The effects of nitrogen form on root morphological and

Research Article

The effects of nitrogen form on root morphological andphysiological adaptations of maize white lupinand faba bean under phosphorus deficiency

Haitao Liu1 Caixian Tang2 and Chunjian Li1

1 Department of Plant Nutrition China Agricultural University Beijing 100193 China2 Department of Animal Plant and Soil Sciences La Trobe University Melbourne Campus Bundoora VIC 3086 Australia

Received 18 January 2016 Accepted 4 August 2016 Published 12 August 2016

Associate Editor Astrid Volder

Citation Liu H Tang C Li C 2016 The effects of nitrogen form on root morphological and physiological adaptations of maize whitelupin and faba bean under phosphorus deficiency AoB PLANTS 8 plw058 doi101093aobplaplw058

Abstract Root morphologicalphysiological modifications are important for phosphorus (P) acquisition of plantsunder P deficiency but strategies differ among plant species Detailed studies on the response of maize roots to Pdeficiency are limited Nitrogen (N) form influences root morphologyphysiology and thus may influence root re-sponses to P deficiency This work investigated adaptive mechanisms of maize roots to low P by comparison withwhite lupin and faba bean supplied with two N forms Plants were grown for 7ndash16 days in hydroponics with sufficient(250 mmol L1) and deficient P supply (1 mmol L1) under supply of NH4NO3 or Ca(NO3)2 Plant growth and P uptakewere measured and release of protons and organic acid anions and acid phosphatase activity in the root were mon-itored The results showed that P deficiency significantly decreased shoot growth while increased root growth andtotal root length of maize and faba bean but not white lupin It enhanced the release of protons and organic acidanions and acid phosphatase activity from the roots of both legumes but not maize Compared with Ca(NO3)2NH4NO3 dramatically increased proton release by roots but did not alter root morphology or physiology of the threespecies in response to low P It is concluded that the N form did not fundamentally change root morphologicalphys-iological responses of the three species to P deficiency Morphological variation in maize and morpho-physiologicalmodifications in white lupin and faba bean were the main adaptive strategies to P deficiency

Keywords Low P availability organic acids rhizosphere root exudation root morphology species variation

Introduction

Low phosphorus (P) availability in the soil is one of the mostlimiting factors for crop production (Schachtman et al 1998Lynch 2007) Plants have evolved different mechanisms inroots in order to increase P acquisition under P-limiting con-ditions These mechanisms include morphological modifica-tions mycorrhizal symbioses rhizosphere acidification

release of carboxylates and phosphatases and up-regulation of P transporters (Neumann and Romheld 1999Raghothama 1999 Hinsinger 2001 Lambers et al 2006)Root morphological variations are beneficial for labile P ac-quisition since P is relatively immobile in soil and enhancedroot growth increases exploration of localized P sources(Barber 1995) while root physiological modifications can

Corresponding authorrsquos e-mail address lichjcaueducn

VC The Authors 2016 Published by Oxford University Press on behalf of the Annals of Botany CompanyThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (httpcreativecommonsorglicensesby40) which permits unrestricted reuse distribution and reproduction in any medium provided the original work is prop-erly cited

AoB PLANTS wwwaobplantsoxfordjournalsorg VC The Authors 2016 100

increase mobilization of non-labile P in soil (Neumann andRomheld 1999 Hinsinger 2001)

Although the adaptive mechanisms of plants to P defi-ciency are well understood relatively few studies haveaddressed the differences in root responses to P defi-ciency among plant species White lupin (Lupinus albus)a model plant to study root response to P deficiencyshows a superior ability to utilize sparingly soluble P andbound P from soil by developing proteoid roots that ex-ude protons (Dinkelaker et al 1989) citrate and acidphosphatases (APase) (Dinkelaker et al 1989 Gerke et al1994) Although faba bean (Vicia faba) does not formproteoid roots under P deficiency its extensive rootsystem a morphological advantage enables explora-tion of a larger volume of soil to access inorganicP (Nuruzzaman et al 2005a b) It also releases largeamounts of protons phenolics and APase which enhancemobilization of soil non-labile P and hence P uptake(Nuruzzaman et al 2005a Li et al 2007)

Maize is widely cultivated for both staple food and indus-trial usage in tropical and temperate soils worldwide(Calderon-Vazquez et al 2011) Results of the adaptiveresponses in maize roots to P deficiency however are con-troversial Maize roots always showed an extensive morpho-logical variation when the roots had restricted growth (Yanet al 2011) and grew in nitrate-rich patches (Yu et al 2013)and under P deficiency (Anghinoni and Barber 1980 Zhanget al 2012) Phosphorus deficiency has been shown to eitherincrease (Gaume et al 2001) or decrease (Liu et al 2004Corrales et al 2007) the release of organic acid anions fromthe roots in solution culture It enhanced (Kummerova1986) or did not change (Corrales et al 2007) the activity ofAPase on root surface Furthermore P deficiency did notchange rhizosphere pH (Neumann and Romheld 1999George et al 2002) or resulted in a strong alkalization in therhizosphere (Li et al 2007)

Nitrogen form influences the balance of cation and an-ion uptake by plant roots and thus alters the rhizospherepH The change of rhizosphere pH caused by differentialuptake of cations and anions is more prominent than thatcaused by root exudates such as protons and organic acidanions (Jaillard et al 2003 Tang and Rengel 2003Marschner 2012) Ammonium nutrition leads to preferen-tial cation uptake and thus net proton excretion by rootscausing rhizosphere acidification whilst nitrate supply in-duces hydroxyl secretion and causes rhizosphere alkaliza-tion (Gahoonia and Nielsen 1992 Mengel et al 2001 Tanget al 2011) In legumes on one hand N2 fixation generallyleads to rhizosphere acidification due to excess uptake ofcations over anions (Tang et al 1997 Hinsinger et al 2003Li et al 2008) On the other hand N source is one of thefactors affecting P supply of plants Ammonium supply of-ten enhances plant uptake of P from soil via rhizosphere

acidification (Miller 1974 Marschne 2012) A lower pH inthe rhizosphere could increase the H2PO4 HPO2

4 ratio andthe solubility of Ca-phosphates However interactions of Nform and P supply on rhizosphere pH are not fully under-stood For example P deficiency could decrease increaseor have no effect on rhizosphere pH in NO3 -fed plants (egDinkelaker et al 1989 Neumann and Romheld 1999 Tanget al 2009) These different responses to P supply and Nform depend on plant species

The aim of this study was to elucidate the adaptivestrategies of maize roots in response to P deficiency andto investigate the influence of N form on these responsesusing white lupin and faba bean as reference species

Methods

Plant materials and treatments

Two hydroponic experiments were conducted in thegreenhouse under natural light with 28 C16 C daynight temperatures and 45ndash55 relative humidity Eachexperiment consisted of three factors which were P sup-ply (LP 1 mmol L1 P and HP 250 mmol L1 P) N forms[NH4NO3 and Ca(NO3)2] and plant species [maize (Zeamays L cv Zhengdan 958) white lupin (Lupinus albus Lcv Amiga) and faba bean (Vicia faba L cv Lincan No 5)]There were four replicates for each treatment

Seeds of the three plant species were surface-sterilized in 10 (vv) H2O2 for 30 min rinsed thoroughlyin distilled water and then germinated between filter pa-pers moistened with saturated CaSO4 solution in thedark After a week seven seedlings of each species weretransferred into 7-L opaque pots (21 cm in diameter and24 cm in height) filled with nutrient solution of the fol-lowing composition (mol L1) K2SO4 75104 MgSO4

65104 KCl 1104 H3BO3 10106 MnSO4

10106 CuSO4 10107 ZnSO4 10106(NH4)6Mo7O24 50109 and Fe-EDTA 20104 (Niuet al 2007) Nitrogen was supplied as Ca(NO3)2 orNH4NO3 at the concentration of 20103 mol L1Phosphorus was supplied as KH2PO4 at 1 or 250 mmolL1 and KCl was added to the LP treatment to keep thesame K concentration between the P treatments (Panget al 2010) The pH of nutrient solution was adjusted to60 using 1 mol L1 HCl or NaOH when the solution wasrenewed The nutrient solution in each pot was continu-ously aerated and renewed every 3 days

The first experiment was harvested 12 days after thecommencement of treatments (DAT) Seven plants ofeach species in each pot were harvested as follows (1)two plants were used to collect root exudates (2) twoplants were used to measure APase activity on root sur-face (3) two plants were used to study rhizosphere acidi-ficationalkalization using the agar technique (4) one

Liu et al ndash Effects of N form on root responses of three plant species to P deficiency

002 AoB PLANTS wwwaobplantsoxfordjournalsorg VC The Authors 2016

plant was used to measure rhizosphere pH using thescanning ion-selective electrode technique

In the second experiment nutrient solution pH wasmeasured at 1000 am daily during the experimental pe-riod using a pH meter (pH Testr30 USA) Plants were har-vested 7 12 and 16 days after the commencement oftreatments At each harvest two roots of each specieswere kept at 4 C for root length analysis Five plantswere separated into shoots and roots and dried for dryweight and P measurements In both experiments nonodules were observed on roots of the two legumes andno cluster roots were observed in white lupin at the lastharvest (Wang et al 2006)

Collection of root exudates and analysis oforganic acids

Plant roots were carefully rinsed three times with deion-ized water and were then immersed in 100 mL collectionsolution in an opaque vessel and aerated for 2 h from1000 am to 1200 noon for collection of root exudatesCollection of root exudates under non-sterile conditionreflects the root exudates in nature (Shen 1998) Thecompositions of the collection solution were (lmol L1)200 MgCl2 100 KCl 600 CaCl2 and 5 H3BO3 These nutri-ents in the collection solution allowed for membrane in-tegrity and minimizing osmotic stress and possiblepassive leakage After collection a subsample of 8 mL ofcollection solution was acidified by adding two drops ofconcentrated H3PO4 Microbe inhibitor (MicropurKatadyn Deutsschl and Gmbh Munchen Germany) wasadded into the collected exudate solutions at 001 g L1

to prevent microbial decomposition of root exudates(Shen et al 2004) The 8 mL of collection solution wasthen filtered through 02-lm membrane and frozen at20 C until analysis

Organic acids were analyzed by a reversed-phase highperformance liquid chromatography (Wang et al 2006)Separation was conducted on a 25046 mm reversedphase column (Alltima C-18) The mobile phase was25 mmol L1 KH2PO4 (pH 25) with the flow rate of 1 mLmin1 at 28 C and UV detection was set at 214 nmOrganic acids in the sample were identified by compari-son with the retention times and absorption spectra ofpure standards including tartaric malic citric fumaricand T-aconitic acids which are the reported major or-ganic acids released by white lupin faba bean and maize(Dinkelaker et al 1989 Neumann and Romheld 1999Gaume et al 2001 Liu et al 2004 Li et al 2007)

Measurement of acid phosphatase activity

Plant roots were washed three times using deionized wa-ter and blotted dry The roots were placed in opaque

vessels containing 05 g L1 p-nitrophenyl phosphate(NPP) as a substrate in acetate buffer (01 mmol L1 pH56) for 1 h under nature illumination Five mL of the reac-tion solution was mixed immediately with 25 mL NaOH(2 mol L1) to terminate the reaction and to develop thecolour (McLachlan 1980) The absorbance of the solutionwas determined at 405 nm using a spectrophotometer(UVmini 1240 Japan) The APase activity in the root wascalculated on a basis of an oven-dried weight

Rhizosphere acidificationalkalization

Rhizosphere acidificationalkalization was detected usingan agar technique modified from Marschner andRomheld (1983) Intact roots of the plants cultured intreatment solutions for 12 days were washed in deion-ized water (pH 59) spread out in a flat tray(310 mm150 mm) and covered immediately with theagar solution (07 38 C) containing pH indicator 001 bromocresol-purple at pH 59 After 30 min imageswere recorded A yellow colour indicates acidificationwhereas purple colour means alkalization

Non-invasive measurement of H1 fluxes on rootsurface

Net Hthornfluxes on root surface were measured using thescanning ion-selective electrode technique as describedby Xu et al (2006) Before measurement microelec-trodes were calibrated with pH 55 and pH 65 measuringsolution only those with a Nernst slope of 58 6 6 mVwere used The Hthornflux was recorded every 6 s for 10 minData were treated using Mageflux software (Version 10)developed by Xuyue Company (httpxuyuenetmageflux Sun et al 2009) The results were presented as pmolcm2 s1

After washing in deionized water the roots wereplaced in a beaker containing measuring solution(01 mmol L1 KCl 01 mmol L1 CaCl2 01 mmol L1

MgCl2 05 mmol L1 NaCl 03 mmol L1 MES 02 mmolL1 NaSO4 pH 60) (Sun et al 2009) After 20 min theroots were transferred into a Petri dish containing freshmeasuring solution Net Hthornfluxes were continuously re-corded for 10 min at 10 mm from the tip of lateral roots

Dry weight and P content measurement

The shoots and roots harvested from the second experi-ment were dried at 105 C for 30 min and then at 70 Cto a constant weight After dry weights were recordedthe materials were ground into powder Subsamples ofground materials were digested with a mixture of con-centrated sulfuric acid and 30 H2O2 (vv) and total Pcontent determined using the molybdivanadate method(Soon and Kalra 1995)

Root length analysis

The harvested roots were digitally scanned (Epson V700Jakarta Indonesia) and the resulting images were ana-lyzed with the WinRHIZO version 50 (RegentInstruments Quebec City Canada) Axial root length wasmeasured by a ruler before scanning it stands for pri-mary root length of legumes and the sum of axial rootlengths of primary root seminal roots and crown roots ofmaize Lateral root density indicated the numbers of thefirst-order lateral roots per unit length of an axial rootHigher order lateral roots were not measured Specificroot length was the ratio of root length to root dryweight

Calculations

Root P influx between the two harvests (7 and 16 DAT) inthe second experiments was calculated according toBrewster and Tinker (1972)

In frac14 P2 P1eth THORNt2 t1eth THORN

loge L2=L1eth THORNL2 L1eth THORN (1)

where In refers to P influx (mg m1 d1) P refers to plantP content (mg plant1) t refers to time (days) and L re-fers to root length (m plant1) The indices 1 and 2 referto 7 and 16 DAT respectively

Phosphorus-use efficiency (PUE) and P-absorption effi-ciency (PAE) were calculated according to Equations (2)and (3) respectively (Jakobsen et al 1992 Corrales et al2007)

PUE frac14 total plant dry weight=total plant P content (2)

PAE frac14 total plant P content=total root length (3)

Rootshoot ratio was calculated according to Claassenand Jungk (1984)

Root=shoot ratio frac14 L=W (4)

where L refers to root length (m plant1) and W refers toshoot dry weight (g plant1)

Statistical analysis

The statistical means of different treatments were com-pared using the least significant difference (LSD) at a005 level of probability using SAS 802 Differences be-tween the P treatments were analyzed using the one-way PROC ANOVA The effects of P supply and N formson the variables and their interactions were tested usinga two-way analysis of variance

Results

Plant growth and P uptake

Low-P supply (1 mmol L1) significantly decreased dryweights of maize at last two harvests and of faba beanat the last harvest under both N forms compared withHP (250 mmol L1) supply (Table 1) These decreasedplant dry weights were the consequence of the de-creased shoot dry weights with the decrease beinggreater for maize than for faba bean [see SupportingInformationmdashFigure S1A and C] In contrast LP in-creased root dry weights of the two species at three har-vests under Ca(NO3)2 and of maize at the first twoharvests under NH4NO3 [see Supporting InformationmdashFigure S1B and D] However LP supply did not affect thedry weights of shoot or roots of white lupin at all threeharvests irrespective of N form [Table 1 see SupportingInformationmdashFigure S1]

Nitrogen forms did not affect whole plant dry weightsof white lupin and faba bean at three harvests with theexception at 12 days for white lupin with significantlower dry weights under NH4NO3 than under Ca(NO3)2 Incomparison the plant dry weight of maize was signifi-cant higher under NH4NO3 than under Ca(NO3)2 The in-teraction between P levels and N forms on plant dryweight was only significant in maize at the last two har-vests (Table 1)

Among the three species total root length (TRL) wasthe greatest in maize and the smallest in white lupinCompared with the HP treatment in maize LP increasedTRL axial root length (ARL) and lateral root density (LRD)at all three harvests under Ca(NO3)2 and increased spe-cific root length (SRL) at the last two harvests underCa(NO3)2 Under NH4NO3 supply however LP increasedTRL ARL and LRD at first two harvests while inhibitedSRL of maize at the last harvest For faba bean LP in-creased TRL due to the increased LRD at the last twoharvests under Ca(NO3)2 while decreased TRL due to thedecreased LRD at the last harvest under NH4NO3 Inmost cases LP did not change the measured root lengthparameters of white lupin under both N forms comparedwith the HP treatment (Table 2)

All three species had significantly lower P content ofthe whole plant supplied with LP than with HP underboth N forms Similar to the influence on plant dryweight N forms influenced plant P content only in maizewith more P uptake under NH4NO3 but not in two le-gumes In maize increasing P supply increased the Pcontent more under NH4NO3 than under Ca(NO3)2 lead-ing to an interaction between P levels and N forms(Table 1) Low P supply significantly decreased wholeplant N and K contents of maize and faba bean at thelast two harvests but not of white lupin at either harvest

compared with the HP supply [see SupportingInformationmdashTable S1] Nitrogen forms significantlyinfluenced whole plant N and K contents of the threespecies with few exceptions at the first harvest NH4NO3

facilitated N uptake of all three species while Ca(NO3)2

facilitated K uptake of legumes Unlike the legumesmaize took up more K under NH4NO3 than underCa(NO3)2 at the last harvest under both P levels [seeSupporting InformationmdashTable S1]

Plant P concentration and P efficiency

Compared with HP supply LP significantly decreasedplant P concentration (g P per 100 g DW) in all three spe-cies under both N forms Maize showed the lowest P con-centration under LP level while the highest value underHP level Consistent with these results maize had thelowest P-influx rate between the first and the third har-vest (Table 3) and P-absorption efficiency (PAE) at threeharvests under LP level and both N forms (Fig 1A and C)when P influx and PAE were calculated per unit of rootlength The P-influx rate was calculated by Equation (1)

using the data in Tables 1 and 2 Because the P contentof maize plants grown at LP did not increase betweenDays 7 and 16 (Table 1) the calculated P-influx rates ofmaize under LP and both N forms were zero (Table 3)However irrespective of N form maize had the highestP-use efficiency among the three plant species when LPwas supplied (Fig 1B and D) Nitrogen form did not affectP concentration in maize or faba bean at a given P level(Table 3)

The supply of LP significantly increased the rootshootratio (m root length g1 shoot DW) of maize under bothN forms and of faba bean under Ca(NO3)2 but did not af-fect the rootshoot ratio of white lupin under either Nform compared with HP supply (Table 3)

Proton release in roots of three plant speciesunder two N forms

When Ca(NO3)2 was used as the N source LP treatmentincreased proton release and decreased solution pH un-der both legumes especially faba bean (Fig 2A and B)In contrast the solution pH under maize was higher than

Table 1 Dry weights and P contents of the whole plant of white lupin faba bean and maize grown with low (LP) (1 mmol L1) and high P (HP)(250 mmol L1) under two N forms [ca(NO3)2 and NH4NO3] for 7 12 and 16 days

N forms P supply White lupin Faba bean Maize

7 d 12 d 16 d 7 d 12 d 16 d 7 d 12 d 16 d

Plant biomass (g dry weight per plant)

Ca(NO3)2-N LP 012 028 047 046 072 111 034 056 072

HP 014 030 053 042 080 138 027 084 157

NH4NO3-N LP 012 018 047 043 071 107 037 061 079

HP 012 021 050 043 077 137 034 101 199

LSD (Pfrac14 005) 001 003 004 004 008 014 003 006 008

P level ns ns ns ns ns

N form ns ns ns ns ns

P N ns ns ns ns ns ns ns

P content (mg P per plant)

Ca(NO3)2-N LP 104 162 167 339 406 433 089 073 075

HP 148 234 396 537 834 1546 438 1029 1990

NH4NO3-N LP 105 118 167 353 460 501 097 087 096

HP 134 188 469 598 862 1579 547 1506 2511

LSD (Pfrac14 005) 012 026 038 060 152 203 062 128 217

P level

P N ns ns ns ns ns ns

P005 Plt001 Plt0001 ns not significant at Pfrac14005

the initial pH value throughout the experimental periodunder both P levels indicating an alkalization process(Fig 2C) When NH4NO3 was supplied the pH of solutiongrown with all three species declined dramatically overtime with an exception of white lupin during the first 6days (Fig 2A) LP treatment further decreased solutionpH under both legumes during the later cultural periodwhile increasing solution pH in maize during the sameperiod In comparison the solution pH was lower withmaize and faba bean than with white lupin underNH4NO3 supply (Fig 2)

Rhizosphere acidificationalkalization and net Hthornfluxwere compared among the three plant species on 12days after treatments (Figs 3 and 4) When Ca(NO3)2 wassupplied yellow colour was observed surrounding theroot tips of white lupin and LP-treated faba bean (Fig3AndashC) indicating rhizosphere acidification while purplecolour along with the root surface of maize and HP-treated faba bean (Fig 3DndashF) revealed a rhizospherealkalization When NH4NO3 was supplied yellow colourappeared on the root tips of maize and on the surface of

the entire root of the two legumes (Fig 3GndashL) The resultswere consistent with those of solution pH measurement(Fig 2) and net Hthornflux determined using the scanningion-selective electrode technique (Fig 4) Proton releasefrom the root tips was greater under NH4NO3 than underCa(NO3)2 (Fig 4)

Root release of organic acid anions and acidphosphatase activity

Release of organic acid anions and APase activity on rootsurface were measured 12 days after treatments The to-tal amounts of organic acid anions released by the le-gume roots under both N forms were greater in the LPthan in the HP treatment where the increases of malatecitrate and tartrate in white lupin and of tartrate in fababean were significant In contrast the release of organicacid anions by maize roots was lower in the LP than inthe HP treatment (Table 4)

Low P supply significantly increased the APase activityon root surface of the legumes but not maize The APase

Table 2 Root length parameters of white lupin faba bean and maize grown with low (LP) (1 mmol L1) and high P (HP) (250 mmol L1) undertwo N forms [ca(NO3)2 and NH4NO3] for 7 12 and 16 days

Parameters Ca(NO3)2 as N source NH4NO3 as N source

White lupin Faba bean Maize White lupin Faba bean Maize

LP HP LP HP LP HP LP HP LP HP LP HP

7 days

TRL (m) 16 b 20 a 46 b 61 a 145 a 124 b 18 a 17 a 47 a 48 a 209 a 149 b

ARL (cm) 283 b 315 a 323 a 360 a 2516 a 2195 b 238 b 269 a 261 a 297 a 2214 a 1899 b

LRD (no cm1) 26 a 24 a 14 b 17 a 40 a 34 b 27 a 21 b 35 a 25 b 39 a 34 b

SRL (m g1) 412 a 414 a 270 a 335 a 1113 a 1136 a 437 a 380 b 285 a 280 a 1590 a 1479 a

12 days

TRL (m plant1) 39 a 49 a 110 a 88 b 336 a 245 b 39 a 43 a 90 a 98 a 474 a 269 b

ARL (cm) 360 a 357 a 387 a 424 a 4974 a 3368 b 349 a 389 a 390 a 412 a 4205 a 3017 b

LRD (no cm1) 39 a 29 b 20 a 16 b 37 a 35 b 32 a 35 a 28 a 29 a 48 a 41 b

SRL (m g1) 860 a 739 b 367 a 430 a 1762 a 1500 b 824 a 807 a 428 a 368 b 2260 a 2161 a

16 days

TRL (m) 62 a 63 a 235 a 131 b 432 a 362 b 58 a 69 a 107 b 182 a 527 a 505 a

ARL (cm) 529 a 486 a 518 a 16 b 5068 a 4543 b 366 b 468 a 468 a 462 a 5083 a 4501 a

LRD (no cm1) 37 a 35 a 18 a 16 b 40 a 35 b 36 a 35 a 21 b 28 a 45 a 44 a

SRL (m g1) 746 a 681 a 736 a 638 b 1969 a 1891 b 797 a 825 a 562 b 727 a 2011 b 2697 a

Values of each pair in rows of individual plant species followed by different letters represent significant differences between the P treatments

(Plt005) Means 6 SE nfrac144 Abbreviations TRL total root length (m plant1) ARL axial root length (cm) LRD lateral root density (number

of lateral roots per unit axial root nocm) SRL specific root length (m g1)

Table 3 Plant P concentration and rootshoot ratio at the final harvest (16 days after treatment) and P influx rate between 7 and 16 days ofwhite lupin faba bean and maize grown with low (LP) and high P supply (HP) under two N forms [ca(NO3)2 and NH4NO3]

Parameters Crops Ca(NO3)2-N NH4NO3-N LSD P level N form P 3 N

LP HP LP HP

P concentration White lupin 035 076 036 094 007

(g P 100 g1 DW) Faba bean 039 112 047 115 009 ns ns

Maize 010 127 012 126 005 ns ns

P influx White lupin 002 007 002 010 001 ns

(mg P m1 root length d1) Faba bean 001 012 002 011 002 ns ns

Maize 000 008 000 008 001 ns ns

rootshoot ratio White lupin 165 156 156 181 18 ns ns ns

(m root length g1 shoot DW) Faba bean 313 122 149 180 35

Maize 1053 280 1126 305 80 ns ns

Figure 1 P-absorption efficiency (PAE) and P-use efficiency (PUE) of white lupin faba bean and maize grown with low (LP) and high P (HP)under two N forms for 7 12 and 16 days Nitrogen was supplied as Ca(NO3)2 on left panels and NH4NO3 on right panels Values are means offour replicates and error bars represent the standard error of the mean

activity on root surface was higher under NH4NO3

than under Ca(NO3)2 for all three species and washigher in white lupin than in faba bean and maize(Fig 5)

Discussion

Morphological modification was the mainadaptive response of maize roots to P deficiency

This study demonstrated that the growth of maize wasmore sensitive to P deficiency than that of the twolegumes This is consistent with the findings that maizehas a high soil-fertility requirement to attain maximalgrain yield (Paponov and Engels 2003) Under P defi-ciency more carbon was distributed to roots and wasused for the formation of new roots but not for in-creases in the release of protons organic acid anionsand APase activity although the two N forms had con-trasting effects on net Hthorn release (Figs 2ndash5 and Table4) These responses were also true for different maizegenotypes (Anghinoni and Barber 1980 Corrales et al2007 Li et al 2012 Fernandez and Rubio 2015) under abroad range of growing conditions including differentP levels and growth media like hydroponics (Anghinoniand Barber 1980 Mollier and Pellerin 1999 Gaumeet al 2001 Li et al 2012 Fernandez and Rubio 2015)sand culture (Corrales et al 2007) and field conditions(Fernandez et al 2009 Zhang et al 2012) Additionallythe density and the length of maize root hairs werealso found to increase under P deficiency (Zhu et al2005) While distributing more carbon to roots in-creases root length and surface area for explorationand acquisition of P from the soil (Tang et al 2009) P-deficiency decreased photosynthesis and reproductivegrowth of the plant (Lynch and Ho 2005) In fact lowP supply dramatically decreased maize shoot growth[Table 1 see Supporting InformationmdashFigure S1] Inother studies continuous P deficiency decreased thegrowth of maize shoot and roots and subsequentlygrain yields (Plenet et al 2000 Zhang et al 2012 Kreyet al 2013 Deng et al 2014) It is not clear whymaize has the priority to invest more carbon for rootmorphological over physiological modifications underP deficiency

Maize responded to P deficiency similarly to wheatand does not increase proton release or exudation of or-ganic acid anions in roots (Neumann and Romheld 1999Li et al 2008) In contrast maize and wheat roots exhibita remarkable increase in total root length and rootshootratio in response to P deficiency (Table 3 Vlek et al 1996Bhadoria et al 2002) A larger root system is obviouslybeneficial for a better access of inorganic P because ofthe low mobility of P in the soil (Hinsinger 2001)Phosphorus uptake by plants depends on the root lengthand surface area and on lateral roots to explore a largesoil volume (Richardson et al 2009 Balemi and Negisho2012 Lambers et al 2015) According to the results inthe literature and the present study it is concluded that

Figure 2 Changes over time in pH of nutrient solutions grown withwhite lupin faba bean and maize supplied with low (LP) and high P(HP) under supply of Ca(NO3)2 or NH4NO3 during the cultural pe-riod The pH was measured at 1000 am daily Arrows indicate thetimes when nutrient solutions were replaced Values are means offour replicates and the bars represent the standard error of themean Asterisks represent significant differences between the Plevels at each measurement (Plt005)

the main adaptive strategy of maize roots to P deficiencyis morphological variation

White lupin and faba bean exhibited morpho-physiological strategy in roots in response to Pdeficiency

Unlike maize low P did not influence shoot and rootgrowth of white lupin under both N forms in the presentstudy [see Supporting InformationmdashFigure S1] althoughthe shoot and root P content and concentration decreased(Tables 1 and 3) In comparison low P increased the rootdry weight total root length and rootshoot ratio of fababean under Ca(NO3)2 supply [Tables 2 and 3 seeSupporting InformationmdashFigure S1] indicating that fababean has the morphological advantage of an extensiveroot system favouring to access P by exploring a larger vol-ume of soil The P concentration of white lupin and fababean was higher than that of maize under low P supply(Table 3) indicating that both legumes have a higher inter-nal P requirement than maize (Fohse et al 1988)

It is well known that P-deficient white lupin adapts Pdeficiency by morphological and physiological variations

ie formation of cluster roots and increased release of or-ganic acid anions and protons (Dinkelaker et al 1989Gerke et al 1994) In the present study white lupin sup-plied with low P had not yet formed cluster roots becausethe duration of low-P treatment was not long enough(Dinkelaker et al 1989 Gerke et al 1994 Neumann andMartinoia 2002 Wang et al 2006) Furthermore malatewas the main organic acid anion released by the roots(Table 4) yet the major organic acid anion released byroot clusters is citrate while that released by root tips in-cluding non-proteoid roots and proteoid roots is malate(Gardner et al 1982 1983 Neumann et al 1999)Apparently white lupin roots adapted P deficiency bymorphologicalphysiological modifications although thecarbon costs for root physiological variation are greaterthan that for morphological modification (Gardner et al1983 Dinkelaker et al 1989 Lynch and Ho 2005) Sincewhite lupin grows in P-impoverished habitats the beststrategy of the P-deficient white lupin is to mine soil P byreleasing protons and organic compounds rather thanmodifying root growth (Neumann and Martinoia 2002Lambers et al 2008 2011)

Figure 3 Effect of P supply (1 and 250 mmol L1) on the intensity of rhizosphere acidificationalkalization in white lupin faba bean and maizegrown with Ca(NO3)2 or NH4NO3 Rhizosphere pH changes were detected by embedding the roots of 12-d-old plants in agar with bromocre-sol-purple as a pH indicator (initial pH 59) The images were recorded 05 h after embedding Yellow colour indicates pHlt52 while purplecolour indicates pHgt68

Although reflecting different aspects of proton releaseby roots the results obtained using solution pH measure-ment agar technique and non-invasive measurement ofnet Hthornfluxes were consistent and showed that low P

stimulated proton release in the two legumes in agree-ment with the previous findings (Dinkelaker et al 1989Nuruzzaman et al 2005a) The proton release induced byP deficiency was greater in faba bean than in white lupin

Figure 4 Net (left) and mean (right) Hthornfluxes over 10 min on root surface of white lupin (A and B) faba bean (C and D) and maize (E and F)that had grown for 12 days with low (LP) and high P (HP) supply under two N forms [Ca(NO3)2 and NH4NO3] Values are means of four repli-cates and the bars represent the standard error of the mean (A C and E) Different letters (lower case) (B D and F) represent significant dif-ferences between the P treatments within a plant species and N form (Plt005) Positive values indicate net efflux while negative valuesindicate net influx

Table 4 Exudation of organic acid anions by roots of white lupin faba bean and maize grown for 12 days with low (LP) and high P supply (HP)under two N forms [ca(NO3)2 and NH4NO3]

Crops Organic acids Root exudation (mmol g21 dry root h21)

Ca(NO3)2-N NH4NO3-N LSD P level N form P 3 N

LP HP LP HP

White lupin Tartaric 096 054 049 050 004

Malic 167 092 166 124 027 ns ns

Citric 059 047 nd nd 004

Fumaric 002 003 nd nd 000 ns ns ns

T-aconitic 004 nd nd 003 000 ns ns

Total organic acids 329 195 215 182 029

Faba bean Tartaric 135 083 287 225 020 ns

Malic 075 081 089 079 039 ns ns ns

Citric 031 042 048 035 015 ns ns ns

Fumaric 005 nd nd nd 002

T-aconitic nd 001 nd 002 000

Total organic acids 247 208 424 381 035 ns

Maize Tartaric 041 362 049 219 108 ns ns

Malic nd 120 nd 091 027 ns ns

Citric nd nd nd nd 000 ns ns ns

Fumaric 003 005 002 002 001 ns ns

T-aconitic 048 029 nd nd 015 ns ns

Total organic acids 092 516 051 312 142 ns ns

P005 Plt001 Plt0001 ns not significant at Pfrac14005 nd not detected

Figure 5 Activity of acid phosphatase (APase) on the root surface of white lupin faba bean and maize grown for 12 days with low (LP) andhigh P (HP) supply under two N forms [Ca(NO3)2 and NH4NO3] Values are means of four replicates and the error bars represent the standarderror of the mean Different letters (lower case) represent significant differences between the P treatments within a plant species and N form(Plt005)

as presented by the intensive rhizosphere acidificationwithin 30 min (Fig 3) and more Hthornefflux on root tips offaba bean (Fig 4) On one hand the increased proton re-lease by the roots of P-deficient faba bean would con-tribute greatly to mobilization of non-labile P in soilespecially in high-pH soils (Li et al 2004) On the otherhand the increased release of protons under P defi-ciency may compensate for both excess uptake of cat-ions (Dinkelaker et al 1989) and a concomitant releaseof organic acid anions (Neumann and Romheld 1999)

Nitrogen form did not affect the rootmorphologicalphysiological response to low P

The present study investigated whether N forms influ-ence root responses to P deficiency NH4NO3 facilitated Nuptake of all three species while Ca(NO3)2 facilitated Kuptake of legumes [see Supporting InformationmdashTableS1] In comparison with Ca(NO3)2 NH4NO3 supply re-sulted in an increase in proton release from the roots ofall three species especially maize under the high-P con-dition Low-P supply increased proton release in white lu-pin and faba bean at the supply of both N forms and inmaize at the supply of NH4NO3 (Figs 2ndash4) The marked in-crease in proton release of the NH4NO3-fed maize underboth P levels could be explained by the preferential up-take of NHthorn4 over NO3 under mixed supply of ammoniumand nitrate which causes more proton release (Clark1982) There is a competition for transport across the to-noplast between chloride and nitrate which affects theiraccumulation and uptake (White and Veneklaas 2012)In the present study KCl was added to the low-P treat-ment to keep the K supply constant between the P treat-ments which increased Cl concentration (250 mmol) inthe low-P solution (see Methods section) In comparisonwith the high-P treatment however low P did not de-crease N uptake in white lupin but decreased it in fababean at the final harvest and maize at the last two har-vests under both N forms [see SupportingInformationmdashTable S1] The increased uptake of Ncould be explained by the larger plant biomass and thushigh demand of the faba bean and maize supplied withhigh P (Table 1)

Conclusions

The study demonstrated that root morphological varia-tion was the main adaptive strategy in maize in responseto P deficiency under the present condition A large rootsystem is therefore important for maize to access avail-able nutrients in soil especially immobile P Breedingcrops such as maize with large root systems and betterarchitecture will also contribute to increasing the use

efficiency of P and other nutrients NH4NO3 enhancedthe magnitude of proton release in white lupin and fababean under P deficiency although N form did not funda-mentally alter the responses of root morphology andphysiology of the three species to P deficiency It is im-portant in practices to use appropriate N forms to maxi-mize rootrhizosphere processes and to enhance P-useefficiency The use of ammonium-based fertilizers mayfacilitate P acquisition of legumes in non-acidic P-defi-cient soils but further work is needed to elucidate the ef-fects of N form on P acquisition strategies of N2-fixinglegumes

Sources of Funding

This research was supported by the State Key BasicResearch and Development Plan of China (No2013CB127402)

Contributions by the Authors

CL conceived and designed research HL performed theexperiments and analyzed data CL coordinated the re-search provided all the technical support and partici-pated in data interpretation HL CT and CL wrote thearticle all authors participated in stimulating discussionand approved the final article

Conflicts of Interest Statement

None declared

Acknowledgements

The authors would like to thank Dr Wei Ma from theChinese Academy of Agricultural Science for providingexperimental equipment for non-invasive measurementof Hthornfluxes on root surface Chao Wang and YapingZhou for their help in the experiments

Supporting Information

The following additional information is available in theonline version of this article mdashFigure S1 Dry weights of shoot and root of white lupinfaba bean and maize grown with low (LP 1 mmol L1)and high P (HP 250 mmol L1) under two N forms for 712 and 16 days Nitrogen was supplied as Ca(NO3)2 onleft panels and NH4NO3 on right panels Values aremeans of four replicates and error bars represent 6 thestandard error of the mean

Table S1 Nitrogen and potassium contents of intactwhite lupin faba bean and maize grown with LP (1 mmolL1) and HP (250 mmol L1) under two N forms [ca(NO3)2

and NH4NO3] for 7 12 and 16 days

Literature citedAnghinoni I Barber S 1980 Phosphorus influx and growth charac-

teristics of corn roots as influenced by phosphorus supplyAgronomy Journal 72685ndash688

Balemi T Negisho K 2012 Management of soil phosphorus andplant adaptation mechanisms to phosphorus stress for sustain-able crop production a review Journal of Soil Science and PlantNutrition 12547ndash562

Barber SA 1995 Soil nutrient bioavailability a mechanistic ap-proach New York John Wiley

Bhadoria PS Steingrobe B Claassen N Liebersbach H 2002Phosphorus efficiency of wheat and sugar beet seedlings grownin soils with mainly calcium or iron and aluminium phosphatePlant and Soil 24641ndash52

Brewster J Tinker P 1972 Nutrient flow rates into roots Soils andFertilizers 35355ndash359

Calderon-Vazquez C Sawers RJ Herrera-Estrella L 2011 Phosphatedeprivation in maize genetics and genomics Plant Physiology1561067ndash1077

Claassen N Jungk A 1984 Bedeutung der KaliumaufnahmerateWurzelwachstum und Wurzelhaare fur das Kaliumaneignungs-vermogen verschiedener Pflanzenarten Zeitschrift FuerPflanzenerneuroahrung Und Bodenkungde 147276ndash289

Clark RB 1982 Nutrient solution growth of sorghum and corn in min-eral nutrition studies Journal of Plant Nutrition 51039ndash1057

Corrales I Amenos M Poschenrieder C Barcelo J 2007 Phosphorusefficiency and root exudates in two contrasting tropical maizevarieties Journal of Plant Nutrition 30887ndash900

Deng Y Chen K Teng W Zhan A Tong Y Feng G Cui Z Zhang FChen X 2014 Is the inherent potential of maize roots efficientfor soil phosphorus acquisition PloS One 9e90287

Dinkelaker B Romheld V Marschner H 1989 Citric acid excretionand precipitation of calcium citrate in the rhizosphere of whitelupin (Lupinus albus L) Plant Cell and Environment 12285ndash292

Fernandez MC Belinque H Boem FHG Rubio G 2009 Comparedphosphorus efficiency in soybean sunflower and maize Journalof Plant Nutrition 322027ndash2043

Fernandez MC Rubio G 2015 Root morphological traits related tophosphorus-uptake efficiency of soybean sunflower and maizeJournal of Plant Nutrition and Soil Science 178807ndash815

Fohse D Claassen N Jungk A 1988 Phosphorus efficiency of plantsI External and internal P requirement and P uptake efficiency ofdifferent plant species Plant and Soil 110101ndash109

Gahoonia TS Nielsen NE 1992 The effects of root-induced pHchanges on the depletion of inorganic and organic phosphorusin the rhizosphere Plant and Soil 143185ndash191

Gardner W Barber D Parbery D 1983 The acquisition of phospho-rus by Lupinus albus L Plant and Soil 70107ndash124

Gardner W Parbery D Barber D 1982 The acquisition of phospho-rus by Lupinus albus L Plant and Soil 6819ndash32

Gaume A Meuroachler F De Leon C Narro L Frossard E 2001 Low-P tol-erance by maize (Zea mays L) genotypes significance of root

growth and organic acids and acid phosphatase root exudationPlant and Soil 228253ndash264

George T Gregory P Robinson J Buresh R 2002 Changes in phos-phorus concentrations and pH in the rhizosphere of some agro-forestry and crop species Plant and Soil 24665ndash73

Gerke J Romer W Jungk A 1994 The excretion of citric and malic acidby proteoid roots of Lupinus albus L effects on soil solution con-centrations of phosphate iron and aluminum in the proteoid rhi-zosphere in samples of an oxisol and a luvisol Zeitschrift FurPflanzenerneuroahrung Und Bodenkunde 157289ndash294

Hinsinger P 2001 Bioavailability of soil inorganic P in the rhizo-sphere as affected by root-induced chemical changes a reviewPlant and Soil 23725ndash41

Hinsinger P Plassard C Tang C Jaillard B 2003 Origins of root-mediated pH changes in the rhizosphere and their responses toenvironmental constraints a review Plant and Soil 24843ndash59

Jaillard B Plassard C Hinsinger P 2003 Measurements of Hthorn fluxesand concentrations in the rhizosphere In Rengel Z edsHandbook of soil acidity New York Dekker 231ndash266

Jakobsen I Abbott L Robson A 1992 External hyphae of vesicular-arbuscular mycorrhizal fungi associated with Trifolium subterra-neum L New Phytologist 120371ndash380

Krey T Vassilev N Baum C Eichler-Lobermann B 2013 Effects oflong-term phosphorus application and plant-growth promotingrhizobacteria on maize phosphorus nutrition under field condi-tions European Journal of Soil Biology 55124ndash130

Kummerova M 1986 Localization of acid phosphatase activity inmaize root under phosphorus deficiency Biologia Plantarum 28270ndash274

Lambers H Clode PL Hawkins HJ Laliberte E Oliveira RS Reddell PShane MW Stitt M Weston P 2015 Metabolic adaptations ofthe non-mycotrophic proteaceae to soils with low phosphorusAnnual Plant Reviews Phosphorus Metabolism in Plants 48289

Lambers H Finnegan PM Laliberte E Pearse SJ Ryan MH ShaneMW Veneklaas EJ 2011 Update on phosphorus nutrition inProteaceae Phosphorus nutrition of proteaceae in severelyphosphorus-impoverished soils are there lessons to be learnedfor future crops Plant Physiology 1561058ndash1066

Lambers H Raven JA Shaver GR Smith SE 2008 Plant nutrient-acquisition strategies change with soil age Trends in Ecologyand Evolution 2395ndash103

Lambers H Shane MW Cramer MD Pearse SJ Veneklaas EJ 2006Root structure and functioning for efficient acquisition of phos-phorus matching morphological and physiological traits Annalsof Botany 98693ndash713

Li H Shen J Zhang F Clairotte M Drevon J Le Cadre E Hinsinger P2008 Dynamics of phosphorus fractions in the rhizosphere ofcommon bean (Phaseolus vulgaris L) and durum wheat(Triticum turgidum durum L) grown in monocropping and inter-cropping systems Plant and Soil 312139ndash150

Li L Li SM Sun JH Zhou LL Bao XG Zhang HG Zhang FS 2007Diversity enhances agricultural productivity via rhizosphere phos-phorus facilitation on phosphorus-deficient soils Proceedings ofthe National Academy of Sciences 10411192ndash11196

Li SM Li L Zhang FS Tang C 2004 Acid phosphatase role in chick-peamaize intercropping Annals of Botany 94297ndash303

Li Z Xu C Li K Yan S Qu X Zhang J 2012 Phosphate starvation ofmaize inhibits lateral root formation and alters gene expressionin the lateral root primordium zone BMC Plant Biology 1289

Liu Y Mi G Chen F Zhang J Zhang F 2004 Rhizosphere effect androot growth of two maize (Zea mays L) genotypes with con-trasting P efficiency at low P availability Plant Science 167217ndash223

Lynch JP Ho MD 2005 Rhizoeconomics carbon costs of phospho-rus acquisition Plant and Soil 26945ndash56

Lynch JP 2007 Turner review no 14 Roots of the second green rev-olution Australian Journal of Botany 55493ndash512

Marschner H Romheld V 1983 In vivo measurement of root-induced pH changes at the soilndashroot interface effect of plantspecies and nitrogen source Zeitschrift Fur Pflanzenphysiologie111241ndash251

Marschner P 2012 Mineral nutrition of higher plants 3rd ednLondon Academic Press

McLachlan K 1980 Acid phosphatase activity of intact roots andphosphorus nutrition in plants 2 Variations among wheat rootsCrop and Pasture Science 31441ndash448

Mengel K Kosegarten H Kirkby EA Appel T 2001 Principles of plantnutrition New York Springer Science and Business Media

Miller MH 1974 Effects of nitrogen on phosphorus absorption byplants In Carson E eds The plant root and its environmentCharlottesville University Press of Virginia 643ndash668

Mollier A Pellerin S 1999 Maize root system growth and develop-ment as influenced by phosphorus deficiency Journal ofExperimental Botany 50487ndash497

Neumann G Martinoia E 2002 Cluster rootsndashan underground adap-tation for survival in extreme environments Trends in PlantScience 7162ndash167

Neumann G Massonneau A Martinoia E Romheld V 1999Physiological adaptations to phosphorus deficiency during pro-teoid root development in white lupin Planta 208373ndash382

Neumann G Romheld V 1999 Root excretion of carboxylic acidsand protons in phosphorus-deficient plants Plant and Soil 211121ndash130

Niu J Chen F Mi G Li C Zhang F 2007 Transpiration and nitrogenuptake and flow in two maize (Zea mays L) inbred lines as af-fected by nitrogen supply Annals of Botany 99153ndash160

Nuruzzaman M Lambers H Bolland MD Veneklaas EJ 2005aPhosphorus benefits of different legume crops to subsequentwheat grown in different soils of Western Australia Plant andSoil 271175ndash187

Nuruzzaman M Lambers H Bolland MD Veneklaas EJ 2005bPhosphorus uptake by grain legumes and subsequently grownwheat at different levels of residual phosphorus fertiliser Cropand Pasture Science 561041ndash1047

Pang J Ryan MH Tibbett M Cawthray GR Siddique KH Bolland MDDenton MD Lambers H 2010 Variation in morphological andphysiological parameters in herbaceous perennial legumes inresponse to phosphorus supply Plant and Soil 331241ndash255

Paponov IA Engels C 2003 Effect of nitrogen supply on leaf traitsrelated to photosynthesis during grain filling in two maize geno-types with different N efficiency Journal of Plant Nutrition andSoil Science 166756ndash763

Plenet D Mollier A Pellerin S 2000 Growth analysis of maize fieldcrops under phosphorus deficiency II Radiation-use efficiencybiomass accumulation and yield components Plant and Soil224259ndash272

Raghothama K 1999 Phosphate acquisition Annual Review ofPlant Biology 50665ndash693

Richardson AE Hocking PJ Simpson RJ George TS 2009 Plantmechanisms to optimise access to soil phosphorus Crop andPasture Science 60124ndash143

Schachtman DP Reid RJ Ayling SM 1998 Phosphorus uptake byplants from soil to cell Plant Physiology 116447ndash453

Shen J 1998 Separation and determination of low-molecular-weight organic acids and Phenolic acids in root exudates PhDThesis China Agricultural University China

Shen J Tang C Rengel Z Zhang F 2004 Root-induced acidificationand excess cation uptake by N2-fixing Lupinus albus grown inphosphorus-deficient soil Plant and Soil 26069ndash77

Soon Y Kalra Y 1995 Short communication a comparison of planttissue digestion methods for nitrogen and phosphorus analysesCanadian Journal of Soil Science 75243ndash245

Sun J Dai S Wang R Chen S Li N Zhou X Lu C Shen X Zheng X HuZ Zhang Z Song J Xu Y 2009 Calcium mediates root KthornNathorn

homeostasis in poplar species differing in salt tolerance TreePhysiology 291175ndash1186

Tang C Barton L McLay C 1997 A comparison of proton excretionof twelve pasture legumes grown in nutrient solution AnimalProduction Science 37563ndash570

Tang C Conyers MK Nuruzzaman M Poile G Li Liu D 2011Biological amelioration of subsoil acidity through managing ni-trate uptake by wheat crops Plant and Soil 338383ndash397

Tang C Han XZ Qiao YF Zheng SJ 2009 Phosphorus deficiency doesnot enhance proton release by roots of soybean [Glycine max (L)Murr] Environmental and Experimental Botany 67228ndash234

Tang C Rengel Z 2003 Role of plant cationanion uptake ratio insoil acidification In Rengel Z eds Handbook of soil acidity NewYork Dekker 57ndash81

Vlek P Luttger A Manske G 1996 The potential contribution ofarbuscular mycorrhiza to the development of nutrient and wa-ter efficient wheat In Tanner DG Payne TS Abdalla OS eds Theninth regional wheat workshop for eastern central and SouthernAfrica Addis Ababa Ethiopia CIMMYT 28ndash46

Wang Z Shen J Zhang F 2006 Cluster-root formation carboxylateexudation and proton release of Lupinus pilosus Murr as af-fected by medium pH and P deficiency Plant and Soil 287247ndash256

White PJ Veneklaas EJ 2012 Nature and nurture the importanceof seed phosphorus content Plant and Soil 3571ndash8

Xu Y Sun T Yin LP 2006 Application of non-invasive microsensingsystem to simultaneously measure both Hthorn and O2 fluxesaround the pollen tube Journal of Integrative Plant Biology 48823ndash831

Yan H Li K Ding H Liao C Li X Yuan L Li C 2011 Root morphologi-cal and proteomic responses to growth restriction in maizeplants supplied with sufficient N Journal of Plant Physiology1681067ndash1075

Yu P Li X Yuan L Li C 2013 A novel morphological response of maize(Zea mays) adult roots to heterogeneous nitrate supply revealedby a split-root experiment Physiologia Plantarum 150133ndash144

Zhang Y Yu P Peng Y Li X Chen F Li C 2012 Fine root patterningand balanced inorganic phosphorus distribution in the soil indi-cate distinctive adaptation of maize plants to phosphorus defi-ciency Pedosphere 22870ndash877

Zhu J Kaeppler SM Lynch JP 2005 Mapping of QTL controlling roothair length in maize (Zea mays L) under phosphorus deficiencyPlant and Soil 270299ndash310

plw058-TF1

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increase mobilization of non-labile P in soil (Neumann andRomheld 1999 Hinsinger 2001)

Although the adaptive mechanisms of plants to P defi-ciency are well understood relatively few studies haveaddressed the differences in root responses to P defi-ciency among plant species White lupin (Lupinus albus)a model plant to study root response to P deficiencyshows a superior ability to utilize sparingly soluble P andbound P from soil by developing proteoid roots that ex-ude protons (Dinkelaker et al 1989) citrate and acidphosphatases (APase) (Dinkelaker et al 1989 Gerke et al1994) Although faba bean (Vicia faba) does not formproteoid roots under P deficiency its extensive rootsystem a morphological advantage enables explora-tion of a larger volume of soil to access inorganicP (Nuruzzaman et al 2005a b) It also releases largeamounts of protons phenolics and APase which enhancemobilization of soil non-labile P and hence P uptake(Nuruzzaman et al 2005a Li et al 2007)

Maize is widely cultivated for both staple food and indus-trial usage in tropical and temperate soils worldwide(Calderon-Vazquez et al 2011) Results of the adaptiveresponses in maize roots to P deficiency however are con-troversial Maize roots always showed an extensive morpho-logical variation when the roots had restricted growth (Yanet al 2011) and grew in nitrate-rich patches (Yu et al 2013)and under P deficiency (Anghinoni and Barber 1980 Zhanget al 2012) Phosphorus deficiency has been shown to eitherincrease (Gaume et al 2001) or decrease (Liu et al 2004Corrales et al 2007) the release of organic acid anions fromthe roots in solution culture It enhanced (Kummerova1986) or did not change (Corrales et al 2007) the activity ofAPase on root surface Furthermore P deficiency did notchange rhizosphere pH (Neumann and Romheld 1999George et al 2002) or resulted in a strong alkalization in therhizosphere (Li et al 2007)

Nitrogen form influences the balance of cation and an-ion uptake by plant roots and thus alters the rhizospherepH The change of rhizosphere pH caused by differentialuptake of cations and anions is more prominent than thatcaused by root exudates such as protons and organic acidanions (Jaillard et al 2003 Tang and Rengel 2003Marschner 2012) Ammonium nutrition leads to preferen-tial cation uptake and thus net proton excretion by rootscausing rhizosphere acidification whilst nitrate supply in-duces hydroxyl secretion and causes rhizosphere alkaliza-tion (Gahoonia and Nielsen 1992 Mengel et al 2001 Tanget al 2011) In legumes on one hand N2 fixation generallyleads to rhizosphere acidification due to excess uptake ofcations over anions (Tang et al 1997 Hinsinger et al 2003Li et al 2008) On the other hand N source is one of thefactors affecting P supply of plants Ammonium supply of-ten enhances plant uptake of P from soil via rhizosphere

acidification (Miller 1974 Marschne 2012) A lower pH inthe rhizosphere could increase the H2PO4 HPO2

4 ratio andthe solubility of Ca-phosphates However interactions of Nform and P supply on rhizosphere pH are not fully under-stood For example P deficiency could decrease increaseor have no effect on rhizosphere pH in NO3 -fed plants (egDinkelaker et al 1989 Neumann and Romheld 1999 Tanget al 2009) These different responses to P supply and Nform depend on plant species

The aim of this study was to elucidate the adaptivestrategies of maize roots in response to P deficiency andto investigate the influence of N form on these responsesusing white lupin and faba bean as reference species

Methods

Plant materials and treatments

Two hydroponic experiments were conducted in thegreenhouse under natural light with 28 C16 C daynight temperatures and 45ndash55 relative humidity Eachexperiment consisted of three factors which were P sup-ply (LP 1 mmol L1 P and HP 250 mmol L1 P) N forms[NH4NO3 and Ca(NO3)2] and plant species [maize (Zeamays L cv Zhengdan 958) white lupin (Lupinus albus Lcv Amiga) and faba bean (Vicia faba L cv Lincan No 5)]There were four replicates for each treatment

Seeds of the three plant species were surface-sterilized in 10 (vv) H2O2 for 30 min rinsed thoroughlyin distilled water and then germinated between filter pa-pers moistened with saturated CaSO4 solution in thedark After a week seven seedlings of each species weretransferred into 7-L opaque pots (21 cm in diameter and24 cm in height) filled with nutrient solution of the fol-lowing composition (mol L1) K2SO4 75104 MgSO4

65104 KCl 1104 H3BO3 10106 MnSO4

10106 CuSO4 10107 ZnSO4 10106(NH4)6Mo7O24 50109 and Fe-EDTA 20104 (Niuet al 2007) Nitrogen was supplied as Ca(NO3)2 orNH4NO3 at the concentration of 20103 mol L1Phosphorus was supplied as KH2PO4 at 1 or 250 mmolL1 and KCl was added to the LP treatment to keep thesame K concentration between the P treatments (Panget al 2010) The pH of nutrient solution was adjusted to60 using 1 mol L1 HCl or NaOH when the solution wasrenewed The nutrient solution in each pot was continu-ously aerated and renewed every 3 days

The first experiment was harvested 12 days after thecommencement of treatments (DAT) Seven plants ofeach species in each pot were harvested as follows (1)two plants were used to collect root exudates (2) twoplants were used to measure APase activity on root sur-face (3) two plants were used to study rhizosphere acidi-ficationalkalization using the agar technique (4) one

Calculations

Results

7 d 12 d 16 d 7 d 12 d 16 d 7 d 12 d 16 d

Ca(NO3)2-N LP 012 028 047 046 072 111 034 056 072

HP 014 030 053 042 080 138 027 084 157

NH4NO3-N LP 012 018 047 043 071 107 037 061 079

HP 012 021 050 043 077 137 034 101 199

LSD (Pfrac14 005) 001 003 004 004 008 014 003 006 008

Ca(NO3)2-N LP 104 162 167 339 406 433 089 073 075

HP 148 234 396 537 834 1546 438 1029 1990

NH4NO3-N LP 105 118 167 353 460 501 097 087 096

HP 134 188 469 598 862 1579 547 1506 2511

LSD (Pfrac14 005) 012 026 038 060 152 203 062 128 217

P level

7 days

12 days

16 days

LP HP LP HP

Maize 010 127 012 126 005 ns ns

Maize 000 008 000 008 001 ns ns

Maize 1053 280 1126 305 80 ns ns

Discussion

LP HP LP HP

Malic 167 092 166 124 027 ns ns

Malic 075 081 089 079 039 ns ns ns

Citric 031 042 048 035 015 ns ns ns

Fumaric 003 005 002 002 001 ns ns

Conclusions

Sources of Funding

None declared

Acknowledgements

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Calculations

Results

7 d 12 d 16 d 7 d 12 d 16 d 7 d 12 d 16 d

Ca(NO3)2-N LP 012 028 047 046 072 111 034 056 072

HP 014 030 053 042 080 138 027 084 157

NH4NO3-N LP 012 018 047 043 071 107 037 061 079

HP 012 021 050 043 077 137 034 101 199

LSD (Pfrac14 005) 001 003 004 004 008 014 003 006 008

Ca(NO3)2-N LP 104 162 167 339 406 433 089 073 075

HP 148 234 396 537 834 1546 438 1029 1990

NH4NO3-N LP 105 118 167 353 460 501 097 087 096

HP 134 188 469 598 862 1579 547 1506 2511

LSD (Pfrac14 005) 012 026 038 060 152 203 062 128 217

P level

7 days

12 days

16 days

LP HP LP HP

Maize 010 127 012 126 005 ns ns

Maize 000 008 000 008 001 ns ns

Maize 1053 280 1126 305 80 ns ns

Discussion

LP HP LP HP

Malic 167 092 166 124 027 ns ns

Malic 075 081 089 079 039 ns ns ns

Citric 031 042 048 035 015 ns ns ns

Fumaric 003 005 002 002 001 ns ns

Conclusions

Sources of Funding

None declared

Acknowledgements

plw058-TF1

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Calculations

Results

7 d 12 d 16 d 7 d 12 d 16 d 7 d 12 d 16 d

Ca(NO3)2-N LP 012 028 047 046 072 111 034 056 072

HP 014 030 053 042 080 138 027 084 157

NH4NO3-N LP 012 018 047 043 071 107 037 061 079

HP 012 021 050 043 077 137 034 101 199

LSD (Pfrac14 005) 001 003 004 004 008 014 003 006 008

Ca(NO3)2-N LP 104 162 167 339 406 433 089 073 075

HP 148 234 396 537 834 1546 438 1029 1990

NH4NO3-N LP 105 118 167 353 460 501 097 087 096

HP 134 188 469 598 862 1579 547 1506 2511

LSD (Pfrac14 005) 012 026 038 060 152 203 062 128 217

P level

7 days

12 days

16 days

LP HP LP HP

Maize 010 127 012 126 005 ns ns

Maize 000 008 000 008 001 ns ns

Maize 1053 280 1126 305 80 ns ns

Discussion

LP HP LP HP

Malic 167 092 166 124 027 ns ns

Malic 075 081 089 079 039 ns ns ns

Citric 031 042 048 035 015 ns ns ns

Fumaric 003 005 002 002 001 ns ns

Conclusions

Sources of Funding

None declared

Acknowledgements

plw058-TF1

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7 d 12 d 16 d 7 d 12 d 16 d 7 d 12 d 16 d

Ca(NO3)2-N LP 012 028 047 046 072 111 034 056 072

HP 014 030 053 042 080 138 027 084 157

NH4NO3-N LP 012 018 047 043 071 107 037 061 079

HP 012 021 050 043 077 137 034 101 199

LSD (Pfrac14 005) 001 003 004 004 008 014 003 006 008

Ca(NO3)2-N LP 104 162 167 339 406 433 089 073 075

HP 148 234 396 537 834 1546 438 1029 1990

NH4NO3-N LP 105 118 167 353 460 501 097 087 096

HP 134 188 469 598 862 1579 547 1506 2511

LSD (Pfrac14 005) 012 026 038 060 152 203 062 128 217

P level

7 days

12 days

16 days

LP HP LP HP

Maize 010 127 012 126 005 ns ns

Maize 000 008 000 008 001 ns ns

Maize 1053 280 1126 305 80 ns ns

Discussion

LP HP LP HP

Malic 167 092 166 124 027 ns ns

Malic 075 081 089 079 039 ns ns ns

Citric 031 042 048 035 015 ns ns ns

Fumaric 003 005 002 002 001 ns ns

Conclusions

Sources of Funding

None declared

Acknowledgements

plw058-TF1

plw058-TF2

plw058-TF3

plw058-TF4

plw058-TF5

plw058-TF6

plw058-TF7

plw058-TF8

plw058-TF9

plw058-TF10

7 days

12 days

16 days

LP HP LP HP

Maize 010 127 012 126 005 ns ns

Maize 000 008 000 008 001 ns ns

Maize 1053 280 1126 305 80 ns ns

Discussion

LP HP LP HP

Malic 167 092 166 124 027 ns ns

Malic 075 081 089 079 039 ns ns ns

Citric 031 042 048 035 015 ns ns ns

Fumaric 003 005 002 002 001 ns ns

Conclusions

Sources of Funding

None declared

Acknowledgements

plw058-TF1

plw058-TF2

plw058-TF3

plw058-TF4

plw058-TF5

plw058-TF6

plw058-TF7

plw058-TF8

plw058-TF9

plw058-TF10

LP HP LP HP

Maize 010 127 012 126 005 ns ns

Maize 000 008 000 008 001 ns ns

Maize 1053 280 1126 305 80 ns ns

Discussion

LP HP LP HP

Malic 167 092 166 124 027 ns ns

Malic 075 081 089 079 039 ns ns ns

Citric 031 042 048 035 015 ns ns ns

Fumaric 003 005 002 002 001 ns ns

Conclusions

Sources of Funding

None declared

Acknowledgements

plw058-TF1

plw058-TF2

plw058-TF3

plw058-TF4

plw058-TF5

plw058-TF6

plw058-TF7

plw058-TF8

plw058-TF9

plw058-TF10

Discussion

LP HP LP HP

Malic 167 092 166 124 027 ns ns

Malic 075 081 089 079 039 ns ns ns

Citric 031 042 048 035 015 ns ns ns

Fumaric 003 005 002 002 001 ns ns

Conclusions

Sources of Funding

None declared

Acknowledgements

plw058-TF1

plw058-TF2

plw058-TF3

plw058-TF4

plw058-TF5

plw058-TF6

plw058-TF7

plw058-TF8

plw058-TF9

plw058-TF10

LP HP LP HP

Malic 167 092 166 124 027 ns ns

Malic 075 081 089 079 039 ns ns ns

Citric 031 042 048 035 015 ns ns ns

Fumaric 003 005 002 002 001 ns ns

Conclusions

Sources of Funding

None declared

Acknowledgements

plw058-TF1

plw058-TF2

plw058-TF3

plw058-TF4

plw058-TF5

plw058-TF6

plw058-TF7

plw058-TF8

plw058-TF9

plw058-TF10

LP HP LP HP

Malic 167 092 166 124 027 ns ns

Malic 075 081 089 079 039 ns ns ns

Citric 031 042 048 035 015 ns ns ns

Fumaric 003 005 002 002 001 ns ns

Conclusions

Sources of Funding

None declared

Acknowledgements

plw058-TF1

plw058-TF2

plw058-TF3

plw058-TF4

plw058-TF5

plw058-TF6

plw058-TF7

plw058-TF8

plw058-TF9

plw058-TF10

LP HP LP HP

Malic 167 092 166 124 027 ns ns

Malic 075 081 089 079 039 ns ns ns

Citric 031 042 048 035 015 ns ns ns

Fumaric 003 005 002 002 001 ns ns

Conclusions

Sources of Funding

None declared

Acknowledgements

plw058-TF1

plw058-TF2

plw058-TF3

plw058-TF4

plw058-TF5

plw058-TF6

plw058-TF7

plw058-TF8

plw058-TF9

plw058-TF10

Conclusions

Sources of Funding

None declared

Acknowledgements

plw058-TF1

plw058-TF2

plw058-TF3

plw058-TF4

plw058-TF5

plw058-TF6

plw058-TF7

plw058-TF8

plw058-TF9

plw058-TF10

plw058-TF1

plw058-TF2

plw058-TF3

plw058-TF4

plw058-TF5

plw058-TF6

plw058-TF7

plw058-TF8

plw058-TF9

plw058-TF10

plw058-TF1

plw058-TF2

plw058-TF3

plw058-TF4

plw058-TF5

plw058-TF6

plw058-TF7

plw058-TF8

plw058-TF9

plw058-TF10

Documents

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