horticulturae Article Rhizosphere Acidification as the Main Trait Characterizing the Differential In Vitro Tolerance to Iron Chlorosis in Interspecific Pyrus Hybrids Claudia Paola Mora-Córdova 1 , Roser Tolrà 1 , Rosa Padilla 1 , Charlotte Poschenrieder 1 , Marie-Helene Simard 2 , Luis Asín 3 , Pere Vilardell 4 , Joan Bonany 4 , Elisabet Claveria 3 and Ramon Dolcet-Sanjuan 3, * 1 Laboratori Fisiologia Vegetal, Facultat de Biociències, Universitat Autònoma Barcelona, E-08193 Bellaterra, Spain; moracordovacp@gmail.com (C.P.M.-C.); roser.tolra@uab.cat (R.T.); rosa.padilla@uab.cat (R.P.); charlotte.poschenrieder@uab.cat (C.P.) 2 INRAE, IRHS, INRAE-AO-UA, CS 60057, CEDEX, 49071 Beaucouzé, France; marie-helene.simard@angers.inra.fr 3 Laboratori Cultiu In Vitro de Plantes, Programa de Fructicultura, IRTA, Fruitcentre, PCiTAL, Parc de Gardeny, E-25003 Lleida, Spain; luis.asin@irta.cat (L.A.); elisabet.claveria@irta.cat (E.C.) 4 Programa de Fructicultura, IRTA, Mas Badia, 17134 La Tallada d’Empordà, Spain; pere.vilardell@irta.cat (P.V.); joan.bonany@irta.cat (J.B.) * Correspondence: ramon.dolcet@irta.cat Abstract: Physiological responses of different interspecific Pyrus hybrids and an open pollinated Pyrus communis ‘Williams’ (Pcw) grown under in vitro culture conditions simulating lime induced chlorosis were studied. The hybrids were derived from crosses between the ‘Pyriam’ pear rootstock and four Citation: Mora-Córdova, C.P.; Tolrà, Pyrus species of the Mediterranean region, namely P. amygdaliformis Vill. (Pa), P. amygdaliformis persica R.; Padilla, R.; Poschenrieder, C.; Simard, M.-H.; Asín, L.; Vilardell, P.; Bornme. (Pap), P. communis cordata (Desv.) Hook. (Pcc), and P. elaeagrifolia Pall (Pe), all known for their Bonany, J.; Claveria, E.; Dolcet- higher field tolerance to iron-chlorosis than P. communis. Twenty hybrids and one open pollinated Sanjuan, R. Rhizosphere Acidification Pcw were micropropagated, and plantlets were in vitro characterized for their physiological responses as the Main Trait Characterizing the to iron-deficiency conditions. Rooted plantlets were transferred to a culture medium with 2 µM Fe3+ Differential In Vitro Tolerance to Iron DTPA and 10 or 20 mM NaHCO3 . These physiological responses were scored at 1, 3, 7, and 28 days Chlorosis in Interspecific Pyrus from the start of the in vitro assay. Leaf total chlorophyll content, the capacity of roots to acidify the Hybrids. Horticulturae 2022, 8, 551. medium, reduced iron, and exudates of phenolic acids and organic acids were analyzed in each media https://doi.org/10.3390/ and time sample. Leaf chlorophyll levels for the clones derived from Pcc were the highest, especially horticulturae8060551 under the highest bicarbonate concentration, followed by those derived from Pap and Pa. The higher Academic Editor: Sergio Ruffo chlorophyll content of Pcc clones were related with their higher capacity to acidify the media but not Roberto with their iron reduction capacity at the root level. On the other hand, hybrid clones derived from Pe showed a higher Fe3+ reduction ability than clones from all the other species during the whole Received: 27 April 2022 Accepted: 14 June 2022 assay but only when the bicarbonate concentration was lower. The exudation of phenolic acids by Published: 18 June 2022 the roots was higher in Pcw than in the other species, and this response might explain why the total chlorophyll levels in Pcw clones are similar to those of Pe and Pa ones. These results with Pyrus spp. Publisher’s Note: MDPI stays neutral bring more evidence in support of the idea that iron reduction capacity at the root level is not directly with regard to jurisdictional claims in related with a higher tolerance to iron deficiency caused by the high pH of calcareous soils. Instead, published maps and institutional affil- the ability to acidify the rhizosphere is the trait of choice for the selection of the pear hybrid clones iations. better adapted to lime induced chlorosis. In addition, the in vitro assay to select the Pyrus clones for tolerance to iron chlorosis could be shortened to one week of culture in 10 mM NaHCO3 , measuring the leaf chlorophyll level, acidification of the culture medium, and exudation of phenolic acids as the Copyright: © 2022 by the authors. physiological responses to predict tolerance to lime-induced chlorosis. Licensee MDPI, Basel, Switzerland. This article is an open access article Keywords: in vitro culture; Pyrus spp.; iron chlorosis; pear rootstocks distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Horticulturae 2022, 8, 551. https://doi.org/10.3390/horticulturae8060551 https://www.mdpi.com/journal/horticulturae Horticulturae 2022, 8, 551 2 of 15 1. Introduction Iron is an essential element for plants and it is vital for photosynthesis, respiration, and plant growth. Despite its high abundance in the Earth’s crust, Fe is frequently inaccessible to plants as it tends to form insoluble ferric hydroxide complexes under aerobic and alkaline conditions. Plants grown in calcareous soils cope with Fe deficiency leading to reductions in growth, crop yield, and quality. Under low Fe availability, iron deficiency chlorosis (IDC) is a common growth limiting condition for plants. This iron chlorosis is caused by the low availability of iron in soils with a high content in calcium carbonate and high pH. In fruit crops, this nutritional disorder limits growth, affects fruit quality, and causes great economic losses in a wide range of fruit crops, such as peach, pear, quince, kiwifruit, and grapes cultivated in the Mediterranean basin [1–6]. The abundance of calcareous soils and limestone in the Mediterranean area and worldwide, buffers the soil pH, neutralizing the acidification response carried out by the roots of some plant species, which aggravates the low availability of iron in the soil [7]. Iron deficiency and a decrease in the iron redistribution capacity from adult leaves to growing parts leads to a reduction in photosynthetic activity, subsequent growth inhibition of the plant, and a decline in the crop [5,8,9]. The use of rootstocks tolerant to lime-induced chlorosis is the best alternative to prevent iron chlorosis in fruit crops [6,10,11]. Starting in 1998 in INRAE [12] and to date, a breeding program is going on for selecting pear hybrid rootstocks, generated from interspecific crosses between species with desirable characteristics to overcome soil and disease problems in Mediterranean environments [13]. Fruit trees are classified as strategy I plants, which under iron deficiency conditions develop morphological and biochemical changes aimed at enhancing its acquisition from the soil [14]. Some morphological changes include the development of lateral roots, for- mation of root hairs, and differentiation of specialized transfer cells to increase the surface area between the plant and the soil for improving the iron uptake. The biochemical and physiological changes can include local rhizosphere acidification, synthesis of organic acids, and reducing the capacity of Fe3+ [3,15–17]. This acidification helps to increase the solubility of Fe(III) complexes in the soil, followed by the reduction of Fe(III) to Fe(II) and the subsequent uptake by the roots. After acidification, this reduction is performed by the membrane-bound ferric chelate reductase FRO2 (ferric reductase oxidase 2). Iron is then taken up into root epidermal cell layers by the specialized root Fe2+ iron transporter IRT1 [18]. The reduction from Fe(III) to Fe(II) appears to be crucial; a tight regulation of Fe homeostasis is essential, and it is strictly controlled by regulating iron uptake, transport, and storage. A temporal solution to iron deficiency in high pH calcareous soils is the periodic application of chelated iron in its reduced form. However, a more desirable permanent strategy is breeding for genotypes tolerant to lime induced chlorosis. In fruit trees, the selection of rootstocks is performed by obtaining genotypes which confer tolerance to the grafted varieties. For this reason, peach varieties are grafted on rootstocks, which are hybrids between almond (Prunus amygdalus Mill.) that are more tolerant to calcareous soils, and peach (P. persica L.), which confer good compatibility with the varieties. The currently existing pear rootstocks are clones of Pyrus or Cydonia. Quince rootstocks can be unattractive because of their incompatibility with some varieties and their suscepti- bility to calcareous soils. Pyrus rootstocks have a better tolerance to these soils, are fire blight tolerant, and compatible with pear varieties. A breeding program using interspe- cific hybridization between ‘Pyriam’ and diploid Pyrus species [19] has been developed. A part of this program [12], dealing with higher tolerance to global warming, is done in collaboration with IRTA, and it is under development to improve the tolerance to lime induced chlorosis in Spain [20,21]. Four Mediterranean species belonging to INRAE Angers genetics resources have been chosen for their tolerance to drought, hot summers, and to iron chlorosis [22]. As reported in [21], and more recently [9], some Pyrus genotypes, compared with quince (Cydonia oblonga), are able to improve the acquisition of Fe through increasing the Horticulturae 2022, 8, 551 3 of 15 enzymatic reduction of Fe(III) through the ferric chelate reductase activity and acidification of the root apoplast, with a higher activity of H+ -ATPase. The possibility of simulating in vitro the Fe-limiting environmental conditions caused by high pH, calcareous soils, and the induction of genetic variation has previously been used [21] to generate quince clones with a higher tolerance to lime-induced chlorosis. Their superior performance was subsequently checked under field conditions [1]. Similar in vitro culture conditions were used in the present work, first to study the differential physiological responses of Pyrus interspecific hybrids to lime-induced chlorosis, and second to simplify and shorten the in vitro selection protocol for future selection of seedlings or clones. 2. Material and Methods Plant Material. Twenty-one clones were established in vitro between 2012 and 2014 from field plots of the pear rootstocks breeding program, oriented to select the most tolerant to iron chlorosis after being grafted with the commercial variety ‘Conference’ and in comparison with Cydonia oblonga ‘BA29’ [2,23]. Twenty of these clones were derived from interspecific crosses between OH11, also named ‘Pyriam’ [24], and four Pyrus species, namely P. amygdaliformis (Pa), P. amygdaliformis spp persica (Pap), P. communis spp cordata (Pcc), and P. elaeagrifolia (Pe). These Pyrus parents were selected for their higher field tolerance to iron-chlorosis, drought, hot summers and sandy soil than P. communis [22]. Two clones (coded 74 and 170) were ‘Pyriam’ x Pa hybrids, eight clones (coded 66, 101, 103, 105, 109, 123, 129, and 162) were ‘Pyriam’ x Pap hybrids, seven clones (coded 25, 32, 36, 38, 45, 87, and 88) were ‘Pyriam’ x Pcc hybrids, and three clones (coded 55, 58, and 111) were ‘Pyriam’ x Pe hybrids. In addition, a clone (coded 12) derived from open pollinated P. communis ‘Williams’ (Pc) was included. In vitro culture micropropagation and rooting. The protocol, culture medium, and con- ditions used for the multiplication were as described in [13]. Basically, the Murashige and Skoog media [25], supplemented with sucrose (30 g·L−1 ), myo-inositol (100 mg·L−1 ), tiamina-HCl (1 mg·L−1 ), nicotinic acid (1 mg·L−1 ), pyridoxine-HCl (1 mg·L−1 ), 5 µM Benzy- laminopurine (BAP), pH adjusted to 5.7, and Difco Bacto-agar (7 g·L−1 ), was used to induce vegetative growth. Cultures were kept at a temperature between 24 and 26 ◦ C and with a photoperiod of 16 h day/8 h night (100–150 µEm−2 s−1 ). Before rooting, shoot elongation was achieved using the double phase culture system as previously described [4]. Rooting of 4- to 6-cm-long shoots was induced by culturing shoots in half strength Murashige and Skoog media, supplemented with 10 mM Fe-EDTA and 10 uM of the auxin Indolebutyric acid (IBA). Cultures were kept in the dark for 7 days and then transferred to an auxin free medium for three more weeks under light conditions. In vitro assays. Plantlets with a developed root system and with three to four new leaves (Figure 1A) were selected for the lime-induced chlorosis in vitro test as earlier reported [21], using a medium described by [26]. Twenty-four plants per genotype and treatment were submitted from 2012 to 2016 to different conditions by adding 10 or 20 mM NaHCO3 and 2 µM Fe(III)DTPA. The conditions of iron deprivation were achieved by transferring the plants to bottles Magentas® (Figure 1B) with chromatography paper bridges (WhatmanTM , GE Heathcare UK Limited, Maidstone, UK) to give stability to the plant as described in [2] and adding 25 mL of liquid medium, which was renewed twice a week. Horticulturae2022, Horticulturae 2022,8,8,551 x FOR PEER REVIEW 5 of 1615 4 of Figure 1. Rooted plant material used for the in in vitro vitro iron iron deficiency deficiency assay (A) (A) in in liquid liquid cultures cultures with a a paper paper bridge bridge (B)(B) at at dayday 0 the 0 of of the assay, assay, andand after after a 28-day-long a 28-day-long culture culture under under ironiron deficiency deficiency con- conditions ditions in medium with 20 mM NaHCO 3 (C,D), showing high leaf chlorophyll levels in the case of in medium with 20 mM NaHCO3 (C,D), showing high leaf chlorophyll levels in the case of ‘Pyriam’ ‘Pyriam’ x Pyrus communis spp cordata hybrid Pcc38 (C) or leaf chlorosis in the open pollinated P. x Pyrus communis spp cordata hybrid Pcc38 (C) or leaf chlorosis in the open pollinated P. communis communis ‘Williams’ Pcw12 (D). Bars are 1-cm-long. ‘Williams’ Pcw12 (D). Bars are 1-cm-long. Horticulturae 2022, 8, 551 5 of 15 Total chlorophyll level. The chlorophyll level was measured, using a SPAD-502 meter (Minolta Co., Osaka, Japan) on the first and the second expanded leaves, starting from the shoot apex. Culture medium pH. The pH of the nutrient solution was measured with a pH-meter on days 1, 3, 7, and 28 of culture when the nutrient solution was renewed with a fresh solution, under sterile conditions. Iron reduction. The ferric chelate reductase (FC-R) activity was monitored in all geno- types following the protocol adapted by [10]. Four plants per genotype and the treatment were incubated in amber vials containing 11 mL of fresh nutrient solution, with 0.3 mM BPDS and 100 µM Fe(III)-EDTA solution pH 6.0 at a temperature of 24–26 ◦ C, photoperiod 16 h light / 8 h dark (100–150 µEm−2 s−1 ) for 24 h. The ferric chelate reductase (FC-R) activity was measured in absorbance at 535 nm with a spectrophotometer. The amount of reduced iron was calculated from the concentration of the complex Fe(II)-(BPDS) using an extinction coefficient of 22.14 mM·cm−1 , and it was expressed in nmol Fe(II) reduced g−1 FW root h−1 [13,23]. This determination was performed on days 1, 3, 7, and 28. Total phenols. Phenolics were extracted according to [27] with modifications [28,29]. In brief, shoots were extracted with 70% methanol and after centrifugation (10 min, 5000× g) the supernatant was reextracted three times with ethyl ether to eliminate ether-soluble lipids. The remaining water phase was treated with 2 M HCl for acid hydrolysis of soluble conjugated phenolics. After extraction with ethyl acetate and drying, the residue was redissolved in 50% methanol. Concentrations of phenolic substances in this extract were determined with an HPLC system (Shimadzu LC-10AT) equipped with a diode-array detec- tor (250–400 nm, Shimadzu SPD-M10Avp), using a C18 Nucleosil® column (Sigma-Aldrich, Madrid, Spain) and a gradient solvent-elution (2% acetic acid in water and acetonitrile- water-acetic acid, 80:18:2). The analysis of total free phenols in the samples was performed according to the method for the determination of total phenols by Folin–Ciocalteu spec- trophotometry [30]. The determination was made by comparing the absorbance to an external reference phenol standard curve of gallic acid. Results were expressed in GAE (Gallic Acid Equivalents). Extraction and analysis of simple organic acids. Root samples of 1 g FW were homogenized in a porcelain mortar with 0.025 N HCl, vortexed, centrifuged at 10,000 rpm for 20 min at 4 ◦ C, and the supernatant was recovered. This supernatant was passed through a Sep-pack Plus C18 cartridge (Waters) previously activated with MeOH, Milli-Q Water and HCl 0.025 N, and then filtered through a Millex 0.22 µm (Millipore, Burlington, MA, USA) directly into a glass HPLC vial [31]. Extracts were analyzed by an HPLC system (LC-10 AT, Shimadzu Corporation, Kyoto, Japan) coupled with a C18 AtlantisTM column (Waters Corporation, Milford, MA, USA). Analytical conditions were as follows: 10 µL of injection volume, 0.5 mL min−1 flow rate, 30 ◦ C for column temperature, NaH2 PO4 (pH 2.7) as mobile phase, 20 min of run time, and 210 nm wavelength detection [32]. Identification of the organic acids in the samples was achieved by comparison of component retention times in standard solutions. To quantify the organic acids in the extracts, the integrate peak area of known concentrations of the standards were used to build a calibration curve. Statistical Analysis of the Results Each experiment with a completely randomized design consisted of four randomly selected rooted plantlets per clone and treatment. The responses of all clones, derived from the same inter-specific hybrid, were pooled together to analyze the results. Statistical tests included analysis of variance, separation of means by Fisher’s protected LSD, and planned contracts [33] using the software JMP® (SAS Institute Inc., Cary, NC, USA). 3. Results and Discussion Total chlorophyll level. Genotypic differences were observed on the leaf chlorophyll lev- els under iron deprivation, indicating a differential susceptibility to lime-induced chlorosis Horticulturae 2022, 8, 551 6 of 15 (Figure 1C,D). Increasing the level of bicarbonate, which rises the pH buffering capacity of the culture medium, resulted in a further decrease in the leaf chlorophyll content, and it better discriminated the most tolerant hybrids. When grown in the lower concentration of NaHCO3 (10 mM), plantlets belonging to hybrids with Pa, Pap, Pcc, and Pe showed similar behavior during the time of conditioning (Figure 2A), significantly (p = 0.006) different to Pcw. While for the former group of hybrids, Horticulturae 2022, 8, x FOR PEER REVIEW 7 of716 the leaf chlorophyll level decreased during the first 3 days, it recovered starting at day and Horticulturae 2022, 8, x FOR PEER REVIEW reached a maximum at the end of the assay. Contrastingly, the Pcw presented a continuous Horticulturae 2022, 8,in decrease x FOR PEERchlorophyll the leaf REVIEW level which never recovered to the initial values. Horticulturae 2022, 8, x FOR PEER REVIEW Horticulturae 2022, 8, x FOR PEER REVIEW Figure Figure 2. Total 2. 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