Annals of Applied Biology ISSN 0003-4746

R E S E A R C H A R T I C L E

Drought resistance and recovery in mature Bituminariabituminosa var. albomarginataK. Foster1,2,3, H. Lambers1, D. Real1,2,3, P. Ramankutty1,2, G.R. Cawthray1 & M.H. Ryan1,2

1 School of Plant Biology and Institute of Agriculture, The University of Western Australia, Perth, Australia2 Future Farm Industries Cooperative Research Centre, The University of Western Australia, Perth, Australia3 Department of Agriculture and Food Western Australia, South Perth, Australia

KeywordsAlfalfa; climate change; osmotic adjustment;paraheliotropism; plant adaptation; Psoraleabituminosa; rehydration; water-use efficiency.

CorrespondenceK. Foster, School of Plant Biology and Institute ofAgriculture, The University of Western Australia,35 Stirling Highway, Crawley (Perth) WA 6009,Australia. Email: kevin.foster@agric.wa.gov.au

Received: 30 March 2014; revised versionaccepted: 10 September 2014.

doi:10.1111/aab.12171

Abstract

Few studies have investigated the response of perennial legumes to droughtstress (DS) and their ability, following rewatering, to regrow and restorephotosynthetic activity. We examined these responses for two genotypes ofdrought-tolerant tedera (Bituminaria bituminosa var. albomarginata) and onegenotype of lucerne (Medicago sativa). Plants were grown outdoors in 1-mdeep PVC pots with a reconstructed field soil profile, regularly watered for8 months (winter to mid-summer), and then moved to a glasshouse whereeither watering was maintained or drought was imposed for up to 47 days,before rewatering for 28 days. Drought stress greatly decreased shoot drymatter (DM) production in both species. Lucerne plants showed severe leafdesiccation after 21 days of withholding water. Relative leaf water content(RWC=42%) and midday leaf water potential (LWP=−6.5 MPa) decreasedin tedera in response to DS, whereas leaf angle (85∘) and lateral root DMboth increased. Proline and pinitol accumulated in tedera leaves during DS,and their concentration declined after rewatering. Nine days after rewatering,previously drought-stressed tedera had similar RWC and LWP to well-wateredcontrol plants. In tedera and lucerne, 28 days after rewatering, photosynthesisand stomatal conductance were greater than in the well-watered controls. Thelateral root DM for one tedera genotype decreased during the recovery phasebut for lucerne, the lateral root DM did not change during either the droughtor the recovery phases. Overall, the root systems in tedera showed greaterplasticity in response to DS and rewatering than in lucerne. In conclusion,tedera and lucerne showed different physiological and morphological strategiesto survive and recover from DS. Proline and soluble sugars may act as acarbon source for regrowth in tedera during recovery. In comparison withlucerne, tedera’s more rapid recovery after rewatering should contribute to agreater aboveground DM yield under alternating dry and wet periods. Tederagenotypes are highly heterogeneous and selecting genotypes with enhancedconcentrations of pinitol and proline could be a valuable tool to improve plantperformance during DS and recovery.

Introduction

There is a need in Australia and elsewhere to develop

pasture plants and grazing systems to address the issue

of climate change (Cullen et al., 2009). It is critical to

understand the potential role perennial plants can play

in a changing climate, and develop systems that candeliver sustainability and, importantly, profitability forthe livestock industries (Nie, 2011). However, with thefluctuations in rainfall patterns predicted from climatechange, there may also be an increase in the occurrenceof episodic drought (IPCC, 2007) where plants will be

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Drought resistance and recovery in tedera K. Foster et al.

repeatedly exposed to drought in the field. While droughtresistance and recuperative potential are known to varywithin several perennial grass species (Su et al., 2008), thecombined responses to water stress and rewatering amongperennial legumes is relatively unknown (Filippou et al.,2011).

Lucerne (Medicago sativa L.) is a widely grown peren-nial legume in southern Australia and is considered tohave strong drought avoidance due to its deep rootingsystem (Li et al., 2010); it survives prolonged drought bylimiting aboveground growth and often sheds its leaves(Loo et al., 2006). While lucerne does have the abilityto respond to summer/autumn rainfall (Moore et al.,2006), the uncertainty of summer rainfall events makesit difficult to predict lucerne growth in any season andto match growth with the demand of livestock (Revellet al., 2012). However, there remains a strong demand fornew summer-active perennials and the need to maintainnitrogen fixation has focused attention on the inclusionof perennial legumes rather than perennial grasses (Dearet al., 2003).

Tedera (Bituminaria bituminosa (L.) C.H. Stirton var.albomarginata) is a promising fodder species and hasrecently been introduced into Australia for evaluationas a pasture legume species in Mediterranean climates(Real et al., 2009). Tedera is native to Lanzarote, CanaryIslands, Spain, where it is found at low altitude and inareas with an annual rainfall of 300 mm, a Mediter-ranean rainfall pattern, a 5–6 month dry season (Realet al., 2009), and high amounts of sunshine and hightemperatures which when combined with strong winds,result in very high evaporation rates (Díaz, 2004). Tederais a drought-resistant perennial legume (Pang et al., 2011;Suriyagoda et al., 2013) and compared to lucerne, isphysiologically better adapted to water deficit, retainingmore of its leaves and having a higher leaf water-useefficiency in summer (Foster et al., 2013). However, thedrought-resistance mechanisms enabling tedera to growin the harsh habitat of the Canary Islands are not known,nor has the recuperative potential of this promisingfodder species, once drought stress (DS) is relieved, beeninvestigated.

Species differences in drought resistance and thedegree of recovery from drought have been associatedwith various physiological, morphological and biochem-ical factors including changes in root distribution, rootdiameter, osmotic adjustment (OA) and accumulationof sugars and organic solutes (DaCosta & Huang, 2006).However, evergreens like tedera that endure severewater stress over summer may also rely on physiologicalchanges that are more permanent, which may prohibitrapid recovery from DS (Mittler et al., 2001). Droughtstress can also result in permanent dysfunction of some

vessels via tyloses or resins that are released in responseto DS (Chen et al., 2010).

Rapid and complete photosynthetic recovery followingrewatering is likely the key to prevent significant declinesin crop yield following episodic drought (Chaves et al.,2009). There is anecdotal information from the field thatthe photosynthetic potential of tedera might be enhancedfollowing rewatering after drought, and so help compen-sate for the loss of dry matter (DM) production due todrought. A better understanding of the mechanisms ofboth drought resistance and recovery following rewater-ing in tedera will allow for specific traits to be targetedin selection/breeding programmes prior to the release ofthe first tedera cultivar and enhance the potential for theadoption of this species into low-medium-rainfall pasturesystems.

We conducted a glasshouse experiment to test ourhypotheses: (1) the accumulation of compatible sugarsand proline will increase in tedera during DS and thendecrease during recovery; (2) the area-based rate of pho-tosynthesis of tedera is enhanced following rewateringafter DS; (3) tedera will exhibit superior recuperativepotential compared with lucerne after DS and rewatering;and (4) the roots of tedera will show greater plasticity inresponse to DS and rewatering than those of lucerne.

Materials and methods

This experiment comprised three drought treatmentsapplied to mature plants of lucerne and two geno-types of tedera. The two tedera genotypes differed ingrowth habit (tedera 4 is an erect genotype and ted-era 6 is a semi-prostrate genotype) and were the mostdrought resistant of seven genotypes in field experimentsat Newdegate (Latitude: 33 06.56 S Longitude: 118 50.15E), Western Australia (K. Foster, personal observation).The legume species used for comparison was lucerne(SARDI TEN) which, where suited, it is highly produc-tive, relatively drought resistant and persistent, and it isthe standard by which other perennial legumes are oftencompared (Dear et al., 2003; Pang et al., 2011; Suriyagodaet al., 2013).

Soil was obtained from a field site at Newdegate inthree layers (0–40, 40–80, 80–100 cm) at the beginningof autumn in April 2009 from a dry profile. This soilrepresents a major soil type of agricultural importancein south-western Australia (Moore et al., 1998). Soil wasmixed completely within each layer, passed through a5-mm sieve and stored dry. This site has a texture-contrastsoil with coarse sand over an alkaline clay loam at80–100 cm. Bulked analyses were performed on thesesamples of soil by CSBP FutureFarm analytical labora-tories (Bibra Lake, Western Australia). In the 0–40 cm

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layer, pH (CaCl2) was 5.5 and the soil chemistry (mg kg−1

dry soil) was 27 for bicarbonate-extractable phosphorus(P; Colwell, 1963), 34 for mineral nitrogen (N), 47 forbicarbonate-extractable potassium (K) and 15.1 for sul-phur (S). In the 40–80 cm soil layer, pH (CaCl2) was 6.2and the soil contained 19 mg kg−1 bicarbonate-extractableP, 7 mineral N, 22 bicarbonate-extractable K and 1.7 S. Inthe 80–100 cm soil layer, pH (CaCl2) was 8.0 and the soilcontained 5 mg kg−1 bicarbonate-extractable P, 9 mineralN, 85 bicarbonate-extractable K and 5.6 S.

The three soil layers that were collected from the fieldsite were later reconstructed in polyvinyl chloride (PVC)cylindrical pots (15 cm diameter, 100 cm high), whichwere closed at the bottom with a solid plastic cap withtwo holes to allow free drainage of water. Each pot wascut lengthwise up to 80 cm on both sides and tapedwith waterproof 50 mm wide tape. Filter paper (WhatmanNo.5) was placed over the holes in the bottom of the pot,and pots were filled with 29.1 kg of dry soil. The soil waspacked in 20-cm layers to a bulk density similar to thatat the field site (approximately 1.40 g cm−3). There wasno significant difference among species in the pot weightbefore or after water was added (data not shown).

Lucerne seeds were scarified with fine sandpaper (P240grit) to overcome seed-coat impermeability. Seeds of ted-era were scarified by cutting the outer seed coat usinga surgical scalpel blade. On 28 May 2009, seeds werepre-germinated in 90-mm Petri dishes with wet filterpaper (Whatman No.1). Seedlings were then transplantedinto plastic pots (Track PK48 clear) with a commercial pot-ting mix [Waldecks premium potting mix, pH (CaCl2) 6]and transferred to a controlled-temperature glasshouse at20/12∘C (day/night) for 4 weeks. Rhizobia were appliedat 0.50 g per tray to the soil surface and then tap water wasapplied [lucerne, AL Group (strain RRI128) from Nodu-laid at Becker Underwood; tedera, WSM4083 from theCentre for Rhizobium Studies, Murdoch University].

All pots were watered to pot capacity (approximately11% v/v) and allowed to drain for 48 h. One seedlingwas transplanted into each pot on 29 June 2009 (earlywinter). Plants then grew in the open air and werewatered each week if necessary. Plants were fertilised inOctober and November with the equivalent of 150 kg ha−1

superphosphate and potash at a 3:1 ratio. Plants were notcut. Hence, plants were grown under close to the usualenvironmental conditions for an establishing perenniallegume pasture for 8 months from winter to mid-summer.

All pots were moved on 12 January 2010 (mid-summer) into a naturally lit glasshouse (to avoid sum-mer rainfall events) at the Department of Agriculture andFood Western Australia (DAFWA) with temperature setat 30∘C/15∘C (day/night), which is the average Januarytemperature for Newdegate, in the southern wheatbelt

of Western Australia (B.O.M., 2008). Plants were allowedto acclimate in the glasshouse for 14 days before impos-ing the treatment, and during this time they were rewa-tered to pot capacity on 12, 19 and 26 January. Bothtedera genotypes and lucerne plants were flowering andsetting seeds while plants were outside. After the lastwatering, drought was imposed in the DS treatments, thatis, no more watering occurred, while the well-watered(WW) control continued to be watered. The impositionof drought in mid-summer mimics what may be expectedto occur in the field.

The experiment was a randomised complete blockdesign of genotype (tedera 4, tedera 6, lucerne) bydrought (DS, WW) by harvest (Treatments 1, 2 and 3).Plants in treatment 1 were harvested after 33 days ofdrought (Fig. 1, Harvest 1). Plants in treatment 2 wereharvested after 47 days of drought (Harvest 2). Days 0–47are therefore referred to as the ‘drought phase’. Plants intreatment 3 were rewatered back to pot capacity after 47days of drought, and rewatered every seven days for thefollowing 28 days before being harvested at day 75 (Har-vest 3). Days 48–75 are therefore referred to as the ‘recov-ery phase’. Well-watered control plants were wateredfrom large plastic containers adjusted to glasshouse tem-perature (i.e. cold tap water was not added to plants) topot capacity once a week and harvested at the same timeas the plants in the DS treatment (i.e. days 33, 47 and75). There were four replicates. Plants in treatment 1 were

Figure 1 Experimental design (one of four replicates shown). Plants wereeither well watered throughout the 75 days of the experiment or experienceddrought for up to 47 days, before being rewatered for 28 days. Plants inharvest 1 (H1) were harvested after 33 days of drought; plants in harvest 2(H2) were harvested after 47 days of drought and plants in harvest 3 (H3)were harvested after 47 days of drought followed by a recovery phase of 28days of weekly rewatering.

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harvested on 27 February and those in treatment 2 on 12March 2010. All plants in the DS treatment 3 and theirWW controls were rewatered back to pot capacity on 12March, and harvested on 12 April 2010. For leaf angle,water potential (pre-dawn and midday), leaf RWC, netphotosynthesis, stomatal conductance, OA and compat-ible solutes and sugars, readings were taken at the startof the treatments (i.e. day 1 of the drought phase). Sub-sequent readings, unless stated otherwise, were taken atregular intervals for treatment 3 plants only.

Measurements

Plant water use

To determine plant water use, all pots were weighed onday 1 (26 January), and every 7 days thereafter, with aspecifically designed pot crane fitted with an electronicbalance.

Photosynthesis and instantaneous leaf water-use efficiency

Net photosynthesis (A) and stomatal conductance (gs)were measured on youngest fully expanded leavesbetween 09:00 h and 12:00 h using a LI-COR 6400portable gas exchange system (LI-COR, Lincoln, NE,USA) with a red/blue LED light source, at a photosyn-thetically active radiation (PAR) of 1500 μmol quantam−2 s−1, a CO2 concentration of 380 μmol mol−1 and aleaf chamber air flow rate of 200 μmol s−1, with blocktemperature set at 30∘C. The humidity of the air cominginto the leaf chamber was kept the same as that in theglasshouse. Plants were measured on days 1, 7, 16, 31and 47 of the drought phase, and then on days 21 and28 of the recovery phase. Leaflets of the trifoliates wereseparated and placed in the standard 6 cm2 cuvette;care was taken to keep the leaf angle close to the in situorientation. Leaves that were folded or rolled due toDS were gently unfolded before measuring. After mea-surement, each leaf was imaged and the leaf area wasdetermined; this was used to calculate A and gs. Intrinsicleaf water-use efficiency (WUEL) was calculated as A/gs

(Ahmadi and Siosemardeh, 2005). Some readings forstomatal conductance, and therefore for WUE, occurredon plants at permanent wilting point (PWP) and thesereadings were therefore zero.

Relative leaf water content, pre-dawn and midday leaf waterpotential

Relative leaf water content (RWC) of fully expandedleaves was calculated as RWC (%)= (FW – DW)/(SFW –DW)×100, where FW is fresh weight, DW is dry weightand SFW is saturated fresh weight. Leaves were removed

(days 1, 7, 14, 30 and 47 of the drought phase and days9, 21 and 28 of the recovery phase) between 12:00 hand 14:00 h and placed in zip-lock plastic bags and thenon ice. Fresh weight was recorded, then the leaves wereimmersed in a 90-mm Petri dish filled with deionisedwater for 24 h at room temperature (20–25∘C) beforereweighing to attain saturated fresh weight (Turner,1981). Turgid leaf weight was measured and leaveswere oven-dried at 80∘C for 48 h to determine dryweight.

Pre-dawn leaf water potential (LWPP) (03:00–05:00 h)and midday leaf water potential (LWPM) (12:00–14:00 h)were measured in a pressure chamber (Scholander Model1002, PMS Instruments, Corvallis, OR, USA) on petiolesof young fully expanded leaves. Pre-dawn LWP was mea-sured for all plants on day one and treatment 3 plantswere measured on days 7, 16 and 23 of the drought phase.Subsequent LWP readings were taken at midday on days30 and 47 of the drought phase, and days 9, 21 and 28 ofthe recovery phase. The pressure chamber used allowedmeasurements to −7 MPa.

Photosynthetically active radiation

Photosynthetically active radiation was measured at thelevel of the terminal trifoliate leaflet of the 2nd or 3rduppermost leaves (two leaves per plant) from treatment3 on 24 February at 12:00–15:00 h (leaf elevation angleswere at maximum for tedera genotypes) with clear skiesand 36∘C outside using a LI-192 Underwater QuantumSensor [Model UWQ 7534, (LI-COR) calibration date 25March 2008] set for reading in air with a LI-1400 datalogger unit (LI-COR). Care was taken to keep the sensorhead parallel to the leaf in its normal orientation.

Osmotic adjustment

Leaf samples were removed at noon (days 1, 7, 31and 47 of the drought phase, and days 21 and 28of the recovery phase) and immediately placed in awater-tight vial, snap frozen in liquid N2 and stored in a−80∘C freezer. Samples were later thawed, sap expressedusing a leaf press, and sap osmolality measured using afreezing point osmometer, which was calibrated against50 and 850 mOsm kg−1 standard solutions (Fiske Asso-ciates, Norwood, MA, USA). Osmotic potential (OP, MPa)of samples was then calculated from osmolality OP as2.447× osmolality/1000. Data on RWC were used to con-vert the OP at the given water content to that at 100%RWC. Osmotic adjustment was calculated as the differ-ence in OP at full turgor (OP 100; i.e. 100% leaf RWC)between DS and WW plants, according to the method ofLudlow et al. (1983).

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Compatible solutes and soluble sugars

Leaf samples within treatment 3 were removed on threeoccasions; pre-drought, day 47 of the drought phase andday 28 of the recovery phase. On day 47, lucerne leaveswere desiccated and could not be sampled. Leaf sampleswere immediately placed in a water-tight vial, snap frozenin liquid N2 and stored in a freezer at −20∘C. Sampleswere placed in a lyophiliser for 72 h (Labconco FreezeDry System, Model LyphLock 12, Kansas City, MO, USA)directly from the −20∘C freezer; samples did not thawbefore lyophilisation. Leaves were ground (60 s) in amodified coffee grinder (Sunbeam Autogrinder ModelEM0415, China) to a fine powder, collected into a 2 mLEppendorf tube and stored at −20∘C.

Ethanol extraction of compatible solutes, soluble sugarsand sugar alcohols from plant tissues

Samples were brought to room temperature and 20 mgwere placed into a 1.5 mL screw-top Eppendorf tube.Samples were extracted twice with 1 mL of 80% (v/v)ethanol (99.5% analytical grade) by incubating at 80∘Cin a water bath for 20 min. Tubes were centrifuged(Model: Biofuge 13, Heraeus Instruments, Thermo Elec-tron Corporation, Langenselbold, Germany) for 20 min at13 793 g. The supernatants were combined in a 2 mL vialand the extract stored at −20∘C. The pellet was dried at60∘C for 24 h and the dry weight measured to calculatethe ethanol-insoluble weight.

The initial HPLC analysis of compatible solutes(glycinebetaine, proline, proline betaine and hydrox-yproline), soluble sugars (fructose, glucose and sucrose)and sugar alcohols (pinitol, sorbitol and mannitol) wasadapted from Slimestad & Vågen (2006). The HPLCsystem (Waters, Milford, MA, USA) consisted of a 600Epump, 717 plus autosampler and a 996 photo-diodearray detector (PDA). As detection of fructose, glucoseand sucrose with the PDA at 195 nm is very insensitive, anAlltech (Deerfield, IL, USA) evaporative light-scatteringdetector (ELSD) was used to improve sensitivity by a min-imum of 100-fold. Separation was achieved at 22± 1.0∘Con a Prevail ES Carbohydrate column (250× 4.6 mmi.d. with 5 μm packing; Alltech) using a gradient elutionprofile of acetonitrile (Eluent A) and water (Eluent B)at 1 mL min−1. Samples in the autoinjector were held at10∘C, the ELSD drift tube was held at 85∘C and eluentnebulisation with high purity nitrogen gas was at a flowrate of 2.6 L min−1.

Quantification based on peak area for compatiblesolutes used the PDA, while peak area from the ELSDwas used for soluble sugars and sugar alcohols. Cali-bration curves were generated from peak area versusthe mass of standard analytes injected, with a linear

relationship for the PDA output and a power relationshipfor the ELSD output. A standard was analysed every 10samples to check for instrument/detector drift. Retentiontimes of standards were used to identify analytes in thesample extracts, with the PDA spectral data and peakpurity used to confirm compatible solutes. Typical sampleinjections were 20 μL and runtime was 20 min per sam-ple, with EMPOWER™ 2 software (Waters) used for dataacquisition and processing.

For the tedera samples under the HPLC conditions usedand described above, pinitol in the extracts co-elutedwith fructose. For pinitol and fructose analyses, 16 sam-ples were re-analysed for the genotype tedera 4 only(due to time constraints and cost), pre-drought, day 47of the drought phase and day 28 of the recovery phase(four replicates each from the DS treatment). In addition,four replicates from the WW control on day 47 of thedrought phase were also analysed for pinitol. To separateand quantify fructose and pinitol, a Sugar-Pak (Waters)column (300× 6.5 mm i.d.) was held at 90±0.5∘C andseparation achieved using a mobile phase of 2.5 mg L−1

Ca-EDTA at 0.6 mL min−1 (Naidu, 1998). Detection andquantification of pinitol were undertaken with the PDAas this offered good sensitivity, as well as peak spectraland purity comparisons with the standards. Not detected(ND) denotes failure to detect any quantity above thedetection limit, where the detection limits were: pro-line (10 μmol g−1 DW), proline betaine (10 μmol g−1 DW),fructose (11 mg g−1 DW), glucose (16 mg g−1 DW), sucrose(3 mg g−1 DW) and pinitol (25 mg g−1 DW).

Shoot and root growth characters

At each of the three harvests (days 33 and 47 of thedrought phase and day 28 of the recovery phase) plantswere cut at the soil surface and shoots dried at 80∘C for48 h and weighed. Plant components were separated intoleaf, stem and reproductive components (seed and flow-ers), and weighed. Leaf mass ratio (LMR) was calculatedas the proportion of the total harvested biomass that wasleaf biomass. The weight of desiccated leaves that haddropped from the plant was not quantified. At each of thethree harvests, pots were cut open; roots were placed on a2-mm sieve, soil was gently washed away from the roots,and then immediately frozen with dry ice. Roots of con-siderable length were found coiled in the bottom 10 cmof the pot, but these were not harvested separately. Nod-ules were visible on lateral roots and the taproots of allgenotypes at all three harvests in the DS treatments andWW controls. Roots (crowns, taproot and laterals) werethen oven-dried at 80∘C for 72 h and DM recorded. Theroot to shoot ratio (r : s ratio) was calculated as total rootDM/total shoot DM.

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Individual leaf angle and plant height

Leaf angle inclination was measured from a horizontalline from the base of the primary pulvinus of the termi-nal leaflet (days 1, 7, 18, 25, 37 and 47 of the droughtphase, and days 6, 21 and 28 of the recovery phase) usinga transparent plastic protractor with a swing arm bladein the centre of the base (Fiskars, Madison, WI, USA).Leaf angles above horizontal (0∘) were represented as pos-itive values; leaf angles below horizontal were assignednegative values. Plant height was measured from the soilsurface of the pot at day 30 of drought and again 28 daysafter rewatering.

Statistical analysis

A preliminary analysis was done using the data at the startof the treatments (26 January 2010 – day 1) to check forthe presence of initial variation among the treatments. Ifvariation was present (i.e. for photosynthetic rates, rela-tive leaf water content), these initial pre-drought readingswere used as covariates. Scatter plots of the response vari-ables against the covariates were examined to ensure thatanalysis of covariance assumptions were met.

A linear mixed model was fitted to all response variatesbecause both fixed and random effects were present. Therandom components were common to all models andwere Block+Block.Genotype. Block was considered ran-dom because the environmental conditions (Blocks) inwhich the plants were grown were used to represent allpossible field environments in which these plants can begrown. Similarly, the genotypes grown in these environ-ments (Block.Genotype) were considered representativeof the growth of the entire genotype in any environment,and not just the specific genotypes examined in thisexperiment.

For the photosynthetic active radiation, the fixedcomponents were: Constant+Genotype+Drought+Genotype. Drought. Responses for destructive harvestmeasurements had the following fixed components:Constant+Genotype+Harvest+Genotype. Harvest+Harvest.Drought+Genotype. Harvest.Drought. Plantheight and OA were analysed using repeated measuresanalysis of variance. Responses for plant heightand other non-destructive measures (excluding OAand LWP) had the following fixed components: Con-stant + Genotype+ Drought + Genotype.Drought + Re-watering+ Genotype.Rewatering+Drought.Rewatering+ Rewatering.Date + Genotype.Drought.Rewatering +Genotype.Rewatering.Date+Drought.Rewatering.Date+Genotype.Drought.Rewatering.Date. Osmotic adjustmenthad the following fixed components: Constant+Geno-type+Rewatering+Genotype. Rewatering+Rewatering.

Date+Genotype. Rewatering. Date. Leaf water potentialhad the following fixed components: Constant+Geno-type+Harvest+Genotype. Harvest+Harvest.Drought+Genotype.Harvest. Drought + Date + Genotype.Date +Date. Harvest+Genotype.Date. Harvest+Date. Harvest.Drought+Genotype. Date.Harvest.Drought. [Correctionadded on 6 November 2014, after first online publication:Several changes were made to the terms in the formulaein this paragraph.].

All models assume that the error terms are normallyand independently distributed with zero mean and con-stant variance. Where necessary, the data were trans-formed before analysis using the natural logarithmictransformation to ensure that the model assumptionswere met. The data were analysed by analysis of variance(ANOVA) or by repeated measures analysis of varianceusing GENSTAT RELEASE 12.1 [(PC/Windows XP) Copyright2009, Lawes Agricultural Trust (Rothamsted Experimen-tal Station)]. Various plots (scatter plots, histograms andnormal probability plots) of the residuals were examinedto ensure that the model assumptions were met. Stan-dard Error of the Differences (SED) are given to allow formeans comparisons (McNicol, 2013).

Results

Soil water

On day 1, soil water content was around 11% (v/v)in all pots (data not shown). Soil water content thencontinuously declined in all DS pots. By day 16 of thedrought phase, soil water content was reduced to 4%(v/v) for all DS pots. By day 21, lucerne was approachingPWP; thereafter, aboveground productivity stopped, withplants neither growing nor using water. However, tederaplants did not reach PWP and continued to functionduring this period. The soil water content slowly declinedfor tedera genotypes, resulting in the lowest soil watercontent [approximately 2% (v/v)] at day 47 of DS. Notethat during the drought and recovery phases, soil watercontent in all WW control pots was returned to potcapacity every 7 days [approximately 11% (v/v)].

Photosynthesis and stomatal conductance

For all genotypes, photosynthetic rates (A) in the DStreatment and WW controls at day 7 were very similar atapproximately 13–15 μmol CO2 m−2 s−1 (Fig. 2). At day16, A in the DS treatment was reduced for all species andby day 31 tedera had reached a low 4–5 μmol CO2 m−2 s−1.At day 47, A for tedera in the DS treatment decreasedto 5% of the WW controls (approximately 0.5 μmol CO2

m−2 s−1). At days 31 and 47, lucerne plants in the DStreatment were at PWP and hence A and gs were zero.

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Figure 2 Photosynthesis for two genotypes of tedera and one genotype oflucerne (n=4, SED=0.9455). There was a three-way interaction among treat-ment, rewatering and day (P <0.001), but no significant effect of genotype.Lucerne plants at days 31 and 47 of the drought phase were at permanentwilting point and hence photosynthesis was zero.

In the WW control, A increased in tedera from day 7and reached a peak at day 31 (16–18 μmol CO2 m−2 s−1)and declined by day 47. On day 1 of the recovery phase,all plants in the DS treatment and WW controls wererewatered back to pot capacity. At day 21 and 28 of therecovery phase, A in the DS treatment was higher forall genotypes, up to 50% for tedera 4, than that in theWW controls. By day 47, A in the WW controls haddeclined in tedera from day 31 by approximately 50%,whereas for lucerne, A declined during the recovery phaseby approximately 30%.

The gs of lucerne and tedera 6 in the WW control andDS treatment at day seven of the drought phase weresimilar; however, gs of tedera 4 was higher in the DStreatment (Fig. 3a). By day 16 of the drought phase,gs for all genotypes in the DS treatment had generallydecreased compared to their WW controls. The gs of tedera

had dropped sharply in the DS treatment by day 31,to approximately 15% of the WW controls. Both tederagenotypes in the DS treatment maintained this level oflow gs from day 31 to 47 of the drought phase. The gs ofall genotypes increased markedly in the recovery phaseand was higher than that of the WW controls. For tederain the DS treatment at day 21 of the recovery phase, gs wasmore than double that in the WW control and the valuefor tedera 4 was higher than that of lucerne. However, byday 28 of recovery, the gs of all genotypes was similar.

Intrinsic leaf water-use efficiency

In the drought phase, the WUEL for all genotypes in theDS treatment and WW control was similar from day 7to 16 (Fig. 3b). The WUEL of tedera genotypes in the DStreatment increased sharply at day 31 compared withthat in the WW control plants, with WUEL of tedera 4higher (50%) than that of tedera 6. At the same time,the values of gs decreased rapidly in tedera. However,between day 31 and day 47, WUEL for both tedera geno-types decreased, particularly for tedera 4, which had alower WUEL than its WW control. At day 28, the WUEL

of all the genotypes was similar in the DS treatment andWW control. The WUEL for lucerne in the DS treatmentafter rewatering was similar to that of the WW controls,although values for the latter decreased at day 21 of therecovery phase. The WUEL of the WW controls variedslightly over time.

Relative leaf water content

Relative leaf water content remained around 80% inthe WW controls throughout the drought and recoveryphases (Fig. 4). In the DS treatment, RWC declined fortedera 6 to a low of 42% by day 47 of the drought phasein green leaf tissues. By day 9 after rewatering, previouslydrought-stressed tedera had a similar RWC as the WWcontrols, whereas for lucerne, RWC was still lower thanthat in the WW controls.

Pre-dawn and midday leaf water potential

The effect of the DS treatment on LWP (pre-dawn andmidday) differed between lucerne and tedera (data notshown). At day 7 of DS, there was no difference in LWPp

among the treatments or genotypes. A decrease in the soilwater content [4% (v/v)] in all pots in the DS treatmentat day 16 resulted in a large decrease in the LWPP inlucerne to −4.2 MPa. At day 23 for lucerne, the LWPP

was −4.5 MPa, and plants were close to PWP, whereas fortedera, LWPp was less negative at approximately −2 MPa.At day 30 of the drought phase, LWPM for tedera in the DS

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Drought resistance and recovery in tedera K. Foster et al.

Figure 3 (A) Stomatal conductance (n=4; SED=0.0390) and (B) intrinsic leaf water-use efficiency (n=4, SED=7.46) for two genotypes of tedera and onegenotype of lucerne. For (A) and (B) there was a four-way interaction among genotype, treatment, rewatering and day (P <0.001). Lucerne plants at days 31and 47 of the drought phase were at permanent wilting point and hence gs and WUEL were zero.

treatment was −4 MPa, whereas xylem sap could not beobtained from lucerne plants at −7 MPa. At day 47 of thedrought phase, the LWPM for tedera in the DS treatmenthad declined to below −6.5 MPa. In the WW control, theLWPM for tedera ranged from −1.2 to −1.7 MPa, while inlucerne it declined to −2.5 MPa during the drought phase.The severity of the DS, measured in terms of LWP (bothpre-dawn and midday), did not differ between the twotedera genotypes. The LWPM in the DS treatment wasthree to four times more negative than that of the WWcontrols; however, neither tedera genotype had reachedPWP by day 47 of DS.

By day 9 of the recovery phase, LWPM for tedera inthe DS treatment had returned to the values of the WWcontrols. However, for lucerne, LWPM in the DS treatment

was higher (−1.6 MPa), that is, less negative, than that inthe WW controls (−2 MPa). However, by day 28, LWPM

in the DS treatment and WW control for lucerne did notdiffer.

Other plant growth responses

Leaf angle and photosynthetic active radiation

At day 7 of the drought phase, there was no difference inleaf angle (≤5∘) among the genotypes or between the DStreatment and WW control (Fig. 5). In the DS treatment,by day 18 the leaf angles of all genotypes increased greatlyand were 80∘–85∘. At day 25, the majority of the lucerneleaves were drooping towards the ground (i.e. −80∘,below horizontal). The leaf angles remained at 70∘–85∘

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K. Foster et al. Drought resistance and recovery in tedera

Figure 4 Relative leaf water content for two genotypes of tedera and onegenotype of lucerne (n=4, SED=0.033). There was a three-way interactionamong treatment, rewatering and day (P <0.001), but no significant effectof genotype.

for tedera 6 during the entire drought phase, whereas fortedera 4 the leaves declined to 45∘–50∘ by day 47. By day6 of the recovery phase the leaf angle for tedera in thepreviously drought-stressed plants was approximately 5∘,less than in the WW controls, all of which had increasedtheir leaf angles just prior to the next rewatering. By day21 of the recovery phase, the leaf angles of all genotypesin both treatments did not differ (≤5∘).

For tedera 4, the increase in leaf angle in the DS treat-ment reduced the upper leaf area exposed to vertical radi-ation when compared to the WW controls. At day 30,this reduced the average PAR on upper leaf surfaces oftedera 4 to approximately 850±55 μmol photons m−2 s−1

(mean±SE) compared to approximately 1860± 36 μmolphotons m−2 s−1 in the WW control (Table S1, Supporting

Figure 5 Mean leaf angle for two genotypes of tedera and one genotype oflucerne (n=4, SED=4.19). There was a four-way interaction (P <0.001) ofgenotype, treatment, rewatering and day. Note that 0∘ is horizontal.

Information), a reduction of approximately 55% com-pared to the WW control plants.

Plant height and pubescence

There was a two-way interaction of time by genotype(P<0.05) for plant height (Table S2). The plant height atday 30 of drought phase differed among the genotypesin the DS treatment with lucerne plants taller (560 mm)than tedera 4 and 6 (266 and 208 mm, respectively). Byday 21 after rewatering, the height of the lucerne plants inthe DS treatment had increased (702 mm), but there wasno change for tedera. Increased pubescence was observedon stems and new leaves of both tedera genotypes atday 30 of the drought phase in the DS treatment whencompared to the WW control.

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Drought resistance and recovery in tedera K. Foster et al.

Table 1 Shoot DM and root DM for two genotypes of tedera and one genotype of lucernea

Drought phase Recovery phase

Day 33 Day 47 Day 28

WW DS WW DS WW DS

Total shoot DM (g)

Tedera 4 26.6 9.2 30.4 9.2 46.4 17.2

Tedera 6 20.5 11.2 27.9 10.3 37.8 15.8

Lucerne 20.3 12.2 22.1 13.2 32.8 20.1

SED 2.7

Total root DM (g)

Tedera 4 19.3 11.6 18.1 14.7 23.9 12.7

Tedera 6 18.8 11.7 21.0 13.1 24.8 11.6

Lucerne 15.3 13.1 21.2 12.9 28.4 15.3

SED 1.3

DM=dry matter; DS=drought-stressed; WW=well-watered controls.aThere was a three-way interaction of genotype, harvest and treatment for shoot DM (P <0.001) and root DM (P <0.05).

Plant growth

Total shoot dry matter and leaf to total mass ratio

The shoot DM of all genotypes in the DS treatment duringthe drought phase was greatly reduced compared to thatof the WW control (Table 1). For tedera genotypes, thisreduction in shoot DM was between 63% and 70% at day47. For lucerne, this reduction was only 40%; while allleaves were desiccated, they had not been shed, as is oftenseen in the field. In the recovery phase in the DS treat-ment, shoot DM for tedera 4 increased by 86%, whereasthat of both tedera 6 and lucerne increased by approxi-mately 50%. However, the total shoot DM for all geno-types was still less (38–60%) than that of their equivalentWW controls. By day 28 of the recovery phase the canopystructure in tedera was similar to that of the WW controls,whereas the canopy of lucerne plants in the DS treatmentcontained both dried and green shoots. Consequently, theleaf to total plant mass ratio (LMR) for tedera genotypesat the end of the recovery phase in the DS treatment washigher (0.48–0.52) than for lucerne plants (0.32).

Root dry matter production

The root DM for all the genotypes in the DS treatmentwas reduced compared to that of the WW control plants(Table 1) at all three harvests. At harvest one, the reduc-tion in root DM was 37–40% for tedera and 14% forlucerne. For tedera genotypes, the root DM in the WWcontrol plants did not change during the drought phase.The total root DM increased at harvest two in the DS treat-ment for tedera 4 only, however the lateral roots contin-ued to grow for both tedera genotypes (data not shown).At harvest three in the rewatered DS treatment, the totalroot DM for tedera and lucerne did not change during

the recovery phase although the lateral roots for tederadecreased but this was significant for tedera 4 only. Forlucerne, the lateral root DM did not change during eitherthe drought or the recovery phases.

Root to shoot ratio

The root to shoot ratios (r : s ratio) for tedera 4 and lucernein the DS treatment at day 33 (Harvest 1) of the droughtphase were higher than those of their WW controls(Fig. 6). However, at day 47 of the drought phase, the r : sratio was higher for tedera only in the DS treatment, whilethere was no change for lucerne. At day 28 of the recoveryphase, the r:s ratio of tedera in the rewatered DS treat-ment was reduced, and all genotypes were now similar totheir respective WW controls. The r : s ratio changed littlefor the genotypes in WW controls over the three harvests.

Osmotic adjustment

At day 7 of the drought phase, there was no evidence ofOA in either lucerne or tedera in the DS treatment (datanot shown). However, by day 30 the OA was −0.12 MPaand −0.20 MPa for tedera 4 and tedera 6, respectively.By day 47, OA increased to −0.5 MPa for both tederagenotypes. Osmotic adjustment for lucerne at day 30 andday 47 was not measured, as plants were at PWP. In theDS treatment at the end of the recovery phase, OA fortedera 4 and 6 had declined to −0.13 MPa and −0.12 MPa,respectively, and for lucerne OA was −0.04 MPa.

Compatible solutes and sugars

Proline was not detected in either species or eithertreatment at day 1 (Table S1), but accumulated to high

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K. Foster et al. Drought resistance and recovery in tedera

Figure 6 Root : shoot ratio for two tedera genotypes and one genotype oflucerne at day 33 and day 47 of the drought phase and day 28 of the recoveryphase (n=4, SED=0.12). There was a three-way interaction of genotype,treatment and harvest (P <0.001).

concentrations (191–205 μmol g−1 DW) in tedera in theDS treatment at the end of the drought phase (day 47).At the end of the recovery phase in the DS treatment(day 28), proline was again not detected in tedera orlucerne. Proline betaine was present in the leaves oflucerne in both treatments at day 1 (28–35 μmol g−1

DW); it is the main betaine in lucerne (Trinchant et al.,2004). However, at the end of the drought phase in theDS treatment, lucerne was at PWP and leaves could notbe measured. In the recovery phase, the proline betaineconcentration for lucerne in the DS treatment was againsimilar to that of the WW controls (58–67 μmol g−1 DW),but still higher than pre-drought levels.

Concentrations of glucose (36–40 mg g−1 DW) andfructose (80–95 mg g−1 DW) which co-eluted with pini-tol for tedera at day 47 in the DS treatment were more

Figure 7 Pinitol and fructose concentrations in leaf tissue of tedera 4 at days1 and 47 of the drought phase and day 28 of the recovery phase (mean± SE,n=4).

than three times greater than at day 1 of the droughtphase. At the end of the recovery phase (day 28), fruc-tose and glucose concentrations for tedera 4 and tedera6 in the DS treatment had declined (48–57 mg g−1 DWand 16–25 mg g−1 DW, respectively) and were similar tothose in the WW control. The fructose concentrations inlucerne in the DS and WW plants were similar at the endof the recovery phase (45–48 mg g−1 DW), but glucoseconcentrations were lower than pre-drought concentra-tions. Sucrose was either not detected or at low concen-trations, with little change observed over time.

Pinitol concentrations in the DS treatment at the endof the drought phase for tedera 4 (Fig. 7) were 68%higher than those in the WW plants (3-fold higherthan pre-drought values). In the recovery phase, tedera4 maintained enhanced pinitol concentrations, abovepre-drought concentrations, but the concentrations weresimilar to those in the WW plants at day 47 of the droughtphase.

Discussion

Key findings from this study are: (a) concentrations ofproline and pinitol in tedera substantially increased inresponse to water deficits; (b) gs and A for both speciesin the DS treatment at days 21 and 28 of the recoveryphase exceeded those of the WW control plants; (c) tedera4 showed more root plasticity in response to DS andrewatering than did tedera 6 or lucerne; (d) OA playsan important role in drought tolerance in tedera; and (e)tedera had a more rapid recovery after rewatering thanlucerne. Overall, tedera and lucerne showed differentphysiological and morphological strategies to survive andrecover from DS.

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Drought resistance and recovery in tedera K. Foster et al.

Physiological and morphological responses to droughtstress

In tedera, the decreasing RWC was also associated witha decrease in A and gs, approximately in parallel. How-ever, the decrease in A in both species is unlikely to arisefrom stomatal closure alone. Stomatal and non-stomatallimitations can both contribute to the reduction of photo-synthesis under severe drought conditions (Flexas et al.,2004). The photosynthetic apparatus in tedera, althoughseverely curtailed by low gs, appears highly resistant to DS,in contrast to that of lucerne (where leaves were brown).Even under extreme DS at day 47, the leaves of tederawere still pliable and green, and gas exchange continued,albeit at a low rate and very low LWPM.

In tedera, reduced stomatal conductance was one ofthe primary mechanisms for acclimating to water stress.Stomatal closure occurred when significant reductionswere observed in RWC and LWPM in the DS treatment,and it is likely the main cause of reduced photosynthesisin tedera under DS. In contrast, lucerne leaves did notshow a reduced gs, by day 31 of DS; instead leaves wiltedand died which reduced transpiration.

Tedera’s WUEL increased as water supply declined inthe DS treatment, but only until day 31, before decliningat day 47 when water deficit was severe. Elevated WUEvalues are commonly observed in water-stressed plants(Pou et al., 2008), and WUE is an important index of aplant’s acclimation to an arid environment (Xing and Wu,2012). However, severe water stress may have resultedin a decrease in the activities of photosynthetic enzymes,resulting in a lower WUEL (Zhao et al., 2004); a decreasein WUE has also been reported in both winter wheat(Shangguan et al., 2000) and spring wheat (El Hafid et al.,1998) subjected to severe stress.

Plant growth responses to drought stress

Total shoot and root dry matter production

The long-term DS drastically reduced aboveground plantproduction in both tedera and lucerne. The reductionin leaf shoot DM under DS for tedera (63–69%) wassimilar to that reported for other legumes (Pandey et al.,1984). Nonetheless, any green leaf retention under DSis a highly desirable trait in a perennial pasture speciesover summer, as leaf biomass is likely to make up mostof the nutritional intake for livestock. In contrast to ted-era, lucerne plants sacrificed green leaves and becamedormant under declining moisture conditions. Overall,long-term severe DS dramatically reduced total root DMin both species. However, for tedera 4, total root DMincreased between Harvest 1 and 2; even though tap-root DM did not change, the lateral roots continued to

grow. Lateral root growth in wheat (Triticum aestivum L.) isalso promoted by water deficit (Ito et al., 2006). Likewise,Pang et al. (2011) reported an increase in the proportion ofroots in the topsoil for tedera in response to DS; the mech-anisms underlying the increase may include OA (Saabet al., 1992). For tedera, enhanced allocation of assimilatesto the lateral roots likely provides a store of assimilates,available after rewatering. Consistent with this interpreta-tion for tedera 4, the lateral root DM decreased after rewa-tering although total root DM did not change, whereaslateral root DM for lucerne did not change during thedrought or recovery phase.

Other aboveground plant growth responses

Morphological responses to DS in tedera were striking.The extent of the leaf movements increased as the RWCand water potential decreased, thereby reducing lightinterception. Therefore, steep leaf angles at midday, withthe consequent reduced radiation interception, wouldconstitute a crucial adaptation in tedera to surviving inarid environments due to reduced leaf temperature andtranspiration. Droughted plants were also increasinglypubescent on stems and new leaves, which would partlyprotect them from excessive light injury and decreasetranspirational water loss (Pfeiffer et al., 2003).

Root to shoot ratio

The increase in r : s ratio under DS reflects the adaptivegrowth balance of the leaf canopy and root system. Manycrop plants divert assimilates to root growth under waterstress, resulting in a high r:s ratio (Whitfield et al., 1986),which is one of the mechanisms involved in the accli-mation of plants to drought (Turner, 1997). Indeed, theincrease in r : s ratio in tedera 4 at day 47 of DS wasdue to root biomass accumulating and not just a decreasein aboveground biomass. This suggests a stronger accli-mation in tedera 4 to prolonged DS than in tedera 6 orlucerne. This should be advantageous because it enhancessoil resource acquisition and suggests a close relationshipwith drought resistance in this species.

Response of compatible solutes and sugars to drought stress

Our observations are consistent with our first hypoth-esis that DS induces the accumulation of compatibleosmolytes in leaf tissue in tedera, at least at the repro-ductive stage. The accumulation of substantial concentra-tions of proline in tedera at the same time as significantchanges occurred in RWC and LWPM suggests it has a rolein OA in leaf tissue during the later phase of water stress,as has been reported for cowpea [Vigna unguiculata (L.)

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K. Foster et al. Drought resistance and recovery in tedera

Walp.] (Oliveira Neto et al., 2009). Osmotic adjustmenthas been found in tedera genotypes previously (Panget al., 2011); our results also suggest it plays an impor-tant role in drought tolerance, at least in the leaves. Intedera, OA may also allow stomata to remain at least par-tially open, and CO2 assimilation to continue, at LWPs thatcould otherwise be inhibitory and thereby enable plantsto extract more water from the soil and adjust their waterpotential. High concentrations of proline in tedera dur-ing drought may also have protected the photosyntheticsystem from permanent damage (Lawlor, 2001). Osmoticadjustment in tedera likely helps assists to maintain leafmetabolism and root growth at very low LWP by main-taining the turgor pressure in the cells.

This is the first report of pinitol in tedera in responseto water deficit and it suggests this sugar alcohol con-tributes to drought tolerance in this species, at least inthe specific genotype tested. Consistent with our results,Ford (1984) concluded that pinitol accumulation mayindicate the ability of a legume to tolerate low LWP.With its slow turnover relative to sugars (Paul & Cock-burn, 1989), and limited reactivity, pinitol is a goodcandidate for an osmoprotectant. Pinitol concentrationsalso increased in the control plants at day 47. Like-wise, Streeter et al. (2001) found pinitol accumulatedin the leaves of WW soybean [Glycine max (L). Merr.]plants. These authors suggested there is some advan-tage for plants that are more likely to experience DS toaccumulate pinitol before the onset of stress and thatpinitol has a key role in stress tolerance of soybean.Silvente et al. (2012) also showed that a drought-tolerantsoybean genotype had higher amounts of pinitol,even under WW conditions, than a drought-sensitivegenotype.

Physiological traits associated with post-drought recovery

Tedera and lucerne quickly returned to pre-drought levelsof physiological activity once DS had been relieved. Fortedera, rewatering following the DS treatment resultedin a rapid reduction in the leaf folding angle to almosthorizontal by day 6. By day 9 after rewatering, both leafRWC and LWPM in the DS treatment and WW controlplants were similar, indicating the quick reversibility ofthe responses to the DS in tedera. The reason for the fullrecovery of leaf water relations in tedera indicates that,after rewatering, embolised xylem vessels were likelyrapidly refilled (Holbrook et al., 2001).

Despite more than 16 days of almost full stomatalclosure in both tedera genotypes in the DS treatment(days 31–47), severe DS did not appear to impair vascularcapacity for water transport, as there was complete recov-ery of photosynthesis and gs after rewatering. Gallé &

Feller (2007) also reported a completely restored photo-synthetic rate 4 weeks after rewatering, but in contrast toour results, gs remained at a lower level. The resumptionof CO2 assimilation of water-stressed tedera plants afterrewatering indicates that the basic mechanisms of pho-tosynthetic photochemistry and biochemistry were notpermanently damaged by water deficit (Cornic, 2000).

Our second hypothesis, that the area-based photosyn-thetic rate of tedera is enhanced during the rewateringphase after drought was also supported. After rewater-ing, A and gs were greater in the DS plants than inthe WW controls, indicating an overcompensation of gasexchange. However, the decline in aboveground DM intedera due to long-term severe DS was not completelyreplaced by enhanced growth following rewatering. Sim-ilar results were found for soybean (Bunce, 1977; Wanget al., 2006; Lobato et al., 2008) and for some grasses (Xuet al., 2010).

On rewatering, tedera was able to rapidly restore LWPand unfold green leaves, and this likely played a criti-cal role in the resumption of plant growth, as previouslyfound in some grasses (Munne-Bosch & Alegre, 2004).Although the recovery phase was limited to 28 days, dur-ing this period, tedera did exhibit superior recuperativepotential to lucerne, confirming our third hypothesis. Incontrast to tedera, lucerne plants in the DS treatmenthad brown desiccated leaves and dry stems upon rewa-tering. For lucerne to recover from DS, new shoots mustbe initiated following rewatering. Consequently, lucernehad fewer green leaves and a lower LMR, and its above-ground growth rate was slower than that of tedera oversubsequent weeks. The new shoot growth resulted in anincrease in plant height after rewatering in lucerne; how-ever, there was no change for tedera.

Root growth responses associated with post-drought recovery

Root DM of tedera and lucerne did not change followingrewatering of the DS plants, however, lateral root DM oftedera 4 decreased and perhaps thereby supplied energyand nutrients for the resumption of new shoot growth intedera. The increase in root DM for tedera and lucernein the WW controls during the recovery phase may be aresponse to the reduction in photoperiod in early autumn(April), with more assimilate investment towards peren-nial organs like taproots for survival. The root system oftedera showed greater phenotypic plasticity in responseto DS and rewatering than that of lucerne which supportsour fourth hypothesis. High levels of root plasticity to DSand rewatering in tedera may be a key factor in deter-mining plant persistence and productivity under climatechange.

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Drought resistance and recovery in tedera K. Foster et al.

Changes in compatible solutes and sugars associatedwith post-drought recovery

Rewatering of the DS plants resulted in proline no longerbeing detected in leaves of tedera at day 28. Prolinehas been reported to act as a source of energy, carbonand nitrogen (Van Heerden & Krüger, 2002; Szabados& Savoure, 2010). This suggests an important role forproline in plant recovery from severe DS in tedera, andis consistent with our first hypothesis. Our results alsosuggest an important role for pinitol in plant recoveryfrom long-term DS in tedera 4. The pinitol concentra-tion decreased substantially in leaves of tedera 4 followingrewatering, but was still higher than pre-drought val-ues, reflecting a slower turnover that likely contributedto metabolism for growth of new shoots. Soluble sug-ars may also act as a carbon source for regrowth in ted-era during recovery, and glucose and fructose in tederadecreased by up to 55% after rewatering. Osmotic adjust-ment in tedera also declined at the end of the recoveryphase and osmotically adjusted leaves likely metabolisedmuch of these solutes. Osmotic adjustment is consid-ered one of the critical processes in plant acclimation todrought, because it sustains tissue metabolic activity andalso enables regrowth upon rewatering (Morgan, 1984).

Concluding comments

The maintenance of plant functions at low LWP in ted-era and their more rapid recovery after rewatering shouldcontribute to greater aboveground DM yield under inter-mittent drought periods than possible for lucerne recover-ing from dormancy. The ability of tedera to rapidly regrowfollowing a period of DS depends on the presence of theresidual photosynthetic leaf area. Selecting tedera geno-types that maximise the number of leaves that survivethe dry season will likely further enhance this recoveryand persistence. Following rewatering, tedera can rapidlyadjust from steep leaf angles to the horizontal, to againobtain maximum photosynthesis. This would also be akey factor in facilitating a rapid growth response to rewa-tering in tedera.

This study contributes to our understanding of stressbiology of plants through the identification of key com-pounds in drought resistance and recovery in peren-nial legumes. The capacity to synthesise and accumulatepinitol could also be an important adaptive feature inother tedera genotypes. Selecting tedera genotypes withenhanced concentrations of pinitol during DS could be avaluable tool to improve plant performance under, as wellas recovery from, DS.

The identification of perennial plants with resistanceto DS and, especially, the physiological properties utilised

by perennial legumes to cope with DS and maximiseresponse to rewatering is of vital importance in the faceof predicted climate change. Tedera has the potential toprovide out-of-season forage across the low-to-mediumrainfall zones in southern Mediterranean Australia and,critically, to quickly respond to rewatering events, tohelp balance the feed supply across the entire season.However, appropriate grazing-management strategies fortedera over autumn and summer are yet to be defined.

Acknowledgements

This research was supported by Department of Agri-culture and Food Western Australia (DAFWA) and theFuture Farm Industries Cooperative Research Centre (FFICRC). We thank Michael Renton for his input into theexperimental design and statistical approach and alsoDaniel Kidd, Dion Nicol, Neil Turner, Kevin Goss, ClintonRevell and Mark Sweetingham for their ongoing supportand advice.

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Supporting Information

Additional Supporting Information may be found in theonline version of this article:

Table S1. Photosynthetically active radiation (PAR)(μmol photons m−2 s−1) for two genotypes of tedera andone genotype of lucerne in the drought-stressed treat-ment and the well-watered control on day 30 in thedrought phase at 12:00–15:00 h (average of two leavesper plant) (mean± SE, n=4).

Table S2. Plant height (mm) for two genotypes of ted-era and one genotype of lucerne in the drought-stressedtreatment at day 30 in the drought phase and day 28 in therecovery phrase (n= 4, SED= 58). There was a two-wayinteraction of time by genotype (P<0.05).

Table S3. Proline, proline betaine, fructose/pinitoland glucose concentrations in leaf tissue for two geno-types of tedera and one genotype of lucerne at day 1and day 47 of the drought phase, and day 28 of therecovery phase in the drought-stressed treatment (DS)and the well-watered control (WW) (mean±SE, n=4).PWP=permanent wilting point, *=not detected, that isfailure to detect any quantity above the detection limit:proline (10 μmol g−1 DW), proline betaine (10 μmol g−1

DW), fructose (11 mg g−1 DW), glucose (16 mg g−1 DW)and pinitol (25 mg g−1 DW).

16 Ann Appl Biol (2014)© 2014 Association of Applied Biologists