In nature, the growth of plants is affected by multiple factors, such as drought stress and arbuscular mycorrhizal fungi (AMF) (Gupta et al., 2020). AMF can increase water and nutrient, and enhance plant tolerance to environmental stresses (Bennett and Groten, 2022; Kakouridis et al., 2022; Wang et al., 2023). Therefore, mycorrhizal plants generally exhibit stronger drought tolerance than those without mycorrhizae (Jongen et al., 2022). The underlying mechanisms mainly include physiological modulation (e.g., the increment of water and nutrient absorption by extraradical hyphae, improvement of photosynthesis and water use efficiency, and activation of antioxidant defense systems) and molecular regulation (e.g., regulation of the expression of genes related to drought response in plants) (Kakouridis et al., 2022; Wang et al., 2023). Plants exposed to mycorrhizal fungi developed a wider root system that increased availability and accessibility of water and nutrients, especially nitrogen, phosphorus, and potash, that is a main factor in growth and development of plant (Sardans et al., 2023). Mycorrhizal hyphae by increase magnesium absorption and, as a result, increase the amount of total chlorophyll and flavonoids in quinoa and maize plants (Benafari et al., 2022). The increase in total chlorophyll synthesis depends on the sufficient absorption of minerals, especially nitrogen, and magnesium (Begum et al., 2019). Phosphorus has an important physiological role in the phytochemical biosynthesis pathway because it has a synergistic effect with nitrogen. Our results determained that using of AMF increased the amount of secondary metabolite in quinoa compared to control treatments. This increase can be due to improved biochemical composition (Benafari et al., 2022).
Secondary metabolites such as flavonoids, which belong to phenylpropanoids, can maintain cell structural and functional integrity and reduce oxidative damage caused by reactive oxygen species (ROS), thus protecting plants against drought stress (Zhao et al., 2021). Mycorrhizae can systemically alter plant primary and secondary metabolism (Xia et al., 2023). For example, AMF markedly increased anthocyanin and total flavonoid concentrations in Medicago truncatula leaves (Adolfsson et al., 2017), although these mycorrhizal effects can be partially attributed to the improved phosphorus (P) status (Gerlach et al., 2015). Xia et al., (2023) demonstrated the common specialized root metabolites accumulated after mycorrhizae, with an increase in flavan-3-ols and a decrease in flavanols regardless of mycorrhizal lifestyles. However, whether and how these secondary metabolites induced by AM symbiosis play roles in improving plant drought tolerance remains largely unknown. The mechanisms by which mycorrhizae modulate plant metabolism can be divided into general and specific aspects. As one of the known benefits of AM symbiosis to the host plant, the substantial improvement of water and nutrient status by AMF can largely explain mycorrhiza-caused changes in carbohydrates and parts of secondary metabolism, which are defined as general mycorrhizal effects (Schweiger and Müller, 2015). Although mycorrhizal effects on plants are often considered highly specific to host plants and, or fungal species, the common traits of mycorrhiza-associated metabolite alterations are present in plant roots (Xia et al., 2023). Similar to our results it has been reported that mycorrhiza often increased the amounts of bioactive phenolic and lignin metabolites compared with non-mycorrhizal plants (Brundrett, 2002; Tekaya et al., 2022). Mycorrhizal symbiosis can also induce plant specific metabolism, especially under stress conditions, that cannot be or can only be partially explained by nutrient effects (Adolfsson et al., 2017). For example, Kaling et al. )2018(, integrated transcriptome and metabolome analyses of poplar plants with mycorrhizae and herbivores and found that mycorrhizal plants can specifically accumulate specialized protective compounds, including protease inhibitors and aldoxime, at the expense of plant constitutive phenol-based compounds against herbivores. Experimental data and meta-analysis revealed high specific effects of mycorrhizal fungi, plant species, and/or their interactions on plant metabolism, let alone on plant-drought stress interactions (Sardans et al., 2021). Previous studies exploring the mechanisms of the interaction between plants and beneficial microbes, such as bacteria and AMF, mainly focused on model and crop plants (Bastías et al., 2022; Wang et al., 2023), the associated transcriptome-metabolome changes induced by AMF to relieve drought stress in non-model plant species have not yet been fully disclosed. Photosynthetic and ability to use soil nutrients determines the growth, development, and metabolic activity (Li and Cai, 2021). Adding phosphorus along with arbuscular mycorrhizal fungi by affecting the levels of available nutrients can alter the metabolic environment of plants, as a result enhancing their absorb and assimilate CO2 from the atmosphere. Mycorrhizal fungi improved biochemical properties such as antioxidant enzyme activity, flavonoids, and phenol above-ground biomass by reducing peroxidase activity (Ren et al., 2019). The hyphae of arbuscular mycorrhizal fungi ability to increase water and other nutrient uptake by plants thereby the symbiotic relationship between plants and arbuscular mycorrhizal fungi (Smith et al, 2010). Some studies determined that maize has serious adverse effects on quinoa during plant growth, and quinoa also has some negative effects on maize. None of the mixture treatments (25%, 50%, or 75% quinoa) containing quinoa showed the same phytochemical values as 100% maize (Erdogan et al., 2020). Compounds that can delay or inhibit the oxidation of lipids and scavenge free radicals by preventing the initiation or spread of oxidation chain reactions are known as antioxidants (Sanders, 2009). Quinoa seeds are rich in bioactive compounds with high antioxidant activity and natural antioxidant compounds (Valencia-Chamorro, 2003). Total phenolic compounds found in quinoa show antioxidant activity (Miranda et al., 2010). The Location effects bioactive compounds. The total phenol content of the extract of the quinoa grown in Japan (148.0 mg g1 equivalent of tannic acid) was higher than the total phenol content (94.3 mg g1) of the quinoa grown in Bolivia (Nsimba et al., 2008). The differences in the phenolic content of both quinoa ecotypes could be due to different environmental factors and agricultural conditions (Miranda et al., 2010). Similar results were observed in our experiment. Our results showed that in Shahrood region the most of phenolic contents was observed in mycorrhiza inoculation alone treatment but in Mayamey the most of phenolic was observed by application of mycorrhiza and phosphorous 50 kg ha− 1 ( Fig. 3). The degradation of phenolic acids has been reported to be dependent on the substituent groups in the ring structure (especially hydroxyl and methoxyl) that can assist in the thermal decarboxylation of phenolic acids and act as an activation group (Lindquist and Yang, 2011). Total phenolic amounts of quinoa seeds changed between 16.8 and 59.7 mg/100 gr, while the content of soluble phenolic contents of samples was measured between 7 and 61 mg/100 g (Repo -Carrasco-Valencia et al., 2010).
Although saponins associated with quinoa have been identified as anti-quality factors, isolated saponins also have some interesting biological properties (Yao et al., 2014). In our study, the saponin amount of quinoa was varied. Our results showed that the content of saponin decreased with increasing the proportion of maize in mixed crops (Fig. 3). Application of 100 kg ha− 1 phosphorous decreased significantly the amounts of saponin than control and 50 kg ha− 1 phosphorous in the proportion of 75% maize. It has been reported that saponins may also have value when considered from a nutritional or pharmacological standpoint and may increase under different conditions (Mohyuddin et al., 2019). Tannins, which can form complexes with other macromolecules, such as undesirable biological effects, belong to the group of polyphenols, which are more common and widely distributed natural substances in the plant kingdom (Santos, 2006). As products of secondary metabolism, phenolic, flavonoids, and saponins have vital antioxidant activities that benefit cardiovascular and cerebrovascular health, and have antiallergenic, antibacterial, anticarcinogenic, and anti-inflammatory effects (Sivakumar et al., 2018). Quinoa has also been used as a functional ingredient in meat products due to its extender, filler, binder, and fat-replacer properties (Fernandez-Lopez et al., 2020). Today, the multi-cropping system is viral in developing countries and among smallholder farmers due to the use of more land and food and the reduction of pests and diseases (Hang et al, 2019). Considering the ever-increasing trend of the world's population and the forecast of a 7.9 billion world population in 2050, and limited agricultural resources, increasing the yield of food products per unit area is inevitable (Anonymous, 2019). Intercropping agricultural systems have proven to be highly effective in increasing production, depending on the specific ecological conditions. Intercropping refers to the practice of growing multiple plant species in the same area within a single year (Portes et al., 2000). In studies conducted by Turk, J (2020) on the forage yield and growth parameters of the maize – quinoa intercropping system, it was shown that the amount of maize dry matter increased in almost all mixtures, and quinoa performed well only in the 50% quinoa-50% maize practice. Intercropping systems are preferred in areas with limited land, workforce, mechanization, etc. due to the better use of available resources than single cropping systems (Takil et al. 2020). Quinoa seeds are rich in protein, lipids, fiber, vitamins, and minerals. In addition to its excellent balance of essential amino acids, quinoa contains phytochemicals, including saponins, phytosterols, phytoecdysteroids, phenolic compounds, polysaccharides, and bioactive proteins and peptides. Recent investigations demonstrating the beneficial effects of these compounds on metabolic, cardiovascular, and gastrointestinal health have made quinoa gain recognition as a functional food and nutraceutical (Hernández-Ledesma, 2019). Quinoa, known for its resistance to arid and saline conditions, exhibits the ability to survive with minimal precipitation, particularly after the seedling stage and throughout the vegetation period. The seeds of quinoa possess high nutritional value, making it a potential alternative agricultural product suitable for human consumption (Takao et al., 2005). Apart from its nutritional content, quinoa also possesses other notable characteristics such as tall plant height (approximately 150 cm), waxy leaves that offer resistance against arid conditions and pests, and a taproot structure that enables its adaptability to dry and saline soil conditions (Geren et al., 2015; Koca et al., 2018; Koca et al., 2017). Phosphorus is one of the essential elements for plant growth and development, with a vital role in crop production, and phosphorus deficiency has become an essential factor limiting present-day agricultural productivity (Efthymiou et al, 2018). Li and Cai, (2021) found that by increasing arbuscular mycorrhizal fungi (AMF), all trate the maize, such as maize biomass, chlorophyll content, plant height, and leaf area, significantly improved. The mycelium of arbuscular mycorrhizal fungi enhances phosphatase and microbial activity in the soil. Phosphorus in the soil is affected by soil microorganisms such as arbuscular mycorrhizal fungi, which promote root growth and convert inorganic phosphorus into organic (Richardson, 2001). AMF are symbiotic with most crops and found on the surface of roots, in, and around the epidermis of root cells. The hyphae of mycorrhizal fungi are thinner than root hairs, so they can extend into soil micropores and transport some minerals such as nitrates and phosphates, and then to their host plant in exchange for carbohydrates (Van et al, 2015). Which was consistent with the results of this study. Therefore, mycorrhizal plants generally exhibit stronger drought tolerance than those without mycorrhizae (Jongen et al., 2022). This difference can be due to geographical and climatic characteristics and soil physiochemical properties in the two locations (Tables 1 and 2).
Phenolic compounds are secondary metabolites that contribute to detoxification; active oxygen species help, and free radicals neutralize it before damaging the plant cell. The antioxidant potential of the plant depends on the concentration of phenolic and tannin compounds. This study showed the amount of phenolic and tannin compounds using AMF increased. In stressful conditions, fungi mycorrhizal plants respond to oxidative stress by increasing phenolic compound synthesis. The increase of phenolic and tannin compounds using AMF has been reported in violets (Zubek et al., 2015), grapes (Eftekhari., 2012), and Commiphora leptophloeos (Cleilton et al., 2017). In studies conducted by Turk J (2020) on the forage yield and growth parameters of the maize–quinoa intercropping system, it was shown that the amount of maize biocompound increased in almost all mixtures, and quinoa performed well only in the 50% quinoa-50% maize practice. Tannins, which can complexes form with some macromolecules and belong to the group of polyphenols with undesirable biological effects, are distributed natural substances in the plants widely (Santos, 2006). Secondary metabolism such as phenolic, saponins, and flavonoids, with having antioxidant activities are profits for cardiovascular and cerebrovascular health. They also have anti-inflammatory, antiallergenic, antibacterial, and anticarcinogenic, effects (Sivakumar et al., 2018).
Acid oleanolic, hederagenin, and acid phytolaccagenic, are main aglycones of saponin in quinoa plant. Based on previous studies, acid serjanic is also called as an aglycone. Nevertheless, the amounts of acid serjanic in organs of quinoa plant compared with other aglycones is relatively low (Ruiz et al., 2017). Though the amounts of saponins in quinoa is an anti-quality factors, the saponins that isolated from quinoa plants have some interesting biological confidants (Yao et al., 2014). In our study, the amount of saponin in quinoa was different. According to my results, the content of saponin decreased with increasing the proportion of maize in mixed crops (Fig. 3). Application of 100 kg ha− 1 phosphorous decreased significantly the amounts of saponin than control and 50 kg ha− 1 phosphorous in the proportion of 75% maize. The saponins in plants may also have useful when considered from a nutritional or pharmacological viewpoint and maybe increase under different conditions (Mohyuddin et al., 2019). When quinoa samples were exposed to different pearling processes, the amounts of saponins decreased (Gómez-Caravaca et al., 2014).
Furthermore, amounts and composition of quinoa plant saponin were not always constant because seeds pericarps and bran were continually removed in the harvest process and make it edible (such as cooking under pressure and toasting (Gómez-Caravaca et al., 2014). The total saponin content of quinoa grown ranged from 3.81 to 27.1 mg gr -1 in Washington State (Medina-Meza et al., 2016; Nickel et al., 2016), consistent with our results. Increasing the N uptake rate per root length promotes saponin accumulation in roots by increasesing the uptake rate per root length, reported that there is a significant positive relationship with content of saponin in the taproots. Adequate nitrogen fertilizer could optimize both root structure and nutrient uptake efficiency, then increases saponin synthesis (Wei et al., 2020). Considering the very important role of nitrogen in the synthesis of saponin, it seems that the decrease of saponin in high concentrations of phosphorus (100 kg ha-1) in our study can be used due to the decrease of nitrogen absorption and thus decrease the amount of saponin.
Andrino et al. (2021) showed the presence of mono-, di-, and tricarboxylic low-molecular-weight organic acids orthophosphate in compartments containing orthophosphate (OP) or goethite-bound- orthophosphate (GOE-PA) and phytic acid (PA or GOE-PA) points toward the occurrence of reductive dissolution and ligand exchange/dissolution reactions. Furthermore, hyphae grown in goethite loaded with OP and PA exhibited an increased content of unsaturated lipids, pointing to an increased membrane fluidity to maintain optimal hyphal functionality and facilitate the p incorporation. The accompaniment arbuscular mycorrhizal fungi may be practically diverse and provide growth-promoting functions metabolit in plant, such as mineralization of phytate, production of siderophore, phosphorus dissolution, and low-molecular-weight organic acids production (Battini et al., 2016). Mycelium of Rhizobium engaged in the phosphorus mobilization from acid phytic (Selvakumar et al. 2016 ). Hara and Saito (2016), reported that arbuscular mycorrhizal fungi cause phytate mineralization and as a result, they cause the transfer of phosphorus. They isolated bacteria from the hyphosphere fertilizer of the arbuscular mycorrhizal fungi that can mineralize phytic acid. The hyphae of arbuscular mycorrhizal fungi can increase water and other nutrient uptake by plants, thereby the symbiotic relationship between plants and arbuscular mycorrhizal fungi (Smith et al., 2010). Some studies determined that maize has serious adverse effects on quinoa during plant growth, and quinoa also has some negative effects on maize. None of the mixture treatments (25%, 50%, or 75% quinoa) containing quinoa showed the same phytochemical values as 100% maize (Erdogan et al., 2020). Previous studies exploring the mechanisms of the interaction between plants and beneficial microbes, such as bacteria and AMF, mainly focused on model and crop plants (Bastías et al., 2022; Wang et al., 2023), the associated transcriptome-metabolome changes induced by AMF to relieve the ecological condition in non-model plant species have not yet been fully disclosed. Photosynthetic and the ability to use soil nutrients determine growth, development, metabolic activity, and fatty acids (Li and Cai, 2021). Zamani et al., 2023, showed the amounts of phenolic compounds and fatty acid profiles in intercropping of Lallemantia iberica and Cicer arietinum L. by use of AMF increased. The phytic acid of quinoa head in the 50% intercropping pattern with AMF increased. AMF improved nutrient accessibility, and thus plant growth and photosynthesis would have affected the production of fatty acid precursor compounds and the activity of associated enzymes such as fatty acid synthase and acetyl-CoA carboxylase (Aid 2019). Other studies have reported the positive effects of intercropping systems on plant nutrient availability, oil productivity, and oil quality. Rezaei Chiyaneh et al. (2021) showed a cropping ratio of 50:50 in intercrop of black cumin with fenugreek using biofertilizer had the highest oil concentration of black cumin due to increased nutrient uptake. Rezapour et al., 2021, showed that the combined application of phosphorus and AMF enhanced fatty acid synthesis in groundnut. As well as the application of phosphorus fertilizer and a groundnut-corn intercropping system with a ratio of 1:2 improved groundnut oil content (Rezapour et al., 2021). In a similar study, researchers showed that using phosphorus chemical fertilizer and biological fertilizer increased safflower oil yield (Saeidi et al., 2018). Oil synthesis needs more energy than other compounds studied in this research. For this reason, using mycorrhiza by increasing the availability and absorption of nutrients by the plant root provides more energy to carry out these processes. It thus leads to an increase in oil synthesis. On the other hand, the energy needed by plant processes is supplied in the form of high-energy ATP molecules; due to the role of phosphorus in these molecules, the availability of phosphorus leads to the production of more high-energy molecules and, as a result, increases oil synthesis. Also, the intercropped groundnut by corn reduced the amount of palmitic acid in peanut oil compared with sole culture, improving the quality of peanut oil (Rezapour Kavishahi et al., 2021). Studies show that increasing the consumption of phosphorus fertilizer increased the content of saturated palmitic fatty acid in peanut oil and reduced the quality of peanut oil (Rezapour Kavishahi et al., 2021), and increased saturated fatty acids in corn (Kaptan et al., 2017).
Polyphenols compounds have antioxidant potential and are found naturally present in plants. They can eliminate free radicals (Gyu Lim et al., 2019). The antioxidant activity is depends on the arrangement and number groups of hydroxyl in the phenols copmound that can decrease the promotion of oxidation by transferring atoms of hydrogen to radicals (Shahidi and Ambigaipalan, 2015).
The amounts of CAT, POX, and SOD enzymes were in the Mayamey location 7.50%, 6.25%, and 16.39% higher than in the Shahrood location, respectively. In perennial ryegrass plants, increasing the antioxidant enzymes activity has been found in inoculation with mycorrhiza (Lee et al., 2012). Polyphenol oxidases are antioxidant enzymes containing copper that are found abundantly in plants. This enzyme uses oxygen molecules to oxidize orthodiphenolic compounds such as caffeic acid and catechol and convert them to quinone (Constabel & Barbehenn., 2008).
Higher amounts of phenolic compounds can cause higher antioxidant activity, and tannins are known to have a positive relationship they have antioxidant activities (Jugran et al., 2013). Also, increasing the activity of catalyze enzymes inoculation with symbiotic fungi reported in some plants such as rise (Kamoshita et al., 2008), rose (Amiri et al., 2015), and citrous (Wu & YN., 2009), which was consistent with our results.
Antioxidants are compounds that can delay and inhibit the lipids oxidation and scavenge of free radicals of oxidation chain reactions (Taylor et al., 2009). Quinoa seeds are rich in bioactive compounds with high antioxidant activity and natural compounds (Valencia-Chamorro, 2003).The location affects bioactive compounds. The total phenol content of the extract of the quinoa grown in Japan (148.0 mg gr − 1 equivalent of tannic acid) was higher than the total phenol content (94.3 mg gr − 1) of the quinoa grown in Bolivia (Nsimba et al., 2008). The differences in the phenolic content of both quinoa ecotypes could be due to different environmental factors and agricultural conditions (Miranda et al., 2010). These results were observed in our experiment similarly. The most phenolic contents were observed in the Shahrood region by use of mycorrhiza inoculation alone treatment, but in Mayamey, the most phenolic was observed by application of mycorrhiza and phosphorous 50 kg ha− 1 (Fig. 3). The degradation of phenolic copmounds dependent on the substituent groups particularly methoxyl and hydroxyl, that can help in the thermal of acid phenolics decarboxylation (Lindquist and Yang, 2011). Total phenolic compound of quinoa seeds is variable between 16.8 and 59.7 mg/100 gr, while the soluble phenolic contents of samples were measured between 7 and 61 mg/100 g (Repo -Carrasco-Valencia et al., 2003).
The seeds of quinoa are rich in protein, lipids, fiber, vitamins, and minerals. In addition to its excellent balance of essential amino acids, quinoa contains phytochemicals, including saponins, phytosterols, phytoecdysteroids, phenolic compounds, polysaccharides, and bioactive proteins and peptides. Recent investigations demonstrating the beneficial effects of these compounds on metabolic, cardiovascular, and gastrointestinal health have made quinoa gain recognition as a functional food and nutraceutical (Hernández-Ledesma, 2019). Adding phosphorus along with arbuscular mycorrhizal fungi by affecting the levels of available nutrients can alter plants' metabolic environment, enhancing their absorption and assimilating CO2 from the atmosphere. Mycorrhizal fungi improved biochemical properties such as antioxidant enzyme activity, flavonoids, and phenol above-ground biomass by reducing peroxidase activity (Ren et al., 2019). Li and Cai (2021) found that by increasing arbuscular mycorrhizal fungi (AMF), all traits of maize, such as maize biomass, some physiological traits, plant height, and leaf area, significantly improved. The mycelium of arbuscular mycorrhizal fungi enhances phosphatase and microbial activity in the soil. Phosphorus in the soil is affected by soil microorganisms such as arbuscular mycorrhizal fungi, which promote root growth and convert inorganic phosphorus into organic (Richardson, 2001). Mycorrhizal symbiosis can also induce plant-specific metabolism, especially under stress conditions, that cannot be or can only be partially explained by nutrient effects (Adolfsson et al., 2017). For example, Kaling et al. )2018(, integrated transcriptome and metabolome analyses of poplar plants with mycorrhizae and herbivores and found that mycorrhizal plants can specifically accumulate specialized protective compounds, including protease inhibitors and aldoxime, at the expense of plant constitutive phenol-based compounds against herbivores. Experimental data and meta-analysis revealed highly specific effects of mycorrhizal fungi, plant species, and/or their interactions on plant metabolism, let alone on plant-drought stress interactions (Sardans et al., 2021). The underlying mechanisms mainly include physiological modulation (e.g., the increment of water and nutrient absorption by extraradical hyphae, improvement of photosynthesis and water use efficiency, and activation of antioxidant defense systems) and molecular regulation (e.g., regulation of the expression of genes related to drought response in plants) (Kakouridis et al., 2022; Wang et al., 2023). Arbuscular mycorrhizal fungi are symbiotic with most plants and found on the roots surface and around root cells epidermis. Mycorrhizal fungi hyphae, can extend into soil micropores ( because of thinner than plants roots hairs) and transport some minerals, such as phosphates and nitrates, and then to their host plant in exchange for carbohydrates (Van et al., 2015), which was consistent with the results of our study.
Quinoa belongs to a family of plants considered non-mycorrhizal (Amaranthaceae) (Muthukumar and Tamilselvi, 2010). AMF associations with quinoa have been observed in a few prior studies (Vestberg et al., 2012), though the rates of colonization were minimal. Vestberg et al. (2012) observed 19% colonization, the greatest of which we know (before our observation), occurred under temperate field conditions. Notably, both vesicles and arbuscules were observed in quinoa roots, indicating that there could have been nutrient exchange occurring (Brundrett, 2009). In studies conducted by Kellog et al. (2021) in 10 quinoa genotypes reported 0 to 3% quinoa root colonization by AMF under greenhouse conditions. If, Benaffari et al. (2022) reported 50% colonization. Based on the results, the intensity and frequency of arbuscular mycorrhizal in roots of quinoa were decreased significantly in mono culture system (p < 0.05). The treatments intercropped with maize (75% maize-25% quinoa), and combined with AMF (AMF + p50 kg ha− 1), showed the highest frequency of mycorrhizal. When the levels of soluble phosphorus in the soil increased, the colonization of arbuscular mycorrhizal fungi with roots decreased. These fungi have specific biochemical and physiological characteristics that can increase roots' phosphorus availability. Fungi acidify the rhizosphere by increasing the release of protons, which can increase the solubility and transfer of phosphorus, especially in alkaline soils such as the soil of the studied areas. In acid soils where phosphorus is mainly bound with iron or aluminum, the neutralization of chelating agents by mycorrhizal fungi can increase the root bioavailability of soil phosphorus (Bender et al., 2015).