leaf water relations and solute accumulation in two grain sorghum lines exhibiting contrasting...

Journal of Experimental Botany, Vol. 46, No. 293, pp. 1833-1841, December 1995Journal ofExperimentalBotany

Leaf water relations and solute accumulation in two grainsorghum lines exhibiting contrasting drought tolerance

Gnanasiri S. Premachandra, Daniel T. Hahn, David Rhodes and Robert J. Joly1

Department of Horticulture, Purdue University, West Lafayette, IN 47907-1165, USA

Received 8 March 1995; Accepted 30 July 1995

Abstract

A drought-tolerant grain sorghum line (K886) main-tained significantly higher relative water content(RWC), osmotic potential at full turgor (y i<x») andturgor pressure (ip^ than did a drought-susceptibleline (CS3541) when the two genotypes were grown incontainers and subjected to severe water stress priorto anthesis. Leaf area expansion was inhibited to agreater extent by water deficit in line CS3541 than inK886. Both the basal ^ 1 0 0 ) and the capacity to accu-mulate solutes upon exposure to stress appear to playimportant roles in the measured genotypic differencesin leaf water relations. Sap osmolarity was greater inline K886 than in CS3541 throughout the entire rangeof water potential induced. With the exception of pro-line, the baseline concentrations of each of eight sol-utes were higher in K886 than in CS3541. Further,when water deficit was imposed, K886 exhibited largerincreases in sap osmolality than did CS3541. Theconcentrations of K + , sugars, Cl~ and P, predominantsolutes contributing to osmotic adjustment, increasedwith increasing stress in K886, but essentiallyremained constant in CS3541. The two lines exhibitedlarge differences in the relative contributions of indi-vidual solutes to osmotic adjustment, and these contri-butions changed markedly during stress developmentboth within and between lines. The most notable dif-ferences between genotypes were with respect to thecontributions of sugars and K+ ions. The capacity toaccumulate K+ ions and to minimize stress-inducedreductions in water content, turgor and leaf expansionappear to be useful traits for inclusion in germplasmscreening programmes for improved drought tolerancein sorghum.

Key words: Drought tolerance, glycinebetaine, osmoticadjustment, Sorghum bicolor, water stress.

Introduction

Crop productivity in a water-limited environment derivesfrom mechanisms that either permit tolerance of episodesof cellular dehydration or that minimize water loss andthereby maintain a favourable water status for leaf devel-opment. Osmotic adjustment, a well-defined componentof adaptation to water deficit in sorghum, has beenshown to be associated with maintenance of protoplastvolume and cell turgor (Kaiser, 1982; Santakumari andBerkowitz, 1991) as well as with the avoidance of lethalrelative water content (Flower and Ludlow, 1986). It alsohas been shown to be an important factor in continuedroot elongation in drying soil (Sharp and Davies, 1979).Potassium ions, sugars, proline, and glycinebetaine havelong been known to be important components of osmoticadjustment. Few studies, however, have evaluated howthese and other solutes contributing to osmotic adjust-ment differ among drought-tolerant and susceptiblegenotypes of any crop species, nor how their relativecontributions to osmotic adjustment change with increas-ing stress severity. The general objective of this work wasto evaluate leaf water balance and growth in conjunctionwith solute profiles, in order to gain a more detailedunderstanding of metabolic responses to water deficit ingrain sorghum.

Two sorghum lines whose agronomic responses towater deficit have been well characterized in field experi-ments were evaluated. These genotypes differ significantlyfor panicle exertion, seed set, kernel weight, and grainyield when subjected to pre-anthesis drought stress underfield conditions (Monyo et ai, 1992). In the present

1 To whom correspondence should be addressed. Fax: +1 317 494 0391.Abbreviations: $„ leaf water potential; <j/,, osmotic potential; i ^ o * osmotic potential at full turgor; ^p, pressure potential; AWC, apoplastic watercontent; RWC, relative water content

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1834 Premachandra et al.

study, the two lines were grown in large containers andsubjected gradually to a severe water stress prior topanicle initiation and differentiation. Responses wereassessed at intervals during the dehydration period. Thespecific objective was to test whether these contrastinglines exhibited differences in relative water content, turgor,intracellular osmolarity, cell sap composition, rate ofosmotic adjustment, and/or the effective range of thatadjustment.

Drought resistance in sorghum is a complex traitinfluenced by many genes (Blum, 1979). Variation atnumerous loci probably govern the responses of thegenotypes studied here and this necessarily limits thepower to ascribe agronomic performance to specific meta-bolic traits. Nevertheless, pairs of genotypes that exhibitsimilar phenological development yet strongly contrast-ing responses to drought may offer the opportunity touncover relationships between osmolyte composition, leafwater relations and growth not previously deduced. Suchknowledge may also lead to new means of assessinggenotypic differences in osmotic adjustment.

Materials and methods

Plant materials and growth conditions

Seeds of two lines of Sorghum bicolor (L.) Moench were sownin vermiculite on 11 September 1991. Lines K886 (developedby the Agricultural Research Cooperative of Sudan) andCS3541 (developed by the All India Sorghum Program) areknown to exhibit tolerance and susceptibility, respectively, whensoil water deficits occur during panicle initiation and differenti-ation (Monyo et al, 1992). Seedlings were transplanted 7dlater into 15.2 1 pots containing a 2:2:1 (by vol.) fertilizer-amended mixture of perlite, peat moss and top soil. Growingconditions and soil fertility management were exactly asdescribed by Premachandra et al. (1994).

Fifty-four seedlings of each line were arranged in a completelyrandomized design and grown on benches in the Horticulturegreenhouse at Purdue University, West Lafayette, Indiana,USA, under approximately 28 °C daytime maximum and 23 °Cnight-time minimum temperatures. Forty days after sowing,two levels of irrigation were assigned; these consisted of (1)irrigation to pot capacity every other day and (2) no waterduring a 24 d soil dry-down period. Leaf water relations wereevaluated 15, 20 and 24 d after the treatments were initiated.On each sampling date, five plants of each line of each treatmentwere selected for uniformity of size and stress developmentfrom among the 54 plants available. Sampled plants were underslight water stress on day 15 (^w —1.1 to — 1.4 MPa), moderatestress on day 20 (i/rw - 1 . 6 to —1.9 MPa), and severe stress onday 24 (0W - 2 . 1 to - 2 . 4 MPa).

An additional experiment, grown under identical culturalconditions and subjected to the treatments described above,was carried out to assess the sensitivity of leaf area expansionto water deficit in the two lines. Beginning on the day thatwater was withheld, total leaf area was monitored at 3—4 dintervals in each of five plants per treatment and line. Lengthand width of each leaf were measured, and leaf area wasestimated by multiplying these dimensions by 0.772, a coefficientestablished from an earlier regression analysis of actual projected

area as measured with a LiCor LI-3000A leaf area meter. Leafarea expansion rate (cm2 d"1) was determined for each line,and the percentage reduction in expansion rate due to waterdeficit was calculated.

Leaf water relations

Leaf water potential (</rw) was measured between 10.00 h and11.00 h on each sample date by use of a pressure chamber(PMS Instruments Co., Corvallis, Oregon, USA). Leaves werethen frozen in sealed polyethylene freezer bags. Leaf sampleswere thawed and centrifuged at lOOOxg for 20min at 6-8 CCto extract cell sap, and the osmotic potential (i/i J of the cell sapwas measured using a Wescor Model 5100C vapour pressureosmometer. Cell sap samples were stored in a freezer at — 20 °Cfor subsequent analysis of solutes. 0, was corrected for thedilution of symplastic sap by apoplastic water which occurswhen sap is expressed from frozen and thawed tissue (Tyree,1976); a constant apoplastic fraction of 0.13 was assumed (afterFlower et al, 1990). Turgor was calculated by subtracting thecorrected 0, from if>w. Relative water content (RWC) wascomputed as: 7?ffC=(fresh wt.-dry wt.)/(turgid wt.-dry wt.),using 1 cm diameter discs excised from the third most recentfully expanded leaf. Osmotic potential at full turgor O/ ioo))was calculated as: 4,A]0O) = <px{RWC-AWC)l{\.Q-AWC), whereAWC is the apoplastic water content (Wilson et al, 1979).Osmotic adjustment was evaluated as the difference betweenthe i/vdoo) values estimated in stressed and non-stressed leaves.

Solute analyses

Total sugar concentration in cell sap was determined by theanthrone method (Yoshida et al, 1976). Na + , K+ , Ca2 + , andMg2+ concentrations were assayed on a 1/100 dilution ofextracted cell sap using a Varian Model Spectra AA-10 atomicabsorption spectrophotometer (Varian Techtron Pty. Ltd.,Mulgrave, Victoria, Australia). Total P was determined by themolybdenum blue method (Jackson, 1958). Chloride ions weredetermined by the mercury thiocyanate method (Adriano andDoner, 1982). Total a-amino nitrogen was determined by theninhydrin method (Yemm and Cocking, 1955). Proline wasdetermined by the method of Bates et al. (1973). Glycinebetainewas quantified by stable isotope dilution plasma desorptionmass spectrometry as described by Yang et al. (1995), using2H9-glycinebetaine as internal standard.

Statistical analysis

Leaf water relations variables and solute concentrations wereeach subjected to analysis of variance, and Fisher's leastsignificant difference test was used to test for differences betweenlines, irrigation treatments and stress levels (SAS InstituteInc., 1985).

Results

Leaf water relations

Leaf tfiw measured in stressed plants did not differ signif-icantly between lines on either day 20 or day 24 afterwithholding water (Table 1). However, leaf <fiw wasapproximately 0.15 MPa lower in line CS3541 than inK886 on day 15. Leaf turgor was lower in line CS3541than in K886 on all three sample dates, regardless of thelevel of irrigation. Turgor reached zero in stressed plantsof line CS3541 between day 15 and day 20, but stressed

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Osmotic adjustment in Sorghum 1835

Table 1. Leaf water potential (</rw), turgor (</ip), osmotic potentialat full turgor (iji^ioo)), osmotic adjustment (OA) and leaf relativewater content (RWC) measured in two sorghum lines (K886 andCS3541) under irrigated and non-irrigated conditions atand 24 d after

Days

15 K886

CS3541

20 K886

CS3541

24 K886

CS3541

LSD*

•LSD (0.05)stress levels.

withholding

Treatment

IrrigatedNon-irrigatedIrrigatedNon-irrigatedIrrigatedNon-irrigatedIrrigatedNon-irrigatedIrrigatedNon-irrigatedIrrigatedNon-irrigated

water

(MPa)

-0.89-1.23-0.88-1.38-0.72-1.73-0.87-1.74-0.78-2.32-0.78-2.31

0.15

(MPa)

0.530.220.220.040.710.150.340.000.750.000.410.000.13

« 1 0 0 )(MPa)

—-

—-

.36

.39

.09

.36

.38

.80

.20

.49

.47-2.01-1.12-1.52

0.14

for comparison between lines, irrigation

OA(MPa)

0.03—0.27—0.42—0.29—0.54—0.400.18

15, 20

RWC(%)

0.960.960.990.960.970.960.990.890.960.920.940.700.06

treatments and

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plants of K886 did not lose turgor until between day 20and day 24. In irrigated controls, </ip increased by approxi-mately 0.2 MPa between day 15 and day 24.

"Aifioo) w a s significantly lower in K.886 than in CS3541,regardless of the level of irrigation; the single exceptionto this pattern was for the comparison of stressed plantson day 15. In addition, although <pn(ioo) decreased withincreasing water deficit in both lines, the rate of decreasewas greater in K886 than in CS3541. The extent ofosmotic adjustment, defined as the difference in tfix{ioo)values between irrigated and non-irrigated plants,increased progressively over time in line K886, but nosignificant increase in osmotic adjustment was measuredin CS3541 after day 15. Osmotic adjustment was approxi-mately 0.24 MPa greater in CS3541 than in K886 on day15, but no significant differences were observed betweenlines on either day 20 or day 24.

Leaves of water-stressed plants of line K886 maintainedsignificantly higher RWC than leaves of CS3541 on day20 and day 24. Further, leaf RWC was lower in stressedplants than in controls in line CS3541 on those sampledates, but no differences between irrigation treatmentswere observed for K886 on any sample date.

The effects of </rB on leaf RWC, ipp and </<„<! oc» are shownin Fig. 1. Line K886 maintained substantially higher leafRWC at low tfjw than did CS3541. In addition, ifip wasnot only higher in K886 than in CS3541 at high leaf </-w,but it was also maintained at higher values as soil waterdeficit intensified and leaf i/rw declined. Turgor lossoccurred at </<w values of approximately —1.6 MPa and- 2 . 2 MPa in plants of CS3541 and K886, respectively.>fiMioo) was lower in line K886 than in CS3541 throughoutthe entire range of leaf ipw induced. Although the relationbetween <pna00) and i/«w was linear in K886, a curvilinear

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Leaf water potential (-MPa)

Fig. 1. Leaf relative water content (RWC), turgor pressure (i p) andosmotic potential at full turgor (i^ioo)) expressed as functions of leafwater potential (< w). Variables were measured in leaves of container-grown grain sorghum lines K886 (closed circles) and CS3541 (opencircles) during a 24 d period in which water was withheld. Slopes oflinear regression equations for K886 and CS3541 differed from eachother at /'<0.05 for each of the three variables.

response was noted in CS3541. I/I IOO) decreased in lineCS3541 as leaf <pw dropped to approximately —2.0 MPa,but further reductions in </<w were associated with highervalues of i

Solute analyses

The contributions of individual solutes to total measuredsap osmolality are shown in Table 2 for each of the threesampling dates. Sugars and K+ were the major contrib-utors to solute concentration, and together accountedfor approximately 51%, on average, of measured osmol-ality. Chloride ions and amino acids also contributed

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Table 2. Solute concentrations and their respective contributions to measured osmolality in two sorghum lines (K886 and CS3541)under irrigated (Irri) and non-irrigated (Non) conditions at 15, 20 and 24 d after withholding water

Days afterwithholdingwater

15

20

24

LSD"

K.886

CS3541

K886

CS3541

K886

CS3541

IrriNonIrriNon

IrriNonIrriNon

IrriNonIrriNon

Sugar

180181104147

147221129144

19222011213424

K

116113130137

115146130146

109183103142

11

Ca

18201011

20231312

212621153

Mg

23251517

23291819

233529224

P

43382327

46412928

474429274

Cl

58544653

57724662

5687506013

Aminoacids*

(mmol 1

56573443

61714357

677453757

Proline

• ' )

2.62.62.02.8

2.64.32.18.0

2.910.92.0

15.41.3

Glycine-betaine

15.620.5

3.36.3

19.022.4

5.28.5

20.225.610.610.04.3

Totalsolutes

512511367444

491630415485

538706410500

Saposmolality

550558437548

558728482601

592811452615

59

Contributionto measuredosmolality(%)

93.191.684.181.0

87.986.586.280.6

90.987.090.681.4

•Total amino acids excluding proline.bLSD (0.05) for comparison between lines, irrigation treatments and stress levels.

substantially to total solutes, with their concentrationsaveraging 58.4 and 57.6 mmol I"1, respectively, over lines,treatments and sample dates. Phosphorus, Mg2+ Ca2 + ,glycinebetaine, and proline contributed, on average, 35.2,23.2, 17.5, 13.9, and 4.9 mmol I"1, respectively. Theconcentration of Na+ was the lowest among meas-ured solutes and averaged only 0.45 mmol 1 ~1 (datanot shown).

Higher concentrations of sugars were measured in lineK886 than in CS3541, regardless of treatment or sampledate. Further, for a given sample date, sugars contributeda higher proportion of measured sap osmolality in K886than in CS3541 in both irrigated and stressed plants. Thesingle exception to this pattern was for irrigated plantsmeasured 20 d after treatments were initiated, when sugarscontributed approximately 26% of sap osmolality in bothK886 and CS3541. Potassium ion concentration washigher in stressed plants relative to irrigated controls onday 20 and day 24 after withholding water in both K886and CS3541. Further, K+ accounted for a higher propor-tion of sap osmolality in CS3541 than in K886 in bothirrigated and stressed plants on day 15 and day 20.Differences between lines were not detected on day 24,however, for irrigated plants.

In general, the concentrations of most of the solutesmeasured in stressed plants tended to be higher in K886than in CS3541, although some exceptions exist forparticular combinations of solute and sample date. Thepattern was clear for P, Ca2 + , Mg2"1", and glycinebetainefor all three sample dates. In contrast, the concentrationof amino acids in stressed plants differed between linesonly on day 15 and day 20, while Cl" concentration

differed only on day 24. In marked contrast to all othersolutes, proline concentration was higher in stressed plantsof CS3541 than in K886; significant differences wereevident on day 20 and day 24.

Leaves of line K886 had significantly higher concentra-tions of total solutes than those of CS3541 for nearly allpairwise combinations of treatment and sample date; thesingle exception was for non-irrigated plants on day 15,where the difference between lines was not significant.Further, when water deficit was imposed, K886 showedlarger increases in sap osmolality than did CS3541. Saposmolality increased from 558 to 728 to 811 mmol P 1 ondays 15, 20 and 24, respectively, in line K886, while thecorresponding values for CS3541 were 548, 601 and615 mmol I"1. The additive contributions of individualsolutes accounted for 81-93% of cell sap osmolality.Solutes responsible for the remaining 7-19% of totalosmolality may have included sugar alcohols, organicacids, sulphate and nitrate ions, as well as other quatern-ary ammonium compounds and soluble nitrogenous com-pounds not measured in this study. It is notable that theproportion of sap osmolality accounted for by the nineindividual solutes was 5-10% higher in K886 than inCS3541 for stressed plants.

The relationships between leaf </rw and both sap osmolal-ity and osmotic adjustment are shown for non-irrigatedplants in Fig. 2. Sap osmolality was greater in line K886than in CS3541 throughout the range of 0W induced.Osmolality increased linearly (r = 0.93) in K886 as tpw

declined to —2.4 MPa (Fig. 2A), but the rise measuredin CS3541 was confined to a narrower range of ipw.Although osmolality increased gradually in CS3541 as

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Fig. 2. The relationship of sap osmolarity (A) and osmotic adjustment(B) to leaf water potential. Variables were measured in leaves ofcontainer-grown grain sorghum lines K886 (closed circles) and CS3541(open circles) during a 24 d period in which water was withheld.

leaf i/«w fell to approximately —2.0 MPa, it failed toincrease beyond approximately 650 mmol I"1 as </>w con-tinued to decline. The relation between </>w and osmoticadjustment shown in Fig. 2B indicates that the two linesbehave similarly throughout the range of ipw measured.

The individual solute contributions to osmotic adjust-ment are illustrated in Fig. 3 for each of the threesample dates. On day 15, osmotic adjustment was evidentonly in CS3541, and sugars were the principal solute.Amino acids, K + , Cl~ and P each contributed less than10 mmol I"1 in CS3541. On day 20, sugars and K+ werethe principal solutes contributing to osmotic adjustmentand, in each case, their contribution was significantlygreater in K886 than in CS3541. Amino acids and Cl"were moderately large components of adjustment, but nodifferences between lines were detected. On day 24, sugars,K + , Ca2 + , Nig2"1", Cl", and glycinebetaine each contrib-uted significantly more to osmotic adjustment in lineK886 than in CS3541. In contrast, only amino acids andproline contributed more in CS3541 than in K886. Asubstantial reversal in the relative contributions of sugarsand K+ in K886 occurred as water deficit intensified,with sugars and K+ being the predominant osmotica onday 20 and day 24, respectively.

The relationships between individual solute concentra-

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24 d

X LSD (0.05)

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Fig. 3. Contributions to osmotic adjustment of sugars (Su), K + , Ca2 + ,Mg2*, P, Cr\ amino acids (except proline) (AA), proline (Pr) andglycinebetaine (Gb) measured in the first and second uppermost, fully-expanded leaves of lines K886 and CS3541 at 15, 20 and 24 d afterwithholding water.

tions and i/«w are illustrated for non-irrigated plants ofK886 and CS3541 in Fig. 4. Three general classes ofresponse can be identified. In the first class, the concentra-tion of sugars, Ca2 + , Mg2 + , P, and glycinebetaine wereeach significantly higher in line K886 than in CS3541throughout the entire range of ipw induced by withholdingwater. Within this group, Ca2+ , Mg2 + and glycinebetaineconcentrations increased in parallel for the two lines aswater deficit intensified; sugar and P concentrations alsoincreased with stress in K886, but they remained constantin CS3541. In the second class of response, solute concen-trations either diverged or converged for the two lines astpv decreased. This was the case for Cl", where concentra-

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Fig. 4. Concentrations of sugars, Ca2 + , Mg^ + , P, glycinebetaine, Cl , K + , amino acids (except proline) and proline expressed as functions of leafwater potential (< w) in leaves of non-irrigated, container-grown sorghum lines K886 (closed circles) and CS3541 (open circles) during a 24-d periodin which water was withheld.

tion did not differ between lines at high </iw but increasedfaster in K886 as </>w fell. The converse was observed foramino acids. K + concentration increased sharply in K886as water deficit increased, but it remained nearly constantin CS3541; concentration in CS3541 exceeded that inK.886 at high values of i/iw, but the reverse was true at i/rwvalues below approximately —2.0 MPa. In the third class,proline concentration was higher in line CS3541 than inK886. Although no difference between lines was observedat high i/«w, proline increased more rapidly in CS3541 as0W declined below approximately —1.6 MPa.

Leaf area expansion

Line CS3541 exhibited a greater percentage reduction intotal leaf area expansion rate relative to K886 duringeach of four arbitrarily-chosen growth intervals betweenday 9 and day 21 (Table 3). For example, between days9 and 12, water deficit caused a 38.2% and 15.7% reduc-tion in the area expansion rate of CS3541 and K886plants, respectively. Values for percentage reduction intotal leaf area expansion rate were greater than 100%between days 19 and 24. This reflects reduction in wholeplant leaf area due to senescence of lower leaves.

Table 3. Water stress-induced reduction (%) in leaf area expan-sion rates during various growth intervals throughout a 24 dperiod in which water was withheld from container-grown plantsof lines K886 and CS3541

Reduction in rates of leaf expansion are given as percentages ofirrigated controls for whole-plant leaf area and for the top three leaves.Values are means of five replicates.

Growth interval(days after withholding water)

0-45-89-12

13-1516-1819-2122-24

Percentage reduction in leafexpansion rate

Whole-plant

K886

1.10.4

15.724.168.0

126.8192.4

CS3541

1.85.2

38.283.180.9

192.8181.8

Top three leaves

K886

1.24.7

21.616.357.884.990.0

CS3541

5.44.2

29.242.365.492.098.2

Leaf area expansion rate of the top three leaves showeda similar differential sensitivity. Between days 9 and 12,the expansion rate of the uppermost leaves of draughtedCS3541 plants was reduced by 29.2%, relative to irrigated

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controls while K886 was reduced by only 21.6%. Thedifference between lines was larger during the nextinterval (days 13 through 15), when CS3541 and K886exhibited stress-induced reductions of 42.3% and 16.3%,respectively.

Discussion

A drought-tolerant grain sorghum line (K886) main-tained significantly higher leaf R WC and turgor pressurethan did a drought-susceptible line (CS3541) when thetwo genotypes were subjected to severe water stress duringpanicle initiation and differentiation (Fig. 1). As waterdeficit intensified, plants of line K886 exhibited a muchsmaller reduction in leaf water content per unit decreasein </iw than did CS3541 (Table 1), and they maintainedhigher leaf turgor at equivalent values of leaf waterpotential higher than — 2.2 MPa (Fig. 1). Leaf areaexpansion was much less severely inhibited by water stressin K886 than in CS3541 (Table 3).

The results for these container-grown plants are consist-ent with a report by Monyo et al. (1992), demonstratingthat K886 and CS3541 exhibit strongly contrasting beha-viour in response to field drought stress. When plantsgrown under a rainout shelter were subjected to anirrigation regime that caused severe plant water deficit todevelop during the panicle initiation and differentiationstages of growth, K886 and CS3541 exhibited reductionsin grain yield of 46.6% and 74.4%, respectively, from thatmeasured in irrigated control plots. In addition, theauthors reported significant differences in 100 kernelweight between lines, with K886 and CS3541 attainingkernel weights of 3.25 g and 2.86 g, respectively.

At any point in time, prevailing solute levels can beconsidered as the product of basal osmotic potential (i.e."Anfioo)) of non-stressed leaves and those solutes whichhave accumulated in response to dehydration or osmoticstress (Morgan, 1984). Both factors appear to playimportant roles in the genotypic differences in leaf waterrelations observed here. The basal i/i^oo) measured inirrigated plants was significantly lower in line K886 thanin CS3541 at each of the three sample dates (Table 1). Inaddition, the level of sap osmolarity was greater in lineK886 than in CS3541 throughout the entire range of </>w

induced (Fig. 2). With the exception of proline, theconcentrations of each of eight solutes were higher inK886 than in CS3541 under water-limited conditions(Fig. 4). Finally, when water deficit was imposed, K.886exhibited larger increases in sap osmolality than didCS3541. In particular, it is notable that the concentrationsof K+ , sugars, Cl~ and P (predominant solutes contribut-ing to osmotic adjustment) increased with increasingstress in K886, but essentially remained constant inCS3541.

The drought-tolerant and -susceptible lines exhibit large


differences in the relative contributions of individualsolutes to osmotic adjustment, and these contributionschange markedly during stress development both withinand between lines (Fig. 3). The most notable differencesbetween the genotypes were with respect to the contribu-tions of sugars and K + ions. In general, sugars con-tributed more to osmotic adjustment during the earlydevelopment of stress, while K + ions accounted for anincreasingly larger fraction of adjustment as the waterdeficit intensified. Similar large increases in sugar concen-tration were noted by Munns et al. (1979) for wheatleaves, where initial drought-induced reductions in </>,,were largely attributable to soluble carbohydrates. Thelower contribution of sugars to osmotic adjustment onday 24 probably reflects inhibition of photosynthesis bylow water potential.

The highly intercorrelated nature of the individualsolute responses shown in Fig. 4 suggests that control ofipK involves tight co-ordination of numerous metabolicprocesses. The results could be interpreted by postulatingeither close linkage of many genes controlling the accumu-lation of particular solutes, or the existence of one ora few regulatory genes that govern overall <(iK. A thirdalternative is that a relatively small set of genes specifiesproduction of one or more key osmolytes, while the levelsof other solutes are governed by pleiotropic effects ofthese genes. In this regard, it is notable that the two linesdiffer significantly in basal levels of glycinebetaine andthat the high-betaine line (K886) had much higher solutelevels overall than did the low-betaine line (CS3541).Grumet and Hanson (1986) reported a very similar resultfor barley isopopulations differing for glycinebetaine con-tent; the authors suggested that betaine levels were con-trolled by 'osmoregulatory' genes with pleiotropic effects,whose alleles govern i/>x. They proposed that betaine couldact as a marker for genetic differences in i/iK, a suppositionwhich is also supported by evidence of such genes inwheat (Morgan, 1983).

These results provide evidence of a primary role ofpotassium in the observed differences in osmotic adjust-ment between these two sorghum genotypes. Jones et al.(1980) showed that K + was the major cation contributingto osmotic adjustment in sorghum, and Morgan (1992)reported that wheat lines exhibiting high osmotic adjust-ment did so largely (78%) through K + accumulation. Theresults from the present study are remarkable, however,in the qualitatively different response between the twolines. K + concentration approximately doubled in leafsap of K886 as </iw declined to —2.4 MPa, but its concen-tration in CS3541 remained virtually unchanged (Fig. 4).The basis for this difference is not known, but it suggeststhe presence of a constitutively expressed trait in CS3541that is incapable of adjusting to limited soil moisture incontrast with an inducible and highly stress-responsivesystem of K + accumulation in K886. It would be useful

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to ascertain rates of K+ uptake by roots of these geno-types and to evaluate their relative sensitivity to soil waterdeficit. A stress-induced reduction in K+ uptake, forexample, could diminish stomatal responsiveness to leafwater deficit.

The response of proline to soiJ drought was uniqueamong the solutes analysed. Both its contribution toosmotic adjustment and its rate of increase with decreas-ing ipv, were greater in the drought-susceptible line CS3541than in the tolerant line K886 (Fig. 4). This result isconsistent with the report by Hanson et al. (1979), whichconcluded that differences in proline accumulation rateamong barley genotypes could be accounted for by differ-ences in leaf water status. Hanson (1980) observed higherproline accumulation in more drought-susceptible barleycultivars and concluded that this was associated with amore rapid decline in </iw. It seems likely that the higherrate of proline accumulation measured in CS3541 relativeto K886 is related to the slightly earlier drop in tpw

observed in this line as well as to the lower leaf watercontent that ensued as stress intensified.

The extent of osmotic adjustment was similar in thetwo lines until </rw fell below —1.8 MPa (Fig. 2). At lowervalues of tpw, osmotic adjustment was only 0.13 to0.14 MPa greater in K886 than in CS3541 (Table 1),suggesting that the substantial differences noted for R WC,leaf turgor and leaf expansion do not derive entirely fromosmotic adjustment per se. Improved water status in K886relative to CS3541 at low </iw may have been related, atleast in part, to its significantly higher constitutive levelsof glycinebetaine. A compatible osmolyte, glycinebetaineis thought to confer osmoprotection by facilitating theformation of a hydration sphere at protein surfaces which,in turn, stabilizes their native structures (Rhodes andHanson, 1993). It may also be involved in the stabilizationof membranes through its interaction with phosphatidyl-choline moieties (Rudolph et al., 1986). Because glycine-betaine is predominantly localized in the cytoplasm(especially in chloroplasts), the concentrations inferredhere (Fig. 4) greatly underestimate the levels in intactorganelles. ChJoroplasts of salinized spinach plants, forexample, contain up to 0.3 M glycinebetaine (Robinsonand Jones, 1986). Thus, although glycinebetaine is aminor component of overall osmotic adjustment in thesesorghum lines, it may be contributing significantly tochJoroplast osmotic adjustment such that volume andphotosynthetic function are maintained at low </<w.

In growing leaves, a drought-induced decrease in therate of tissue volume expansion would reduce the rate ofdilution of cell solutes. Leaf cells might simultaneouslyincrease their rate of synthesis or uptake of osmoticum.Either of these events would be interpreted as osmoticadjustment. The latter mechanism could contribute togrowth maintenance (or at least could slow the rate ofreduction) at low </rw. In this regard, it is interesting to

note that line K886, which exhibited larger increases insap osmolarity when water deficit was imposed, alsoshowed significantly less reduction in leaf area expansionbetween days 5 and 15 than did CS3541 (Table 3). Thissuggests that the solutes that contributed to osmoticadjustment in K886 were not simply diverted from cellexpansion, but rather were the result of an active mechan-ism of solute accumulation. The possibility can not beruled out, however, that solute import may have beenless inhibited in K886 than in CS3541 as growth wasslowed; in this case, the accumulated solutes would stillhave been diverted from cell expansion.

Sorghum lines K886 and CS3541 present a remarkablearray of contrasting behaviours in response to soildrought. Hybrids of these two lines have been constructed,and a large number of F6 recombinant inbred (RI)families have been derived from the cross. Future workwill utilize the existing sorghum restriction fragmentlength polymorphism (RFLP) genetic map (Hulbert et al.,1990) to screen these RI families for RFLPs which maybe associated with specific drought resistance/suscept-ibility traits. The capacity to accumulate K+ ions andto minimize stress-induced reductions in water content,turgor and leaf area expansion appear to be usefulcandidate traits for the screening programme and forfuture efforts to map the major genes responsible fordrought resistance in sorghum.

Acknowledgements

This research was supported by the McKnight FoundationInterdisciplinary Research Project in Plant Biology. This isJournal Paper No. 14 227 of the Purdue University AgriculturalExperiment Station. We thank Dr Gebisa Ejeta for suggestingthe comparison of these genotypes.

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