Fungal endophytes promote wheat (Triticum aestivum L., genotype-PBW-343) growth and enhance salt tolerance through improvement of ascorbate-glutathione cycle and gene expression

doi:10.21203/rs.3.rs-4874583/v1

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Fungal endophytes promote wheat (Triticum aestivum L., genotype-PBW-343) growth and enhance salt tolerance through improvement of ascorbate-glutathione cycle and gene expression

https://doi.org/10.21203/rs.3.rs-4874583/v1

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Background and aims

Wheat (Triticum aestivum L.) faces considerable challenges in terms of growth and productivity due to soil salinity, which is a major constraint to agricultural success. This study investigated the potential of fungal endophytes to enhance wheat growth and improve salt tolerance by influencing the ascorbate-glutathione cycle and gene expression.

Methods

Experiments were conducted, usingwheat seedlings (PBW-343) inoculated with endophytic fungi (Cladosporium parahalotolerant and Aspergillus medius)isolated from salt-tolerant wheat genotypes (KRL-210, KRL-213 and KRL-19) fromthe previous study. Endophytic fungi were used individually and in combination. Fifteen days seedlings exposed to 100 mM NaCl in the presence and absence of fungal endophytes. To elucidate the molecular mechanism, gene expression analysis was performed on key genesAPX, SOD, GR, DHAR, and MDHAR.

Results

Seedlings treated with endophytic fungi (consortia form)significantly enhanced the sugar, protein, chlorophyll, carotenoid content, and chlorophyll fluorescence (Fv/Fm)compared to control under salt stress. The O2- and lipid peroxidation levels were significantly reduced in plants inoculated with fungal endophytes. Salt stress increased APX, SOD activities and decreased GR, MDHARand DHAR activities.Endophytic fungi inoculated with salt-stressed seedlings enhanced the above-mentioned indicators compared to the salt-stressed plants without fungal endophytes, as well as in the ratios of AsA/DHA and GSH/GSSG.Endophytic fungienhanced the transcript levels of SOD, DHAR, APX, GR, and MDHAR genes compared to the control

Conclusions

The present study found that the expression levels of several genes associated with the ascorbate-glutathione cycle were upregulated in endophyte-inoculated plants, indicating a more efficient antioxidant system capable of scavenging reactive oxygen species.

Triticum aestivum L.

Fungal endophyte

Salt stress

Reactive oxygen species (ROS)

Ascorbate-glutathione cycle

Defensive system gene expression

Wheat (Triticum aestivum L.) is one of the world’s most essential cereal crops, constituting a significant portion of the global food supply and serving as a staple for millions of people. However, its productivity is severely affected by various environmental stresses, among which soil salinity is a critical challenge. Soil salinity as a major abiotic stress factor adversely affects plant growth, leading to reduced yield and economic losses in agricultural systems worldwide (Elbagory 2023; Kaya et al. 2023; Munns and Tester 2008; Sharma 2023; Wang et al. 2023). As global climate change continues to exacerbate soil salinization, it is imperative to develop sustainable strategies to enhance crop tolerance and improve agricultural productivity.

At present, salt affects over 6% of the world's arable land primarily in the dry and semiarid regions (Bui 2013). Thus, food security and the sustainability of agriculture worldwide are gravely threatened. It is imperative, therefore, to devise effective and efficient methods for mitigating the detrimental effects of salt stress on plant growth and development. Although novel cultivars with better salt resistance have been created using conventional breeding and transgenic technology, breeding for salt tolerance has not been successful (Phang et al. 2008). Difficulties arise because of the lengthy breeding cycle and low breeding efficiency of the quantitative trait. Using transgenic technology, salt-tolerant genes can be introduced in new plant materials (Lu et al. 2007; Otaibi et al. 2024; Sairam and Tyagi 2004; Sahi et al. 2006), but it has been criticized due to gene loss, high cost, and other regulatory concerns (Glick 2007).

The use of exogenous compounds to counteract the negative effects of abiotic stress increases the plant tolerance to salt stress. Examples of such compounds include chitooligosaccharides (Zou et al. 2015), oligochitosan (Ma et al. 2012), nitric oxide and calcium nitrate (Tian et al. 2015), jasmonic acid (Qiu et al. 2014), gibberellic acid and calcium chloride in linseed (Linum usitatissimum; Khan et al. 2010), and ascorbic acid in broad beans (Vicia faba; Younis et al. 2010). It has been demonstrated that these exogenous substances increase the ability of plants to withstand salt stress; however, the precise physiological processes involved remain unclear. The use of bacteria and fungi that promotes plant development to induce tolerance to abiotic stress is a novel and highly recent technology that has garnered much interest. This is a useful strategy for increasing plant resistance to salt stress and could aid in the creation of sustainable agricultural systems.In recent years, plant-microbe interactions have become a viable approach for solving the problems caused by salt in the soil. Among these interactions, the symbiotic association between plants and fungal endophytes has gained significant attention because of its potential to promote plant growth and confer tolerance to abiotic stresses including salinity (Chowdhary et al. 2024; Chauhan et al. 2024; Koudadaria et al. 2023; Prajapati et al, 2024; Rodriguez et al. 2008). Microorganisms, known as fungal endophytes, live inside plant tissues and have a significant impact on host plant physiology and stress responses, without exhibiting any outward signs of disease (Pertini 1991).

Fungal endophytes have various beneficial effects on host plants, including nutrient acquisition, enhanced water uptake, and protection against pathogens and herbivores (Compant et al. 2010). Additionally, they play a crucial role in regulating plant responses to abiotic stress by inducing stress-related genes and modulating antioxidant defence systems (Asaf et al. 2023; Aizaz et al. 2023; Cao et al. 2023; EL-Sayed et al. 2022; Lubna et al. 2022; Rodriguez et al. 2009; Shen et al. 2023; Zhang et al. 2016; Zou et al. 2023). One of the most important antioxidant defence mechanisms in plants is the ascorbate-glutathione cycle, which scavenges reactive oxygen species (ROS) generated under stressful conditions and preserves cellular redox homeostasis (Noctor et al. 2012; Popovic et al. 2024). Salt stress causes plant cells to produce ROS, including superoxide, hydrogen peroxide, and hydroxyl radicals (Mittler 2002). An overabundance of ROS can cause oxidative damage to proteins, lipids, and nucleic acids, ultimately hindering the growth and development of plants (Foyer and Noctor 2005).

In order to resist the oxidative damage brought out by salt stress, plants need a robust antioxidant system(Foyer and Kunert 2024; Liu et al. 2024). In a previous study (Ghorai et al. 2023;Khan et al. 2015; Li et al. 2023; Ren et al. 2021; Sadeghi et al. 2019; Wang et al. 2017), fungal endophytes are involved in improving the ascorbate-glutathione cycle in plants under abiotic stress conditions. Endophytes can affect the activity of important AsA-GSH cycle enzymes including superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR). This can result in an increased ability to scavenge reactive oxygen species (ROS) (Li et al. 2023; Rong et al. 2022; Waqas et al. 2012; Wang et al. 2023; Zou et al. 2023).

Furthermore, fungal endophytes can modulate gene expression in host plants, thereby influencing various physiological processes including stress responses. Several studies have reported changes in the expression of stress-responsive genes such as those encoding heat shock proteins, late embryogenesis abundant (LEA) proteins, and various transcription factors in endophyte-inoculated plants under salt stress conditions (Varma et al. 2012). These changes in gene expression likely contribute to the enhanced stress tolerance observed in endophyte-associated plants (Aizaz et al. 2023; Zhan et al. 2016; Zou et al. 2023). Despite a growing body of research on the beneficial effects of endophytic fungi in enhancing plant development and stress tolerance, there is still a knowledge gap regarding their specific roles in promoting wheat growth and conferring salt tolerance. The present study is, thus, aimed to address this gap by investigating the effects of selected fungal endophytes on wheat plants grown under saline conditions. We hypothesized that these endophytes could improve wheat growth and enhance salt tolerance by positively modulating the ascorbate-glutathione cycle and regulating stress-responsive gene expression.

Endophytic fungi

We previously isolated and identified endophytic fungi from tissues of salt-tolerant wheat genotypes (KRL-19, KRL-213 and KRL-210), such as leaves, stems, and roots, in our laboratory. Two out of 20 fungal endophytes were found potential, that are, C.parahalotolerant L. isolate KRL213/150 (accession number: ON714989) and A.medius isolate KRL-19/200 (accession number: ON753782). For the purposes of this study, these two isolates were used individually and in combination (Prajapati et al. 2024).

Endophyte inoculum preparation

A. medius and C. parahalotolerant were inoculated on PDA and conidial suspensions were prepared using the method described by Zhang et al. (2014). Two-week-old culture plates were flooded with 10 ml of sterile distilled water consisting of 0.1per cent (v/v) Tween 80 to maintain a final suspension of 1.0x10⁸ spores ml^-1, which was stored at 4^oC.

Plant material and treatment conditions

Under ideal humidity, temperature and light conditions (25°C, 60 percent relative humidity, and 600 molm^-2s^-1) the experiment was conducted in a glasshouse chamber during the wheat growing season (November-February). Eight groups were formed for the experiment: a control group that did not receive any treatment from the fungus or the 100 mM NaCl solution; a negative control group that received treatment from the 100 mM NaCl solution; three positive control groups comprised of C. parahalotolerant, A. medius, and C. parahalotolerant + A. medius; and a stressed group that received treatment from the fungus and 100 mM NaCl and contained C. parahalotolerant + A. medius and C. parahalotolerant+ NaCl, A. medius + NaCl.

Wheat seeds were collected from an endophyte spore solution and left to dry in the open after being submerged overnight. Twenty fungal-coated seeds were placed in a perforated pot filled with autoclaved soil, all of which had three replicates, and were organized in a completely randomized block arrangement. After a week of wheat plant growth, NaCl treatment was initiated in accordance with previously described methodology (Prajapati et al. 2024; Sheng et al. 2008). To prevent salts from interfering with the growth of different endophytic fungi and causing osmotic shock to the roots of the plants, 50 ml of a solution containing the recommended concentration of NaCl was applied to the soil every seven days in each pot. Plants were routinely irrigated with distilled water twice a week (Fig. 1).

Endophytic fungi establishment in inoculated plants

To verify the presence of endophytic fungi in the plants, tissue samples (stems, leaves, and roots) from ten plants were collected 16 d after inoculation. The same reference was used for the identification and isolation of fungal endophytes (Prajapati et al. 2024).

Quantification of photosynthetic pigments

By extracting 0.5 g of leaf material in 10 ml of 80 per cent acetone, chlorophyll (Chl) and carotenoids (Car) were measured spectrophotometrically using the method described by Agrawal and Rathore (2007).

Measurement of chlorophyll fluorescence

The photochemical effectiveness of photosystem II (PSII) was determined by measuring the variable fluorescence to maximum fluorescence (Fv/Fm) ratio of chlorophyll fluorescence using a portable fluorometer (Pocket PEA, Hansatech, England)as summarized by Rivero et al. (2009).,

Measurement of malondialdehyde (MDA) level

The Hodges et al. (1999) procedure was used to measure the MDA levels in the leaves. Two hundred mg of leaf powder and 2.5 mL of 0.1 percent TCA (Trichloroacetic Acid) were combined, and the mixture was centrifuged at 10,000×g for 15 min at 4^oC. Then, 4 ml of 0.5 per cent TBA (Thiobarbituric acid) and 1 mL of 20 per cent TCA were mixed with the supernatant. The blend was cooled in an ice bath for approximately 20 min in a water bath at 90°C. The mixture was then centrifuged for 10 min at 15,000 × g, and 532 nm was used to calculate the absorbance.

O₂^- deposition detection using nitrobluetetrazolium (NBT) staining

Wang et al. (2007) stated that the detection of O₂^-requires the ability to decrease Nitro Blue Tetrazolium (NBT). Under salt stress conditions, wheat leaves were treated with fungal endophytes. After 15 days, the NaCl-treated and control samples were used to monitor O₂^- buildup. The leaf samples were submerged in an amber-colored vial that contained 100 mg of NBT in 0.05M sodium phosphate buffer (pH 7.5). The NBT solution (0.2%) was prepared by adding 50 mL to the final volume, and the sample was stored for 8h in a dark environment. After eight hours of incubation, the leaves were placed on a filter paper and cooked in 96 per cent ethanol to eliminate chlorophyll. Superoxide anion (O₂^-) accumulation was visible in the dark blue color of the leaf tissue when viewed using an Olympus compound microscope. Three replicates of each treatment were maintained andthe experiments were conducted in triplicate.

Soluble sugar and protein content

To determine the soluble protein and sugar contents in the leaves of wheat seedlings, the leaf samples obtained from the treatment and control groups were cut into small pieces and thoroughly cleaned with distilled water thrice. Next, the tiny wheat seedling leaf fragments were dried, weighed, and placed one at a time into glass vials containing 10 mL of 80% (v/v) ethanol. The vials were heated in a water bath for 30 min at 60°C. The filtered extracts were diluted with the 80 per cent (v/v) ethanol to a final volume of 20 ml. According to Giannakoula et al. (2008), the amount of soluble sugar present in the extract was determined by comparing its content with a standard curve and glucose criteria. The soluble protein content was assayed using the Bradford method (1976). To ascertain the amount of soluble protein present in wheat seedlings leaves, the Coomassie Brilliant Blue G-250 reagent containing BSA was utilized as the standard.

Assay of antioxidant metabolites

The ASA content was determined according to the procedure described by Gillespie and Ainsworth (2007). 2.5 mL of 6% (w/v) TCA was combined with 50 mg of leaf powder and centrifuged at 10,000g for 10 min at 4°C. The amount of oxidized ascorbate (DHA) was calculated by subtracting the concentration of AsA from that of tASA, while the GSH and GSSG contents were assessed using the method described by Rahman et al. (2006). Following the addition of 50 mg of frozen leaf powder to an extraction buffer of 2.5 mL (which contained 50 mM potassium phosphate, 23mM sulfosalicylic acid, pH 8.0, and 5mM EDTA), the mixture was centrifuged at 10,000×g for the time of 10 min at 4°C temp. The GSSG concentration was subtracted from the total glutathione (tGSH) to estimate the concentration of GSH.

Assay of antioxidant enzyme activities

The leaf powder (500 mg per antioxidant enzyme) was homogenized in an extraction buffer containing 1% (w/v) polyvinylpyrrolidone (PVP) and 50 mM potassium phosphate buffer at 7.5 pH. The mixture was centrifuged at 10,000×g for 15 min at 4°C. Antioxidant enzymes were tested in the supernatant.

The ability of superoxide dismutase SOD (EC 1.15.1.11) to prevent photoreduction of NBT was used to evaluate its activity, as described by Becana et al. (1986). To a 50 μl of enzyme extract, 1 mL reaction mixture, 14.3 mM methionine, 50 mM K₃PO₄ buffer (pH 7.5), 0.1mM EDTA (Ethylenediamine tetraacetic acid), 82.5 mM NBT and 2.2 mM riboflavin were added. Fluorescent tungsten bulb (15 W) was used to illuminate the process. Ten minutes later, the reaction was terminated And the absorbance was measured at 560 nm.

Ascorbate peroxidase activity (EC 1.11.1.11) was measured using the protocol described by Asada (1984). To the reaction mixture (1.0 mL) consisting of 33 μL of enzyme extract, 0.17 mM ascorbate, and 50 mM K₃PO₄ buffer (pH 7.0), addition of 5 mM H₂O₂ started the reaction. For three minutes, the absorbance was determined at 290 nm (ε=2.8 mM^-1 cm^-1).

The activity of monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) which is based on MDHA-dependent oxidation of NADH was evaluated using Drew et al. (2007). One hundred microliters of enzyme extract, 50 mM K₃PO₄, 0.128 units of ascorbate oxidase and 2.5 mM ascorbate were included in the standard reaction mixture (1.0mL). The reaction was initiated by the addition of 0.2 mM) And the absorbance was measured at 340 nm.

The Asada (1984) method was used to assess the dehydroascorbate reductase activity (DHAR, EC 1.8.5.1). The standard reaction mixture (1.0 mL) was comprised of 50 mM K₃PO₄ buffer (pH 7.5), 5 mM GSH, 75 μL enzyme extract, and 0.11 mM EDTA. 0.5 mM DHA was then added to initiate the reaction. The quantity of enzyme required to produce one mol of ascorbate per minute at 25°C was measured and designated as one DHAR unit. Absorbance was measured at 265 nm (ε =14.5 mM^-1 cm^-1).

The glutathione activity (EC 1.6.4.2) was measured using the methodology described by Smith et al. (1988). Fifty mM K₃PO₄ (potassium phosphate) buffer (pH 7.5), 1mM GSSG, 100 μL crude enzyme extract, and 0.75mM DTNB (5,5′-dithiobis-(2-nitrobenzoic acid) were all present in the reaction mixture (1.0 mL). The reaction was initiated by adding 0.1 mM NADPH. At 412 nm (ε = 14.15mM^-1cm^-1), the increase in absorbance resulting from the production of TNB (5-thio-2-nitrobenzoic acid) was measured.

Extraction of total RNA and analysis of gene expression by quantitative real-time reverse transcriptase-PCR (qRT-PCR)

The100 mg leaves were taken separately from 15-days-old stressed and unstressed wheat seedlings, instantly frozen in liquid N2 and stored at -80°C for use in subsequent studies. The leaves were finely ground and TRIzol (Invitrogen) was used to extract total RNA. The SuperScriptTM III First-Strand Synthesis System (Invitrogen, USA) was used to synthesize cDNA from the extracted RNA, according to the manufacturer’s protocol. Synthesized cDNA was used as a template for qPCR. The NCBI candidate protein wheat EST sequences (Qiu et al. 2014; Zou et al. 2015)” were used to design specific primers. Table 1 lists the DNA sequences of primers used for SOD, APX, MDHAR, DHAR, and GR. The reaction was carried out with Lightcycler 480 SYBR green Master mix, 2X-10 μl (Roche, USA); PCR primers (Forward and Reverse), 10 mM-1μl each; 40 ng/μl-5μl; cDNA template, and PCR grade water-3 μl by utilizing a C1000TM Thermal Cycler CFX96TM Real-Time System (BIO-RAD, USA). At the conclusion of each PCR, melting curve analysis of the amplified products was performed to verify that a single PCR product was amplified. Gene expression was measured by using the delta-delta Ct method in relation to each sample's level of actin expression, an internal reference gene (Livak and Schmittgen 2001). Each reaction was performed in triplicate.

Statistical analyses

GraphPad Prism 8.0 was used to evaluate the data that were collected for each experimental variety. The data were subjected to a statistical analysis of the SD using IBM SPSS STATISTICS 20. The mean values have been then compared using Duncan's multiple range test (DMRT) at P < 0.05 (ANOVA SAS release 9, SAS Cary, North Carolina). PCA (Principal component analysis) was carried out by utilizing the XLSTAT software, version 2016 (www.xlstat.com, Addinsoft SARL).

Chlorophyll (Chl) and carotenoid (Car) contents

The levels of carotenoid and chlorophyll reduced in comparison to the control values of plants after treatment with 100 mM NaCl (Table 2). Irrespective of whether endophyte-associated plants were subjected to salt stress, their chlorophyll content enhanced (p < 0.05) (Table 2). Compared to the endophyte-free plants (control), the plants inoculated with a combination of the two fungal endophytes (C. parahalotolerant + A. medius) showed a higher chlorophyll content under salt stress conditions, with values of 51.72% and 49.34%, respectively (Table 2).

The maximum increase in chlorophyll content was observed after inoculation with a combination of A. medius and C. parahalotolerant in comparison to the control. Similar to chlorophyll, the amount of carotenoids decreased as the salt stress levels increased. Also, plants that were inoculated with endophytes had higher carotenoid contents than those not inoculated with the endophytes (Table 2). Plants treated with C. parahalotolerant and A. medius fungal endophytes exhibited the highest carotenoids content with 57.14% and 50% differences from the control groups, respectively.

Chlorophyll fluorescence

The highest possible photochemical efficiency or potential quantum yield of PSII was determined by using the Fv/Fm ratio. Both, endophyte colonization and salt stress affected the Fv/Fm ratio. Plants inoculated with a mixture of the two fungal endophytes showed the highest Fv/Fm ratio, with a deviation of 35.71% compared to the control group (Table 2).

Soluble sugar and protein contents

Compared to the control, the application of fungal endophytes (A. medius and C. parahalotolerant) increased the amounts of protein and soluble sugar in wheat seedlings grown under salt stress and non-saline stress. However, following the application of NaCl alone, the protein and soluble sugar levels of the wheat seedlings dramatically decreased (Table 2). In contrast to the NaCl-stressed plants, the soluble sugar and protein contents in the wheat leaves increased by 44.99%, 47.76%, 60.05%, and 63.67% when treated with C. parahalotolerant and A. medius in isolation, and by 32.26%, 35.24%, 41.58%, and 44.63%, respectively when treated with A. medius and C. parahalotolerant in combination with 100 mM NaCl. The levels of soluble sugar and protein in wheat seedlings increased by priming with C. parahalotolerant and A. medius, the impact on soluble protein content being higher than that on the soluble sugar content (Table 2).

MDA content

The concentrations of MDA and O₂- in the wheat seedlings were significantly elevated by salt stress (Fig. 2). When exposed to salt stress, plants primed with fungal endophytes showed distinct reaction patterns. Plants under salt stress and those free of endophytes exhibited the highest MDA contents (control). The consortia application of the two fungal endophytes (A. medius + C. parahalotolerant) decreased MDA concentrations by 25.16%, 43.13%, and 28.15% (Fig. 2).

O₂^-deposition detection in leaves

The formation of a dark blue formazan color due to the reduction of NBT is indicative of the concentration of superoxide anion (O₂^-). The dark blue color of the formazan compound was more noticeable on leaves that had been treated with salt than with fungi. Superoxide anion deposition was greatest in stressed samples, followed by A. medius, C. parahalotolerant, and C. parahalotolerant + A. medius treated samples under stress conditions (Fig. 3).

Contents of ascorbate and glutathione

Salt stress dramatically reduced the levels of the antioxidant molecules (GSH and AsA) in wheat plants (Table 3). Treatment with the two fungal endophytes considerably boosted the AsA and DHA contents even under salt stress. Under salt stress conditions, the most successful therapy colonization with the two fungal endophytes substantially (p < 0.05) increased the AsA/DHA ratio by 57.81% compared to endophyte-free plants. Salt stress also significantly increased the GSH and GSSG levels (Table 3).

In contrast, plants inoculated with fungal endophytes exhibited reduced GSSG levels and increased GSH levels. The plants underwent considerable reduction in the GSH/GSSG ratio upon exposure to extreme salt stress (100 mM). However, in comparison to salt-stressed plants alone, plants colonized with the two fungal endophytes showed a considerable improvement in the GSH/GSSG ratio by 74.17% (Table 3).

Activities of antioxidant enzymes

Antioxidant enzyme activities vary in several ways when plants are exposed to salt stress. As shown in Fig 4, plants under stress exhibited decreased MDHAR, DHAR and GR (Fig. 4-c,d,e) activities, but higher SOD and APX (Fig 4, a,b) activities compared to control. Under salt stress, there was a positive correlation between SOD and APX activities. Fungal endophytes treated with salt stress showed elevated activities of SOD, DHAR, APX, MDHAR, and GR. The most effective treatment i.e. colonizing plants with both fungal endophytes substantially raised SOD activity in wheat leaves by 23.63%, 8.83%, and 5.47% (Fig. 4A), APX activity by 51.3%, 21.3%, and 39.4% (Fig. 4B), MDHAR activity by 13.3%, 29.3%, and 53.2% (Fig. 4C, DHAR activity by 9.6%, 29.45%, and 41.4% (Fig. 4D), and the GR activity by 19.6%, 32.85%, and 79.1%, respectively (Fig. 4E) in comparison to non-colonized plants.

Antioxidants differentially react to the intensity of salt stress and endophyte inoculation

PCA was carried out for the different factors considered to identify the largest variability components to better understand the antioxidant variations related to endophyte inoculation as well as to the salt intensity (Fig. 5 A). Two components were identified by all parameter analyses in relation to the endophyte inoculation and salt stress conditions (Fig. 5, A). PC1 accounted for 66.04% of the overall variance, whereas PC2 accounted for 18.02% of the variance. Significant correlations were observed among SOD, GSH, MDHAR, GR, GSH/GSSG, APX, AsA, DHAR, ASA/DHA and GSSG under salt stress (Fig. 5, A).

While DHA, ASA, SOD, GSH, MDHAR, GR, GSH/GSSG, APX, AsA, DHAR, ASA/DHA and GSSG were found to be linked to component 1 in PCA (Fig. 5, A), MDA, DHA, ASA/DHA, GR, SOD, MDHAR, and GSH/GSSG were linked to component 2. The PCA score plots (Fig. 5, B) showed a distinct separation of plants under salt stress. Plants that were not inoculated with endophytes showed high MDA and GSSG values, whereas those colonized by fungal endophytes showed higher levels of APX, SOD, MDHAR, DHAR, and GR (Fig. 5,B). Fungal-endophyte-colonized plants exhibited high ASA/DHA and GSH/GSSG ratios (Fig. 5, B).

Measurements of the transcript levels in salt-stressed wheat seedlings using quantitative real-time PCR (qPCR) of the five genes encoding ASA-GSH cycle enzymes

In this study, five genes encoded by the AsA-GSH cycle enzymes were measured in salt-stressed wheat seedlings to explore the influence of fungal endophytes on the AsA-GSH cycle at the molecular level. Transcript levels of SOD and APX genes increased under salt stress conditions as compared to the control [Fig. 6 (A and B)]. In contrast, the transcript levels of DHAR, MDHAR and GR genes decreased under salt stress conditions and all other treatments compared to the control. Under salt stress, the fungal endophyte treatment significantly increased the transcript levels of SOD, DHAR, APX, GR and MDHAR genes compared to the control (Figure 6, A, B, C, D, and E). Plants treated with both endophytic fungi significantly upregulated transcript levels of SOD, APX, MDHAR, DHAR and GR genes compared to plants treated with individual fungus.

According to earlier studies (Chauhan et al. 2024; Mahmood et al. 2012; Otaibi et al. 2024; Prajapati et al. 2022; Prajapati et al. 2024; Zhang et al. 2016), higher salinity levels are among the primary environmental stressors that affect plant’s biochemically, restrict plant growth and reduce plant productivity. The potential of these symbionts to produce broad-spectrum resistance to plant diseases and their amazing interactions with host plants are well-known (Harman et al. 2004; Naseby et al. 2000; Yedidia et al. 2003; Zhang et al. 2016). Fungal endophytes have been shown to be capable of boosting plant growth and systemic reactions in plants when they are exposed to salt stress.

Our findings showed that after 15 days, NaCl treatment dramatically reduced the growth and development of wheat seedlings, and this effect was greatly mitigated by the inoculation of fungal endophytes. To the best of our knowledge, this study is the 1st to identify the function of the plant growth-promoting fungi A. medius and C. parahalotolerant in increasing the salt stress tolerance of wheat seedlings. Additionally, our investigation identified a potential mechanism by which A. medius and C. parahalotolerant mitigated the detrimental effects of NaCl stress on wheat seedlings. The physiological, biochemical, and molecular resistance of wheat seedlings to salt stress was increased by the application of endophytic fungi.

In the present study, we discovered that inoculation with a fungal endophyte enhanced plant growth under salt stress conditions compared to non-inoculated plants. According to our earlier research, the most commonly observed response to endophyte inoculation in wheat plants is an increase in root and shoot system growth (Prajapati et al. 2024). Endophytes secrete secondary metabolites to protect themselves from biotic and abiotic stressors. Host plants develop significantly more when exposed to secondary metabolites released by fungi, particularly hormones such as gibberellins and auxins (Bilal et al. 2018; Gul et al. 2023; Jan et al. 2019; Lubna et al. 2022; Nanda et al. 2018; Prajapati et al 2024; Waqas et al. 2012).

Our research indicated that the increasing levels of salt stress are correlated with decreasing amounts of carotenoids and chlorophyll. Chlorophyll breakdown and pigment photooxidation may be the cause of this oxidative stress indicator. Photosynthetic capacity is affected by decrease in chlorophyll content because it is a necessary component of photosynthesis. Stimulation of the net photosynthetic rate was observed in the C. parahalotolerant and A. medius endophyte-treated plants. Similar outcomes were observed in NaCl-stressed wheat (Triticum aestivum L.), tomato (Lycopersicum), soybean (Glycine max), cotton (Gossypium spp.), pepper (Capsicum annuum), and mung bean (Vigna radiata L.) seedlings by other researchers (Chauhan et al. 2024; Liu et al. 2014; Moghaddam et al. 2021; Simaei et al. 2011; Zhang et al. 2016; Zou et al. 2023).

Tariq et al. (2011) discovered that the instability of the pigment-protein complex under salt stress conditions and the oxidation of chloroplasts and chlorophyll pigments were the likely causes of the decrease in chlorophyll content in wheat seedling leaves subjected to salinity stress. Regardless of whether wheat seedlings experienced salt stress, we found that the application of fungal endophytes dramatically increased their chlorophyll content. Subsequent application may prevent ROS accumulation and production in plant tissues. Qi et al. (2012) reported comparable results in cucumber (Cucumis sativus) seedlings treated with NaCl. Carotenoids act as radical scavengers and singlet oxygen quenchers while also maintaining and shielding the lipid phase (Bu et al. 2012).

Wheat treated with endophytes may be more resistant to salt stress because of their increased carbohydrate content. Fv/Fm ratios are frequently used as markers to demonstrate the induction of stress in plants. A plant is considered healthy and free of photosynthetic stress if its Fv/Fm ratio is 0.7 or higher (Bu et al. 2012; Ogbe et al. 2023; Sadeghi et al. 2020). Salt stress resulted in a decrease in the Fv/Fm ratio, which indicated a reduction in PSII activity and maximal quantum yield. Inactivation of the PSII donor side and certain structural alterations in PSII centers may be connected to the decline in the Fv/Fm ratio (Azad and Kaminskyj 2016; Irshad et al. 2023; Tóth et al. 2005). It has been discovered that Fv/Fm is dramatically increased in endophyte-colonized wheat and other crop plants under severe salt stress, although the potential interactions between chlorophyll fluorescence and fungal endophytes are still poorly understood (Siddiqui et al. 2022). Our findings showed that the maximum fungal endophyte inoculation increased quantum yield and PSII activity.

In the present study, colonization by fungal endophytes inhibited lipid peroxidation at all stress levels, whereas salt stress led to a significant increase in MDA concentration (lipid peroxidation). This might be due to the fact that endophytes protect plants from oxidative stress by preventing membrane damage and eliciting antioxidant responses. Comparable outcomes of endophyte treatment on various agricultural plants have also been reported (Azad and Kaminskyj 2016; Gul et al. 2023; Li et al. 2023; Prajapati et al. 2024; Siddiqui et al. 2022; Zhang et al. 2016).

The accumulation of O₂^- following NaCl treatment, as demonstrated by the findings of the histochemical experiments, indicated the deposition of blue coloration and dark brown coloration on the surface of leaves. Owing to the extensive buildup of ROS, blue and dark brown diformazan precipitates were much more noticeable in the leaves under NaCl stress. Plants treated with fungal endophytes showed a considerable reduction in ROS accumulation after 15 days. Similar results have been observed under abiotic stress conditions in crops treated with fungal endophytes (Cao et al. 2023; Chowdhury et al. 2024; Sadeghi et al. 2020; Zou et al. 2023). Superoxide radicals (O₂^-) are neutralized by SOD to O₂^- and H₂O, which serves as the first line of defence against ROS (Azad and Kaminskyj 2016).

Osmotic molecules such as soluble carbohydrates and proteins are generally acknowledged as significant markers of plant responses to abiotic stress (Alscher et al. 2002; Azevedo-Neto et al. 2006). An extremely effective defensemechanism against oxidative damage caused by high salinity and drought stress in plant environments is usually demonstrated by increased levels of sucrose and glucose accumulation in plants. (Bartels and Sunkar 2005; Chauhan et al. 2024; Ogbe et al. 2023;Murakeozy et al. 2003; Zhang et al. 2016). However, the majority of earlier studies have identified the physiological functions of soluble sugars and their uptake by plants. We discovered that, in both salt stress and non-saline environments, fungal endophytes had a significant impact on the amount of soluble protein and sugar in wheat seedlings.

The primary defence mechanism of the chloroplasts, cytosol, mitochondria, peroxisomes and apoplasts against the harmful effects of ROS is the AsA-GSH cycle. The AsA-GSH cycle, which is an efficient detoxification process against oxidative stress, contains five enzymes: SOD, DHAR, APX, GR, and MDHAR, in addition to GSH, AsA and NADPH (Asada 1999; Gill et al. 2013). SOD converted superoxide to H₂O₂ in this cycle. APX, by decreasing H₂O₂ to water with AsA oxidation to MDHA, decreases to AsA via the monodehydroascorbate reductase (MDHAR) enzyme and disproportionates nonenzymatically to AsA and DHA. DHAR, which uses GSH as a reductant to convert DHA to AsA (Ahmad et al.2008). As a result, GSSG is produced and the GR can convert it back to GSH. (Gill et al. 2013) (Fig. 7).

Under salt stress, the AsA content, DHA and AsA/DHA ratio drastically decreased in plants. Chen and Gallie (2006) also reported that reducing DHAR and MDHAR activity in salt-stressed plants led to greater DHA levels consistent with less AsA recycling; this, in turn, led to a low (i.e., more oxidized) AsA redox state. However, Table 3 clearly demonstrates that the reduction in the AsA/DHA ratio caused by salt stress was greatly reduced by inoculation with fungal endophytes. According to our study, fungal endophytes control the redox state of AsA to induce salt tolerance in wheat plants. Increasing the antioxidant enzyme activity of the AsA-GSH cycle may increase the amount of decreased AsA in the cells and its redox state. The AsA-GSH cycle plays a role in either direct or indirect elimination of ROS while GSH is a non-enzymatic antioxidant similar to AsA (Noctor and Foyer 2011).

Table 3 shows that despite the decrease in the ratio of GSH to GSSG, GSSG concentration increased under salt stress conditions. However, inoculation of plants under salt stress with fungal endophytes causes the GSH pool to be transformed into reduced forms, which greatly increases the GSH/GSSG ratio. The stressed plants appeared to take up a sizable amount of GSH, which corrected the redox status, most likely triggered cellular defence systems, and prevented cellular damage. As previously stated, boosting the antioxidant enzyme activity in the AsA-GSH cycle in stressed plants might help to maintain levels of glutathione and ascorbate, the two essential antioxidants against ROS-damaging effects.

Earlier studies on fungal-treated crops such as onion, maize, and wheat (Harman et al. 2004; González-Teuber as al. 2022; Prajapati et al. 2022), have demonstrated similar results. According to Rawat et al. (2011), metabolites produced by Trichoderma inoculation function as quenchers during ROS generation under environmental stress, thereby protecting the plant from oxidative damage. Our results are corroborated by earlier studies described by Bagheri et al. 2013; Hamilton et al. 2012;Irshad et al. 2023; Kumar et al. 2009; Li et al. 2023; Lubna et al. 2022; Moghaddam et al. 2021; Nath et al. 2016; Popovic et al. 2024; Sadeghi et al. 2020.

In the current study, salinity stress increased the SOD and APX activities, while reducing MDHAR, DHAR, and GR activities in a dose-dependent manner. In other studies, Cao et al. (2023), Chaudhry et al. (2021), Lubna et al. (2022), Popovic et al. (2024), Wang et al. (2023), Zhang et al. (2016), Zou et al. (2023) illustrated that the expression of antioxidant enzyme genes provides tolerance to abiotic stresses, including salinity by reducing the oxidative stress caused by ROS in Citrus trifoliate, Piper nigrum, Poncirus trifoliata, Glycine max L., Triticum aestivum L., and A. cepa cultivars. These results are consistent with earlier researches on Brassica parachinensis (Kamran et al. 2020) and Brassica juncea (Ahmad et al. 2015). According to researches on Fragaria sp. (Christou et al. 2014), further salinity-induced reductions in MDHAR, GR, and DHAR activities may be caused by reduced transcription of these genes, whereas decreased GR activity may be caused by NADPH deficiency under extreme stress (Gupta and Seth 2020; Prajapati et al. 2022).

Comparing the same activities to those that were solely influenced by salinity, fungal endophytes enhanced APX, SOD, GR, MDHAR, and DHAR. This may be due to the overexpression of transcription and the gene translation responsible for synthesis, as observed under various abiotic stress conditions in Solanum melongena, Solanum lycopersicum (Singh and Prasad 2019), and Triticum aestivum (Sun et al. 2015; Zhang et al. 2016).Notably, MDHAR and DHAR work together to preserve the concentration of AsA and the redox state during stressful situations (Kaya et al. 2020). In contrast to the treatments using salt alone, the current study’s findings on the effects of fungal supplementation on MDHAR and DHAR activities have been found associated with an increase in AsA levels. A higher GSH/GSSG ratio is necessary for the DHAR and other GSH-dependent enzymes associated to the antioxidant defence mechanisms and fungal inoculation increasing GR activity under salinity stress contributed to this maintenance (Fig. 7). Our results corroborate the increased AsA-GSH cycle upregulation resulting from fungal endophyte inoculation, as observed in Triticum aestivum (Zhang et al. 2016; Tripathi et al. 2017), and Citrus reticulata L. (Sadeghi et al. 2020) under various abiotic stresses. Under biotic and abiotic stresses, fungal endophytes can cause significant alterations in gene expression in several plant species (Zhang et al. 2016). According to the current study, elevated ROS levels in stressed plants may be the cause of elevated APX and SOD activities because they increase the expression of genes that code for these enzymes (Bowler et al.1992; Shekhawat et al. 2010). Thus, we assessed the possible influence of fungal endophytes on the expression of genes encoding antioxidant enzymes (APX, SOD, GR, MDHAR and DHAR).

We found that the expression of genes encoding APX, SOD, DHAR, MDHAR and GR led to the elevated transcription levels, which correlated with an increase in the activity of the corresponding enzymes and augmentation of APX, SOD, MDHAR, DHAR and GR. According to earlier studies (Bailey et al. 2006; Alfano et al. 2007; Zhang et al. 2016; Wang et al.2023; Popovic et al. 2024), plants treated with fungal endophytes exhibited higher expression levels of genes related to plant defense against abiotic stresses. However, comprehensive research on gene expression in fungal-endophyte-treated wheat seedlings is lacking. We have also shown that increased transcription and activity of ROS-scavenging enzymes under salt stress are correlated with ROS generation in wheat plants treated with fungal endophytes, indicating that ROS are important for regulating plant acclimation and detoxifying cellular survival (Miller et al. 2010).

Furthermore, ROS serve as vital signaling molecules for cell survival and multiplication. Similar studies indicate that salinity is an environmental factor that can alter the normal homeostasis of plant cells and increase ROS production in plants. According to Miller et al. (2010), ROS are hazardous byproducts of stress metabolism and play a key role in signaling transduction molecules that are activated in response to salt stress.

Our research concluded that A. medius and C. parahalotolerant, the two endophytic fungi, have a remarkable ability to mitigate the detrimental effects of salt stress on the development and growth of wheat seedlings. Using a variety of experiments, this study enabled us to investigate the potential physiological and molecular processes by which fungal endophytes mitigate the suppressive effects of salt stress. The mechanisms could be (i) fungal endophytes boosting the antioxidative defense system activity of wheat seedlings to withstand salt stress, or (ii) elevating the relative expression levels of antioxidant genes in stressed plants. Future researches must address a few difficulties, such as the effectiveness of endophytic fungi against other plant species and abiotic pressures. Further comprehensive investigations are required to ascertain as to which substance functions as a signaling component in endophytic fungi, causing systemic modifications in the expression of genes and antioxidant enzymes that encode them.

APX Ascorbate peroxidase

SOD Superoxide dismutase

GR Glutathione reductase

MDHAR Monodehydroascorbate reductase

DHAR Dehydroascorbate reductase

AsA Ascorbate

GSH Glutathione

DHA Dihydroascorbic acid

GSSG Oxidized glutathione

ROS Reactive oxygen species

TCA Trichloroacetic acid

TBA Thiobarbituric acid

NBT Nitrobluetetrazolium test

Declaration of competing interest

The writers affirm that there is no conflict of interest between them.

Acknowledgments

The Head and Coordinator, CAS and DST-FIST in Botany, Institute of Science, BHU, Varanasi, India, are gratefully acknowledged by the authors for providing necessary research facilities. The Institute of eminence (IoE) (R/Dev/IoE/ Incentive/2021-22/32181), BHU, Varanasi, is gratefully acknowledged by RNK for its financial support. PP is grateful to the BHU administration for financial assistance in the form of a BHU RET fellowship. Prashasti acknowledges Prime Minister Research fellowship (PMRF) for financial support in the form of fellowship.

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Table 1. DNA sequences of PCR primers were used for qPCR determination of the five AsA-GSH biosynthesis related gene in wheat seedlings, F, Forward primer, R, reverse primer

Gene

Accession number

Primer pairs

Expected amplification size (bp)

SOD

JQ613154.1(GenBank, Zhang et al. 2016)

Forward: CATTGTCGATAGCCAGATTCCTTT

Reverse: AGTCTTCCACCAGCATTTCCAGTA

138

APX

EF55121.1 (GenBank, Popovic et al., 2024)

Forward: TGGAAGACGTGATTCGTCAG

Reverse: TCAAACCCAGACCTTTCAGG

174

MDHAR

AK371371(GenBank, Li et al. 2013)

Forward: AGAAGTTTACGCCCTTCGGC

Reverse: TTGGAATGTCATCGCCATC

132

DHAR

AYO74784(GenBank, Li et al. 2013)

Forward: GTGCCTGTGTATAACGGTG

Reverse: ACAAGTGATGGAGTTGGGT

TC84151(GenBank, Popovic et al. 2024)

Forward: TGCGTCCCGAAGAAGATACT

Reverse: GTTGATGTCCCCGTTGATCT

Table 2. Changes in Sugar, Protein content, total chlorophyll (mg/g FW), Carotenoid (mg/g FW) and chlorophyll fluorescence (Fv/Fm) in wheat seedlings inoculated with endophytic fungi, Cladosporium parahalotolerant and Aspergillus medius inoculation individually and in combination with each other, under salt stress condition (100 mM NaCl). Control was treated with sterile water

Treatments

Soluble sugar (mg/g FW)

Protein (mg/g FW)

Total chlorophyll (mg/g FW)

Carotenoid (mg/g FW)

Fv/Fm

Control

14.37^e± 0.51

6.59^e± 0.58

7.51^e± 0.28

0.33^d± 0.00

0.64^d± 0.02

NaCl

10.16^f± 0.41

4.13^f± 0.21

5.45^f± 0.10

0.21^e± 0.01

0.54^e± 0.01

C. parahalotolerant

18.47^c± 0.26

10.34^c± 0.48

11.29^b± 0.69

0.49^b± 0.06

0.74^b,c± 0.02

C. parahalotolerant+NaCl

15.0^d,e± 0.86

7.07^d,e± 0.26

8.31^d± 0.24

0.38^c,d± 0.00

0.64^d± 0.02

Medius

19.45^b± 0.21

11.37^b± 0.30

10.76^b± 0.64

0.42^c± 0.03

0.73^c± 0.01

A. medius+NaCl

15.69^d± 0.15

7.46^d± 0.13

7.82^d,e± 0.16

0.36^d± 0.00

0.67^d± 0.02

C. parahalotolerant+ A. medius

22.12^a± 0.14

16.23^a± 0.23

12.71^a± 0.38

0.68^a± 0.03

0.84^a± 0.02

C. parahalotolerant +A. medius+NaCl

18.42^c± 0.22

11.18^b+ 0.17

9.27^c± 0.06

0.48^b± 0.02

0.763^b± 0.02

Table 3. Change pattern of glutathione (GSH), oxidized glutathione (GSSG), ascorbate (AsA), and dehydroascorbate (DHA) and the ratios of GSH/GSSG and AsA/DHA in wheat seedlings inoculated with endophytic fungi, C. parahalotolerant and A. mediusindividually and in combination with each other under salt stress (100 mM NaCl). Control was treated with sterile water

Treatments

AsA(µg/g FW)

DHA(µg/g FW)

AsA/DHA

GSH(µg/g FW)

GSSG(µg/g FW)

GSH/GSSG

Control

1.96^f ± 0.21

1.33^a± 0.11

1.48± 0.17

0.83^g± 0.03

0.43^b ± 0.07

1.96 ± 0.43

NaCl

1.36^h ± 0.31

1.03± 0.12

1.35± 0.47

0.72^h± 0.02

0.46^a ± 0.01

1.57 ± 0.03

C. parahalotolerant

2.16^e ± 0.25

1.46± 0.23

2.7± 1.13

1.3^b± 0.06

0.30^c ± 0.09

4.64 ± 1.44

C.parahalotolerant+NaCl

1.56^g ± 0.31

1.33^a± 0.55

1.46± 1.08

1.21^d± 0.05

0.21^f ± 0.05

5.84 ± 1.77

A. medius

3.43^b ± 0.21

1.36^a ± 0.45

2.75± 1.13

1.20^e ±0.02

0.27^d,e ± 0.02

4.52 ± 0.28

A. medius+NaCl

2.33^d ± 0.25

1.33± 0.32

1.83± 0.52

1.09^f± 0.04

0.19^h ± 0.04

5.70 ± 1.21

C. parahalotolerant+ A. medius

3.9^a ± 0.10

1.56± 0.31

3.20± 0.81

1.47^a± 0.03

0.24^d,e ± 0.01

6.04 ± 0.21

C. parahalotolerant+ A. medius+NaCl

3.16^c ± 0.31

1.43± 0.26

2.51± 0.58

1.30^c± 0.02

0.20^g ± 0.03

6.64 ± 1.09

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Fungal endophytes promote wheat (Triticum aestivum L., genotype-PBW-343) growth and enhance salt tolerance through improvement of ascorbate-glutathione cycle and gene expression

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