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Article

Effect of Pisolithus tinctorious on Physiological and Hormonal Traits in Cistus Plants to Water Deficit: Relationships among Water Status, Photosynthetic Activity and Plant Quality

by
Beatriz Lorente
,
Inés Zugasti
,
María Jesús Sánchez-Blanco
,
Emilio Nicolás
and
María Fernanda Ortuño
*
Centro de Edafología y Biología Aplicada del Segura (CEBAS-CSIC), Department of Irrigation, P.O. Box 164, 30100 Espinardo-Murcia, Spain
*
Author to whom correspondence should be addressed.
Plants 2021, 10(5), 976; https://doi.org/10.3390/plants10050976
Submission received: 12 April 2021 / Revised: 25 April 2021 / Accepted: 10 May 2021 / Published: 13 May 2021
(This article belongs to the Special Issue Plant Metabolites and Regulation under Environmental Stress)
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Abstract

:
Cistus species can form ectomycorrhizae and arbuscular mycorrhizal fungus that can bring benefits when plants are under water stress conditions. However, the application of some ectomycorrhizae on the water uptake under drought through physiological traits and hormonal regulation is less known. The experiment was performed during three months in a growth chamber with Cistus albidus plants in which the combined effect of the ectomycorrhiza Pisolithus tinctorious inoculation and two irrigation treatments (control and water-stressed plants) were applied. Irrigation absence caused significant decrease in aerial growth and tended to decrease soil water potential at the root surface, leading to a decrease in leaf water potential. Under these conditions, the abscisic acid and salicylic acid content increased while the precursor of ethylene decreased. Although the mycorrhization percentages were not high, the inoculation of P. tinctorious improved the water status and slightly cushioned the rise in leaf temperature of water-stressed plants. The ectomycorrhiza decreased the scopoletin values in leaves of plants subjected to deficit irrigation, indicating that inoculated plants had been able to synthesize defense mechanisms. Therefore, Pisolithus tinctorious alleviated some of the harmful effects of water scarcity in Cistus plants, being its use a sustainable option in gardening or restoration projects.

1. Introduction

Cistus albidus L. is a typical shrub of the Mediterranean climate. They are widely cultivated both for reforestation and for its use in Mediterranean gardening, especially in Xeriscape, where water management for saving this resource is one of the best options. These plants began to be widely used in gardening and landscaping due to their rusticity and attractive blooms.
Cistus albidus responds to water deficit by developing avoidance mechanisms for the regulation of transpiration based on stomatal closure, reduction of the leaf area and epinastia [1]. Furthermore, tolerance to water stress can be explained by other functional and structural adaptations of plants, such as osmotic adjustment, changes in the elasticity of the cell wall, and mineral and hormonal balance [2].
It is widely known that mycorrhizae confer physiological characteristics to plants that help them improve their water and nutritional status, especially in adverse conditions such as water stress. However, the response of these plants will depend on both the species of the plant and the type of fungus with which it is inoculated, as well as the extension, duration, and type of stress [3,4].
Cistus species can form both ectomycorrhizae (ECM) and arbuscular mycorrhizal fungus (AMF); they are dualistic plants [5,6,7]. The dual mycorrhization state is also present in other species such as Populus, Salix, Eucalyptus [5,6]. In an earlier experiment we studied whether an AMF, Glomus iranicum var. Tenuihypharum, despite being a genus (Cistus) that generally forms ectomycorrhizae, could bring benefits, especially when plants are under water stress conditions. In that experiment, mycorrhizae helped improve the water status of the plant when irrigation was removed, as demonstrated by the less negative leaf water potential values; it also minimized the decrease in chlorophyll levels, showing plants with higher ornamental value and increased in quantum yield (Y(II)) and photochemical quenching (qP) were related to the greater photosynthetic efficiency recorded [8].
Several studies have shown beneficial effects of ectomycorrhizal symbioses on the performance of tree species under drought stress [9]. For example, seedlings of Picea colonized by ECM fungi exhibited increased stomatal conductance and photosynthesis, shoot water potential and growth compared to non-mycorrhizal plants [10]. These ameliorative effects have, at least partly, been ascribed to the increasing in the absorbing surface area and the exploration of larger soil volumes by the extramatrical mycelia, as well as to the role of aquaporins which function is related to post-translational regulation and to the coordination of the phytohormones [11,12]. Transport of water and nutrient uptake through hyphae to host plants improves plant water and nutrient supply improving the performance of plants during periods of drought stress [9]. Furthermore, drought-stressed ECM-colonized plants show increased hydraulic conductance compared with non-mycorrhizal plants [13,14]. Concretely, Pisolithus tinctorius is one of the most widespread ECM fungi and can establish associations with a broad variety of species and has been recorded in a wide range of habitats [15]. P. tinctorius has great mycorrhizal capacity and provides the plant with greater efficiency in the absorption of water and nutrients [16], so it has been used in reforestation program sand and in commercial ECM inoculum production [17].
In general, mycorrhizal fungi plays an important role in regulating phytohormones such as jasmonic acid (JA), salicylic acid (SA), and ethylene (ET), which are involved in the host defense response, and abscisic acid (ABA), gibberellin (GA), and citokinin (CK), which modulate the growth of the plants. However, while the role of hormones in AMF-inoculated plants is widely known [18,19], less is known in ECM-inoculated plants, although, fundamentally, studies of the effect of ABA and SA in ECM-inoculated plants under adverse conditions have been carry out. For example, Rincón et al. [20] evaluated whether two ectomycorrhizal fungi (Laccaria bicolorthe and Cenococcum geophilum) affected ABA production in larch during osmotic stress and Luo et al. [21] determined the phytohormone changes in plants inoculated with Paxillus involutus under salinity. However, in the case of P. tinctorius, the relationships between water status and hormonal production in plants subjected to water stress is less known.
For all the above, in this study we examine how the interaction of Cistus albidus with the ectomycorrhizal fungus Pisolithus tinctorious affects water uptake under drought through physiological traits and hormonal regulation. The objectives of the study were (i) to study physiological and hormonal response to water deficit; (ii) to evaluate the effect of P. tinctorious on physiological traits and hormonal regulation of Cistus plants; and (iii) to analyze the changes in the relationships among physiological, morphological, and ornamental parameters under water deficit and mycorrhizal inoculation. Comparative data relating these physiological processes to water stress may prove beneficial toward understanding the drought tolerance of plants.

2. Results

2.1. Mycorrhization Percentage and Growth

At the end of the experiment, mycorrhizal inoculation produced a colonization of around 30% in root systems of well-irrigated Cistus plants. The level of colonization in roots of mycorrhizal plants decreased near to 20% with the suppression of the irrigation (Table 1). In both cases, the mycorrhization percentages were not high.
Water stress caused significant decrease in aerial growth parameters such as height and leaf DW, being the latter the one that decreases the most (30% respect to C) (Table 2). The inoculation of Pisolithus tinctorious increased almost all growth parameters for both irrigation conditions, as observed when we compare CM with C and WSM with WS. Indeed, inoculated plants of both irrigation treatments (CM and WSM) were taller and their leaf and root DW were higher than those corresponding to non-inoculated plants (C and WS) (Table 2).

2.2. Leaf Mineral Ontent

Water stress had a significant effect on leaf mineral content decreasing almost the entire mineral measured, when comparing C and WS, having a highly significant effect in Ca, Na, Mg, and Cu. The effect of the mycorrhiza treatment was hardly significant. Under well-watered conditions, only the leaf content of Ca and S in CM plants increased their values both in around 30% respect C plants (Table 3). Under water stress conditions, ECM produced a slight tendency to decrease almost all the elements of WSM plants respect to WS (Table 3).

2.3. Water Relations

Soil water potential at the root surface (Ψr) tended to decrease in WS plants due to water stress, causing greater resistance to water absorption, which leads to a decrease in leaf water potential. No differences were observed due to the effect of mycorrhizae (Figure 1A).
In well-watered plants, both inoculated and non-inoculated (CM and C, respectively), the values of leaf water potential (Ψl) remained at −1.0 MPa, showing a good water status of the plants (Figure 1B). When stress was applied, non-inoculated plants (WS) decreased Ψl significantly, reaching minimum values of −3.0 MPa. The use of Pisolithus tinctorius alleviated significantly the negative effect of water stress and the fall in values was not so severe (−2.1 MPa) (Figure 1B).
Figure 1C shows the contribution to dehydration to change in osmotic water potential (ΔΨSS). Under well-watered conditions, ΔΨSS was almost negligible for both inoculated and non-inoculated plants (C and CM, respectively). Water stress caused a decrease in ΔΨSS, reaching minimum values of around −0.27 MPa in non-inoculated plants (WS), while the ECM dampened this drop (−0.12 MPa).

2.4. Gas Exchange

Under water stress conditions, the stomatal conductance (gs) fell drastically, almost reaching the stomatal closure in WS plants. Differences of 150 mmol m−2 s−1 between C and WS treatments were observed (Figure 2A). Pisolithus tinctorious did not favor opening in either case, neither under conditions of good irrigation nor under water deficit (CM and WSM). Net photosynthesis (Pn) showed a behavior very similar to that of gs (Figure 2B). Leaf temperature (Tl) of plants subjected to water stress (WS) was 1.5 °C higher than C plants (Figure 2C).

2.5. Phytohormones

At the end of the experiment period, the abscisic acid (ABA) and salicylic acid (SA) content in WS leaves were approximately 5 and 3 times as high as those in control leaves, respectively (Figure 3A,B). By contrast, the precursor of ethylene (ACC) content was decreased significantly by the irrigation suppression (45% of C) (Figure 3C). Mycorrhizae only had a significant effect on scopoletin (SC) under water stress conditions (WSM), significantly decreasing their values by approximately 46% respect to non-inoculated plants (WS) (Figure 3D).

2.6. Relationships between Physiological, Morphological and Ornamental Parameters

The relationship between gs and Pn was well represented by an exponential function when all the values of the four treatments obtained during the experimental period were considered (Figure 4A). Highly significant linear regressions between gs versus Pn can be obtained when considering control treatments (C and CM) and water stress treatments (WS and WSM) individually, suggesting that gs explain differences in Pn within each irrigation treatment. A strong correlation (R2 = 0.86) between gs and leaf water potential (Ψl) was observed, fitting with an exponential sigmoid model. Ψl of plants subjected to water stress (WS and WSM) declined sharply (variation between −1.0 and −3.2 MPa) in a narrow range of gs (Figure 4B). Relationship between Tl and gs fits with an exponential decay model. Leaf temperature was maintained between 23.3 and 24.3 °C when the stomatal conductance exceeded approximately 65 mmol m−2 s−1 (Figure 4C).
With increasing water stress, the dependence of Ψl and Tl on gs increased, as well as the dependence of the relative chlorophyll content (RCC) on Pn. WS as compared to C promoted intrinsic water use efficiency (WUE, Pn/gs). However, Pn was less dependent on shoot dry weight when the plants were subjected to irrigation suppression (Table 4). Under water stress conditions, Pisolithus tinctorious only had effect on the relation between RCC and Pn, being WSM higher than WS (Table 4).

3. Discussion

The percentage of mycorrhization of Pisolithus tinctorius was not excessively high, despite being an ectomycorrhiza, which has special relevance in this genus with high mycorrhizal capacity [6]. In our conditions, the cistus seedlings were cultivated in pots, which prevented the extension of the fungal mycelium and of the ECM roots. Imposed water stress meant a reduction in the percentage of mycorrhization, like plants, fungi are also affected by water limitation [22]. Studies of dually colonized plant species such as members of the genus Cistus indicate that ECM may be more sensitive to drought than AMF [23,24]. In fact, in a previous study with AMF (Glomus iranicum var. tenuihypharum) in Cistus albidus plants under water stress [8], the percentage of mycorrhization was 65%, more than double that obtained with Pisolithus tinctorius.
Drought is one of the main adverse factors for seasonal plants, especially for plants grown in pots [1,25,26]. At the end of the experiment, water stress caused a decrease in leaf dry weight, which could be interpreted as a conservative strategy to reduce transpiration and maintain hydraulic conductivity under water depletion [27,28,29]. Despite the low percentage of mycorrhization, ECM improved both shoot and root growth of the plants under well-irrigated conditions. Furthermore, unlike what occurred in Cistus plants inoculated with AMF [8], Pisolithus tinctorious increased root growth under water stress. Usually, more root branching has been found in ECM species than AMF, what is related to higher ability to absorb more water and nutrient, improving plant water relations under low water conditions [30,31,32,33,34].
Leaf water potential and actual osmotic potential are good indicators of water stress [35,36]. In our study, plants subjected to water stress showed no osmotic adjustment, perhaps, the speed of the development of water stress and the low inorganic solutes accumulation, such as potassium and sodium, could not contribute to osmo-regulation [8,37]. The fact that ΔΨSS at the end of the experiment in control treatment were almost null indicated that dehydration was the major mechanism involved in ΔΨSS, while the more noteworthy ΔΨSS in WS treatment seems to indicate that the dehydration could be produced by the transpiration [38]. Soil water potential at the root surface (Ψr) reflects the accumulation of net solutes allowing reduction of Ψl (around −3.0 MPa), in order to guarantee water transport to the leaves [37,39,40,41]. Concerning ECM effect, it improved the plant water status under stress conditions [42]. This ameliorative effect has been ascribed to the extended external mycelia of the root systems of ECM fungi, which reach soil pores inaccessible to the roots in water [5,43,44].
As a consequence of the reduction of the substrate water potential at the root surface and the leaf water potential in plants subjected to water stress, the stomatal conductance decreased drastically, acting as a mechanism to prevent excessive loss of water [25,26,45,46,47]. In our conditions, the relationship between gs and Ψl was adjusted to an exponential curve, so that under water-stress plants exhibited progressively lower Ψl, but almost no change in gs. Likewise, it has been well established that plants regulate rates of transpiration and photosynthesis in parallel, maintaining a balance between gs and Pn [48]. A strong correlation between stomatal conductance with net photosynthesis observed in the current study appears to reflect the gas exchange limitation of photosynthesis [49,50,51]. At the end of the experiment, the imposed water stress induced an increase in intrinsic WUE, which is in agreement with Costa et al. [46], who reported that deficit irrigation strategies can be successfully applied to different crops, in order to improve water savings. Water deficit by closing stomata, causes increasing leaf temperature, considering the major determinant of leaf temperature is the rate of evaporation or transpiration from the leaf [52,53,54]. This behavior was observed at the end of our experiment.
ABA synthesis is one of the fastest responses of plants to abiotic stress causing stomatal closure. In addition, SA is involved in the regulation of drought responses [55], inducing the generation of reactive oxygen species (ROS) and, consequently, causing the stomatal closure [56]. In our experiment, endogenous ABA and SA levels in stressed plants increased up to of six and almost twofold than those of control plants, respectively, while endogenous 1-aminocyclopropane1-carboxylic acid (ACC) levels in leaves decreased. ACC is the precursor for the production of ethylene and under drought, a close relationship between ACC expression and ethylene synthesis has been demonstrated [57]. In these conditions, ACC levels decrease and, therefore, plant growth is reduced. However, some studies suggest that ABA and ethylene are antagonists and have described decreases in ethylene production as a consequence of an increased concentration of ABA in water stressed plants, regulating some drought responses in plants, such as root and leaf growth [58,59,60,61,62]. This is in accordance with our results, which show that in conjunction with a decrease in stomatal conductance and leaf water potential, ABA in leaves increased, while ethylene decreased, increasing root growth.
Hormonal profiles are also altered to alleviate the negative effects of water stress on mycorrhizated plants [32,63,64,65]. Several studies confirmed that inoculation with mycorrhizal fungi results in decreasing the endogenous ABA and SA [20,66]. However, in our assay ECM treatments (CM and WSM) had not significant changes in any of both leaf hormones neither under well-watered nor water stress conditions. Since ABA is known to induce stomatal closure [67], its lower concentration in ECM plants could play a role in the stomatal conductance, allowing no increase in net CO2 assimilation rate in ECM inoculated plants [17]. This fact leads no significant improvement in intrinsic water use efficiency (Pn/gs).
Nevertheless, ECM significantly decreased leaf scopoletin (SC). Scopoletin (6-methoxy-7-hydroxycoumarin) is a typical phytoalexin which is an important secondary metabolite synthesized in plants as a defense mechanism against various environmental stresses [68]. Its synthesis is activated once some kind of infection has occurred in plants, but it can also be triggered due to various types of abiotic stresses. Scopoletin has also been found in many other plant species (e.g., Solanaceae, such as tobacco or potato, and sunflower, among others), showing antifungal and antibacterial activity [68,69,70]. Its accumulation has been correlated with resistance to microbial attack and other stresses, as well as mechanical damage and dehydration [71]. Furthermore, it appears to be the product that most increases in concentration in infected plants compared to other coumarins and coumaric glycosides, such as scopolin, esculetin, and esculin [72,73,74]. In our study, SC decreased significantly in mychorrhizal plants indicating that ECM plants have been able to synthesize defense mechanisms against abiotic and biotic stresses against non-mycorrhizal plants, and therefore SC is lower in ECM plants.
In conclusion, the mechanism of C. albidus to avoid water stress was related to its ability to decrease aerial growth and to modify leaf gas exchange, increasing water use efficiency. Water stress positively stimulated the levels ABA and SA, who strongly enhanced drought tolerance. On the other hand, despite the low percentages of mycorrhization, Pisolithus tinctorious inoculation in addition to improving plant growth, and slightly gas exchange and quality plant parameters, it improved the water status of Cistus under water stress conditions, probably due to a decrease in SC values, suggesting that the selected ECM can alleviate some of the harmful effects of water scarcity. Therefore, the use of Cistus plants inoculated with Pisolithus tinctorious in gardening or restoration projects can be a sustainable economic and environmental option.

4. Materials and Methods

4.1. Plant Material and Experimental Conditions

Forty-eight plants of Cistus albidus L. from the nursery were used. On January 18, 2017, these plants were transplanted into 1.5 L pots with a commercial substrate based on Sphagnum peat, coconut fiber and perlite in an 8:7:1 ratio and were placed in a controlled growth chamber. The climatic conditions of the chamber were those necessary for optimal growth: temperature (23 °C/18 °C, day/night), photosynthetic photon flux density (350 μmol m−2 s−1), photoperiod (16h/8h, light/dark), and relative humidity (RH) (60%). Plants were watered at field capacity.

4.2. Treatments

After two weeks of acclimatization, half of the plants (24) were inoculated with the ectomycorrhizal fungus Pisolithus tinctorius. Two months later, once the plants were stabilized, the irrigation of half of inoculated and non-inoculated plants were removed for a month, while the remaining plants continued to be irrigated at field capacity. Thus, the following treatments were established: C, Control, well-watered plants; CM, well-watered and inoculated plants; WS, plants subjected to withholding; WSM, inoculated plants subjected to water stress. Each treatment included three replications. Each replication consisted of four plants. The experiment lasted three months.

4.3. Fungal Colonization

At the end of the experiment, roots samples with the surrounding rhizosphere soil were collected to treat to evaluate fungal development. Three root samples were used in each replication. The percentage of mycorrhizal root colonization was estimated as following: Once cleaned, the roots were immersed in KOH (100 °C for 7 min), followed by a bath in H2O2 (100 °C, 5–6 min) and finally, trypan blue staining (4 min). The percentage of colonization was calculated using the methodology proposed by Kormanik and McGraw [75]. The colored roots were placed on specialized plates for counting and were observed under the magnifying glass by counting 100 fields neatly (positive and negative). The percentage of colonization was calculated using the following formula proposed by Sieverding [76]:
%colonization = (number of colonized fields/total number of fields observed) × 100

4.4. Leaf Mineral Content

At the end of the experimental period, the inorganic mineral content of dry leaves was determined in three plants per replication by means of emission spectrophotometry. The nutrient concentrations were determined in a digestion extract with HNO3:HClO4 (2:1, v/v) by Inductively Coupled Plasma optical emission spectrometer (ICP-OES IRIS INTREPID II XDL, Thermo Fisher Scientific Inc., Loughborough, UK).

4.5. Biomass and Height

At the end of the experiment, three plants per replication were selected and all the substrate was gently washed from their roots. After washing the substrate from the roots, the plants were individually separated into leaves, stems and roots and the fresh weight of each organ was determined. After that, each sample was dried in an oven at 80 °C, until samples reached a constant weight, and the dry weights (DW) were obtained.
Every 15 days, the height of three plants per replication was measured. Plant height was measured from the base of the plant at the substrate surface to the most distal growth.

4.6. Water Relations

Leaf water potential (Ψl), leaf osmotic potential (Ψs) and osmotic water potential at full turgor (Ψ100s) were determined in three plants per replication throughout the experimental period. The leaf water potential was determined during light hours according to the technique described by Scholander et al. [77], using a pressure chamber (Model 3000; Soil Moisture Equipment Co., Santa Barbara, CA, USA), the leaves were pressurized at a rate of 0.03 MPa s-1. Ψs and Ψ100s was measured using a Wescor 5520 vapor pressure osmometer (Wescor Inc., Logan, UT, USA), according to Gucci et al. (1991) [78]. For Ψ100s the leaf samples were previously subjected to a rehydration treatment by dipping their petioles in distilled water for 24 h to achieve complete saturation.
Changes in Ψs (ΔΨs) and in Ψ100S (ΔΨ100S) were calculated according to Girma and Krieg [79] as the difference in Ψs and Ψ100S measured at the initial (i) and at the end (e) of water stress period.
ΔΨS = (Ψs)i − (Ψs)e
ΔΨ100S = (Ψ100S)i − (Ψ100S)e
The contribution of dehydration to changes of ΨS (ΔΨSS) was calculated using the following equation:
ΔΨSS = ΔΨS − ΔΨ100S
Soil water potential at the soil-root interface (Ψr) was computed according to Jones (1983) [80]:
Ψr = (ΨlWS − ΨlC) × gsWS/gsC
where, ΨlWS and ΨlC correspond to the mean value of the leaf water potential in the WS and C treatments, respectively, and the gsWS and gsC correspond to the mean value of the respective treatments. A value of Ψr was calculated for each of the three drought pots using the value of ΨlC and gsC. The Ψr is assumed to be zero for control plants.

4.7. Gas Exchange and Thermography

Stomatal conductance (gs) and net photosynthesis rate (Pn) were measured with the LI-COR 6400 portable meter (LI-COR Inc., Lincoln, NE, USA). The flow rate of circulating air within the system was approximately 200 mmol s−1, with a leaf vapour pressure deficit to air of about 2 KPa. The CO2 concentration was fixed at 380 ppm and the photosynthetically active radiation (PAR) at 600 µmol m−2 s−1. The measurements were carried out in three plants per replication on the same days as the water relations.
At the same time and in the same plants that gas exchange was determined, leaf temperature (Tl) was measured with an infrared camera (FLIR-e50 System, Inc., Danderyd, Sweden) which consisted of a 240 × 180 pixels line scan imager operating in the 7.5e13 mm region, with a noise equivalent temperature difference of 0.05 °C at 30 °C and an accuracy of 2 °C or 2% of the reading. The background temperature, distance of the camera from the canopy, air temperature, emissivity, and relative humidity were used as input at the start of each series of measurements; so, the camera automatically corrects for atmospheric transmission based on these data. Background temperature was determined as the temperature of a crumpled sheet of aluminium foil in a similar position to the leaves of interest with the emissivity set at 1.0. Emissivity for leaf measurements was set at 0.96 [81] and the distance at which images were taken was 0.5 m. The images were processed with ThermaCam Researcher Professional 2.10 software (FLIR Quick Report, Danderyd, Sweden).

4.8. Relative Chlorophyll Content

The relative chlorophyll content (RCC) was determined periodically in three plants per replication with a Minolta SPAD-502 chlorophyll meter (Konica Minolta Sensing Inc., Osaka, Japan), a non-destructive method.

4.9. Hormonal Determination

The extraction and analysis of the plant hormones were developed in agreement with Albacete et al. [82] including slight modifications in the protocol, such as the measurement of scopoletin (phytoalexin). Firstly, 100 mg of fresh plant material (leaf) was homogenized in liquid nitrogen and placed in 0.5 mL of cold (−20 °C) extraction mixture of methanol/water (80/20, v/v). The mix was centrifuged at 15,000 r.p.m. (20,627× g), 4 °C for 15 min. The supernatants were kept at 4 °C and the remaining plant material (pellet) was re-extracted with additional 0.5 mL of the same extraction solution. The mix was re-centrifuged at 15,000 r.p.m. (20,627× g), 4 °C for 15 min. Pooled supernatants were passed through a Chromafix C18 cartridge (previously activated with 3 mL methanol/water (80/20, v/v) to remove interfering lipids and plant pigments and evaporated to dryness (3 h, approximately). The residue was dissolved in 200 µL metanol/water (80/20, v/v), then sonicated during 8 min and filtered through Millex filters with 0.45 µm pore size nylon membrane (Millipore, Bedford, MA, USA). The final sample extracted was injected into an ultra-high-performance liquid chromatography (UHPLC) coupled triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS).
The separation of plant hormones and phytoalexins was developed with slight modifications in accordance with Albacete et al. [82] by using UHPLC coupled to a 6460 triple quadrupole mass spectrometer (Agilent Technologies, Waldbronn, Germany), and a BEH C18 column (2.1 × 50 mm, 1.7 µm) (Waters, Milford, MA, USA) with a guard column (2.1 × 5.0 mm, 1.7 µm). The column temperature was 40 °C. Water/acetic acid (99.99:0.01, v:v) (solvent A) and acetonitrile (solvent B) were used as mobile phases at the flow rate of 0.2 mL min−1. The injection volume was 10 µL. The gradient program used was: 19% B at 0 min, 90% B at 2.5 min, 90% B at 4.5 min, 19.0% B at 6 min, and 19% B at 8.0 min for column equilibration. The electrospray interface (ESI) was set up in the negative and positive mode and the mass spectrometry analysis were run in the multiple reaction monitoring modes (MRM). The ionization and fragmentation conditions were as follows: gas temperature 325 °C, gas flow 8 L/min, nebulizer 45 psi, sheath gas temperature 375 °C, jet stream gas flow 11 L/min, capillary voltage 4000 V and 2750 V (positive and negative mode, respectively), and nozzle voltage 1000 V and 1500 V (positive and negative mode, respectively) according to the most abundant product-ions. The quantitative evaluation of plant hormones and scopoletin was carried out using authentic standards. The standards used are included in Table 5 and were diluted in water/methanol (20:80, v/v) for the quantification curves.

4.10. Statistical Analysis

In the experiment, 48 plants were randomly attributed to each treatment, with three replications for each treatment. The data were analysed by one-way ANOVA using Statistical Package for the Social Sciences (IBM SPSS Statistics 26 for Windows, CA, USA). Treatment means were separated with Duncan’s Multiple Range Test (p ≤ 0.05). Prior to the statistical analysis, percentage of root colonization was subjected to an arcsine square-root transformation to ensure the homogeneity of the variance. Relationships between parameters were fitted to different regressions using SigmaPlot v. 14.5 software (SPSS Inc., Chicago, IL, USA).

Author Contributions

Conceptualization, M.F.O.; Data curation, B.L.; Formal analysis, B.L.; Funding acquisition, M.J.S.-B. and M.F.O.; Investigation, I.Z. and E.N.; Methodology, I.Z. and E.N.; Project administration, M.J.S.-B.; Resources, M.J.S.-B. and M.F.O.; Validation, E.N.; Visualization, B.L.; Writing—original draft, B.L. and M.F.O.; Writing—review and editing, M.J.S.-B. and M.F.O. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Seneca Foundation of Murcia, grant number 19903/GERM/15 and by the Ministry of Science, Innovation, and Universities of Spain, and the European Regional Development Fund, grant number RTI2018-093997-B-I00.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

  1. Sánchez-Blanco, M.J.; Álvarez, S.; Navarro, A.; Bañón, S. Changes in leaf water relations, gas exchange, growth and flowering quality in potted geranium plants irrigated with different water regimes. J. Plant. Physiol. 2009, 166, 467–476. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Álvarez, S.; Rodríguez, P.; Broetto, F.; Sánchez-Blanco, M.J. Long term responses and adaptive strategies of Pistacia lentiscus under moderate and severe deficit irrigation and salinity: Osmotic and elastic adjustment, growth, ion uptake and photosynthetic activity. Agric. Water Manag. 2018, 202, 253–262. [Google Scholar] [CrossRef] [Green Version]
  3. Ruiz-Lozano, J.; Azcón, R.; Gomez, M. Effects of arbuscular mycorrhizal Glomus species on drought tolerance: Physiological and nutritional plant responses. Appl. Environ. Microb. 1995, 61, 456–460. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Mardukhi, B.; Rejali, F.; Daei, G. Arbuscular mycorrhizas enhance nutrient uptake in different wheat genotypes at high salinity levels under field and greenhouse conditions. C. R. Biol. 2011, 334, 564–571. [Google Scholar] [CrossRef]
  5. Smith, S.; Read, D. Mycorrhizal Symbiosis, 3rd ed.; Academic Press: New York, NY, USA, 2008; ISBN 978-0-12-370526-6. [Google Scholar]
  6. Comandini, O.; Contu, M.; Rinaldi, A.C. An overview of Cistus ectomycorrhizal fungi. Mycorrhiza 2006, 16, 381–395. [Google Scholar] [CrossRef]
  7. Caravaca, F.; Alguacil, M.M.; Hernández, J.A.; Roldán, A. Involvement of antioxidant enzyme and nitrate reductase activities during water stress and recovery of mycorrhizal Myrtus communis and Phillyrea angustifolia plants. Plant. Sci. 2005, 169, 191–197. [Google Scholar] [CrossRef]
  8. Ortuño, M.F.; Lorente, B.; Hernández, J.A.; Sánchez-Blanco, M.J. Combined effect of mycorrhizal inoculation and substrate on the plant- water relations, gas exchange, photosynthetic efficiency and nutrient content of Cistus albidus plants submitted to water stress. Braz. J. Bot. 2018, 41, 299–310. [Google Scholar] [CrossRef]
  9. Lehto, T.; Zwiazek, J.J. Ectomycorrhizas and water relations of trees: A review. Mycorrhiza 2011, 21, 71–90. [Google Scholar] [CrossRef]
  10. Lehto, T. Mycorrhizas and drought resistance of Picea sitchensis (bong) car. I. In conditions of nutrient deficiency. New Phytol. 1992, 122, 661–668. [Google Scholar] [CrossRef]
  11. Navarro-Ródenas, A.; Bárzana, G.; Nicolás, E.; Carra, A.; Schubert, A.; Morte, A. Expression analysis of aquaporins from desert truffle mycorrhizal symbiosis reveals a fine-tuned regulation under drought. Mol. Plant. Microbe Interact. 2013, 26, 1068–1078. [Google Scholar] [CrossRef] [Green Version]
  12. Xu, H.; Kemppainen, M.; El Kayal, W.; Lee, S.H.; Pardo, A.G.; Cooke, J.E.; Zwiazek, J.J. Overexpression of Laccaria bicolor aquaporin JQ585595 alters root water transport properties in ectomycorrhizal white spruce (Picea glauca) seedlings. New Phytol. 2015, 205, 757–770. [Google Scholar] [CrossRef]
  13. Muhsin, T.M.; Zwiazek, J.J. Colonization with Hebeloma crustuliniforme increases water conductance and limits shoot sodium uptake in white spruce (Picea glauca) seedlings. Plant. Soil 2002, 238, 217–225. [Google Scholar] [CrossRef]
  14. Bogeat-Triboulot, M.B.; Bartoli, F.; Garbaye, J.; Marmeisse, R.; Tagu, D. Fungal ectomycorrhizal community and drought affect root hydraulic properties and soil adherence to roots of Pinus pinaster seedlings. Plant. Soil 2004, 267, 213–223. [Google Scholar] [CrossRef]
  15. Carney, J.W.G.; Chambers, S.M. Interactions between Pisolithus tinctorius and its hosts: A review of current knowledge. Mycorrhiza 1997, 7, 117–131. [Google Scholar] [CrossRef]
  16. Marx, D.H.; Cordell, C.E. The use of specific ectomycorrhizas to improve artificial forestation practices. In Biotechnology of Fungi for Improving Plant Growth: Symposium of the British Mycological Society Held at the University of Sussex, United Kingdom, September 1988; Whipps, J.M., Lumsden, R.D., Eds.; Cambridge University Press: Cambridge, UK, 1989; pp. 1–25. [Google Scholar]
  17. Sebastiana, M.; Pereira, V.; Alcântara, A.; Pais, M.; da Silva, A. Ectomycorrhizal inoculation with Pisolithus tinctorius increases the performance of Quercus suber L. (cork oak) nursery and field seedlings. New For. 2013, 44, 937–949. [Google Scholar] [CrossRef]
  18. Quiroga, G.; Erice, G.; Aroca, R.; Zamarreño, A.M.; García-Mina, J.M.; Ruiz-Lozano, J.M. Arbuscular mycorrhizal symbiosis and salicylic acid regulate aquaporins and root hydraulic properties in maize plants subjected to drought. Agric. Water Manag. 2018, 202, 271–284. [Google Scholar] [CrossRef]
  19. Sánchez-Romera, B.; Calvo-Polanco, M.; Ruíz-Lozano, J.M.; Zamarreño, A.M.; Arbona, V.; García-Mina, J.M.; Gómez-Cadenas, A.; Aroca, R. Involvement of the def-1 mutation in the response of tomato plants to arbuscular mycorrhizal symbiosis under well-watered and drought conditions. Plant. Cell Physiol. 2018, 59, 248–261. [Google Scholar] [CrossRef] [Green Version]
  20. Rincón, A.; Priha, O.; Lelu-Walter, M.A.; Bonnet, M.; Sotta, B.; Le Tacon, F. Shoot water status and ABA responses of transgenic hybrid larch Larix kempferi × L. decidua to ectomycorrhizal fungi and osmotic stress. Tree Physiol. 2005, 25, 1101–1108. [Google Scholar] [CrossRef] [Green Version]
  21. Luo, Z.B.; Janz, D.; Jiang, X.; Göbel, C.; Wildhagen, H.; Tan, Y.; Rennenberg, H.; Feussner, I.; Polle, A. Upgrading root physiology for stress tolerance by ectomycorrhizas: Insights from metabolite and transcriptional profiling into reprogramming for stress anticipation. Plant. Physiol. 2009, 151, 1902–1917. [Google Scholar] [CrossRef] [Green Version]
  22. Dell’Amico Rodríguez, J.M.; Torrecillas, A.; Rodríguez Hernández, P.; Morte, A.; Sánchez-Blanco, M.J. Responses of tomato plants associated with the arbuscular mycorrhizal fungus Glomus clarum during drought and recovery. J. Agric. Sci. 2002, 138, 387–393. [Google Scholar] [CrossRef]
  23. Gehring, C.A.; Mueller, R.C.; Whitham, T.G. Environmental and genetic effects on the formation of ectomycorrhizal and arbuscular mycorrhizal associations in cottonwoods. Oecologia 2006, 149, 158–164. [Google Scholar] [CrossRef] [PubMed]
  24. Querejeta, J.I.; Egerton-Warburton, L.M.; Allen, M.F. Topographic position modulates the mycorrhizal response of oak trees to interannual rainfall variability. Ecology 2009, 90, 649–662. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Álvarez, S.; Bañón, S.; Sánchez-Blanco, M.J. Regulated deficit irrigation in different phenological stages of potted geranium plants: Water consumption, water relations and ornamental quality. Acta Physiol. Plant. 2013, 35, 1257–1267. [Google Scholar] [CrossRef] [Green Version]
  26. Álvarez, S.; Gómez-Bellot, M.J.; Acosta-Motos, J.R.; Sánchez-Blanco, M.J. Application of deficit irrigation in Phillyrea angustifolia for landscaping purposes. Agric. Water Manag. 2019, 218, 193–202. [Google Scholar] [CrossRef]
  27. Limousin, J.M.; Rambal, S.; Ourcival, J.M.; Rocheteau, A.; Joffre, R.; Rodriguez-Cortina, R. Long-term transpiration change with rainfall decline in a Mediterranean Quercus ilex forest. Glob. Chang. Biol. 2009, 15, 2163–2175. [Google Scholar] [CrossRef]
  28. Barbeta, A.; Mejía-Chang, M.; Ogaya, R.; Voltas, J.; Dawson, T.E.; Peñuelas, J. The combined effects of a long-term experimental drought and an extreme drought on the use of plant-water sources in a Mediterranean forest. Glob. Chang. Biol. 2015, 21, 1213–1225. [Google Scholar] [CrossRef] [Green Version]
  29. Sánchez-Blanco, M.J.; Rodríguez, P.; Morales, M.A.; Ortuño, M.J.; Torrecillas, A. Comparative growth and water relations of Cistus albidus and Cistus monspeliensis plants during water deficit conditions and recovery. Plant. Sci. 2002, 162, 107–113. [Google Scholar] [CrossRef]
  30. Liese, R.; Alings, K.; Meier, I.C. Root branching is a leading root trait of the plant economics spectrum in temperate trees. Front. Plant. Sci. 2017, 8, 315. [Google Scholar] [CrossRef] [Green Version]
  31. Wu, Q.S.; Srivastava, A.K.; Zou, Y.N.; Malhotra, S.K. Mycorrhizas in citrus: Beyond soil fertility and plant nutrition. Indian J. Agric. Sci. 2017, 87, 427–432. [Google Scholar]
  32. Liu, C.Y.; Zhang, F.; Zhang, D.J.; Srivastava, A.K.; Wu, Q.S.; Zou, Y.N. Mycorrhiza stimulates root-hair growth and IAA synthesis and transport in trifoliate orange under drought stress. Sci. Rep. 2018, 8, 1978. [Google Scholar] [CrossRef] [Green Version]
  33. Zou, Y.N.; Wu, Q.S.; Huang, Y.M.; Ni, Q.D.; He, X.H. Mycorrhizal-Mediated Lower Proline Accumulation in Poncirus trifoliata under Water Deficit Derives from the Integration of Inhibition of Proline Synthesis with Increase of Proline Degradation. PLoS ONE 2013. [Google Scholar] [CrossRef] [Green Version]
  34. Morte, A.; Zamora, M.; Gutiérrez, A.; Honrubia, M. Desert truffle cultivation in semiarid Mediterranean areas. In Mycorrhizas Functional Processes and Ecological Impact; Azcón-Aguilar, C., Barea, J.M., Gianinazzi, S., Gianinazzi-Pearson, V., Eds.; Springer: Berlin/Heidelberg, Germany, 2009; pp. 221–233. [Google Scholar]
  35. Álvarez, S.; Sánchez-Blanco, M.J. Changes in growth rate, root morphology and water use efficiency of potted Callistemon citrinus plants in response to different levels of water deficit. Sci. Hortic. 2013, 156, 54–62. [Google Scholar] [CrossRef] [Green Version]
  36. Álvarez, S.; Sánchez-Blanco, M.J. Comparison of individual and combined effects of salinity and deficit irrigation on physiological, nutritional and ornamental aspects of tolerance in Callistemon laevis plants. J. Plant. Physiol. 2015, 185, 65–74. [Google Scholar] [CrossRef] [Green Version]
  37. Acosta-Motos, J.R.; Ortuño, M.F.; Bernal-Vicente, A.; Diaz-Vivancos, P.; Sánchez-Blanco, M.J.; Hernández, J.A. Plant Responses to Salt Stress: Adaptive Mechanisms. Agronomy 2017, 7, 18. [Google Scholar] [CrossRef] [Green Version]
  38. Sánchez-Blanco, M.J.; Morales, M.; Torrecillas, A.; Alarcón, J.J. Diurnal and seasonal osmotic potential changes in Lotus creticus creticus plants grown under saline stress. Plant. Sci. 1998, 136, 1–10. [Google Scholar] [CrossRef]
  39. Álvarez, S.; Gómez-Bellot, M.J.; Castillo, M.; Bañón, S.; Sánchez-Blanco, M.J. Osmotic and saline effect on growth, water relations, and ion uptake and translocation in Phlomis purpurea plants. Environ. Exp. Bot. 2012, 78, 138–145. [Google Scholar] [CrossRef] [Green Version]
  40. Acosta-Motos, J.R.; Diaz-Vivancos, P.; Álvarez, S.; Fernández-García, N.; Sanchez-Blanco, M.J.; Hernández, J.A. Physiological and biochemical mechanisms of the ornamental Eugenia myrtifolia L. plants for coping with NaCl stress and recovery. Planta 2015, 242, 829–846. [Google Scholar] [CrossRef] [Green Version]
  41. Acosta-Motos, J.R.; Ortuño, M.F.; Alvarez, S.; López-Climent, M.F.; Gómez-Cadenas, A.; Sánchez-Blanco, M.J. Changes in growth, physiological parameters and the hormonal status of Myrtus communis L. plants irrigated with water with different chemical compositions. J. Plant. Physiol. 2016, 191, 12–21. [Google Scholar] [CrossRef]
  42. Augé, R.M. Water relations, drought and vesicular-arbuscular mycorrhizal symbiosis. Mycorrhiza 2001, 11, 3–42. [Google Scholar] [CrossRef]
  43. Dixon, J.B.; Arora, H.S.; Hons, F.M.; Askenasy, P.E.; Hossner, L.R. Chemical, physical, and mineralogical properties of soils, mine spoil, and overburden associated with lignite mining. In Reclamation of Surface-Mined Lignite Spoil in Texas; Research Monograph No. 10: Texas Agricultural Experiment Station; Hossner, L.R., Ed.; Texas A & M University: College Station, TX, USA, 1980; pp. 13–21. [Google Scholar]
  44. Futai, K.; Taniguchi, T.; Kataoka, R. Ectomycorrhizae and Their Importance in Forest Ecosystems. In Mycorrhizae: Sustainable Agriculture and Forestry; Siddiqui, Z.A., Akhtar, M.S., Futai, K., Eds.; Springer: Dordrecht, The Netherlands, 2008. [Google Scholar] [CrossRef]
  45. Costa, J.M.; Ortuño, M.F.; Lopes, C.; Chaves, M.M. Grapevine varieties exhibiting differences in stomatal response to water deficit. Funct. Plant. Biol. 2012, 39, 179–189. [Google Scholar] [CrossRef]
  46. Costa, J.M.; Ortuño, M.F.; Chaves, M.M. Deficit irrigation as a strategy to save water: Physiology and potential application to horticulture. J. Integr. Plant. Biol. 2007, 49, 1421–1434. [Google Scholar] [CrossRef]
  47. Chaves, M.M.; Costa, M.; Zarrouk, O.; Pinheiro, C.; Lopes, C.; Pereira, J. Controlling stomatal aperture in semi-arid regions—The dilemma of saving water or being cool? Plant. Sci. 2016, 251, 54–64. [Google Scholar] [CrossRef]
  48. Lawson, T.; von Caemmerer, S.; Baroli, I. Photosynthesis and stomatal behaviour. In Progress Inbotany; Luttge, U., Beyschlag, W., Beudel, B., Francis, D., Eds.; Springer: Berlin/Heidelberg, Germany, 2011; pp. 265–304. [Google Scholar]
  49. Meng, F.R.; Arp, P. Net photosynthesis and stomatal conductance of red spruce twigs before and after twig detachment. Can. J. Res. 1993, 23, 716–721. [Google Scholar] [CrossRef]
  50. Sánchez-Blanco, M.J.; Ortuño, M.F.; Bañón, S.; Álvarez, S. Deficit irrigation as a strategy to control growth in ornamental plants and enhance their ability to adapt to drought conditions. J. Hortic. Sci. Biotechnol. 2019, 94, 137–150. [Google Scholar] [CrossRef]
  51. Chaves, M.M.; Flexas, J.; Pinheiro, C. Photosynthesis under drought and salt stress: Regulation mechanisms from whole plant to cell. Ann. Bot. 2009, 103, 551–560. [Google Scholar] [CrossRef] [Green Version]
  52. Jones, H.G.; Serraj, R.; Loveys, B.R.; Xiong, L.; Wheaton, A.; Price, A.H. Thermal infrared imaging of crop canopies for the remote diagnosis and quantification of plant responses to water stress in the field. Funct. Plant. Biol. 2009, 36, 978–979. [Google Scholar] [CrossRef] [Green Version]
  53. Costa, J.M.; Grant, O.M.; Chaves, M.M. Thermography to explore plant-environment interactions. J. Exp. Bot. 2013, 64, 3937–3949. [Google Scholar] [CrossRef]
  54. Gómez-Bellot, M.J.; Nortes, P.A.; Sánchez-Blanco, M.J.; Ortuño, M.F. Sensitivity of thermal imaging and infrared thermometry to detect water status changes in Euonymus japonica plants irrigated with saline reclaimed water. Biosyst. Eng. 2015, 133, 21–32. [Google Scholar] [CrossRef]
  55. Munné-Bosch, S.; Penuelas, J. Photo- and antioxidative protection, and a role for salicylic acid during drought and recovery in field-grown Phillyrea angustifolia plants. Planta 2003, 217, 758–766. [Google Scholar] [CrossRef]
  56. Melotto, M.; Underwood, W.; Koczan, J.; Nomura, K.; He, S.Y. Plant stomata function in innate immunity against bacterial invasion. Cell 2006, 126, 969–980. [Google Scholar] [CrossRef] [Green Version]
  57. Gómez-Cadenas, A.; Tadeo, F.R.; Talón, M.; Primo-Millo, E. Leaf abscission induced by ethylene in water-stressed intact seedlings of cleopatra mandarin requires previous abscisic acid accumulation in roots. Plant. Physiol. 1996, 112, 401–408. [Google Scholar] [CrossRef] [Green Version]
  58. Wright, S.T.C. The effect of plant growth regulator treatments on the levels of ethylene emanating from excised turgid and wilted wheat leaves. Planta 1980, 148, 381–388. [Google Scholar] [CrossRef]
  59. Tan, Z.-Y.; Thimann, K.V. The roles of carbon dioxide and abscisic acid in the production of ethylene. Physiol. Plant. 1989, 75, 13–19. [Google Scholar] [CrossRef]
  60. Chaves, M.M.; Maroco, J.P.; Pereira, J.S. Understanding plant responses to drought—From genes to the whole plant. Funct. Plant. Biol. 2003, 30, 239–264. [Google Scholar] [CrossRef]
  61. Olivella, C.; Vendrell, M.; Savé, R. Abscisic acid and ethylene content in Gerbera jamesonii plants submitted to drought and rewatering. Biol. Plant. 1998, 41, 613–616. [Google Scholar] [CrossRef]
  62. Spollen, W.G.; LeNoble, M.E.; Samuels, T.D.; Bernstein, N.; Sharp, R.E. Abscisic acid accumulation maintains maize primary root elongation at low water potentials by restricting ethylene production. Plant. Physiol. 2000, 122, 967–976. [Google Scholar] [CrossRef] [Green Version]
  63. Fernández-Lizarazo, J.C.; Moreno-Fonseca, L.P. Mechanisms for tolerance to water-deficit stress in plants inoculated with arbuscular mycorrhizal fungi. A review. Agron. Colomb. 2016, 34, 179–189. [Google Scholar] [CrossRef]
  64. Miransari, M.; Abrishamchi, A.; Khoshbakht, K.; Niknam, V. Plant hormones as signals in arbuscular mycorrhizal symbiosis. Crit. Rev. Biotechnol. 2014, 8551, 1–12. [Google Scholar] [CrossRef]
  65. Miransari, M. Contribution of arbuscular mycorrhizal symbiosis to plant growth under different types of soil stress. Rev. Plant. Biol. 2010, 12, 563–569. [Google Scholar] [CrossRef]
  66. Aroca, R.; Ruiz-Lozano, J.M.; Zamarreño, A.M.; Paz, J.A.; García-Mina, J.M.; Pozo, M.J.; López-Ráez, J.A. Arbuscular mycorrhizal symbiosis influences strigolactone production under salinity and alleviates salt stress in lettuce plants. J. Plant. Physiol. 2013, 170, 47–55. [Google Scholar] [CrossRef]
  67. Zhang, J.; Schurr, U.; Davies, W.J. Control of stomatal behavior by abscisic acid which apparently originates in roots. J. Exp. Bot. 1987, 38, 1174–1181. [Google Scholar] [CrossRef]
  68. Siwinska, J.; Kadzinski, L.; Banasiuk, R.; Gwizdek-Wisniewska, A.; Olry, A.; Banecki, B.; Lojkowska, E.; Ihnatowicz, A. Identification of QTLs affecting scopolin and scopoletin biosynthesis in Arabidopsis thaliana. BMC Plant. Biol. 2014, 14, 280. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  69. Tal, B.; Robeson, D.J. The metabolism of sunflower phytoalexins ayapin and scopoletin: Plant-fungus interactions. Plant. Physiol. 1986, 82, 167–172. [Google Scholar] [CrossRef] [PubMed]
  70. Costet, L.; Fritig, B.; Kauffmann, S. Scopoletin expression in elicitor-treated and tobacco mosaic virus-infected tobacco plants. Physiol. Plant. 2002, 115, 228–235. [Google Scholar] [CrossRef]
  71. Tanaka, Y.; Data, E.S.; Hirose, S.; Taniguchi, T.; Uritani, I. Biochemical changes in secondary metabolites in wounded and deteriorated cassava roots. Agric. Biol. Chem. 1983, 47, 693–700. [Google Scholar]
  72. Uritani, I. Biochemistry on postharvest metabolism and deterioration of some tropical tuberous crops. Bot. Bul. Acad. Sin. 1999, 40, 177–183. [Google Scholar]
  73. Buschmann, H.; Rodriguez, M.X.; Tohme, J.; Beeching, J.R. Accumulation of hydroxycoumarins during post-harvest deterioration of tuberous roots of cassava (Manihot esculenta Crantz). Ann. Bot. 2000, 86, 1153–1160. [Google Scholar] [CrossRef] [Green Version]
  74. Giesemann, A.; Biehl, B.; Leiberei, R. Identification of scopoletin as a phytoalexin of the rubber tree Hevea brasiliensis. J. Phytopathol. 2008, 117, 373–376. [Google Scholar] [CrossRef]
  75. Kormanik, P.P.; McGraw, A. Quantification of vesicular-arbuscular mycorrhizae in plant roots. In Methods and Principles of M~corrhizal Research; Schenck, N.C., Ed.; American Phytopathological Society: St. Paul, MN, USA, 1982; pp. 37–45. [Google Scholar]
  76. Sieverding, E. Manual de Métodos para la Investigación de la Micorriza Vesículo-Arbuscular en el Laboratorio; Centro Internacional de Agricultura Tropical (CIAT): Cali, Colombia, 1983; 121p. [Google Scholar]
  77. Scholander, P.F.; Hammel, H.T.; Bradstreet, E.D.; Hemmingsen, E.A. Sap pressure in vascular plants. Science 1965, 148, 339–346. [Google Scholar] [CrossRef]
  78. Gucci, R.; Xiloyannis, C.; Flore, J.A. Gas exchange parameters water relations and carbohydrate partitioning in leaves of field-grown Prunus domestica following fruit removal. Physiol. Plant. 1991, 83, 497–505. [Google Scholar] [CrossRef]
  79. Girma, F.S.; Krieg, D.R. Osmotic Adjustment in Sorghum. Plant. Physiol. 1992, 99, 583–588. [Google Scholar] [CrossRef] [Green Version]
  80. Jones, H.G. Estimation of an effective soil water potential at the root surface of transpiring plants. Plant. Cell Environ. 1983, 6, 671–674. [Google Scholar]
  81. Leinonen, I.; Grant, O.; Tagliavia, C.P.P.; Chaves, M.; Jones, H. Estimating stomatal conductance with thermal imagery. Plant. Cell Environ. 2006, 29, 1508–1518. [Google Scholar] [CrossRef]
  82. Albacete, A.; Ghanem, M.E.; Martínez-Andújar, C.; Acosta, M.; Sánchez-Bravo, J.; Martínez, V.; Lutts, S.; Dodd, I.C.; Pérez-Alfocea, F. Hormonal changes in relation to biomass partitioning and shoot growth impairment in salinized tomato (Solanum lycopersicum L.) plants. J. Exp. Bot. 2008, 59, 4119–4131. [Google Scholar] [CrossRef]
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Figure 1. Soil water potential at the root surface (Ψr) (A), leaf water potential (Ψl) (B) and total seasonal leaf osmotic water potential changes due to dehydration (ΔΨSS) (C) of Cistus albidus plants under well-irrigated and non-irrigated conditions, with and without Pisolithus tinctorious inoculation, at the end of the experiment. Values are means of three replications. The vertical bars indicate standard errors. Different lowercase letters indicate significant differences between treatments according to Duncan0.05 test. Absence of letter s in rows indicates no significant difference between treatments. C, well-watered plants; WS, non-irrigated plants; CM, well-watered and inoculated plants; WSM, non-irrigated and inoculated plants.
Figure 1. Soil water potential at the root surface (Ψr) (A), leaf water potential (Ψl) (B) and total seasonal leaf osmotic water potential changes due to dehydration (ΔΨSS) (C) of Cistus albidus plants under well-irrigated and non-irrigated conditions, with and without Pisolithus tinctorious inoculation, at the end of the experiment. Values are means of three replications. The vertical bars indicate standard errors. Different lowercase letters indicate significant differences between treatments according to Duncan0.05 test. Absence of letter s in rows indicates no significant difference between treatments. C, well-watered plants; WS, non-irrigated plants; CM, well-watered and inoculated plants; WSM, non-irrigated and inoculated plants.
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Figure 2. Stomatal conductance (gs) (A), net photosynthetic rate (Pn) (B) and leaf temperature (Tl) (C) of Cistus albidus plants under well-irrigated and non-irrigated conditions, with and without Pisolithus tinctorious inoculation, at the end of the experiment. Values are means of three replications. The vertical bars indicate standard errors. Different lowercase letters indicate significant differences between treatments according to Duncan0.05 test. C, well-watered plants; WS, non-irrigated plants; CM, well-watered and inoculated plants; WSM, non-irrigated and inoculated plants.
Figure 2. Stomatal conductance (gs) (A), net photosynthetic rate (Pn) (B) and leaf temperature (Tl) (C) of Cistus albidus plants under well-irrigated and non-irrigated conditions, with and without Pisolithus tinctorious inoculation, at the end of the experiment. Values are means of three replications. The vertical bars indicate standard errors. Different lowercase letters indicate significant differences between treatments according to Duncan0.05 test. C, well-watered plants; WS, non-irrigated plants; CM, well-watered and inoculated plants; WSM, non-irrigated and inoculated plants.
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Figure 3. Abscisic acid (ABA) (A), salicylic acid (SA) (B), precursor of ethylene (ACC) (C), and scopoletin (SC) (D) of Cistus albidus plants under well-irrigated and non-irrigated conditions, with and without Pisolithus tinctorious inoculation, at the end of the experiment. Values are means of three replications. Different lowercase letters indicate significant differences between treatments according to Duncan0.05 test. The vertical bars indicate standard errors. C, well-watered plants; WS, non-irrigated plants; CM, well-watered and inoculated plants; WSM, non-irrigated and inoculated plants.
Figure 3. Abscisic acid (ABA) (A), salicylic acid (SA) (B), precursor of ethylene (ACC) (C), and scopoletin (SC) (D) of Cistus albidus plants under well-irrigated and non-irrigated conditions, with and without Pisolithus tinctorious inoculation, at the end of the experiment. Values are means of three replications. Different lowercase letters indicate significant differences between treatments according to Duncan0.05 test. The vertical bars indicate standard errors. C, well-watered plants; WS, non-irrigated plants; CM, well-watered and inoculated plants; WSM, non-irrigated and inoculated plants.
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Figure 4. Solid lines represent the relationships between stomatal conductance (gs) and net photosynthesis (Pn) (A), gs and leaf water potential (Ψl) (B) and gs and leaf temperature (Tl) (C) in Cistus albidus plants under well-irrigated and non-irrigated conditions, with and without Pisolithus tinctorious inoculation, during the experimental period. Dotted lines represent the linear regressions of the control (C and CM) and water stress treatments (WS and WSM) individually. Values are means of three replications. C, well-watered plants (closed circles); WS, non-irrigated plants (open circles); CM, well-watered and inoculated plants (closed triangles); WSM, non-irrigated and inoculated plants (open triangles). *** significant at p < 0.001.
Figure 4. Solid lines represent the relationships between stomatal conductance (gs) and net photosynthesis (Pn) (A), gs and leaf water potential (Ψl) (B) and gs and leaf temperature (Tl) (C) in Cistus albidus plants under well-irrigated and non-irrigated conditions, with and without Pisolithus tinctorious inoculation, during the experimental period. Dotted lines represent the linear regressions of the control (C and CM) and water stress treatments (WS and WSM) individually. Values are means of three replications. C, well-watered plants (closed circles); WS, non-irrigated plants (open circles); CM, well-watered and inoculated plants (closed triangles); WSM, non-irrigated and inoculated plants (open triangles). *** significant at p < 0.001.
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Table
Table 1. Percentage of root colonization in plants subjected to control and water stress with Pisolithus tinctorious (CM and WSM, respectively) at the end of the experiment. Values are means of three root samples per replication.
Table 1. Percentage of root colonization in plants subjected to control and water stress with Pisolithus tinctorious (CM and WSM, respectively) at the end of the experiment. Values are means of three root samples per replication.
Mycorrhization Percentage (%)
CM29.3 a
WSM21.05 b
Different letters indicate significant differences according Duncan 0.05 test.
Table
Table 2. Dry weight of leaf, stem, root and shoot, root/shoot ratio, and height of Cistus albidus plants under well-irrigated and non-irrigated conditions, with and without Pisolithus tinctorious mycorrhiza, at the end of the experiment. Values are means ± SEM (n = 3).
Table 2. Dry weight of leaf, stem, root and shoot, root/shoot ratio, and height of Cistus albidus plants under well-irrigated and non-irrigated conditions, with and without Pisolithus tinctorious mycorrhiza, at the end of the experiment. Values are means ± SEM (n = 3).
TREATMENTS
CWSCMWSM
Leaf DW (g)3.32 ± 0.39 b2.37 ± 0.31 c5.59 ± 1.44 a4.06 ± 0.41 ab
Stem DW (g)2.25 ± 0.302.05 ± 0.223.58 ± 1.052.80 ± 0.15
Root DW (g)1.64 ± 0.15 c1.55 ± 0.20 c2.05 ± 0.13 b3.17 ± 0.30 a
Shoot DW (g)5.58 ± 0.68 bc4.43 ± 0.54 c8.50 ± 0.89 a6.85 ± 0.55 ab
Root/shoot ratio0.30 ± 0.03 b0.35 ± 0.01 ab0.22 ± 0.03 b0.48 ± 0.09 a
Height (cm)33.42 ± 1.35 b30.50 ± 1.08 c35.83 ± 1.46 a33.23 ± 1.49 b
DW, dry weight; C, well-watered plants; WS, non-irrigated plants; CM, well-watered and inocu-lated plants; WSM, non-irrigated and inoculated plants. Different letters in rows indicate significant differences between treatments according Duncan0.05 test. Absence of letters in rows indicates no significant difference between treatments.
Table
Table 3. Leaf mineral content of Cistus albidus plants under well irrigated and non-irrigated conditions, with and without Pisolithus tinctorious mycorrhiza, at the end of the experiment. Values are means ± SEM (n = 3).
Table 3. Leaf mineral content of Cistus albidus plants under well irrigated and non-irrigated conditions, with and without Pisolithus tinctorious mycorrhiza, at the end of the experiment. Values are means ± SEM (n = 3).
TREATMENTS
CWSCMWSM
Macronutrients
Ca (%)0.86 ± 0.02 b0.60 ± 0.03 c1.00 ± 0.05 a0.49 ± 0.05 c
K (%)3.19 ± 0.23 a2.72 ± 0.50 ab3.02 ± 0.32 a2.18 ± 1.28 b
P (%)0.67 ± 0.06 ab0.59 ± 0.03 ab0.73 ± 0.06 a0.54 ± 0.04 b
Na (%)0.14 ± 0.02 a0.08 ± 0.01 b0.16 ± 0.01 a0.05 ± 0.01 b
S (%)0.17 ± 0.03 b0.16 ± 0.02 b0.22 ± 0.01 a0.15 ± 0.01 b
Mg (%)0.35 ± 0.02 a0.26 ± 0.02 b0.37 ± 0.01 a0.24 ± 0.01 b
Micronutrients
Cu (mg/Kg)12.95 ± 1.29 a4.97 ± 0.82 b10.85 ± 2.23 a4.15 ± 0.45 b
Fe (mg/Kg)68.78 ± 7.44 ab54.68 ± 4.96 b82.97 ± 10.13 a102.85±35.34 a
Mn (mg/Kg)117.13 ± 3.41 ab123.52 ±28.55 ab153.05 ± 7.71 b108.93 ± 23.75a
B (mg/Kg)165.35 ± 3.29158.55 ± 1.40162.75 ± 3.98161.10 ± 7.69
Zn (mg/Kg)87.95 ± 13.71 a59.58 ± 9.09 ab72.73 ± 5.42 ab41.97 ± 7.37 b
C, well-watered plants; WS, non-irrigated plants; CM, well-watered and inoculated plants; WSM, non-irrigated and inoculated plants. Different letters in rows indicate significant differences between treatments according Duncan0.05 test. Absence of letters in rows indicates no significant difference between treatments.
Table
Table 4. Water indices related to gas exchange and photosynthetic efficiency at the end of the experiment. Values are means ± SEM (n = 3).
Table 4. Water indices related to gas exchange and photosynthetic efficiency at the end of the experiment. Values are means ± SEM (n = 3).
TREATMENTS
CWSCMWSM
Pn/gs (μmol CO2/mol H2O)26.21 ± 4.41 b38.58 ± 3.87 a23.31 ± 8.62 b40.55 ± 2.10 a
−Ψl/gs (MPa/mol H2O m−2 s−1)5.40 ± 0.23 b170.10 ± 45.75 a4.73 ± 1.05 b205.97 ± 33.10 a
Tl/gs (°C/mol H2O m−2 s−1)166.2 ± 32.61 b2215.92 ± 82.63 a130.34 ± 27.72 b2576 ± 50.02 a
RCC/Pn (%/μmol CO2 m−2 s−1)10.10 ± 0.57 c102.60 ± 8.24 b10.20 ± 2.17 c127.95 ± 11.16 a
Pn/Shoot DW (μmol CO2 m−2 s−1/g)0.63 ± 0.05 a0.10 ± 0.01 c0.34 ± 0.02 b0.06 ± 0.02 c
Pn, net photosynthesis; gs. stomatal conductance; Ψl, leaf water potential; Tl, leaf temperature; RCC, relative chlorophyll content; Shoot DW, shoot dry weight; C, well-watered plants; WS, non-irrigated plants; CM, well-watered and inoculated plants; WSM, non-irrigated and inoculated plants. Different letters in rows indicate significant differences according Duncan0.05 test.
Table
Table 5. Plant hormones and phytoalexins: reference, brand, and solubility.
Table 5. Plant hormones and phytoalexins: reference, brand, and solubility.
HormoneReferenceLaboratorySolubility
Jasmonic acid14631-10 MGSIGMADMSO (16 mg/mL)
trans-ZeatinZ0876-5 MGSIGMADMSO (3 mg/mL)
trans-Zeatin001030n (5 mg)OLCHEMLMDMSO (3 mg/mL)
[2H5]-trans-Zeatin030030n (1 mg)OLCHEMLMDMSO (3 mg/mL)
CamalexinSML1016-5 MGSIGMADMSO (20 mg/mL)
IsopentenyladenineSC-279669 (1G)SANTA CRUZDMSO (20 mg/mL)
trans-Zeatin ribosideSC-20846 (5 MG)SANTA CRUZETHANOL/DMSO (78.95:21.05, v/v) (50 mg/mL)
trans-Zeatin riboside001031n (5 mg)OLCHEMLMDMSO
trans-Zeatin glucosideSC-237225 (1MG)SANTA CRUZETHANOL/DMSO (75.00:25.00, v/v) (50 mg/mL)
trans-Zeatin 9-glucoside001047n (1 mg)OLCHEMLMDMSO
Abscisic acidSC-238015 (50 MG)SANTA CRUZETHANOL (50 mg/mL)
3-indoleacetic acidSC-254494 (5 G)SANTA CRUZETHANOL (50 mg/mL)
1-aminocyclopropane-1-carboxylic acid (ACC)SC-202393 (500 MG)SANTA CRUZWATER (437 mg/mL)
Giberellic acid (GA1)012249n (1 mg)OLCHEMLMETHANOL (50 mg/mL)
Giberellic acid (GA3)SC-257556 (500 MG)SANTA CRUZETHANOL (50 mg/mL)
Giberellic acid (GA4)SC-235248 (5 MG)SANTA CRUZETHANOL (50 mg/mL)
Giberellic acid (GA5)SC-490117 (25 MG)SANTA CRUZETHANOL (50 mg/mL)
Giberellic acid (GA7)012254n (1 mg)OLCHEMLMETHANOL (50 mg/mL)
Salycilic acidSC-203374 (100 G)SANTA CRUZETHANOL (50 mg/mL)
ScopoletinSC-206059 (50 MG)SANTA CRUZDMSO (30 mg/mL)
All the phytohormones were determined in three plants per replication at the end of the experimental period.
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Lorente, B.; Zugasti, I.; Sánchez-Blanco, M.J.; Nicolás, E.; Ortuño, M.F. Effect of Pisolithus tinctorious on Physiological and Hormonal Traits in Cistus Plants to Water Deficit: Relationships among Water Status, Photosynthetic Activity and Plant Quality. Plants 2021, 10, 976. https://doi.org/10.3390/plants10050976

AMA Style

Lorente B, Zugasti I, Sánchez-Blanco MJ, Nicolás E, Ortuño MF. Effect of Pisolithus tinctorious on Physiological and Hormonal Traits in Cistus Plants to Water Deficit: Relationships among Water Status, Photosynthetic Activity and Plant Quality. Plants. 2021; 10(5):976. https://doi.org/10.3390/plants10050976

Chicago/Turabian Style

Lorente, Beatriz, Inés Zugasti, María Jesús Sánchez-Blanco, Emilio Nicolás, and María Fernanda Ortuño. 2021. "Effect of Pisolithus tinctorious on Physiological and Hormonal Traits in Cistus Plants to Water Deficit: Relationships among Water Status, Photosynthetic Activity and Plant Quality" Plants 10, no. 5: 976. https://doi.org/10.3390/plants10050976

APA Style

Lorente, B., Zugasti, I., Sánchez-Blanco, M. J., Nicolás, E., & Ortuño, M. F. (2021). Effect of Pisolithus tinctorious on Physiological and Hormonal Traits in Cistus Plants to Water Deficit: Relationships among Water Status, Photosynthetic Activity and Plant Quality. Plants, 10(5), 976. https://doi.org/10.3390/plants10050976

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