Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 1

Effect of wheat phosphorus status on leaf surface properties and permeability to foliar-applied phosphorus

Victoria Fernaacutendez Paula Guzmaacuten Courtney A E Peirce Therese M McBeath Mohamed Khayet Mike J McLaughlin V Fernaacutendez () P Guzmaacuten Genetics and Eco-physiology Research Group School of Forest Engineering Technical University of Madrid Ciudad Universitaria sn 28040 Madrid Spain For correspondence E-mail vfernandezupmes Tel +34 913367113 Fax +34 913572293 C A E Peirce M J McLaughlin School of Agriculture Food and Wine The University of Adelaide PMB 1 Waite Campus Glen Osmond SA 5064 Australia T M McBeath CSIRO Sustainable Agriculture Flagship CSIRO Ecosystem Sciences PMB 2 Glen Osmond SA 5064 Australia M Khayet Department of Applied Physics I Faculty of Physics University Complutense of Madrid Avda Complutense sn 28040 Madrid Spain M J McLaughlin CSIRO Sustainable Agriculture Flagship CSIRO Land and Water PMB 2 Glen Osmond SA 5064 Australia

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 2

Abstract Aims This study aimed to analyse the effect of phosphorus (P) nutritional status on wheat leaf surface properties in relation to foliar P absorption and translocation Methods Plants of Triticum aestivum cv Axe were grown with three rates of root P supply (equivalent to 24 8 and 0 kg P ha-1) under controlled conditions Foliar P treatments were applied and the rate of drop retention P absorption and translocation was measured Adaxial and abaxial leaf surfaces were analysed by scanning and transmission electron microscopy The contact angles surface free energy and work-of-adhesion for water were determined Results Wheat leaves are markedly non-wettable the abaxial leaf side having some degree of water drop adhesion versus the strong repulsion of water drops by the adaxial side The total leaf area stomatal and trichome densities cuticle thickness and contact angles decreased with P deficiency while the work-of-adhesion for water increased Phosphorous deficient plants failed to absorb the foliar-applied P Conclusions Phosphorous deficiency altered the surface structure and functioning of wheat leaves which became more wettable and had a higher degree of water drop adhesion but turned less permeable to foliar-applied P The results obtained are discussed within an agronomic and eco-physiological context Keywords Cuticle Foliar absorption Plant surfaces Trichomes Stomata Wettability

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 3

Introduction Grains from wheat plants provide an important food staple and phosphorus (P) is the second most limiting nutrient after nitrogen for wheat production (Batten 1992) Many soils of the world naturally maintain soil solution P at levels below the optimum for productive crop plants (Hedley and McLaughlin 2005) To sustain productive crops soils are commonly supplemented with P fertilisers at varying levels of efficiency as reviewed by Syers et al (2008) Wheat requires starter P at sowing to provide essential P for early growth and to replace P exported in the previous grain crop (Batten et al 1986 Grant et al 2001) Using starter P fertiliser often provides sufficient P to grow crops to tillering but in seasons of higher yield potential it is possible that supplemental in-season P application will increase yields (Noack et al 2010)

Soil application of supplemental in-season P is difficult to achieve efficiently as movement of P in soil is diffusion limited and therefore surface-applied P is not positioned for access by growing roots and in-season drilling of P would cause considerable crop damage This leaves foliar P fertilisation as the most practical avenue for in-season application of P to broadacre crops The most common in-field use of foliar P fertiliser is for horticultural crops There have been several studies of the effectiveness of foliar P for broadacre crops but this work has shown variable outcomes (Alston 1979 Mosali et al 2006 Girma et al 2007)

Foliar fertilisation is a widely used agricultural tool for the sustainable management of crops (Kannan 2010) Many factors which are currently not fully understood influence the response of plants to foliar-applied nutrient solutions (Fernaacutendez and Eichert 2009) For simplicity they may be grouped under physico-chemical properties of the foliar fertiliser formulation (eg point of deliquescence or surface tension) the environmental conditions under which sprays are applied (eg light relative humidity or temperature) and plant biology-related aspects (eg leaf surface structure and composition or plant physiological status) All of these factors may interact to alter the absorption and translocation of foliar-applied nutrients and ultimately the plant response to the treatments (Fernaacutendez and Eichert 2009 Fernaacutendez and Brown 2013)

The epidermis of aerial plant parts is generally covered with a cuticle and may contain specialised cells including trichomes or stomata This extra-cellular protective layer is chiefly made of a biopolymer matrix of cutin andor cutan with waxes deposited onto and intruded into it in addition to variable amounts of polysaccharides and phenolics (Domiacutenguez et al 2011) The inner structure and chemical composition of the cuticle of most plant species and organs remain unclear (Khayet and Fernaacutendez 2012) and they have been observed to change in response to environmental and physiological variations during plant growth and development (Domiacutenguez et al 2011) Fernaacutendez et al (2011) recently introduced the use of the three-liquids method for evaluating the physico-chemical properties of plant surfaces quantitatively Using a peach cv as model of a pubescent surface they calculated the surface free energy polarity and work-of-adhesion as derived from contact angle measurements of water glycerol and diiodomethane While water contact angles have been often recorded for assessing plant surface wettability (eg Holloway 1969 Enkisat et al 2011) they do not take into consideration the retention or repulsion of drops by the surfaces a phenomenon with important eco-physiological and practical implications (Ahmad and Wainwright 1976 Aryal and Neuner 2010 Ensikat et al 2011) After applying a foliar nutrient spray the higher the retention and contact area between the liquids drops and the leaf surfaces the greater the chance for fertiliser uptake to occur (Fernaacutendez and Brown 2013) The rate of droplet retention by plant surfaces has been measured by different methodologies (eg Brewer et al 1991 Enkisat et al 2011) which

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 2

Abstract Aims This study aimed to analyse the effect of phosphorus (P) nutritional status on wheat leaf surface properties in relation to foliar P absorption and translocation Methods Plants of Triticum aestivum cv Axe were grown with three rates of root P supply (equivalent to 24 8 and 0 kg P ha-1) under controlled conditions Foliar P treatments were applied and the rate of drop retention P absorption and translocation was measured Adaxial and abaxial leaf surfaces were analysed by scanning and transmission electron microscopy The contact angles surface free energy and work-of-adhesion for water were determined Results Wheat leaves are markedly non-wettable the abaxial leaf side having some degree of water drop adhesion versus the strong repulsion of water drops by the adaxial side The total leaf area stomatal and trichome densities cuticle thickness and contact angles decreased with P deficiency while the work-of-adhesion for water increased Phosphorous deficient plants failed to absorb the foliar-applied P Conclusions Phosphorous deficiency altered the surface structure and functioning of wheat leaves which became more wettable and had a higher degree of water drop adhesion but turned less permeable to foliar-applied P The results obtained are discussed within an agronomic and eco-physiological context Keywords Cuticle Foliar absorption Plant surfaces Trichomes Stomata Wettability

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 3

Introduction Grains from wheat plants provide an important food staple and phosphorus (P) is the second most limiting nutrient after nitrogen for wheat production (Batten 1992) Many soils of the world naturally maintain soil solution P at levels below the optimum for productive crop plants (Hedley and McLaughlin 2005) To sustain productive crops soils are commonly supplemented with P fertilisers at varying levels of efficiency as reviewed by Syers et al (2008) Wheat requires starter P at sowing to provide essential P for early growth and to replace P exported in the previous grain crop (Batten et al 1986 Grant et al 2001) Using starter P fertiliser often provides sufficient P to grow crops to tillering but in seasons of higher yield potential it is possible that supplemental in-season P application will increase yields (Noack et al 2010)

Soil application of supplemental in-season P is difficult to achieve efficiently as movement of P in soil is diffusion limited and therefore surface-applied P is not positioned for access by growing roots and in-season drilling of P would cause considerable crop damage This leaves foliar P fertilisation as the most practical avenue for in-season application of P to broadacre crops The most common in-field use of foliar P fertiliser is for horticultural crops There have been several studies of the effectiveness of foliar P for broadacre crops but this work has shown variable outcomes (Alston 1979 Mosali et al 2006 Girma et al 2007)

Foliar fertilisation is a widely used agricultural tool for the sustainable management of crops (Kannan 2010) Many factors which are currently not fully understood influence the response of plants to foliar-applied nutrient solutions (Fernaacutendez and Eichert 2009) For simplicity they may be grouped under physico-chemical properties of the foliar fertiliser formulation (eg point of deliquescence or surface tension) the environmental conditions under which sprays are applied (eg light relative humidity or temperature) and plant biology-related aspects (eg leaf surface structure and composition or plant physiological status) All of these factors may interact to alter the absorption and translocation of foliar-applied nutrients and ultimately the plant response to the treatments (Fernaacutendez and Eichert 2009 Fernaacutendez and Brown 2013)

The epidermis of aerial plant parts is generally covered with a cuticle and may contain specialised cells including trichomes or stomata This extra-cellular protective layer is chiefly made of a biopolymer matrix of cutin andor cutan with waxes deposited onto and intruded into it in addition to variable amounts of polysaccharides and phenolics (Domiacutenguez et al 2011) The inner structure and chemical composition of the cuticle of most plant species and organs remain unclear (Khayet and Fernaacutendez 2012) and they have been observed to change in response to environmental and physiological variations during plant growth and development (Domiacutenguez et al 2011) Fernaacutendez et al (2011) recently introduced the use of the three-liquids method for evaluating the physico-chemical properties of plant surfaces quantitatively Using a peach cv as model of a pubescent surface they calculated the surface free energy polarity and work-of-adhesion as derived from contact angle measurements of water glycerol and diiodomethane While water contact angles have been often recorded for assessing plant surface wettability (eg Holloway 1969 Enkisat et al 2011) they do not take into consideration the retention or repulsion of drops by the surfaces a phenomenon with important eco-physiological and practical implications (Ahmad and Wainwright 1976 Aryal and Neuner 2010 Ensikat et al 2011) After applying a foliar nutrient spray the higher the retention and contact area between the liquids drops and the leaf surfaces the greater the chance for fertiliser uptake to occur (Fernaacutendez and Brown 2013) The rate of droplet retention by plant surfaces has been measured by different methodologies (eg Brewer et al 1991 Enkisat et al 2011) which

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 3

Introduction Grains from wheat plants provide an important food staple and phosphorus (P) is the second most limiting nutrient after nitrogen for wheat production (Batten 1992) Many soils of the world naturally maintain soil solution P at levels below the optimum for productive crop plants (Hedley and McLaughlin 2005) To sustain productive crops soils are commonly supplemented with P fertilisers at varying levels of efficiency as reviewed by Syers et al (2008) Wheat requires starter P at sowing to provide essential P for early growth and to replace P exported in the previous grain crop (Batten et al 1986 Grant et al 2001) Using starter P fertiliser often provides sufficient P to grow crops to tillering but in seasons of higher yield potential it is possible that supplemental in-season P application will increase yields (Noack et al 2010)

Soil application of supplemental in-season P is difficult to achieve efficiently as movement of P in soil is diffusion limited and therefore surface-applied P is not positioned for access by growing roots and in-season drilling of P would cause considerable crop damage This leaves foliar P fertilisation as the most practical avenue for in-season application of P to broadacre crops The most common in-field use of foliar P fertiliser is for horticultural crops There have been several studies of the effectiveness of foliar P for broadacre crops but this work has shown variable outcomes (Alston 1979 Mosali et al 2006 Girma et al 2007)

Foliar fertilisation is a widely used agricultural tool for the sustainable management of crops (Kannan 2010) Many factors which are currently not fully understood influence the response of plants to foliar-applied nutrient solutions (Fernaacutendez and Eichert 2009) For simplicity they may be grouped under physico-chemical properties of the foliar fertiliser formulation (eg point of deliquescence or surface tension) the environmental conditions under which sprays are applied (eg light relative humidity or temperature) and plant biology-related aspects (eg leaf surface structure and composition or plant physiological status) All of these factors may interact to alter the absorption and translocation of foliar-applied nutrients and ultimately the plant response to the treatments (Fernaacutendez and Eichert 2009 Fernaacutendez and Brown 2013)

The epidermis of aerial plant parts is generally covered with a cuticle and may contain specialised cells including trichomes or stomata This extra-cellular protective layer is chiefly made of a biopolymer matrix of cutin andor cutan with waxes deposited onto and intruded into it in addition to variable amounts of polysaccharides and phenolics (Domiacutenguez et al 2011) The inner structure and chemical composition of the cuticle of most plant species and organs remain unclear (Khayet and Fernaacutendez 2012) and they have been observed to change in response to environmental and physiological variations during plant growth and development (Domiacutenguez et al 2011) Fernaacutendez et al (2011) recently introduced the use of the three-liquids method for evaluating the physico-chemical properties of plant surfaces quantitatively Using a peach cv as model of a pubescent surface they calculated the surface free energy polarity and work-of-adhesion as derived from contact angle measurements of water glycerol and diiodomethane While water contact angles have been often recorded for assessing plant surface wettability (eg Holloway 1969 Enkisat et al 2011) they do not take into consideration the retention or repulsion of drops by the surfaces a phenomenon with important eco-physiological and practical implications (Ahmad and Wainwright 1976 Aryal and Neuner 2010 Ensikat et al 2011) After applying a foliar nutrient spray the higher the retention and contact area between the liquids drops and the leaf surfaces the greater the chance for fertiliser uptake to occur (Fernaacutendez and Brown 2013) The rate of droplet retention by plant surfaces has been measured by different methodologies (eg Brewer et al 1991 Enkisat et al 2011) which

Fernaacutendez et al Plant and Soil (2014) in press

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provided a rough estimate and were difficult to perform and reproduce especially on water repellent surfaces In this regard the estimation of the work-of-adhesion of a liquid constitutes an easy and valuable tool for quantifying the degree of drop retention or repellence of a particular plant surface The measurement of the contact angles of liquids with different polarity provides additional information about the combined effects of surface chemistry and physical structure and enables the calculation of intrinsic physico-chemical characteristics of the solid surface such as the surface free energy and polarity The application of this membrane science approach (eg Khayet et al 2007) to plant surfaces may improve our understanding of plant surface phenomena and facilitate the optimisation of foliar-applied agrochemical treatments when supplied to different crops

Scientific progress over the last few decades has provided a better though incomplete understanding of the processes affecting plant responses to foliar nutrient sprays (Fernaacutendez and Brown 2013) Foliar fertiliser absorption is a complex process initially affected by the interactions between the sprayed agrochemical drops and plant surfaces (Fernaacutendez and Brown 2013) A major degree of surface micro- and nano-structure variations have been observed in relation to different plant surfaces (Barthlott and Neinhuis 1997) and it has been recognised that this is a key factor influencing the deposition of agrochemical spray drops (Holloway 1969 Khayet and Fernaacutendez 2012) Additionally the affinity (solubility) between foliar formulation components (ie active ingredients solvents and adjuvants) and epidermal materials (ie the cuticle and the cell wall) will affect the permeability of plant surfaces to foliar sprays (Khayet and Fernaacutendez 2012)

Many cuticular permeability trials carried out over the past 60 years enabled the development of the ldquodissolution-diffusion modelrdquo for the cuticular penetration of apolar lipophilic compounds (Riederer and Friedmann 2006) In contrast the mechanisms of penetration of hydrophilic polar solutes through the cuticle are still not fully characterised (Fernaacutendez and Eichert 2009) For at least some plant species and in the absence of an external pressure or surfactants there is evidence for the stomatal uptake of water and solutes (Eichert and Burkhardt 2001 Eichert et al 2008 Burkhardt el al 2012) The overall contribution of stomata to the absorption of foliar sprays remains unclear but can be highly significant (Eichert et al 2008) and may vary according to factors such as the plant species and variety leaf phenological stage and stomatal functionality and frequency or due to the prevailing environmental conditions during plant growth and development (Fernaacutendez and Brown 2013) Solutes penetrating stomata have been suggested to follow a diffusion pathway along the pore walls which appears to be less size selective than in cuticular permeability (Eichert et al 2008) The potential mechanisms of absorption of foliar fertilisers by alternative epidermal structures such as trichomes have not yet been investigated in detail (Fernaacutendez and Brown 2013)

The foliar uptake of P has been measured directly using radioactive tracer studies with a range of approaches from leaf dipping in radioactive solutions to spraying with radioactive solutions The resultant foliar P absorption efficiency varied dependent on a number of experimental factors (including method of application plant type formulation type and plant P status) Koontz and Biddulph (1957) measured an absorption efficiency of 60 when spraying radioactive solution on the leaf Bouma (1969) measured an absorption efficiency of 30 using a leaf dipping approach while McBeath et al (2012) measured an absorption efficiency of 60-99 using multiple 3microL drops added to leaves The influence of P status and leaf physiology on the uptake and translocation of foliar P fertiliser requires exploration to underpin the development of sensible foliar P fertilisation strategies

Given the commercial significance of wheat and the great potential of P fertilisers as a strategy to preserve yields of dryland grain crops in soils with limited P availability (Noack et al 2010) a study was carried out to characterise the effect of P nutrition on leaf surface

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properties and in relation to the absorption of foliar-applied P solutions In this investigation we aimed to (i) characterise the properties of wheat leaf surfaces that may affect their interaction with foliar P spray drops (ii) analyse the effect of plant P status on wheat leaf surface features and (iii) measure the effect of P status on the uptake and translocation of a foliar-applied P formulation

Materials and methods

Soil Properties

Soil was collected from Black Point in the grain producing region of Southern Australia (S34deg36776rsquo E137deg48599) to 10 cm depth air-dried and sieved to less than 2 mm prior to characterisation and use for a plant growth experiment in the growth chamber Soil pH (H2O) and electrical conductivity (EC) were measured in a 1 soil5 solution suspension (Rayment and Higginson 1992) with calcium carbonate content measured according to Martin and Reeve (1955) Field capacity was measured according to Klute (1986) and total organic carbon according to the method of Matejovic (1997) Cation exchange capacity was measured using method 15E1 and Colwell P using method 9B2 of Rayment and Higginson (1992) The diffusive gradient in thin film phosphorus soil test (DGT-P) was measured using the method outlined by Mason et al (2010)

Black Point soil is an alkaline (pH 85) loam with no surface salinity issues or detectable calcium carbonate an organic carbon content of 16 and cation exchange capacity of 179 cmol+ kg-1 The soil is deficient for P according to both the Colwell (measured 3 vs critical concentration 25 mg kg-1 Moody 2007) and DGT-P (measured 4 vs critical concentration 60 ug L-1 Mason et al 2010) soil tests

Plant culture

The growth chamber experiment comprised one soil with soil P fertiliser added at three rates (equivalent to 0 8 and 24 kg P ha-1) replicated 14 times This level of replication was required due to the extensive number of destructive measurements made on each treatment (described below) to allow each measurement to be adequately replicated A total of 15 kg of air-dry sieved (lt2 mm) soil was used in each pot The pots were black 15 L pots and were not free-draining The following basal nutrients were added to each pot one day prior to sowing nitrogen at 50 mg N kg -1 as urea (CO(NH2)2) potassium at 67 mg K kg-1 as potassium sulfate (K2SO4) magnesium at 17 mg Mg kg-1 as magnesium sulfate (MgSO47H20) zinc at 10 mg Zn kg-1 as zinc sulfate (ZnSO4 7H2O) manganese at 13 mg Mn kg-1 as manganese chloride (MnCl2) copper at 8 mg Cu kg-1 as cupric sulfate (Cu2SO4 5H20) and total sulphur applied in these reagents of 58 mg kg-1 Pots were watered to 80 field capacity following basal nutrient application At four weeks after sowing an additional 17 mg kg-1 of nitrogen 2 mg kg-1 of zinc 03 mg kg-1 of manganese and 16 mg kg-1 copper were applied in solution to the surface and watered in All reagents were sourced from Sigma Aldrich (St Louis USA) The soil P fertiliser treatments were applied immediately prior to sowing as reagent grade phosphoric acid from a 0016 v v-1 H3PO4 solution (VWR International Pennsylvania USA) administering 47 and 142 mg kg-1 for the 8 and 24 kg P ha-1 treatments respectively The 0 kg P ha-1 treatment received no solution Pots were watered to 80 field capacity following P treatment application

Four pre-germinated seeds of wheat (Triticum aestivum cv Axe) were sown in each pot at 10-15 mm depth The seedlings were thinned to two per pot at the 2-leaf growth stage by leaving the two most uniform seedlings in each pot Immediately after sowing the soil

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surface in each pot was covered with 70 g of polyethylene granules to minimise evaporation Pots were watered to weight with reverse osmosis water every 2-3 days in order to maintain 80 field capacity Pots were placed in a growth chamber with a daynight cycle of 12 hour at 20oC15oC for 10 days and then at 23oC20oC and 60 to 80 relative humidity for 39 days with experiment duration of 49 days The pots were arranged in a completely randomised design and the positions of pots were re-randomised every week

Four replicate pots of each treatment were selected for treating leaves with radiolabelled P fertiliser Previously ten pots with plants supplied with 24 kg P ha-1 were selected for a preliminary foliar blotting experiment Both trials were carried out four weeks after planting at ear in the boot (Zadoks growth stage (GS) 39 Zadoks et al 1974) At mid-anthesis (at GS 65) undamaged and comparable second leaves (ie the second youngest emerged blades) of the different P treatments were collected for microscope observation soluble cuticular lipid extraction and contact angle determination

The remaining ten replicate pots were harvested at GS 80 (early dough development) the heads and leaves were removed from the stems and the stems were cut 1 cm above soil level and oven-dried at 70degC for 48 h Then the dry weight for heads and stems for each pot was recorded (leaf dry weight was not recorded as some leaves had been sampled for the aforementioned procedures)

Foliar treatments

A preliminary blotting experiment to assess the deposition onto adaxial and abaxial leaf sides of drops of either 2 ammonium phosphate (NH4H2PO4 Sigma Aldrich) in water or in combination with 01 Genapol X-80 (Sigma Aldrich surface tension of 27 mN m-1 Khayet and Fernaacutendez 2012) was performed on plants at GS 39 (ear in the boot) Three microl drops (10 repetitions per leaf surface) of both P solutions were applied with a 3 ml micro-dosing syringe and the number of drops deposited onto adaxial and abaxial leaf surfaces 5 min after treatment was recorded This experiment was carried out on leaves of 24 kg P ha-1 supplied plants

At the same GS four replicate pots of each treatment were treated with 32P labelled fertiliser solution as described by Fernaacutendez et al (2008a) with some modifications Each 40 ml dipping solution contained 195 mg P mL-1 with P supplied as 2 NH4H2PO4 plus 01 Genapol and 7 Kbq ml-1 of 32P Approximately 13 of the length of leaf section was marked on the flag leaf and the second leaf on the main stem The area of treated leaf was measured by photographing the marked area of treated leaf and calculating the treated area (ImageJ 145s NIH USA) The leaf was then dipped in the solution for 30 s then removed from the solution and left for 48 h At the end of the experimental period the plant was harvested in the following sections treated area of treated leaf non-treated area of treated leaf other leaves and stems

Plant tissue 32P analysis

Leaves treated with 32P were washed first in 30 ml of 005 w w-1 Triton X-100 (Sigma Aldrich) plus 01M HCl gently rubbing the tissues with gloved fingers and 40 ml of both tap (230 microS cm-1) and deionised (25 microS cm-1) water Each washing solution was retained in order to measure the amount of 32P removed during each step Then the plant parts and leaves were oven-dried at 70degC for 48 h and tissue dry weights were recorded Tissues were subsequently cut finely using stainless steel scissors and a subsample (asymp05 g) was digested by boiling in 5 mL of HNO3 making to 20 mL volume in 01 w v-1 HNO3 and then filtered through 042 μm filters A subsample of 2 mL of each digest solution and 10 mL of National

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Diagnostics Ecoscintreg A (Georgia USA) scintillant were placed in a glass scintillation vial and homogenised The 32P activities in digest filtrates were measured using a Rackbeta II Wallac Liquid Scintillation Counter (Finland) All counts were corrected for decay to a single time point The amount of foliar P absorbed was expressed as percentage absorption calculated by dividing the amount of 32P absorbed by the plant by the total 32P radioactivity recovered (wash + total absorbed) The translocation was expressed as the percentage of the total 32P that was measured in untreated sections of the plant as a proportion of the 32P absorbed This is similar to the approach used by Singh and Singh (2008)

Tissue electron microscopy examination

Second leaves collected at GS 65 (mid-anthesis) which had been supplied 0 8 and 24 kg P ha-1 were selected for evaluating the effect of P nutritional status on leaf surface properties using scanning electron (SEM) and transmission electron microscopy (TEM)

Platinum-sputtered intact adaxial and abaxial surfaces corresponding to the middle part of the second leaves were examined by SEM (FEI Quanta 450 FEG acceleration potential 5 kV working distance 10-11mm) Stomatal and trichome densities were obtained after analysing SEM micrographs

For TEM tissues were fixed in 25 glutaraldehyde-4 paraformaldehyde (both from Electron Microscopy Sciences (EMS) Hatfield USA) for 6 h at 4ordmC rinsed in ice-cold phosphate buffer pH 72 four times within a period of 6 h and left overnight Samples were post-fixed in a 11 2 aqueous osmium tetroxide (TAAB Laboratories Berkshire UK) and 3 aqueous potassium ferrocyanide (Sigma-Aldrich) solution for 1 h They were then washed with distilled water (x3) dehydrated in a graded series of 30 50 70 80 90 95 and 100 ethanol (x2 15 min each concentration) and infiltrated at room temperature with ProcureAraldite epoxy resin (ProSciTech Townsville Australia) solutions (31 2h 11 2h 13 3h) and pure resin overnight Blocks were polymerized at 70degC for 3 d and ultra-thin sections were consequently cut with an ultra-microtome using a diamond knife Prior to TEM observation tissue sections were post-stained with Reynolds lead citrate for 5 min

Wax extraction

Soluble cuticular lipids were extracted by immersing 20 second leaves of 0 8 and 24 kg Pmiddotha-

1 treated plants at GS 65 in 250 ml of chloroform for 1 min using two replicates per sample Extracts were initially evaporated in a glass beaker and then in a watch glass until dryness in a laboratory fume cupboard The amount of soluble cuticular lipids was expressed gravimetrically on a leaf surface area basis The average leaf surface area of the three P treatments was calculated after scanning fresh leaves and analyzing the images with ImageJ software

Contact angle measurements and estimation of leaf surface properties

Advancing contact angles of drops of double-distilled water glycerol and diiodomethane (both 99 purity Sigma-Aldrich) were measured at room temperature (25ordmC) using a OCAH 200 contact angle meter (DataPhysics Filderstadt Germany) Contact angles were determined on intact adaxial and abaxial wheat leaves collected from plants grown with 0 8 and 24 kg Pmiddotha-1 (30 repetitions at GS 65) Second leaf sections of approximately 2 x 05 cm2 were cut with a scalpel from the middle part of the leaf discarding the mid vein and were mounted on a microscope slide with double-sided adhesive tape in such a way that the veins laid in the direction of the camera Two μl drops of each liquid (30 repetitions) were

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deposited on to the leaf surfaces with a 1 ml syringe having a 05 mm diameter needle Contact angles were automatically calculated by fitting the captured drop shape to the one calculated from the Young-Laplace equation

The total surface free energy (or surface tension γ) and its components ie the Lifshitz-van der Waals (LW) acid (+) and base (-) components in addition to the work-of-adhesion for water were calculated for all the leaf samples analysed (ie two leaf sides and three P treatments) as described by Fernaacutendez et al (2011)

Briefly γ can be divided into their components as 2LW AB LW

i i i i i iγ γ γ γ γ γ+ minus= + = + (1)

where i denotes either the solid or the liquid phase and the acid-base component ( ABiγ ) breaks

down into the electron-donor ( iγminus ) and the electron-acceptor ( iγ

+ ) interactions (van Oss et al 1987 1988) For a solid-liquid system the following expression is given (van Oss et al 1987 1988)

1 2 1 2 1 2(1 cos ) 2( ) 2( ) 2( )LW LWl s l s l s lθ γ γ γ γ γ γ γ+ minus minus ++ = + + (2)

where the three components of the solid γ ( andLWs s sγ γ γ+ minus ) can be obtained from measuring

the contact angles (θ) of the three liquids employed which have known γ components (120574119897119871119882 120574119897 + and 120574119897 minus) Additionally the degree of surface polarity was calculated as the ratio between the acid-base component and the total γ (γABγ -1) The total work-of-adhesion for water (Wa) was determined for each wheat leaf surface following the Young-Dupre equation

( )1 cosa s lv sl lW γ γ γ θ γ= + minus = + (3) where γs is the surface free energy of the solid γlv is the interfacial tension of the liquid and γsl corresponds to the interfacial tension between the solid and the liquid

Statistical Analysis

Analysis of variance (ANOVA) was undertaken using Genstatreg V13 and SPSS statistical packages to estimate treatment effects The level of significance between the treatments was determined using Duncanrsquos Multiple Range Test (5 significance)

Results

Wheat responsiveness to increasing doses of soil P and the addition of foliar P

Wheat grown in Black Point soil was highly responsive to soil P addition with a ten-fold dry matter increase between 0 and 24 kg P ha-1 equivalent applied (Table 1) Phosphorus deficient (0 kg P ha-1) and marginal (8 kg P ha-1) plants had second leaf areas 74 and 30 less than plants in the 24 kg P ha-1 treatment (Table 1) Except for the lowest P rate (0 kg P ha-1 applied at sowing) leaf P concentration was elevated in the foliar-treated area of the treated leaf while the untreated area of the treated leaves and other untreated leaves had P concentrations that were not different from each other (Table 2) The absorption of foliar P was dependent on P nutritional status with 20 absorption for soils treated with 8 kg P ha-1 at sowing and 33 absorption for soils treated with 24 kg P ha-1 at sowing while the most deficient plants were found to not absorb any detectable amounts of foliar-applied P (Table 2) Most (65-75) of the P absorbed was retained within the treated leaf area with the remaining 35 being translocated to the untreated part of the treated leaf No significant amounts of foliar applied P appeared to be transported to other leaves or stems within the 2 days since foliar application (Table 2)

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deposited on to the leaf surfaces with a 1 ml syringe having a 05 mm diameter needle Contact angles were automatically calculated by fitting the captured drop shape to the one calculated from the Young-Laplace equation

The total surface free energy (or surface tension γ) and its components ie the Lifshitz-van der Waals (LW) acid (+) and base (-) components in addition to the work-of-adhesion for water were calculated for all the leaf samples analysed (ie two leaf sides and three P treatments) as described by Fernaacutendez et al (2011)

Briefly γ can be divided into their components as 2LW AB LW

i i i i i iγ γ γ γ γ γ+ minus= + = + (1)

where i denotes either the solid or the liquid phase and the acid-base component ( ABiγ ) breaks

down into the electron-donor ( iγminus ) and the electron-acceptor ( iγ

+ ) interactions (van Oss et al 1987 1988) For a solid-liquid system the following expression is given (van Oss et al 1987 1988)

1 2 1 2 1 2(1 cos ) 2( ) 2( ) 2( )LW LWl s l s l s lθ γ γ γ γ γ γ γ+ minus minus ++ = + + (2)

where the three components of the solid γ ( andLWs s sγ γ γ+ minus ) can be obtained from measuring

the contact angles (θ) of the three liquids employed which have known γ components (120574119897119871119882 120574119897 + and 120574119897 minus) Additionally the degree of surface polarity was calculated as the ratio between the acid-base component and the total γ (γABγ -1) The total work-of-adhesion for water (Wa) was determined for each wheat leaf surface following the Young-Dupre equation

( )1 cosa s lv sl lW γ γ γ θ γ= + minus = + (3) where γs is the surface free energy of the solid γlv is the interfacial tension of the liquid and γsl corresponds to the interfacial tension between the solid and the liquid

Statistical Analysis

Analysis of variance (ANOVA) was undertaken using Genstatreg V13 and SPSS statistical packages to estimate treatment effects The level of significance between the treatments was determined using Duncanrsquos Multiple Range Test (5 significance)

Results

Wheat responsiveness to increasing doses of soil P and the addition of foliar P

Wheat grown in Black Point soil was highly responsive to soil P addition with a ten-fold dry matter increase between 0 and 24 kg P ha-1 equivalent applied (Table 1) Phosphorus deficient (0 kg P ha-1) and marginal (8 kg P ha-1) plants had second leaf areas 74 and 30 less than plants in the 24 kg P ha-1 treatment (Table 1) Except for the lowest P rate (0 kg P ha-1 applied at sowing) leaf P concentration was elevated in the foliar-treated area of the treated leaf while the untreated area of the treated leaves and other untreated leaves had P concentrations that were not different from each other (Table 2) The absorption of foliar P was dependent on P nutritional status with 20 absorption for soils treated with 8 kg P ha-1 at sowing and 33 absorption for soils treated with 24 kg P ha-1 at sowing while the most deficient plants were found to not absorb any detectable amounts of foliar-applied P (Table 2) Most (65-75) of the P absorbed was retained within the treated leaf area with the remaining 35 being translocated to the untreated part of the treated leaf No significant amounts of foliar applied P appeared to be transported to other leaves or stems within the 2 days since foliar application (Table 2)

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 9

Effect of P nutritional status on leaf surface properties

Surface structure

For all the treatments highly significant differences were observed for trichome and stomatal densities in adaxial and abaxial leaf surfaces Phosphorus shortage decreased stomatal and trichome frequencies with the biggest impact on trichome frequencies and larger differences between P treatments on adaxial leaf surfaces (Table 1)

The adaxial leaf side of leaves from the highest P treatment (24 kg P ha-1 equivalent) was found to have approximately 77 stomata and 59 trichomes per mm2 For the same treatment lower values were recorded for the abaxial leaf surface which contained an average of 59 stomata and 7 trichomes per mm2 (Table 1) Reducing the root P supply had a drastic effect on the trichome densities of both leaf sides which decreased between 30 to 60 for the 8 kg P ha-1 treatment and 90 to 100 for the 0 kg P ha-1 treatment on the adaxial and abaxial surfaces respectively Adaxial and abaxial stomatal frequencies were also affected by plant P supply from soil although to a lesser extent compared to trichome densities being reduced between 29 to 34 for the 8 kg P ha-1 treatment and 51 to 53 for the 0 kg P ha-1 treatment

The wheat leaf epidermis was found to be sinuous having alternate concave (furrows) and convex (ridges) epidermal areas running parallel with each other along the long axis of the leaf (Fig 1) Ridges correspond to the vascular bundles which vary in diameter in relation with major or secondary veins For both leaf sides stomata were located in the furrows generally aligned in two rows per furrow Trichomes occurred chiefly over the ridges and on either side of the rows of stomata (Fig 1) While the stomatal layout was observed to be unaffected by decreasing doses of root P supply trichomes appeared in a more disordered manner in P deficient treatments especially over the furrows where the decrease of density with root P supply was more noticeable

The epicuticular wax layer mainly occurred as a network of platelets and was often 2 to 4 times thicker (ie sometimes up to 240 nm) than the cuticular layer underneath (see Fig 2a as an example) No significant differences were observed in the amount of soluble cuticular lipids extracted from second leaves collected from 0 8 and 24 kg P ha-1 plants which varied between 196 and 220 microg cm-2 Similarly no significant differences were found when comparing the amount of soluble wax on adaxial vs abaxial leaf sides (data not shown) Epicuticular waxes were found to be unevenly distributed on the leaf surfaces occurring sometimes as a thick surface layer (Fig 2a) Wax deposits were often identified in the spaces between the veins (furrows) chiefly in association with adaxial leaf surfaces (Fig 2b)

No remarkable cuticle structure differences were derived when analysing TEM micrographs of abaxial vs adaxial leaf surfaces (data not shown) A thin whitish mainly reticulate cuticle with no distinguishable cuticle proper was observed underneath the epicuticular waxes (Fig 2) Enzymatic wheat leaf cuticle extraction in 2 cellulase and 2 pectinase led to the disintegration of the tissues and the cuticle could not be obtained as an intact layer

While the cuticular and cell wall ultra-structure of plants treated with root P was observed to be similar (data not shown) the lack of root P supply decreased cuticle thickness from approximately 80-100 nm down to 40-50 nm (disregarding the thickness of the epicuticular wax layer) and affected the cell walls which had a heterogeneous appearance and randomly distributed dark patches in a lighter grey cell wall (Figs 2c d)

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 10

Leaf surface wettability surface free energy and work-of-adhesion

Both for adaxial and abaxial surfaces the highest contact angles (θ) were obtained for water followed by those of glycerol and then diiodomethane (Table 3) For the three liquids and P treatments the highest θ were recorded for the adaxial leaf surfaces

A significant leaf surface effect was also observed in association with P nutrition with differences in the water and diiodomethane θ measured on the adaxial and abaxial leaf sides respectively The water θ of the adaxial surface of P sufficient leaves (ie those supplied 24 kg P ha-1) showed that these surfaces had the highest degree of hydrophobicity Glycerol and diiodomethane θ values were lower than those of water and both leaf sides followed a similar trend regarding θ measurements

For all the root P treatments approximately 2 times higher total surface free energy (γ) values were estimated for the abaxial leaf surfaces compared to the corresponding adaxial leaf surfaces chiefly due to the contribution of the Lifshitz van der Waals component ie the dispersive component (γLW in Table 4) Phosphorus deficient leaves (0 kg P ha-1 P treatment) had a slightly lower γ and an increased work-of-adhesion for water as compared to P treated plants Regardless of the plant P status all the surfaces had a higher work-of-adhesion for water on the abaxial leaf sides In general a gradual increase of γLW γAB in the adaxial sides and of γ on both leaf surfaces occurred with rising root P concentrations whereas the work-of-adhesion (Wa) for water decreased for the two leaf sides

In the absence of a surfactant most of the deposited aqueous 2 NH4H2PO4 drops rolled off the wheat leaf surfaces which had repellence for the drops (adaxial leaf side) None of the pure aqueous solution drops (ie without surfactant) deposited onto the adaxial leaf surfaces were retained and only 22 of the drops remained deposited onto the abaxial leaf sides which showed some degree of adhesion for the P solution drops The repellence or adhesion of drops of aqueous 2 NH4H2PO4 was in accordance with the contact angles and work-of-adhesion for water of P-sufficient leaves which was 63 lower for the adaxial surface compared to the abaxial surface (Tables 3 4) Plant P deficiency increased the adhesion (ie increased the work-of-adhesion for water) of aqueous drops by both leaf sides with leaves from the P deficient treatment (0 kg P ha-1) reaching the highest work-of-adhesion values (ie the lowest degree of water drop repellence Table 4) Discussion

In this study the effect of P deficiency on leaf wettability and subsequent absorption of a foliar-applied P fertiliser has been quantified for the first time Phosphorus is an important macro-element which often limits plant growth in many soils of the world (Hawkesford et al 2012) Foliar P fertilisation of dryland crops such as wheat may prove advantageous to ensure grain yield and quality (Noack et al 2010) provided that the foliar P formulations are optimised in terms of improved wetting spreading and retention suggested in previous studies (eg Fernaacutendez et al 2006 Blanco et al 2010)

In agreement with previous studies increasing the soil P supply increased wheat dry weight tissue P concentrations and leaf area (Singh et al 1977 Rodriacuteguez et al 1998) The wheat leaves analysed were amphistomatous and increasing plant P status resulted in higher stomatal and trichome frequencies especially on the adaxial side Although P deficiency reduced both stomatal and trichome densities a greater impact on trichome frequencies was observed An increased number of trichomes on wheat leaves is often observed for drought-resistant cultivars (Doroshkov et al 2011) The wheat cultivar Axe is a short season and non-glaucous variety according to the predominance of epicuticular wax platelets on the leaf wheat surfaces (Bianchi and Figini 1986 Koch et al 2006) The adaxial and abaxial stomata

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 10

Leaf surface wettability surface free energy and work-of-adhesion

Both for adaxial and abaxial surfaces the highest contact angles (θ) were obtained for water followed by those of glycerol and then diiodomethane (Table 3) For the three liquids and P treatments the highest θ were recorded for the adaxial leaf surfaces

A significant leaf surface effect was also observed in association with P nutrition with differences in the water and diiodomethane θ measured on the adaxial and abaxial leaf sides respectively The water θ of the adaxial surface of P sufficient leaves (ie those supplied 24 kg P ha-1) showed that these surfaces had the highest degree of hydrophobicity Glycerol and diiodomethane θ values were lower than those of water and both leaf sides followed a similar trend regarding θ measurements

For all the root P treatments approximately 2 times higher total surface free energy (γ) values were estimated for the abaxial leaf surfaces compared to the corresponding adaxial leaf surfaces chiefly due to the contribution of the Lifshitz van der Waals component ie the dispersive component (γLW in Table 4) Phosphorus deficient leaves (0 kg P ha-1 P treatment) had a slightly lower γ and an increased work-of-adhesion for water as compared to P treated plants Regardless of the plant P status all the surfaces had a higher work-of-adhesion for water on the abaxial leaf sides In general a gradual increase of γLW γAB in the adaxial sides and of γ on both leaf surfaces occurred with rising root P concentrations whereas the work-of-adhesion (Wa) for water decreased for the two leaf sides

In the absence of a surfactant most of the deposited aqueous 2 NH4H2PO4 drops rolled off the wheat leaf surfaces which had repellence for the drops (adaxial leaf side) None of the pure aqueous solution drops (ie without surfactant) deposited onto the adaxial leaf surfaces were retained and only 22 of the drops remained deposited onto the abaxial leaf sides which showed some degree of adhesion for the P solution drops The repellence or adhesion of drops of aqueous 2 NH4H2PO4 was in accordance with the contact angles and work-of-adhesion for water of P-sufficient leaves which was 63 lower for the adaxial surface compared to the abaxial surface (Tables 3 4) Plant P deficiency increased the adhesion (ie increased the work-of-adhesion for water) of aqueous drops by both leaf sides with leaves from the P deficient treatment (0 kg P ha-1) reaching the highest work-of-adhesion values (ie the lowest degree of water drop repellence Table 4) Discussion

In this study the effect of P deficiency on leaf wettability and subsequent absorption of a foliar-applied P fertiliser has been quantified for the first time Phosphorus is an important macro-element which often limits plant growth in many soils of the world (Hawkesford et al 2012) Foliar P fertilisation of dryland crops such as wheat may prove advantageous to ensure grain yield and quality (Noack et al 2010) provided that the foliar P formulations are optimised in terms of improved wetting spreading and retention suggested in previous studies (eg Fernaacutendez et al 2006 Blanco et al 2010)

In agreement with previous studies increasing the soil P supply increased wheat dry weight tissue P concentrations and leaf area (Singh et al 1977 Rodriacuteguez et al 1998) The wheat leaves analysed were amphistomatous and increasing plant P status resulted in higher stomatal and trichome frequencies especially on the adaxial side Although P deficiency reduced both stomatal and trichome densities a greater impact on trichome frequencies was observed An increased number of trichomes on wheat leaves is often observed for drought-resistant cultivars (Doroshkov et al 2011) The wheat cultivar Axe is a short season and non-glaucous variety according to the predominance of epicuticular wax platelets on the leaf wheat surfaces (Bianchi and Figini 1986 Koch et al 2006) The adaxial and abaxial stomata

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DOI 101007s11104-014-2052-6 11

and trichome frequencies determined for cv Axe are within the range reported in previous studies for different wheat cultivars (Malone et al 1993 Doroshkov et al 2011)

The wheat leaf cuticle was found to have two layers with a sometimes prominent epicuticular wax layer which may be two to four times thicker than the cuticular layer beneath Hence the thickness of the leaf epicuticular wax layer in wheat (a herbaceous monocot with a short life cycle) may be a fast and more metabolically cost-effective strategy to protect such perishable leaf tissues from dehydration in contrast to developing a more complex layered and thicker leaf cuticular membrane as observed in several evergreen and deciduous woody and annual plant leaves (eg Jeffree 2006) Phosphorus deficiency did not seem to have an effect on the amount of leaf soluble cuticular lipids but significantly decreased the thickness of the cuticular layer underneath the epicuticular waxes The wheat leaf cuticle could not be isolated via pectinase and cellulase digestion possibly due to its chemical composition and structure which may be strongly linked to the epidermal cell wall (Guzmaacuten et al 2014)

The reduction in trichome and stomatal densities and cuticle thickness in relation to P deficiency indicate that such wheat leaf epidermal traits were affected by P shortage probably at early stages of plant development Concerning the effect of nutritional disorders at the leaf epidermal level iron deficiency has been reported to decrease the amount of soluble cuticular lipids and cuticle weights per unit surface in peach and pear leaves respectively (Fernaacutendez et al 2008b)

The epidermal alterations observed as a result of P deficiency influenced the wettability and hydrophobicity of the abaxial and adaxial leaf surfaces as assessed by the three liquid method (Fernaacutendez et al 2011) Interestingly leaves with a better P status had higher water contact angles which led to a lower work-of-adhesion for water on both leaf surfaces This implies a higher degree of water drop repellence of P-sufficient leaf surfaces especially with regard to the adaxial side where there are higher trichome densities It is concluded that trichomes are largely responsible for the major hydrophobic character of the wheat leaf adaxial surface as also observed in other plant surfaces containing hairs (eg Brewer et al 1991 Fernaacutendez et al 2011) A similarly low work-of-adhesion for water to that of the adaxial surface of P-sufficient wheat leaves has been determined for the almost super-hydrophobic juvenile Eucalyptus globulus leaf (θ ~143ordm Wa= 15 mJ m-2 Khayet and Fernaacutendez 2012)

Given the markedly non-wettable and in general water-repellent character of the wheat leaf it will be necessary to apply agrochemical treatments with a lower surface tension and a higher rate of retention as a prerequisite for foliar uptake to occur This can be achieved by adding to the foliar fertiliser formulations surfactants and adjuvants which may improve the rate of leaf surface wetting retention and spreading as shown in some studies (eg Fernaacutendez et al 2006 Blanco et al 2010) Otherwise fertiliser drops sprayed as pure water solutions and in the absence of adjuvants will roll off the wheat leaf surface and will have a limited efficacy Our results indicate that different plant surfaces such as the wheat or eucalypt leaf or the peach or pepper fruit surface (Khayet and Fernaacutendez 2012) may have a different degree of water drop adhesion or repellence as determined by the work-of-adhesion for water In practical terms this implies that plant surfaces that have an increased work-of-adhesion for water may have a higher contact area between the drop and the surface and could theoretically but not necessarily be more permeable (this will be affected by various factors such as cuticular structure and composition or the presence of surface features like stomata or trichomes) These plant surfaces may be treated with moderate success with simpler foliar nutrient formulations (ie adjuvants are not so crucial for improving solid-liquid interactions) as compared to more water-repellent surfaces like the wheat leaf

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Plant P deficiency improved the adhesion of water drops onto the adaxial leaf side The increased level of water drop adhesion and decreased repellence of P-deficient leaf surfaces may yield them more vulnerable to stress factors such as water-soluble contaminants (eg atmospheric aerosols) or pathogens However the higher wettability of P-deficient leaf surfaces and increased work-of-adhesion for water did not contribute to increasing the rate of absorption of foliar-applied 32P Transport of 32P from the point of application was not detected after foliar application of P to plants not supplied with P via the root system Phosphorus-deficient leaves failed to take up the radiolabelled P solution which may be related to stomatal malfunctioning and a failure to open following a normal diurnal rhythm (eg Yu et al 2004) This indicates that extreme P deficiency may not be easily corrected by foliar P fertilisation In addition to the observed reduction in cuticle thickness changes in the chemical composition and ultra-structure of the cuticle may be expected as a result of P deficiency which may consequently affect the rate of foliar uptake We observed that P deficient plants failed to take up the irrigation water during the growth chamber trial already at the GS45 stage of development (ear was emerged) which indicates that plants were not able to significantly transpire The importance of the stomatal pathway for the uptake of leaf-applied nutrients even in the absence of surfactants has been shown in several investigations (eg Eichert et al 1998 2008) The limited transpiration of P deficient leaves is likely linked to stomatal closure which will hinder the penetration of leaf-applied P solutions through stomata

Fertilisers applied to wheat leaves may penetrate via the cuticle (including the occurrence of cuticular cracks and imperfections) and also through stomata and trichomes but the relative contribution of each pathway is not easy to ascertain experimentally There is evidence that the cuticular and stomatal uptake route can be equally important but their relative contribution may depend on multiple factors such as fertiliser formulation properties leaf surface characteristics and also the prevailing environmental conditions during plant treatment (Eichert and Fernaacutendez 2011) Many studies showed that the presence of stomata may increase the rate of foliar penetration of nutrient solutions chiefly under conditions favoring stomatal aperture (eg Wallihan et al 1964 Will et al 2012) In soybean plants boron deficiency was found to reduce the rate of uptake of foliar-applied B due to stomatal malfunctioning (Will et al 2011)

The role of trichomes concerning the uptake of foliar applied P cannot be neglected The permeability of the foliar applied P fertiliser may be facilitated by the lower cuticle thickness over the trichomes and also due to the occurrence of cracks and discontinuities in the trichome base (data not shown) Schlegel and Schoumlnherr (2001 2002) observed that CaCl2 was preferentially taken up by leaves of several species and apple fruits of different developmental stages when trichomes were present in the epidermis The contribution of Phlomis fruticosa leaf trichomes to the absorption of water has also been suggested by Grammatikopoulos and Manetas (1994) Furthermore some species of Bromeliaceae have developed leaf trichomes which are capable of absorbing water and nutrients (eg Pierce et al 2001 Papini et al 2010) In conclusion P deficiency significantly altered the structure and function of wheat leaves which became more wettable less water repellent but poorly permeable to the foliar-applied P solution It is likely that a severe P deficiency cannot be corrected only using foliar P fertilisers as leaves need to be healthy with higher stomatal and trichome frequencies and a normal stomatal functioning for increased foliar P fertiliser absorption While these leaves with better P nutrition are almost super-hydrophobic and have a higher degree of repellence for solutes foliar treatment with P solutions having a low surface tension will be taken up by the foliage as shown in this investigation

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Acknowledgements The authors acknowledge funding from the CSIRO Sustainable Agriculture Flagship Fellowship Fund Victoria Fernaacutendez is supported by a ldquoRamoacuten y Cajalrdquo contract (MINECO Spain) co-financed by the European Social Fund Paula Guzmaacuten is supported by a pre-doctoral grant from the Technical University of Madrid Courtney A E Peirce is supported by the Grains Research and Development Corporation of Australia and the Fluid Fertilizer Foundation (USA) References Ahmad I Wainwright S (1976) Ecotype differences in leaf surface properties of Agrostis

stolonifera from salt marsh spray zone and inland habitats New Phytol 76(2)361-366 doi 101111j1469-81371976tb01471x

Alston AM (1979) Effects of soil water content and foliar fertilization with nitrogen and phosphorus in late season on the yield and composition of wheat Aust J Agr Res 30577-585 doi 101071AR9790577

Aryal B Neuner G (2010) Leaf wettability decreases along an extreme altitudinal gradient Oecologia 1621-9 doi101007s00442-009-1437-3

Barthlott W Neinhuis C (1997) Purity of the sacred lotus or escape from contamination in biological surfaces Planta 2021-8 doi 101007s004250050096

Batten GD Wardlaw IF Aston MJ (1986) Growth and distribution of phosphorus in wheat developed under various phosphorus and temperature regimes Aust J Agr Res 37459-469 doi101071AR9860459

Batten GD (1992) A review of phosphorus efficiency in wheat Plant Soil 146163-168 Bianchi G Figini ML (1986) Epicuticular waxes of glaucous and nonglaucous durum wheat

lines J Agr Food Chem 34(3)429-433 doi 101021jf00069a012 Blanco A Fernaacutendez V Val J (2010) Improving the performance of calcium-containing

spray formulations to limit the incidence of bitter pit in apple (Malus x domestica Borkh) Sci Hort 12723-28 doi 101016jscienta201009005

Bouma D Dowling EJ (1976) Relationship between phosphorus status of subterranean clover plants and dry weight responses of detached leaves in solutions with and without phosphate Aust J Agr Res 27 53-62 doi101071AR9760053

Brewer CA Smith WK Vogelmann TC (1991) Functional interaction between leaf trichomes leaf wettability and the optical properties of water droplets Plant Cell Environ 14955ndash962 doi 101111j1365-30401991tb00965x

Burkhardt J Basi S Pariyar S Hunsche M (2012) Stomatal penetration by aqueous solutions ndash an update involving leaf surface particles New Phytol 196774-787 doi 101111j1469-8137201204307x

Domiacutenguez E Heredia-Guerrero JA Heredia A (2011) The biophysical design of plant cuticles an overview New Phytol 189938-949 doi 101111j1469-8137201003553x

Doroshkov AV Pshenichnikova TA Afonnikov DA (2011) Morphological characterization and inheritance of leaf hairiness in wheat (Triticum aestivum L) as analyzed by computer-aided phenotyping Russ J Genet 47(6)739-743 doi 101134S1022795411060093

Eichert T Goldbach HE Burkhardt J (1998) Evidence for the uptake of large anions through stomatal pores Botan Acta 111461ndash466

Eichert T Burkhardt J (2001) Quantification of stomatal uptake of ionic solutes using a new model system J Exp Bot 52(357)771-781 doi 101093jexbot52357771

Eichert T Kurtz A Steiner U Goldbach HE (2008) Size exclusion limits and lateral heterogeneity of the stomatal foliar uptake pathway for aqueous solutes and water-

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 14

suspended nanoparticles Physiol Plantarum 134151-160 doi 101111j1399-3054200801135x

Eichert T Fernaacutendez V (2012) Uptake and release of elements by leaves and other aerial plant parts In Marschner P (ed) Marschnerrsquos mineral nutrition of higher plants 3rd edn Elsevier London pp 71-84

Ensikat HJ Ditsche-Kuru P Neinhuis C Barthlott W (2011) Superhydrophobicity in perfection the outstanding properties of the lotus leaf Beilstein J Nanotech 2152-161 doi 103762bjnano219

Fernaacutendez V Del Riacuteo V Abadiacutea J Abadiacutea A (2006) Foliar iron fertilization of peach (Prunus persica (L) Batsch) effects of iron compounds surfactants and other adjuvants Plant Soil 289239-252 doi 101007s11104-006-9132-1

Fernaacutendez V Del Riacuteo V Pumarintildeo L Igartua E Abadiacutea J Abadiacutea A (2008a) Foliar fertilization of peach (Prunus persica (L) Batsch) with different iron formulations effects on re-greening iron concentration and mineral composition in treated and untreated leaf surfaces Sci Hort 117241-248 doi 101016jscienta200805002

Fernaacutendez V Eichert T Del Riacuteo V Loacutepez-Casado G Heredia JA Abadiacutea A Heredia A Abadiacutea J (2008b) Leaf structural changes associated with iron deficiency chlorosis of field-grown pear and peach - physiological implications Plant Soil 311161-172 doi 101007s11104-008-9667-4

Fernaacutendez V Eichert T (2009) Uptake of hydrophilic solutes through plant leaves current state of knowledge and perspectives of foliar fertilization Crit Rev Plant Sci 28(1)36-68 doi 10108007352680902743069

Fernaacutendez V Khayet M Montero-Prado P Heredia-Guerrero JA Liakoloulos G Karabourniotis G Del Riacuteo V Domiacutenguez E Tacchini I Neriacuten C Val J Heredia A (2011) New insights into the properties of pubescent surfaces peach fruit as model Plant Physiol 156(4)2098-2108 doi 101104pp111176305

Fernaacutendez V Brown PH (2013) From plant surface to plant metabolism the uncertain fate of foliar-applied nutrients Front Plant Sci 4289 doi 103389fpls201300289

Girma K Martin KL Freeman KW Mosali J Teal RK Raun WR Moges SM Arnall B (2007) Determination of the optimum rate and growth stage for foliar applied phosphorus in corn Commun Soil Sci Plant Anal 381137-1154 doi 10108000103620701328016

Grant CA Flaten DN Tomasiewicz DJ Sheppard SC (2001) The importance of early season phosphorus nutrition Can J Plant Sci 81211-224 doi 104141P00-093

Grammatikopoulos G Manetas Y (1994) Direct absorption of water by hairy leaves of Phlomis fruticosa and its contribution to drought avoidance Can J Bot 72(12)1805-1811 doi 101139b94-222

Guzmaacuten P Fernaacutendez V Garciacutea ML Khayet M Fernaacutendez A Gil L (2014) Localization of polysaccharides in isolated and intact cuticles of eucalypt poplar and pear leaves by enzyme-gold labelling Plant Physiol Bioch 76 1-6 doi 101016jplaphy201312023Hawkesford M Horst W Kichey T Lambers H Schjoerring J Moslashller IS White P (2012) Functions of macronutrients In Marschner P (ed) Marschnerrsquos mineral nutrition of higher plants 3rd edn Elsevier London pp 135-189

Hedley MJ McLaughlin MJ (2005) Reactions of phosphate fertilizers and by-products in soils In Sharpley AN (ed) Phosphorus Agriculture and the Environment American Society of Agronomy Crop Science Society of America Soil Science Society of America Madison WI pp 181-252

Holloway PJ (1969) The effects of superficial wax on leaf wettability Ann Appl Biol 63145-153 doi 101111j1744-73481969tb05475x

Jeffree CH (2006) The fine structure of the plant cuticle In Riederer M Muumlller C (eds) Biology of the plant cuticle Annual Plant Reviews Vol 23 Blackwell Oxford pp 11-125

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 14

suspended nanoparticles Physiol Plantarum 134151-160 doi 101111j1399-3054200801135x

Eichert T Fernaacutendez V (2012) Uptake and release of elements by leaves and other aerial plant parts In Marschner P (ed) Marschnerrsquos mineral nutrition of higher plants 3rd edn Elsevier London pp 71-84

Ensikat HJ Ditsche-Kuru P Neinhuis C Barthlott W (2011) Superhydrophobicity in perfection the outstanding properties of the lotus leaf Beilstein J Nanotech 2152-161 doi 103762bjnano219

Fernaacutendez V Del Riacuteo V Abadiacutea J Abadiacutea A (2006) Foliar iron fertilization of peach (Prunus persica (L) Batsch) effects of iron compounds surfactants and other adjuvants Plant Soil 289239-252 doi 101007s11104-006-9132-1

Fernaacutendez V Del Riacuteo V Pumarintildeo L Igartua E Abadiacutea J Abadiacutea A (2008a) Foliar fertilization of peach (Prunus persica (L) Batsch) with different iron formulations effects on re-greening iron concentration and mineral composition in treated and untreated leaf surfaces Sci Hort 117241-248 doi 101016jscienta200805002

Fernaacutendez V Eichert T Del Riacuteo V Loacutepez-Casado G Heredia JA Abadiacutea A Heredia A Abadiacutea J (2008b) Leaf structural changes associated with iron deficiency chlorosis of field-grown pear and peach - physiological implications Plant Soil 311161-172 doi 101007s11104-008-9667-4

Fernaacutendez V Eichert T (2009) Uptake of hydrophilic solutes through plant leaves current state of knowledge and perspectives of foliar fertilization Crit Rev Plant Sci 28(1)36-68 doi 10108007352680902743069

Fernaacutendez V Khayet M Montero-Prado P Heredia-Guerrero JA Liakoloulos G Karabourniotis G Del Riacuteo V Domiacutenguez E Tacchini I Neriacuten C Val J Heredia A (2011) New insights into the properties of pubescent surfaces peach fruit as model Plant Physiol 156(4)2098-2108 doi 101104pp111176305

Fernaacutendez V Brown PH (2013) From plant surface to plant metabolism the uncertain fate of foliar-applied nutrients Front Plant Sci 4289 doi 103389fpls201300289

Girma K Martin KL Freeman KW Mosali J Teal RK Raun WR Moges SM Arnall B (2007) Determination of the optimum rate and growth stage for foliar applied phosphorus in corn Commun Soil Sci Plant Anal 381137-1154 doi 10108000103620701328016

Grant CA Flaten DN Tomasiewicz DJ Sheppard SC (2001) The importance of early season phosphorus nutrition Can J Plant Sci 81211-224 doi 104141P00-093

Grammatikopoulos G Manetas Y (1994) Direct absorption of water by hairy leaves of Phlomis fruticosa and its contribution to drought avoidance Can J Bot 72(12)1805-1811 doi 101139b94-222

Guzmaacuten P Fernaacutendez V Garciacutea ML Khayet M Fernaacutendez A Gil L (2014) Localization of polysaccharides in isolated and intact cuticles of eucalypt poplar and pear leaves by enzyme-gold labelling Plant Physiol Bioch 76 1-6 doi 101016jplaphy201312023Hawkesford M Horst W Kichey T Lambers H Schjoerring J Moslashller IS White P (2012) Functions of macronutrients In Marschner P (ed) Marschnerrsquos mineral nutrition of higher plants 3rd edn Elsevier London pp 135-189

Hedley MJ McLaughlin MJ (2005) Reactions of phosphate fertilizers and by-products in soils In Sharpley AN (ed) Phosphorus Agriculture and the Environment American Society of Agronomy Crop Science Society of America Soil Science Society of America Madison WI pp 181-252

Holloway PJ (1969) The effects of superficial wax on leaf wettability Ann Appl Biol 63145-153 doi 101111j1744-73481969tb05475x

Jeffree CH (2006) The fine structure of the plant cuticle In Riederer M Muumlller C (eds) Biology of the plant cuticle Annual Plant Reviews Vol 23 Blackwell Oxford pp 11-125

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 15

Kannan S (2010) Foliar fertilization for sustainable crop production Sustain Agric Rev 4371-402 doi 101007978-90-481-8741-6_13

Khayet M Vazquez Alvarez M Khulbe KC Matsuura T (2007) Preferential surface segregation of homopolymer and copolymer blend films Surf Sci 601885-895 doi 101016jsusc200611024

Khayet M Fernaacutendez V (2012) Estimation of the solubility parameter of model plant surfaces and agrochemicals a valuable tool for understanding plant surface interactions Theor Biol Med Model 945 doi 1011861742-4682-9-45

Klute A (1986) Water retention laboratory methods In Klute A (ed) Methods of soil analysis Part 1 Physical and mineralogical methods 2nd edn American Society of Agronomy Inc Soil Science Society of America Inc Madison WI pp 635-662

Koch K Barthlott W Koch S Hommes A Wandelt K Mamdouh W De-Feyter S Broekmann P (2006) Structural analysis of wheat wax (Triticum aestivum cv lsquoNaturastarrsquo L) from the molecular level to three dimensional crystals Planta 223(2) 258-270 doi 101007s00425-005-0081-3

Koontz H Biddulph O (1957) Factors affecting absorption and translocation of foliar applied phosphorus Plant Physiol 32 463-470

Malone SR Mayeux HS Johnson HB Polley HW (1993) Stomatal density and aperture length in four plant species grown across a subambient CO2 gradient Am J Bot 80(12)1413-1418

Martin AE Reeve R (1955) A rapid manometric method for determination of soil carbonate Soil Sci 79187-198

Mason SD McNeill AM McLaughlin MJ Zhang H (2010) Prediction of wheat response to an application of phosphorus under field conditions using diffusive gradients in thin-films (DGT) and extraction methods Plant Soil 337243-258 doi 101007s11104-010-0521-0

Matejovic I (1997) Determination of carbon and nitrogen in samples of various soils by the dry combustion method Commun Soil Sci Plant Anal 281499-1511 doi 10108000103629709369892

McBeath TM McLaughlin MJ Kirby JK Armstrong RD (2012) The effect of soil water status on fertiliser topsoil and subsoil phosphorus utilisation by wheat Plant Soil 358(1-2)337-348 doi 101007s11104-012-1177-8

Moody PW (2007) Interpretation of a single-point P buffering index for adjusting critical levels of the Colwell Soil P Test Aust J Soil Res 4555-62 doi 101071SR06056

Mosali J Desta K Teal RK Freeman KW Martin KL Lawles JW Raun WR (2006) Effect of foliar application of phosphorus on winter wheat grain yield phosphorus uptake and use efficiency J Plant Nutr 292147-2163 doi

10108001904160600972811 Noack SR McBeath TM McLaughlin MJ (2010) Potential for foliar phosphorus fertilisation

of dryland cereal crops a review Crop Pasture Sci 61(8)659-669 doi 101071CP10080 Papini A Tani G Di Falco P Brighigna L (2010) The ultrastructure of the development of

Tillandsia (Bromeliaceae) trichome Flora 205(2)94-100 doi 101016jflora200902001 Pierce S Maxwell K Griffiths H Winter K (2001) Hydrophobic trichome layers and

epicuticular wax powders in Bromeliaceae Am J Bot 88(8)1371-1389 doi 1023073558444

Rayment GE Higginson FR (1992) Australian laboratory handbook of soil and water chemical methods Inkata Press Melbourne

Riederer M Friedmann A (2006) Transport of lipophilic non-electrolytes across the cuticle In Riederer M Muumlller C (eds) Biology of the plant cuticle Annual Plant Reviews Vol 23 Blackwell Oxford pp 250-279

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DOI 101007s11104-014-2052-6 16

Rodriacuteguez D Keltjens WG Goudriaan J (1998) Plant leaf area expansion and assimilate production in wheat (Triticum aestivum L) growing under low phosphorus conditions Plant Soil 200227-240 doi 101023A1004310217694

Schlegel TK Schoumlnherr J (2001) Selective permeability of cuticles over stomata and trichomes to calcium chloride In International Symposium on Foliar Nutrition of Perennial Fruit Plants 594 pp 91-96

Schlegel T K Schoumlnherr J (2002) Stage of development affects penetration of calcium chloride into apple fruits J Plant Nut Soil Sci 165738ndash745 doi 101002jpln200290012

Singh R Chadha RK Verma HN Singh Y (1977) Response of dryland wheat to phosphorus fertilizer as influenced by profile water storage and rainfall J Agric Sci Camb 88591-595 doi101017S0021859600037266

Singh D Singh M (2008) Absorption and translocation of glyphosate with conventional and organosilicone adjuvants Weed Biol Manag 8104-111 doi 101111j1445-6664200800282x

Syers JK Johnston AE Curtin D (2008) Efficiency of soil and fertilizer phosphorus use Reconciling changing concepts of soil phosphorus behaviour with agronomic information FAO Fertilizer and Plant Nutrition Bulletin 18 Food and Agriculture Organization of the United Nations Rome

van Oss CJ Chaudhury MK Good RJ (1987) Monopolar surfaces Adv Colloid Interf Sci 2835-64

van Oss CJ Chaudhury MK Good RJ (1988) Interfacial Lifshitz-van der Waals and polar interactions in macroscopic systems Chem Rev 88927-941 doi 101021cr00088a006

Wallihan E F Embleton TW Sharpless RG (1964) Response of chlorotic citrus leaves to iron sprays in relation to surfactants and stomatal apertures Proc Amer Soc Hort Sci 85 210-217

Will S Eichert T Fernaacutendez V Moumlhring J Muumlller T Roumlmheld V (2011) Absorption and mobility of foliar-applied boron in soybean as affected by plant boron status and application as a polyol complex Plant Soil 344283-293 doi 101007s11104-011-0746-6

Will S Eichert T Fernaacutendez V Muumlller T Roumlmheld V (2012) Boron foliar fertilization of soybean and lychee Effects of side of application and formulation adjuvants J Plant Nut Soil Sci 175180ndash188 doi 101002jpln201100107

Yu Q Zhang Y Liu Y Shi P (2004) Simulation of the stomatal conductance of winter wheat in response to light temperature and CO2 changes Ann Bot 93(4)435-441 doi 101093aobmch023

Zadoks JC Chang TT Konzak CF (1974) A decimal code for the growth stages of cereals Weed Res 14415-421 doi 101111j1365-31801974tb01084x

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 17

Tables

Table 1 Total plant weight at maturity and leaf surface area trichome and stomatal densities of upper and lower leaf sides of wheat plants in response to root P supply equivalent to 0 8 and 24 kg P ha-1 Data are means plusmn SE Within columns values marked with different letters are significantly different according to Duncanrsquos Multiple Range Test (P le 005) Leaves not included due to sampling for other measurements

P treatment (kg P ha-1)

Plant maturity dry weight

(g pot-1)

Total leaf surface area

(cm2)

Stomatal densities per leaf side (Ndeg mm-2)

Trichome densities per leaf side (Ndeg mm-2)

adaxial abaxial adaxial abaxial

24 56plusmn01 a 261plusmn07 c 772plusmn16 c 594plusmn10 c 587plusmn13 c 68plusmn05 c

8 33plusmn01 b 182plusmn19 b 549plusmn13 b 392plusmn13 b 408plusmn08 b 28plusmn04 b

0 041plusmn001 c 68plusmn14 a 360plusmn11 a 290plusmn06 a 51plusmn05 a 00plusmn00 a

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 18

Table 2 Wheat phosphorous concentration and leaf absorption and translocation in response to foliar-applied P as affected by soil P addition (48 h after treatment) Data are means plusmn SE Within each measurement values marked with different letters are significantly different according to Duncanrsquos Multiple Range Test (P le 005)

Soil P addition

(kg P ha-1) Whole plant Treated leaf-

treated area

Treated leaf-untreated

area Other leaves Stems

Phosphorus concentration (mg kg-1) 24 7434plusmn452 a 4289 plusmn381 c 3713 plusmn 62 cd 3819 plusmn 178 c 8 5221plusmn586 b 2907plusmn143 e 2323 plusmn 46 ef 2998 plusmn 65 de 0 1731plusmn248 fg 1351 plusmn192 gh 842 plusmn 47 h 1784 plusmn 107 fg

Foliar P absorption

( of foliar P recovered)

Foliar P translocation ( of foliar P absorbed)

24 33 plusmn 2 a 75 plusmn 10 a 34 plusmn 7 b nd nd 8 20 plusmn 4 b 65 plusmn 4 a 35 plusmn 4 b nd nd 0 nd nd nd nd nd

nd not detected

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 19

Table 3 Contact angles of water (θw) glycerol (θg) and diiodomethane (θd) with upper and lower wheat leaf surfaces collected from plants supplied equivalent to 24 8 or 0 kg P ha-1 P via the root system Data are means plusmn SD Within columns values marked with different letters are significantly different according to Duncanrsquos Multiple Range Test (P le 005)

P treatment (kg P ha-1)

θw (ordm) θg (ordm) θd (ordm)

adaxial abaxial adaxial abaxial adaxial abaxial

24 1432 plusmn51b 1177plusmn107 a 1251plusmn88 a 1101plusmn70 a 1040 plusmn59 a 753plusmn54 a

8 1398plusmn77 ab 1114plusmn97 a 1239plusmn80 a 1004plusmn65 a 1026plusmn38 a 752plusmn61 a

0 1232plusmn109 a 1032plusmn141 a 1236plusmn97 a 954plusmn84 a 1054plusmn50 a 876plusmn91 b

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 20

Table 4 Total surface free energy (γ) with the corresponding Lifshitz van der Waals component (γLW) and acid-base component (γAB) and work-of-adhesion for water (Wa) of upper and lower wheat leaf surfaces collected from plants supplied equivalent to 24 8 or 0 kg P ha-1 P via the root system

P treatment γLW (mJ m-2) γAB (mJ m-2) γ (mJ m-2) Wa (mJ m-2)

(kg P ha-1) adaxial abaxial adaxial abaxial adaxial abaxial adaxial abaxial

24 730 1994 446 087 1175 2081 1452 3898

8 776 2002 273 015 1049 2018 1717 4619

0 686 1380 125 176 811 1556 3299 5621

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 21

Figures

Fig 1 Scanning electron micrographs (x300) of adaxial (a c e) and abaxial (b d f) wheat leaf surfaces in relation to the different root P treatments (a b) 24 kg P ha-1 (c d) 8 kg P ha-

1 and (e f) 0 kg P ha-1

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 22

Fig 2 Adaxial wheat leaf TEM micrographs (a) conspicuous epicuticular wax layer (24 kg P ha-1 leaf bar 1 microm) (b) epicuticular waxes may be unevenly distributed accumulate in the furrows (arrows 24 kg P ha-1 leaf bar 200 nm) (c) cuticular structure of a 0 kg P ha-1 leaf (bar 500 nm) and (d) cuticular structure of a 24 kg P ha-1 leaf (bar 200 nm) cu cuticle cw cell wall

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 17

Tables

Table 1 Total plant weight at maturity and leaf surface area trichome and stomatal densities of upper and lower leaf sides of wheat plants in response to root P supply equivalent to 0 8 and 24 kg P ha-1 Data are means plusmn SE Within columns values marked with different letters are significantly different according to Duncanrsquos Multiple Range Test (P le 005) Leaves not included due to sampling for other measurements

P treatment (kg P ha-1)

Plant maturity dry weight

(g pot-1)

Total leaf surface area

(cm2)

Stomatal densities per leaf side (Ndeg mm-2)

Trichome densities per leaf side (Ndeg mm-2)

adaxial abaxial adaxial abaxial

24 56plusmn01 a 261plusmn07 c 772plusmn16 c 594plusmn10 c 587plusmn13 c 68plusmn05 c

8 33plusmn01 b 182plusmn19 b 549plusmn13 b 392plusmn13 b 408plusmn08 b 28plusmn04 b

0 041plusmn001 c 68plusmn14 a 360plusmn11 a 290plusmn06 a 51plusmn05 a 00plusmn00 a

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 18

Table 2 Wheat phosphorous concentration and leaf absorption and translocation in response to foliar-applied P as affected by soil P addition (48 h after treatment) Data are means plusmn SE Within each measurement values marked with different letters are significantly different according to Duncanrsquos Multiple Range Test (P le 005)

Soil P addition

(kg P ha-1) Whole plant Treated leaf-

treated area

Treated leaf-untreated

area Other leaves Stems

Phosphorus concentration (mg kg-1) 24 7434plusmn452 a 4289 plusmn381 c 3713 plusmn 62 cd 3819 plusmn 178 c 8 5221plusmn586 b 2907plusmn143 e 2323 plusmn 46 ef 2998 plusmn 65 de 0 1731plusmn248 fg 1351 plusmn192 gh 842 plusmn 47 h 1784 plusmn 107 fg

Foliar P absorption

( of foliar P recovered)

Foliar P translocation ( of foliar P absorbed)

24 33 plusmn 2 a 75 plusmn 10 a 34 plusmn 7 b nd nd 8 20 plusmn 4 b 65 plusmn 4 a 35 plusmn 4 b nd nd 0 nd nd nd nd nd

nd not detected

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 19

Table 3 Contact angles of water (θw) glycerol (θg) and diiodomethane (θd) with upper and lower wheat leaf surfaces collected from plants supplied equivalent to 24 8 or 0 kg P ha-1 P via the root system Data are means plusmn SD Within columns values marked with different letters are significantly different according to Duncanrsquos Multiple Range Test (P le 005)

P treatment (kg P ha-1)

θw (ordm) θg (ordm) θd (ordm)

adaxial abaxial adaxial abaxial adaxial abaxial

24 1432 plusmn51b 1177plusmn107 a 1251plusmn88 a 1101plusmn70 a 1040 plusmn59 a 753plusmn54 a

8 1398plusmn77 ab 1114plusmn97 a 1239plusmn80 a 1004plusmn65 a 1026plusmn38 a 752plusmn61 a

0 1232plusmn109 a 1032plusmn141 a 1236plusmn97 a 954plusmn84 a 1054plusmn50 a 876plusmn91 b

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 20

Table 4 Total surface free energy (γ) with the corresponding Lifshitz van der Waals component (γLW) and acid-base component (γAB) and work-of-adhesion for water (Wa) of upper and lower wheat leaf surfaces collected from plants supplied equivalent to 24 8 or 0 kg P ha-1 P via the root system

P treatment γLW (mJ m-2) γAB (mJ m-2) γ (mJ m-2) Wa (mJ m-2)

(kg P ha-1) adaxial abaxial adaxial abaxial adaxial abaxial adaxial abaxial

24 730 1994 446 087 1175 2081 1452 3898

8 776 2002 273 015 1049 2018 1717 4619

0 686 1380 125 176 811 1556 3299 5621

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 21

Figures

Fig 1 Scanning electron micrographs (x300) of adaxial (a c e) and abaxial (b d f) wheat leaf surfaces in relation to the different root P treatments (a b) 24 kg P ha-1 (c d) 8 kg P ha-

1 and (e f) 0 kg P ha-1

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 22

Fig 2 Adaxial wheat leaf TEM micrographs (a) conspicuous epicuticular wax layer (24 kg P ha-1 leaf bar 1 microm) (b) epicuticular waxes may be unevenly distributed accumulate in the furrows (arrows 24 kg P ha-1 leaf bar 200 nm) (c) cuticular structure of a 0 kg P ha-1 leaf (bar 500 nm) and (d) cuticular structure of a 24 kg P ha-1 leaf (bar 200 nm) cu cuticle cw cell wall

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 18

Table 2 Wheat phosphorous concentration and leaf absorption and translocation in response to foliar-applied P as affected by soil P addition (48 h after treatment) Data are means plusmn SE Within each measurement values marked with different letters are significantly different according to Duncanrsquos Multiple Range Test (P le 005)

Soil P addition

(kg P ha-1) Whole plant Treated leaf-

treated area

Treated leaf-untreated

area Other leaves Stems

Phosphorus concentration (mg kg-1) 24 7434plusmn452 a 4289 plusmn381 c 3713 plusmn 62 cd 3819 plusmn 178 c 8 5221plusmn586 b 2907plusmn143 e 2323 plusmn 46 ef 2998 plusmn 65 de 0 1731plusmn248 fg 1351 plusmn192 gh 842 plusmn 47 h 1784 plusmn 107 fg

Foliar P absorption

( of foliar P recovered)

Foliar P translocation ( of foliar P absorbed)

24 33 plusmn 2 a 75 plusmn 10 a 34 plusmn 7 b nd nd 8 20 plusmn 4 b 65 plusmn 4 a 35 plusmn 4 b nd nd 0 nd nd nd nd nd

nd not detected

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 19

Table 3 Contact angles of water (θw) glycerol (θg) and diiodomethane (θd) with upper and lower wheat leaf surfaces collected from plants supplied equivalent to 24 8 or 0 kg P ha-1 P via the root system Data are means plusmn SD Within columns values marked with different letters are significantly different according to Duncanrsquos Multiple Range Test (P le 005)

P treatment (kg P ha-1)

θw (ordm) θg (ordm) θd (ordm)

adaxial abaxial adaxial abaxial adaxial abaxial

24 1432 plusmn51b 1177plusmn107 a 1251plusmn88 a 1101plusmn70 a 1040 plusmn59 a 753plusmn54 a

8 1398plusmn77 ab 1114plusmn97 a 1239plusmn80 a 1004plusmn65 a 1026plusmn38 a 752plusmn61 a

0 1232plusmn109 a 1032plusmn141 a 1236plusmn97 a 954plusmn84 a 1054plusmn50 a 876plusmn91 b

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 20

Table 4 Total surface free energy (γ) with the corresponding Lifshitz van der Waals component (γLW) and acid-base component (γAB) and work-of-adhesion for water (Wa) of upper and lower wheat leaf surfaces collected from plants supplied equivalent to 24 8 or 0 kg P ha-1 P via the root system

P treatment γLW (mJ m-2) γAB (mJ m-2) γ (mJ m-2) Wa (mJ m-2)

(kg P ha-1) adaxial abaxial adaxial abaxial adaxial abaxial adaxial abaxial

24 730 1994 446 087 1175 2081 1452 3898

8 776 2002 273 015 1049 2018 1717 4619

0 686 1380 125 176 811 1556 3299 5621

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 21

Figures

Fig 1 Scanning electron micrographs (x300) of adaxial (a c e) and abaxial (b d f) wheat leaf surfaces in relation to the different root P treatments (a b) 24 kg P ha-1 (c d) 8 kg P ha-

1 and (e f) 0 kg P ha-1

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 22

Fig 2 Adaxial wheat leaf TEM micrographs (a) conspicuous epicuticular wax layer (24 kg P ha-1 leaf bar 1 microm) (b) epicuticular waxes may be unevenly distributed accumulate in the furrows (arrows 24 kg P ha-1 leaf bar 200 nm) (c) cuticular structure of a 0 kg P ha-1 leaf (bar 500 nm) and (d) cuticular structure of a 24 kg P ha-1 leaf (bar 200 nm) cu cuticle cw cell wall

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 19

Table 3 Contact angles of water (θw) glycerol (θg) and diiodomethane (θd) with upper and lower wheat leaf surfaces collected from plants supplied equivalent to 24 8 or 0 kg P ha-1 P via the root system Data are means plusmn SD Within columns values marked with different letters are significantly different according to Duncanrsquos Multiple Range Test (P le 005)

P treatment (kg P ha-1)

θw (ordm) θg (ordm) θd (ordm)

adaxial abaxial adaxial abaxial adaxial abaxial

24 1432 plusmn51b 1177plusmn107 a 1251plusmn88 a 1101plusmn70 a 1040 plusmn59 a 753plusmn54 a

8 1398plusmn77 ab 1114plusmn97 a 1239plusmn80 a 1004plusmn65 a 1026plusmn38 a 752plusmn61 a

0 1232plusmn109 a 1032plusmn141 a 1236plusmn97 a 954plusmn84 a 1054plusmn50 a 876plusmn91 b

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 20

Table 4 Total surface free energy (γ) with the corresponding Lifshitz van der Waals component (γLW) and acid-base component (γAB) and work-of-adhesion for water (Wa) of upper and lower wheat leaf surfaces collected from plants supplied equivalent to 24 8 or 0 kg P ha-1 P via the root system

P treatment γLW (mJ m-2) γAB (mJ m-2) γ (mJ m-2) Wa (mJ m-2)

(kg P ha-1) adaxial abaxial adaxial abaxial adaxial abaxial adaxial abaxial

24 730 1994 446 087 1175 2081 1452 3898

8 776 2002 273 015 1049 2018 1717 4619

0 686 1380 125 176 811 1556 3299 5621

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 21

Figures

Fig 1 Scanning electron micrographs (x300) of adaxial (a c e) and abaxial (b d f) wheat leaf surfaces in relation to the different root P treatments (a b) 24 kg P ha-1 (c d) 8 kg P ha-

1 and (e f) 0 kg P ha-1

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 22

Fig 2 Adaxial wheat leaf TEM micrographs (a) conspicuous epicuticular wax layer (24 kg P ha-1 leaf bar 1 microm) (b) epicuticular waxes may be unevenly distributed accumulate in the furrows (arrows 24 kg P ha-1 leaf bar 200 nm) (c) cuticular structure of a 0 kg P ha-1 leaf (bar 500 nm) and (d) cuticular structure of a 24 kg P ha-1 leaf (bar 200 nm) cu cuticle cw cell wall

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 20

Table 4 Total surface free energy (γ) with the corresponding Lifshitz van der Waals component (γLW) and acid-base component (γAB) and work-of-adhesion for water (Wa) of upper and lower wheat leaf surfaces collected from plants supplied equivalent to 24 8 or 0 kg P ha-1 P via the root system

P treatment γLW (mJ m-2) γAB (mJ m-2) γ (mJ m-2) Wa (mJ m-2)

(kg P ha-1) adaxial abaxial adaxial abaxial adaxial abaxial adaxial abaxial

24 730 1994 446 087 1175 2081 1452 3898

8 776 2002 273 015 1049 2018 1717 4619

0 686 1380 125 176 811 1556 3299 5621

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 21

Figures

Fig 1 Scanning electron micrographs (x300) of adaxial (a c e) and abaxial (b d f) wheat leaf surfaces in relation to the different root P treatments (a b) 24 kg P ha-1 (c d) 8 kg P ha-

1 and (e f) 0 kg P ha-1

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 22

Fig 2 Adaxial wheat leaf TEM micrographs (a) conspicuous epicuticular wax layer (24 kg P ha-1 leaf bar 1 microm) (b) epicuticular waxes may be unevenly distributed accumulate in the furrows (arrows 24 kg P ha-1 leaf bar 200 nm) (c) cuticular structure of a 0 kg P ha-1 leaf (bar 500 nm) and (d) cuticular structure of a 24 kg P ha-1 leaf (bar 200 nm) cu cuticle cw cell wall

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 21

Figures

Fig 1 Scanning electron micrographs (x300) of adaxial (a c e) and abaxial (b d f) wheat leaf surfaces in relation to the different root P treatments (a b) 24 kg P ha-1 (c d) 8 kg P ha-

1 and (e f) 0 kg P ha-1

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 22

Fig 2 Adaxial wheat leaf TEM micrographs (a) conspicuous epicuticular wax layer (24 kg P ha-1 leaf bar 1 microm) (b) epicuticular waxes may be unevenly distributed accumulate in the furrows (arrows 24 kg P ha-1 leaf bar 200 nm) (c) cuticular structure of a 0 kg P ha-1 leaf (bar 500 nm) and (d) cuticular structure of a 24 kg P ha-1 leaf (bar 200 nm) cu cuticle cw cell wall

Fernaacutendez et al Plant and Soil (2014) in press

DOI 101007s11104-014-2052-6 22

Fig 2 Adaxial wheat leaf TEM micrographs (a) conspicuous epicuticular wax layer (24 kg P ha-1 leaf bar 1 microm) (b) epicuticular waxes may be unevenly distributed accumulate in the furrows (arrows 24 kg P ha-1 leaf bar 200 nm) (c) cuticular structure of a 0 kg P ha-1 leaf (bar 500 nm) and (d) cuticular structure of a 24 kg P ha-1 leaf (bar 200 nm) cu cuticle cw cell wall