Biochemical Response and Nutrient Uptake of Two Arbuscular Mycorrhiza-Inoculated Chamomile Varieties Under Different Water Potentials

Water-decit stress is one of the most important sources of damage to crop production worldwide. Adopting appropriate varieties using soil microorganisms such as arbuscular mycorrhiza(AM) can signicantly reduce theadverseeffectsofwater deciency.This study is aimed to evaluate the role of Funneliformismosseaeon nutrients uptake and some physiological traits of two chamomile varieties namely Bodgold (Bod) and Soroksári(Sor) under water-decit stress. The pot experiment was performed in a hydroponic system within a completely randomized design considering four replications. Three levels of water-decit stress (PEG 6000) were taken into account at water potentials of -0.4 and -0.8MPa. The second factor was AM inoculation.


Introduction
The use of herbal medicinal products and supplements has tremendously increased over the past three decades. More than 80% of the world's population rely on these products as a part of primary healthcare (Sharma 2004;Ekor et al. 2014). German chamomile (Matricaria chamomilla L.) belongs to the family of Asteraceae, one of the most common medicinal plants (Wichtl 2004). Chamomile is a prominent medicinal plant whose compounds are considered safe (Shari et al. 2014). German chamomile ower and its extracts have antimicrobial and antioxidant activity and have been used as a painkiller, antianxiety, antispasmodic, anti-in ammatory, and anti-gastrointestinal agent (Rehmat et al. 2020). With the increase in the global demand for medicinal plants, there is an urgent need to increase their cultivation and production. Water-de cit stress (due to global warming and climate change) is the main cause of the decremented annual plant performance. In arid and semi-arid regions, plants are exposed to water-de cit stress due to the simultaneous increase in the rate of transpiration and temperature and the reduction of the root access to water (Halo et al. 2020). Iran has a dry and semi-arid climate with an average annual rainfall below a third of the global average (Madani et al. 2016). The increasing demand for medicinal plants, especially chamomile, necessitates deeper knowledge to adopt drought-resistant varieties. In addition to water management, the selection of the right genotype can also contribute to preventing water-de cit stress damages and promoting sustainable use of water resources. Concerning medicinal plants, although water-de cit stress increases the synthesis of secondary metabolites, it can also decline the growth of the plant especially its vegetative and reproductive organs (biomass) which generally contain medicinal compounds (Selmar et al. 2017). It also reduces the nutrient uptake of chamomile (Salehi et al. 2018), which plays a vital role in its total dry weight and essential oil content (Andrzejewska et al. 2014). Under water-de cit stress, lipids, proteins, and nucleic acids are damaged due to the rise in the content of reactive oxygen species (ROS) (Uzilday et al. 2012). Plants exploit effective systems such as antioxidants enzymes catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX) and osmolytes such as soluble sugars to combat toxic ROSs and reduce their consequent damages (Al-Arjani et al. 2020).
As one of the commercial varieties of chamomile tetraploid, Bodgold (Bod) has shown favorable performance in terms of total dry weight and essential oil among other types of diploid and tetraploid chamomile (Banatska (2x), Lutea (4x), Zloty Lan (4x), and Goral (4x)) (Tsivelika et al. 2018). Soroksári (Sor) is another important and diploid variety of chamomile with a desirable essential oil content compared to Lutea, Goral (tetraploid), and Bona (diploid) varieties according to Gosztola et al. (2010). Heretofore, no comparative study has been carried out to explore the activity of antioxidant enzymes, absorption of nutrients, and dry weight of these varieties under stress conditions. Nonetheless, a study reported an increase in proline and antioxidant activity of the Bod variety under water-de cit stress (Benabdellah et al. 2011). Arbuscular mycorrhizal (AM) fungi coexist with the root of most plants and have exhibited great potential for counteracting environmental stresses, as they can increase the availability of plants to a larger volume of the rhizosphere and also improve water and nutrients uptake (Zhang et al. 2018) via morphological change of root volume and through their hyphae (Hameed et al. 2014). On the other hand, these fungi enhance the nutrient uptake by increasing the synthesis of compounds and enzymes involved in the absorption process, such as phosphatase (Hu et al. 2013). Improving the nutrients and water absorption by AM promotes plant growth and reduces the adverse  (Doubková et al. 2013). AM also improves growth and yield in Echinacea angustifolia by increasing the defensive level of antioxidant (catalase and peroxidase) and osmolyte (proline) enzymes (Attarzadeh et al. 2019). This study is an attempt to study the effect of water-de cit stress on nutrients levels (roots and shoots) and the activity of antioxidant enzymes and osmolytes in two chamomile varieties. Moreover, the in uence of this factor was compared in these two varieties to determine a more resistant one. Additionally, the effect of Funneliformis mosseae on reducing the impact of water-de cit stress was explored in terms of physiological traits and nutrients uptake.

Experimental design
A pot experiment was performed in the factorial arrangement within a completely randomized design with four replications in the research greenhouse of the Faculty of Agriculture, Yasouj University. The greenhouse temperature was 25 ± 2°C. Water-de cit stress was tested at three water potentials (control, -0.4, and − 0.8 MPa). Different levels of water-de cit stress were prepared using polyethylene glycol 6000 (PEG) via the formula proposed by Michel and Kaufman (1973) and applied to the Hoagland solution. The second studied factor was the use of arbuscular mycorrhiza (AM) fertilizer (Funneliformis mosseae species (fungal and non-fungal)) which was initially inoculated in the culture medium. In mycorrhizal plants, each pot of mycorrhizal treatment received 40 g of AM inoculant (containing spore numbers of 120 g − 1 substrate) at a depth of 5 cm and incorporated well with the soil below. Two German chamomile varieties (Bodgold (Bod) and Soroksari (Sor)) were considered. The Bod variety seeds were purchased from Isfahan Agricultural and Natural Resources Research Center while the Sor variety was supplied from Yasouj Zardband Company. To produce seedlings, the seeds were rst transplanted in a bed of peat moss and cocopeat (1: 2) in a 72-cell (30 cc) seedling tray. At the 4-6-leaf stage, they were transferred to plastic pots with a height of 25 cm and a diameter of 18 cm (6 seedlings per pot), lled with perlite. During the transplanting phase, a quarter of Hogland's solution was used for irrigation. Drought stress was gradually applied after transferring 4-6 leaf seedlings in full Hogland solution with irrigation water for one week. From the second week, the stress levels were applied once every three days for 21 days. The pH of the solutions was adjusted to 5.8 ± 0.1 before each irrigation. It should be noted that all the seed-starting containers, pots, and seedling beds were decontaminated with a greenhouse autoclave, and distilled water was used to make all the nutrient solutions.
2.2 Sampling to measure dry weight, physiological, and nutrients traits 21 days after water-de cit stress, two plants were selected from each pot, and young upper leaves were sampled to measure physiological traits. The samples were transferred to the laboratory after being placed in a liquid nitrogen container where they were stored at -40°C. To measure the traits associated with the dried sample, four plants were harvested from each pot and dried at 70°C for 48 hours.

Enzyme activity
To prepare the enzymatic extract, 3 ml of extraction buffer (100 mM potassium phosphate at pH = 7.8, 0.1 M EDTA, and 0.1 M PVP) was homogenized with 0.1 g of leaf sample using a mortar in an ice bath. The obtained homogeneous samples were centrifuged for 30 minutes (14000 rpm at 4 ° C) and the supernatant was used to measure the enzymes activity. Catalase (CAT) activity was evaluated by monitoring the reduction of the absorption of hydrogen peroxide in the reaction mixture at 240 nm using a spectrophotometer (Aebi 1984). Peroxidase (POD) activity was also assessed according to the absorption of the reaction mixture (enzymatic extract, potassium phosphate buffer, and guaiacol along with 30% H 2 O 2 ) at 470 nm (Zhou and Leu 1999). Polyphenol oxidase (PPO) activity was measured based on the intensity of the orange color of methyl catechol at a wavelength of 420 nm produced in the reaction mixture (Kahn 1975). The CAT, POD, and PPO activity of the extract was expressed as enzyme unit mg − 1 protein min − 1 . One unit of enzyme activity is de ned as the amount required to decompose µl mol of the substrate within one min.

Determination of proline content
To determine the amount of proline in the shoot, 0.1 g of fresh tissues were homogenized with 10 ml of 3 % aqueous sulfosalicylic acid followed by centrifugation. Two milliliters of the supernatant were blended with acid ninhydrin and glacial acetic acid (two milliliters of each). The mixture was kept in a water bath for 1 h at 100°C. The reaction mixture was then extracted with toluene (four milliliters) whose absorbance was determined at 520 nm after cooling down to room temperature (Lechasseur and Paquine 1979).

Determination of total soluble sugar
Total soluble sugar was determined based on the method speci ed by Irigoyen et al. 1992. Fresh leaves (0.1g) were added to 5 ml of 80 % ethanol in a water bath and heated for 1 hour at 80 ºC. Then, 1 ml of the sample extract was taken to another set of test tubes and mixed with 1 ml each of 18 % phenol and distilled water. They were then allowed to stand at room temperature for an hour. Finally, 5 ml of sulfuric acid was added and the whole mixture was vortexed. The absorbance was read at 490 nm using a UV spectrophotometer. Ethanol 80 % was used as a blank sample. Absorbance was recorded at 625 nm using a spectrophotometer.

Measurement of nutrients
The extract for measuring nutrients was prepared based on digestion by the H 2 SO 4 -salicylic acid- Statistical analysis was carried out by SAS software 9.1 and the graphs were drawn by Excel 2013 software while correlation matrices were prepared using xlstat. Comparison of means was performed using the LSD at a P-value of 5%. In the case of signi cant interaction, LS means procedure was used to compare signi cant interactions.

Content of macro-nutrients (N, P, and K) in roots and shoots
The three-way interaction between different levels of drought stress, mycorrhiza, and variety was signi cant on the nitrogen (N) and phosphorus (P) contents of the shoot (Table 1). However, the P and N contents of roots and potassium (K) level of the shoot and root were evaluated based on their signi cance at the 5% level as interaction and main effects (Table 1). Water-de cit stress reduced N accumulation in the shoot. High levels of water stress (-0.8 MPa) signi cantly declined these nutrients uptake in Bodgold (Bod) variety. Regardless of AM application, the N content of the shoot of the Soroksári (Sor) variety was much higher than the Bod variety; nonetheless, the difference got more evident under water stress; so that the N content of the shoot of Bod was 4% lower than Sor variety for + AM treatment. Under stress at an osmotic potential of -0.4 and − 0.8 MPa, this value reached 13 and 35%, respectively ( Table 3). The water stress also reduced the N content of the root, but AM partially increased the content of this nutrient uptake in the roots under stress and control conditions. Also, the N content of the root of both varieties was almost the same. However, the effect of AM on increasing the content of this nutrient was higher in Sor as compared with the Bod variety (Table 4). AM reduced the adverse effects on the P content of shoot. At the water potential of -0.4 and − 0.8 MPa, the impact of AM on the amount of P in shoot was higher in the Bod variety. Regarding the + AM treatment, the P content of shoot of the Bod variety was 20 and 27.5% at the stress level of -0.4 and − 0.8 MPa, respectively. In the case of Sor variety, this parameter was 12 and 10% higher during the -AM treatment. In general, the P level of the shoot of the Bod was lower than the Sor variety, so that the highest P content of the shoot of the Sor variety was observed upon AM inoculation ( Table 3). The P content of the root also decreased with a reduced amount of osmotic potential. The P amount of root was higher in Sor variety. With increasing stress, the mentioned difference declined as the P content of Sor root was 25% under control condition, which decremented to 11 and 3% at osmotic potentials of -0.4 and − 0.8 MPa, respectively (Table 5). According to Table 4, AM caused a 12% enhancement in the P content of the root. Water-de cit stress increased the K level of shoots and roots. At non-stress conditions, the K content of the shoot of the Sor was 10% higher than the Bod variety. By enhancing the stress rate, the difference narrowed so that the K content of the shoot did not signi cantly differ between the two varieties at a water potential of -0.8 MPa. According to the mean comparison of the main effects of the treatments (Table 4), AM generally increased the K content of shoot in the chamomile by 9%. Both water stress and AM treatments enhanced the K content of the root; where the Sor variety exhibited higher root K content. As already stated, AM increased the root K content. Under stress conditions, this increase was more profound, so that the potassium content of the AM-inoculated roots was respectively 17%, 25%, and 19% higher than the non-AM treatment under normal (Control) and water potential of -0.4 and − 0.8 MPa (Table 5).

Content of micronutrients (Fe, Zn, Mn, and Cu) in roots and shoot
The three-way interaction between different water-de cit stress levels, AM, and the variety was signi cant only on the Fe content of the shoot (Table 1). However, comparisons between iron (Fe), zinc (Zn), and copper (Cu), and manganese (Mn) contents of the roots and other micro-elements in the shoot were evaluated based on the signi cance level of 5% for the main effects and the two-way interaction effects (Tables 4 and 5). Water-de cit stress reduced the Fe content of the shoot in both varieties; however, AM signi cantly increased the amount of this nutrient at all levels. In comparison with -AM, + AM enhanced the Fe content of the shoot of the Bod variety by 12%, 12.5%, and 44% under water-de cit water potentials of 0, -0.4, and − 0.8 MPa, respectively. Moreover, the Fe content of Sor was correspondingly increased by 5%, 22%, and 29 % (Table 3). AM also incremented the accumulation of Fe in the roots of these plants under stress conditions. On the other hand, the Fe content of the roots of the two studied varieties was not signi cantly different under normal conditions. A reduction in the osmotic potential declined the Fe content of root in both varieties, nonetheless, its effect was more signi cant on the Bod variety (Table 5).
Water-de cit stress reduced the Cu content of the shoots (Table 3). At normal conditions, there was no signi cant difference between the Mn content of the shoots of the two varieties. A decline in Mn of the shoot of the Bod variety was observed at all levels. The reduction of this nutrient was observed in the shoot of the Sor variety only at the level of -0.8 MPa. The Mn and Cu levels of the shoots in both varieties did not signi cantly differ, but AM increased the content of both nutrients, with a higher rate of increase in the shoot of the Sor variety. Water-de cit stress reduced Mn and Cu contents of the root. Under both stress and normal conditions, the level of these nutrients was higher in Sor root (Table 5). AM also increased the Zn level of the shoot under osmotic stress conditions. The Zn content of the shoots was higher at a water potential of -0.4 and − 0.8 MPa as compared with the control condition. Regarding the root, the trend was the opposite, and under water-de cit stress, the root content of these nutrients was less than the controls. Also, the Zn level of shoot was higher in the Sor variety under all conditions, nonetheless, this was signi cant in normal and water potential of -0.4 MPa (Table 5). AM inoculation increased the Zn content of chamomile by 16% in (Table 3). There was no signi cant difference between the Mn contents of the shoots of the two varieties under normal conditions. A decrease was, however, observed in the Mn level of the Bod at all stress levels, nonetheless, the decrease in Mn content of the shoot of Sor was observed only at the stress level of -0.8 MPa. AM enhanced the Mn and Cu levels of the shoots of the Sor and Bod varieties although their difference was not signi cant. Water-de cit stress reduced Mn and Cu contents of the root, the level of these nutrients was higher in Sor roots than Bod under stress and control conditions. AM also increased the Zn content of the shoot under osmotic stress conditions. Result showed, the Zn amount of the shoot was higher than that of controls at a water potential of -0.4 and − 0.8 MPa. Concerning the root, the trend was the opposite, as the amount of these nutrients in the root was less than the controls. Also, the Zn content of Sor shoot was higher than that of Bod in all conditions, which was signi cant under normal and water potential of -0.4 MPa.

Osmolytes
According to the result (Table 2), the three-way interaction of variety, water-de cit stress and AM signi cantly affected the proline level, whereas, the amount of total soluble sugar was affected by the interaction of water-de cit stress × variety and water-de cit stress × AM. The water-de cit stress generated by PEG increased the levels of proline and total soluble sugar in both varieties. The highest level of these osmolytes was observed in the osmotic potential of -0.8 MPa. The proline level of Sor was higher than Bod variety in all treatments. Also, in stress and non-stress conditions, the total soluble sugar content of Sor was more than that of Bod. Under stress conditions, AM increased the amount of proline in both varieties, however, its effect on increasing the osmolyte was more evident in the case of Bod variety. An increase was also observed in the amount of total soluble sugar of the AM-inoculated samples at different osmotic potentials (Fig. 1).

The activity of antioxidant enzymes
The three-way interaction of water-de cit stress, AM, and variety was signi cant on the levels of POD, PPO, and CAT enzymes ( Table 2). Water-de cit stress increased the activity of all three enzymes relative to the non-stress conditions. Under stress conditions, the activities of CAT and PPO were higher in Sor as compared with Bod variety. Under stress conditions, the activity of the POD enzyme in Sor variety was higher. AM also enhanced CAT and POD at control conditions as well as water potential levels of -0.4 and − 0.8 MPa. The uppermost activity of these enzymes was observed in both varieties upon AM inoculation under the stress potential of -0.8 MPa. For both varieties, the uppermost activity of these enzymes was observed in AM inoculation and the stress potential of -0.8 MPa. Although the level of PPO enzyme in the stress condition was higher than the control, the highest activity was observed under water-de cit conditions (-0.4 MPa) in AM-inoculated plants (Fig. 2).

Dry weight of root and shoot
A signi cant three-way interaction was observed regarding water-de cit stress, AM, and variety on the dry weight of root and the shoot ( Table 2). The water-de cit stress reduced the dry weight of the root and shoot (Fig. 3). On the other hand, AM + decremented the adverse effect of osmotic potential and increased root dry weight. AM + treatment, at all levels, led to higher shoot and root dry weight as compared with AM-treatment. In general, AM improved the dry weight of chamomile at different water potential levels. With decreasing the osmotic potential, shoot dry weight was declined so that the highest shoot dry weight was obtained in PEG 0, AM+, and Sor variety (Fig. 3).
The correlation analysis (Fig. 4) showed a strong correlation between different nutrients uptake and the dry weight of chamomile. For example, P and N contents of the shoot and Mg, Fe, and Cu levels of root showed a slightly positive and signi cant correlation with shoot dry weight. In chamomile, the content of each nutrient showed some relations with the uptake of other nutrients and most of these correlations were synergistic (positive). For example, a slightly positive and signi cant correlation was observed between the Mg, P, N, Cu, and Fe nutrients of the shoot. Moreover, the Mg and P contents of the root, in addition to being highly correlated with each other, showed a positive correlation with other nutrients uptake such as N, Fe, and Cu of shoots and roots. Although the effect of the nutrients on each other was more synergistic, but a signi cant negative correlation was also observed between the shoot and root as well as between K and N contents of the shoot.

Discussion
Under water-de cit stress, the uptake of many nutrients declines due to reduced nutrient mass ow and diffusion (Zhao et al. 2020). Our results and other studies showed that osmotic stress caused by PEG impairs the uptake of micro and macronutrients (Mouradi et al. 2016). Although AM improved nutrient levels and reduced the stress damage by expanding root depth and more soil access through their hyphae, the variation trends of the uptake, accumulation, and transfer of nutrients vary in different species of plants and AM under water-de cit stress condition. In many studies, AM has reported to increases the uptake of nutrients such as N (Hashem et  mosseae (Amiri et al. 2015) and other species has been reported in many plants under water-de cit stress (Aalipour et al. 2020; Al-Al-Arjani et al. 2020). Osmolytes such as total soluble sugars and proline increased under water-de cit stress; playing a signi cant role in regulating the osmotic potential of the plant (Khan et al. 2015). Proline is an amino acid and can be stored in the cytoplasm, which in addition to osmotic regulation of the cell, detoxi es ROS and protects membrane integrity and stabilizes proteins/enzymes, and serve as one of the plant's solutions to reduce stress damage (Ashraf et al. 2007). In the current study, the increased leaf proline level was observed by Funneliformis mosseae under waterde cit stress.
The increase in proline content can be assigned to the effect of AM on increasing the N content of the plants under water-de cit stress (Augé 2001). High N levels in the plant under water stress can signi cantly in uence the genes involved in proline biosynthesis which nally increase proline (Monreal et al. 2007;Wang et al. 2011). In another study, an increase was reported in total soluble sugar under drought stress conditions, which is consistent with our results (Al-Arjani et al. 2020). AM increased the level of total soluble sugar in plants as it increased the activity of sucrose-metabolized enzymes which had a positive and signi cant relationship with glucose, fructose, and sucrose contents (total soluble sugars) (Wu et al. 2017). As observed, under water-de cit stress, plant growth decreased due to reduced osmotic regulation ability, disruption of the solute uptake system, disturbance of osmotic balance, and excessive energy requirements to produce osmolytes (Munns et al. 1993). Based on the ndings of this study, a loss was observed in the dry weight of shoots, roots, and owers of chamomile under waterde cit stress (Baghalian et al. 2011). One of the causes of reduced chamomile growth under stress may be the osmoregulation imbalance and the disruption in the salt absorption system or the high level of energy required for counteracting the stress (Salehi et al. 2018). An increment was also detected in the dry weight of shoots, roots, and owers of chamomile (Bączek et al. 2019) due to the improved absorption, distribution of nutrients, the increment of proline, total soluble sugars, and antioxidant enzymes by AM, which improved the growth performance, lowered the stress damage, enhanced the plant growth and elevated the dry weight (Al-Arjani et al. 2020).
According to the results, the dry weight of chamomile shoots, roots, and owers reduced under drought stress (Baghalian et al. 2011). Under drought stress, plant growth was reduced due to the reduction of osmotic regulation, osmotic imbalance, and the requirement of excessive energy needs to cope with stress (i.e. the production of osmolites and disruption of the nutrient uptake) (Munns et al. 1993). All nutrients play a vital role in plant growth; the nutrients (macro and micro) were positively correlated with the plant growth was positive (Daur et al. 2011). The effects of each nutrient on the uptake of other nutrients are very complex. According to the correlation shown in Fig. 4, the synergistic effect between many nutrients in chamomile re ects the diverse roles of these nutrients in the growth, yield, and uptake of other nutrients by chamomile. For example, su cient Mg causes a proportional distribution of carbohydrates in the root and shoot; promoting the chamomile root growth (He et al., 2020). On the other hand, Mg affects biomass production and plant growth by proper distribution of carbohydrates and the appropriate allocation of hydrocarbons to different parts of plants (Verbruggen and Hermans 2013) or improving the plant access to N (Haberman et al. 2019) and iron (due to its vital role in photosynthesis) (Dong et al. 2019) with an effective role in vegetative growth and ultimately the accumulation of plant dry weight. This is consistent with a positive and high correlation of the dry weight of chamomile with the mentioned elements. According to the results, one of the most important causes of reduced growth of chamomile under stress conditions is the disturbed nutrients uptake (Salehi et al. 2018). The level of nutrients in the plant and the ability to uptake these nutrients are important factors in selecting the best cultivar under stress. In this regard, the Sor cultivar was almost superior to Bod in terms of both factors. On the other hand, improved absorption and distribution of elements as well as increased proline, total soluble sugar and antioxidant enzymes by mycorrhiza inoculation enhanced the growth while reducing stress-induced damages (Al-Arjani et al. 2020). In line with previous reports (Bączek et al. 2019), mycorrhiza increased the dry weight of shoots, roots, and owers of chamomile.

Conclusions
Water-de cit stress increased the levels of the total soluble sugar, proline, and the activity of antioxidant enzymes (CAT, POD, and PPO) in both chamomile varieties. The amount of these enzymes and osmolytes was higher in the Soroksári variety as compared with the Bodgold variety. Water-de cit stress also reduced the uptake and transport of many nutrients from the roots to the shoots, which resulted in decreased content of nutrients such as N, P, Fe, and Mn in the shoots of both varieties. Such a reduction of nutrients in the plant declined the plant dry weight under water-de cit stress, however, the dry weight of the shoot was higher in Soroksári variety under the control treatment and water potential of -0.4 MPa as compared with the Bod variety. AM + reduced the negative effects of drought stress on the plant through increasing the nutrients uptake, osmolytes contents and the activity of antioxidant enzymes.  Means within a column followed by the different letter are signi cantly different at P< 0.05. Standard error of the mean (n = 3)