Morpho-physiology and Pht1 gene expressions in native maize plants with AM fungi and phosphorus

Maize is a crop important worldwide, but its production is limited to phosphorus availability in soil. Plants form a symbiotic association to improve their nutrition with arbuscular mycorrhizal fungi (AMF), which increase to absorption phosphorus (P) and the expression of transporters of the family Pht1. Few studies have focused on native maize plants and AMF. Thus, the objective of this study was to determine the morphophysiological response and expression of phosphate Pht1 transporters in two native maize plants inoculated with Claroideoglomus etunicatum and P concentrations. The height, leaf area, dry biomass, CO2 assimilation rate, stomatal conductance, transpiration rate, intercellular CO2, water potential, greenness index, total chlorophyll, and ZEAma; Pht1;3 and ZEAma; Pht1;6 transporter expressions in maize plants under P (0.01 and 1 mM) concentrations were evaluated. The results showed that each native maize plant had a differential response in morpho-physiology and transporter expressions when they were inoculated with AMF and P. The response of maize plant was related with its genotype and phenotype plus environmental factor that influenced the AMF-host interaction, mycorrhizal colonization and soil nutrient absorption.


Introduction
Phosphorus (P) represents from 0.1-0.5% of plant dry weight, and it is an element that participates in the synthesis of nucleic acids, phospholipids, membranes, adenosine triphosphate (ATP), and forms part of enzyme activation/inactivation (Malhotra et al., 2018). Inorganic phosphate (Pi) is the chemical way that plants assimilate this macronutrient. Nonetheless, it shows low mobility and availability in soil due to its ion retention in soil particles charged positively (mineral oxides and clays) and/or to its precipitation in insoluble mineral complexes by its combination with calcium, iron and aluminum . Low Pi availability has turned out to be a limiting factor for agriculture production globally.
Mexico is the center of maize (Zea mays L.) origin, because it has the greatest diversity of native species in worldwide. Nevertheless, one of the main limitations in maize production is low availability of Pi in soil in the tropical/subtropical regions (Lie et al., 2019), which has allowed plants to develop different strategies to improve their nutrition. The strategy that stands out is its symbiotic association with arbuscular mycorrhizal fungi (AMF) (Saia et al., 2020).
As a fundamental soil microbial component, AMF belonging to the phylum Glomeromycotina, are the most common type of mycorrhizal fungi and form mycorrhizal symbioses with 80% of all terrestrial plant species (Spatafora et al., 2016;Cheng et al., 2020;Rimington et al., 2020). Within the main AMF species, those that stand out are Funilliformis mossea, Rhizophagus intraradices, Claroideoglomus etunicatum, among others (Higo et al., 2019a(Higo et al., , 2019b(Higo et al., , 2020a(Higo et al., , 2020b. In this symbiotic association between AMF-plant, the benefits that endophytes obtain are carbon sources that used for synthesizing their lipids and cellular membranes (Thirkell et al., 2020), and the plants increase their defenses toward soil or foliage phytopathogens, mitigate environmental stress, increase water absorption and acquire mineral nutrients, mainly Pi though Pht1 (Attarzadeh et al., 2019;Jain and Pundir, 2019) gene activation. In plants, the Pht1 gene family codifies phosphate transporters of high affinity, which participate actively in Pi absorption and transport, expressed preferably in root cells (Victor Roch et al., 2019). Several Pht1 transporters have demonstrated to be regulated in plants by AMF (Zhang et al., 2019).
Six Pht1 transporters have been characterized in maize plants, of which those that stand out are ZEAma;Pht1;3 related with direct Pi soil absorption (DPU, Direct Pathway Uptake) and the transporter ZEAma;Pht1;6 associated with Pi soil absorption through mycorrhizal symbiosis (MPU, Mycorrhizal Pathway Uptake) (Smith et al., 2011). In maize varieties or hybrids, different studies have assessed responses in growth, physiology and expressions of the family of Pht1 genes associated to AMF colonization (Liu et al., 2016;Liu et al., 2018). Nevertheless, no information has been found on the morpho-physiological expressions of the Pht1 genes in native maize plants. Therefore, the objective of this study was to determine the differential response in morpho-physiology and expressions of Pht1 transporters of two native maize plants inoculated with Claroideoglomus etunicatum and Pi concentrations in soil.

Materials and Methods
Maize seed and AMF inoculum Zea mays L. seeds of two native maize plants -catalogued as PEE004-AC and LPO-km100 that belong to the germplasm collection of the School of Agriculture at Universidad Veracruzana, Xalapa, Veracruz, México-were used. The AMF (Claroideoglomus etunicatum) inoculum -provided by Universidad Veracruzana-contained as substrate sand and soil (5:1, v/v) and 20 spores g -1 of dry soil.

Zea mays seedlings
The seeds were treated with NaClO at 10% for 20 min, then washed thrice with sterile distilled water and deposited in a bedding with sterile sand for 10 days. Subsequently, seedlings with two true leaves were transplanted to 10 L pots previously disinfected with NaClO at 10%, which contained 7 L of a substrate sand and peat moss (5:2, v/v) previously sterilized by autoclaving for 15 min at 120 °C. The amount of phosphorus in the substrate was 59 mg kg -1 and determined with the method proposed by Jackson (1976). Plants were kept in a greenhouse for 75 days.

Inoculation of maize with AMF
During transplant, each plant was inoculated with 10 g of substrate (sand and soil, 5:1, v/v) containing C. etunicatum and irrigated with K2HPO4 using phosphorus as source. The concentrations of 0.01 and 1 mM, equivalent to 0.309 mg L -1 and 30.9 mg L-1 of P were used. As control treatment, a group of plants was not inoculated with AMF and only fertilized. Ten replicates were used per treatment and the experiment was repeated twice.

Microbiological variables
The number of C. etunicatum spores were determined following the technique described by INVAM (2016), and for the mycorrhizal colonization percentage, McGonigle et al. (1990) method was used. From each plant per treatment, twenty root fragments of 1 cm were taken, previously stained with trypan blue (Phillips and Hayman, 1970) and observed in the compound microscope (Nikon Eclipse, Model E200). The estimation of the mycorrhizal colonization percentage (%CM) was determined with the following formula: %CM = (# of observations with AMF/# of total observations) x 100.

Pht1 gene expressions in maize
Five grams of root tissue were collected from each treatment; they were washed with sterile distilled water, wrapped in aluminium foil and stored in liquid nitrogen at -80 °C. RNA extraction was performed by the TRIzol® Reagent (Thermo Fisher Scientific) method. To eliminate the genomic DNA residual, RNA was purified by DNase I (Thermo Fisher Scientific) enzyme. RNA visual integrity was assessed with the methodology described by Aranda (2012). Total RNA concentration ng μL -1 was performed by spectrophotometry (NanoDrop, ThermoScientific); 100 ng of total RNA were used for cDNA synthesis starting from the reverse transcriptase enzyme SuperScript® II (Thermo Fisher Scientific); cDNA was stored at -20 °C.

Pht1 gene expression
For the real time quantitative polymerase chain reaction (RT-qPCR) gene expression assays, the reactive SsoFastTMEvaGreen®SuperMix (Bio-Rad) was used. Each reaction contained 5 μL of SsoFastTMEvaGreen®SuperMix (2X); 0.3 μL of each primer (10 μm of ZEAma:Pht1;3 and ZEAma:Pht1;6), 1.5 μL cDNA and the final volume was adjusted to 10 μL with sterile ultrapure H2O. The amplification was one cycle at 95 °C for 30 s, 40 cycles at 95 °C for 5 s and 60 °C for 30 s. To confirm that the primers did not generate unspecific products, a dissociation curve was performed with a cycle of 65 at 95 °C with increments of 0.5 °C each 5 s, using a real time thermocycler "C1000TouchTM" (Bio-Rad CFX96TM Real-Time System).
To normalize expression levels, the eukaryotic initiation factor-4A (EIF4A) (Lin et al., 2014) (Table 1) was used. Amplification efficiency was confirmed with the following formula: PCR efficiency percentage = 10 -1/slope x 100 For the relative expression analysis, the 2 -ΔΔCq (Livak and Schmittgen 2001) method was performed. The results were expressed as the change in units of a target gene expression level in a group of treatments related to a calibrator group.
Change in expression level = 2 -ΔΔCq Where ΔΔCq = (Cqobjetive -Cqendogenous) calibrator -(Cqobjetive -Cqendogenous) treatment Statistical analysis Data were processed by a one-way analysis of variance (ANOVA). The statistical data analysis was performed using the software STATISTICA v. 10.0 (StatSoft software package, Tulsa, OK, U.S.A.) and Tukey's test (P<0.05) was used for comparison of means. Prior to ANOVA, mycorrhization percentages were arcsine-square root transformed.

Results
Effect of P, AMF and native maize plants on morphologic variables P, AMF, native maize plants and the interaction between AMF-plant increased morphologic variables ( Table 2). The highest Pi (+) concentration increased leaf area, root length, dry stem weight, and aerial biomass in the two native maize plants compared to those fertilized with the lowest Pi (-) dose. Moreover, the inoculation of both maize with AMF only increased the leaf area when compared to the plants without AMF. Among the native maize plants, LPO-km100 increased leaf area, dry stem, leaf and root weight, and total aerial biomass compared with maize PEE004-AC. In the AMF × Native Maize Plant interaction, the maize LPO-km100 only increased leaf dry weight ( Figure 1). The treatments Pi × AMF, Pi × Native Maize Plant and Pi × AMF × Native Maize Plant did not show differences in morphologic variables.

Effect of P, AMF and native maize plants on physiologic variables
The response of the native maize plants and interactions between P-plant and AMF-plant increased physiological variables (Table 3). The maize EPP004-AC with P (+) dose and AMF only increased total chlorophyll (Figure 2a, 2d) with respect to maize LPO-km100. The CO2 assimilation rate and stomatal conductance were increased in the maize LPO-km100 with the lowest Pi (-) dose (Figure 2b, 2c). Data are shown as the mean ± standard deviation (SD) (n = 10). The experiment was repeated twice under greenhouse conditions. Columns with different letters were significantly different according to Tukey's test (P<0.05)   Figure 4). The expression of both genes was not conditioned to the presence or absence of AMF in plants.
The lowest expression levels of the ZEAma;Pht1;3 and ZEAma;Pht1;6 genes was in the plants fertilized with the highest Pi (+) dose. With respect to LPO-km100, the ZEAma;Pht1;3 and ZEAma;Pht1;6 gene expressions were significantly greater with the presence of AMF in plants and the lowest Pi (-) ( Figure 5). The lowest expression levels for the ZEAma;Pht1;3 gene was in the plants fertilized with the highest Pi (+) dose.

Discussion
Studies have shown the benefits that AMF contribute to plants, mainly in promoting growth, productivity (Rocha et al., 2019;Scrase et al., 2019), and gene expressions related with P and N absorption, among others . When plants are colonized by AMF, they increase their capacity of exploring the soil surface and achieving crossing boundaries beyond the nutrient depletion zone, allowing them to absorb more water, macro and micronutrients (especially P, Zn, and Cu) that improve plant vigour (Liu et al., 2018).
Nevertheless, the benefits of AMF colonized plants depend mainly on the interaction plant genotype, soil, environment and AMF species, which could affect the symbiotic root-endophyte relationship directly, observing differences in growth response among plants of the same species (Kim et al., 2017). Moreover, in agricultural management practices, tillage and cover cropping such as crop histories can alter the indigenous AMF community structure and diversity in soil and roots (Higo et al., 2018a(Higo et al., , 2018bMorimoto et al., 2018).
In this study, we observed a different response in the Pht1 gene expression and morpho-physiological parameters of native maize plants when inoculated with AMF because each genotype showed differences in its phenotype and root architecture, which influenced in mycorrhizal symbiosis and the beneficial effects of AMF on plant growth (Londoño et al., 2019). Additionally, among maize genotypes, different plant-dependence levels exist that influence mainly on the host-endophyte interaction and expression of the genes that codify phosphate transporters (Sawer et al., 2017). The physiological response of the inoculated plants with AMF is generally affected during abiotic stress conditions, such as temperature, soil fertility (Mathur and Jajoo, 2020), salinity (Hashem et al., 2019) or hydric stress (Mickan et al., 2019). These conditions have a bearing on chlorophyll concentration, relative leaf water content, gas exchange capacity carbon demand, enzymatic activity, changes in hormonal level, among others (Chandrasekaran et al., 2019). In particular, maize plants inoculated with AMF have shown to increase photosynthetic efficiency and chlorophyll content associated to an increase in soil Mg absorption by the extraradical mycelium in plants colonized by the endophyte (Londoño et al., 2019). Moreover, morpho-physiological maize cultivars of plants synthetically fertilized with P, particularly those that grow on soils with low P content, have increased their acid phosphatase and phytase, peroxidase and superoxide dismutase activities and AMF colonization, showing an influence on mycorrhized plant morphology and physiology (Lie et al., 2019).
The effect of Pi on AMF colonization in maize plants with the highest Pi (+) dose reduced the colonization. Moreover, the application of low P (-) dose increased the association between AMF and host. The high phosphorus concentration in soil induced direct P absorption in plants through root epidermis cells, autoregulating mycorrhizal colonization starting from a decrease in endophyte carbon delivery to maintain cellular homeostasis (Chu et al., 2020). Finally, a differential response was observed in the Pht1 transporter expressions in native maize plants inoculated with AMF. Nagy et al. (2009) and Tian et al. (2013) discussed that a high P availability in soil for plants eliminates the transcription of Pht1 phosphate transporters implied in the MPU route, affecting mycorrhizal colonization indexes. In this study, the differences in Pht1 transporter expressions assessed in native maize plants inoculated with AMF were related with genotype and environmental factors that have an influence on genomic and phenotypic expression in plants during different growth stages (Loth-Pereda et al., 2011). Additionally, root architecture -one of the structures that characterizes each plant-has a direct influence in AMF-host interaction, mycorrhizal colonization and soil nutrient absorption (Deng et al., 2014).

Conclusions
The results in this study showed a differential response in morpho-physiology and Pht1 transporter expressions of two native maize (PEE004-AC and LPO-km100) plants inoculated with Claroideoglomus etunicatum and under Pi in soil. The response of maize plant was related with its genotype and phenotype plus environmental factor that influenced the AMF-host interaction, mycorrhizal colonization and soil nutrient absorption. Subsequent studies focused on genomic and phenotypic response of each maize plant inoculated with AMF should allow knowing in depth the endophyte-host-environment interactome. Furthermore, future research should investigate how and whether different AMF inoculations show functional diversity in Pi uptake by maize.
Authors' Contributions SVL, RZR, and LGHM, designed the experiment. SVL, EEQA, LLC, and CA contributed in the assembly of experiment in agricultural area, collection of samples and transfer to the laboratory. SVL, EEQA, and LGHM participated with laboratory analysis, data and preparation of manuscripts. CA and PPR contributed with reagents another laboratory and field materials. RZR, PPR, and LGHM analyzed the data. SVL and LGHM wrote and edited the first draft of the manuscript. LGHM wrote the final version of the manuscript. RZR and LGHM are the main thesis advisors of SVL, a Master of Science student. All authors read and approved the final manuscript.