Changes in root characteristics, gas exchange and water use efficiency following water stress and rehydration of Alfalfa and Sorghum

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1 JCS 5(12): (2011) ISSN: Chnges in root hrteristis, gs exhnge n wter use effiieny following wter stress n rehyrtion of lflf n Sorghum Wenro Li 1, 2, Suiqi Zhng * 2, Lun Shn 1, 2, Egriny Eneji 3 1 College of Life Sienes, Institute of Eologil Siene n Tehnology, Henn University,Kifeng, Henn , Chin 2 Northwest Siene n Tehnology University of griulture n Forestry, Stte Key Lortory of Soil Erosion n Dryln Frmings on Loess Plteu, Institute of Soil n Wter Conservtion Chinese emy of Sienes, Yngling, Shnxi, , Chin 3 Deprtment of Soil Siene, University of Clr, PMB 1115, Nigeri *Corresponing uthor: sqzhng@ms.isw..n strt Soil wter stress hs prmount effets on wter uptke y roots n its use y shoots. In this stuy, we etermine hnges in root hyruli onutivity (Lp r ) n morphology uner rought stress n how they regulte shoot gs exhnge n wter use effiieny in lflf n sorghum, two rops with ontrsting pttions to rought, the former exhiiting strong rought voine n the lter strong rought tolerne. Seelings of two lflf ultivrs iffering in rought voine n one highly rought tolernt sorghum ultivr were sujete to PEG-6000-inue wter stress n then rehyrte. The Lp r eline rpily fter the PEG tretment ut lrgely reovere within 48 hours of rehyrtion; however, inter-speifi n ultivr ifferenes were signifint. The rpi hnge in Lp r lso le to eqully rpi hnges in lef wter potentil, gs exhnge n onsequently, the instntneous wter use effiieny (WUE i ) in oth speies. resonle orreltion ws foun etween Lp r n WUE i. Tretment with Hg 2+ inite tht the wter stress-inue hnges in Lp r were ue to the involvement of quporins. One yer ol lflf n sorghum ultivrs sujete to moerte n severe rought stress t the fiel level h ltere root morphology n reue iomss proution n wter issiption ut inrese wter use effiieny of iomss proution (WUE ). These hnges were epenent on stress level, speies n ultivrs. However, not ll the root morphologil hnges ontriute to improve wter use. For exmple, the erese in tproot length negtively ffete the WUE of lflf wheres the inrese in root surfe re ws positively relte only to the WUE of sorghum. The ifferene in rought tolerne etween speies or ultivrs ws relte to their ility to reover the lost Lp r n CO 2 ssimiltion fter rehyrtion, s well s the ility to effetively regulte root morphologil hnges to inrese WUE. Keywors: Root hyruli onutivity, rought tolerne (DT), gs exhnge, wter use effiieny(wue), lflf() n sorghum (S). revitions: Lp r -root hyruli onutivity; WUE i -instntneous wter use effiieny; WUE -wter use effiieny of iomss proution; R/S-rtio of root n shoot; MC-merury hlorie; β-me-β-merptoethnol; RVR-the reltive vrition rte; Ψ lef -lef wter potentil; Pn-inluing net CO 2 ssimiltion rte; Gs-stomtl onutne; Tr-trnspirtion rte; C i -interellulr CO 2 -onentrtion; WDT-wter issiption y trnspirtion; DSI ove or DSI elow -the ove- or elowgroun ry sustne stress inex; RGR-the reltive growth rte root length, SR-root surfe re; NR-numer of roots (Dimeter 1mm); WR-with of tp/seminl roots; LTR-the length of tproot; TLR-totl length of roots; MS-Moerte stress; SS-Severe stress Introution Wter shortge is worlwie hllenge n its verse effets on wter uptke y roots n uses y shoots results to low-proution n even eth of foo or forge rops. Generlly, some ehviors in plnts relte to wter sorption n use, suh s xylem wter onutne, hnges in root omponents n stomtl movement n reflet ifferent responses to rought n help explin the strength of plnt s rought-tolerne or rought-voine. Wter flow long the soil-plnt ontinuum, esrie with iret physiologil prmeter, i.e. root hyruli onutivity (Lp r ), vrie with speies n onitions (Steule,2000; Miymoto et l. 2001; Mtsuo et l. 2009). For exmple, the ttenution of Lp r ws oserve in whet, mize, lupin, olive, et., uner wter stress (Steule n Peterson 1998;Zhng et l. 2002; Siemens n Zwizek 2004; Belr et l. 2007). The nlysis of soil-plnt hyrulis n e use to explin n preit the lrge ifferenes mong speies or ultivrs (Hur et l. 2001). Speies or ultivrs etter-pte to rought stress h higher Lp r s the soil rie thn those less-pte (Zho et l. 2004; Mu et l. 2006; Belr et l. 2007). However, there is unertinty out the effet of wter uptke y roots on wter use y shoots, espeilly uner rought euse of srity of 1521

2 regulte mehnisms. itionl response to rought y roots ours through their morphologil omponents. ntomy, inluing ortex thikness, xylem vessel imeter n root sueriztion ply mjor role in root hyrulis (Brmley et l. 2009). For root omponents, it is more importnt to trnsport wter to root surfe from soil. Deep rooting is strtegy for esition voine in nturl vegettion n in rops like rie n sorghum (Hun et l. 2009). But enormous roots re not lwys enefiil to wter use y shoots even though lrger rtio of root n shoot (R/S) uner rought ontriutes to etter WUE to some extent (Morison et l. 2008). lrge root system woul onsume more photosyntheti en prouts for their growth n iniretly negte shoot growth uring wter efiit (Brmley et l. 2009). The ilemm for wter-stresse plnts is how to shre their ron ssimiltions for root formtion n nopy growth. ommon response of plnts to wter efiieny is n inrese in R/S (Shrp n Dvies 1979; Morison et l. 2008). However, ifferent omponents of roots, suh s root length n root numer, might hnge ifferently uner rought oring to speies responses n pttion to wter stress. These iversities resulting from speies n eo-physiologil properties shoul e stuie to etermine n efine reltionships etween ifferent omponents of roots n WUE. The min effet of rought stress on shoot (wter use) re the erese in lef wter potentil, photosynthesis n growth, ue to stomtl losure n lower CO 2 supply to the roxylting enzymes (Brori n Holrook 2003; Silv n rr 2004; Volker n Wolfgng 2005). In response, there is n inrese in rought-inue hemil-signls- suh s B (Cohr et l. 2002; Brori n Holrook 2003), or eletril-signls-meite hnges in shoots (Grms et l. 2007). Different stomtl ehviours hve lso een foun in some speies in response to hnges in hyruli onutivity inue y xylem vition (Betul oientlis), root pruning (ies mnilis) n efolition (Pinus poers) (Hur et l. 2001). Therefore, stomtl ontrol n root hyruli onutivity or vritions in root morphology re onsiere the key ontrolling ftors for WUE uner rought. The inrese in WUE uner wter stress ws srie to iomss proution eing reue less y rought thn wter use (iomss proution WUE, WUE ) (Belr et l. 2007; Brmley et l. 2009) or to net CO 2 ssimiltion rte eing erese less y wter stress thn trnspirtion rte (instntneous WUE, WUE i ) (Li et l. 2007; Belr et l. 2007). Thus, there is hypothesis tht higher-wue speies hve etter pttion to rought. More reserh informtion is neessry to unerstn the stomtl or photosyntheti responses of ifferent speies or ultivrs with iffering rought-resistne perhps, simultneously with hnges in root hyruli onutivity n morphology uner onstnt rought. This unerstning will e very useful to illustrte the mehnism(s) of resistne of plnts to wter stress. To ress these issues, we exmine the hyruli onutivity n morphologil omponents of roots s well s physiologil hrteristis of shoots relte to wter use in two ontrsting speies: lflf (Meigo stiv L) n sorghum (Sorghum iolor (L.) Moenh). lflf hs ominnt tproot tht n evelop lots of lterl roots, wheres sorghum evelops root system of severl extensively rnhe iniviul roots. Tritionlly, lflf is onsiere to hve strong rought voine euse of n enormous n eep rooting system (Bi et l. 2001; Li et l. 2010). But lflf hs speil hrteristi of strong wter uptke n severely exhusting soil wter (Li et l. 2010). Therefore, it is neessry to lrify rought-tolerne in lflf. In ontrst, sorghum is onsiere to e strong physiologil rought-tolernt plnt with efinite rought tolerne mehnisms (Khizzh et l. 1995; li et l. 2009). One of our erlier works on the threshol of soil wter potentil neee for seeling emergene in lflf n sorghum suggeste signifintly higher moisture environment for lflf (Shn et l. 2008). When we ompre the ility for osmoti justment in oth speies, we foun tht sorghum ws etter thn lflf (Shn et l. 2008). In ition, sorghum h higher root osmoti pressure ( MP), stronger root system n more stle proution uner wter stress or rought (Jose et l. 1990; li et l. 2009). However, ifferenes in wter sorption n use, proutivity level n ility to enure rought uner the sme wter stress re poorly unerstoo. We hypothesize tht ifferenes exist n proeee to etermine them y exmining hnges in root hyruli onutivity n morphology uner soil wter stress n their regultive role in shoot physiology of lflf n sorghum seelings. To unerstn the mehnism of rought tolerne etween ultivrs, we lso exmine the root hyruli n morphologil hrteristis n shoot properties relte to wter use in two lflf speies: n,the former eing Chinese ntive ultivr n the ltter hving higher proution n pttion to wier ulturl prties. Our ojetive ws to gin new insight into the physiologil n morphologil responses tht my ontriute to rought tolerne in lflf n sorghum. Results Root hyruli onutivity (Lp r ) of lflf n sorghum Mrke n progressive eline in root hyruli onutivity (Lp r ) of lflf ( n ) n sorghum (Kng-Si) seelings uner wter stress ws oserve (Fig.1). The lowest Lp r ourre fter 48 h of wter stress, uring whih the Lp r of ws 10.4%, tht of ws 17.9% n tht of Kng-Si 10.4% of ontrol vlues. omplete reovery of Lp r i not our in n, whih just ttine 61.5 n 71.4% of ontrol vlues t the en of rehyrtion. However, the Lp r of Kng-Si reovere to ontrol level only fter 36 h of rehyrtion. Therefore, wter stress h muh more signifint effet on the Lp r of lflf thn of sorghum. There ws slight ifferene etween n in the response of Lp r to PEG tretment. Chnges in gs exhnge uring wter stress n rehyrtion For oth lflf vrieties n sorghum, the net CO 2 ssimiltion rte (Pn), stomtl onutne (Gs) n trnspirtion rte (Tr) showe fst elines within the initil 24 h of wter stress, prlleling the hnges in Lp r ; no signifint further elines ourre in the next 24h of stress. fter stress for 48h, the Pn eline y 59.4%, Gs y 66.4% n Tr y 62.6% for Kng-Si; the elines were 82.5, 86.4 n 78.8% for n 81.5, 88.3 n 85.0% respetively for (Fig.2 I, II n IV). However, the Pn, Gs n Tr of Kng-Si (sorghum) h lesser 1522

3 Tle 1. Effet of MC n MC+β-ME on Lp r in n Tretments Lp r ( 10-7 m s -1 MP -1 ) Without wter stress Uner wter stress CK 9.69± ± ± µm/l β-me ± µm/l β-me ± µm/l MC 3.59±2.08 D 4.61± ± µm /L MC+ 500 µm /L β-me 5.03±0.58 C 5.26± ± µm /L MC µm L β-me 6.67±2.08 B 7.58± ± ųm /L MC 2.33±1.15 E 3.63± ± µm /L MC+ 500 µm /L β-me 3.00±1.00 DE 5.05± ± µm /L MC µm /L β-me 5.00±1.00 C 7.44± ±0.90 β-me i not ffet root wter uptke (Lp r ) in lflf. However, MC (Hg + ) erese Lp r signifintly in n in onentrtion epenent response. signifint reovery effet inue y β-me ws shown. fter 24h wter stress, Lp r ws erese y 40.72% in. itionl tretment with 50µM (or 100µM) MC further reue Lp r y 13.7% (or 28.1%). The itionl eline in Lp r ue to MC ws sustntilly reverse y β-me, suggesting tht OPs might e present in lflf root ells n ws responsile for the eline in Lp r, espeilly uner wter stress. Dt re shown s men ± SD of six inepenent mesurements (P<0.05). Root system hyruli onutivity Lp r (x10-7 m.s -1.MP -1 ) B CD DE DEF EF Kng-Si EF F EF EF BC CD BCD 0 CK S2 S6 S12 S24 S36 S48 R2 R6 R12 R24 R36 R48 Stress n rehyrtion time (h) Fig 1. Effet of wter stress on root system hyruli onutivity(lp r )of lflf ( n ) n sorghum (Kng-Si). S n R represent stress n rehyrtion, respetively. For this n susequent figures, rs (with stnr errors) with the sme letters re not signifintly ifferent (P<0.05): e pitl letters refer to, smll letters to n itli letters to Kng-Si. elines n etter reovery (P<0.05) thn those of lflf uring wter stress n rehyrtion. The reovery rtes for t the en of the rehyrtion were 53.7% for Pn, 38.8% for Gs n 66.2% for Tr; for, the rtes were 51.8, 59.7 n 61.0% while for Kng-Si the rtes were 93.9, 89.5 n 94.3% respetively. However, the interellulr CO 2 onentrtion (Ci) of only n Kng-Si reovere to the ontrol level y the en of rehyrtion (Fig.2 III). Therefore, there ws signifint ifferene mong gs exhnge prmeters in response to PEG tretment etween lflf n sorghum. Chnges in lef wter potentil (Ψ lef ) n instntneous wter use effiieny (WUE i ) uring wter stress n rehyrtion In response to wter stress, the lef wter potentil (Ψ lef ) showe typil n time-epenent eline to the lowest vlues of MP for, MP for n MP for Kng-Si, t the 48 th h of wter stress (Fig.4). During rehyrtion, the Ψ lef inrese slowly n reovere to 2.14, 1.56 n 1.28 times the respetive ontrol in, n Kng-Si, respetively. The Ψ lef of n Kng-Si i reover to their ontrol levels. Wter efiit ws less influentil on the WUE i of sorghum thn lflf. In ition, wter stress promote instntneous wter use effiieny (WUE i ), efine s the rtio of Pn to Tr (Fig.3), with pek vlues out 1.8 n 1.7 times higher thn respetive ontrols in n t 36h of wter stress n out 1.2 times higher thn ontrol in Kng-Si following 48h of wter stress. fter rehyrtion, the WUE i of sorghum reovere grully to the ontrol level ut tht of lflf i not, suggesting tht the WUE i of sorghum ws ffete less thn tht of lflf y wter stress. lthough the WUE i hnge 1523

4 Tle 2. Effet of rought stress on length of tp/seminl roots, totl root length, numer of roots (D 1 mm), with of tp/seminl roots n surfe re of roots of lflf n sorghum Root morphology Kng-Si CK MS SS CK MS SS CK MS SS Length of tp/ Brnhing stge 27.48± 22.54± 14.16± 26.85± 21.4± 13.2± 38.58± 37.77± 36.6± seminl root B 2.72 C (m/pot) Hrvesting stge 43.55± 37.1± 32.1± 32.92± 29.89± 27.74± 55.2± 59.7± 56.14± B 0.36 B RVR (µm.m ) 51.16± ± ± ± 8.58B 39.13± 15.24B 93.29± ± ± ± 21.2 Totl root length Brnhing stge 0.48± 0.73± 1.01± 0.69± 0.83± 0.96± 1.12± 1.97± 1.55± (m/pot) C 0.22 B Hrvesting stge 1.11± 1.14± 1.42± 0.91± 1.01± 1.12± 7.17± 8.74± 8.95± B 0.05 B RVR (mm.m ) 9.29± ± ± ± ± ± ± ± ± 1.23 Numer of roots Brnhing stge 12±1 19±7 28±5 12±5 B 13±4 B 21±4 18±3 30±2 38±2 (D 1 mm, Hrvesting stge 32±13 40±9 42±10 32±4 B 35±2 B 41±4 57±4 78±5 88±4 piee/pot) RVG ( 10-3 piee ± 8.88± 5.06± 10.92± 11.69± 8.34± 12.96± 11.31± 10.51± piee ) B With of tproot Brnhing stge 0.30± 0.31± 0.28± 0.30± 0.29± 0.29± 0.35± 0.38± 0.38± or seminl root (m) Hrvesting stge 0.62± 0.49± 0.47± 0.63± 0.57± 0.43± 0.41± 0.48± 0.51± B C RVR (µm.m ) 8.13± 5.59± 6.31± 8.12± 8.08± 5.02± 1.69± 2.63± 4.16± B Surfe re of Brnhing stge 13.47± 19.01± 16.50± 10.73± 16.45± 15.33± 35.72± 48.84± 36.10± roots (m 2 /pot) B Hrvesting stge 24.97± 34.98± 26.97± 32.40± 42.11± 37.34± ± 162.4± ± C B RVR ( 10-3 m ± 8.85± 4.46± 12.37± 11.89± 9.12± 14.92± 14.15± 17.95± m ) B Drought inue inonsistent hnges in root morphologil omponents of soil-ulture lflf n sorghum. Signifint effets of genotypes n moisture levels were oserve on some morphologil prmeters. Dt re shown s men ± SD of four inepenent mesurements (P<0.05). Net photosyntheti rte (Pn umol CO2.m-2.s-1) B C ef e D g I h gh fg fg h EF EF i F EF EF j i h fg e e D E f E C B CD EF e e e GH f Kng-Si C FG DE f H GH fg H GH f e ef g g f GH g h II Stomtl onutne (Gs mol H 2 O.m -2.s-1) Interellulr CO 2 onentrtion (Ci umol CO2.mol-1) CD B III BC e e BCD CD D BCD BC BCD B B e e f efg gh h ef fg e B C DE D D DE gh EF e f DE e EFG e g h FG f G FG i ij IV (Tr mmol H 2 O.m -2.s -1 ) Trnspirtion rte 0 CK S2 S6 S24 S12 S36 S48 R6 R24 R48 R2 R12 R36 CK S2 S6 S24 S12 S36 S48 R6 R24 R48 R2 R12 R Stress n rehyrtion time (h) Fig 2. Effet of wter stress n rehyrtion on photosyntheti prmeters in lflf ( n ) n sorghum (Kng-Si). Dt re shown s men ± SD of six inepenent mesurements (P<0.05). 1524

5 in similr fol in oth lflf ultivrs, the solute vlue for ws higher thn. Inhiition or reversl of wter flow in lflf seelings through QPs with (HgCl 2, MC) or/n β-merptoethnol (CH 3 CH 2 -SH, β-me) No hnge ws foun in the Lp r of fter treting with β-me solution (Tle 1). This showe tht β-me h no effets on root wter uptke in lflf. However, t higher onentrtion (100 µm), MC resulte in greter reution of Lp r thn ontrols (uner 100 µm, it eline 76.0 n 65.6% for n ut uner 50 µm MC, it erese only 63.0% n 56.3%, respetively). It n lso e seen tht the reovery effets of β-me were signifint. ition of β-me to nutrient solution t 500µM (or 1000 µm) resulte in Lp r reovery of 47.9% (or 71.8%) in n 31.0% (or 68.8%) in. There ws ifferene in the response of Lp r to MC n/or β-me tretments in the two ultivrs. fter 24h of wter stress, the Lp r ws erese y 40.72% in. itionl tretment with 50µM (or 100µM) MC further reue the Lp r y 13.7% (or 28.1%). Strikingly, the further eline in Lp r use y MC ws sustntilly rreste y tretment with β-me. For exmple, n ition of 500µM β-me inrese the Lp r to 92.79% of the ontrol vlue (fter 24h wter stress; see Tle 1), while the ition of 1000µM β-me ompletely restore the Lp r. This fining ws ifferent from tht note for plnts not ffete y wter stress, in whih the 1000µM β-me tretment oul only reover Lp r y 71.8% t the mximum. Drought inue hnges in root morphologil omponents of soil-ulture lflf n sorghum Signifint effets of genotypes n wter vililities were oserve on some morphologil prmeters of roots, inluing root length, root surfe re (SR), numer of roots (Dimeter 1mm, NR) n with of tp/seminl roots (WR) in lflf n sorghum (Tle 2). Not surprisingly, the length of tproot (LTR) of rought-stresse lflf (MS n SS ) ws signifintly less thn ontrol (P<0.05), ut its totl length of roots (TLR) ws greter (Tle 2). Unlike lflf, there ws no signifint ifferene in the LTR n TLR etween rought-stresse n well-wtere sorghum t the jointing stge, ut the seminl n totl root lengths of rought-stresse sorghum were longer t hrvesting stge (P <0.05, Tle 2). These hnges were epenent on stress level in lflf ut not in sorghum. However, the reltive vrition rte (RVR) of LTR n TLR inite n improvement in growth of seminl root n n inhiition of totl roots in oth speies y rought stress, though sorghum h igger (P<0.05) RVR of TLR thn lflf t the sme wter level. Thus, rought inhiite the elongtion of roots muh more in lflf thn in sorghum n muh more in thn in (P < 0.05). lso, rought-stresse lflf n sorghum h igger NR n SR, wheres rought-stresse lflf h less WR n wter-stresse sorghum higher WR, epening on stress levels, genotypes n growth stges. On the other hn, the RVR of NR, WR n SR erese to the lowest rte in severe rought-stresse lflf; vlues in severe rought-stresse sorghum were muh higher thn well-wtere sorghum. Comine with hnges in root length, this inite tht morphologil hnges in sorghum roots were less ffete y wter stress thn those in lflf. Chnges in ove- n elowgroun iomss proution in rought-stresse lflf n sorghum Drought stress, espeilly if severe, signifintly (P< 0.05) reue (exept t the jointing stge of sorghum) the oven unergroun iomss in lflf n sorghum (Tle 3). This ws lso onfirme from hnges in ove- n elowgroun ry sustne stress inex (DSI ove n DSI elow ). For rought-stresse sorghum, the ereses in ove- n unergroun iomss proution were less pronoune (P<0.05) thn for lflf ut the ifferene etween n ws not sttistilly signifint. For instne t hrvest, the ovegroun iomss of moerte-stresse lflf erese y 22.47, 54.89, n tht for severe-stresse lflf y 55.03, n 25.42% ompre with respetive ontrol vlues for, n Kng-Si. The elowgroun iomss of moerte- n severe-stresse Kng-Si erese y 2.41 n 14.88%, followe y tht of (10.51 n 24.05%) n (9.72% n 37.77%). Only severe rought-stresse sorghum n lflf h n ovious inrese in the rtio of roots to shoots (R/S), espeilly t hrvest (Fig.5 I); the R/S of lflf inrese more thn tht of sorghum (P< 0.05), whih ws onsistent with hnges in their iomss proution. Of speil note ws tht only the reltive growth rte (RGR) of the ovegroun iomss in rought-stresse sorghum n lflf eline signifintly, implying tht the effet of wter shortge on shoots ws higher thn on roots n lso onfirming tht the eline in shoots iomss ws minly responsile for vritions in R/S. Wter use effiieny of iomss proution (WUE ) The elining effet of rought stress on wter issiption y trnspirtion (WDT) vrie mong speies (P< 0.05, Tle 3). Sorghum h greter WDT ut fewer elines in WDT uner rought stress thn lflf. The WDT in moerte-stresse, n Kng-Si erese y n 71.43, n 30.26%; tht in severe-stresse erese y 69.65, n 45.43% respetively, suggesting ifferenes in rought tolerne. These trens plus the vrition in ovegroun iomss proution uner rought stress suggeste signifint ifferenes (P< 0.05) in wter use effiieny of iomss proution (WUE ) mong speies (Fig.5 II). Sorghum h higher WUE thn lflf. For moerte- n severe rought-stresse, the WUE were 1.20 n 1.27 times greter; for, vlues were 1.10 n 1.20 times while for Kng-Si vlues were 1.27 n 1.36 times greter. This is nother onfirmtion of ifferenes in rought tolerne mong speies. Disussions s oserve in other plnts (Zhng et l. 2002; Siemens n Zwizek 2004; Belr et l. 2007), when the vilility of wter ws limite y PEG-6000, the Lp r firstly erese n then the stomt lose rpily; susequently, photosyntheti ssimiltion ws ffete through elines in Pn, Tr n Ci. s result, the Ψ lef strte to fll grully n WUE i inrese slightly, espeilly t the initil perio of wter stress in lflf n sorghum seelings. These hnges were time-n speiesepenent response to wter stress. Susequent rehyrtion tretment for 48h inue inomplete reovery of these prmeters in lflf ut omplete reovery in sorghum. It is inevitle tht hnges in hyruli onutivity will iniretly rive hnges in stomtl onutne n trnspirtion (Hur et l., 2001). The eline in Lp r oul e ue to 1525

6 Tle 3. Effet of rought stress on ove- n elow-groun iomss, rought stress inex (DSI) n wter issiption y trnspirtion (WDT) in lflf n sorghum Kng-Si CK MS SS CK MS SS CK MS SS ove- Brnhing stge 4.25± 3.57± 2.73± 3.34± 3.28± 2.29± 19.16± 20.67± 17.65± groun B iomss Hrvesting stge 18.66± 14.46± 8.42± 19.41± 16.24± 8.73± 96.60± 86.49± 72.04± (g/pot) B 0.34 C RGR (mg 16.46± 14.70± 11.26± 19.64± 17.82± 14.15± 18.24± 16.91± 13.95± g ) B 0.94C Below- Brnhing stge 0.57± 0.49± 0.46± 0.57± 0.55± 0.35± 8.68± 7.59± 7.54± groun B iomss Hrvesting stge 5.45± 4.88± 4.14± 6.38± 5.76± 3.97± 21.66± 21.14± 18.44± (g/pot) B 0.55 B RGR (mg 25.11± 27.18± 27.61± 27.03± 27.67± 30.36± 10.17± 11.45± 11.16± g ) DSI ove ± 0.36± ± 0.36± ± 0.75± DSI elow ± 0.76± ± 0.62± ± 0.85± WDT 19.02± 10.61± 5.43± 16.90± 11.78± 5.13± 25.74± 18.42± 14.05± (Kg/pot) B 0.76 C Similr ereses were oserve for ove- n elow-groun iomss proution n wter issiption y trnspirtion (WDT) in rought-stresse lflf n sorghum. This ws lso onfirme from hnges in ove- n elow-groun ry sustne stress inex (DSI ove n DSI elow ). For wter-stresse sorghum, the erese in ove- n elow-groun iomss ws less ut tht of WDT ws higher thn lflf (P< 0.05); the ifferene etween n ws no sttistilly signifint. Dt re shown s men ± SD of four inepenent mesurements (P<0.05). 7 WUE i (umol CO 2.mmolH2O.m -2.s -1 ) CK S2 S6 S24 S12 S36 Kng-Si S48 R6 R24 R48 R2 R12 R36 Stress n rehyrtion time (h) Fig 3. Effet of wter stress n rehyrtion on WUE i of lflf ( n ) n sorghum (Kng-Si). Dt re shown s men ± SD of six inepenent mesurements (P<0.05). physiologil hnges in the root system inue y wter shortge, suh s the losure of wter hnnel or the eline in QPs expression, whih ws onfirme y MC n /or β-me test (Tle 1). The HgCl 2 reue Lp r y % in well-wtere n % in wter-stresse lflf, whih ompres resonly with vlues foun in literture (Klenhoff et l. 2008). The 1000µM β-me oul nerly restore the Lp r of wter-stresse lflf to 71.8% of tht in well-wtere lflf. Therefore, QPs might e present in lflf root ells n ws responsile for the eline in Lp r, espeilly uner wter stress, lthough there ws no greement s to prtiiption of QPs tivities (Crvjl et l. 1996; Brmley et l. 2009). In ition to the eline in osmoti justment we reporte erlier (Li et l. 2007), n inrese in ortex thikness n eline in root imeter were oserve (these hnges were not signifint, n re thus not shown in this pper). Brmley et l. (2009) inite tht ntomy plye mjor role in root hyrulis, influening xil onutne n the istriution of wter uptke long the root, with more lolize role for QPs in whet n lupines. Influene on photosynthesis n gs exhnge hrteristis y wter stress ws nother importnt reson inuing Lp r eline n inomplete reovery in rehyrte lflf. Signifintly, ll these hnges my e the min resons why Lp r in wter-stresse sorghum lso erese. The oviously positive n non-liner reltionship etween Lp r n WUE i ientifie 1526

7 0.0 Lef wter potentil (MP) B BC BC D ef DE e Kng-Si ef Wter Stress n Rehyrtion CK S2 S6 S12 S24 S36 S48 R2 R6 R12 R24 R36 R48 Stress n rehyrtion time (h) Fig 4. Chnges in lef wter potentil (Ψ lef ) of lflf ( n ) n sorghum (Kng-Si). Dt re shown s men ± SD of six inepenent mesurements (P<0.05). f F ef f BC C BC D f DEF EF e ef ef e Rtio of root n shoot (R/S, %) I B B WUE (g/kg) (wter use effiieny of iomss proution) II Brnhing or jointing stge Vritions from rnhing/jointing to hrvesti Stnr errors WUE C B Tretments 1 75% (CK) 4 75% (CK) 7 Kng-Si 75% (CK) 2 55% (MS) 5 55% (MS) 8 Kng-Si 55% (MS) 3 35% (SS) 6 35% (SS) 9 Kng-Si 35% (SS) Fig 5. Effet of rought stress on rtio of roots n shoots (R/S, I) n wter use effiieny of iomss proution (WUE, II) of lflf ( n ) n sorghum (Kng-Si). Dt re shown s men ± SD of four inepenent mesurements (P< 0.05). hyruli limits s the use of prtil photosyntheti mge in response to rought,whih in turn use the fee-k inhiitions in reovery of Lp r in lflf n sorghum. We hve foun tht stomtl n non-stomtl limittions were responsile for the eline in photosynthesis of lflf t the initil perio of wter stress (Li et l. 2007). In ition, the WUE i showe gentle hnge se on the hnges in Pn n Tr uner wter stress n reovere to ontrol levels fter rehyrtion. Therefore, the eline in wter sorption inue slight photosyntheti mge. It ws possile the photosyntheti funtions of CO 2 fixtion, RUBP roxyltion n inorgni phosphorus trnsformtion were inhiite y wter stress to some extent (Prry et l. 2002; Bot et l. 2004), resulting to inomplete reovery of Pn, Gs n Tr, then Lp r fter 48h rehyrtion. Consiering the response of Ci to vrition in wter stress, this effet on Lp r ws more importnt t the eginning of wter stress n rehyrtion n the inhiition of root wter uptke ws ue mostly to metoli inhiition, espeilly t lter stges of wter stress. In terms of wter sorption, wter stress ffete the Lp r of lflf more severely thn tht of sorghum n wter uptke ility ws weker in lflf thn in sorghum. Gullo et l. (1998) reporte tht the Lp r of olive roots experiening gentle n meium wter stress reovere ompletely to ontrol level t 24 n 48 h fter rehyrtion. Lrge ifferenes in wter use etween speies n e ttriute in prt to ifferenes in their 1527

8 hyruli equipment (Hur et l. 2001). Therefore, wter stress h muh more effet on gs exhnge n photosynthesis in lflf thn in sorghum. Furthermore, there ws etter orreltion etween Lp r n WUE i in sorghum thn in lflf (Fig.6), suggesting stronger reltionship etween root wter uptke n sving wter use in sorghum when wter vilility iminishe n lflf ws more sensitive to wter stress thn sorghum. The onsierle erese in iomss of rought-stresse lflf n sorghum ws ttriute to reue net CO 2 ssimiltion n susequently, wekene growth of leves s reporte lso for mny plnts suh s olive, whet, mize, soyen, western lrh, tomto n so on (Xiong et l. 2006; Hung et l. 2007; Belr et l. 2007). s onsequene, wter onsumption ws reue, while the rtio of roots to shoots (R/S) n WUE inrese sustntilly in rought-stresse lflf n sorghum ompre with well-wtere plnts (T.3). Stuies on the reltionship etween iomss n WUE hve shown tht hnges in root n shoot weights were losely relte to those in WUE (Hung et l. 2007). Moerte rought stress oul improve plnt WUE through eline in the ost of proution (Grimes et l. 1992; Shn et l. 2008); to some extent, the inrese in R/S uner soil rought reflets the ptive growth lne of root system n nopy. Comine with the greter erese in RGR in ovegroun iomss thn elowgroun iomss uner rought stress, there ws n inition tht more ompetitive rohyrtes were llote to roots, resulting in greter eline in lflf n sorghum shoots yiels thn roots, in orer to mintin root funtion, suh s promoting the sorption pity to use eep soil moisture, meet the nees of evportion n plnt growth n then reue rought losses (Bi et l. 2001; Li et l. 2010). Therefore, erese in nopy growth plye ritil role in inresing the R/S, whih ws responsile muh more thn iniviul root extension n growth, to enuring rought stress in lflf n sorghum s reporte for soyen (Grimes et l. 1992; Li et l. 2010). The higher erese in elow- n ovegroun iomss (RGV, Tle 2) n inreses in R/S (RVR, Fig.5) suggeste sustntilly stronger pttion to rought for sorghum thn lflf. In ition to the inrese in R/S, the other morphologil omponents of root vrie in suh wy s to ope with prolonge rought in soil-ulture lflf n sorghum, suggesting their lose reltionship with rought resistne of the speies. It ws tritionlly unerstoo tht plnts with strong n well-evelope root system hve exellent ility to resist rought stress (Hun et l. 2009). Uner prolonge wter efiieny, prt from n inrese in totl length of roots, lflf tene to inrese its tproot n lterl root length (Li et l. 2010) n NR while sorghum tene to vry its WR, SR n NR, suggesting ifferenes in root morphologil pttion to rought etween sorghum n lflf. Uner rought onitions, erese root with n inrese root length, surfe re n numer of fine roots were enefiil to the norml growth of lflf n sorghum. These hnges oul improve soil moisture, expn the sope for wter uptke, erese istne from roots to soil n espeilly improve the effetive use of eep soil wter, s reporte in Bi et l. (2001). More importntly, sorghum root morphologil RVR were less ffete y wter stress thn those of lflf n muh more in thn in. This might e ttriute to ifferenes in rought resistne in sorghum n lflf s well s n. Mtthis n Smith (1997) foun smller stems n leves of lflf fter the esstion of irrigtion in summer ut lflf root growth inrese t the sme time, whih ws inonsistent with our results. However, we etermine tht the hnges in root morphology n shoot wter use n gs exhnge were losely relte in plnts suh s lflf n sorghum. oring to Pssiour s (1982) theory on WUE, there re two types of wter-use ehvior in plnts (Belr et l. 2007). Sorghum with higher WUE ppere to use onservtive wter-use strtegy, wheres proigl wter-use strtegy ws use y lflf, whether uner well-wtere or rought-stresse onition. Similr result ws reporte in Jefferson n Cutforth (2005) for nturl lflf fiel. The reltionship etween ifferent root morphologil omponents n WUE lso resse ifferent wter use n rought tolerne mehnisms. For instne from Fig.7, the tproot length ws positively relte only to lflf WUE while root surfe ws only positively orrelte with sorghum WUE. In ition, the with of tproot n WUE were positively orrelte in sorghum ut negtively orrelte in lflf. Totl length of roots, numer of roots (D 1mm) n R/S were negtively orrelte with WUE in oth speies. These reltionships were loser, i.e. higher orreltion oeffiient, in sorghum thn in lflf. This might e ttriute to the ifferent root morphology, in whih lflf hs ominnt tproot with lots of lterl roots, wheres sorghum evelops root system of severl extensively rnhe iniviul roots. lthough soil wter efiit promote the istriution of rohyrtes to roots, resulting to ereses in proution of lflf n sorghum n onsumption of wter, it enhne ptive hnges in root morphologil omponents n oherent reltionship etween root n shoot, whih ultimtely inrese the use of eep soil moisture n WUE. Uner soil rought, physiologil hnges in sorghum i iffer from those in lflf ue to their ifferent iologil hrteristis, whih expline their ifferenes in rought enurne. Vritions in iomss, WUE n root morphology were less ovious in sorghum thn lflf when mei wter potentil ontinue to eline, onfirming etter pttion to rought-stress for sorghum. Comprtive nlysis of the teste lflf ultivrs showe tht uner soil rought, the tproot n totl root lengths were greter n root surfe re n with of tproot eline more in uner stress. Therefore, n iffer in root morphologil evelopment, whih might le to ifferent resistne to rought even though their iomss n wter use showe no ovious ifferenes. From the ove results, it n e onlue tht lflf n sorghum respone to wter shortge y eveloping ifferent enuring mehnisms though these were se on stomtl losing, reuing trnspirtion, eresing wter sorption n use n ltere root morphology, whih inhiite growth of nopy n roots (reution in photosyntheti ssimiltion). Our t lso showe tht the hnges in hyruli onutivity of roots t the initil perio of vrile wter stress were ritil to the regultion of wter use n gs exhnge in lflf n sorghum seelings. In ition, sorghum hs stronger rought resistne thn lflf euse of etter root hyruli onutivity n R/S, less ffete iomss n gs exhnge, higher WUE n superior pttion of root morphologil omponents to prolonge rought stress. However, se on our t, we onsier lflf to e very promising for ultivtion in semi-ri n semi-wet or irrigte res. To lrify the pity for rought tolerne in lflf, it is neessry to relte the results otine in this stuy to extene (two or severl yer s ol lflfs) ulture in fiel onitions. Mterils n methos Plnt mteril n growth onitions lflf (ultivrs: n ) n sorghum 1528

9 6 Instntneous wter use effiieny WUEi (umol CO2.mmol H2O.m -2.s-1) Root system hyruli onutivity Lp r (x10-7 m.s -1.MP -1 ) y=-0.32x X R 2 =0.76* y=-6.07x10-4 X X R 2 =0.67* Kng-Si y=-0.49x x R 2 =0.90** Fig 6. Reltionship etween Lp r n WUE i in lflf n sorghum. (ultivr: Kng-Si) sees germinte in limti hmer t 25 fter eing sterilize with 75% (v/v) ethnol solution for 2 min. Seelings were trnsferre to the growth hmer (Moel: PGV-36, Cn) n ulture with 1/2-strength Hogln nutrient solution (ph: ) when the otyleon (lflf) or first euphyll (sorghum) ws fully expne with the following onitions: y/night temperture 23/18 (lflf) or 30/25 (sorghum), reltive humiity 65%, photoperio 12 h with photosyntheti photon flux ensity of 260 µmol m -2.S -1. Culture meium ws ompletely renewe every 3 n n oxygen pump ws use to erte the solution twie y t 4 h eh time. Potte sorghum (two plnts per pot) n lflf (five plnts per pot) were ulture on 11.3 Kg soil per pot mixe with ure (4.4 g), potssium sulfte (4.4 g), triple phosphte (7.2g) n orgni fertilizer (50g) for lflf or y 2.2, 4.4, 2.8 n 170g, respetively for sorghum. Eh of the pots mesure m 3. Sees were sown on 1 My 2006 (sorghum) n 30 pril 2006 (lflf). The potte plnts were grown in rin-proof she uring the experiment n irrigte to 75%±5% of fiel pity y using tp wter until erly June Eight non-plnte- pots were use to quntify the evportion from soil surfe. Experimentl tretments The wter stress tretments (wter potentil: -0.2 MP; stress time: 48 h) were estlishe y ing PEG-6000 to the nutrient solution when eh hyroponi seeling h grown for 40 ys (t lest 10 mture leves in lflf or 4 mture leves in sorghum); stresse seelings were then rewtere for 48 hours y putting them k into 1/2-strength non-peg Hogln solution. Control seelings grew ontinuously in nutrient solution without PEG The tretments were replite four times. Roots of seelings were immerse for 20 min in merury hlorie (HgCl 2 ; MC) or β-merptoethnol (CH 3 CH 2 -SH; β-me) solutions s follows: µmol/l β-me solution; µmol/L β-me solution; 3 50µmol/L MC solution; 4 100µmol/L MC solution; 5 50µmol/L MC solution, then in 500µmol/L or 1000µmol/L β-me solution; 6 100µmol/L MC solution, then in 500µmol/L or 1000µmol/L β-me solution; The roots were lso immerse in PEG-6000 solution for 24 h, n then trete s 3, 4, 5 n 6 ove. In the ontrol group, seelings were ultivte in the unltere (non-peg) Hogln nutrient solution. From 10 June 2006, when soil-ulture lflf h 7-8 fully-expne leves n sorghum h 3 fully-expne leves, three levels of soil moisture with 75%±5% (Control, CK), 55%±5% (Moerte stress, MS) n 35%±5% (Severe stress, SS) of fiel pity were impose on the potte plnts. These moisture levels were mintine y perioi weighing of pots n orreting for soil moisture until the plnts were hrveste. The ily wter onsumption per pot ws note. The ove- n elow-groun smples were tken t rnhing (25 June to 4 July 2006) n nthesis (50% flowering, 18 Septemer to 3 Otoer 2006) of lflf or t jointing (2 July to 10 July 2006) n hrvesting stges (13 Septemer to 1 Otoer 2006) of sorghum. Root hyruli onutivity (Lp r ) Eight hyroponi seelings of eh speies or ultures were selete to mesure root hyruli onutivity (Lp r ) y using pressure hmer (Moel: 3005, Soil Moisture Equipment Co. U.S.) oring to Miymoto et l. (2001) with minor moifition. The whole root system of hyroponiseelings ws immerse in 1/2-strength Hogln nutrient solution within the pressure hmer when the stems were ut uner otyleons (pproximtely 2.5 m ove emerging roots) through the pressure hmer li. Then, the pressure in the hmer ws rise in steps of 0.05 MP up to 0.40 MP ove tmospheri level. Root flow rte ws llowe to stilize (in 1-2 min per pressure) n the flow rte ws mesure for 1 min t eh pressure (t). For given gs pressure (P gs in MP), the volume exue from the root system (V in m 3 ) ws plotte ginst time. The slopes of these reltionships were lulte n use s unit surfe re (m 2 ) whih ws mesure y the CI-400 root imge nlysis system (CID In. U.S.). This yiele the volume flow, Jv r in m 3 m -2 s -1. Root hyruli onutivity, Lp r (m 3 m -2 s -1 MP -1 ) ws lulte s the slope of the regression line of Jv r plotte ginst hyrostti pressures of MP oring to the eqution: Jv r = V/(S T); Lp r = Jv r / P gs. Root morphologil prmeters The potte plnt roots were wshe len with root wshing evie fter hrvesting. Every root ws expne on piee of trnsprent pper n snne with snner. Root surfe re n totl root length were mesure with CI-400 root imge nlysis system (CID In. U.S.). Ruler n vernier liper were use to mesure tproot (or seminl root of sorghum) length n imeter. t the sme time, the numer of roots (imeter 1mm) ws reore. Root volume ws etermine 1529

10 Length of tproot/seminl root (m/pot) I y=-19.92x r 2 =0.46* y=-19.48x r 2 =0.63* Kng-Si y=1.13x r 2 =0..07 V y=-0.35x+0.92 r 2 =0..55* y=-0.66x+1.37 r 2 =0.67** Kng-Si y=0.068x+0.16 r 2 =0.84** With of tproot or seminl root (m) Totl length of roots (m/pot) Surfe re of roots (m 2 /pot) Numer of roots (Dimeter ws more thn1 mm, piee/pot) II III y=0.50x+0.64 r 2 =0.26* y=1.01x-0.26 r 2 =0.81** Kng-Si y=1.27x+2.5 r 2 =0.84** y=26.84x-7.35 r 2 =0.55* y=64.21x r 2 =0.73** Kng-Si y=21.16x r 2 =0.92** y=10.65x r 2 =0.15 y=22.09x+8.54 r 2 =0.29 Kng-Si y=11.34x r2=0.49* IV VI VII VIII y=-2.92x+8.08 r 2 =0.56* y=-9.38x r 2 =0.75** Kng-Si y=-2.14x r 2 =0.58* y=-8.31x r 2 =0.08 y=-36.34x r 2 =0.57* Kng-Si y=-16.43x r 2 =0.84** y=0.59x-0.27 r 2 =0.48* y=0.60x-0.36 r 2 =0.46* Kng-Si y=0.020x+0.15 r 2 =0.65** Dry weight of roots (g/pot) Dry weight of shoots (g/pot) Rtio of roots n shoots WUE (g/kg) (wter use effiieny of iomss proution) WUE (g/kg) (wter use effiieny of iomss proution) Fig 7. Reltionship etween WUE n root morphologil omponents, inluing tproot length (I), totl length of roots (II), surfe re of roots (III), numer of roots (D 1mm, IV), with of roots (V), ry weight of roots (VI), ry weights of shoots (VII) n R/S (VIII) in lflf n sorghum. y ringe metho. The root morphology ws ssesse y lulting the vritions in root length, root surfe re, numer of roots (imeter 1mm) n with of tp/seminl root from rnhing (jointing stge) to hrvesting stges of lflf (or sorghum). The reltive vrition rte (RVR) of eh omponent ws lulte s: RVR = [ ln (V 2 /V 1 )] /, where V 1 n V 2 re the vlues t rnhing/ jointing n hrvesting stges n is numer of ys from rnhing/ jointing to hrvesting. Lef wter potentil, gs exhnge n instntneous wter use effiieny (WUE i ) The mesurement of fully expene lef wter potentil (Ψ lef ) ws rrie out with pressure hmer (Moel: 3005, Soil Moisture Equipment Co. U.S.) s esrie in Belr et l. (2007). The seon n thir leves from the top were mesure for lflf while the top two fully expne leves were mesure for sorghum. Lef gs exhnge prmeters, inluing net CO 2 ssimiltion rte (Pn), stomtl onutne (Gs), trnspirtion rte (Tr) n interellulr CO 2 onentrtion (Ci) in the top two fully expene leves of lflf or sorghum were mesure with Li-6400 portle photosyntheti system (Li-or Co. U.S.) t 9:30-11:00 m. Mesurements of lef gs exhnge prmeters of hyroponi seelings were performe in the growth hmer. Instntneous wter use effiieny (WUE i ) ws lulte from the rtio of Pn to Tr. This mesurement ws repete 10 times. Biomss proution, wter issiption y trnspirtion (WDT) n wter use effiieny of iomss proution (WUE ) The soil-ulture plnts were hrveste t rnhing n nthesis stges of lflf n jointing n hrvesting stges of 1530

11 sorghum to ssess iomss. The hrveste ove- n elow-groun iomss were rie first t 105 o C for 30 min n then t 80 o C to onstnt weight. The ove- or elow-groun ry sustne stress inex ws etermine s the rtio of ry sustne of rought-stressese plnts to tht of well-wtere plnts. The reltive growth rte (RGR) ws lulte oring to Belr et l. (2007) n Boughlle et l. (2009) with slight moifition: RGR = [ ln (DW 2 /DW 1 )] /, where DW 1 n DW 2 re the iomss t rnhing/ jointing n hrvesting stges n is numer of ys from rnhing/ jointing to hrvesting. Wter issiption y trnspirtion (WDT) per pot ws etermine s the ifferene etween wter onsumption per pot for mintining 75%±5%, 55%±5% n 35%±5% of fiel pity n the evportion from soil surfe. t the eginning of this mesurement, plnt weight per pot ws etermine to voi prejuiing the mount of wter use per pot. The umultive mount of WDT per pot -1 uring the whole growing seson ws the totl WDT per potte plnts. Wter use effiieny of iomss proution (WUE ) ws etermine for eh potte plnt y iviing totl iomss proution (ove- n elowgroun iomss) y umultive wter use throughout the growing perio (i.e. the totl WDT). Sttistil nlysis ll t otine from the mesurements were sujete to nlysis of vrine (NOV) using the SigmPlot 8.0 Dome sttistil pkge n the sttistil nlysis system (SS) softwre. Mens were ompre using Dunn s multiple rnge tests t the 5% level of proility. Moel nlysis ws use to etermine reltionships etween vriles n ifferenes etween prmeters of fitte moels were evlute with the t-test or F-test. knowlegements The stuy ws supporte y Ntionl Key Bsi Reserh Progrm of Chin (No. 2009CB118604), Ntionl Nturl Siene Fountion of Chin (No ) n reserh projets of Ministry of Eution n Henn University (No. SBGJ090405). We thnk Prof. J H Zhng of Hong Kong Bptist University n Prof. Q Ye for the tehnil reing of this mnusript. 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