Arbuscular mycorrhizal fungus alters root-sourced signal (abscisic acid) for better drought acclimation in Zea mays L. seedlings

Environmental and Experimental Botany 167 (2019) 103824

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Arbuscular mycorrhizal fungus alters root-sourced signal (abscisic acid) for better drought acclimation in Zea mays L. seedlings

T

Ai-Tian Rena,1, Ying Zhub,1, Ying-Long Chenc,d, Hong-Xu Rene, Ji-Yuan Lia, Lynette Kay Abbottc,d, ⁎ You-Cai Xionga, a

State Key Laboratory of Grassland Agro-ecosystems, Institute of Arid Agroecology, School of Life Sciences, Lanzhou University, Lanzhou, 730000, China Key Laboratory of Microbial Resources Exploitation and Application of Gansu Province, Institute of Biology, Gansu Academy of Sciences, Lanzhou, 730000, China c UWA School of Agriculture and Environment, The University of Western Australia, Perth, WA, Australia d Institute of Agriculture, The University of Western Australia, Perth, WA, Australia e The Institute of Botany, Chinese Academy of Sciences, Xiangshan, Beijing, 100093, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Arbuscular mycorrhizal symbiosis Drought tolerance Stomatal conductance Non-hydraulic signals Antioxidant enzymes

Inoculation with arbuscular mycorrhizal (AM) fungi can modify stomatal behavior and increase antioxidant enzyme activities and therefore play a pivotal role in plant growth. We hypothesised that inoculation with AM fungi postpones the non-hydraulic root signal and alters physiological and biochemical traits, which enhances drought tolerance and water-use efficiency (WUEB) for plant biomass. Two pot experiments (including progressive soil drying and partial root-zone drying) were conducted to reveal how mycorrhizal colonization altered root signal and its effects on plant growth, biochemical traits and WUEB in maize seedlings in drying soil. In our experiments, inoculation with Funneliformis mosseae improved water absorption and reduced the sensitivity of roots to drought. In addition, it decreased leaf abscisic acid (ABA) content of inoculated plants. Regardless of water conditions, plant biomass production, antioxidant enzyme activity, net photosynthetic rate, stomatal conductance and WUEB were elevated in AM fungal treatments compared to non-AM fungal treatments. Under water-stressed conditions, inoculation with F. mosseae greatly reduced leaf ABA content, and postponed the decline in photosynthetic rate, stomatal conductance and osmotic adjustment. Malondialdehyde (MDA) level was significantly lower in mycorrhizal plants than in non-inoculation plants. However, inoculation with F. mosseae increased antioxidant enzyme activities including peroxidase (POD) and superoxide dismutase (SOD). In this study, inoculation with F. mosseae reduced ABA accumulation that acts as a non-hydraulic root signal and thereby postponed a decline in stomatal conductance and photosynthetic rate, improved water use efficiency and antioxidant enzymes activities, and accordingly reduced proline and MDA content. Thus, inoculation with AM fungi played a role in effective defense for better drought acclimation in water-stressed maize seedlings.

1. Introduction Drought stress is considered to be one of the most important abiotic factors limiting plant growth, yield and altering water relations in many regions (Asrar and Elhindi, 2011; Asrar et al., 2012). Symptoms of drought included wilting of the plants, and reductions in net photosynthesis rate (pn), stomatal conductance (gs), water use efficiency (WUE) and leaf relative water content (Abbaspour et al., 2012). However, crop plants have evolved a number of biochemical and physiological mechanisms to cope with temporary or terminal water shortage (Turner and Asseng, 2005; Du et al., 2012). One of the mechanisms of plant tolerance to drought stress may be formation of the symbiosis

with arbuscular mycorrhizal (AM) fungi (Doubková et al., 2013). Mycorrhizal associations have led to improved water relations and enhanced plant resistance to water deficit (Stevens et al., 2011; Asrar et al., 2012; Bitterlich et al., 2018). Stomatal conductance and WUE are among the most-studied water relations parameters in the mycorrhizal literature, there are several reports showing that gs and transpiration are often higher in mycorrhizal plants compared to those without mycorrhizas (Wu and Xia, 2006; Ruiz-Sánchez et al., 2010; Lee et al., 2012). Mycorrhizal plants demonstrated postponed declines in leaf water potential during drought stress (Porcel and Ruiz-Lozano, 2004) and several direct and indirect mechanisms may be involved (Mickan, 2014). The most important mechanism is the direct uptake and transfer

⁎

Correspondence author. E-mail address: [email protected] (Y.-C. Xiong). 1 Aitian Ren and Ying Zhu equally contributed to this work. https://doi.org/10.1016/j.envexpbot.2019.103824 Received 7 April 2019; Received in revised form 19 July 2019; Accepted 22 July 2019 Available online 25 July 2019 0098-8472/ © 2019 Elsevier B.V. All rights reserved.

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lipid membrane peroxide levels in plant tissues; (2) AM fungal inoculation improves the gs, pn, and leaf relative water content; and (3) inoculation with AM fungi can reduce plant ABA content and postpone a decline in nHRS response. The hypotheses were tested using maize (Zea mays L.) and the AM fungus Funneliformis mosseae (T.H. Nicolson & Gerd.) C. Walker & A. Schüßler.

of water through the fungal hyphae to the host plant (Smith et al., 2010; Borowicz, 2010). In nutrient-poor or dry soil, nutritional benefits of mycorrhization lie mainly in enhancing uptake of P and other immobile nutrients (Subramanian et al., 2006; Doubková et al., 2013). Many hypotheses unrelated to nutrition have been suggested to explain how the mycorrhizas might influence host gs during drought (Augé and Duan, 1991). The most popular notion is that soil hyphae greatly increase the absorptive area of root systems, acting as conduits for water movement into roots (Faber et al., 1991) and regulating root hydraulic properties, including root hydraulic conductivity (Bárzana et al., 2012). The discovery of non-hydraulic root to shoot communication (Saab and Sharp, 1989; Duan et al., 1996) reveals another possibility. Plants may sense drought around roots and respond with root-sourced chemical signals to the shoot thus eliciting several adaptive responses including decreased leaf expansion and stomatal closure (Duan et al., 1996). This early-warning response to soil drying is characterized as a non-hydraulic root signal (nHRS) (Xiong et al., 2006). The relationship between gs and leaf water potential may also be changed by mycorrhization. AM fungi can inhibit stomatal closure that occurs when only a portion of the root system is dried and before drying affects leaf water potential, suggesting an effect on non-hydraulic rootto-shoot communication of soil drying (Augé and Duan, 1991; Ebel et al., 1996). Root-to-shoot regulation of stomatal behaviour during soil drying may occur via a multiple chemical signal, including abscisic acid (ABA) (Druge and Schonbeck, 1992; Goicoechea et al., 1997). Indeed, Goicoechea et al. (1997) noted that higher gs and transpiration of mycorrhizal alfalfa plants was associated with altered ABA/cytokinins ratios in their leaves. It is also noticeable that mycorrhizal and nonmycorrhizal plants have shown different critical points or thresholds of stomatal behaviour during drought episodes. At low soil water contents, AM plants maintained higher gs rates and leaf relative water content than non-mycorrhizal plants, and these higher foliar water status characters were associated with lower ABA fluxes to leaves in AM plants at low water contents (Ruiz-Lozano and Aroca, 2010; Duan et al., 1996). Additional mechanisms have been proposed for drought tolerance, such as enhanced osmotic adjustment and/or reduced oxidative damage caused by the reactive oxygen species generated during drought. AM fungi may increase drought resistance of plants by promoting antioxidant enzymes, such as SOD and POD and increase photosynthesis under water-stress (Huang et al., 2011; Essahibi et al., 2018). Waterstress is always associated with an increase in ABA, which mediates most plant responses to drought (Du et al., 2012). Jiang and Zhang (2002) showed that ABA is an essential mediator in triggering a drought-induced antioxidative defense response against oxidative damage in maize seedlings. Therefore, there is a link between ABA, root signals, and antioxidant defense in plants exposed to a water deficit. Many studies have focused on the physiological and biochemical of plant to drought. At times, it has been reported that gs and transpiration are often higher in AM fungi plants compared to those in non-mycorrhizal ones during soil drying (e.g. Ruiz-Sánchez et al., 2010). Partial root-zone drying was demonstrated that allows the induction of the ABA-based root to-shoot chemical signaling system to regulate growth and water use and thereby increase WUE (Liu et al., 2006). At the same time, partial root-zone drying was usually used to study the relationship between gs and leaf relative water content. However, there was no relationship between ABA and soil water, if the root systems were only partially droughted (Ebel et al., 1996; Dodd et al., 2008). Therefore, we conducted the progressive soil drying to test whether mycorrhizal symbiosis altered stomatal sensitivity to ABA or changed the accumulation of ABA in the leaf across a range of soil moisture. Furthermore, we investigated the regulation of inoculation with AM fungi on root signal and physiological and biochemical traits under water-stress conditions with both progressive soil drying and partial root-zone drying. The aim of the study was to test the hypotheses that: (1) inoculation with AM fungi increases the antioxidant defense and reduces

2. Materials and methods 2.1. Experimental design The experiments were conducted at the Yuzhong Experiment Station of Lanzhou University in Yuzhong County, Gansu Province (35°51′ N, 104°07′ E, altitude 1620 m). This design allowed to induce a physiological drought affecting the whole plant or a non-physiological drought affecting only a part of the root system (Bárzana et al., 2015). The non-physiological drought (partial root-zone drying) was used as a tool to ascertain if the AM regulate ABA signaling in the absence of changing leaf water status. So we conducted the partial root-zone drying was a supplement of progressive drying in order to verify the results of progressive soil drying. The set-up of Experiment 1 had progressive soil drying (physiological drought) and consisted of two water conditions (well-watered, WW; water-stress, WS) and two AM fungal treatments (soil amended with either a living mixture of AM fungal inoculum (+AMF) or an autoclaved AM mixture). Experiment 2 had partial root-zone drying (non-physiological drought) and included two water treatments (well-watered, WW/WW; water stress, WW/WS) and two AM treatments (soil amended with either a living mixture of AM fungal inoculum (+AMF) or an autoclaved AM mixture).

2.1.1. Experiment 1: progressive soil drying This experiment was designed to test the hypothesis that AM fungal inoculation reduced the gs and leaf relative water content and postpone declines in nHRS response. The inoculation with AM fungi was expected to increase antioxidative enzymes activities and reduce the lipid membrane peroxide levels. All pots were watered to bring the soil content to 80% field water capacity (FWC) by weighing every 2–3 days until two watering treatments were imposed after 50 days: (i) half of the pots were well-watered every day to maintain soil water content close to 80% FWC (wellwatered, WW); (ii) water was withheld from the other half of the pots to impose a slow dry down of the soil water content by re-watering the pots so that a maximum of 100 ml of soil water was lost each day until water loss was below 100 ml (water-stress, WS) (Kong et al., 2015).

2.1.2. Experiment 2: partial root-zone drying This experiment sought to verify the effect of inoculation with AM fungi on ABA signaling, leaf gas exchange, physiological changes and non-hydraulic root-to-shoot communication of maize during the soil drying. The pots were evenly separated into two compartments with plastic sheets such that water exchange between the two compartments was prevented. A piece of plastic [width (1 cm) × height (2 cm)] was removed from the middle of the sheet where the seeds were planted. Before planting, maize seedlings were pre-germinated on sterilized vermiculite and then transferred to containers prepared ad hoc for a split-root assay. Roots from the seedlings were evenly distributed between the two separated compartments. Plants were kept well-watered during the first 50 days after planting. Then plants were subjected to two irrigation treatments: (1) full irrigation (WW/WW) in which both soil compartments were watered daily, and (2) partial root drying (WW/WS) in which half of the plants were allowed to dry for 16 d before harvest, while the other half was maintained at field capacity by daily watering, and the irrigation was not shifted between sides. 2

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decreased simultaneously (the onset of hydraulic signals, HRS) (Xiong et al., 2006). These criteria were used to determine the SWC at which nHRS and HRS were induced, compared with the well-watered plants.

2.2. Plant materials and growth conditions A silty-loam loess soil collected from Yuzhong Experiment Station of Lanzhou University, Gansu Province, and mixed with vermiculite (soil: vermiculite = 2:1, v/v). The soil at Yuzhong had a pH of 7.8, organic carbon of 9.54 g kg−1, total nitrogen of 0.48 g kg−1, total phosphorus of 0.46 g kg−1. The soil was autoclaved twice on two consecutive days at least 24 h in between in order to ensure absence of viable microbial propagules before the experiment. Each plastic pot (23 cm diameter×24 cm high) was filled with 7 kg of mixture soil in two experiments. Inoculum of F. mosseae was propagated with Trifolium repens L. in sterilized soil in growth chamber for 4 months. The density of spores in the inocula of F. mosseae was estimated by microscopic examination after wet-sieving and centrifugation. Spore numbers were 50–80 per 10 g soil. For the AM fungal treatment, F. mosseae inoculum was added to each treatment. The inoculum containing spores, hyphae and infected clover root fragments was mixed throughout the pot soil before the experiment started. The non-AM fungal treatments were treated with a microbial filtrate obtained from the AM fungal inocula to exclude AM fungal spores, to minimize the possible effects caused by mineral and non-mycorrhizal microbial components in the living inoculum. A local widely-planted maize variety (cv. Xianyu 335) was used in this study. Seeds of maize were sterilized in 5% sodium hypochlorite for 30 s, washed several times with sterile water, and sown directly into the soil in Experiment 1. After seedling emergence plants were thinned to three plants per pot in Experiment 1 and two plants per pot in Experiment 2. Each treatment was replicated ten times and arranged in a complete randomized design. In some cases, the abbreviations are combined. For example WS + AMF (WW/WS + AMF) and WS (WW/ WS), respectively indicate treatments with AM fungal inoculum and water-stress and autoclaved inoculum and water-stress. The greenhouse conditions had average temperatures of 30 °C/15 °C (day/night), with a mean relative humidity of 65–90 %, and photon flux density of 600–1000 μmol m−2 s-1.

2.4. Analysis of lipid peroxidation, proline content and antioxidant enzyme activity Frozen leaf segments (0.5 g) were crushed into fine powder with a mortar and pestle under liquid N2. The soluble proteins were extracted by homogenizing with 10 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 1% polyvinylpyrrolidone (PVP), with the addition of 1 mM ascorbate acid (ASC) for the APX assay. The homogenate was centrifuged at 12 000 g for 1200s at 4 °C and the supernatant used for the following enzyme assays. Total superoxide dismutase (SOD) activity was assayed by monitoring the inhibition of the photochemical reduction of nitro blue tetrazolium (NBT) according to the method of Giannopolitis and Ries (1977). One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the reduction of NBT as monitored at 560 nm. The activity of peroxidase (POD) was assayed according to the method described by Kwak et al. (1995), using pyrogallol as a substrate. One unit of POD activity was defined as the amount of enzyme necessary to obtain 1 mg of purpurogallin from pyrogallol in 20 s, at 420 nm. The level of lipid peroxidation was determined by the content of malondialdehyde (MDA) in the leaf segment homogenates, prepared in 10 ml of 10% trichloroacetic acid (TCA) containing 0.5% thiobarbituric acid (TBA) as described by Zhao et al. (1994). The mixture was heated in a water bath at 100 °C for 900 s and then quickly cooled in an ice bath. After centrifugation at 4000 g for 900 s, the absorbance of the supernatant was recorded at 532, 600, and 450 nm. The MDA content was calculated by its absorbance (Zhao et al., 1994) and expressed as nmol MDA g−1 dry weight. For proline determination, fresh samples were extracted with 3% sulfosalicylic acid, placed in a boiling water bath for 10 min and filtered through filter paper. Two milliliters of the extract was added to 6 ml (final volume) assay media containing 2 ml ninhydrin solution and 2 ml acetic acid and incubated for 30 min at 100 °C and then cooled. The colored product formed was extracted with 4 ml toluene by shaking and the absorbance of resultant organic layer was measured at 520 nm (Troll and Lindsley, 1955).

2.3. Soil water content (SWC), stomatal conductance (gs) and relative water content (RWC) After the implementation of treatments in both experiments, the effects of AM fungi on the physiological and biochemical responses of the maize were investigated after the two watering treatments had been imposed from 50 days after sowing (DAS), when plants were at the jointing stage. Soil water content (SWC) is expressed as a percentage of water available between FWC and dry soil. Therefore, once the drying treatment was imposed, all pots were weighed daily to determine the SWC, and the stomatal conductance and net photosynthesis rate were measured on one mature non-senescent leaf in each of the three replicate pots between 08.30 h and 10.30 h using an LI-6400 portable photosynthesis system (Li-Cor, Lincoln, NE, USA). The stomatal conductance for each leaf was a mean of five readings per leaf. After measuring gs, the leaf RWC was measured on three leaves in each of the three replicate pots at a similar position as the leaf used to measure gs. Two leaf discs (5 mm in diameter) were cut with a cork borer from each leaf and weighed immediately for fresh weight (FW). The discs were floated in freshly-distilled water for 4 h, then blotted dry and weighed to obtain the saturated weight (SW). Dry weight (DW) was measured after drying at 80 °C in a forced-draught oven for 24 h. leaf RWC was calculated as RWC= [(FW–DW)/(SW–DW)]×100. Leaf RWC and gs were monitored for 3 days prior to the start of soil drying to ensure that equilibrium with chamber conditions were established (Saab and Sharp, 1989). The data collected for the plants were used to infer relations between leaf RWC or gs and SWC. Non-hydraulic root signals (nHRS) were judged to have been induced when there was a significant reduction in leaf gs (compared with gs in the well-watered plants) without change in leaf RWC, and to end when gs and RWC both

2.5. Abscisic acid (ABA) extraction, purification, and quantification The methods for extraction and purification of ABA were modified from those described by Bollmark et al. (1988). The leaf samples (0.5 g) were ground in liquid N2 using a mortar and pestle, extracted with icecold 80% methanol (v/v) containing 1 mM butylated hydroxytoluene to avoid oxidation, and then stored overnight at 4 °C. The extracts were then centrifuged at 10 000 g for 900 s at 4 °C. The residues were suspended in the same ice-cold extraction solution and stored at 4 °C for 1 h, and then centrifuged again at 10 000 g for 900 s at 4 °C. The supernatants were combined and passed through Chromosep C18 columns (C 18 Sep-Park Cartridge, Waters, Millford, MA, USA), prewashed with 10 ml of 100% and 5 ml of 80% methanol, respectively. The efflux was collected and dried by evaporation with nitrogen. The residues were dissolved in 1.6 ml of phosphate-buffered saline (PBS) containing 0.1% (v/v) Tween 20 and 0.1% (w/v) gelatin (pH 7.5) for analysis by enzyme-linked immunosorbent assay (ELISA). The mouse monoclonal antigen and antibody against ABA and the immunoglobulin G-horse radish peroxidase (IgG-HRP) used in ELISA were produced at the Phytohormones Research Institute, China Agricultural University, Beijing, China. The method for quantification of ABA by ELISA has been described previously (Yang et al., 2001). In the current study, the percentage recovery of each hormone was calculated by adding known quantities of standard hormone to a split 3

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extract. The recovery percentage of ABA in leaves was 85.6 ± 5.2%. The specificity of the monoclonal antibody was confirmed and the possibility of other non-specific inhibitors was excluded in previous studies (Yang et al., 2001). 2.6. Plant biomass production and mycorrhizal colonization After 16 days of water treatment, all seedlings were harvested. Roots were washed carefully with each treatment. Leaves, stems and roots were separated and dry weights of leaves, stems and washed roots were determined. Fresh roots were carefully washed, fine roots (0.5 g fresh weight) were picked by hand from the samples and cut into 1 cm long fragments, and stained with Trypan Blue to investigate AM fungal colonization (Phillips and Hayman 1970; Abbott and Robson, 1981). AM fungal colonization was quantified using the magnified grid line intersects method (Giovannetti and Mosse, 1980). This method uses a compound microscope (200 to 400×) to measure the percentage root length colonized by intramatrical hyphae, vesicles, coils and arbuscules.

Fig. 1. Developments of the soil water content under progressive soil drying treatment (n = 6). WS + AMF, water stress and inoculation of mycorrhizal; WW + AMF, well water and inoculation of mycorrhizal; WS, water stress and non-inoculation of mycorrhizal; WW, well water and non-inoculation of mycorrhizal. Values are means ± 1 standard error of the mean.

2.7. Data analysis The values of leaf gs, pn and leaf RWC were expressed as percentages of the values measured in the controls (100%). Variables of root colonization rate, leaves area, above ground biomass and root to shoot ratio were analyzed using two-way ANOVA. The differences among treatments were examined using LSD test (P ≤ 0.05). The data analysis and figures were prepared using SPSS 13 and Origin 9.

significant different between the well-watered non-mycorrhizal plants and water-stressed mycorrhizal plants in both experiments. The WUEB was significantly increased by inoculation with AM fungi under waterstressed conditions in Experiment 1 (Table 1). The WUEB of waterstressed mycorrhizal plants were 46% higher than that of water-stressed non-mycorrhizal plants and 24% higher than that of well-watered nonmycorrhizal plants. Plant leaf area was lower for water-stressed plants than for well-watered plants. However, mycorrhizal plants under wellwatered and water-stressed conditions had significantly higher leaf area compared with non-mycorrhizal plants in Experiment 1, but in Experiment 2, leaf area was no significantly different between treatments (Table 1).

3. Results 3.1. Plant biomass and AM fungi root colonization The roots of maize were colonized by the inoculated F. mosseae, but no mycorrhizas were observed in the non-mycorrhizal treatment (Table 1). Water-stress markedly decreased shoot and root dry weights, but did not significantly affect the AM fungal colonization. However, mycorrhizal plants under well-watered and water-stressed conditions had significantly higher shoot and root dry weights than the corresponding non-mycorrhizal plants. Under water stress condition, the shoot DW of AM plants significantly increased by 10.3% and 33.0% compared with non-mycorrhizal treatments in Experiment 1 and Experiment 2, respectively. However, plant shoot dry weight was not

3.2. Stomatal conductance, leaf relative water content and photosynthetic rates Changes of soil water content in WS-treated plants during the experimental periods are shown in Fig. 1. The soil water content of the drying treatment declined slower during the experimental period, and was about 20% and 19% by the end of treatment in WS and WS + AMF,

Table 1 Mycorrhizal colonization, biomass allocation, water use efficiency and leaf area in Zea mays L. subjected to progressive soil drying (WS) and partial root drying (WW/ WS) at the end of the experimental period. Treatments

Colonization (%)

Shoot DM (g pot−1)

Root DM (g pot−1)

root/shoot (g g−1)

WUE (g/Kg H2O)

Leaf area (cm2/plant)

WS + AMF WW + AMF WS WW ANOVA AMF(A) Drought(D) A×D WW/WS + AMF WW/WW + AMF WW/WS WW/WW ANOVA AMF(A) Drought(D) A×D

22a 25a — — — — — 25a 26a — — — — —

48.0b 61.0a 43.5c 49.8b *** *** ** 50.0b 57.0a 33.5c 48.9b *** *** **

27.6b 33.0a 24.8b 28.3b *** *** ns 27.8a 29.8a 17.4c 24.6b *** *** **

0.57a 0.54a 0.54a 0.57a ns ns ns 0.56a 0.52a 0.52a 0.50a ns ns ns

25.1a 23.9a 17.2b 20.2b *** ns ns — — — — — — —

896.5a 566.2c 466.9bc 799.7ab ns *** ns 801.6a 860.5a 767.3a 886.2a ns ns ns

Notes: WS + AMF, water stresse and inoculation of mycorrhizal; WW + AMF, well water and inoculation of mycorrhizal; WS, water stress and non-inoculation of mycorrhizal; WW, well water and non-inoculation of mycorrhizal. WW/WS + AMF, only one root fraction subjected to water stress and both root compartments inoculation of mycorrhizal; WW/WW + AMF, well water and both root compartments inoculation of mycorrhizal; WW/WS, only one root fraction subjected to water stress and both root compartments non- inoculation of mycorrhizal; WW/WW, well water and both root compartments non- inoculation of mycorrhizal. DM is plant dry mass. Means ± SE with the same letter in a column are not significantly different at the 0.05 level using the LSD test. ***, significant at p < 0.01; **, significant at p < 0.05; ns, not significant at p < 0.05. 4

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Fig. 2. Change of stomatal conductance (gs) as a percentage of the controls (A, D), photosynthetic rates (pn) as a percentage of the controls (B, E) and leaf relative water content (RWC) as a percentage of the controls (C, F) with time after water was withheld in Zea mays L. in a progressive drying (A, B, C) and partial root drying (D, E, F) system. WS + AMF, water stress and inoculation of mycorrhizal; WS, water stress and non-inoculation of mycorrhizal; WW/WS + AMF, only one root fraction subjected to water stress and both root compartments inoculation of mycorrhizal; WW/WS, only one root fraction subjected to water stress and both root compartments non-inoculation of mycorrhizal. Arrows indicate the time that gs and pn decline under different treatments. Values are means ± 1 standard error of the mean.

3.3. ABA content in leaves

respectively. While there was no statistical difference between mycorrhizal plants and no-mycorrhizal plants. Moreover, the relative leaf gs, RWC and pn in drought-affected plants with or without mycorrhizal treatment were plotted as function of time (Fig. 2). Leaf gs and pn in non-mycorrhizal plants decreased earlier than in AM fungal plants (Fig. 2A, B and D, E). However, the addition of AM fungi did not influence the Leaf RWC of mycorrhizal plants and non-mycorrhizal plants under water stress in two experiments (Fig. 2C, F). Furthermore, in experiment 1, AM fungal inoculation was not significantly affected the gs during the first 8 days and had significantly enhanced the gs thereafter. Eleven days after the water stress, AM fungal plants significantly improved the pn compared with non-mycorrhizal plants. Although the leaf RWC of mycorrhizal plants was generally higher than that of the non-mycorrhizal plants, there was no statistical difference between them, except for the last harvest (Fig. 2C). In experiment 2, the gs and pn were not significantly affected by the AM inoculation. However, leaf RWC of mycorrhizal plants was generally lower than that of the nonmycorrhizal plants during the experimental period (Fig. 2D–F). At higher soil water content, gs was stable. When the SWC gradually decreased, there was a significant decrease in leaf gs for mycorrhizal plant and non-mycorrhizal plant at its own critical SWC (Fig. 3A, B). The critical SWC at which nHRS is triggered varied consistently with the AM fungi inoculation and growing period. Non-hydraulic root signals appeared first in the non-mycorrhizal plant at 38.8% FWC, then in the mycorrhizal plant at 33.5% FWC (Fig. 3C). With further decline in SWC, the gs generally decreased for the inoculated plant and non-inoculated plants with successively lower SWC, often without a significant change in leaf RWC, demonstrating that the nHRS was operative throughout. With still further decrease in SWC, the leaf RWC started to fall significantly. This indicated that soil water deficit caused a marked decrease in shoot water content, and that the HRS was triggered. For non-inoculated plants, this happened at SWC as high as 22.7% FWC, but for inoculated plants, it did not occur until the SWC decreased to about 21.6% FWC (Fig. 3C). In the Experiment 2, the nHRS appeared first in the non-inoculated plants and non-hydraulic root signals did not appear in the inoculated plants (Fig. 3D, E). With still further drying, the leaf RWC started to fall significantly. This indicated that soil drought caused a marked decrease in shoot water content and that the HRS was triggered (Fig. 3D, E).

AM fungal colonization did not affect the ABA concentration in the leaves under well water condition in the two experiments. However, with intensification of drought stress, water stress significantly increased the ABA concentration. At nHRS appeared, lower values were recorded for the AM fungi-treatment (6.30 mg/g) compared to the nonmycorrhizal treatment (8.37 mg/g). Compared with the ABA concentration of non-mycorrhizal plant, the ABA concentration of mycorrhizal plant decreased by 24.6% under water stress conditions, but ABA concentration was no affected by the drought × inoculation interaction (Fig. 4A). In the experiment 2, the ABA concentration of mycorrhizal plant decreased by 17.2% compared to the non-mycorrhizal treatment under water stress conditions, but ABA concentration was affected by the drought × inoculation interaction (Fig. 4B). Meanwhile, mycorrhizal plants displayed low ABA concentration compared to the nonmycorrhizal treatment at HRS appeared (Fig. 4A, B). 3.4. Accumulation of osmoregulatory compounds, oxidative damage to lipids and antioxidant enzyme activities The accumulation of drought-induced antioxidant enzymes are shown in Fig. 5. The POD and SOD activities in all plants increased due to water stress conditions in the experiment 1, in general, enzyme activities were higher in mycorrhizal plants than in the corresponding non-mycorrhizal control, regardless of the water conditions (Fig. 5A, B). The POD and SOD activities were significantly increased by AM fungal colonization under water stress conditions. Compared with the nonmycorrhizal plants, the POD and SOD activity of mycorrhizal plants increased by 23.0% and 9.15% under water stress conditions, respectively. While the POD and SOD activities were not significantly increased by the water stress and AM fungal inoculation under the partial root drying systems (Fig. 5E, F). The POD activity was significantly affected by the drought × inoculation interaction, but SOD activity was not affected by the drought × inoculation interaction (Fig. 5A, B). Water stress condition significantly increased malondialdehyde concentration and proline concentration of leaves (Fig. 5C, G and 5D, H). But the malondialdehyde and proline concentration were significantly decreased by AM fungal colonization under water stress conditions. Compared with the non-mycorrhizal plants, the malondialdehyde and proline concentration of mycorrhizal plants reduced by 24.8% and 5

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Fig. 3. Soil moisture trend in leaf stomatal conductance (leaf gs, indicated by filled circles) and leaf relative water content (leaf RWC, indicated by filled triangle) of Zea mays L., during progressive soil drying (A, B) and partial root drying (D, E), and (C) the change in SWC a threshold between the threshold for a decrease in gs and the threshold for a decrease in RWC for Zea mays L. with (+) and without AM fungi. WS + AMF, water stress and inoculation of mycorrhizal; WS, water stress and non-inoculation of mycorrhizal; WW/WS + AMF, only one root fraction subjected to water stress and both root compartments inoculation of mycorrhizal; WW/WS, only one root fraction subjected to water stress and both root compartments non-inoculation of mycorrhizal. * indicate the points at which significant differences begin to occur for Leaf gs and leaf RWC at P < 0.05.

32.7% under water stress conditions in the experiment 1, respectively (Fig. 5C, D). The malondialdehyde and proline concentration of mycorrhizal plants reduced by 28.9% and 26.0% under water stress conditions in the experiment 2, respectively (Fig. 5G, H). Malondialdehyde and proline concentration were significantly affected by the drought × inoculation interaction (Fig. 5C, D and G, H). The severity of drought determined the strategies of plant adaptation to water stress. The role of the different studied parameters during the response to drought was elucidated in a model figure (Fig. 6). During the progressive soil dying, the nHRS was at first responsive to the soil drought, which reduced water loss by decreasing stomatal aperture. With the commencement of HRS, the regulative role of nHRS for stomatal could be weakened. While inoculation with AM fungi reinforced the ability of plants defense to drought by accumulating the POD and SOD activity in the leaves, and meanwhile, it reduced MDA and proline concentrations in the leaves. In the partial root drying experiment, the AM fungi colonization also greatly reduced the leaf ABA concentration, and then postpone declines in pn, gs and the osmotic adjustment, malondialdehyde were lower in mycorrhizal plants compared with that in non-mycorrhizal plants under water-stressed condition. Thus, AM fungal inoculation stimulated growth, enhanced drought tolerance of maize, through alterations in root signal, physiological and biochemical traits.

4. Discussion 4.1. Effect of drought and AM fungi inoculation on biomass and mycorrhizal colonization

Fig. 4. Leaf ABA concentration in Zea mays L. under progressive soil drying (A) and partial root drying (B) at three different stages including the well-watered stage, the commencement of non-hydraulic root-sourced signal and hydraulic signal operation respectively (WS + AMF, water stresse and inoculation of mycorrhizal; WW + AMF, well water and inoculation of mycorrhizal; WS, water stress and non-inoculation of mycorrhizal; WW, well water n and noninoculation of mycorrhizal. WW/WS + AMF, only one root fraction subjected to water stress and both root compartments inoculation of mycorrhizal; WW/ WW + AMF, well water and both root compartments inoculation of mycorrhizal; WW/WS, only one root fraction subjected to water stress and both root compartments non- inoculation of mycorrhizal; WW/WW, well water and both root compartments non- inoculation of mycorrhizal). Note: vertical bars on each histogram represent means ± SE. ***, significant at p < 0.01; *, significant at p < 0.05; ns, not significant.

Physiological and biochemical aspects related to water relations and drought tolerance in mycorrhizal and non-mycorrhizal treated plants subjected to the water stress were investigated. Mycorrhizal inoculation enhanced shoot and root biomass under both well-watered and waterstressed conditions compared to non-mycorrhizal plants. AM fungal colonization was not affected by water stress in either of the two experiments. This was contrary to the finding of Wu and Xia (2006), who reported that water stress significantly decreased the AM fungal colonization by Glomus versiforme in tangerine (Citrus tangerine). In addition, as a whole, in viewing the literature, root colonization is more often increased than decreased under water stress conditions (Augé, 2001). The results of our research may be that drought stress time was not too long. On the other hand, it has been reported that the effects of 6

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Fig. 5. The activity of POD (A, E) and SOD (B, F), and the concentration of malondialdehyde (MDA) (C, G) and proline (D, H) of the leaf of Zea mays L. under progressive soil drying (A, B, C and D) and partial root drying (E, F, G and H) at the end of the experimental period. WS + AMF, water stresse and inoculation of mycorrhizal; WW + AMF, well water and inoculation of mycorrhizal; WS, water stress and non-inoculation of mycorrhizal; WW, well water n and non-inoculation of mycorrhizal. WW/WS + AMF, only one root fraction subjected to water stress and both root compartments inoculation of mycorrhizal; WW/ WW + AMF, well water and both root compartments inoculation of mycorrhizal; WW/ WS, only one root fraction subjected to water stress and both root compartments non- inoculation of mycorrhizal; WW/WW, well water and both root compartments non- inoculation of mycorrhizal. Note: vertical bars on each histogram represent means ± SE. ***, significant at p < 0.01; *, significant at p < 0.05; ns, not significant.

(Asrar et al., 2012).

AM fungi on plant physiology are not directly linked to the extent of AM root colonization (Bárzana et al., 2012). The higher WUEB in mycorrhizal compared with non-mycorrhizal plants grown under water stress conditions could indicate that AM fungi increased the ability of root to absorb soil moisture. Enhanced water conductivity has been attributed to area increase for water uptake produced by AM fungi hyphae in soil (Augé, 2001; Asrar et al., 2012). Moreover, the AM fungal plants in this study produced more root biomass, decreased ABA production, increased gs, pn and antioxidative defence at the leaf level. This might partially explain why mycorrhizal plants had higher WUEB than the non-mycorrhizal plants

4.2. AM fungi inoculation increased antioxidant defense and reduced proline and MDA content In order to tolerate drought stress and improve the WUEB, the plants accumulate a high concentration of low molecular-mass organic solutes such as proline to regulate the osmotic potential of cells aiming at improving absorption of water under drought stress (Zhang et al., 2010). Our study indicated that the leaves of mycorrhizal plants had lower levels of proline, so mycorrhizas could improve plant’s resistance 7

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4.3. AM fungal inoculation decreased drought-induced ABA accumulation, reduced gs and soil moisture threshold range of non-hydraulic root signalling Under water stressed, mycorrhizal plants showed higher levels of gs, pn and RWC than did non-mycorrhizal plants, which could be related to the maintenance of root biomass (Morte et al., 2001), increased the leaf area (Liu et al., 2006) and the increase of the water absorbed in stressed mycorrhizal plants. In this sense, many authors (Nelsen and Safir, 1982; Augé et al., 1986) have reported that AM fungi can affect the stomatal behaviour of host plants under well-watered conditions with these effects being often associated with an altered root or whole plant hydraulic conductivity or an increased water uptake by extraradical hyphae (Faber et al., 1991), resulting in changes in leaf physiology and changes in biochemical properties (Augé, 2001; Barros et al., 2018). Duan et al. (1996) and Augé et al. (1986) found high values of leaf gs, pn and RWC in inoculated water-stressed plants due to a high rate of water absorption. On the other hand, Subramanian et al. (1995) found higher values of pn and leaf water content in AM water stressed plants than in non-mycorrhizal stressed plants. In our experiment, gs and pn decreased earlier in non-mycorrhizal plants than mycorrhizal plants, and mycorrhizal plants exhibited the higher gs, pn and RWC than in non-mycorrhizal plants in the late experimental period under progress soil drying. While it is notable that inoculation with AM fungi not affected the gs, pn and RWC under partial root drying, compared with the non-mycorrhizal plants during the experimental period. Therefore, the amount of water given to the wet side was important in maintaining a high leaf RWC in partial root drying (Liu et al., 2006). However, inoculation with AM fungi postponed the decline the gs and pn, and nonmycorrhizal plant significantly decreased the RWC at the end of experiment. In our study, stomatal conductance decreased steadily with the decreasing soil water content of drying treatment. The chemical signals, mainly ABA, produced in the root tips are most likely to have caused stomatal closure (Liu et al., 2006). ABA involvement in signaling and control of stomatal closure also has been proposed as one of the possible non-nutritional explanations of the mycorrhizal promotion effect on drought-stressed plants. Increasing intensity of drought stress was also marked by a gradually rising content of ABA in leaf tissues (Wang et al., 2017). Accumulation of ABA is a signal for initialization of adaptive mechanisms against drought, indicating root sensitivity to soil water status and stomatal closure (Sauter et al., 2001). At low soil water contents, mycorrhizal plants maintained lower ABA concentration than non-mycorrhizal plants (Ruiz-Lozano and Aroca, 2010). It is noteworthy that ABA of HRS was lower than that observed of nHRS in the progressive soil drying. Previous research showed that ABA in the leaf continued to increase as the soil dried (Du et al., 2012).The method for measure ABA concentration may be critical. In our study, we using an older ELISA technique rather than more modern analytical methods. On the other hand, a possible explanation for the ABA was lower in HRS is that when the soil drying is prolonged and becomes more severe, more ABA concentration was accumulated in the older leaves, which wilted earlier than the younger leaves, the samples were from the young leaves (Zhang et al., 2006). Moreover, mycorrhizal plants exhibited the higher ABA concentration in well-watered condition than in water stress under partial root drying. Our results showed that AMF colonization postponed triggering of the non-hydraulic root signal (nHRS), so the soil water content was lower when the nHRS trigger. Ultimately the decrease in sap flow from partially dry roots as the soil dries will limit ABA transport to the shoot, and the leaf ABA accumulation of WW/WS + AMF was lower than mycorrhizal well-watered plants. (Dodd et al., 2008). Considering the declined of ABA concentration induced by inoculation with AM fungi, the results suggest that the AM fungi decreased the sensitivity of stomatal to ABA. Some studies have noted that higher gs characters were associated with lower ABA and in AM fungi plants at low water contents (Ruiz-Lozano and Aroca, 2010; Duan et al.,

Fig. 6. A model figure displaying the impact of the inoculation with arbuscular mycorrhizal (AM) fungi on plants during the response to drought. During the soil drying, arbuscular mycorrhizal fungi inoculation can improve peroxidase (POD) and superoxide dismutase (SOD) activity, the malondialdehyde (MDA), proline (OA) and abscisic acid (ABA) will be reduced with AM fungal inoculation. Meanwhile, AM fungal colonization postponed trigger the non-hydraulic root signal (nHRS). As a consequence of all of these factors, water use efficiency for biomass and root and shoot biomass is enhanced when plants are inoculation with AM fungi. The sizes of the arrows indicate the direction of change (i.e., increased, decreased), but they are not drawn to scale.

to drought stress and reduce the proline content (Tang et al., 2009). At same the time, AM fungal-treated maize had a lower content of MDA in the leaves under drought stress, indicating that AM fungal infection might alleviate or decrease lipid damage, at severe water shortage (Abbaspour et al., 2012). These results may be attributed to either greater drought resistance of AM fungal plants or less injury in mycorrhizal plants grown under drought stress conditions (Wu and Xia, 2006). It is well documented that water deficit in plants increases the concentration of free radicals in cells, resulting in oxidative stress (RuizLozano, 2003; Essahibi et al., 2018). Plants possess a number of antioxidant mechanisms (including enzymatic antioxidant) to protect themselves against the production of reactive oxygen species. Huang et al. (2011) reported that AM fungi may increase the drought resistance of plants by promoting antioxidant enzymes such as POD and SOD in plants under water stress. But previous reports have indicated that the formation of mycorrhizas could reduce the activity of POD (Liu et al., 2007). In the present study, POD and SOD activities of maize inoculated with Glomus monosporum was increased significantly compared to non-inoculated plants, when the host plants were under progressive soil drying. Two possibilities may suggested to explain the low oxidative damage found in the mycorrhizal plants: (1) mycorrhizal colonisation can induce gene expression or enhance the activities of a set of defense enzymes, especially POD and SOD, involved in the elimination of reactive oxygen species and (2) plants suffered less drought stress due to a primary drought avoidance effect by the symbiosis (e.g. by direct water uptake by fungal hyphae and transfer to the host plant, which has a direct effect on the RWC) which kept plants protected against the generation of reactive oxygen species (Abbaspour et al., 2012). In our study, although the RWC was improved by the AMF inoculation, while AM fungi acted as a role of amplifying effects of POD and SOD activity. In contrast to progressive soil drying, while the POD and SOD activities were not significantly increased when the maize was under partial root drying. Increased water uptake from roots in comparatively wet soil may contribute to the maintenance of RWC (Dodd et al., 2008), which would reduce the effect of AM inoculation.

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Technology Plan Funding of Gansu Province (18JR3RA254), Chinese Academy of Sciences “Western Light”, and Project Open Program of Chinese National Key Laboratory of Forest Genetics and Breeding (TGB2018001), Application Development Project of Gansu Province (2018JK-15).

1996). Thus, non-mycorrhizal treatments did not significantly alter the leaf RWC at which the gs declined firstly, the soil water threshold range of nHRS was broadened from 22.7% FWC to 38.8% FWC observed in non-mycorrhizal plants. Previous research showed that the threshold range of nHRS was associated with increased drought resistance (Wang et al., 2008), but this has not been previously reported in maize with inoculation AM fungal. However, in the present study, the nHRS did not appear in the inoculated plants under partial root-zone drying. Increased water uptake from roots in comparatively wet soil may contribute to the maintenance of leaf RWC (Liu et al., 2006), the gs and pn in partial root-zone drying were higher than that of progressive soil drying. On the other hand, AM fungal inoculation reduced the ABA concentration. Thus, nHRS was not triggered until the HRS appeared. The results in the present study show that the enhancement of drought resistance and WUEB was associated with a narrow threshold range of nHRS with mycorrhizal treatment. At the same time, previous reports have indicated that AM fungi can trigger the root signal in advance (Augé and Duan, 1991), but we found out that AM fungal colonization greatly reduced the leaf ABA concentration, then postpone declines in pn, gs, and narrowed the threshold range of nHRS.

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5. Conclusion Under the water stress condition in the two experiments, AM inoculation greatly reduced the leaf ABA concentration, and then postponed a decline in photosynthetic rate and stomatal conductance in Zea mays L. plants. Furthermore, inoculation with AM fungi decreased ABA accumulation and then reduced generation of reactive oxygen species, meanwhile, increased antioxidant enzymes activities. This led to the stimulation of plant growth as reflected mainly in biomass and WUEB. Author contribution Aitian Ren and Ying Zhu had the main responsibility for data collection, analysis and writing, Jiyuan Li had a significant contribution to data analysis, Hongxu Ren had a significant contribution to the interpretation of data and manuscript preparation, Lynette Kay Abbott and Yinglong Chen assisted in revising the manuscript, and Youcai Xiong (the corresponding author) had the overall responsibility for experimental design and project management. Author statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Aitian Ren and Ying Zhu had the main responsibility for data collection, analysis and writing, Jiyuan Li had a significant contribution to data analysis, Hongxu Ren had a significant contribution to the interpretation of data and manuscript preparation, Lynette Kay Abbott and Yinglong Chen assisted in revising the manuscript, and Youcai Xiong (the corresponding author) had the overall responsibility for experimental design and project management. Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by the Natural Science Foundation of China (31570415), State Technology Support Program (2015BAD22B04), National Specialized Support Plan for Outstanding Talents (“Ten Thousand People Plan”), Overseas Masters Program of Ministry of Education (Ms2011LZDX059), Basic Research Innovation Group Project of Gansu Province (1606RJIA325), Innovation Team Project of Gansu Academy of Sciences (2019CX004-01), Science and 9

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