Leaf surface features of maize cultivars and response to foliar phosphorus application: effect of leaf stage and plant phosphorus status

ORIGINAL RESEARCH Leaf surface features of maize cultivars and response to foliar phosphorus application: effect of leaf stage and plant phosphorus status Jon Niklas Henningsen 1 | Martin David Venturas 2 | José Manuel Quintero 3 | Ramiro Recena Garrido 3 | Karl Hermann Mühling 1 | Victoria Fernández 2 1 Institute of Plant Nutrition and Soil Science, Kiel University, Kiel, Germany 2 Systems and Natural Resources Department, School of Forest Engineering, Madrid, Spain 3 Departamento de Agronomía, ETSIA, Universidad de Sevilla, Sevilla, Spain Correspondence Jon Niklas Henningsen, Email: [email protected]; Victoria Fernández, Email: [email protected] Edited by S. Husted Abstract Soil phosphorus (P) application is the most common fertilisation technique but may involve constraints due to chemical fixation and microbial immobilisation. Furthermore, excessive P fertilisation leads to P runoff into water bodies, threatening ecosystems, so targeted foliar P fertilisation is an interesting alternative. This study aimed to determine the importance of leaf surface characteristics for foliar P uptake in P-deficient maize (Zea mays L.). The leaf surface of four maize cultivars was characterised by electron microscopy, Fourier transform infrared spectroscopy and contact angle measurements. Uptake of foliar-applied P by maize cultivars was estimated, measuring also leaf photosynthetic rates after foliar P spraying. Plants of cultivar P7948 were found to be wettable from the 4th leaf in acropetal direction, whereas other cultivars were unwettable until the 6th leaf had developed. Minor variations in stomatal number and cuticle composition were recorded, but no differences in foliar P absorption were observed between cultivars. Nevertheless, cultivars showed variation in the improvement of photosynthetic capacity following foliar P application. Phosphorus deficiency resulted in ultrastructural disorganisation of mesophyll cells and chloroplasts, which impaired photosynthetic performance, yet there was no effect on stomatal frequency and leaf wettability. This study provides new insights into the influence of P deficiency and cultivar on leaf surface characteristics, foliar P uptake and its effect on physiological processes. Understanding the relationships between leaf characteristics and P uptake allows a more targeted evaluation of foliar P fertilisation as an application technique and contributes to the understanding of foliar uptake mechanisms. 1|INTRODUCTION In crop production systems, phosphorus (P) is mostly provided to plants by soil application at the time of sowing or in spring, at the beginning of vegetative growth. After fertilisation, large amounts of P are accumulated in the soil, of which only about 15–30% is taken up by plants in the year of application due to biological and chemical adsorption processes (Syers et al., 2008; Cordell & White, 2015). Conventional fertilisation practices thus threaten the stability of globally significant ecosystems due to potentially high losses of P, mostly through surface runoff and erosion (Withers et al., 2014; Steffen et al., 2015; Campbell et al., 2017). Furthermore, low plant availability often leads to P deficiency and limits the growth and quality parameters of crops (Hawkesford et al., 2023). Foliar fertilisation provides the Received: 21 June 2023 Revised: 6 November 2023 Accepted: 10 November 2023 DOI: 10.1111/ppl.14093 Physiologia Plantarum This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2023 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society. Physiologia Plantarum. 2023;175:e14093. wileyonlinelibrary.com/journal/ppl 1of13 https://doi.org/10.1111/ppl.14093 potential to complement conventional soil fertilisation with a more targeted and thus more sustainable, but also more demand-oriented application of nutrients. This technique is increasingly used worldwide (Fernández et al., 2021) and for P, foliar fertilisation has been investigated in several studies in recent years (Noack et al., 2010;Peirce et al., 2014;Görlachetal.,2021;Görlach&Mühling,2021; Arsic et al., 2022; Henningsen et al., 2022,2023). Despite the increasing knowledge, there is still substantial uncertainty about the underlying mechanisms of foliar nutrient uptake into plants (Read et al., 2020), as complex interrelationships of physico-chemical, physiological and environmental factors are involved (Fernández et al., 2021). To be absorbed and subsequently utilised by the plant, foliarapplied nutrients have to penetrate the plant surface (Fernández & Eichert, 2009). Aerial plant parts with primary growth are covered with a cuticle (Jeffree, 2006; Shellakkutti et al., 2022). This specialised part of the primary cell wall is generally rich in lipids such as waxes and cutin, but may also contain cell wall polysaccharides and small amounts of phenolics and mineral elements (Guzmán et al., 2014; Guzmán-Delgado et al., 2016; Philippe et al., 2020; González Moreno et al., 2023). While lipophilic components i.e., cutin and waxes, are hydrophobic, polysaccharides have high polar and hydrogen-bonding components which can adsorb water, leading to swelling of the cuticle, potentially facilitating the transport of nutrients and electrolytes (Chamel et al., 1991; Riederer, 2006; Fernández et al., 2016,2017). Stomata and trichomes are epidermal structures which often occur in plant leaves and may play a major role in foliar uptake (Fernández et al., 2021). Regarding the absorption of solutions/suspensions through stomata, substances most probably diffuse along a thin liquid film via the guard cell walls into the leaf interior (Eichert & Goldbach, 2008; Eichert et al., 2008). Uptake via trichomes potentially depends on many factors which have not been systematically analysed yet due to their physico-chemical complexity. While in soybean leaves glandular trichomes have taken up zinc (Zn), this could not be confirmed for trichomes of tomato leaves (Li et al., 2018a). Recently however, especially for trichomes located on leaf veins/bundle sheath extensions, nutrient uptake and subsequent transport via the vascular bundles has been demonstrated (Li et al., 2019; Schreel et al., 2020). On the other hand, few studies performed with beech, barley and tomato showed that veins can be the major foliar uptake pathway in some leaves (Bahamonde et al., 2018; Arsic et al., 2020,2022; Henningsen et al., 2023). Cuticle and epidermal structures may not only influence foliar uptake but also spray drop-leaf surface interactions as a result of surface chemical composition and structure (Fernández et al., 2017). In this regard, a major effect of leaf surface on leaf wettability has been reported due to micro-scale, owing to the occurrence of e.g., trichomes, stomata, papillae or cuticular folds (Almonte et al., 2022) and nano-scale roughness, due to crystalised epicuticular waxes (Koch et al., 2008; Koch & Barthlott, 2009; Barthlott et al., 2017). The combination of surface chemistry and structure will determine whether foliar spray droplets will adhere or be repelled by plant surfaces (Fernández et al., 2021). Spray drop adhesion to the foliage is a prerequisite for leaf penetration, and leaf surface physico-chemical attributes for each species can be estimated by measuring static and dynamic contact angles, surface free energies and other variables which help to optimise the design of foliar spray formulations in the future (Fernández et al., 2017,2021; Henningsen et al., 2023). Nutrient deficiencies can alter leaf structure-chemical composition and thus, the absorption capacity of leaf surfaces as shown in some studies (Fernández et al., 2014; Arsic et al., 2022). Stomatal and trichome densities may be affected by nutrient status, leading to e.g., reduced stomatal frequencies in lemon (Eichert & Fernández, 2023) or decreased stomatal pore sizes and apertures in peach and pear as a consequence of iron (Fe) deficiency (Fernández et al., 2008). Boron (B)-deficient coffee (Rosolem & Leite, 2007) and soybean (Will et al., 2011) leaves had an altered stomatal morphology and in sunflower, stomatal and trichome densities were reduced as a result of Zn deficiency (Li et al., 2019). Lower foliar nutrient uptake of the limiting nutrient supplied to the foliage have been reported in K-deficient olive (Restrepo-Diaz et al., 2008), B-deficient soybean (Will et al., 2011) and Zn-deficient sunflower (Li et al., 2019) leaves compared to adequately nourished leaves (Eichert & Fernández, 2023). Recent studies have also investigated the effects of P deficiency on leaf structure and P uptake. Fernández et al. (2014) were able to show that although the leaves of highly P-deficient wheat plants were more wettable, the permeability for P was reduced and thus no benefit could be achieved by foliar fertilisation. In barley, too, nutrient functionality in the plant was not restored by foliar P fertilisation (Arsic et al., 2022). This also applies to maize plants, which absorbed significant amounts of P via the leaves, but as a result the photosynthetic rate only rose above the level of P-deficient control plants for a limited period of time (Henningsen et al., 2022). However, these studies have focused on one variety of the respective plant species. But species can differ considerably in their morphological and physiological characteristics so that comparisons between them are influenced by many factors. Different varieties of the same species, on the other hand, are fundamentally very similar, but can also show differences, for example in the composition of the leaf surface. Therefore, identifying variety differences in leaf surface properties and characterising their influence on the uptake of foliar-applied P could provide revealing insights into foliar solute uptake mechanisms. Taking into consideration the potential variations in leaf surface characteristics of different crop varieties which may ultimately affect the rate of absorption of foliar sprays, an issue which has basically been ignored so far, the objective of this study was to determine the influence of P deficiency on the leaf surface of four commercial cultivars, using maize as model species. This globally significant arable crop is particularly affected by P deficiency during the juvenile developmental stage. Thereby, in addition to characterising the leaf surface features of the selected varieties and their response to P deficiency, we aimed at identifying potential correlations between leaf surface traits and P uptake, also following and ecophysiological approach. The hypotheses to be tested were: (I) Leaf surface features dynamically change between different leaf developmental stages also in response to P deficiency, (II) leaf surface properties will differ between maize varieties both when cultivated under P sufficiency and P deficiency 2of13 HENNINGSEN ET AL. Physiologia Plantarum 13993054, 2023, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ppl.14093 by Readcube (Labtiva Inc.), Wiley Online Library on [16/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License conditions, and (III) foliar P absorption will be affected by differences in maize leaf surface properties. 2|MATERIALS AND METHODS 2.1 |Experimental setup The experiment was conducted in the greenhouse of the School of Forest Engineering at the Technical University of Madrid (4027005.3”N, 343022.5”W). Maize plants of four varieties (cv. Keops, KWS Saat SE & Co. KGaA, Einbeck, Germany; cv. DKC 3096, Bayer CropScience AG, Monheim, Germany; cv. P7948, cv. P8834, Pioneer Hi-Bred International Inc., Johnston, IA, USA) were grown under semi-controlled conditions from May to June. Radiation and relative humidity (RH) corresponded to the ambient environment, so the plants were exposed to a day length of 14.5–15 h and a RH of 35–95%. The air temperature was regulated to 20C at night and a maximum of 30C during the day. Seeds were germinated in perlite and transferred to soil. The soil was an alkaline (pH 8.34), sandy loam (Sl4) and had a P concentration of 0.35 mg P 100 g 1 soil (determined by Olsen P extraction) and was thus to be classified as P-deficient. For each pot, two litres of air-dried, sieved (<2 mm) soil (2890 g) were homogeneously mixed with one litre of perlite (112 g) to improve the soil structure and avoid waterlogging. Three uniform maize seedlings were then placed at a depth of 30–35 mm. For avoiding nutrient deficiencies excepting P in some of the treatments, all essential elements were added to each pot in the following amounts (in g pot 1 ): 0.5 N (as NH 4 NO 3 ), 0.6 K (as KCl), 0.6 K (as K 2 SO 4 ), 0.25 Ca (as CaSO 4 ), 0.17 Mg (as MgSO 4 ), 0.008 Fe (as Fe-EDDHA), 0.005 Cu (as CuSO 4 ), 0.008 Zn (as ZnSO 4 ), 0.015 Mn (as MnSO 4 ), 0.005 B (as H 3 BO 4 ), 0.001 Mo (as (NH 4 ) 6 Mo 7 O 24 ). Two weeks after the first application (21 days after sowing, DAS), a follow-up supply of nutrients was carried out in the same quantity. Potassium dihydrogen phosphate (KH 2 PO 4 ) was primarily used as a P source and its supply was adjusted depending on the variant. The pots were arranged following a completely randomised design and were re-randomised every week. Since this study focussed on characterising the surface properties and qualitative composition of maize leaves as affected by P nutrition and their influence on foliar P uptake, four commercial varieties (see above) with different characteristics were selected out of a preliminary trial with 16 varieties. When transferred to the soil (7 DAS), all four varieties were subdivided into four further treatments –(1) plants with sufficient P supply (0.3 g P pot 1 as KH 2 PO 4 , P-sufficient), (2) plants without additional P supply (P-deficient) which developed P deficiency symptoms throughout the entire growing period, (3) P- sufficient plants (as described for treatment 1) but sprayed with P 28 DAS (see details in Section 2.2.), (4) P-deficient plants (as described for treatment 2) but also sprayed with P 28 DAS, as described below. One day after P foliar application, plants were sampled for nutrient analysis (n =4). Further investigations to characterise the leaf surface as well as physiological processes were carried out as described hereafter. 2.2 |Foliar treatment Four weeks after sowing, at BBCH 13 for P-deficient plants and BBCH 14 for P-sufficient plants (Lancashire et al., 1991), i.e. three/four leaves unfolded, plants were sprayed with 200 mM KH 2 PO 4 and 0.1% Genapol X-80 (pH 6.6) as described by Henningsen et al. (2022). Genapol X-80 is a non-ionic surfactant that significantly reduces surface tension approximately 27 mN m 1 , thus significantly decreasing the contact angle of aqueous solutions to low values (Bahamonde et al., 2023). Genapol X-80 has been used at a 0.1% concentration in previous foliar fertilisation studies (e.g. Bahamonde et al., 2023; Barlas et al., 2023). To prevent soil P contamination due to KH 2 PO 4 spraying, the pots were covered with aluminium foil and absorbent paper. Then, in the early morning, the whole aerial parts of maize plants were sprayed by carefully applying the P fertiliser solution onto the upper (adaxial) and lower (abaxial) leaf side until complete leaf surface wetting. 2.3 |Contact angle measurements and estimation of leaf surface properties Equilibrium contact angles were measured by using a drop shape analysis system (DSA 100, Krüss). Leaf segments from the middle of intact, youngest fully expanded leaves (YFEL) were cut out with a scalpel, excluding the midrib and leaf margin, and were attached to microscope slides with double-sided tape with the adaxial or abaxial leaf side facing the air interface. Drops of approximately 1.5 μl of deionised water and diiodomethane (99% purity, Sigma-Aldrich) were deposited onto these leaf segments with a 1 ml syringe and a 0.5 mm diameter needle (n =16). The YFEL corresponded to the 3rd leaf in P-deficient and the 4th leaf in P-sufficient variants. The total surface free energy (γ) and its components were calculated as described by Fernández et al. (2011,2014). At a later stage, in addition to the YFELs, the contact angles of all leaves, from the 3rd/4th to the 8th, were measured on plants of variety DKC 3096 and P7948 (n =14). 2.4 |Electron microscopy Segments from the centre of intact 3rd/4th leaves were selected to characterise the leaf surface and anatomy of leaves in relation to P nutritional status using SEM and TEM (n =3). Samples of adaxial and abaxial leaf surfaces were analysed either intact or after critical point drying. For this purpose, leaf segments of about 10 mm 2 were cut out with a scalpel and immediately immersed in phosphate buffer (pH 7.2). After 4 h the segments were dehydrated in a series with pure ethanol (30, 50, 70, 80, 90, 100%; Merck) and distilled water. After completion of the dehydration series, critical point drying (Leica EM CPD300, Leica Microsystems) was performed. The dried samples were then coated with gold (Au; Sputter Quorum Q150T-S, Quorum Technologies) and observed by SEM (Jeol JSM6400, Jeol Ltd.) at the National Electron Microscopy Centre (CNME, Complutense University, Madrid, Spain). Intact, fresh leaf segments were also observed using a HENNINGSEN ET AL.3of13 Physiologia Plantarum 13993054, 2023, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ppl.14093 by Readcube (Labtiva Inc.), Wiley Online Library on [16/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License field emission SEM (Carl Zeiss, Sigma 300 VP) at the facilities of Miguel Hernández University (Elche, Spain). For TEM, leaf samples were cut into 4 mm 2 pieces and fixed in phosphate buffer for 6 h to be then post-fixed in a 1:1 solution of osmium tetroxide (2%, TAAB Laboratories) and potassium ferrocyanide (3%, Sigma-Aldrich) for 1.5 h. After washing several times with distilled water, the samples were then dehydrated in a series of acetone (30, 50, 70, 80, 90, 95, 100%) and subsequently immersed in an acetone-Spurr's resin (TAAB Laboratories) mixture of increasing concentration (3:1 for 2 h, 1:1 for 2 h, 1:3 for 3 h, pure resin for 12 h). Finally, the pieces were placed in blocks of pure Spurr's resin and left at 70C until complete polymerisation. Ultra-thin sections were then cut (Leica Ultracut E, Leica Microsystems) and observed with a Jeol JEM 1400plus (Jeol Ltd.) at the CNME. 2.5 |Physiological parameters A portable open-flow gas exchange system (LI-6400, LI-COR Biosciences) was used to measure the net photosynthetic rate in lightadapted plants (n =4). An area of 2 cm 2 was measured in the middle of the 3rd/4th leaf excluding the midrib. Gas exchange measurements were performed at 2000 μmol m 2 s 1 photosynthetic photon flux density (PPFD) using the LED light source (LI-6400-02, LI-COR Biosciences). The flow rate was set to 400 μmol s 1 and the CO 2 concentration of the incoming air was regulated to 415 μmol mol 1 by CO 2 injection (LI-6400-01, LI-COR Biosciences). The temperature in the measuring cuvette was set to 20C. 2.6 |Fourier transform infrared (FTIR) spectroscopy Infrared spectra of critical point dried leaf samples were obtained with an attenuated total reflectance (ATR) accessory (MIRacle ATR; PIKE Technologies) coupled to an FTIR spectrometer (Nicolet Nexus 670–870; Thermo Fisher Scientific). Spectra of intact samples were recorded in transmission mode in the range 4000–400 cm 1 with 4 cm 1 resolution and accumulating 64 scans and analysed with Omnic v4.1b (Thermo Fisher Scientific) software. Two replicates per sample were analysed. 2.7 |Leaf P determination One day after the foliar P spraying, one plant per replicate (n =4) was sampled for tissue P analysis. Leaves were first cleaned following a three-step washing procedure which involved leaf surface scrubbing with acidulated 0.1% detergent, followed by a thorough rinse in tap and distilled water (Bahamonde et al., 2023). Subsequently, leaves were oven-dried at 65C until they were constant in weight and were then ground, dry-ashed and digested with acid prior to mineral element determination by inductively coupled plasma optical emission spectroscopy (ICP-OES, Optima 3000, PerkinElmer), following the UNE-EN ISO/IEC 17025 standards for calibration and testing laboratories (CEBAS-CSIC Analysis Service) as previously described by Henningsen et al. (2023). 2.8 |Statistical analysis The statistical software R (version 4.0.2, R foundation for statistical computing) was used for result analysis. Data evaluation started with the definition of an appropriate statistical mixed model (Pinheiro & Bates, 2000). Based on this model, a Pseudo R 2 was calculated (Nakagawa & Schielzeth, 2013) and an analysis of variances (ANOVA) was conducted, followed by multiple contrast tests (Hothorn et al., 2008; Bretz et al., 2016). 3|RESULTS 3.1 |Leaf surface features 3.1.1 | Stomatal and trichome density and plant biomass The stomatal density in maize plants was dependent on leaf side and variety, but independent of soil P supply and thus plant P nutrient status. While the stomatal density of the adaxial leaf side ranged between 35 and 46 stomata mm 2 depending on the variety, there were significantly (Ø 65%) more stomata on the abaxial side with a number of 58–75 stomata mm 2 . Significant differences between the varieties occurred exclusively on the abaxial leaf side (Figure 1, Table S2). Thereby, the stomatal density in P-deficient plants was significantly lower in variety DKC 3096 than in P7948 and P8834. In the case of sufficient P nutrition, DKC 3096 also had the lowest stomatal density, but only P7948 had significantly more (Figure 1A, Table S2). Thus, on the abaxial side of the leaf, DKC 3096 had the fewest stomatal numbers and P7948 the highest. At the sampling stage, hardly any trichomes had developed yet. FIGURE 1 Stomatal densities on adaxial and abaxial leaf surfaces of four maize varieties. (A) 3rd leaf of P-deficient and (B) 4th leaf of P-sufficient plants. Letters (A, a-b, X or x-y) show differences between the varieties of a same leaf side and P supply according to multiple contrast test (P ≤0.05). Data are means ± SD (n =3). 4of13 HENNINGSEN ET AL. Physiologia Plantarum 13993054, 2023, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ppl.14093 by Readcube (Labtiva Inc.), Wiley Online Library on [16/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 2 Representative SEM micrographs of the adaxial leaf surface of maize YFEL. (A, B) variety Keops, (C, D) variety DKC 3096, (E, F) variety P7948, and (G, H) variety P8834. YFEL represent the the 3rd leaf of deficient (A, C, E, G) and 4th leaf of sufficient (B, D, F, H) supplied maize plants with P. For each leaf surface, representative schematical droplets are additionally shown, making it clear that the leaf surface without wax crystalloids has resulted in lower contact angles. HENNINGSEN ET AL.5of13 Physiologia Plantarum 13993054, 2023, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ppl.14093 by Readcube (Labtiva Inc.), Wiley Online Library on [16/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Note in the following sections that the 4th leaf was sampled for P-sufficient plants and the 3rd leaf was sampled for P-deficient plants. Both the 3rd and 4th leaves corresponded to the YFEL of their P supply level. If it is not explicitly mentioned, these leaf stages have always been sampled. The sampling of different leaf stages can be explained by the retarded growth and organ development of P-deficient plants. The dry weight (DW) was significantly lower in P-deficient plants than in P-sufficient plants, but within the different P supply regimes there were no significant differences between the cultivars, except for P-deficient Keops having a lower dry weight than DKC 3096 and P7948 (Figure S1). 3.1.2 | Cuticle structure The surface of P-deficient leaves was covered with crystallised, nanoscale epicuticular waxes (Figure 2A, C, E, G). The structure of wax crystalloids could be characterised as membranous platelets or plates and they were fixed to the cuticle in an unstructured way (Barthlott et al., 1998). In the varieties Keops, DKC 3096 and P8834, no significant changes in the wax structure occurred with sufficient P nutrition (Figures 2B, D, H), whereas in P7948 the crystalloids disappeared completely and only a smooth layer of wax remained (Figure 2F; Barthlott et al., 1998). Examination of the 5th leaf of all cultivars also showed the absence of epicuticular wax crystals exclusively in cultivar P7948, indicating that cultivar and leaf stage rather than P status had an influence on the development of epicuticular waxes (Figure S2). Only the adaxial side of the leaf was shown here, as the leaf sides did not differ except for the stomatal density. In further analyses, varieties Keops, DKC 3096 and P8834 showed very similar characteristics, as in wax morphology, which is why only one of these varieties (DKC 3096) is compared with the differing variety P7948 in the following paragraphs. 3.1.3 | Contact angles and surface free energy In Figure 3, the contact angles of P-deficient plants from the 3rd–8th leaf (Figure 3A, B) and for P-sufficient plants for the 1st and from the 4th–8th leaf (Figure 3C, D) are shown. The contact angles of 3rd leaves of varieties DKC 3096 and P7948 were within the same range (138.4for DKC 3096, 137.2for P7948). For variety P7948, the contact angle and thus the wettability of the 4th leaf decreased significantly compared to the 3rd leaf, whereas for variety DKC 3096 the contact angle remained constantly high until the 5th leaf and FIGURE 3 Contact angles of water (θ w ) with the adaxial leaf surfaces of six leaf levels. (A, C) variety DKC 3096 and (B, D) variety P7948 with (A, B) deficient and (C, D) sufficient P supply. Letters (A-C or W-Z) show differences between the leaf stages of a same varieties according to multiple contrast test (P ≤0.05). Data are means ± SD (n =14). 6of13 HENNINGSEN ET AL. Physiologia Plantarum 13993054, 2023, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ppl.14093 by Readcube (Labtiva Inc.), Wiley Online Library on [16/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License only decreased significantly from the 6th leaf in acropetal direction (Figures 2C-F,3). As the contact angles for P-deficient and -sufficient plants were similar (Figure 3), the wettability seemed to be less dependent on P supply and was rather influenced by variety and leaf stage as major factors. Thus, the variety without epicuticular wax crystal formation from the 4th leaf (i.e., P7948; Figure 2F,S2C)alsoshowedstronglydeviating contact angles and thus wettability (Figure 3B, D). However, the change in surface characteristics and contact angles was not radical but rather involved a gradual transition between wettable and unwettable lamina zones, so that these areas were occasionally located close to each other on the same leaf (4th leaf for P7948, 6th leaf for DKC 3096; Figures 4,S3). These transition zones were mostly found at the leaf tips or extended from the midrib to the leaf lamina and were even visible to the bare eye (Figure 4A,S3). This heterogeneity resulted in different liquid drop adsorption characteristics within the same leaf. Thereby, the liquid application technique played a major role. While carefully placing droplets led to their adherence to areas having high water contact angles, rapid deposition of drops onto the surface led to repellence, droplets rolled off the leaves. In areas with low contact angles, on the other hand, the drops adhered to the leaf surface, regardless of the application technique. The total surface free energy and its components (dispersive and non-dispersive) also differed for the plants supplied with P (4th leaf) of variety P7948 compared to the rest of the varieties. Both for the adaxial and abaxial leaf surfaces, the surface free energy was about 4–5 times higher for P7948. When comparing Keops, DKC 3096 and P8834, no major differences were found between these varieties, nor within the varieties when comparing P supply regimes or leaf sides (Table S5). 3.2 |Influence of P deficiency on chemical composition of cuticles and cell ultrastructure 3.2.1 | Chemical composition of the cuticle Critical point dried leaf sections were analysed by FTIR. Cuticle spectra (Figure 5) indicated a higher presence of polysaccharides in cultivar P7948 than in DKC 3096, with P-deficient leaves of both varieties having higher presence than P-sufficient ones. Furthermore, peaks linked to long-chain aliphatic compounds which can be attributed to the occurrence of cutin and waxes were higher for P-deficient plants than for P-sufficient plants, independently of the variety analysed, this difference being particularly pronounced in variety DKC 3096. Absorbance at 1625 cm 1 also differed depending on the variety and was related phenolic compounds or cutan (Heredia-Guerrero et al., 2014; Guzmán-Delgado et al., 2016; Zhuang et al., 2020). 3.2.2 | Plant cell ultrastructure The cross-section ultrastructure of maize leaves as affected by P deficiency is shown for variety DKC 3096 as an example (Figure 6). Leaves were covered by a thin cuticle of approximately 50 nm thickness depending on the epidermal cell wall area and no effect of P deficiency was noticed regarding cuticular ultrastructure (Figures 6A, B). However, a limited supply of P had a major impact on leaf mesophyll and bundle sheath cells which had fewer, smaller and malformed chloroplasts (Figure 6C). Indeed, some mesophyll protoplasts were separated from their surrounding cell wall and cell lysis was also observed in some leaf areas of P-deficient plants (Figure 6C). Similar results are shown for Keops in Figure S4. Biomass formation was reduced as a result of P deficiency, so that P-deficient plants only reached an average of 21% (Control) and 19% (Foliar P treatment) of the dry matter (DM) of P-sufficient plants. 3.3 |Foliar phosphorus absorption and physiological response 3.3.1 | Foliar phosphorus absorption A concentration of 200 mM KH 2 PO 4 plus 0.1% Genapol was applied to the plants to determine the foliar P uptake. The P concentration in the total plant depended on P supply (i.e., P-deficient versus -sufficient) and foliar P application, but was not influenced by maize plant variety FIGURE 4 Wettable and unwettable areas on the adaxial surface of the 4th leaf of maize variety P7948. Images in (A) original size and (B) 120x magnification by SEM showing areas covered with epicuticular wax crystals (high contact angle; ) next to areas without epicuticular wax crystals (low contact angle; ). HENNINGSEN ET AL.7of13 Physiologia Plantarum 13993054, 2023, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ppl.14093 by Readcube (Labtiva Inc.), Wiley Online Library on [16/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License (Figure 7A, B;TableS6). Consequently, for both varieties and P availability regimes, tissue P concentrations significantly increased as a result of foliar P application, with no significant foliar P uptake differences recorded between varieties (Figures 7A, B;TableS6). Furthermore, P concentrations show a similar absolute increment (i.e., the difference between the leaf P concentration in P sprayed and non-foliar treated plants) of tissue P for P-deficient and -sufficient plants after foliar P application (around 2 mg g 1 DW; Figure 7A, B). However, the rate of foliar P uptake of P-deficient and -sufficient plants cannot be derived from this, owing to the major leaf area and plant biomass difference between large P-sufficient and stunted P-deficient plants. The relative increase in P concentration (i.e. the share of the P concentration increase due to foliar spraying considering the total P concentration of the foliar P sprayed plants) is way higher for P-deficient plants with 240% (DKC 3096) and 211% (P7948) compared to P-sufficient plants which had a 40% (DKC 3906) and 39% (P7948) relative P concentration FIGURE 5 Averaged and normalized ATR-FTIR spectra of critical point dried YFEL for maize variety DKC 3096 and P7948 with deficient and sufficient P supply. FIGURE 6 TEM micrographs of variety DKC 3096. (A, B) cuticle and (C, D) leaf mesophyll and bundle sheath cells of (A, C) P-deficient and (B, D) -sufficient leaves. C =Cuticle, CL =Cell lysis. 8of13 HENNINGSEN ET AL. Physiologia Plantarum 13993054, 2023, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ppl.14093 by Readcube (Labtiva Inc.), Wiley Online Library on [16/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License change due to P spraying. Phosphorus-deficient plants thus took up high amounts of P, despite the negative effects of P deficiency on plant growth and leaf anatomical features (Figure 7A, B). In order to take the influence of biomass formation into account, the P content was also determined (Figure S5). Phosphorus-sufficient plants which have a larger biomass and a greater leaf area that can be covered by the foliar P spray, show a higher increase in P content compared to P-deficient plants (Figure S1,S5). As with P concentration, the difference between the varieties is not significant (Figures 7A, B,S5;TablesS6,S7). Foliar P uptake or absorption means in the following sections, if not mentioned else, the difference in tissue P concentration between untreated control plants and foliar P applied plants. 3.3.2 | Rate of photosynthesis Gas exchange measurements revealed a significant influence of P supply regime, foliar P application and maize variety on leaf net photosynthetic rates. Phosphorus-sufficient plants had a significantly higher net photosynthetic rate compared to P-deficient plants (Figures 7C, D). Foliar P application to P-deficient plants significantly increased the photosynthetic rate for variety P7948, but not for DKC 3096. However, despite the positive effect of P spraying on variety P7948 P-deficient plants, photosynthetic rates were still significantly lower than those of P-sufficient plants (Figures 7C, D). On the other hand, foliar P application to P-sufficient plants seemed to induce a slightly negative effect on leaf net photosynthesis (Figure 7D;TableS8). 4|DISCUSSION In this study, we examined the leaf physico-chemical properties of different commercial maize varieties, as affected by leaf stage and P status, assessing also the effect of foliar P spraying on leaf P concentrations and net photosynthesis. Out of the four cultivars analysed, three were found to follow a similar pattern of wettability, surface free energy and other leaf surface features also during plant development, which made us focus more in detail only on two varieties with markedly different properties, i.e., unwettable DKC 3096 and wettable P7948. An array of variations was recorded regarding the effect of P supply, variety type and response to foliar P application as described in the following paragraphs. 4.1 |P deficiency did not change stomatal density The P status of maize plants did not influence adaxial and abaxial leaf side stomatal frequencies (Figure 1) and only slight differences in the appearance of stomata were observed between the varieties. Regarding leaf trichomes, only small amounts of them were formed at the time of sampling which reduced their influence on the leaf parameters analysed in this investigation. Previous studies have linked a higher number of stomata (Schönherr & Bukovac 1978; Eichert & Burkhardt 2001; Eichert & Goldbach 2008)andthe presence of trichomes (Li et al., 2018b,2019,2021)withhigher nutrient uptake. As P deficiency did not affect the number of stomata, stomatal frequency was not a factor affecting foliar P absorption in P-deficient maize plants. However, P-deficient plants had a lower photosynthesis (Figure 7C, D) and stomatal conductance rate (data not shown) and we cannot discard the possibility that stomatal functionality in P-deficient plants may be altered as described for P-deficient wheat (Fernández et al., 2014), but this will require further investigation. The contribution of trichomes to foliar P uptake and leaf surface interactions in maize should be examined in future studies using maize plants at more advanced developmental stages, when more leaf trichomes have developed, as observed to occur in the studied cultivars from the 6th leaf upwards. They may play a major role in maize leaf wettability and be key for the adaptation of this species to different climatic areas of the world (Revilla et al., 2016). FIGURE 7 Tissue P concentrations and net photosynthesis of maize varieties DKC 3096 and P7948 one day after foliar application of 200 mM KH 2 PO 4 +0.1% surfactant. (A, B) P concentration and (C, D) net photosynthesis of (A, C) P-deficient and (B, D) P-sufficient maize plants. The dotted line is the lower limit for the tissue P concentration of P-sufficient maize according to Bergmann (1983). Differences between control plants and foliar P applied plants within the same variety and P supply are shown according to multiple contrast test. Data are means ± SD (n =4). * =p≤0.05; *** =p≤0.001. HENNINGSEN ET AL.9of13 Physiologia Plantarum 13993054, 2023, 6, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ppl.14093 by Readcube (Labtiva Inc.), Wiley Online Library on [16/01/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License