Running head: Utility of root cortical cell file number under drought Corresponding author: Jonathan P. Lynch, Department of Plant Science, The

Transcription

1 Running hed: Utility of root corticl cell file numer under drought Corresponding uthor: Jonthn P. Lynch, Deprtment of Plnt Science, The Pennsylvni Stte University, University Prk, PA, USA 16802, , jpl4@psu.edu Journl reserch re: Ecophysiology nd sustinility 1

2 Reduced root corticl cell file numer improves drought tolernce in mize Joseph G. Chimungu, Kthleen M. Brown, Jonthn P. Lynch* Summry: Reduced root corticl cell file numer sustntilly reduces root respirtion, permitting greter root growth nd explortion of deep soil domins, therey improving wter cquisition, plnt growth, nd yield under drought. 2

3 This reserch ws supported y the Ntionl Science Foundtion/Bsic Reserch to Enhnce Agriculturl Development (grnt numer: 4184 UM NSF 5380) nd Agriculture nd Food Reserch Inititive competitive grnt numer: of the USDA Ntionl Institute of Food nd Agriculture. Deprtment of Plnt Science, The Pennsylvni Stte University, University Prk, PA, 16802, USA 3

4 Astrct We tested the hypothesis tht reduced root corticl cell file numer (CCFN) would improve drought tolernce in mize (Ze mys L.) y reducing the metolic costs of soil explortion. Mize genotypes with contrsting CCFN were grown under well-wtered nd wter-stressed conditions in greenhouse mesocosms nd in the field in the USA nd Mlwi. CCFN rnged from 6 to 19 mong mize genotypes. In mesocosms reduced CCFN ws correlted with 57% reduction of root respirtion per unit root length. Under wter stress in the mesocosms, genotypes with reduced CCFN hd etween 15% nd 60% deeper rooting (D 95 ), 78% greter stomtl conductnce, 36% greter lef CO 2 ssimiltion, nd etween 52% to 139% greter shoot iomss thn genotypes with mny cell files. Under wter stress in the field, genotypes with reduced CCFN hd etween 33% nd 40 %deeper rooting (D 95 ), 28% lighter stem wter δ 18 O signture signifying deeper wter cpture, etween 10% nd 35% greter lef reltive wter content, etween 35% nd 70% greter shoot iomss t flowering, nd etween 33% nd 114% greter yield thn genotypes with mny cell files. These results support the hypothesis tht reduced CCFN improves drought tolernce y reducing the metolic costs of soil explortion, enling deeper soil explortion, greter wter cquisition, nd improved growth nd yield under wter stress. The lrge genetic vrition for CCFN in mize germplsm suggest tht CCFN merits ttention s reeding trget to improve the drought tolernce of mize nd possily other cerel crops. Key words: Corticl cell file numer, root costs, Ze mys L., (Mize), drought 4

5 Introduction Drought is primry constrint to glol crop production (Schmidhuer nd Tuiello, 2007) nd glol climte chnge is likely to increse the risk of drought, especilly in rin-fed griculture (Bttisti nd Nylor, 2009; Burke et l., 2009; Mishr nd Cherkuer, 2010; Loell et l., 2011). The development of crops with greter drought tolernce is therefore n importnt glol ojective. Yield under drought is often not n efficient selection criterion in drought reeding progrms, since yield is ffected y mny elements of the phenotype nd the environment, intercting in complex nd often unknown wys. Trit-sed selection or ideotype reeding is generlly more efficient selection strtegy, permitting the identifiction of useful sources of vrition mong lines tht hve poor gronomic dpttion, elucidtion of genotype y environment interctions, nd informed trit stcking (Arus, 2002; Mnschdi et l., 2006; Lynch, 2007; Arus et l., 2008; Lynch, 2011, York et l., 2013). In most groecosystems the topsoil dries efore the susoil s drought progresses. In such environments plnts with deeper roots re le to cquire wter ville in deeper soil domins tht my not e ville to plnts with shllower roots (Ludlow nd Muchow, 1990; Ho et l., 2005; Hmmer et l., 2009). An ideotype hs een proposed to guide the reeding of crops with deeper roots nd therefore greter wter cquisition from drying soil clled steep, chep nd deep, integrting rchitecturl, ntomicl, nd physiologicl phenes (Lynch, 2013). The term chep denotes phenes tht reduce the metolic cost of soil explortion, which is n importnt limittion to the cquisition of scrce soil resources, including wter in dry soil (Fn et l., 2003; Lynch, 2007; Zhu et l., 2010; Postm nd Lynch, 2010; Postm nd Lynch, 2011; Jrmillo et l., 2013). Plnt resource lloction to root growth typiclly increses under drought to enhnce wter cquisition, nd therefore the metolic cost of root growth ecomes significnt component of plnt fitness nd dpttion under drought (Lynch, 2007; Lynch, 2013). Therefore, plnt tht is le to ccess wter in deep soil domins t reduced metolic cost will hve superior productivity, ecuse it will hve more metolic resources ville for further resource cquisition, growth, nd reproduction. Evidence in support of this hypothesis comes from empiricl nd modelling studies for mize under wter nd edphic stress (Lynch, 2007; Zhu et l., 2010; Postm nd Lynch, 2010; Postm nd Lynch, 2011; Jrmillo et l., 2013). Root corticl erenchym (RCA) is the enlrged ir spce in the root cortex tht forms either through cell deth or cell seprtion (Evns, 2004). RCA is ssocited with disproportionte reduction of root respirtion in mize y converting living corticl tissue to ir volume (Fn et l., 2003; Zhu et l., 2010). Reduction of root metolic costs permits more internl resources to e llocted to greter root growth nd consequently greter soil resource cquisition. RCA formtion is lso ssocited with reduction of phosphorus content in root tissue on volume sis, since ir spces do not contin phosphorus (P) (Fn et l., 2003), nd with improved growth in low-p soil (Lynch, 2011). RCA lso reduces the N content of root tissue, nd is eneficil for N cpture nd mize growth on low N soils (Sengwili 2013). Modelling studies suggest tht RCA improves crop dpttion to suoptiml nutrient vilility y reducing the metolic costs of soil explortion (Postm nd Lynch, 2010; Postm nd Lynch, 2011). Under drought Zhu et l. (2010) found tht mize genotypes with more RCA hd 5 times greter iomss nd 8 times greter yield thn genotypes with less RCA. Living Corticl Are (LCA) is totl trnsverse root corticl re minus RCA re. Jrmillo et l. (2013) found tht root respirtion is positively correlted with LCA, nd 3.5-fold reduction in LCA is ssocited with 5

6 fold improvement in plnt growth under drought. These results indicte tht the metolic demnd of living corticl tissue is primry determinnt of root growth, soil explortion nd resource cquisition in soil environments with suoptiml resource vilility. The current study uilds on erlier studies indicting tht sustntil reduction of root metolic cost is ssocited with vrition in LCA. The cortex of the mize root is composed of severl concentric lyers of prenchym cells, the numer of which we refer to s corticl cell file numer (CCFN). Recently Burton et l., (2013) reported tht there is 3-fold vrition for CCFN in Ze species. In tht study the vrition ws wider in lndrces (6-16 cell files) thn in teosinte (7-13 cell files). It hs een proposed tht reduced CCFN would decrese the metolic costs of root growth nd mintennce, oth in terms of the cron cost of root respirtion s well s the nutrient content of living tissue, y reducing the proportion of root volume occupied y living corticl tissue, which hs greter metolic demnds thn the stele (Lynch, 2013). However, the physiologicl utility of CCFN hs not een explored. The ojective of this study ws to test the hypothesis tht reduced CCFN would reduce root respirtion, permitting greter rooting depth, therey enhncing wter cquisition nd improving oth plnt growth nd yield under wter stress. Results We oserved sustntil phenotypic vrition for CCFN within mize recominnt inred lines (RILs) (Fig 1). In mesocosms (GH1), CCFN rnged from 8 to 17 in the IBM popultion (Fig 2A). In the field in Mlwi (MW2011), CCFN rnged from 6 to 19 mong lines from the mize reeding progrm t LUANAR (Lilongwe University of Agriculture nd Nturl Resources; Fig 2B). The stility of CCFN cross environments ws estimted s the correltion coefficient etween CCFN mesured on the sme genotypes in mesocosms (30 dy old plnts, GH2) nd in the field (70 dy old plnts, PA2011) nd cross environments in the field, Bund (BU2012) nd Chitl (CH2012). Strong positive correltions were found etween CCFN in mesocosms (GH2) nd in the field (PA2011) (r = 0.85, P <0.05), nd etween CCFN mesured in two environments in Mlwi (BU2012 nd CH2012) (r =0.68, P<0.05). To understnd the effects of CCFN on root respirtory costs, CO 2 production from excised root segments ws mesured in diverse sets of mize lines in mesocosms (GH 1,2,3). Reduced CCFN ws correlted with reductions of specific root respirtion y 57% (GH1-IBM), 46% (GH1- NyH), 52% (GH2), nd 69% (GH3) (Fig. 3). However, there ws no significnt difference in respirtion rtes etween well-wtered nd wter-stressed roots in GH2 nd GH3 (Tle 1). In GH1, CCFN ws correlted with specific root length (SRL) oth in IBM lines (r= -0.55, p<0.05) nd NyH lines (r= -0.48, p<0.05). CCFN ws etter predictor of root respirtion thn SRL (Tle 2). In well-wtered mesocosms, CCFN hd no reltionship with rooting depth, stomtl conductnce, photosynthesis rte or plnt iomss. Under wter stress, genotypes with reduced CCFN hd 15% (GH1) nd 60% (GH2) deeper rooting, 78% greter stomtl conductnce (GH3), 36% greter lef photosynthetic rte (GH3), nd 52% (GH2) nd 139% (GH3) greter iomss thn genotypes with mny cell files (Tle 1, Figs. 4, 5, 6). Reduced CCFN genotypes proliferted more roots in soil domins elow 60 cm compred to mny CCFN genotypes under wter stressed conditions (Supplementl figure S1A). 6

7 In the field t Rock Springs, PA, under wter stress genotypes with reduced CCFN hd 33% (PA2011) nd 40% (PA2012) deeper rooting depth (D 95 ), nd 10% (PA2011) nd 35% (PA2012) greter lef reltive wter content thn genotypes with mny cell files (Tle 3, Fig. 7, 9 A&B). D 95 is the depth ove which 95% of totl root length is locted in the soil profile. In ddition, genotypes with deeper D 95 hd greter lef wter sttus thn genotypes with shllow D 95, while there ws no reltionship in well wtered conditions (PA2011) (r=0. 51, p<0.000). In ddition reduced CCFN genotypes proliferted more roots in soil domins elow 30 cm compred to mny CCFN genotypes under wter stressed conditions (Supplementl figure S1B). Anlysis of soil wter δ 18 O showed progressively lighter isotopic signture of wter with incresing depth in wter stress conditions (Fig 8). However, the mjority of chnge in this signture ws in the top two lyers: 0-10 nd cm depth (pproximtely 2.09 ). The vlues of soil wter signture elow 30 cm depth showed no significnt difference with depth (Fig 8) nd were ggregted s deep wter for further nlysis. The verge vlues of xylem wter δ 18 O for genotypes vried y 3.19 (Tle 4). Genotypes with reduced CCFN hd collective xylem wter signture tht ws 28% lighter thn tht of genotypes with mny cell files (Tle 4). Soil wter δ 18 O vlues were used in n isotopic mixing model to determine wter sources contriuting to the δ 18 O signture for xylem wter, ssuming tht ny wter cquired elow 30 cm depth ws deep wter. Genotypes with reduced CCFN hd greter verge relince on deep wter nd were the lest relint on shllow wter from top two soil lyers thn genotypes with mny cell files (Tle 4). The proportion of deep wter cquired y genotypes with reduced CCFN rnged from 21 to 81% while for two genotypes with mny cell files this vlue ws zero. The only exception ws genotype 181 which ws clssified s hving mny cell files ut hd reltively greter dependency on deep wter of 32%. Wter stress reduced shoot iomss y 30% (PA2011) nd 33% (PA2012), nd reduced yield from 26% to 68% (PA2011) nd from 33% to 75% (PA2012) compred with well-wtered plnts (Tle 3, Fig 9 C, D, E, F). Genotypes with reduced CCFN hd 35% (PA2011) nd 45% (PA2012) greter shoot iomss, nd 38% (PA2011) nd 114% (PA2012) greter yield thn lines with mny cell files under wter stress (Tle 3, Fig 9 C, D, E, F). In the field cross two mize growing environments in Mlwi, wter stress reduced lef reltive wter content y 22% (BU2012) nd 25% (CH2012), shoot iomss y 43% (BU2012) nd 54% (CH2012), nd grin yield y 59% (BU2012) nd 53% (CH2012) (Fig. 9). Under wter stress genotypes with reduced CCFN hd 20% (BU2012) nd 19% (CH2012) greter lef reltive wter content, 70% (BU2012) nd 57% (CH2012) greter shoot iomss, nd 93% (BU2012) nd 33% (CH2012) greter yield thn genotypes with mny cell files under wter stress (Tle 5, Fig. 10) DISCUSSION We hypothesized tht reduced CCFN would reduce root respirtion per unit root length, permitting greter root growth nd explortion t depth, therey enhncing wter cquisition, improving plnt growth nd yield under drought. Our results extend from oservtions of young plnts in greenhouse mesocosms to mture plnts in the field in the USA nd two environments in Mlwi. Our results entirely support our hypotheses: CCFN vried sustntilly mong mize genotypes, nd genotypes with reduced CCFN hd lower specific root respirtion, nd under 7

8 wter stress genotypes with reduced CCFN hd greter rooting depth, greter cquisition of deep soil wter, etter plnt wter sttus, greter lef photosynthesis, etter growth, nd etter yield. The utility of CCFN ws evluted using diverse sets of genotypes contrsting in CCFN in greenhouse mesocosms, in the field using movele rinout shelters, nd with differentil irrigtion in Mlwi. The greenhouse mesocosms nd movle rinout shelters in the field llowed us to simulte terminl drought y the progressive reduction of soil wter content (Fig. 10). The mesocosms lso permit detiled nlysis of root distriution y depth nd root respirtion, since entire root systems cn e recovered. The field environments in Mlwi were nturl drought environments in which rinfll vried ut ws insufficient to meet plnt wter requirements. The comintion of results from the field nd mesocosms lends credence to our conclusions, s the field includes vrile environmentl fctors such s soil temperture, iot, nd soil physicl properties, while mesocosms permit greter environmentl control nd more detiled mesurement of root properties. RILs shring the sme genetic linege were employed to minimize the effects of genetic interction, epistsis, nd pleiotropy, which my confound the interprettion of results from comprison of unrelted lines (Zhu nd Lynch, 2004). CCFN is quntittive trit ssocited with multiple genetic loci in mize (Sengwili, 2013). For evlution of the utility of quntittive trits such s CCFN, RILs re useful since they permit the comprison of lines differing in CCFN expression mong set of genotypes shring common prents. Mize hs sustntil genetic vrition for root rchitecturl nd ntomicl phenes (Hochholdinger, 2009; Byuelo-Jiménez et l., 2011; Trchsel et l., 2011; Burton et l., 2013; Lynch, 2013). Genotypic vriility oserved here ws consistent with previous studies which found tht CCFN vries in the rnge from 7-16 for CCFN in Ze species (including mize lndrces nd teosinte) (Burton et l., 2013). Corticl cell files re formed y severl successive symmetric periclinl divisions in the root picl meristem (Bum et l., 2002; Chpmn et l., 2003; Lux et l., 2004). It hs een documented tht the numer of such periclinl divisions vries mong species, genotypes nd root types (Lux et l., 2004; Coudert et l., 2010), which might generte differences in CCFN s oserved here. Mize, like other monocots, hs no secondry growth in its roots (Esu, 1965). Hence, CCFN vrition long the longitudinl xis of root represent the rdil ptterning in the root picl meristem. Therefore CCFN vrition oserved in this study ws lrgely due to genotypic differences. However, the genetic nd physiologicl mechnism of this vrition in mize is not yet known nd deserves further explortion. CCFN cn e esily oserved with microscope nd is therefore menle to direct phenotypic selection in crop improvement progrms. In this study we showed tht CCFN mesured on young plnts from greenhouse mesocosms 30 dys fter plnting were ccurte reflections of CCFN mesured on mture plnts in the field 70 dys fter plnting. We lso oserved correltion etween CCFN mesured in the field cross two contrsting mize growing environments in Mlwi. These results indicte tht CCFN ws stle cross environments in this study. We hve proposed tht reduced CCFN my e useful dpttion to drought y reducing the metolic costs of soil explortion (Lynch 2013). Previous studies hve ssocited reduction of root respirtion with RCA formtion (Fn et l., 2003; Zhu et l., 2010). Jrmillo et l. (2013) 8

9 found tht reduced LCA sustntilly reduces root respirtion in mize. In tht study, it ws concluded tht LCA is stronger predictor of root respirtion thn either RCA or root dimeter, since it tkes into ccount the differing corticl res mong root clsses. Two key determinnts of LCA re cell file numer nd cell size, nd ltering either one my ffect the size of LCA, consequently ffecting root metolic costs. We hve recently shown tht lrge corticl cell size in mize is ssocited with reduced root respirtion, nd greter root depth, wter cquisition, plnt growth, nd yield under drought (Chimungu et l 2014). As shown in this study, decresing CCFN from 16 to 8 ws ssocited with 57% reduction of root respirtion (Fig 3). This respirtory pttern my reflect the effect of decresing the proportion of metoliclly ctive cells in the cortex nd incresing the proportion of nonrespiring tissues such s sclerenchym nd xylem vessels. Root respirtion ssocited with growth, mintennce, nd ion uptke re mjor components of root metolic costs (Lmers, 1979; vn der Werf et l., 1988; Peng et l., 1993; Lmers et l., 2002; Lynch nd Ho, 2005). In this study, root respirtion ws mesured in the mture region of the root, therefore, totl respirtion in this region is primrily the respirtion for tissue mintennce. Root construction cost is ssumed to e one-time cost tht occurs when the root is formed (Yni et l., 1995). In contrst, mintennce costs ccumulte over time, nd cn quickly exceed initil construction costs, nd therefore mintennce cost re importnt determinnts of root metolic cost (Eissenstt nd Yni, 1997; Lynch nd Ho, 2005; Lynch nd Brown, 2008). For exmple Postm nd Lynch (2011) reported tht mize plnts without mintennce respirtion hd up to 72% greter growth under nutrient limiting conditions thn plnts with root mintennce respirtion. The importnce of mintennce costs is clerly shown y the cse of root corticl erenchym (RCA), which reduces the mintennce respirtion nd nutrient content of mture root tissue y converting living corticl cells to ir spce. Differentil RCA formtion mong mize genotypes is ssocited with reduced mintennce respirtion of root tissue, which when plnts re stressed y suoptiml vilility of wter, N, P, or K, results in greter root growth, greter cquisition of soil resources, greter plnt growth, nd greter yield (Fn et l. 2003; Zhu et l. 2010; Postm nd Lynch 2010; Postm nd Lynch 2011; Sengwili et l., 2014). Since RCA is formed in mture regions of the cortex it ffects root mintennce costs rther thn root construction costs. Although CCFN ffects oth construction nd mintennce costs, we elieve tht y nlogy with RCA, the effects of CCFN on mintennce costs re more importnt for plnt dpttion to stress thn effects on construction costs. A more detiled nlysis of this issue would e possile using the structurl-functionl plnt model SimRoot (Lynch et l., 1997). SimRoot is structurl-functionl plnt model tht simultes the three-dimensionl rchitecture nd soil resource cquisition of root system s it develops over time. It is difficult to quntify oth construction nd mintennce costs in greenhouse nd field studies, ecuse of the tightly coupled integrtion etween the two costs. SimRoot my provide useful insights in this context y llowing the quntifiction nd independent mnipultion of mintennce nd construction costs in plnts contrsting for CCFN. SimRoot hs provided such insights in the context of the effects of RCA on mintennce nd construction costs in mize (Postm nd Lynch 2010; Postm nd Lynch 2011) CCFN ws stronger predictor thn SRL for root segment respirtion, with slightly greter coefficient of determintion (Tle 2). Generlly, greter SRL permits more efficient soil explortion (Eissenstt, 1992). SRL is influenced y root dimeter s well s root ntomy, or tissue mss density (Whl nd Ryser, 2000). However, specific root length vries widely with 9

10 environmentl conditions nd the direction of chnge in SRL is not lwys predictle sed on resource supply (Eissenstt et l., 2005). In ddition, SRL is corse metric tht ggregtes mny distinct phenes to provide n overll estimte of mss per unit root length, without indicting how mss vries or the composition nd hence energy content of the mss. SRL lso does not indicte whether the root mss is living or ded tissue nd therefore it is not well correlted with vrition for mintennce respirtion mong root clsses nd ges. Therefore, CCFN should e more direct predictor of root respirtory costs thn SRL (Tle 2) since it tkes into ccount the differing corticl res which generlly hve high metolic rte. For exmple (Hll et l., 1971) working with mize showed tht fresh isolted cortex hd greter respirtion thn fresh steles. CCFN therefore is n importnt determinnt of root metolic cost. Lynch (2013) proposed tht lrge corticl cells my lso sustntilly reduce root respirtion, since lrger cells hve higher rtio of vcuolr to cytoplsmic volume nd hence reduced respirtion per unit of tissue volume. For this reson we propose tht the enefit of reduced CCFN should e strongest in roots with smll corticl cells. The enefits of reduced metolic cost of soil explortion were greter under wter stress (Figs. 3, 4, 5, 6, 7, 9, & 10). The greter utility of reduced CCFN under drought is ssocited with the fct tht the genotypes with less costly root tissue hd deeper rooting, etter ccess to wter nd therefore extr cron gin through photosynthesis, which in turn will increse root growth further, creting positive feedck for plnt growth under wter stress. We found tht reduced CCFN ws ssocited with incresed rooting depth (D 95 ) in the field under wter stress, ut did not ffect rooting depth in well-wtered conditions (Fig, 7, Supplementl figure S1 A&B). In ddition, our results show tht genotypes with reduced CCFN nd deeper D 95 were le to mintin greter RWC in the field nd stomtl conductnce in mesocosms under wter stress thn genotypes with mny cell files (Fig. 5, 9 A&B, 10 A&B). These results suggest tht incresed vilility of cron from reduced respirtion llows the plnt to grow more roots under drought. Root growth in deep soil domins under wter stress resulted in incresed wter cquisition, greter plnt wter sttus, nd greter photosynthesis, which enefits overll plnt growth nd yield. Xylem wter reflects the oxygen isotopic composition of wter cquired y the plnt from the soil s no isotopic frctiontion occurs during wter uptke nd trnsport (Ehleringer nd Dwson, 1992; Dwson nd Pte, 1996; Ehleringer et l., 2000). In this study we used nturl vrition in the isotopic signture of soil wter to provide insight into the potentil etween root depth nd wter cquisition (Fig 4, 7, & 8). The isotopic signture of soil wter oserved in this study is determined y evportion from the soil, precipittion nd irrigtion. Becuse soil cores were collected 30 dys fter the lst irrigtion or rinfll, the surfce soil wter ws isotopiclly enriched due to evportion. For the susoil wter, the isotope signture could e ttriuted to the comintion of the evportion effect nd the isotopic signtures of irrigtion wter nd rinfll, resulting in grdient of δ 18 O with soil depth (Fig. 8). Xylem wter δ 18 O signtures showed tht genotypes with reduced CCFN hd lighter isotope signtures nd greter dependency on deep soil wter thn genotypes with mny cell files (Tle 3). The difference in the depths of root wter cquisition etween genotypes with reduced CCFN nd mny cell files could e ttriuted to their rooting depth (Fig 4 & 7). The dditionl enefit of reducing root costs in nnul crops like mize is tht extr resources from reduced root metolic demnd cn contriute to crop yield, y enhncing plnt 10

11 reproductive growth, since reproduction nd roots re competing sinks for current photosynthte. In this study we found tht irrespective of mize popultion, environment, soil type, nd tril mngement, genotypes with reduced CCFN hd oth greter shoot iomss nd grin yield thn genotypes with mny cell files under wter stress (Fig. 6, 9 (C,D,E,F), & 10 (C,D,E,F)). These results support the hypothesis tht genotypes with less costly root tissue could develop the extensive, deep root systems required to fully utilize soil wter resources in drying soil without s much yield penlty. The physiologicl utility of phene my depend on interctions with other phenes in integrted phenotypes (York et l., 2013). These interctions mong phenes could result in synergistic or ntgonistic effects on resource cquisition. Understnding phene synergisms is essentil in developing ideotypes for reeding crops with greter tolernce of edphic stress. We lso recognize tht phene cn e eneficil for multiple stresses. Root phenes, such s reduced CCFN, tht influence the metolic cost of soil explortion my e importnt to plnts in lowinput systems y incresing rooting depth. Rooting depth is importnt for the cquisition of moile nutrients including nitrte nd sulfte, prticulrly in soils with high leching potentil. Evidence for this comes from modelling studies, where it hs een shown tht deeper roots could significntly improve cquisition of nitrogen (Dunin et l., 2004; Postm nd Lynch, 2011; Dthe et l., 2013). Reduced CCFN my lso ffect root hydrulic conductivity, ecuse smller rdil pth is ssocited with greter hydrulic conductivity nd consequently greter wter cquisition (Rieger nd Litvin, 1999). We nticipte tht root rdil hydrulic conductivity would increse with reduced CCFN. Reduced CCFN my exhiit trdeoffs when soil hrdness restricts root penetrtion, since the cpcity to penetrte hrd soil is ssocited with lrger root dimeter (Mterecher et l., 1992; Bengough et l., 2006; Bengough et l., 2011). Further reserch is needed to understnd these potentil trdeoffs nd synergisms efore the deployment of this trit in crop improvement progrms. In this study we hve demonstrted the utility of reduced CCFN for mize under wter stress. We propose tht the utility of reduced CCFN my e pplicle to the cquisition of N in leching environments, nd should e pplicle to other plnt species, especilly grminceous species lcking secondry root growth, including rice (Oryz stiv), whet (Triticum estivum L.), rley (Hordeum vulgre L.), ots (Aven stiv), sorghum (Sorghum icolor), millet (Pennisetum glucum) etc. These results support the hypotheses tht phenes nd phenes sttes tht reduce the metolic cost of soil explortion improve the cquisition of limiting soil resources (Lynch nd Ho, 2005; Lynch, 2014; Lynch et l., 2014). Such phenes include production of n optiml numer of root xes, iomss lloction to metoliclly efficient root clsses, nd reduced tissue respirtion (Miller et l., 2003; Jrmillo et l., 2013; Lynch, 2014; Sengwili et l., 2014). CCFN is n exmple of the third ctegory, i.e. n ntomicl phene tht ffects the metolic costs of soil explortion y ffecting tissue respirtion. Another exmple in this ctegory is root corticl erenchym (RCA), which destroys living corticl cells. Mize genotypes with undnt RCA hve reduced root respirtion, greter rooting depth, greter wter cquisition under drought (Zhu et l., 2010), greter N cquisition under N limittion (Postm nd Lynch, 2011; Sengwili et l., 2014), nd greter P nd K cquisition in soils with suoptiml vilility of those resources (Postm nd Lynch, 2011). Similrly, mize genotypes with greter corticl cell size (CCS) hve less root respirtion, greter rooting depth, nd greter wter cquisition under 11

12 drought (Chimungu et l., 2014). The deployment of root phenotypes with greter metolic efficiency of soil explortion represents novel, unexploited prdigm to develop crops with greter resource efficiency nd resilience (Lynch, 2014). Mterils nd methods Plnt mterils Mize genotypes from IBM, NyH popultions nd dvnced lines from the Mlwi mize reeding progrm were utilized in this study. The IBM lines re from the intermted popultion of B73xMo17 nd were otined from Shwn Keppler, University of Wisconsin, Mdison, WI, USA (Genetics Coopertion Stock Center, Urn, IL, USA) nd designted s Mo (Supplementl tle S1). The NyH lines re from the Ny821xH99 popultion (Shwn Keppler, University of Wisconsin, Mdison, WI, USA). Bsed on previous experiments crried out under optimum growing conditions (Burton, 2010), suset of 15 IBM lines nd 11 NyH lines ws selected to ssess the phenotypic vrition of CCFN nd its impct on root respirtion (GH1). A set of six IBM lines contrsting in CCFN ws used in 2011 experiments (GH2 nd PA2011) nd nother set of six IBM lines lso contrsting in CCFN for 2012 experiments (GH3 nd PA2012) to evlute utility of CCFN under wter limited conditions (Supplementl tle S1). In Mlwi, set of 70 reeding lines ws used to ssess phenotypic vrition of CCFN in Mlwin germplsm (MW2011). These lines originted from the Mlwi ntionl mize reeding progrm nd were selected to represent rod rnge of gene pools. A suset of 33 lines contrsting in CCFN ws used in two field experiments (BU2012 nd CH2012) to evlute the utility of CCFN under wter limited conditions nd cross sites in Mlwi (Supplementl tle S1). In ll experiments genotypes were clssified s reduced CCFN ( 10 cell files) nd s mny CCFN ( 13 cell files) (Supplementl tle S1). Greenhouse experiments Three experiments were conducted under the sme conditions in two consecutive yers (GH1,2,3) (Supplementl tle S1). The experiments were conducted in greenhouse t University Prk, PA, USA (40 o 48 N, 77 o 51 W,) under constnt conditions (14-h/10-h dy/night: 23 o C/20 o C dy/night: 40-70% reltive humidity), with mximum illumintion of 1200 µmol photons m -2 s -1 nd dditionl light ws provided when necessry with 400-W metl-hlide uls (Energy Technics, York, PA, USA). Plnts were grown in mesocosms (Supplementl figure S2) consisting of PVC cylinders 1.5 m in height y 0.15 m in dimeter, with plstic liners mde of 4- mil (0.116-mm) trnsprent hi-density polyethylene film, which ws used to fcilitte root smpling. The growth medium consisted of (y volume) 50% commercil grde snd (Quikrete Compnies Inc. Hrrisurg, PA, USA), 35% vermiculite (Whittemore Compnies Inc., Lwrence, MA, USA), 5% Perlite (Whittemore Compnies Inc., Hrrisurg, PA, USA), nd 10% topsoil (Hgerstown silt lom top soil (fine, mixed, mesic Typic Hpludlf)). Minerl nutrients were provided y mixing the medi with 70g per column of OSMOCOTE PLUS fertilizer consisting of (in %); N (15), P (9), K (12), S (2.3), B (0.02) Cu (0.05), Fe (0.68), Mn (0.06), Mo (0.02), nd Zn (0.05) (Scotts-Sierr Horticulturl Products Compny, Mrysville, Ohio, USA) for ech column. The seeds were germinted y plcing them in drkness t 28 ± 1 C in germintion chmer for two dys prior to trnsplnting two seedlings per mesocosm, thinned to one uniform seedling per mesocosm 5 dys lter fter plnting. 12

13 At hrvest, the shoot ws removed, nd the plstic liner ws pulled out of the PVC column nd plced on wshing ench. The plstic liner ws cut open nd the roots were wshed crefully y rinsing the medi wy with wter. This llowed us to recover the entire plnt root system. Smples for root respirtion mesurement were collected cm from the se of three representtive second whorl crown roots per plnt. Root respirtion (CO 2 production) ws mesured using n infrred gs nlysis system (LI-COR 6400 Biosciences, Lincoln, NE, USA) equipped with custom 56 ml closed chmer of plstic tuing (1.5 cm dimeter) hving connection points seled with silicon grese. The chnge in CO 2 concentrtion in the chmer ws monitored for 3 minutes. During the time of mesurement the chmer ws plced in temperture controlled wter th t 27± 1 C to mintin constnt temperture. Following respirtion mesurements, root segments were preserved in 75% ethnol for ntomicl nlysis s descried elow. Root length distriution ws mesured y cutting the root system into 7 segments of 20 cm depth increments. Roots from ech increment were spred in 5 mm lyer of wter in trnsprent plexiglss trys nd imged with flted scnner equipped with top lighting (Epson Perfection V700 Photo, Epson Americ, Inc. USA) t resolution of 23.6 pixel mm -1 (600 dpi). Totl root length for ech segment ws quntified using WinRhizo Pro (Regent Instruments, Quéec, Cnd). Following scnning the roots were dried t 70 o C for 72 hours nd weighed. To summrize the verticl distriution of the root length density we used the D 95 (Schenk nd Jckson, 2002), i.e. the depth ove which 95 % of the root length ws locted in the column or soil profile. Specific root length ws clculted y dividing root length with corresponding weight. Root segments were lted using lser ltion tomogrphy (LAT) (Hll et l, unpulished) to otin imges for ntomicl nlysis. In rief, LAT is semi-utomted system tht uses lser em (Avi 7000, 355 nm pulsed lser) to vporize or sulimte the root t the cmer focl plne hed of n imging stge. The smple is incremented, vporized or sulimted, nd imged simultneously. The cross-section imges were tken using Cnon T3i cmer (Cnon Inc. Tokyo, Jpn) with 5X micro lens (MP-E 65 mm) on the lser-illuminted surfce. Root imges were nlyzed using RootScn, n imge nlysis tool developed for nlyzing root ntomy (Burton et l., 2012). CCFN ws determined from three different imges per root segment. CCFN ws otined y counting the cell lyers from the epidermis to the endodermis. Experiment I (GH1) The im of this experiment ws to ssess the reltionship etween phenotypic vrition for CCFN nd root respirtion. The experiment ws lid out s rndomized complete lock design (RCBD) replicted three times with time of plnting s locking fctor. A set of 26 genotypes ws plnted in the mesocosms nd wter stress ws imposed y withholding wter strting 14 dys fter plnting. Plnts were hrvested for root respirtion mesurements nd ntomicl nlysis 35 dys fter plnting. Experiment II (GH2) nd III (GH3) Two experiments were conducted, one in Fll 2011 (GH2) nd Summer 2012 (GH3) nd were lid out using s rndomized complete lock design (RCBD), replicted four times with time of plnting s locking fctor. Plnting ws stggered y seven dys. A set of six genotypes contrsting in CCFN ws plnted in ech experiment (Supplementl tle S1). These genotypes 13

14 were selected sed on phenotypes from previous experiments (Burton nd Lynch, 2010; Chimungu, 2014). In oth experiments, the irrigted mesocosms (control) ech received 200 ml of wter every other dy, to replenish wter lost y evpotrnspirtion, nd in stressed mesocosms, wter ppliction ws withheld strting 5 dys fter plnting to llow the plnts to exploit residul moisture to simulte terminl drought.. Lef gs exchnge of the third fully expnded leves ws mesured with LI-6400 Infrred Gs Anlyzer (LI-COR, Lincoln, NE, USA) using red-lue light t PAR intensity of 1200 µmol photons m -2 s -1 nd constnt CO 2 concentrtion of 400 ppm 28 dys fter plnting. The mesurements were done etween 9:00 nd 11:00h. Plnts were hrvested 30 dys fter plnting for root respirtion mesurements, root growth distriution nd shoot iomss. The dry mtter of the shoot nd root were mesured fter drying t 70 o C for 72 h nd root length distriution ws determined s descried ove. Field experiments Assessing phenotypic vrition of CCFN (MW2011) The experiment ws conducted t Bund College reserch frm, Lilongwe, Mlwi (33 o 48 E, 14 o 10 S,) in 2011 under optiml conditions (i.e. the plots were rinfed ut only rrely were they severely moisture stressed). The soil is Lilongwe series sndy cly lom (Oxic Rhodustlf). The experiment ws rrnged s rndomized complete lock design (RCBD) with three replictions. Ech plot consisted of single 6 m long row with 25 plnts. Root crowns were excvted y shovelomics (Trchsel et l., 2010). In rief, roots were excvted y removing soil cylinder cm in dimeter with the shoot t its center nd depth of cm. The excvted root crowns were shken riefly to remove lrge frction of the soil dhering to the root crown. The root crowns were immersed in sopy wter for 5-10 minutes in order to fcilitte removl of the remining soil. Three 8-cm root segments were collected cm from the se of representtive second whorl crown root of ech plnt, nd used to ssess CCFN. The segments were preserved in 75% ethnol efore eing processed s descried ove. Utility of CCFN under wter stress In the USA two experiments were conducted in rinout shelters (Supplementl figure S3) locted t the Russell E. Lrson Agriculturl Reserch Center in Rock Springs, PA, USA (40 42 N, W,), during the summer of 2011 (PA2011) nd 2012 (PA2012). In Mlwi experiments were conducted t the Chitl Agriculture Reserch sttion of the Ministry of Agriculture, Slim, Mlwi (13 o 28 S, 33 o 59 E) (CH2012), nd the Bund Reserch frm of the Lilongwe University of Agriculture nd Nturl Resources (14 o 10 S, 33 o 48 E,) (BU2012). The soil is Hgerstown silt lom (fine, mixed, mesic Typic Hpludlf) in Rock Springs, nd sndy cly lom (Oxic Rhodustlf) oth t Chitl nd Bund. The experiments (PA2011, PA2012, CH2012 nd BU2012) were rrnged s rndomized complete lock split plot design with four replictions. The min plots were composed of two moisture regimes nd the suplots contined genotypes contrsting in CCFN in ech experiment. The experiments were hnd-plnted on 15 th June 2011 nd 25 th June 2012 in Rock Springs nd 3 rd nd 4 th Septemer in Bund nd Chitl respectively. In Rock Springs, ech suplot consisted of three rows, with ech row eing 2.5 m long, with 25 cm etween plnts nd 75 cm etween rows. In Mlwi the experiments were plnted in single 6 m row plot with 25 cm nd 75 cm spcing etween plnting sttions nd rows respectively. The drought tretment ws initited strting dys fter plnting using n utomted rinout shelter in Rock Springs nd y withholding wter ppliction in Mlwi. The 14

15 shelters (10 y 30 m) were covered with cler greenhouse plstic film (0.184 mm) nd were utomticlly triggered y rinfll to cover the plots, excluding nturl precipittion. Adjcent non-sheltered control plots were rinfed nd drip-irrigted when necessry to mintin the soil moisture close to field cpcity throughout the growing seson. At ech loction, the recommended fertilizer rte ws pplied efore plnting. Soil wter content for oth well wtered nd wter stressed tretments ws monitored regulrly during the experiment (Fig 11). Soil wter content ws monitored using the TRIME FM system (IMKO Micromodultechnik GmH, Ettlingen, Germny) t three depths (20, 35 nd 50 cm) t six points inside the shelter nd three points outside the rinout shelter. Seven redings were tken etween 30 to 120 dys fter plnting. Plnt mesurements In ll field experiments, middy lef reltive wter content (RWC) ws mesured nd used s physiologicl indictor of plnt wter sttus. To mesure lef RWC, fresh lef discs (3 cm in dimeter) were collected from the third fully expnded lef for three representtive plnts per plot 60 dys fter plnting nd weighed immeditely to determine fresh weight (FW). After which the discs were immeditely hydrted to full turgidity (6 h) y soking them distilled wter. Following soking, the discs were lotted dry nd gin weighed to determine turgid weight (TW). Discs were then oven dried t 70 o C for 72 h, nd dry weight (DW) ws determined. Lef RWC ws clculted ccording to the eqution: 100 /. In PA2011 nd PA2012 soil cores were collected 80 dys fter plnting to determine root distriution in the soil profile. A soil coring tue (Giddings Mchine Co., Windsor, CO, USA) 5.1 cm in dimeter nd 60 cm long ws used for smpling, the core ws tken midwy etween the plnts within row. The cores were sectioned into 6 segments of 10 cm depth increments nd wshed. Susequently the wshed roots were scnned using flted scnner (Epson, Perfection V700 Photo, Epson Americ, Inc. USA) t resolution of 23.6 pixel mm -1 (600 dpi) nd nlyzed using imge processing softwre WinRhizo Pro (Regent Instruments, Quéec, Cnd). Root distriution in the soil profiles ws clculted s descried ove. Shoot nd roots were evluted 75 dys fter plnting. To ccomplish this, three representtive plnts in ech plot were cut t soil level. The collected shoot mteril ws dried t 70 o C for 72 hours nd weighed. Root crowns were excvted y shovelomics (Trchsel et l., 2010). Three 8-cm root segments were collected cm from the se of representtive second whorl crown root of ech plnt for ntomicl nlysis. The segments were preserved in 75% lcohol efore eing processed s descried ove. At physiologicl mturity grin yield ws collected ech plot. Soil nd plnt smpling of δ 18 O nlysis In PA2011, soil smples were collected djcent to plnts in the rinout shelter 65 dys fter plnt using 5 cm dimeter soil core. Soil cores were tken to the mximum chievle depth of 60 cm. The cores were immeditely seprted into 10 cm increments; 10, 20, 30, 40 50, nd 60 cm. The corresponding mize stems were collected t the sme time when soil ws smpled, pproximtely 8-10 cm of the stem ws collected just oveground level nd the epidermis ws immeditely removed. Soil nd mize stem smples were put in snp vils, seled with prfilm to prevent evportion, nd refrigerted immeditely. Cryogenic vcuum distilltion 15

16 (West et l., 2006; Koeniger et l., 2010) ws used to extrct soil wter nd crop stem wter. In cryogenic vcuum distilltion, two glss tues were ttched to vcuum pump. The smple ws plced in one tue nd frozen y sumerging the tue in liquid nitrogen, nd then oth tues were evcuted y vcuum pump to crete closed U-shpe configurtion. After tht, the tue contining smple ws heted, while the collection tue ws still immersed in liquid nitrogen to ctch the vpor. Smples were weighed nd oven dried fter extrction to ensure the extrction time ws sufficient to vporize ll the wter in smples. The wter smples were nlyzed t the Penn Stte Institutes of Energy nd the Environment (PSIEE). Stle isotopic nlyses were performed using PICARRO L2130-i δd/δ18o Ultr High Precision Isotopic Wter Anlyzer (PICARRO Inc, CA, USA). Results were expressed s prts per thousnd devitions from the Vienn Stndrd Men Ocen Wter (VSMOW). To determine the percent contriution of soil wter t depth to the signture of wter within the plnt s xylem, n isotopic mixing model ws used (Phillips et l., 2005). IsoSource Version (Phillips nd Gregg, 2003) ws used to evlute the reltive contriution of ech soil lyer to plnt xylem wter signture. The frctionl increment ws set t 1%, nd tolernce t 0.1. Dt nlysis The dt from ech yer were nlyzed seprtely considering tht different sets of genotypes were used. For greenhouse dt, for comprisons of genotypes, irrigtion levels nd their interction effects two-wy nlysis of vrince (ANOVA) ws used. Field dt were nlyzed s rndomized complete lock split plot designs to determine the presence of significnt effects due to irrigtion level, genotype nd interction effects on the mesured nd clculted prmeters. Men seprtion of genotypes for the different prmeters ws performed y Tukey-HSD test. Unless otherwise noted, HSD 0.05 vlues were only reported when the F test ws significnt t P Liner regression nlysis ws used to estlish reltionships etween CCFN nd mesured nd clculted prmeters. Acknowledgements We thnk Jennifer Yng nd Eric Nord for criticl reding of the mnuscript nd Krol E. Confer, Roert Synder, Moses Mliro, Ptson Nlivt nd Andrew Mkopoliw for technicl ssistnce in the course of this study. 16

17 LITERATURE CITED Arus JL, Slfer GA, Royo C, Serret MD (2008) Breeding for Yield Potentil nd Stress Adpttion in Cerels. CRC Crit Rev Plnt Sci 27: Arus JL, Slfer GA, Reynolds MP, Royo C (2002) Plnt reeding nd drought in C3 cerels. Wht should we reed for? Ann Bot 89: Bttisti DS, Nylor RL (2009) Historicl wrnings of future food insecurity with unprecedented sesonl het. Science (80- ) 323: Bum SF, Durovsky JG, Rost TL (2002) Apicl orgniztion nd mturtion of the cortex nd vsculr cylinder in Aridopsis thlin (Brssiccee) roots. Am J Bot 89: Byuelo-Jiménez JS, Gllrdo-Vldéz M, Pérez-Decelis V., Mgdleno-Arms L, Ocho I, Lynch JP (2011) Genotypic vrition for root trits of mize (Ze mys L.) from the Purhepech Plteu under contrsting phosphorus vilility. F Crop Res 121: Bengough AG, Brnsy MF, Hns J, McKenn SJ, Roerts TJ, Vlentine TA (2006) Root responses to soil physicl conditions; growth dynmics from field to cell. J Exp Bot 57: Bengough AG, Mckenzie BM, Hllett PD, Vlentine TA (2011) Root elongtion, wter stress, nd mechnicl impednce: review of limiting stresses nd eneficil root tip trits. J Exp Bot 62: Burke MB, Loell DB, Gurino L (2009) Shifts in Africn crop climtes y 2050, nd the implictions for crop improvement nd genetic resources conservtion. Glo Environ Chng 19: Burton AL (2010) Phenotypic evlution nd genetic sis of ntomicl nd rchitecturl root trits in the genus Ze. Penn Stte University Burton AL, Brown KM, Lynch JP (2013) Phenotypic diversity of root ntomicl nd rchitecturl trits in Ze species. Crop Sci 53: Burton AL, Lynch JP (2010) Phenotypic evlution nd genetic sis of ntomicl nd rchitecturl trits in the genus Ze. The Pennsylvni Stte University, University Prk, PA Burton AL, Willims MS, Lynch JP, Brown KM (2012) RootScn: Softwre for highthroughput nlysis of root ntomicl trits. Plnt Soil 357: Chpmn K, Groot EP, Nichol S., Rost TL (2003) Primry root growth nd the pttern of root picl meristem orgniztion re coupled. J Plnt Growth Regul 21:

18 Chimungu J, Brown K, Lynch J (2014) Lrge root corticl cell size improves drought tolernce in mize (Ze mys L.). Plnt Physiol. DOI:10.110: Chimungu JG (2014) Root ntomicl trits for efficient wter cquisition under drought in mize (Ze mys L.). The Pennsylvni Stte University Coudert Y, Perin C, Courtois B, Khong NG, Gntet P (2010) Genetic control of root development in rice, the model cerel. Trends Plnt Sci 15: Dthe A, Postm JA, Lynch JP (2013) Modeling Resource Interctions Under Multiple Edphic Stresses. In D Timlin, LR Ahuj, eds, Enhncing Underst. Quntif. Soil Root Growth Interct. ASA, CSSA, SSSA, 5585 Guilford Rd., Mdison, WI , USA, pp Dwson TE, Pte JS (1996) Sesonl wter uptke nd movement in root systems of Austrlin phretophytic plnts of dimorphic root morphology: stle isotope investigtion. Oecologi 107: Dunin V, Rengel Z, Diggle AJ (2004) Simulting form nd function of root systems: efficiency of nitrte uptke is dependent on root system rchitecture nd the sptil nd temporl vriility of nitrte supply. Funct Ecol 18: Ehleringer JR, Dwson TE (1992) Wter uptke y plnts: perspectives from stle isotope composition. Plnt Cell Environ 15: Ehleringer JR, Roden J, Dwson TE (2000) Assessing ecosystem-level wter reltions through stle isotopes rtio nlyses. In OE Sl, RB Jckson, HA Mooney, RW Howrth, eds, Methods Ecosyst. Sci. Springer-Verlg, pp Eissenstt DM (1992) Costs nd enefits of constructing roots of smll dimeter. J Plnt Nutr 15: Eissenstt DM, Volder A, Cldwell MM, Heldmier G, Jckson RB, Lnge OL, Mooney HA, Schultze E-D, Sommer U (2005) The efficiency of nutrient cquisition over the life of root. In H BssiriRd, ed, Nutr. Acquis. y plnts. An Ecol. Perspect. Springer-Verlg, Berlin, pp Eissenstt DM, Yni RD (1997) The ecology of root lifespn. Adv Ecol Res 27: 1 60 Esu K (1965) Plnt ntomy. John Wiley, New York, USA Evns DE (2004) Aerenchym formtion. New Phytol 161: Fn M, Zhu J, Richrds C, Brown KM, Lynch JP (2003) Physiologicl roles for erenchym in phosphorus-stressed roots. Funct Plnt Biol 30:

19 Hll J, Sexton R, Bker D (1971) Metolic chnges in wshed, isolted steles. Plnt 61: Hmmer GL, Dong Z, McLen G, Doherty A, Messin C, Schussler J, Zinselmeier C, Pszkiewicz S, Cooper M (2009) Cn chnges in cnopy nd/or root system rchitecture explin historicl mize yield trends in the U.S. corn elt? Crop Sci 49: 299 Ho MD, Ross JC, Brown KM, Lynch JP (2005) Root rchitecturl trde-offs for wter nd phosphorus cquisition. Funct Plnt Biol 32: Hochholdinger F (2009) The Mize Root System: Morphology, Antomy, nd Genetics. In JL Bennetzen, SC Hke, eds, Hnd. Mize Its Biol. Springer, New York, pp Jrmillo RE, Nord EA, Chimungu JG, Brown KM, Lynch JP (2013) Root corticl urden influences drought tolernce in mize. Ann Bot 112: 1 9 Koeniger P, Leiundgut C, Link T, Mrshll JD (2010) Stle isotopes pplied s wter trcers in column nd field studies. Org Geochem 41: Lmers H (1979) Efficiency of root respirtion in reltion to growth rte, morphology nd soil composition. Physiol Plnt 46: Lmers H, Atkin OK, Millenr FF (2002) Respirtory ptterns in roots in reltion to their functioning. In Y Wisel, A Eshel, K Kfkki, eds, Plnt Roots, Hidden Hlf, Third Edit. Mrcel Dekker, Inc, New York, New York, pp Loell DB, Bänziger M, Mgorokosho C, Vivek B (2011) Nonliner het effects on Africn mize s evidenced y historicl yield trils. Nt Clim Chng 1: Ludlow MM, Muchow RC (1990) A criticl evlution of trits for improving crop yields in wter-limited enviroments. Adv Agron 43: Lux A, Luxov M, Ae J (2004) Root cortex: structurl nd functionl vriility nd responses to environmentl stress. Root Res 13: Lynch JP (2007) Roots of the second green revolution. Aust J Bot 55: Lynch JP (2007) Rhizoeconomics: The roots of shoot growth limittions. Hortscience 42: Lynch JP (2011) Root phenes for enhnced soil explortion nd phosphorus cquisition: tools for future crops. Plnt Physiol 156: Lynch JP (2013) Steep, chep nd deep: n ideotype to optimize wter nd N cquisition y mize root systems. Ann Bot 112: