REGULAR ARTICLE

Contributions of root uptake and remobilization to grain zincaccumulation in wheat depending on post-anthesis zincavailability and nitrogen nutrition

Umit Baris Kutman & Bahar Yildiz Kutman &

Yasemin Ceylan & Emir Ali Ova & Ismail Cakmak

Received: 15 March 2012 /Accepted: 14 May 2012 /Published online: 29 May 2012# Springer Science+Business Media B.V. 2012

AbstractBackground and aims Whether root Zn uptake duringgrain filling or remobilization from pre-anthesis Znstores contributes more to grain Zn in wheat is subjectto an on-going debate. This study investigated theeffects of N nutrition and post-anthesis Zn availabilityon the relative importance of these sources.Methods Durum wheat plants were grown in nutrientsolution containing adequate Zn (0.5μM) and threedifferent N levels (0.5; 1.5; 4.5mM). One third of theplants were harvested when they reached anthesis. Onehalf of the remaining plants were grown tomaturity withadequate Zn, whereas the Zn supply to the other halfwas discontinued at anthesis. Roots, straw and grainswere harvested separately and analyzed for Zn and N.Results Depending on the N supply, Zn remobilizationfrom pre-anthesis sources provided almost all of grainZn when the Zn supply was withheld at anthesis;otherwise up to 100 % of grain Zn could be accountedfor by Zn taken up post-anthesis. By promoting tiller-ing and grain yield and extending the grain filling,

higher N supply favored the contribution of Zn uptaketo grain Zn accumulation.Conclusion Remobilization is critical for grain Zn ac-cumulation when Zn availability is restricted dur-ing grain filling. However, where root uptake cancontinue, concurrent Zn uptake during grain devel-opment, favored by higher N supply, overshadowsnet remobilization.

Keywords Nitrogen . Post-anthesis . Remobilization .

Uptake .Wheat . Zinc

Introduction

Zinc (Zn) deficiency is one of the most commonmicronutrient malnutrition problems affecting thehealth of at least two billion people (Hotz and Brown2004; Welch and Graham 2004). Low dietary intakeand insufficient dietary diversity have been identifiedas the main reasons behind this global problem (Bouis2003; White and Broadley 2009; Cakmak et al.2010a). Biofortification appears to be a promising,cost-effective and sustainable strategy to overcomemicronutrient deficiencies including Zn deficiency(Pfeiffer and McClafferty 2007; Cakmak 2008; Whiteand Broadley 2009). Wheat, a common staple crop, isthe most important source of calorie intake in a num-ber of developing countries (Hotz and Brown 2004;Cakmak 2008; Gibson et al. 2008). As wheat grain istypically too low in Zn to meet the Zn demand of

Plant Soil (2012) 361:177–187DOI 10.1007/s11104-012-1300-x

Responsible Editor: Michael A. Grusak.

Umit Baris Kutman and Bahar Yildiz Kutman are equallycontributing authors.

U. B. Kutman : B. Y. Kutman :Y. Ceylan : E. A. Ova :I. Cakmak (*)Faculty of Engineering and Natural Sciences,Sabanci University,34956 Istanbul, Turkeye-mail: cakmak@sabanciuniv.edu

consumers, it is one of the most important target cropsin biofortification programs. A better understanding ofthe physiological factors affecting Zn homeostasis andgrain Zn accumulation in wheat will contribute to bothgenetic and agronomic biofortification efforts. Therelative contributions of potentially limiting steps onthe path of Zn to developing grains and the effects ofenvironmental and nutritional factors on them are stilldebated.

There are principally two sources of Zn accumulat-ed in the wheat grain: concurrent uptake during thegrain-filling period and net remobilization from thepre-anthesis stores of source tissues (Stomph et al.2009; Waters et al. 2009). In the literature, there isdisagreement about the relative contributions ofthese two sources to grain Zn deposition. Garnettand Graham (2005) stated that, in wheat, most ofthe Zn allocated to grains could be accounted forby Zn entering the shoot post-anthesis in theirexperiment. This statement contradicts the resultspublished by Palmgren et al. (2008), who sug-gested that in wheat and barley, Zn remobilizationcontributes to grain Zn accumulation more thancontinued uptake. However, in a study by Waterset al. (2009), ample Zn and Fe supply during thegrain development could totally supersede the need fornet remobilization in hydroponically-grown wheat,whereas all the grain Zn could be provided by netremobilization in case the Zn supply was withheldpost-anthesis. Stomph et al. (2009) argued that differentcereal species may behave differently in this respect, andthat concurrent uptake during the grain-filling is moreimportant in rice. However, according to Wu et al.(2010), the Zn concentration in rice grains is closelyassociated with the extent of Zn retranslocation fromsource tissues to grains. Waters and Grusak (2008)claimed that continued uptake and translocation of min-erals during the seed-filling stage is at least as importantas net remobilization in Arabidopsis thaliana.

Wheat is widely grown as a rain-fed crop in semi-arid environments, where the top soil often dries as aresult of dry weather during the grain-filling period(Graham and Rengel 1993; Elias and Manthey 2005).In Mediterranean-type climates, the end of the wheat-growing season is characterized by hot weather andrestricted water availability (Distelfeld et al. 2007).When the top soil is dry, the roots in the fertilizer zoneare inactive. Then, the plants must rely on deeper rootsfor water and nutrient uptake, and because deeper soil

layers are typically poor in nutrients, remobilization ofnutrients becomes critical for nutrient supply (Grahamand Rengel 1993). Moreover, nearly half of the soils incereal-growing environments have low Zn availability(Alloway 2004; Cakmak 2008), and Zn fertilizers,applied to the top soil, have low mobility in the soilprofile (Marschner 1993). Drought may therefore of-ten restrict Zn uptake during the grain filling stage ofwheat under field conditions. Efficient remobilizationinduced by accelerated senescence can improve thegrain Zn accumulation as well as the grain protein con-tent, indicating that the remobilization of Zn is linked tothe remobilization of N (Uauy et al. 2006a, b; Distelfeldet al. 2007).

High positive correlations between seed protein andZn concentrations found in several wheat germplasms(Zebarth et al. 1992; Feil and Fossati 1995; Morgounovet al. 2007; Peleg et al. 2008), co-localization of high-protein and high-Zn regions in grains (Lott et al.1995; Ozturk et al. 2006) and speciation data sug-gesting that Zn in the grain is mainly bound to peptides(Persson et al. 2009) indicated the presence of a linkbetween Zn and N in cereals. Recent studies estab-lished that improving the N nutritional status of wheatenhances the grain Zn accumulation under both green-house and field conditions (Cakmak et al. 2010b;Kutman et al. 2010; Shi et al. 2010). This effect of Non grain Zn is dependent on sufficient Zn availabilityand enhanced by high Zn supply (Kutman et al. 2010).Although both the uptake and the remobilization of Znare positively affected by N nutrition (Erenoglu et al.2011), the share of concurrent uptake during grain fillingin grain Zn deposition is increased by higher N supply(Kutman et al. 2011).

Based on the on-going debate in literature, it seemsthat the answer to the question of whether root uptakeor retranslocation is more important in terms of grainZn accretion is dependent on environmental con-ditions as well as N and Zn availability. In this experi-ment, by discontinuing the Zn supply to hydroponically-grown wheat plants at anthesis, an extreme modelenvironment was created, which mimicked the fieldconditions restricting Zn uptake during the grain de-velopment. This model experiment enabled thestudy of the effects of N nutritional status andpost-anthesis Zn availability on the relative contribu-tions of concurrent Zn uptake and net Zn remobili-zation during grain-filling to grain Zn accumulationin wheat.

178 Plant Soil (2012) 361:177–187

Materials and methods

Plant growth and sample preparation

Durum wheat plants (Triticum durum cv. Balcali2000)were grown in solution culture under controlled cli-matic conditions (light/dark periods: 16/8 h; tempera-ture (light/dark): 22°C/18°C; relative humidity (light/dark): 60 %/70 %; photosynthetic flux density:400 μmol m−2 s−1). Seeds were imbibed in saturatedCaSO4.2H2O solution for half an hour and germinatedin perlite moisturized with saturated CaSO4.2H2O so-lution for 5 days at room temperature before beingtransferred to solution culture. The experiment wasdesigned as a 4 pot-replicate experiment with 4 singleplants in each pot.

Seedlings were grown in plastic pots containing 3 Lof nutrient solution consisting of 0.9 mM K2SO4,0.2 mM KH2PO4, 1 mM MgSO4.7H2O, 0.1 mMKCl, 100 μM Fe-EDTA, 1 μM H3BO3, 0.5 μMMnSO4.H2O, 0.2 μM CuSO4.5H2O, 0.2 μMNiCl2.6H2O and 0.14 μM (NH4)6Mo7O24.4H2O.Plants were grown at three N levels throughout thedevelopment: Low N pots were supplied with 0.5 mMN, medium N pots with 1.5 mM N, and high N potswith 4.5 mM N in the form of Ca(NO3)2.4H2O. LowerN pots were supplemented with CaSO4.2H2O to com-plement missing Ca. Until the main stems of the low Nplants reached the Zadoks stage 65 (anthesis half-way), 0.5 μM Zn in the form of ZnSO4.7H2O wasadded to the nutrient solutions in all pots. At this stage,the roots and shoots (straw) of plants were harvestedin one third of the pots. Zinc was continuously sup-plied to one half of the remaining pots until maturity,whereas the Zn supply to the other half was discon-tinued at this stage. When the grains matured and theplants completely senesced, the spikes, straw and rootswere harvested. Grains were manually separated fromhusk, and the husk samples were combined with thecorresponding straw samples. Throughout the experi-ment, nutrient solutions were continuously aerated andrefreshed every 2 (at late growth stages)—4 (at earlygrowth stages) days.

Harvested roots were washed in 0.5 mM CaSO4

solution for 10 min and then with deionized water. Allharvested plant samples were dried at 60°C for 2 days.Dried samples were weighed at room temperature andthen ground to fine powders in an agate vibrating cupmill (Pulverisette 9; Fritsch GmbH; Germany). They

were analyzed for mineral concentrations as describedbelow.

Mineral analysis

The N concentrations in the samples were measuredby using LECO TruSpec C/N Analyzer (Leco Corp.,St Joseph, MI, USA). For Zn analysis, ground sampleswere subjected to acid-digestion (ca. 0.2 g sample in2 ml 30 % H2O2 and 5 ml 65 % HNO3) in a closedvessel microwave system (MarsExpress; CEM Corp.,Matthews, NC, USA). After digestion, the total sam-ple volume was finalized to 20 ml by adding double-deionized water. Zinc concentrations were determinedby inductively coupled plasma optical emission spec-trometry (ICP-OES) (Vista-Pro Axial, Varian Pty Ltd,Mulgrave, Australia). Measurements were checked byusing certified standard reference materials obtainedfrom the National Institute of Standards and Technology(Gaithersburg, MD, USA).

Calculations and statistical analysis

For calculating the mineral contents for a given plantpart, the mineral concentrations were multiplied bythe dry weight of that plant part. Similarly, the grainmineral yields, i.e. the total amounts of minerals ofinterest deposited in the grains, were determinedby multiplying the grain yield by the grain mineralconcentrations.

The JMP software (Release 5.0.1a) was used forstatistical analysis. Reported values are means of 4replicates. The significance of the effects of the treat-ments and their interactions on the reported traits wasevaluated by analysis of variance (ANOVA). Then,significant differences between means were deter-mined by Tukey’s protected honestly significant dif-ference (HSD) test at 95 % confidence (p≤0.05).

Results

The N application levels affected the root and strawdry weights at both anthesis and maturity significantly,as revealed by ANOVA (Table 1A, B), whereas the Znregime and its interaction with N had significanteffects on only the root dry weight, but not the strawdry weight at maturity (Table 1B). Higher N supplyresulted in higher root biomass production at maturity

Plant Soil (2012) 361:177–187 179

under both discontinued and continued Zn supplyconditions; but at anthesis, medium N plants had thegreatest root biomass (Table 2). Continued Zn supplyincreased the root dry weight only at the high Ntreatment, but did not have any significant effect atthe other N levels. The straw biomass production wasconsistently and markedly enhanced by increasing Nsupply at both anthesis and maturity. The higher the Nsupply level, the greater was the post-anthesis increasein straw dry matter.

The grain yield was affected by all sources ofvariation significantly (Table 1B). When the N supplywas increased from low to medium, the grain yieldwas more than doubled under both discontinued andcontinued Zn regimes (Table 2). Increasing the N levelfrom medium to high again almost doubled the grainyield at continued Zn treatment, whereas it significant-ly reduced the grain yield at discontinued Zn treat-ment. When the N level was low or medium, the grainyield was not affected by the Zn regime, but at thehigh N treatment, continued Zn supply increased thegrain yield tremendously.

As shown in Fig. 1, higher N application did notonly enhance tillering and thus biomass production,but also delayed senescence under continued Zn re-gime. However, the effect of high N on senescencewas largely negated by discontinuation of Zn supplyafter anthesis.

The root Zn concentration and content results forthe anthesis stage did not exhibit clear responses to theN supply, but at maturity, both the N supply and theZn regime had significant positive effects on the rootZn results (Tables 1 and 3). When compared to

anthesis, the root Zn content was markedly reducedat maturity under low or medium N conditions. How-ever, at the high N treatment, there was a net increasein the root Zn content after anthesis, especially underthe continued Zn condition. At anthesis, the straw Znconcentration was significantly reduced by higher Nlevels, whereas the straw Zn content was enhanced byincreasing N supply. In completely senesced straw, theZn concentration responded positively to continuedZn supply after anthesis, but decreased down to ca.5 μg.g−1 in response to higher N treatments. The strawZn content at maturity was also improved by bothincreasing N and continued Zn treatments. A net in-crease in the straw Zn content was after anthesis onlyobserved in the case of high N and continued Zn.Under all other conditions, there was a net export ofZn from straw pre-anthesis stores, which was morepronounced at discontinued Zn supply than at contin-ued Zn supply.

As expected, the N contents of both the root andstraw at both anthesis and maturity were enhanced byincreasing N levels (Tables 1 and 3). Under all Nregimes, the root N content at maturity was higherthan the root N content at anthesis. Similarly, the strawgained net N after anthesis when the N level was high,but it exported nearly half of its pre-anthesis N storewhen the N supply was low or medium.

The effects of the N application level, Zn regimeand their interaction on the grain Zn concentration andyield were all significant (Table 1B). On one hand,discontinued Zn supply after anthesis reduced thegrain Zn concentration by 40–55 %, depending onthe N level (Table 4). On the other hand, increasing

Table 1 Analysis of variance (ANOVA) of the effects of nitro-gen level, zinc regime and their interactions on reported traits(A: at anthesis; B: at maturity) of durum wheat (Triticum durum

cv. Balcali2000) grown in solution culture: Degrees of freedom(DF) and F value probabilities (F Pr.) at 95 % confidence

Source ofVariation

DF RootDW

StrawDW

RootZnConc.

StrawZnConc.

RootZnContent

StrawZnContent

RootNContent

StrawNContent

GrainYield

GrainZnConc.

GrainZnYield

GrainNConc.

GrainNYield

F Pr.

(A) Anthesis

NitrogenLevel

2 0.002 <0.001 0.013 <0.001 0.149 <0.001 <0.001 <0.001

(B) Maturity

NitrogenLevel (a)

2 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.029 <0.001 <0.001

Zn Regime (b) 1 <0.001 0.152 0.003 <0.001 <0.001 <0.001 0.018 0.669 <0.001 <0.001 <0.001 0.480 <0.001

a x b 2 <0.001 0.403 0.413 <0.001 <0.001 <0.001 0.002 0.862 <0.001 <0.001 <0.001 0.895 <0.001

180 Plant Soil (2012) 361:177–187

N supply resulted in marked reductions in grain Znconcentrations. At all N levels, the grain Zn yieldunder the discontinued Zn regime was significantlylower than that under the continued Zn regime. Whenthe Zn supply was withheld post-anthesis, the grain Znyield was severely decreased by the high N treatment,whereas increasing the N level from low to medium orhigh led to slightly improved grain Zn yields.

The grain N concentration reached its maximum atthe high N treatment and was not significantly affectedby the Zn supply (Tables 1B and 4). When the N levelwas increased from low to medium, the grain N yieldwas more than doubled, irrespective of the Zn regime;but when the N level was further increased to high, thegrain N yield was unaffected at the discontinued Zntreatment while it was again more than doubled at thecontinued Zn treatment.

Figure 2 shows how the shares of post-anthesis Znuptake and net remobilization of Zn from pre-anthesisstores of the root and straw on the grain Zn yield are

affected by the N and Zn nutrition. At the discontinuedZn treatment, the share of Zn remobilization from rootpre-anthesis stores in grain Zn decreased, while theshare of remobilization from straw pre-anthesis storesincreased, when the N supply was increased. Theapparent small share of continued Zn uptake underthe discontinued Zn regime indicates a minor contam-ination. In contrast, under the continued Zn supplyregime, post-anthesis Zn uptake contributed to grainZn much more than net remobilization from pre-anthesis stores. When the N supply was high, 100 %of the grain Zn could be explained by post-anthesis Znuptake.

Discussion

The N supply level affected the biomass of vegetativetissues more strongly than the Zn regime (Table 2). HighN application stimulated the straw dry matter production

Table 2 The root dry weight (anthesis/maturity), straw dryweight (anthesis/maturity) and grain yield (maturity) of durumwheat (Triticum durum cv. Balcali2000) grown in solution cul-ture at different N (low: 0.5 mM; medium: 1.5 mM; high:

4.5 mM) levels and with standard (Std.) (0.5 μM) Zn beforeanthesis and discontinued (Disc.) or continued (Cont.) Zn sup-ply after anthesis

Stage Zn Supply Low N Medium N High N

Root Dry Weight, g plant−1

Anthesis Std. 3.5 4.3 3.2

Maturity Disc. 4.2 4.9 5.3

Cont. 4.3 5.1 7.7

Straw Dry Weight, g plant−1

Anthesis Std. 6.4 12.3 16.4

Maturity Disc. 8.4 20.9 43.0

Cont. 8.1 19.8 38.2

Grain Yield, g plant−1

Maturity Discont. 6.4 15.6 12.8

Cont. 6.6 15.4 27.6

ANOVA results (F Pr. values) are reported in Table 1.

Root Dry Weight:

Anthesis: HSD0.05 (N)00.6

Maturity: HSD0.05 (N; Zn; NxZn)00.5; 0.3; 0.8

Straw Dry Weight:

Anthesis: HSD0.05 (N) 0 2.5

Maturity: HSD0.05 (N; Zn; NxZn)04.3; n.s.; n.s.

Grain Yield:

Maturity: HSD0.05 (N; Zn; NxZn)02.7; 1.8; 4.6

n.s. Not significant

Plant Soil (2012) 361:177–187 181

and tillering (Fig. 1) markedly, confirming previousresults (Ewert and Honermeier 1999; Salvagiottiand Miralles 2007; Kutman et al. 2010). The Nsupply also had a clear effect on senescence.Higher N plants remained green longer than lowerN plants and had longer grain-filling periods, as reportedby Yang and Zhang (2006) and Kutman et al. (2010).However, this effect of high N almost disappeared underthe discontinued Zn regime, probably because of theretranslocation of all of the mobile Zn out of sourcetissues (Table 3) to growing parts, which triggeredsenescence.

The increases in the grain yield due to improved Nnutrition were parallel to the increases in the tiller andspike number under all conditions except the high N—discontinued Zn condition (Fig. 1). The reason behind the

significant reduction in the grain yield by the highN treatment under the discontinued Zn regime wasthe severe Zn dilution associated with enhancedvegetative growth (Table 3), which continued afteranthesis (Table 2). Dilution of Zn may have af-fected the grain yield by two mechanisms: Firstly,Zn deficiency may have impaired the reproductivedevelopment as documented in the literature (Sharmaet al. 1990; Cakmak and Engels 1999). In the pres-ent study, the Zn supply was discontinued whenthe main stems were at the anthesis stage. How-ever, at the high N treatment, most of the tillers thatcontributed to the grain yield were formed laterthan the discontinuation of the Zn supply, which isnot expected to occur under water-limited fieldconditions. It is very likely that the reproductivedevelopment in later spikes was adversely affectedby Zn deficiency. Secondly, since the senescence-delaying effect of high N was negated by Zn deficien-cy (Fig. 1), the grains had not yet completed theirdevelopment, when these plants completely senesced.Consequently, most of the grains harvested from theseplants were shriveled (not shown).

Higher N treatment increased the total plant Zncontent, which can be calculated from the data inthe Tables 3 and 4, by up to 33 % at anthesis andby up to 60 % at maturity under the continued Znregime. This supports the previous reports aboutthe positive effects of improved N nutrition on Znuptake (Cakmak et al. 2010b; Kutman et al. 2011),and Zn uptake can continue for a longer time dueto delayed senescence. Zinc uptake studies con-ducted with 65Zn revealed that improved N nutri-tional status is associated with significantly higherroot Zn uptake and root-to-shoot Zn transport ratesin wheat plants grown in nutrient solution (Erenoglu etal. 2011). Here, it is important to highlight thatunder solution culture conditions, where the nutri-ent solutions are refreshed every 3–4 days, evenlow-N plants can consume all of the Zn in the nutrientsolution before its refreshment, while adequate-Nplants deplete Zn in nutrient solution much quickerthan low-N plants (Erenoglu et al. 2011). There-fore, solution culture conditions, where Zn is availablein free ionic form, may not represent the real soil con-ditions, where most of the potentially plant-available Znis adsorbed to soil particles or complexed with variousligands (Alloway 2004). In this respect, the decreases instraw and grain Zn concentrations observed here in

Fig. 1 Effect of N (low: 0.5 mM; medium: 1.5 mM; high:4.5 mM) and Zn (continued vs. discontinued after anthesis)regimes on 86-day-old durum wheat (Triticum durum cv.Balcali2000) plants grown in solution culture under growthchamber conditions

182 Plant Soil (2012) 361:177–187

Tab

le3

The

Znconcentrations

andZnandNcontentsof

therootandstrawof

durumwheat(Triticum

durumcv.B

alcali2

000)

atanthesisandmaturity,w

hengrow

ninsolutio

ncultu

reatdifferentN(low

:0.5mM;medium:1.5mM;high

:4.5mM)levelsandwith

standard

(Std.)(0.5

μM)Znbefore

anthesisanddiscon

tinued(D

isc.)or

continued(Con

t.)Znsupp

lyafteranthesis

Roo

tZnCon

centratio

n,μgg−

1Straw

ZnCon

centratio

n,μgg−

1Roo

tZnCon

tent,μg

Plant

−1Straw

ZnCon

tent,μg

Plant

−1Roo

tN

Con

tent,mg

Plant

−1Straw

NCon

tent,mg

Plant

−1

Stage

Zn

Sup

ply

Low

NMedium

NHigh

NLow

NMedium

NHigh

NLow

NMedium

NHigh

NLow

NMedium

NHigh

NLow

NMedium

NHigh

NLow

NMedium

NHigh

N

Anthesis

Std.

18.2

13.1

15.6

27.3

19.0

16.4

6456

5117

522

626

734

5464

7922

753

3

Maturity

Disc.

4.1

4.4

11.0

9.5

5.6

4.9

1721

5977

116

201

3761

116

37111

641

Con

t.6.8

5.4

13.0

20.3

8.3

9.4

2927

101

167

164

357

3858

141

37111

595

ANOVA

results

(FPr.values)arerepo

rted

inTable

1.

Roo

tZnCon

c.:

Anthesis:HSD0.05(N

)03.8

Maturity

:HSD0.05(N

;Zn;

NxZ

n)01.8;

1.2;

n.s.

Straw

ZnCon

c.:

Anthesis:HSD0.05(N

)01.9

Maturity

:HSD0.05(N

;Zn;

NxZ

n)01.4;

1.0;

2.5

Roo

tZnCon

tent:

Anthesis:HSD0.05(N

)0n.s.

Maturity

:HSD0.05(N

;Zn;

NxZ

n)010

;6;

19

Straw

ZnCon

tent.:

Anthesis:HSD0.05(N

)038

Maturity

:HSD0.05(N

;Zn;

NxZ

n)031

;21

;53

Roo

tN

Con

tent:

Anthesis:HSD0.05(N

)09

Maturity

:HSD0.05(N

;Zn;

NxZ

n)09;

6;16

Srtaw

NCon

tent:

Anthesis:HSD0.05(N

)065

Maturity

:HSD0.05(N

;Zn;

NxZ

n)010

9;n.s.;n.s.

n.s.Not

sign

ificant

Plant Soil (2012) 361:177–187 183

response to higher N treatments (Tables 3 and 4) can beexplained by the conditions of this model environment.The dilution effect overshadowed the positive effects ofN on Zn uptake, translocation and remobilization. Theseresults are in agreement with the previous finding thatthe contribution of N fertilization to grain Zn accumu-lation is dependent on Zn availability (Kutman et al.2010).

In all cases, where the straw Zn content atmaturity was lower than that at anthesis, the rootZn content at maturity was either also lower thanor almost the same as the root Zn content atanthesis (Table 3). The net remobilization of Znfrom the root pre-anthesis stores at the low andmedium N levels shows that part of the Zn enter-ing the shoot post-anthesis is in fact Zn remobi-lized from the roots, as speculated by Garnett andGraham (2005). Waters et al. (2009) also demon-strated that net remobilization from the root storescan occur, depending on the nutritional status ofthe plants.

In the discontinued Zn group, the net amount ofZn remobilized from the straw pre-anthesis storeswas slightly increased when the N level was in-creased from low to medium, but significantlydecreased when the N level was further increasedto high (Table 3). When the Zn supply was

withheld after anthesis, the straw Zn concentrationof medium or high N plants decreased to about5 μg g−1 at maturity (Table 3). A small amount ofZn is probably incorporated into structural mole-cules such as cell walls and thus becomes unavailablefor remobilization (Marschner 2011; Waters and Grusak2008). Then, remobilization is only possible if theamount of Zn stored in source tissues is higherthan this minimal amount (Waters and Grusak2008). It seems very likely that this minimal con-centration for Zn is ca. 5 μg g−1 for wheat straw,at least under given conditions of the study. As thestraw Zn concentration approaches this minimal valuedue to net export of Zn to the grains, the remobi-lization ceases. Because of severe Zn dilution inthe high-N plants, which produced a great portionof their vegetative biomass after their main spikereached anthesis, the straw of these plants couldprobably export less Zn to the grains before reach-ing the critical value.

In plants continuously supplied with Zn until ma-turity, the amount of Zn remobilized from the strawpre-anthesis stores was significantly increased by themedium N as compared to the low N level, whereas nonet remobilization from the straw was observed at thehigh N level (Table 3). This apparently contradictoryeffect of increasing N supply on Zn remobilization

Table 4 The grain Zn and N concentrations and yields ofdurum wheat (Triticum durum cv. Balcali2000) at maturity,when grown in solution culture at different N (low: 0.5 mM;

medium: 1.5 mM; high: 4.5 mM) levels and with discontinued(Disc.) or continued (Cont.) Zn supply after anthesis

Grain Zn Concentration, μgg−1

Grain Zn Yield, μg plant−1 Grain N Concentration, % Grain N Yield, mg plant−1

Zn Supply Low N Medium N High N Low N Medium N High N Low N Medium N High N Low N Medium N High N

Disc. 28.2 10.8 6.3 160 162 70 2.43 2.25 2.79 156 350 353

Cont. 46.9 23.8 12.9 310 354 352 2.20 2.23 2.90 147 345 806

ANOVA results (F Pr. values) are reported in Table 1.

Grain Zn Conc.:

HSD0.05 (N; Zn; NxZn)02.8; 1.8; 4.8

Grain Zn Yield:

HSD0.05 (N; Zn; NxZn)041; 28; 71

Grain N Conc.:

HSD0.05 (N; Zn; NxZn)00.18; n.s.; n.s.

Grain N Yield:

HSD0.05 (N; Zn; NxZn)081; 53; 139

n.s. Not significant

184 Plant Soil (2012) 361:177–187

may be explained by the well documented associationof Zn remobilization with senescence. Zinc retranslo-cation to developing grains is enhanced by senescence(Longnecker and Robson 1993; Marschner 1995;Uauy et al. 2006b; Distelfeld et al. 2007), althoughZn can also be retranslocated from non-senescentleaves (Hajiboland et al. 2001; Erenoglu et al. 2002).In contrast to the high N—discontinued Zn condition,where the source tissues senesced before the grainscould complete their development, the source tissueswere still green under the high N—continued Zn con-dition, when the grains matured (Fig. 1). This distinctdelay in senescence under the high N—continued Zncondition may have severely reduced the amount ofZn remobilized from the straw to the grains and thusresulted in the apparent zero net remobilization fromthe pre-anthesis stores (Table 3). As revealed by ge-netic studies on the wheat Gpc-B1 locus associatedwith accelerated senescence, micronutrient remobili-zation to developing grains is linked to nitrogen remo-bilization (Uauy et al. 2006b; Distelfeld et al. 2007). Inagreement with this finding, the straw N concentrationof high N plants was at maturity ca. 3 times higherthan that of low or medium N plants under the con-tinued Zn supply regime (Table 3). So, not only the Znbut also N remobilization was impaired by delayedsenescence.

It is a long-lasting debate whether uptake during theseed development or remobilization from the sourcetissues contributes more to seed accretion of minerals(Waters and Grusak 2008; Stomph et al. 2009; Waters

et al. 2009; Wu et al. 2010). The results of this exper-iment demonstrated that the answer to this questiondepends very much on the Zn and N nutrition regimesand that both extremes are possible: Zinc remobilizedfrom pre-anthesis reserves of the straw can supplyalmost 100 % of the grain Zn if the plants cannot takeup Zn by the root system during the grain develop-ment, whereas up to 100 % of the grain Zn can beattributed to Zn uptake during the seed-filling stage ifthe Zn uptake can continue and a high N supply favorsthe uptake more than net remobilization by delayingsenescence and extending the stay-green period (Figs. 1and 2).

Conclusion

It is a long-lasting debate whether concurrent up-take of Zn during grain development or net Znremobilization from pre-anthesis stores of sourcestissues contributes more to grain Zn accumulation.This knowledge is critical to determine which traitto target in breeding programs for biofortificationof wheat with Zn and develop agronomic strategies tomaximize grain Zn concentrations. Apparently, the an-swer to this question depends on post-anthesis availabil-ity of Zn in the growth medium for uptake as well as thenitrogen nutritional status of wheat. While a high Znavailability for uptake during grain development togeth-er with a sufficient N supply favors the contribution ofroot uptake, net remobilization can account for almost

Fig. 2 Effect of N (low:0.5 mM; medium: 1.5 mM;high: 4.5 mM) supply andpost-anthesis Zn regime(discontinued vs. continued)on the shares of (U) post-anthesis uptake, (R1) remo-bilization from root pre-anthesis stores, and (R2) re-mobilization from straw pre-anthesis stores in the grainZn accumulation of durumwheat (Triticum durum cv.Balcali2000) plants grownin solution culture undergrowth chamber conditions

Plant Soil (2012) 361:177–187 185

all of grain Zn when the Zn availability is restricted, as itis the case under dry field conditions.

Acknowledgments This study was financially supported bythe HarvestPlus Program (www.harvestplus.org) and thesponsors of the HarvestPlus Global Zinc Fertilizer Project(www.harvestzinc.org) including Mosaic Company, K + S KaliGmbH, International Zinc Association, Omex Agrifluids, Interna-tional Fertilizer Industry Association and International Plant Nu-trition Institute. We would like to thank Veli Bayir for hisinvaluable contributions to mineral analyses in the present study.

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