Leaf wettability is the main driver for foliar P uptake in P-deficient maize

Plan Physiology and Biochemis y 205 (2023) 108170 A ailable online 16 No embe 2023 0981-9428/© 2023 The Au ho s. Published by Else ie Masson SAS. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). Lea we abili y is he main d i e o olia P up ake in P-de icien maize Jon Niklas Henningsen a , * , B uno Maximilian G¨ o lach a , d , Jos´ e Manuel Quin e o b , Rami o Recena Ga ido b , Ka l He mann Mühling a , Vic o ia Fe n´ andez c , ** a Ins i u e o Plan Nu i ion and Soil Science, Kiel Uni e si y, He mann-Rodewald-S aße 2, 24118, Kiel, Ge many b Depa amen o de Ag onomía, ETSIA, Uni e sidad de Se illa, C a. de U e a, km 1, 41013, Se illa, Spain c Sys ems and Na u al Resou ces Depa men , School o Fo es Enginee ing, Uni e sidad Poli ´ ecnica de Mad id, 28040, Mad id, Spain d Ge man Ag icul u al Socie y e.V., 60489, F ank u am Main, Ge many ARTICLE INFO Handling Edi o : Shi end a Sahi Keywo ds: Folia P up ake Lea we abili y Maize cul i a s P de iciency Poin o deliquescence Poin o e lo escence Sal hyg oscopici y ABSTRACT Folia e ilisa ion is an al e na i e o m o nu ien applica ion, which is o pa icula in e es o phospho us (P), whe e he e iciency o soil e ilisa ion is low. Howe e , he up ake o olia -applied nu ien s is insu i- cien ly cha ac e ised. The aim o his s udy was o in es iga e he indi idual and combined signi icance o we abili y, olia e ilise p ope ies and su ac an on olia P up ake in P-de icien maize (Zea mays L.). So p ion and deso p ion p ope ies o wo P sal s used as olia e ilise s (KH 2 PO 4 , K 2 HPO 4 ) we e de e mined wi h dynamic apo so p ion iso he ms. Lea su aces and olia sp ay deposi ions o wo di e en ly we able maize cul i a s we e in es iga ed by scanning elec on mic oscopy and con ac angle measu emen . Phospho us up ake was hen linked o lea and e ilise solu ion p ope ies and i s e ec on cell ul as uc u e was cha - ac e ised by ansmission elec on mic oscopy. We abili y was he key ac o o P abso p ion, as all olia e ilise s we e aken up eaching a issue-P le el o adequa ely nou ished plan s. Fo unwe able lea es, only solu ions wi h su ac an , especially he combina ion o su ac an and hyg oscopic P sal (K 2 HPO 4 ) we e aken up. This s udy p o ides no el insigh s in o he signi icance o lea su ace and e ilise p ope ies, which can hus con ibu e o an imp o emen o P e ilisa ion s a egies. 1. In oduc ion Phospho us (P) is conside ed one o he mos limi ing plan nu ien s wo ldwide and is equi ed a concen a ions o 3–5 mg g −1 plan d y ma e o op imal g ow h (Hawkes o d e al., 2023). A e plan up ake, P emains in he plan ei he as ino ganic P (Pi), es e i ies o es e s/- dies e s, o o ms ene gy- ich py o- and polyphospha e bonds (Penn and Cambe a o, 2019; Hawkes o d e al., 2023). This mac o-elemen has se e al unc ions, e.g. as an ATP and NADPH componen , as a s uc u al elemen o memb anes o as a componen o glycolysis, espi a ion and pho osyn hesis (Ca s ensen e al., 2019; Hawkes o d e al., 2023). Howe e , abou 30% o he wo ld’s land a eas ha e acidic soils and ano he 30% a e calca eous, esul ing in P soil ixa ion and p ecipi a ion and hus low P plan a ailabili y ( on Uexküll and Mu e , 1995; Oki e al., 2004; Hawkes o d e al., 2023). As a esul , many a able egions ace P de iciency and hus educed c op biomass and quali y (Lynch e al., 1991), caused by poo ille ing, delayed g ow h o p ema u e senescence as in majo ce eals such as ice and whea , o addi ionally by i egula g ain de elopmen as in maize (Be gmann, 1983). Fu he - mo e, bound P emaining in he soil could con amina e su ace wa e s h ough su ace uno , endange ing he s abili y o ecosys ems (Heck- a h e al., 1995; Jeppesen e al., 1998; Hayga h e al., 1998). Folia P e ilisa ion is an al e na i e o m o nu ien applica ion ha can complemen soil e ilisa ion, po en ially educing P losses and p o iding plan s wi h P du ing pe iods o high demand. Despi e some s udies on olia P e ilisa ion ha e been de eloped in ecen yea s (e.g. A sic e al., 2020, 2022; Talboys e al., 2020; G¨ o lach e al., 2021a; b; G¨ o lach and Mühling, 2021; Henningsen e al., 2022, Henningsen e al., 2023a) he e is s ill much unce ain y abou he e icacy o olia -applied elemen s (Fe n´ andez e al., 2021). Abb e ia ions: DAS, Days a e sowing; DRH, Deliquescence ela i e humidi y; ERH, E lo escence ela i e humidi y; POD, Poin o deliquescence; POE, Poin o e lo escence. * Co esponding au ho . ** Co esponding au ho . E-mail add esses: [email p o ec ed] (J.N. Henningsen), [email p o ec ed] (B.M. G¨ o lach), [email p o ec ed] (J.M. Quin e o), [email p o ec ed] (R.R. Ga ido), [email p o ec ed] (K.H. Mühling), [email p o ec ed] (V. Fe n´ andez). Con en s lis s a ailable a ScienceDi ec Plan Physiology and Biochemis y jou nal homepage: www.else ie .com/loca e/plaphy h ps://doi.o g/10.1016/j.plaphy.2023.108170 Recei ed 25 July 2023; Recei ed in e ised o m 31 Oc obe 2023; Accep ed 6 No embe 2023 Plan Physiology and Biochemis y 205 (2023) 108170 2 The up ake o nu ien s in o he plan ia he lea depends on nume ous ac o s, which can be oughly g ouped in o hose ela ed o plan s (e.g., plan physiology, plan su ace composi ion and s uc u e), en i onmen al condi ions (e.g. ligh , empe a u e, ela i e humidi y (RH)) and physico-chemical and olia -sp ay- o mula ion- ela ed p op- e ies (e.g. chemical p ope ies o nu ien sal , su ac an ; Fe n´ andez and Eiche , 2009; Fe n´ andez e al., 2020, 2021). The composi ion and s uc u e o he lea su ace a e essen ial o whe he applied d ople s o ag ochemicals adhe e o i o whe he hey a e epelled and hus no abso bed (Fe n´ andez e al., 2021). The s uc u al ea u es a e de e - mined by he appea ance o ichomes, s oma a, papillae, cu icula olds (mic o-scale oughness) and epicu icula waxes (nano-scale oughness) (Ba hlo e al., 1998, 2017; Koch and Ba hlo , 2009; Almon e e al., 2022). Since mos ae ial plan pa s a e co e ed wi h a cu icle (Je ee, 2006; Shellakku i e al., 2022), i s physico-chemical p ope ies will a ec he pe o mance o olia sp ays (Ba las e al., 2023). A e being conside ed an independen laye om he unde lying cell wall ma e ial o a long ime, ecen s udies a e add essing he cu icle as a specialised pa o he p ima y cell wall consis ing mos ly o lipophilic componen s (i.e., cu in and waxes), bu also o cell wall polysaccha ides (Guzm´ an e al., 2014; Philippe e al., 2020; Gonz´ alez Mo eno e al., 2023). The combined in luence o lea su ace s uc u es and chemical composi ion de e mines he in e ac ion o he su ace wi h wa e o aqueous solu es applied o i and esul s in di e en con ac angles, p o ided adso p ion occu s (Fe n´ andez e al., 2017, 2021). High con ac angles educe he con ac a ea be ween liquid and su ace, while low con ac angles in- c ease he con ac a ea and hus po en ially enhance olia abso p ion, making we abili y an impo an ac o o conside o olia pene a ion (Fe n´ andez e al., 2021). Besides he composi ion and s uc u e o plan su aces, he chemical p ope ies o he olia sp ay o mula ions a e impo an o up ake, o en in ela ion o en i onmen al condi ions (Fe n´ andez and Eiche , 2009). The impo ance o hyg oscopic compounds o be supplied as olia sp ays in esponse o RH has been emphasised in some in- es iga ions (Sch¨ onhe , 2001; Fe n´ andez and Eiche , 2009). Folia -applied solu ions a e usually no in equilib ium wi h he wa e po en ial o he a mosphe e and he e o e e apo a e quickly om he lea (Eiche and Fe n´ andez, 2023). Once equilib ium wi h he a mo- sphe e is eached, he solu ions may emain liquid o d y ou , depending on ambien RH (Eiche and Fe n´ andez, 2023). Each solu e has a speci ic deliquescence RH (DRH) o poin o deliquescence (POD) a which i becomes liquid due o he abso p ion o wa e om he a mosphe e (Allan and Maue , 2016; Fe n´ andez e al., 2020; Eiche and Fe n´ andez, 2023). Con e sely, as he RH dec eases, solu ions also ha e an e lo- escence RH (ERH) o poin o e lo escence (POE) a which he p e i- ously sa u a ed solu ion becomes solidi ied by c ys allisa ion (Fe n´ andez e al., 2020; Ba las e al., 2023). Thus, when e ilise d op solu ions a e deposi ed on o he lea , ERH de e mines i s d ying and DRH he ehyd a ion due o, o example, inc eased RH, dew o og (Fe n´ andez e al., 2020; Ba las e al., 2023). Fu he mo e, he pene a- ion o olia -applied ag ochemicals depends on he concen a ion g adien be ween he solu ion on he lea su ace and he lea in e io , which wo ks bes wi h he exis ence o an aqueous di usion medium, i. e. when he deposi ed compound is hyd a ed (Fe n´ andez e al., 2020). Deliquescence RH and ERH may he e o e be impo an pa ame e s o de e mining he po en ial o solu es o be aken up by he plan as a esul o olia applica ion (Eiche and Fe n´ andez, 2023). Fu he mo e, addi i es (e.g. su ac an s, humec an s o s icke s) can change he p ope ies o he olia e ilise solu ions such as inc easing/dec easing he DRH o educing su ace ension and he eby imp o ing we abili y and ul ima e olia up ake (Fe n´ andez and Eiche , 2009). A e olia sp ays ha e been deposi ed on o he lea su ace, he subsequen up ake in o he lea in e io depends on he physico- chemical ea u es o he cu icle and o he epide mal s uc u es. Espe- cially s oma a (Eiche and Goldbach, 2008; Eiche e al., 2008), and ichomes (Li e al., 2018a,b, 2019; 2021; A sic e al., 2022), bu also ascula bundle issues (Bahamonde e al., 2018; A sic e al., 2020, 2022; Henningsen e al., 2023a) ha e been iden i ied as being in ol ed in he up ake o nu ien s ia he lea . Wi hin he cu icle, he embedded polysaccha ides a e assumed o be key o he cu icle pene a ion o olia solu es (Fe n´ andez e al., 2017). Polysaccha ides ha e high pola and hyd ogen-bonding componen s and can he e o e adso b wa e a high ambien RH, esul ing in swelling and sh inking o he cu icle, supposedly enabling up ake (Chamel e al., 1991; Fe n´ andez and Eiche , 2009; Fe n´ andez e al., 2016). Nu ien de iciency can lead o al e a ion o su ace composi ion and s uc u es (Rosolem and Lei e, 2007; Fe n´ andez e al., 2008; Will e al., 2011; Li e al., 2019) and hus o educed up ake o olia applied nu- ien s (Res epo-Diaz e al., 2008; Will e al., 2011; Li e al., 2019; Eiche and Fe n´ andez, 2023). Phospho us de iciency was also ound o educe he occu ence o epide mal s uc u es in whea (Fe n´ andez e al., 2014) while in ba ley, he cu icle was hicke and he abundance o polysaccha ides was diminished, leading o educed P up ake ia he lea (A sic e al., 2022). In maize, on he o he hand, i was shown ha no change in he su ace occu ed wi h di e en P nu i ion. Howe e , di e en maize a ie ies had di e en epicu icula wax opog aphy also du ing plan on ogeny which in ol ed we abili y changes (Henningsen e al., 2023a,b). Compa ison o cul i a s o he same c op species wi h ega d o hei su ace ea u es and in luence on he up ake o olia -applied nu ien s ha e been basically igno ed so a , bu ha e he po en ial o be mo e accu a e and enligh ening han he compa ison o wo species. Awa e ha lea we abili y and he cha ac e is ics o e ilise sp ays may be c ucial o he up ake o olia -applied nu ien s, he objec i e o his s udy was o de e mine he in luence o maize lea we abili y (using a we able e sus an unwe able a ie y) on he up ake o a hyg oscopic (K 2 HPO 4 ) and a non-hyg oscopic (KH 2 PO 4 ) P sal , supplied as olia sp ay wi h and wi hou su ac an . The hypo heses o be es ed we e: (i) The cul i a s analysed ha e di e en lea we abili y a es, wi h olia P abso p ion being highe o we able e sus unwe able lea es, (ii) olia P e ilise s e alua ed ha e di e en DRH and ERH alues, he sal leading o he highes olia P abso p ion a es being he one ha ing he lowes DRH/ERH, (iii) lea P up ake is highe o olia e ilise solu ion wi h su ac an ega dless o he P o m supplied as olia sp ay. 2. Ma e ials and me hods 2.1. Plan ma e ial Seeds o maize (Zea mays L., c . DKC 3096, Baye C opScience AG, Monheim, Ge many; c . P7948, Pionee Hi-B ed In e na ional, Inc., Johns on, IA, USA) we e placed in an alkaline (pH 8.34) P-de icien (0.35 mg P 100 g −1 soil, de e mined by Olsen P ex ac ion) sandy loam (Sl4) a a dep h o 30 mm in po s wi h a olume o 3 L. Eigh days a e sowing (DAS), plan s had ge mina ed and all nu ien s equi ed o plan g ow h we e applied o he soil in dissol ed o m in he ollowing amoun s (in g po −1 ): 0.5 N (as NH 4 NO 3 ), 0.6 K (as KCl), 0.6 K (as K 2 SO 4 ), 0.25 Ca (as CaSO 4 ), 0.17 Mg (as MgSO 4 ), 0.008 Fe (as Fe- EDDHA), 0.005 Cu (as CuSO 4 ), 0.008 Zn (as ZnSO 4 ), 0.015 Mn (as MnSO 4 ), 0.005 B (as H 3 BO 4 ), 0.001 Mo (as (NH 4 ) 6 Mo 7 O 24 ). Depending on he ea men , P was applied in an amoun o 0.3 o 0.075 g po −1 in he o m o KH 2 PO 4 . 14 days a e he i s nu ien supply, eapplica ion o he same amoun s was ca ied ou . The plan s we e cul i a ed in a g eenhouse a he School o Fo es Enginee ing a he Technical Uni e si y o Mad id (40◦27 ′ 05.3 ″ N, 3◦43 ′ 22.5 ″ W) om Oc obe o No embe . The day/nigh cycle was 16/ 8 h and he empe a u e was 16 ◦C a nigh and a mos 30 ◦C du ing he day. The ela i e humidi y (RH) a ied be ween 25% and 100%, wi h an a e age o a ound 85%. Po s we e a anged in a comple ely andomised design and we e e-a anged e e y week. Fo compa ison o di e en lea su ace p ope ies, he abo e- men ioned maize a ie ies we e selec ed based on he esul s o J.N. Henningsen e al. Plan Physiology and Biochemis y 205 (2023) 108170 3 (Henningsen e al., 2023b). Immedia ely a e ge mina ion, he plan s o bo h a ie ies we e di ided in o wo P soil-supply le els, namely su i- cien (0.3 g po −1 ) and de icien (0.075 g po −1 ). The de icien ly P supplied plan s o bo h cul i a s we e u he subdi ided in o i e ea men s, o which ou di e en P applica ion ea men s we e selec ed in addi ion o a con ol ea men (desc ibed in chap e 2.2). Be o e and a e he olia P applica ion, which was ca ied ou 41 DAS, se e al plan samples we e collec ed o de ailed cha ac e isa ion o he lea p ope ies and analysed using he me hods desc ibed below. 2.2. Folia P ea men and cha ac e isa ion o P sal esponse o humidi y Folia ea men s con ained 200 mM P supplied as KH 2 PO 4 o K 2 HPO 4 , bo h wi h and wi hou 0.1% Genapol X-80 (Sigma-Ald ich, S . Louis, MO, USA) su ac an . Folia P sp ays we e applied o he lea es o bo h maize a ie ies, esul ing in ou di e en olia applica ion ea - men s. When applying P ia he lea , i was ensu ed ha bo h he uppe (adaxial) and lowe (abaxial) lea su aces we e ully sp ayed. Co e ing he po s and w apping he s ems o he plan s p e en ed con amina ion o he soil by di ec applica ion o s em un-o (Henningsen e al., 2022). To de e mine he DRH and ERH o he P sal s, a clima ic chambe wi h adjus able ela i e humidi y (RH) and empe a u e was used (MKF 56, Binde , Tu lingen, Ge many). Fo assessing he DRH, 1 g o each sal was i s placed in a Pe i dish and hen kep in he clima ic chambe a 25 ◦C and ini ially a a low RH. Subsequen ly, he RH was inc eased in small s eps un il he hyd a ion o sal c ys als became isible using an op ical mic oscope. This RH ma ked he DRH and was hen main ained o abou an hou o ensu e ha he sal con inued o lique y due o wa e so p ion and dissolu ion. The samples we e le o e nigh a he maximum RH le el o 95%. In he case o non-hyg oscopic KH 2 PO 4 , which was no lique ied e en a 95% RH, a concen a ed solu ion was p epa ed close o i s solubili y alue. In a second s ep, he RH was g adually dec eased again and he solu ions we e obse ed wi h an op ical mic oscope o de e mine he poin a which deso p ion and c ys allisa ion lead o a ansi ion o a solid phase, hus ma king he ERH (Fe n´ andez e al., 2020). In o de o u he cha ac e ise he so p ion and deso p ion and hus also he DRH and ERH o KH 2 PO 4 and K 2 HPO 4 , dynamic apo so p ion (DVS) iso he ms we e pe o med a 25 ◦C wi h a TGA Q5000 ins umen (TA ins umen s, USA; The mal Analysis and Calo ime y Se ice IQAC- CSIC, Ba celona, Spain). App oxima ely 10 mg o he sal was placed in he TGA which was p elimina y kep a 20 ◦C and 0% RH o 300 min. A e wa ds, he RH was inc eased a 0.1% min −1 up o 95% RH o de e mine he DRH and, a e eaching he maximum RH, i was dec eased un il 0% RH by 0.1% min −1 o de e mine he ERH (Fe n´ andez e al., 2020). 2.3. Plan issue sampling and elemen al analysis Maize plan s we e ha es ed one (42 DAS; BBCH 14), se en (48 DAS; BBCH 14–15) and 14 (55 DAS; BBCH 15) days a e olia P applica ion o de e mina ion o lea issue P concen a ions. Ini ially, ou plan s (n =4) o each ea men we e di ided in o 4 h lea and emaining shoo . This pa i ioning o he shoo was done due o di e ences in he we abili y o he wo selec ed a ie ies, which only occu om he 4 h lea upwa ds as p e iously disco e ed (Henningsen e al., 2023b). Subsequen ly, all plan pa s we e washed in an acidi ied (0.1 N HCl) solu ion added wi h 0.1% de e gen (Fai y, P&G, Cincinna i, OH, USA) be o e being washed wice wi h ap wa e and once wi h dis illed wa e (Bahamonde e al., 2023b). The samples we e hen o en-d ied a 65 ◦C un il cons an weigh was eached, weigh and g ound (Cyclo ec 1093, Foss Teca o , H¨ ogan¨ as, Sweden). 200 mg o each plan sample was hen dissol ed wi h 10 mL o 69% HNO 3 (ROTIPURAN Sup a o ICP 69%, Ca l Ro h GmbH & Co. KG, Ka ls uhe, Ge many) in a mic owa e-o en (MARS 6 Xp ess; CEM, Ma hews, MC, USA) a 1800 W o 55 min. Subsequen ly, a e dilu ion wi h double-dis illed wa e o 100 mL, an aliquo o he sample was analysed by induc i ely coupled plasma op- ical emission spec oscopy (ICP-OES, Agilen Technologies 5800 RV, San a Cla a, CA, USA) (Henningsen e al., 2022). 2.4. Con ac angle measu emen s Equilib ium con ac angles we e measu ed wi h a d op shape anal- ysis sys em (DSA 100, K üss, Hambu g, Ge many). The adaxial and abaxial side, as well as he mid ib o he 4 h lea o bo h a ie ies, was moun ed on a mic oscope slide and d ops o dis illed wa e , KH 2 PO 4 , KH 2 PO 4 +0.1% su ac an , K 2 HPO 4 and K 2 HPO 4 +0.1% su ac an we e applied. D ops (app oxima ely 1.5 μ L) we e deposi ed wi h a 1 mL sy inge ha ing a 0.5 mm ip and he con ac angle was calcula ed using he angen me hod (Bahamonde e al., 2023a). 2.5. Elec on mic oscopy Adaxial and abaxial su ace samples o P-de icien and P-su icien 4 h lea es o bo h a ie ies we e collec ed on he day o olia applica- ion, and we e examined by scanning elec on mic oscopy (SEM). Immedia ely a e sampling, lea pieces (10 mm 2 ) we e placed in phospha e bu e (pH 7.2). Subsequen ly, he samples we e dehyd a ed in a se ies wi h pu e e hanol (Me ck KGaA, Da ms ad , Ge many) and dis illed wa e , ollowed by c i ical poin d ying (Leica EM CPD300, Leica Mic osys ems, We zla , Ge many). A e gold (Au) spu e ing, he samples we e obse ed by SEM (Jeol JSM6400, Jeol L d., Akishima, Japan; Na ional Elec on Mic oscopy Cen e (CNME), Mad id, Spain). In o de o cha ac e ise lea deposi ions o he di e en olia P applica ion ea men s, also by Ene gy-dispe si e X- ay spec oscopy (EDX), seg- men s o he 4 h lea we e addi ionally in es iga ed in a ield emission scanning elec on mic oscope (Sigma 300 VP, Ca l Zeiss, Jena, Ge - many; Miguel He n´ andez Uni e si y o Elche, Spain) eigh days a e olia applica ion wi hou p io dehyd a ion and spu e ing. T ansmission elec on mic oscopy (TEM) was used o assess he in- luence o P nu i ional s a us and olia P ea men on cell and su ace s uc u es in c oss-sec ion. The e o e, pieces (4 mm 2 ) o he 4 h lea we e selec ed 14 days a e olia applica ion and again imme sed in phospha e bu e o be hen pos - ixed in a 1:1 solu ion o osmium e- oxide (2%, TAAB Labo a o ies, Alde mas on, UK) and po assium e ocyanide (3%, Sigma-Ald ich, S . Louis, MO, USA). The samples we e subsequen ly dehyd a ed in a se ies wi h ace one and dis illed wa e be o e being imme sed in an ace one-Spu ’s esin (TAAB Labo- a o ies, Alde mas on, UK) mix u e o inc easing concen a ion un il pu e esin was p o ided. The lea pieces we e ans e ed in o blocks, embedded in pu e Spu ’s esin and s o ed in an o en a 70 ◦C un il comple e polyme isa ion. Ul a- hin sec ions we e hen cu (Leica Ul acu E, Leica Mic osys ems, We zla , Ge many) and obse ed wi h a Jeol JEM 1400plus (Jeol L d., Akishima, Japan; CNME, Spain) (Hen- ningsen e al., 2023a). 2.6. S a is ical analysis The s a is ical so wa e R ( e sion 2022.12.0, R ounda ion o s a- is ical compu ing, Vienna, Aus ia) was used o e alua e he da a (R Co e Team, 2022). Da a e alua ion s a ed wi h he de ini ion o an app op ia e s a is ical model based on gene alized leas squa es (model 1, 2 and 3; Ca oll and Ruppe , 1988; Box e al., 2015). Model 1 included he ac o s a ie y, ea men and hei in e ac ion e m. No e ha ea men is a pseudo ac o , ep esen ing he combina ion o he ac ual ac o s lea pa and P supply. This pseudo ac o was necessa y as he ac ual ac o s a e no o hogonal (Schaa schmid and Vaas, 2009). Model 2 included he ac o s a ie y, P supply, plan pa and ha es , as well as all in e ac ion e ms. Also, he co ela ions o he measu emen alues due o he se e al plan pa s we e aken in o accoun . In model 3, he ac o s a ie y, P supply and measu emen - ime, as well as all J.N. Henningsen e al. Plan Physiology and Biochemis y 205 (2023) 108170 4 in e ac ion e ms we e included. Also, he co ela ions o he measu e- men alues due o he se e al measu emen - imes we e aken in o ac- coun . The esiduals we e assumed o be no mally dis ibu ed and o be homoscedas ic (model 1) o he e oscedas ic wi h espec o he ac o s a ie y, P supply and plan pa (model 2) o a ie y, P supply and measu emen - ime (model 3). These assump ions a e based on a g aphical esidual analysis. Based on hese models, a Pseudo R 2 was calcula ed (Nakagawa and Schielze h, 2013) and an analysis o a i- ances (ANOVA) was conduc ed, ollowed by mul iple con as es s (Ho ho n e al., 2008; see also B e z e al., 2016) in o de o compa e he se e al le els o he in luence ac o s, espec i ely. 3. Resul s 3.1. Hyd a ion and dehyd a ion cha ac e is ics o P sal s Based on DVS iso he ms and supplemen a y clima e chambe ex- pe imen s, he DRH and ERH as key pa ame e s o he cha ac e isa ion o so p ion and deso p ion p ocesses we e ob ained o KH 2 PO 4 and K 2 HPO 4 (Fig. 1). Acco ding o he esul s o he DVS iso he m, he DRH o K 2 HPO 4 is a abou 46.9% RH (Fig. 1B). Fo KH 2 PO 4 which is no hyg oscopic as de i ed om he lack o hys e esis o he DVS iso he m which also had a Y axis anging be ween 0 and 2% mass change (i.e., he sal had a low wa e so p ion capaci y), his poin could no be clea ly iden i ied, indica ing ha he DRH is a >95% RH (Fig. 1A). The o e all inc ease in mass was signi ican ly g ea e o K 2 HPO 4 (132.9%) han o KH 2 PO 4 (−0.04%), also indica ing ha KH 2 PO 4 did no abso b any wa e and hus did no each he DRH in he 0–95% RH in e al analysed (i.e., he DRH is >95%; Fig. 1A and B). The inc ease in weigh o K 2 HPO 4 co esponds o he mola mass o h ee H 2 O molecules, indi- ca ing ha he hyd a ion s a e o he o iginally anhyd ous compound may ha e changed o h ee wa e molecules a e deso p ion. In clima e chambe expe imen s, he indings om he so p ion iso he m o KH 2 PO 4 we e con i med, because also in his case no hy- d a ion o he sal could be de ec ed a 95% RH (Fig. 1C and D). Addi- ionally, o K 2 HPO 4 , he DRH o 46.9% RH (DVS) was e i ied, as he onse o so p ion was obse ed in a simila RH ange (Fig. 1I). When deso bing he sa u a ed KH 2 PO 4 solu ion, he o ma ion o sal c ys als Fig. 1. Dynamic apo so p ion (DVS) iso he ms o KH 2 PO 4 (A) and K 2 HPO 4 (B) a 25 ◦C and images o he hyd a ion and dehyd a ion p ocesses o KH 2 PO 4 (C–G) and K 2 HPO 4 (H–L) o clima e chambe ials. Fo DVS iso he ms, blue a ows ep esen he DRH and ed ones he ERH. Fo hyd a ion and dehyd a ion images, ed a ows indica e he beginning o c ys al o ma ion a e eaching he ERH o KH 2 PO 4 (F) and he beginning o hyd a ion/change o liquid phase a e eaching he DRH o K 2 HPO 4 (I). Black numbe s indica e he ela i e humidi y [%] when he image was aken. J.N. Henningsen e al. Plan Physiology and Biochemis y 205 (2023) 108170 5 began a app oxima ely 72% RH, which is he ERH o his compound a 25 ◦C (Fig. 1F). The ERH o K 2 HPO 4 is below 15% RH as isually obse ed in clima e chambe ials (Fig. 1L). 3.2. Lea su ace cha ac e is ics 3.2.1. Lea su ace we abili y When d ops o pu e wa e we e applied on o he su ace o he 4 h lea o a ie y P7948 wi h a sp aye , hey adhe ed and he en i e lea was co e ed wi h small o medium-sized d ops (Fig. 2A). The su ace o a ie y DKC 3096, on he o he hand, e ained only a small amoun o ine d ops on he lea lamina. Howe e , mo e d ops adhe ed o he mid ib (Fig. 2B). These obse a ions can be con i med by con ac angle measu e- men s, which a e shown in Fig. 2C–F. When applying solu ions o KH 2 PO 4 and K 2 HPO 4 wi h pu e wa e (wi hou su ac an ), a ie y P7948 always had lowe con ac angles compa ed o DKC 3096, ega dless o he plan pa (Fig. 2C, E, Supplemen a y Table S1). Howe e , by adding a su ac an o he sp ay solu ion, his ela ionship was e e sed on he abaxial lea side and on he adaxial side he e was no longe any we abili y di e ence be ween maize a ie ies (Fig. 2D, F, Supplemen a y Table S1). Fu he mo e, he con ac angles d opped subs an ially due o he addi ion o a su ac an (e.g. om 135◦ o 42◦in DKC 3096; Fig. 2). While he adaxial and abaxial lea sides basically showed he same we abili y, he con ac angle o he mid ib was signi ican ly lowe (Fig. 2C, E, Supplemen a y Table S1). 3.2.2. Phospho us sal deposi ion in d ops Lea su aces o all ea men s and he KH 2 PO 4 and K 2 HPO 4 d op deposi s o med a e olia sp aying a e shown in Supplemen a y Figs. S1. To p o ide a close look a he lea su ace and e ilise d op deposi s o med on o olia -sp ayed lea es, SEM obse a ions we e ca ied ou (Fig. 3, Supplemen a y Fig. S2). Fi s o all, i can be no ed ha he 4 h lea o all ea men s o a ie y DKC 3096 we e co e ed wi h epicu ic- ula waxes (Fig. 3A, C, E, G, I, K). These waxes do no appea in a ie y P7948, which explains i s highe we abili y (Fig. 3B, D, F, H, J, L). While ha dly any deposi s we e isible o he naked eye as a esul o KH 2 PO 4 and K 2 HPO 4 olia applica ion in a ie y DKC 3096 (Supple- men a y Fig. S1), magni ica ion clea ly shows ha he e we e deposi s o be ound on o lea es (Fig. 3E, I, Supplemen a y Fig. S2C). O en, howe e , e ilise esidues adhe ed o he epicu icula waxes and did no appea o ha e ex ensi e con ac wi h he cu icle. The p e e en ial adso p ion o olia sp ay solu ions by he mid ib o a ie y DKC 3096, which was isible o he naked eye, can be con i med by Supplemen a y Fig. S2A. In a ie y P7948, esidues we e o en cha ac e ised by hei c ys alline s uc u e (Fig. 3F, H), some o which we e e y oluminous and elabo a e (Supplemen a y Figs. S2B and D) and o K 2 HPO 4 also by la , dense pa ches, as well as dend i ic c ys als (Fig. 3J, L). To check hei chemical composi ion, SEM-EDX analyses we e addi ionally ca - ied ou . F om he quali a i e a omic pe cen age o he obse ed de- posi s, we no ed ha he e we e changes in hei po assium (K) and P a ios and hus in hei chemical s uc u e (Supplemen a y Fig. S3, Table S2). The addi ion o a su ac an changed he shape o he deposi s, so ha in a ie y DKC 3096, i espec i e o he ype o P sal , p edomi- nan ly hin, od-shaped sal c ys als we e o med, some o which lied on he waxes and some o which we e embedded in hem (Fig. 3G, K). In he case o a ie y P7948, he d op deposi s we e again di e en , wi h c us s o a ying hickness being o med, lying la agains he lea su ace. These appea ed i m and enc us ed wi h KH 2 PO 4 +su ac an (Fig. 3H) and less i m and pa ly somewha luid wi h K 2 HPO 4 +su ac an (Fig. 3L). 3.3. Folia P abso p ion and ul as uc u al esponse 3.3.1. Folia P abso p ion One day a e olia applica ion, he we able 4 h lea o a ie y P7948 had a highe issue P concen a ion han he unwe able one o DKC 3096 o e e y olia P e ilise solu ion applied (Fig. 4A, D, Supplemen a y Table S3). The eby, all olia ea men s in a ie y P7948 eached he P concen a ion le el o su icien con ol plan s (Fig. 4D), whe eas in DKC 3096, only he applica ion o K 2 HPO 4 + su ac an led o signi ican ly inc eased P concen a ions (Fig. 4A). A e se en days, an appa en ly highe amoun o P om nea ly all olia sp ays was aken up by he 4 h lea es o a ie y DKC 3096 (Fig. 4B). Signi ican ly mo e lea issue P could be de e mined by applying solu- ions mixed wi h 0.1% su ac an , whe eby again, DKC 3096 plan s supplied wi h K 2 HPO 4 +su ac an clea ly abso bed he mos P and we e hus he only ones o each he le el o P-su icien con ol plan s (Fig. 4B). In he case o a ie y P7948, P concen a ion in he 4 h lea es o di e en ea men s emained cons an om he i s o he second Fig. 2. Di e en ly p onounced d ople adhesion on he well-we able 4 h lea o maize a ie y P7948 (A) and on he poo ly we able 4 h lea o maize a ie y DKC 3096 (B). Con ac angles om d ops o 200 mM KH 2 PO 4 (C), 200 mM KH 2 PO 4 +0.1% su ac an (D) 200 mM K 2 HPO 4 (E) and 200 mM K 2 HPO 4 +0.1% su ac an (F) applied o he adaxial and abaxial lea lamina as well as o he mid ib in a ie y DKC 3096 and P7948. Di e ences be ween a ie ies wi hin he same lea pa and solu e a e shown acco ding o mul iple con as es . Da a a e means ±SD (n =6). *** =p ≤0.001. J.N. Henningsen e al. Plan Physiology and Biochemis y 205 (2023) 108170 6 Fig. 3. Scanning elec on mic og aphs wi h 1.000×magni ica ion o he lea su ace o maize cul i a DKC 3096 (A, C, E, G, I, K) and P7948 (B, D, F, H, J, L) showing he P-su icien (A, B) and P-de icien con ol (C, D) as well as he deposi s o med on he lea su ace as a esul o olia applica ion o 200 mM KH 2 PO 4 (E, F), 200 mM KH 2 PO 4 +0.1% su ac an (G, H), 200 mM K 2 HPO 4 (I, J) and 200 mM K 2 HPO 4 +0.1% su ac an (K, L). J.N. Henningsen e al. Plan Physiology and Biochemis y 205 (2023) 108170 7 measu emen da e (Fig. 4E). Howe e , a e he hi d ha es , 14 days a e olia sp aying, he p e iously only sligh ly indica ed end owa ds highe P concen a ions in he +su ac an ea men s was now clea ly ecognisable and e en signi ican . Ne e heless, all olia P applied plan s s ill had signi ican ly highe P concen a ions han P-de icien con ol plan s (Fig. 4F). In DKC 3096, issue P concen a ions o he 4 h lea es also dec eased a e 14 days and only he +su ac an ea men s emained signi ican ly highe han he P-de icien con ol plan s (Fig. 4C). The emaining shoo (whole shoo excep o 4 h lea ) had a gene - ally highe P concen a ion le el han he 4 h lea (Fig. 5, Supplemen a y Table S3). The di e ences be ween he P ea men s in a ie y DKC 3096 we e simila o hose in he 4 h lea (Fig. 5A–C). Also o his pa o he plan , he K 2 HPO 4 +su ac an olia sp ay esul ed in he highes P concen a ions a all ha es da es, bu he le el o su icien ly P sup- plied plan s could only be eached se en days a e he olia ea men (Fig. 5B). While in he 4 h lea , he issue P concen a ions ollowing olia applica ion we e mos ly signi ican ly highe in a ie y P7948 han in DKC 3096, o he emaining shoo his was only he case in he KH 2 PO 4 +su ac an ea men on day 1 and 14 and in he KH 2 PO 4 ea men on day 14 a e olia sp ay (Fig. 5B, C, E, F, Supplemen a y Table S3). The highes P up ake was again de e mined by he applica ion o solu ions wi h su ac an s (Fig. 5D–F). In con as o a ie y DKC 3096, howe e , also he applica ion o pu e nu ien solu ions (wi hou su ac an ) in a ie y P7948 esul ed in a signi ican ly highe issue P concen a ion compa ed o he P-de icien con ol plan s on he 7 h and 14 h day (Fig. 5E and F). 3.3.2. Lea ul as uc u e The c oss-sec ion ul as uc u e o he 4 h lea o maize plan s is shown wi h a ie y P7948 as an example (Fig. 5). P de iciency has led o a signi ican ly educed numbe and size as well as o he mal o ma ion o chlo oplas s (Fig. 6B). Phospho us olia applica ion (Fig. 6C), in his case wi h he well-abso bed KH 2 PO 4 +su ac an , imp o ed he numbe and s uc u e o chlo oplas s compa ed o P-de icien plan s (Fig. 6B), bu could no achie e he ul as uc u e o P-su icien plan s. 4. Discussion In his s udy, we examined he in luence o a we able and an unwe able maize a ie y on he olia up ake o a hyg oscopic and a non-hyg oscopic P sal supplied as a olia sp ay wi h and wi hou su - ac an o P-de icien plan s. In addi ion, he in luence o olia P applica ion on he ul as uc u e o mesophyll and bundle shea h cells was examined. An a ay o a ia ions was eco ded ega ding he e ec o P supply, a ie y and esponse o olia P applica ion as desc ibed in he ollowing pa ag aphs. 4.1. P sal s di e in DRH and ERH and hus in P-sal deposi ion Acco ding o Sch¨ onhe (2002), nu ien sal s used o olia e il- isa ion should ha e a low DRH. The eason o his is he ac ha nu- ien s a e abso bed by lea es/isola ed cu icles a highe a es when supplied as solu es (i.e., dissol ed in wa e ) (Fe n´ andez e al., 2020). The e o e, a compound wi h a high DRH alue means ha a e solu ion Fig. 4. Plan issue P concen a ions [P] o he 4 h lea om maize a ie y DKC 3096 (A–C) and P7948 (D–F) ha es ed one day (A, D), se en days (B, E) and 14 days (C, F) a e olia applica ion o 200 mM KH 2 PO 4 , 200 mM K 2 HPO 4 , 200 mM KH 2 PO 4 +0.1% su ac an and 200 mM K 2 HPO 4 +0.1% su ac an . Le e s indica e di e ences be ween he ea men s wi hin each a ie y and ime o ha es acco ding o mul iple con as es (P ≤0.05). Da a a e means ±SEM (n =4). J.N. Henningsen e al. Plan Physiology and Biochemis y 205 (2023) 108170 8 d ying, i can only be abso bed i plan lea es a e exposed o a high RH, i.e. only in a na ow RH ange. Folia sp ays om compounds wi h a low DRH, on he o he hand, may po en ially pene a e lea es a a wide RH ange. E lo escence RH, which is an impo an indica o o he dehy- d a ion p ope ies o solu ions, has mos ly been neglec ed in s udies dealing wi h sal hyg oscopici y wi h ew excep ions (Fe n´ andez e al., 2020; Ba las e al., 2023; Bahamonde e al., 2023b). The P sal s used as olia e ilise s in his s udy ha e a DRH o 47% (K 2 HPO 4 ) and >95% (KH 2 PO 4 ) and an ERH o <15% (K 2 HPO 4 ) and 72% (KH 2 PO 4 ; Fig. 1). The a ying hyd a ion and dehyd a ion p ope ies o he P sal s ana- lysed, as indica ed by hei espec i e DRH and ERH alues, a e help ul o cha ac e ising hei di e en esponse o humidi y (i.e., hei hy- d a ing and d ying cycle) as a esul o he en i onmen al RH changes measu ed in he g eenhouse du ing he expe imen . In he s udy by Bu kha d e al. (2012) on he p ope ies o NaCl and NaClO 3 , i was shown ha hese cycles led o a change in he dis ibu ion o he applied compound. This was pa icula ly no iceable a e se e al we ing and d ying cycles, when he dend i ic g ow h o c ys als occu ed Fig. 5. Plan issue P concen a ions [P] o he whole shoo (excep o he 4 h lea ) om maize a ie y DKC 3096 (A–C) and P7948 (D–F) ha es ed one day (A, D), se en days (B, E) and 14 days (C, F) a e olia applica ion o 200 mM KH 2 PO 4 , 200 mM K 2 HPO 4 , 200 mM KH 2 PO 4 +0.1% su ac an and 200 mM K 2 HPO 4 +0.1% su ac an . Le e s indica e di e ences be ween he ea men s wi hin each a ie y and ime o ha es acco ding o mul iple con as es (P ≤0.05). Da a a e means ±SEM (n =4). Fig. 6. T ansmission elec on mic og aphs o he lea mesophyll and bundle shea h cells o a ie y P7948 o P-su icien 4 h lea (A), P-de icien 4 h lea (B), and P- de icien 4 h lea wi h olia P applica ion o KH 2 PO 4 +su ac an (C) 14 days a e olia applica ion. J.N. Henningsen e al. Plan Physiology and Biochemis y 205 (2023) 108170 9 (Bu kha d e al., 2012). This could also be obse ed wi h K 2 HPO 4 in a ie y P7948 (Fig. 3J, L) and could indica e ha K 2 HPO 4 has unde gone se e al cycles o c ys allisa ion-dissolu ion which may lead o s uc u al changes in c ys als also associa ed wi h di e en sal hyd a ion s a es. Howe e , RH mos ly luc ua ed in he ange om 65 o 100%. Thus, K 2 HPO 4 was mos ly dissol ed, which may a ou olia up ake (Eiche and Fe n´ andez, 2023). On he o he hand, KH 2 PO 4 was always close o i s DRH/ERH alues. Jus p io o eaching he DRH/ERH h eshold, he sal solu ion is a i s highes concen a ion, which may enhance he olia pene a ion p ocess ha is d i en by concen a ion g adien s (Eiche and Fe n´ andez, 2023). Rela i e humidi ies abo e he DRH, on he o he hand, will dilu e he solu ion, po en ially in luencing he p ocess o olia abso p ion. Howe e , i seems ha no only he dis i- bu ion and P concen a ion o deposi s, bu also he composi ion o he ini ially applied compounds was subjec o changes ollowing e ilise d op d ying and ehyd a ion. Lea SEM-EDX analyses indica e om he a omic pe cen age ha some deposi s expe ienced changes in hei K and P a ios and hus, in hei chemical s uc u e. Fo example, he a omic composi ion o he c ys al shown in Supplemen a y Fig. S2D is mo e likely o be KH 2 PO 4 , e en hough K 2 HPO 4 was ini ially supplied o he oliage (Supplemen a y Fig. S3, Table S2). Fu he mo e, Bu kha d e al. (2012) epo ed ha in he cou se o hyd a ing and d ying cycles, he ini ial DRH o compounds changed, which may addi ionally suppo he obse ed change in chemical s uc u e. Alike he wa e so p ion and deso p ion pe o mance o K 2 HPO 4 as obse ed in he DVS diag am (Fig. 1B), Fe n´ andez e al. (2020) epo ed molecula hyd a ion s a e changes o CaCl 2 a e subjec ing i o a hyd a ion-d ying cycle. The esul ing po en ial modi ica ions in s uc u al wa e molecules ha occu du ing he days a e olia e ilise sp aying may lead o physico-chemical changes in pa ame e s such as solubili y o DRH, also including he pa e ns o sal e-c ys allisa ion on o he oliage. The inc easingly b oad dis ibu ion due o dend i ic g ow h o c ys als as epo ed by Bu kha d e al. (2012) may acili a e he p ocess o hy- d a ion and dissolu ion o sal pa icles, bu he chemical na u e o he esul ing compounds (i.e., po en ial changes in s uc u al wa e mole- cules) will de e mine he dynamics o concen a ed sal solu ions and c ys allised d op deposi s on o lea es. No such change was de e mined o KH 2 PO 4 (Fig. 1A). 4.2. Va ie y, lea pa and P sal in luence he con ac angles o olia sp ay solu ions The occu ence o epicu icula wax c ys als in he o m o memb a- nous pla ele s (Ba hlo e al., 1998) on he 4 h lea o cul i a DKC 3096 esul ed in signi ican ly highe con ac angles o pu e wa e olia e ilise solu ions (i.e., wi hou su ac an ) compa ed o cul i a P7948, whe e no such nano-s uc u ed wax c ys als we e o med (Fig. 2C, E, 4) (Henningsen e al., 2023b). This ela ionship be ween he occu ence o wax c ys als and we abili y has been shown in se e al s udies (e.g., Koch and Ba hlo , 2009; Ba hlo e al., 2016, 2017; Henningsen e al., 2023b). Di e ences in we abili y esul ed in he 4 h lea o P7948 adso bing olia sp ays o a la ge ex end e en wi hou he addi ion o a su ac an , whe eas he 4 h lea o DKC 3096 was mos ly epellen and was only ully we ed by e ilise solu ions con aining a su ac an (Fig. 2A and B). Howe e , in bo h a ie ies, he mid ib had a lowe con ac angle han he lea laminae, so e en in he ac ually unwe able 4 h lea o a ie y DKC 3096 he mid ib was we able, as con i med by deposi ions (Supplemen a y Figs. S1C and S2A). The eby, he absence o c ys allised epicu icula waxes (as seen in Supplemen a y Fig. S2A) canno be he only eason o lowe con ac angles, as e en in a ie y P7948, whe e he e we e no wax c ys als on he su ace anyway, he con ac angle o he mid ib was smalle compa ed o he lea lamina (Fig. 2C, E, Supplemen a y Table S1). Ano he eason may be a chemi- cally and s uc u ally di e en su ace o eins and bundle shea h ex- ensions (Fe n´ andez e al., 2021). Bahamonde e al. (2018) also obse ed lowe con ac angles on eins o beech, hus inc easing he up ake o olia -applied nu ien s, which can also be con i med by ecen s udies on ba ley and oma o (A sic e al., 2020, 2022; Henningsen e al., 2023a). Folia sp ay esidues we e no only in luenced by hei chemical p ope ies bu also by di e en lea we abili y. While a b oad dis i- bu ion was achie ed on he we able lea su ace, especially wi h pu e K 2 HPO 4 and bo h P-sal -solu ions wi h su ac an (Fig. 3H,J, L), a p o- nounced, spa ially delimi ed c ys al o ma ion was mo e e iden on he unwe able su ace and wi h K 2 HPO 4 on he we able one (Fig. 3F, G, K, Supplemen a y Fig. S2B). An excep ion o unwe able su aces ega ding deposi dis ibu ion was K 2 HPO 4 +su ac an , whe e also b oadly dis ibu ed deposi s we e p esen (Fig. 3K). The dis ibu ion o he deposi ed solu es, which will also be in luenced by hyd a ion and dehyd a ion cycles (Bu kha d e al., 2012), hus seems o be enabled only by ce ain olia solu e cha ac e is ics and lea su ace p ope ies, in his case, he lack o epicu icula wax c ys als and hus we abili y, whe eby su ac an s only seem o ha e a majo in luence on his p ocess in combina ion wi h speci ic hyg oscopic sal s, in ou case K 2 HPO 4 (Fig. 3G, K). 4.3. Folia P abso p ion is la gely in luenced by we abili y a he han P sal hyg oscopici y Lea we abili y, chemical p ope ies o olia e ilise s and he use o su ac an s a e ac o s ha ha e been iden i ied as impo an pa- ame e s o he up ake o olia -applied nu ien s (Sch¨ onhe , 2001, 2002; Fe n´ andez and Eiche , 2009; Pei ce e al., 2019; Fe n´ andez e al., 2021). Howe e , he indi idual signi icance in he combina ion o hese ac o s emained unclea o da e. Fac o s in luencing olia abso p ion ha e o en been in es iga ed in isola ion and unde a i icial condi ions (e.g. isola ed cu icles; Fe n´ andez e al., 2021). In his s udy, he com- pa ison o wo maize cul i a s unde g eenhouse condi ions showed ha we abili y is he majo ac o in luencing he up ake o olia applied P (Fig. 4). I espec i e o he olia P ea men (KH 2 PO 4 ±su ac an , K 2 HPO 4 ±su ac an ), P concen a ions o adequa ely nou ished plan s (3.5–6 g P kg −1 d y ma e acco ding o Be gmann (1983)) we e ob- ained in we able lea es a e olia sp aying o P-de icien plan s (Fig. 4D and E). This inding con adic s he s a emen by Sch¨ onhe (2002), ha sal s wi h high DRH (e.g. KH 2 PO 4 ) a e gene ally no sui - able as olia e ilise s. Bea ing in mind ha hyd a ion and dehyd a ion cycles can e en al e DRH (Bu kha d e al., 2012; Fe n´ andez e al., 2020), his s a emen is weakened e en mo e. Howe e , i he lea su ace was unwe able, he chemical p ope ies o he hyg oscopic/non-hyg oscopic P sal in combina ion wi h he use o a su ac an became a majo ac o (Fig. 4A–C). E en hough bo h P sal s we e abso bed signi ican ly mo e by he addi ion o a su ac an , he abso p ion o K 2 HPO 4 +su ac an was again signi ican ly highe and e en eached he le el o P-su icien con ol plan s, se en days a e olia applica ion (Fig. 4B). The signi ican inc ease in P concen a ion one day a e olia applica ion showed ha he du a ion o he liquid phase o he sp ayed P solu ion, due o a low ERH, in combina ion wi h a b oad dis ibu ion by su ac an , we e key d i e s o olia P up ake (Fig. 4A). A e a ew days, he impo ance o hyd a ing-d ying cycles also became appa en , wi h ea men s con aining a su ac an ha ing signi ican ly highe up ake (Fig. 4B). Howe e , i should be no ed ha low DRH/ERH alone had li le e ec on P up ake in he unwe able 4 h lea o DKC 3096, because only in combina ion wi h a su ac an a high P up ake a e may be achie ed (Fig. 4A–C). Highe P abso p ion a es we e u he con i med by he occu ence o ligh lea bu n, which is a consequence o , o example, excessi e nu ien concen a ions o he addi ion o su ac an s ha may al e he lea su ace (Fe n´ andez and Eiche , 2009; Bu kha d , 2010). P e iously i was e ealed, ha only he 4 h and 5 h lea es o P7948 we e mo e we able han hose o DKC 3096 (Henningsen e al., 2023b). The e o e, when compa ing bo h a ie ies, he up ake a es o he emaining shoo wi hou 4 h lea we e mo e simila han hose o he 4 h J.N. Henningsen e al.