REGULAR ARTICLE

Contrasting responses to drought stress in herbaceousperennial legumes

Jiayin Pang & Jiyun Yang & Phil Ward &

Kadambot H. M. Siddique & Hans Lambers &

Mark Tibbett & Megan Ryan

Received: 4 March 2011 /Accepted: 5 July 2011 /Published online: 15 July 2011# Springer Science+Business Media B.V. 2011

AbstractBackground and aims Medicago sativa L. is widelygrown in southern Australia, but is poorly adapted todry, hot summers. This study aimed to identifyperennial herbaceous legumes with greater resistanceto drought stress and explore their adaptive strategies.Methods Ten herbaceous perennial legume species/accessions were grown in deep pots in a sandy, low-phosphorus field soil in a glasshouse. Drought stresswas imposed by ceasing to water. A companion M.sativa plant in each pot minimised differences in leafarea and water consumption among species. Plantswere harvested when stomatal conductance of stressedplants decreased to around 10% of well watered plants.

Results A range of responses to drought stress wereidentified, including: reduced shoot growth; leafcurling; thicker pubescence on leaves and stems; anincreased root:shoot ratio; an increase, decrease or nochange in root distribution with depth; reductions inspecific leaf area or leaf water potential; and osmoticadjustment. The suite of changes differed substantiallyamong species and, less so, among accessions.Conclusions The inter- and intra-specific variabilityof responses to drought-stress in the plants examinedsuggests a wide range of strategies are available inperennial legumes to cope with drying conditions,and these could be harnessed in breeding/selectionprograms.

Plant Soil (2011) 348:299–314DOI 10.1007/s11104-011-0904-x

Responsible Editor: Caixian Tang.

J. Pang (*) : P. Ward :H. Lambers :M. RyanSchool of Plant Biology,The University of Western Australia,35 Stirling Highway,Crawley, WA 6009, Australiae-mail: Jiayin.Pang@uwa.edu.au

J. YangInstitute of Grassland Science,Key Laboratory of Vegetation Ecology,Ministry of Education, Northeast Normal University,Changchun 130024, China

J. Pang : P. WardCSIRO Plant Industry,Centre for Environment and Life Sciences,Private Bag No. 5,Wembley, WA 6913, Australia

J. Pang :K. H. M. Siddique :H. Lambers :M. RyanThe UWA Institute of Agriculture,The University of Western Australia,35 Stirling Highway,Crawley, WA 6009, Australia

M. TibbettSchool of Earth and Environment,The University of Western Australia,35 Stirling Highway,Crawley, WA 6009, Australia

M. TibbettNational Soil Resources Institute,Department of Environmental Science and Technology,Cranfield University,Bedfordshire, England, UK

Keywords Drought . Morphology . Osmoticadjustment . Perennial legumes . Photosynthesis .

Root distribution .Water potential

Introduction

South-western Australia has a typical Mediterraneanclimate, characterised by hot, dry summers that restrictplant growth. In such regions, the replacement of deep-rooted native perennial species by annual crop andpasture species has led to rising water tables and drylandsalinity (Dear and Ewing 2008; Lambers 2003) and,thus, to a call for greater inclusion of perennials intofarming systems (Cocks 2001; Ward et al. 2006). Inrecent decades, the perennial pasture legume lucerne(Medicago sativa L.) has been more widely sown insouth-western Australia (Cocks 2001). However, it ispoorly adapted to the dry, hot summers in areas with<350 mm average annual rainfall (Loo et al. 2006) andto acidic sandy soils (Humphries and Auricht 2001).Furthermore, reliance on a small diversity of specieswithin agricultural systems is considered inadequate torespond to the multitude of environmental conditionspresent (Dear et al. 2008). So, there is an urgent need toexpand the range of perennial legumes that grow wellin areas with <350 mm average annual rainfall and canbe included in grazing systems where lucerne is eitherpoorly adapted or has reached the limits of adaptation.The provision of out-of-season (summer/autumn) greenfeed from these novel perennial legumes in a perma-nent grazing situation or in rotation with crops wouldprovide considerable impetus for adoption.

A large research effort has recently evaluated novel,including undomesticated, native and exotic perennial

legumes as alternatives to lucerne in south-westernAustralia and across Australia (Bell et al. 2006; Li etal. 2008; Snowball et al. 2010). Traits viewed asfavourable in novel germplasm include better droughtresistance than lucerne and tolerance of low-nutrient,especially low-phosphorus, soils. Novel species, espe-cially Australian natives in the genera Cullen and theexotic Bituminaria bituminosa var. albo-marginatahave performed favourably (Bennett et al. 2011; Panget al. 2010a; Pang et al. 2010b; Suriyagoda et al. 2010).There are few detailed studies on the response ofherbaceous perennial legumes to drought stress. Thepresent glasshouse study examined the plant growthresponse to an imposed drought stress under low-Pconditions of a range of perennial legumes with potentialfor use in Mediterranean pasture systems: B. bituminosavar. albomarginata (3 accessions), C. australasicum (2accessions), C. pallidum (1 accession), C. cinereum (1accession), Macroptilium atropurpureum (1 cultivar),Kennedia prostrata (1 accession) and M. sativa(lucerne, 1 cultivar). The specific aims were to (1)identify species with greater tolerance of drought stressthan M. sativa, (2) establish whether variation inresistance to drought stress is present among accessionsof B. bituminosa var. albomarginata and C. austral-asicum, and (3) identify the range of strategiesemployed by the plants to cope with drought stress.

Material and methods

Plant material and growth conditions

Plants (See Table 1 for species details) were grown in19.1-L free-draining PVC pots (1 m deep and 16 cm

Table 1 Species and varietal names, geographic origin and rhizobial inoculants of perennial legumes used in this study

Species, authority and accession number Geographic origin Rhizobiumstrain number

Bituminaria bituminosa var. albomarginata (L.) C.H. Stirt, accessions 6, 10 and 22 Canary Islands, Spain WSM4083

Cullen australasicum (Schltdl.) J.W. Grimes, accessions SA44239 and SA42762 Australia RRI2780

Cullen pallidum (N.T.Burb.) J.W.Grimes SA41740 Australia RRI2770

Cullen cinereum (Lindl.) J.W.Grimes AusTRCF320100 Australia RRI2832

Macroptilium atropurpureum (DC.) Urb. cv Aztec America CB756

Kennedia prostrata R.Br JUP001 Australia RRI2353

Medicago sativa L. cv SARDI 10 Asia minor, Iran, Turkmenistanand Transcaucasia

RRI128

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diameter). Each pot was cut lengthwise on both sidesand securely taped. Pots were filled with yellowloamy sand soil which was air-dried and passedthrough a 2-mm sieve. Soil analyses on subsamplesof soil were conducted by CSBP FutureFarm analyt-ical laboratories (Bibra Lake, Australia). The loamysand contained 1 μg g−1 of nitrate-N, 1 μg g−1 ofammonium-N, 1 μg g−1 of bicarbonate-extractable P,and had a pH (CaCl2) of 5.6 and a phosphorus-retention index of 4.6. Thus, the soil was extremelylow in N and P and had a high P-retention index. Ineach pot, 400 g of coarse gravel was placed at thebottom and 15 kg of air-dried soil, without extranutrient supply, was added above the gravel. Another12 kg of soil mixed thoroughly with basic nutrientswas then added on the top. Soil in the pots had a bulkdensity of 1.41 gcm−3. The basic nutrients providedwere 126.6 mg kg−1 Ca(NO3)2.4H2O, 42.8 mg kg−1

NH4NO3, 43.9 mg kg−1 KH2PO4, 178 mg kg−1

K2SO4, 101 mg kg−1 MgSO4.7H2O, 11 mg kg−1

CaCl2.2H2O, 12 mg kg−1 MnSO4.H2O, 8.8 mg kg−1

ZnSO4.7H2O, 1.96 mg kg−1 CuSO4.5H2O,0.68 mg kg−1 H3BO3, 1.01 mg kg−1 NaMoO4.2H2Oand 32.9 mg kg−1 FeNaEDTA. Thus, only 10 mg Pkg−1 dry soil was provided in order to mimic a low Pagricultural soil, while providing adequate supply ofother nutrients. The provision of N, as a mixture ofNH4NO3 and Ca(NO3)2, was to ensure an initialadequate supply of N after germination, prior tonodulation by rhizobia.

Before planting, seeds were scarified and pre-germinated in Petri dishes at staggered times accord-ing to their pre-determined germination time. Threegerminated seedlings were planted in each pot andthinned to one plant after 1 week. In each pot, threeM. sativa seedlings were planted at the same time onthe other side of the pot, and later thinned to one plantafter 1 week. The purpose of planting lucerne in eachpot was to minimise differences in leaf area betweenpots and thus, once watering ceased, to cause eachspecies, irrespective of plant size, to experience asimilar degree of drought stress. All seedlings wereinoculated with an appropriate strain of rhizobiumprovided by the Rutherglen Centre, Department ofPrimary Industries, Victoria, Australia (Table 1). Theexperiment was carried out in a naturally littemperature-controlled glasshouse at The Universityof Western Australia, Perth, Australia. The tempera-ture in the glasshouse ranged from 17°C to 35°C in

January 2008 and from 12°C to 28°C in June 2008,and relative humidity was maintained at ~70% duringthe experiment period.

For each species/accession, 12 pots were set up.Four months after planting, just before drought stresswas imposed, four replicate pots of each species/accession were harvested. Of the eight remainingpots, four continued to be well watered while water-ing ceased for the other four pots. When originallyfilling pots with soil, in some pots (2 per species/accession per treatment, 40 pots in total), twofrequency domain reflectometer (FDR) probes (ModelCS615, Campbell Scientific Ltd, Logan, Utah, USA)were installed vertically between 0.05 and 0.35 m and0.5–0.8 m from the top to obtain hourly changes involumetric soil water content. The FDR sensorconsists of two stainless steel rods (0.3 m long,0.0032 m in diameter, 0.032 m spacing). For eachspecies, all pots were watered to field capacity everyother day as determined by the change in watercontent in the two pots containing the FDR probes.All pots were watered to field capacity on theafternoon of 11 May 2008; watering was then stoppedfor the drought-stressed plants. Well watered controlpots continued to be watered to field capacity.

Physiological measurements

Predawn leaf water potential was measured (0300–0500 h) in a pressure chamber (Soil moisture EquipmentCorp., Santa Barbara, CA, USA) on petioles of youngfully expanded leaves. The same leaf samples wereimmediately placed in a water-tight vial, snap frozen inliquid N2 and stored in a freezer at −20°C. Sampleswere later thawed, sap expressed using a leaf press, andsap osmolality measured using a freezing pointosmometer, which was calibrated against 50 and850 mOsm kg−1 standard solutions (Fiske Associates,Norwood, MA, USA). Osmotic potential (Ψπ, MPa) ofsamples was then calculated from osmolality (Eq. 1).

yp ¼ 2:447� osmolality=1; 000 ð1Þ

Osmotic adjustment was calculated as the differ-ence in Ψπ at full turgor (Ψπ 100; Eq. 2; i.e. 100%relative leaf water content) between drought-stressedand well watered plants according to the method ofLudlow et al. (1983). Relative leaf water content wasdetermined as outlined by Turner (1981). Whole

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leaves were removed adjacent to those used for waterpotential measurements and fresh weight (Leaf FW)measured immediately. Turgid weight (Leaf TW) wasmeasured after whole leaflets were floated on deion-ised water overnight (approximately 16 h) in a Petridish placed in a laboratory with ambient air temper-ature of 25°C. Dry weight (Leaf DW) was measuredafter samples were dried in an oven at 70°C for2 days.

Ψp100 ¼ Ψp � Leaf FW � Leaf DWð Þ= Leaf TW � Leaf DWð Þð2Þ

Measurements of gas exchange on the youngestfully expanded leaves were carried out between 0900and 1200 h using a LICOR–6400 with red/blue LEDlight source (LI-COR, Lincoln, NE, USA). Photosyn-thetic photon flux density at the leaf surface was set at1,500 μmol m−2 s−1, leaf temperature at 25°C, flowrate at 500 μmol s−1 and ambient CO2 concentrationof incoming gas stream at 380 μmol mol−1.

Plant analyses

After drought stress was imposed, all plants wereharvested when stomatal conductance of the youngestfully expanded leaf in the drought-stressed plants wasaround 10% of that of well watered plants, as stomatalconductance was used as an integrative parameterreflecting the drought stress experienced by plants(Medrano et al. 2002). Hence plants of each specieswere harvested at different times, but when thedrought-stressed plants were showing a similar levelof stress reaction.

At harvest, the tape was removed and the pots splitopen which allowed root distribution to be examinedwith minimal soil disturbance. Roots in each pot werewashed carefully in a water tub and separatedcarefully from the top of roots into the legume ofinterest and M. sativa. The root system was then laiddown on a flat surface and divided into 0.1 msegments. Shoots were separated from roots at thecrown and partitioned into leaves and stems (includ-ing petiole). The shoots and roots were dried at 70°Cfor 72 h and dry weight (DW) was measured. Greenleaf area was determined using a WinRHIZO scannerand software (Regent Instruments Inc., Quebec City,QC, Canada). Specific leaf area was calculated asgreen leaf area per unit dry weight (m2 kg−1).

Statistical analysis

The experiment was a two-factorial (species/accessionand water treatment) randomised complete blockdesign. Data for growth and other parameters wereanalysed by general analysis of variance (ANOVA) inGenstat version 13.1 (Lawes Agricultural Trust,Rothamsted Experimental Station, UK, 2007).

Results

Soil water depletion and time for final harvestafter drought stress

At the start of drought stress, soil water content wasmaintained at around 12% (v/v) in all pots (Fig. 1).The imposition of drought stress reduced soil watercontent continuously in all drought-stressed pots(Fig. 1). Fourteen days after drought stress wasimposed, water content was reduced to 6–8% (v/v)

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Fig. 1 Volumetric soil water content in pots with drought-stressed plants after cessation of watering. Note, not allstandard errors are presented to avoid crowding. Data for eachspecies/accession are the average of soil moisture data from thetop and bottom of pots installed with moisture probes. Speciesabbreviations are BB (B. bituminosa var. albomarginata), CA(C. australasicum), CC (C. cinereum), CP (C. pallidum), KP(K. prostrata), MS (M. sativa), MA (M. atropurpureum)

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in all species/accessions. After that, soil water contentonly decreased further in M. atropurpureum, resultingin the lowest soil water content (~4% (v/v)) at harvest.During this period, soil water content in all wellwatered pots was maintained at ~12% (v/v) (data notshown).

The time of final harvest after drought stressdiffered among species/accessions as it occurred whenstomatal conductance of the drought-stressed plantswas reduced to ~10% of that of the well wateredplants. B. bituminosa var. albomarginata accession 6and accession 10, C. australasicum SA42762, C.pallidum, C. cinereum, K. prostrata and M. sativawere harvested 16 days after drought stress wasimposed, C. australasicum SA44239 and M. atropur-pureum were harvested 21 days after drought stresswas imposed and B. bituminosa accession 22 washarvested 35 days after drought stress was imposed.

Phenological difference of species/accessions

There were differences in phenology among species/accessions at the start of drought stress. Whilstexperiencing the drought stress, some species/acces-sions were vegetative, e.g. all accessions of B.bituminosa var. albomarginata and K. prostrata,while the remaining species were flowering and/orsetting seeds.

Shoot and root dry biomass

Initial shoot DW, prior to the imposition of droughtstress, differed among species (Fig. 2a, Table 2). Atthis time, both accessions of C. australasicum, alongwith C. pallidum and M. atropurpureum had muchmore shoot DW than M. sativa, while only M.atropurpureum had more root DW than M. sativa(Fig. 2b). B. bituminosa var. albomarginata accession22 had the lowest shoot DW among the species. Therewas a significant interaction between species/acces-sions and water treatment at final harvest (Fig. 2a,Table 2). After drought stress was imposed, B.bituminosa var. albomarginata accession 22 and M.atropurpureum gained little shoot DW in both wellwatered and drought-stressed plants (Fig. 2a). In thesespecies, drought-stressed plants had similar shootDWs to well watered plants at the final harvest. Littleshoot DW was also gained in drought-stressed plantsof B. bituminosa var. albomarginata accession 6 and

M. sativa, while shoot DW increased in well wateredplants until final harvest. Shoot DW increased indrought-stressed plants of B. bituminosa var. albo-marginata accession10 and both C. australasicumaccessions after watering ceased, but increased morein well watered plants. For C. cinereum, C. pallidumand K. prostrata, shoot DW of drought-stressed plantscontinued to increase after drought stress was im-posed and these species had similar shoot DWs indrought-stressed plants and well watered plants at thefinal harvest.

Initial root DW varied greatly and there was asignificant interaction between species/accessions andwater treatment at final harvest (Fig. 2b, Table 2).After drought stress commenced, root DW changedlittle in B. bituminosa var. albomarginata accessions 6and 22, C. cinereum, C. pallidum and K. prostrata. Incontrast, root DW continued to increase after droughtstress was imposed in B. bituminosa var. albomargi-nata accession 10, but it remained less than the wellwatered plants at the final harvest. Root DW ofdrought-stressed plants was close to that of wellwatered plants for both accessions of C. austral-asicum, along with M. sativa and M. atropurpureum(Fig. 2b).

Root:shoot ratio at final harvest

Root:shoot ratio and its response to drought stressvaried greatly among species at the final harvest(Fig. 2c). There was a significant interaction betweenspecies/accessions and water treatment (Table 2,Fig. 2c). M. sativa and M. atropurpureum had thehighest root:shoot ratios (> 1.75), while that of theother species varied between 1.35 and 0.25. Somespecies/accessions, e.g. B. bituminosa var. albomargi-nata accession 22, both accessions of C. austral-asicum and, most notably, M. sativa, increased theirroot:shoot ratio in response to drought stress. Incontrast, root:shoot ratio decreased in B. bituminosavar. albomarginata accession 10 and C. cinereum inresponse to drought stress (Fig. 2c).

Root distribution down the profile at final harvest

Normalised root DW (percentage of total root drybiomass present in each depth increment of the pot) atthe final harvest was affected by the interaction ofspecies/accessions, root depth and water treatment

Plant Soil (2011) 348:299–314 303

(Fig. 3, Table 2). For well watered plants, B.bituminosa var. albomarginata accession 22, M.sativa and M. atropurpureum had 53–62% of rootsin the top 30 cm and thus the proportion of root DWdecreased with depth. In B. bituminosa var. albomar-ginata accessions 6 and 10, both accessions of C.australasicum and C. cinereum, roots were distributedrelatively evenly with depth. For K. prostrata, roots inthe top 15 cm contributed~35% of total root DW,with roots distributed evenly across other depths. C.pallidum also had ~35% of roots in the top 15 cm,

with the proportion of root DW then decreasing withdepth before increasing at 90–105 cm (Fig. 3). Inresponse to drought stress, the proportion of roots atgreater depths increased for C. australasicum acces-sion SA44239, C. pallidum and, most notably, for B.bituminosa var. albomarginata accession 22 where theproportion of root DW at 90–105 cm changed from 3to 23% although the absolute root biomass of thisaccession remained very low. The proportion of rootsat shallow depths increased for B. bituminosa var.albomarginata accession 10 and, in particular, for C.

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Fig. 2 Shoot dry weight(a), root dry weight (b) androot/shoot ratio (c) at thestart of drought stress (whitebars), in well watered plants(grey bars) and drought-stressed plants (black bars)at final harvest. Data aremeans ± s.e. (n=4). Speciesabbreviations are BB (B.bituminosa var. albomargi-nata), CA (C. australasicum),CC (C. cinereum), CP (C.pallidum), KP (K. prostrata),MS (M. sativa), MA(M. atropurpureum)

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cinereum (28 to 45%) in response to drought stress;there was little change for the remaining species(Fig. 3).

Green leaf area, senesced leaves and leaf morphologyat final harvest

For green leaf area and the percentage of senescedleaf to total leaf DW at final harvest, there wassignificant interaction between species/accessions andwater treatment (Fig. 4, Table 2). For well wateredplants, C. cinereum had the lowest green leaf area(~100 cm2), followed by B. bituminosa var. albomar-ginata accession 22 and M. sativa. The leaf area ofthe other species/accessions ranged from 540 to1235 cm2. In response to drought stress, green leaf

area decreased in all species except M. sativa and M.atropurpureum (Fig. 4a). All species under droughtstress increased the percentage of senesced leaf tototal leaves (Fig. 4b). The highest percentage of leafsenescence in drought-stressed plants occurred in K.prostrata and the lowest for M. sativa and M.atropurpureum. A relatively high percentage of leafsenescence was also observed in well watered B.bituminosa var. albomarginata accession 22 and C.cinereum.

A number of leaf morphological responses to waterdeficit were observed and are summarised in Table 3.Specific leaf area (average of all green leaves onplants) varied among legume species in response todrought stress (Fig. 4c, Table 2). In well watered plants,K. prostrata and M. sativa had the highest specific leafarea, while C. cinereum and C. pallidum had the lowest(Fig. 4c). In response to drought stress, both accessionsof C. australasicum and C. pallidum had ~30% lessspecific leaf area while M. sativa had 17% less; theother species did not show a response. Thick stands ofleaf hair were observed on stems and leaves of M.atropurpureum and C. pallidum which were enhancedin the drought-stressed treatment. In all Cullen species,inward leaf curling was observed after drought stress.In M. sativa, leaflets folded to form a cup.

Physiological adaptations

The effect of drought treatment on pre-dawn leafwater potential at final harvest differed amongspecies/accessions (Fig. 5a, Table 2). Leaf waterpotential was reduced most by drought stress, beingless than −2.5 MPa, in both C. australasicumaccessions, along with C. pallidum, K. prostrata andM. sativa, while it remained higher than −1.0 MPa inB. bituminosa var. albomarginata accession 6 and 10,and M. atropurpureum.

The effect of drought treatment on osmotic potentialat final harvest also differed among species/accessions(Fig. 5b, Table 2). Osmotic potential was reducedgreatly in response to drought stress in all speciesexcept C. cinereum and M. atropurpureum. In drought-stressed plants, osmotic potential was lower than−3.0 MPa in C. australasicum SA44239 and K.prostrata, maintained at around −1.0 MPa in C.cinereum and M. atropurpureum and ranged from−2.0 to −3.0 MPa in the remaining species. The leafwater potential of all species/accessions except B.

Table 2 Significance of different sources of variability for arange of parameters

Variable Sp W Sp*W

Shoot dry weight (start of drought stress) *** – –

Shoot dry weight (final harvest) *** * *

Root dry weight (start of drought stress) *** – –

Root dry weight (final harvest) *** ** *

Root:shoot ratio (final harvest) *** * ***

Green leaf area *** *** ***

Specific leaf area *** ** *

Percentage of senesced leaf to total *** *** ***

Leaf water potential *** *** ***

Osmotic potential *** *** ***

Osmotic potential at full turgor *** *** ***

Photosynthetic rate (start of drought stress) *** – –

Photosynthetic rate (7 d after drought stress) *** n.s. ***

Photosynthetic rate(14 d after drought stress)

*** *** ***

Photosynthetic rate(18 d after drought stress)

** ** ***

Photosynthetic rate(21 d after drought stress)

*** *** ***

Sp D W

Normalised root DW (percentage of totalroot dry biomass present in each depthincrement of the pot)

n.s. *** n.s.

Significant effects are indicated for species/accessions (Sp),water treatment (W), root depth (D) and their interactions (n.s.,no significant difference; *, P<0.05; **, P<0.01; ***, P<0.001); For normalised root DW, there were significantinteraction of Sp*D (P<0.001), D*W (P<0.01) and Sp*D*W(P<0.001) while no significant interaction of Sp*W

Plant Soil (2011) 348:299–314 305

bituminosa var. albomarginata accessions 6 and 10, andM. atropurpureum was close to, or lower than, theosmotic potential, which indicated that leaf turgor wasclose to zero or lost at the final harvest (Fig. 6a and b).

The effect of drought treatment on osmoticpotential at full turgor at the final harvest differedamong species/accessions (concentration effects onchanges in leaf osmotic potential were removed by

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C. australasicumSA42762 C. australasicum

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K. prostrataM. sativa

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Fig. 3 Effect of water availability on the normalised root dryweight (percentage of total root dry biomass present in eachdepth increment of pot) in well watered plants (grey bars) and

drought-stressed plants (black bars) at final harvest. Data aremeans ± s.e. (n=4)

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presenting osmotic potential at full turgor) (Fig. 5c,Table 2). Osmotic potential at full turgor was clearlylower in drought-stressed than well watered plantsof B. bituminosa var. albomarginata accessions 6and 10, both C. australasicum accessions and K.prostrata.

Photosynthetic responses

Prior to imposition of drought stress, photosyntheticrate varied among species (Fig. 6, Table 2) with the

highest rates (~25 μmol m−2 s−1) recorded for allCullen species/accessions (Fig. 6). At all subesquentdates, there was a significant interaction betweenspecies/accession and water treatment (Table 2). Uponimposition of drought stress, C. pallidum, M. sativaand K. prostrata quickly, i.e. within 7 days, reducedtheir photosynthetic rate (Fig. 6). In contrast, M.atropurpureum and B. bituminosa var. albomarginataaccession 22 maintained their pre-stress photosyn-thetic rate for 14 and 18 days, respectively. The otherspecies showed an intermediate response.

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800

1000

1200

1400

1600

BB-6 BB-10 BB-22 CA 42762

CA 44239

CC CP KP MS MA

BB-6 BB-10 BB-22 CA 42762

CA 44239

CC CP KP MS MA

BB-6 BB-10 BB-22 CA 42762

CA 44239

CC CP KP MS MA0

5

10

15

20

25

30

35

40

b

c

a

Gre

en le

af a

rea

per

plan

t(cm

2 )P

erce

ntag

eof

sen

esce

d le

af to

tota

l S

peci

fic le

af a

rea

(m2

kg-1

)

Fig. 4 Green leaf area (a),the percentage of senescedto total leaf dry weight (b),and specific leaf area (c) inwell watered plants (greybars) and drought-stressedplants (black bars) at finalharvest. Data are means ± s.e. (n=4). Species abbrevia-tions are BB (B. bituminosavar. albomarginata), CA(C. australasicum), CC (C.cinereum), CP (C. pal-lidum), KP (K. prostrata),MS (M. sativa), MA (M.atropurpureum)

Plant Soil (2011) 348:299–314 307

Tab

le3

Sum

maryof

morph

olog

ical

andph

ysiologicalrespon

sesto

drou

ghtstress;high

lighted

speciesexhibitedagreaterability

tomaintainph

otosyn

thesis

afterim

positio

nof

drou

ghtstress

Species

Shoot

dry

weightpriorto

droughtstress

Leafmorphological

response

Proportionof

leaves

senesced

atharvest

Daysto

photosynthesis

decline

Shoot

grow

thrate

Harvest

day

afterWSa

Root:shoot

ratio

Specificleaf

area

Leafwater

potential

Osm

otic

adjustment

B.bituminosavar.

albomarginata

accession6

low

leaves

wilted

from

the

botto

mof

theplantand

flattened

medium

9decrease

16no

change

nochange

high

yes

B.bituminosavar.

albomarginata

accession10

low

asabove

medium

9decrease

16reduced(increasein

rootsat

surface)

nochange

high

yes

B.bituminosavar.

albomarginata

accession22

very

low

asabove

medium

18decrease

35increased(increase

inrootsat

depth)

nochange

low

no

C.australasicum

SA42762

medium

leaves

wilted

from

the

botto

mof

theplant,

curled,then

droppedfrom

thebotto

mof

theplant

medium

7decrease

16increased

muchreduced

very

low

yes

C.australasicum

SA44239

high

medium

9decrease

21increased(increase

inrootsat

depth)

muchreduced

very

low

yes

C.cinereum

low

asabove

high

7no

change

16reduced(increasein

rootsat

surface)

nochange

low

no

C.pallidum

high

asabove

low-m

edium

1–7

nochange

16no

change

(increase

inrootsat

depth)

muchreduced

low

no

K.prostrata

low

dead

leaves

still

attached

tothestem

atharvest

very

high

1–7

nochange

16no

change

nochange

very

low

yes

M.sativa

low

leafletsform

edacupand

didnotdrop

off

immediately

low

1–7

decrease

16very

high,then

increased

reduced

low

no

M.atropurpureum

medium

leaves

yello

wed

from

the

botto

mof

theplantand

laterdroppedoff

low-m

edium

14no

dryweightgain

ineither

treatm

ent

21very

high,no

change

nochange

high

no

aStomatal

cond

uctanceof

drou

ght-stressed

plantsarou

nd10

%of

that

ofwellwatered

controlplants

308 Plant Soil (2011) 348:299–314

Discussion

The 10 perennial legume species/accessions in thepresent study showed large and contrasting differ-ences in growth, and physiological and morphologicaltraits, when exposed to drying soil under conditionsof low available soil P. The implications of thesediverse responses, and other major findings of thisstudy, are discussed below.

Physiological adaptations to drought stress

Osmotic adjustment is a commonly reported responseto drought stress. The accumulation of osmoticallyactive compounds such as sucrose, glucose, fructose,potassium and chloride ions lowers the osmoticpotential of cells which in turn allows the mainte-nance of full or partial turgor pressure at a low leafwater potential (Morgan 1984). In our study onlysome species showed osmotic adjustment, that is, B.bituminosa var. albomarginata accessions 6 and 10,both C. australasicum accessions and K. prostrata.Differences in osmotic adjustment among species ofperennial legumes were also reported by Bell et al.(2007), who observed an accumulation of osmoticsolutes in Dorycnium hirsutum and D. rectum inresponse to drought stress, but no accumulation in M.sativa. However, others did report that osmoprotec-tants accumulated in leaves and phloem of M. sativaexposed to drought stress (Girousse et al. 1996;Irigoyen et al. 1992). Bell et al. (2007) hypothesisedthat the absence of osmotic solutes in M. sativa wasdue to the rapid development of drought stress (seeTurner and Jones 1980) or reflected genetic differ-ences among M. sativa cultivars. In the present study,relatively rapid soil water depletion due to the sandysoil and large plant size when watering ceased mightbe the reason for limited osmotic adjustment in somelegume species/accessions.

The ability to maintain leaf turgor which may beaided by osmotic adjustment, is considered importantfor maintaining stomatal conductance and photosyn-thesis under low soil water potential (Jones et al.1980). For instance, leaf turgor was maintained untilfinal harvest in M. atropurpureum which may havecontributed to its relatively long period of continuingphotosynthesis (18 days after the imposition ofdrought stress). Although B. bituminosa var. albo-marginata accessions 6 and 10 also maintained leaf

turgor until final harvest, stomatal conductancedecreased dramatically and these accessions did notmaintain a high photosynthetic rate (i.e. there was arapid reduction of photosynthesis 9 days after thesupply of water ceased). As the leaf water potential inthese two accessions was maintained at a high level,we hypothesise that leaves of these two speciestended towards being isohydric through a strongfeedforward stomatal control, which allowed mainte-nance of a high leaf water potential at a low soil watercontent (Lambers et al. 2008). Abscisic acid (ABA) isa chemical signal to control stomatal behaviour, anddifferences between species/accessions in mainte-nance of photosynthesis under drought stress mightbe due to differential sensitivity of stomata to ABA(Lambers et al. 2008). C. australasicum accessionSA44239 also continued to photosynthesise, albeit ata low rate, at very low leaf water potential. In thisaccession, osmotic adjustment would have assistedthe maintenance of very low leaf water potentials. Incontrast, Bell et al. (2007) found that osmoticadjustment had little effect on maintaining leafphotosynthesis or transpiration in both D. hirsutumand M. sativa, as it occurred when leaf turgor was lostand later than the reduction of stomatal conductanceand photosynthesis.

Shoot and root morphological adaptations to droughtstress

Morphological adaptations to drought stress wereobserved in some species/accessions. For instance,a thicker layer of white leaf hairs was observed onstems and leaves of M. atropurpureum and C.pallidum. Leaf hairs may help to reflect solarradiation, thus increasing resistance to transpiration-al water loss and moderating leaf temperature(Ehleringer and Björkman 1978; Grammatikopouloset al. 1994). In addition, leaf curling (cupping) wasobserved in all Cullen species and M. sativa. Such aphenomenon has been recorded previously in pe-rennial legumes in response to reduced leaf waterpotential (Bell et al. 2007; Travis and Reed 1983).Cupping also reduces light interception by leavesthrough reducing the angle at which leaves areexposed to direct solar radiation (Koller 1990). Leafhairs and leaf curling also reduce transpiration ratesby increasing the thickness of the laminar layer overthe leaf.

Plant Soil (2011) 348:299–314 309

Root systems can show great plasticity in responseto environmental stress, and an increase in root massratio is a typical response to drought (Gregory et al.1996). In the present study, M. sativa, B. bituminosavar. albomarginata accession 22 and both accessions

of C. australasicum increased root:shoot ratio inresponse to drought stress. The increased root:shootratio in these species/accessions was due to rootbiomass in drought-stressed plants accumulating atthe same rate as the well watered plants, while shoot

-4.0

-3.0

-2.0

-1.0

0.0

-4.0

-3.0

-2.0

-1.0

0.0

-1.6

-1.2

-0.8

-0.4

0.0

Leaf

wat

er p

oten

tial (

MP

a)O

smot

ic p

oten

tial (

MP

a)O

smot

ic p

oten

tial a

t ful

l tur

gor

(MP

a)

a

b

c

Fig. 5 Predawn leaf waterpotential (a), osmoticpotential (b), and calculatedosmotic potential at fullturgor (c) in leaves of wellwatered plants (grey bars)and drought-stressed plants(black bars) at final harvest.Data are means ± s.e. (n=4).Species abbreviations areBB (B. bituminosa var.albomarginata), CA (C.australasicum), CC(C. cinereum), CP (C.pallidum), KP (K.prostrata), MS (M. sativa),MA (M. atropurpureum)

310 Plant Soil (2011) 348:299–314

growth was reduced. Skinner and Comas (2010)present similar results in temperate forage speciesunder drought stress. M. sativa has also been shownin two soil types to have consistently lower rootdevelopment under drought stress, associated with ahigher root:shoot ratio, compared with favourableconditions when grown in large boxes with thedimension of 55 cm×12 cm×75 cm (Annicchiarico2007). The high root:shoot ratio of M. sativa wasmainly due to the presence of a large tap-root whichcould have a major function as a storage organ. Anincreased root:shoot ratio is presumably advantageousbecause it enhances soil resource acquisition, but itdoes so at the expense of photosynthetic carbon gain(Ho et al. 2005). As a result, a carbon cost forincreased allocation to roots could ultimately reduceplant growth (Nielsen et al. 2001).

Deep root systems may allow plants to maintainwater uptake as top soil dries (Ho et al. 2005). In thisstudy, all perennial species/accessions reached thebottom of the pots, i.e. 1.05 m. However, there werelarge differences in the distribution of root systemsdown the profile among species/accessions, with rootsof some species (e.g. B. bituminosa var. albomargi-

nata accessions 6 and 10, both accessions of C.australasicum and C. cinereum) being evenly distrib-uted and those of other species (e.g. B. bituminosavar. albomarginata accession 22, M. sativa and M.atropurpureum) having a high concentration in thetopsoil. In response to drought stress, only three outof 10 species/accessions, C. australasicum accessionSA44239, C. pallidum and most particularly, B.bituminosa var. albomarginata accession 22, in-creased the proportion of roots at depth. In the presentstudy, pots dried out throughout the profile duringdrought stress. Suriyagoda et al. (2010) reported thatC. australasicum accessions SA 42762 and SA4966did not enhance the proportion of deep roots whenpots dried throughout the profile, while the proportionof deep roots increased when only the top half of thepots dried while the bottom half was kept moist.These authors used washed river sand, which has alower water-holding capacity than the field soil usedin the present study, so pots dried out even morequickly. Skinner and Comas (2010) found that in 50-cm deep pots drought stress had no effect on theproportion of deep roots in legumes and forbs, butsignificantly increased it in grasses.

Pho

tosy

nthe

tic r

ate

(µm

ol m

-2s-1

)

-7 1 7 9 11 14 18 21 25 29 32

-7 1 7 9 11 14 18 21 25 29 32

BB-6 BB-10 BB-22 CA 42762 CA 44239

0

5

10

15

20

25

30

35

CC CP KP MS MA

0

5

10

15

20

25

30

Days after start of water stress (d)

Fig. 6 Photosynthetic ratebefore (−7 days) and after(1–32 days) the start of thedrought-stress treatment.Data are means ± s.e. (n=4).Species abbreviations areBB (B. bituminosa var.albomarginata), CA (C.australasicum), CC(C. cinereum), CP(C. pallidum), KP (K.prostrata), MS (M. sativa),MA (M. atropurpureum)

Plant Soil (2011) 348:299–314 311

Two species (B. bituminosa var. albomarginataaccessions 10 and, in particular, C. cinereum)increased the proportion of roots in the top soil inresponse to drought stress. In this study, sincenutrients were only supplied in the top 40 cm of soil,to mimic a field situation, and P supply was low, anincreased proportion of roots in the top soil may havehelped plants acquire more nutrients under droughtstress, especially relatively immobile nutrients such asP. Root foraging in top soil is considered an effectivestrategy to acquire P from low-P soils as P levels intop soil are generally higher due to decaying litter,higher organic matter and microbial activity (Ho et al.2005; Lynch and Brown 2001). Top soil root foragingfor P has been reported in a similar set of perenniallegumes (Denton et al. 2006; Suriyagoda et al. 2010)and further study is warranted on the interactionsbetween low-P and drought stress.

Root system responses probably also contributedto drought-stressed and well watered plants of C.pallidum and K. prostrata accumulating approximatelythe same amount of shoot DW by final harvest inspite of photosynthetic rate decreasing immediatelyupon drought stress being imposed. Both thesespecies accumulated little additional root dry weightafter drought stress was imposed, thus maybe wereable to invest more C in shoot growth for bothdrought-stressed and well watered plants. In addi-tion, a range of changes associated with theimposition of drought stress then allowed shootgrowth to continue under water limitation untilharvest. For C. pallidum an increased proportion ofroots at depth may have enhanced access to soilwater at depth, and a large reduction in leaf areaprobably reduced water loss. For K. prostrata,osmotic adjustment may have helped plants toextract water from the soil and adjust their waterpotential. In addition, K. prostrata had an initiallyhigh leaf area ratio (the ratio of leaf area and totalplant weight) before the initiation of drought stressand a high specific leaf area, which might havecontributed to its relatively high accumulation ofshoot biomass during drought stress and high shootDW of this species at final harvest. These results areconsistent with those of Poorter and Remkes (1990)who found a very strong positive correlation betweenrelative growth rate and leaf area ratio, and betweenrelative growth rate and specific leaf area in 24herbaceous C3 species.

Variation among accessions

A key finding from the present experiment was differ-ences between accessions of both B. bituminosa var.albomarginata and C. australasicum. For example, B.bituminosa var. albomarginata accessions 6 and 10 inresponse to drought stress accumulated osmotic solutesand maintained a higher leaf water potential at finalharvest than accession 22. Instead, accession 22increased root:shoot ratio and the proportion of rootsdeep in the profile. Whilst differences betweenaccessions within species were generally not as markedas differences between species, it may be possible tomatch them with field performance and hence theycould provide useful information for future selection/breeding programs.

Extrapolating results to field conditions

Some caution is necessary when using the results ofthis glasshouse experiment to predict responses in afield setting. In the field, drought stress may beimposed more slowly and last for longer, and roots ofwell-established plants may have access to stores ofwater deep in the soil profile. In addition, in the fieldplants may be preconditioned by experiencing periodsof milder drought prior to such a serious period ofdrought stress. Additionally, climatic conditions in thegreenhouse may differ substantially from the field,particularly in terms of wind. Also, for a perennialpasture on a commercial farm, in addition to theimportant issue of ability to grow and providelivestock feed under dry conditions which wasassessed in the current experiment, the ability tosurvive an extended dry period is also crucial. Thiswas not assessed in this experiment.

Conclusions

The present glasshouse study has shown that novelperennial legumes use a range of quite contrastingmorphological and physiological strategies, whichoften differ from those used by M. sativa, to copewith drought stress. The companion M. sativa plantsin each pot in this study minimised the difference insoil water reduction. M. atropurpureum extractedmore water from the soil and achieved the lowestsoil water content at harvest of all species/accessions.

312 Plant Soil (2011) 348:299–314

Leaf adaptations upon drought stress were observedin all four Cullen species and in M. sativa (leafcurling), and in M. atropurpureum and C. pallidum(denser layers of long trichomes on stems and leaves).B. bituminosa accession 22, both accessions of C.australasicum and M. sativa increased their root:shootratio in response to drought stress. Leaf osmoticadjustment in response to drought stress was observedin B. bituminosa var. albomarginata accessions 6 and10, both accessions of C. australasicum and K.prostrata. Leaf water potential was maintained at ahigh level in B. bituminosa var. albomarginata acces-sions 6 and 10 and M. atropurpureum. The inter- andintra-specific variability of responses to drought-stressin the plants examined here suggests a wide range ofstrategies in perennial herbaceous legumes to copewith drying conditions. These findings offer greatencouragement for the development of new perenniallegume forages for a wide range of drought-proneagricultural regions in breeding/selection programs.

Acknowledgements This work was funded by the AustralianResearch Council (ARC), Rural Industries Research and Devel-opment Corporation (RIRDC), the Department of Agriculture andFoodWestern Australia (DAFWA), Heritage Seeds, the ChemistryCentre of Western Australia, and the Facey Group and MingenewIrwin Group. We thank Dr Daniel Real (DAFWA), Dr MatthewDenton (DPI, Victoria) and Mr Richard Bennett (UWA) whoprovided legume seeds and rhizobia, and Mr. Darryl McClements(DAFWA) who provided technical help.

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