maintain K + /Na + balance in salt-exposed Vicia faba plant
Salt treatment disturbed K + /Na + balance in broad bean leaves, as indicated by the increase in Na + content by 10% in leaves, and obvious reduction by 42% in K + content of broad bean leaves (Fig. 3A, B). Thus, K + /Na + ratios decreased significantly with respect to control (Fig. 3C). On the other hand, at 15% VC-exposed broad bean plants reinforced K + / Na + ratios homeostasis by increasing Na + content by 5% in leaves, while K + content elevation was 85% causing K + / Na + ratio increase. Moreover, VC and salt stress treatment caused insignificant reduction in Na + content, but K + content was elevated in leaves by 54 and 77% in 10 and 15% VC, respectively, leading to increases in the K + /Na + ratios of broad bean leaves.
The current results showed that magnesium content in broad bean leaves significantly increased in 5, 10, and 15% VC by about 17, 11, and 11%, respectively, with respect to the control. Under stressed condition (200 mM NaCl), Mg 2+ content in leaves decreased by 1.6-fold as compared with control. Significant increase of Mg 2+ in leaves amended with  Fig. 3 Effect of vermicompost, salinity stress, and their interaction on, sodium (A), potassium contents (B), K + /Na + ratio (C), and Mg +2 (D) in Vicia faba plants. Values are means ± SD based on triplicate independent determinations, and different letters means significant difference as evaluated by Duncan's multiple comparison test 2.5, 5, and 10% VC by about 14, 18, and 36%, respectively, as compared with salt-treated broad bean leaves.

Vermicompost modulate the ultrastructure in leaves of salt-exposed Vicia faba plant
Transmission electron microscopy (TEM) demonstrated that typical mature cells with well-defined cell wall and chloroplasts were elongated with ellipsoid organization. These healthy chloroplasts have stacked closely thylakoids, stroma-thylakoid layers had a distinct structure and arranged in order, chloroplast membrane are distinctly legible, and starch grains are present (Fig. 4A, B). Well-developed mitochondria and clear nucleus surrounded with nuclear membrane containing a dense chromatin material (Fig. 4C). Differential modifications in chloroplast were noticeable in the ultrastructure of the leaf cells treated with 200 mM NaCl, the disorganization of chloroplast was most common, i.e., swelling of the chloroplast and structural alterations causing chloroplasts moving to the cell center and small-reduced mitochondria (Fig. 5A). Salt stress also accompanied by disconnection among cell membranes and chloroplasts. Further examination of the composition of the salt-stressed thylakoid membrane showed that grana thylakoid lamellae were disordered and stroma-thylakoid lamellae were shattered. Chloroplast envelope degenerated and the grana thylakoid partially dissipated with diminished grana packing, characterized by the presence of expanded plastoglobuli and starch grains (Fig. 5B). The nuclei appear to be unchanged, the nucleus containing a dense chromatin material with an occasional absence of the nucleoli (Fig. 5C), with few blocked cytoplasmic connections (Fig. 5D).
The results of the influence of 10% VC and 200 mM NaCl on TEM micrographs revealed that the cell structure of broad bean leaf relatively retained its integrity compared to their corresponding stressed ones. The ultrastructure also showed that mitochondria almost exhibited normal shape, size, and distribution (Fig. 6A). Moreover, healthy chloroplasts were observed which retained its elongated and ellipsoid organization with arrangement of grana and stroma. The thylakoids are stacked closely, and plastoglobuli were present having centrally located (Fig. 6B). Regular-shaped clear nucleus surrounded with nuclear membrane associated with distribution of dense chromatin material, along with presence of nucleolus in some cells (Fig. 6C). A minor increase in cell wall thickness with introverted tissues developing various vesicles and cytoplasmic connections was recorded (Fig. 6D).
Vermicompost modulate proteomics in leaves of salt-exposed Vicia faba plant Molecular mechanisms of broad bean leaves response to salinity were assessed by proteomic analysis. There are 13 proteins which are identified in the leaf samples of control plant (Table 4). The expression of protein was affected by salinity treatment with detection of 16 proteins in salt-treated leaf samples (Table 5). Application of 10%VC resulted in identification of 18 proteins in salt-treated broad bean leaf samples (Table 6).
Amax Quant analysis revealed that majority of proteins downregulated by salt treatment. Along with the upregulated proteins are glucose-1-phosphate adenylyl transferase with higher abundance. The protein ribulose bisphosphate carboxylase large subunit and chloroplastic peptidyl-prolyl cis-trans isomerase showed slight abundance under salt stress (Fig. 7). Chloroplastic ribosomal protein exhibited lower abundance compared with the control. Chloroplatic ferredoxin-NADP reductase, plastocyanin, polyphenol oxidase, alpha-glucan phosphorylase H isozyme, and maturase are downregulated under salt stress (Fig. 8).
A max Quant analysis revealed that majority of proteins upregulated by VC and salt treatment are involved in the processes of photosynthesis, antioxidants and ROS scavenging, metabolism, stress-responsive, and molecular adaptation. The first category of the NaCl-responsive proteins under VC treatment was related with photosynthesis, e.g., plastocyanin and chloroplastic ferredoxin-NADP reductase. The defense/detoxification-related proteins corresponded to the second category of NaCl-responsive proteins, and those are antioxidants and ROS scavenging polyphenol oxidase (Fig. 8). The metabolism-related proteins corresponded to the third category of NaCl-responsive proteins as alpha-glucan phosphorylase and peptidyl-prolyl isomerase (PPIase). The responsive and molecular adaptation-related proteins corresponded to the fourth category of NaCl-responsive proteins as chloroplastic maturase.

Hierarchical clustering
Hierarchical clustering method was applied to identify proteins with similar expression patterns, and two main clusters of protein expression were found, the first consisting of proteins that were more abundant in control and salt-treated broad bean leaves and the second containing proteins that were highly expressed in VC application along with salt stress (Fig. 8); Within this cluster, it was possible to recognize six proteins with different biological function (Fig. 8A) chloroplastic ribosomal protein, plastocyanin, chloroplastic ferredoxin-NADP, polyphenol oxidase, chloroplastic ribosomal protein, and maturase. They are downregulated under salt-treated representing 66.6%. Three proteins were upregulated of which glucose-1-phosphate adenylyl transferase, ribulose bisphosphate carboxylase large subunit, and chloroplastic peptidyl-prolyl cis-trans isomerase under salttreated representing 33.4% (highlighted in red).
The second cluster contains proteins that were abundant under vermicompost and salt stress; within this cluster, it consisted of three groups; first group that is chloroplastic ferredoxin-NADP and plastocyanin both involved in    (Fig. 8B). The second group is polyphenol oxidase which may be involved in ROS scavenging and redox regulation representing 11.1%. The third group is chloroplastic glucose-1-phosphate adenylyl transferase small subunit 1, peptidyl-prolyl cis-trans isomerase, which may be involved in metabolism and stressresponsive representing 22.2% and maturase which may be involved in molecular adaptation representing 11.1%. Three proteins were downregulated, ribulose bisphosphate carboxylases large subunit, chloroplastic ribosomal proteins, and glucose-1-phosphate adenylyl transferase under the interaction of salinity and VC.

Discussion
Vermicomposting is an innovative eco-biotechnological process using earthworms as natural bioreactors for recycling organic wastes to the soil. This process is now acceptable for converting organic material into stabilized eco-environmental humus-like product vermicompost (Tammam et al. 2022a). Vermicompost, the solid excreta of earthworms, helps in recovery the negative properties of abiotic factors and improves plant adaptation under these conditions (Benazzouk et al. 2019). Salinity affects food yield significantly, nutrients imbalance, formation of photosynthetic pigments, efficiency of photosynthesis process, and productivity of field crops (Negrão et al. 2017). The reduction of plant growth by osmotic potential and ionic imbalance can be triggered by shortage in energy supply which causing a limitation of photosynthesis process (Munns and Gilliham 2015). Present study investigated the roles of VC to improve saline environment in broad bean leaves by evaluating several constraints connected with leaf growth. Concurrently, broad bean plants presented a diminish in leaf area index under saline environment, thereby declining photosynthetic pigments consequently, reduction in growth potential, instability of the pigment protein complex, and decrease in Rubisco activity. Magnesium ions uptake is reduced under salt factor; it is an indispensable function in the chlorophyll structure (Hosseinzadeh et al. 2016). Salt damage also increases the production of active oxygen species that can seriously interrupt normal metabolism through oxidative damage to proteins, lipid, DNA, and, ultimately, cellular structures of Chl a as well as the biosynthesis of some compatible metabolites (Flexas and Medrano 2008).
The protective effects of VC on the levels of chlorophylls pigment displayed that VC possess vast prospects which are yet to be explored completely owing to the great diversity and the respective mechanisms via magnesium content ( Table 2). The effectiveness of VC has been attributed to the porous structure, in addition to relatively high water holding capacity, hormones, and organic ions that improve the water   content in leaves (Beykkhormizi et al. 2016). Vermicompost also improves the physical properties in the soils affected by salinity which considers as innovating technology to face plant growth requirements and cost-effective renovation (Ibrahium et al. 2015). Vermicompost helps stressed broad bean leaves to maintain Rubisco content, increases PS II efficiency, and leads to better photosynthetic capacity to compensate biomass loss. Vermicompost contains considerable levels of essential macro-nutrients (Sharma and Banik 2014) and supplements plants with some important micronutrients which serve as prosthetic groups for antioxidant enzymes that affect the essential role ROS decomposition and prevents chlorophyll molecules from degradation (Suthar 2009). Increased carotenoid contents by VC treatments in broad bean leaves can protect photosynthetic pigments by scavenging ROS and play a dynamic role in the shelter of chlorophyll oxidation. Carotenoids also act as photo-protection of cellular structures, especially photosystem I and II, from the damage of high light intensity by quenching excess energy and scavenging 1 O 2 (Sedoud et al. 2014).
In the present study, the efficiency of photochemical apparatus was strongly limited by salinity factor, especially Fv/F 0 of Vicia faba, indicating that the efficiency to capture excitation energy by open PSII reaction centers declined and that the PSII photochemical reaction centers were seriously damaged (Lu et al. 2002). The lower value of Fv/Fm indicated that salinity stress had adverse effect on lightutilization efficiency of broad bean plants and disturbed the regeneration of CO 2 acceptor that needs adequate electron from photosystem II to electron acceptors (Kafil 2009). Application of vermicompost at 2.5% (a desirable concentration) increased the ratios of Fv/F 0 and Fv/Fm, and these clearly explain an increase stability of broad bean PSII by VC application. Photoprotective mechanisms involved in the photosynthetic electron transport have also been achieved in stressed Vicia faba at 10% VC by reducing damage of photosynthetic apparatus, ROS scavenging, and enhancement of Rubisco content under saline conditions (Tammam et al. 2022b). Therefore, the selective discriminating characters were related to photosynthesis and have the potential to improve the salt tolerance of broad bean under saline environment.
Rubisco is an abundant soluble protein of most leaves, and the holoenzyme consists of eight nuclear-encoded SSU together with eight chloroplast-encoded LSU. Rubisco activase plays a regulatory role in its activity (Spreitzer and Salvucci 2002). Results revealed a decline in the structure in Rubisco subunits protein of broad bean leaves by salinity stress probably due to (1) damage extent in many organelles that participate in biosynthesis of both SSU and LSU, (2) decline in Mg 2+ uptake which is essential for Rubisco enzyme, (3) diminishes in chlorophylls biosynthesis and inhibition in the system of electron transport, and (4) augmentation of NaCl concentration inside the cell which leading to inactivate ATP synthesis, and consequently decreasing intracellular ATP content (Allakhverdiev et al. 2005). We proposed also that salt stress may indirectly inhibit protein synthesis by enhancing ROS formation that causing  malformation of chloroplast thylakoid membranes. Oukarroum et al. (2015) reported that both PSI and PSII activities are inhibited at high salinity level, this leading to interference of electron transport system. The current results imply a positive effect of VC at salinity stress by improving protein subunits of Rubisco. This may be related to an increase of chlorophyll content, efficiency of PSII, maintenance of chloroplast membrane, and decrease in Na 2+ uptake. Hosseinzadeh et al. (2016) reported that VC application significantly enhanced total chlorophyll content, increased efficiency of PSII photochemistry (F v /F m ), and photosynthetic and transpiration rates for salt-stressed chickpea plant. Plants synthesizing compatible metabolites such as free amino acids and soluble sugars (SS) to adjust the osmotic potential caused by high salt stress (Chen and Jiang, 2010). We observed that salt-stressed broad bean plants accumulated higher level of SS in leaves with respect to nonstressed plants. Accumulation of SS in broad bean plants under salt stress may lead to reduction of osmotic potential of the root cells and perform an important role in increasing water uptake from the soil to maintain turgor pressure for osmoregulation (Parvaiz and Satyawati 2008), free radical scavenging, and carbon storage (Parida and Das, 2005). Nevertheless, VC suppressed SS accumulation in leaves of saltstressed plants (Fig. 2), suggesting that VC acted to reduce salt toxicity independently of SS and improved the crucial role of VC in mitigating the negative salt effects due to the availability of nutrients in alleviating salt stress effects. Rady et al. (2016) recorded that vermicompost preferred leaf growth and a modern sink developed which would diminish soluble sugar in Phaseolus vulgaris.
The current study revealed that salt stress exhibited significant accumulation of Na + in broad bean leaves, obviously explaining the relation of ion toxicity with salt stress on the leaf area. Moreover, Na + toxicity often causes a significant reduction in the K + content and consequently an impaired K + /Na + ratio (Assaha et al. 2017), as detected in this study. The shortage of K + can cause obvious increase in Na + toxicity by disturbing many physiological metabolisms, such as stomatal movement, performance of photosynthesis, metabolism of secondary metabolites, water status, enzyme activation, osmotic balance, and membrane turgidity (Hniličkova et al. 2019). Potassium defect can enhance salt injuriousness of photosynthesis in maize seedling under salt stress (Qu et al. 2012). Almeida et al. (2017) reported that the maintenance of Na + and K + homeostasis in plant cells is associated with plant survival under salt stress.
In this connection, lower Na + and higher K + contents in stressed leaves of broad bean which improved with VC indicated that VC enhanced the capability of plants to decrease Na + uptake; meanwhile, better level of potassium achieved to maintain a favorable ratio of K + /Na + under this conditions, and this has been ascribed to the porous structure of VC leading to better K + absorption and reducing Na + uptake. Our findings demonstrate the significance of K + / Na + ratio to maintain a favorable ion homeostasis, which is generally observed in salt-tolerant varieties (Siddiqui et al. 2017), and sustaining a favorable K + /Na + ratio, especially in leaves, improved growth performance of vermicompost broad bean plants under salinity stress.
The ultra-structural alterations in the leaf cells of broad bean were mainly concentrated in the chloroplast. Metabolic imbalances caused by ionic toxicity and osmotic stress inhibit the photosynthetic electron transfer process and efficiency of PSII. The alterations in the ultrastructure of broad bean chloroplast by salt stress reflected a rise in ROS levels. Yamane et al. (2004) stated that excessive production of ROS under saline factor caused peroxidation, destabilization of cellular membranes, and DNA and protein damage. This is reliable with destruction of chloroplast membrane, swelling of thylakoid, aberrations in the thylakoid membrane, and stroma which is attributed to the production of ROS. These changes are like disorganization in thylakoid membrane in Zea mays as reported by Hasan et al. (2018). In broad bean leaves, salinity modifies the reliability and functionality of chloroplasts, which opportunity controls cell function, induces thylakoid alterations, and rises in amount and extent of plastoglobuli, that acting on the synthesis and recycling of lipophilic during oxidative metabolism.
Number and extent of starch grains in chloroplast of salt-stressed broad bean leaves were diminished; it may be due to the decrease in water uptake and inhibition of photosynthesis process (El Dakak et al. 2021). Inhibition of photosynthetic CO 2 fixation by salinity factor may be also related to decrease in Rubisco content and activity. The most obvious changes of the mitochondria in salt-stressed broad bean specified distinction in its dimension and shape as well as increase in electron optical contrast in the cytosol and matrix of the mitochondria. This result suggests that role of mitochondria-formed ROS under saline factor and may cause perceptible mitochondrial and cellular injury. Luo et al. (2017) recorded that mitochondrial ultrastructure in black locust (Robinia pseudoacacia L.) becomes deficient in cristae, swollen, vacuolated and damaged under salt stress.
Vermicompost reversed NaCl-maintained an orderly arrangement of grana thylakoids, chloroplast organization, and membrane integrity probably due to increase in absorption of Ca 2+ , which can displace Na + , thereby maintaining membrane stability and lessening ion leakage (Badr, 2020). Membrane stability is essential for differential intracellular ion distribution that has an impact on cell operation as membrane damage is correlated with severe physiological dysfunctions and disturbance of ion homeostasis. These results recommend that salinity triggered oxidative damage to broad bean plants; meanwhile, vermicompost elicited antioxidant power of broad bean plants under saline condition by classification is according to assignment of protein putative function by searching Uniport NCBI improving the production of chromatin materials that involve rearrangement of the genome. Modification of histone and DNA methylation are matched with modifications in the gene expression of salt stress-responsive genes. Also, several chromatin granules monitor to control of stress-responsive gene networks to fine-tune the acetylation status of histone for plant adaptation to salt condition (Kim et al. 2015). The obtained results suggest that VC controls expression of protein by the initiation of transcription and thereby control the number of proteins synthesized during translation and stabilize photosynthetic apparatus to achieve detrimental effects in Vicia faba plants under saline condition.
The obtained results indicate folding and inward coils of the plasmalemma are essential for the adjustment of the cell volume alterations under VC application and salinity for helping salt tolerance in broad bean plant. The same results are reported by Kim and Park (2010), whereas conflicting to Queirós et al. (2011) who reported the absence of vesicles in potato leaves under salt adaptation. Chalbi et al. (2015) found biochemical changes in the plasma membrane of Brassica leaves and roots in response to abiotic factor. In salt-stressed broad bean, VC restored the cytoplasmic connections between cell membranes that help in cytoplasmic streaming and create flow through its cytosol for materials, such as nutrients and metabolites, to pass through cell to cell (Fig. 4D).
Despite progression in understanding the molecular signaling and mechanisms of salt tolerance, much remains to be learned about ribosomal proteins that involved in translation of some stress-responsive proteins.
The current study revealed that proteomic analysis by MS identified 16 proteins. We found that differentially expressed proteins are low within an experimental condition compared to those with other studies. The comparison between the patterns revealed 9 varied proteins, three of which were upregulated. Chloroplastic glucose-1-phosphate adenylyl transferase small subunit participates in synthesis of starch, synthesis of the activated glycosyl donor, synthesis of ADPglucose, and involved in the pathway of Glycan biosynthesis. Therefore, the upregulation of this protein is important for starch synthesis which acts as osmoregulation during salt stress. We speculated that broad bean plants produce starch to store energy for cell metabolism when cell requires energy, and releases enzymes to degrade part of the starch chain to release carbon for producing sucrose.
Proteomic approaches indicated that Rubisco-LSU is upregulated in broad bean leaf under saline conditions; however, the protein content of LSU and SSU was downregulated. The discrepancy may be due to that LSU undergoes post-translational modifications such as methylation at the N-terminal methionine of Rubisco-LSU by large subunit methyl transferase enzyme which appear in many plant genomes (Mininno et al. 2012). Trievel et al. (2003) demonstrated that during Rubisco methylation, replacing hydroxyl by methoxy group will consequence in increasing the enzyme size, stability, hydrophobicity, and decreasing its mobility. They also reported that Rubisco-LSU may interrelate with the chloroplast membrane avoid passing due to its large size and immobilization.
Chloroplastic peptidyl-prolyl cis-trans isomerase (PPIase) is slightly upregulated under saline factor in broad bean leaf tissues. Edvardsson et al. (2007) reported that all peptidyl-prolyl isomerase activity in the thylakoid lumen from Arabidopsis thaliana is involved in protein-folding processes. The upregulation of PPIase activity within the thylakoid lumen may be elaborated in biogenesis of photosynthetic complexes of Arabidopsis thaliana under salt stress (Ingelsson et al. (2009). The bulk of the differentially proteins is downregulated, in which enormous group of these downregulated proteins was correlated with photosynthetic proteins as chloroplastic ribosomal proteins. Many proteins implicated in photosystems I and II polypeptide subunits were diminished by chloroplastic ribosomal proteins. The reduction of these principle components of photosynthetic proteins was described with Arabidopsis thaliana by Tiller et al. (2012) is supposedly responsible for defects in photosynthetic performance on explanation of reduced translational efficiency. In contrast, Soares et al. (2018) recorded that those ribosomal proteins were more abundant in salttreated maize genotype.
In this study, we compared the proteomes of salt-affected broad bean plant under VC treatment. The results indicated that plastocyanin and chloroplastic ferredoxin-NADP reductase were accumulated under VC and saline environment. Pathway enrichment analysis revealed that they involved on the modulation of the transcription and translation of related proteins especially with photosynthesis. A reliable upregulation of proteins in the first group elaborated for photosynthesis and concomitant with broad bean salt tolerance, and these proteins have been suggested to enhance an increased demand for energy, and thus act as a tradeoff alongside some metabolic pathways, prevent acute damage photosynthetic apparatus and conserve energy for plant progression (Ford et al. 2011). Sustaining a high photosynthetic rate is connected with sugarcane genotypes salt tolerance (Pacheco et al. 2013).
The recent advance in the transcriptome and proteome created a rapid comparison of profiles functionally related proteins that have a similar regulation pattern, VC was found to upregulate the defense/detoxification-related proteins correspond to polyphenol oxidase which detoxify ROS and oxidize phenols which represent second category of NaCl-responsive proteins. This protein might cope with oxidative stress and offer fortification for broad bean against salt stress injury and function of redox-signaling proteins. Wang et al. (2007) focused on 23 specific salt-responsive ROS scavenging enzymes such as ascorbate peroxidase, dehydroascorbate reductase and peroxiredoxin, superoxide dismutase in Lycopersicon esculentum plant. Arefian et al. (2019) stated that improving ROS scavenging enzymes as superoxide dismutase, ascorbate peroxidase, and glutathione S transferase in chickpea genotypes under salt stress. Thus, the activity of such detoxifying enzymes is one of the mechanisms sharing to the acclimation of broad bean under VC application and saline condition (El-Dakak et al. 2021).
Proteins forming the alpha-glucan phosphorylase are upregulated under VC application and saline condition; it correlated with the degradation of starch in plants. Zeeman et al. (2004) recorded that phosphorylase provides sugar phosphates for oxidative pentose phosphate pathway to supply reductant for many direct routes of biosynthesis and metabolic energy that drives cellular processes in Arabidopsis leaves. Phosphorylase is also critical for controlling ROS levels through ascorbate and glutathione route that has a potential for detoxification. Thus, the upregulation of alpha-glucan phosphorylase can be proposed when reducing power in the chloroplast is high, other routes that could provide substrates for the oxidative pentose phosphate pathway whenever insufficient, so it probably increases the salt stress tolerance of broad bean plants under VC application.
Our study revealed that one of the induced proteins which were obvious in broad bean leaves is PPIase activity, consistent upregulation of proteins associated with metabolism under VC application and salt condition. Arefian et al. (2019) concluded that the upregulation of PPIase activity in chickpea genotypes was due to presence of both cyclophilins (Cyps) and FK506-binding proteins (FKBPs), indicating their involvement in protein-folding processes. The increased new proteins PPIase are directly related to overcome salinity effects under VC treatment for conserving the photosynthetic machinery from salt damage. Vermicompost along with salt upregulates chloroplast-encoded maturase for the translation and expression of group II intron maturase that play a role in regulation methods concerning with plant growth and ultimately photosynthesis (Barthet and Hilu 2007).