Potential improvement of photosynthetic CO<sub>2</sub> assimilation in crops by exploiting the natural variation in the temperature response of Rubisco catalytic traits

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Potential improvement of photosynthetic CO

₂

assimilation in crops by exploiting the natural variation in the temperature response of Rubisco catalytic traits Jeroni Galme´s

¹

, Sebastia` Capo´-Bauc¸ a`

¹

, U ¨ lo Niinemets

^2,3

and Concepcio´n In˜iguez

¹

Theenhancementofthephotosyntheticcapacityofcropsby theexpressionofmoreefficientRubiscoversionshasbeena maintargetinthefieldofplantphotosynthesisimprovement.

However,suchanincreaseinthephotosyntheticefficiencywill dependontheenvironmentalconditionsandonthe

responsivenessofRubiscototemperatureandCO2availability.

Afteranexhaustivecompilationandstandardizationofthedata publishedsofar,alargenaturalvariabilityinthethermal responsesofRubiscokineticparametersinhigherplant specieswasrevealed.Thevariabilityobservedwasrelatedto thephotosynthetictypebutalimitedadaptationtothespecies thermalenvironmentwasfound.Weprovidetheoretical evidencethattheexistenceofdistinctiveRubiscoresponsesto varyingtemperatureandCO2concentrationconstitutesa promisingavenueforincreasingthephotosyntheticcapacityof importantcropsunderfutureclimaticconditions.

Addresses

1ResearchGrouponPlantBiologyunderMediterraneanConditions, UniversitatdelesIllesBalears–INAGEA,Palma,BalearicIslands,Spain

2ChairofCropScienceandPlantBiology,InstituteofAgriculturaland EnvironmentalSciences,EstonianUniversityofLifeSciences, Kreutzwaldi1,51006Tartu,Estonia

3EstonianAcademyofSciences,Kohtu6,10130Tallinn,Estonia Correspondingauthor:Galme´s,Jeroni([email protected])

CurrentOpinioninPlantBiology2019,49:60–67 ThisreviewcomesfromathemedissueonPhysiologyand metabolism

EditedbyElizabethAAinsworthandElizabeteCarmoSilva

https://doi.org/10.1016/j.pbi.2019.05.002

1369-5266/ã2019ElsevierLtd.Allrightsreserved.

Introduction

The enzyme ribulose-1,5-bisphosphate carboxylase/

oxygenase (Rubisco) catalyzes the addition of CO2 to ribulose-1,5-bisphosphate(RuBP),whichisquantitatively themostsignificantreactionconvertinginorganiccarbonto organiccarbon,therebysustainingthevastmajorityoffood webs on Earth. Despite being one of the most studied

proteinssince its discoveryin 1957[1], somebasic aspects of itscatalyticfunctioning remainunresolved.Notoriously, westillcannotfullyexplainwhyoneofthemostimportant enzymesforlifeontheplanetisincapableofdifferentiating betweenCO2andO2asthesubstrate.Somestudiesargue thatRubisco isfullyoptimized to givethebestpossible performance depending on the environment [2,3], and otherssuggestthatthecarboxylase–oxygenasedualityof the enzyme has been a key factor in the atmospheric dynamicsofEarth[4].Even,arecentstudyindicatesthat Rubiscocatalyticperformanceisnotsopromiscuouswhen comparedtootherchemicallyrelatedenzymes[5].Either way,thereactionofRubiscowithO2leadstoreleasingpart ofpreviouslyfixedcarbonandnitrogenthroughphotores- piration, requiring an extra energyinvestment [6].This imperfectdiscriminationbetweenCO2andO2,together witharelativelyslowcarboxylationturnoverrateandalow affinity for CO2 implies that photosynthetic organisms maintainRubiscoatveryhigh concentrationstosupport autotrophy[7].Itisnot,therefore,surprisingthatengineer- ingRubiscoforbetterperformanceintermsofCO2fixation rateshasbeenoneofthemaintargetsinthefieldofplant improvementinthelastyears[8,9,10,11,12].

The estimates of theoretical models endorse the hopes of Rubisco improvement, predicting very significant increasesinthephotosyntheticcapacityof cropsiftheir nativeRubiscowasreplacedbyforeignnaturallyoccurring Rubiscosdisplayingbettercatalyticproperties[13,14].An importantresearchtargetisthereforetofindmoreefficient naturallyoccurringversionsofRubisco,andtounderstand themolecularcausesofthevariabilityinthecatalytictraits, allowing the design of the improved Rubiscos in the laboratory.Thedataavailablesofarsuggestthatenviron- mentaltemperatureandavailabilityofCO2andO2arethe environmental factors that have induced changes along evolution in the catalytic properties of Rubisco when comparingspeciesfromcontrastingecologyandphylogeny [14,15,16].Actually,thequantitativeimpactofRubisco kinetictraitsonthecarbonfixationratedependsontem- perature, [CO2] and [O2] at the enzyme catalytic sites.

Hence,thepredictedincreasein theatmospheric[CO₂] willfavorRuBPcarboxylationoveroxygenation,butthis effectwillbelowerinwarmertemperatures,sincetheratio ofdissolved[O2]/[CO2]inchloroplastsincreaseswithtem- perature.Furthermore,temperatureitselfhasalsoadirect impact on the catalytic performance of Rubisco. The

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kcat

increases up to 50–60C, while the Rubisco specificity factor(Sc/o)decreasesandtheMichaelis–Mentenconstant for CO2 (Kc) increases with increasing temperature [17,18,19].Studiesreportingtemperatureeffectsonthe Michaelis–MentenconstantforO2(Ko)showcontradictory results,withKobeingalmostindependentoftemperature in some cases,whileincreasing or even decreasingwith increasingtemperatureinothercases[20,21–24,25,26].

Despitethegeneraltrendsforkcat

c,Sc/oandKc,differences in the thermal dependencies of Rubisco kinetics have been reported between different photosynthetic organisms, even among closely related species [18,19,27].

Furthermore, the variation in Rubisco temperature responseshasbeenshowntodependonspecies’climate and photosynthetic mechanism, with lower energy of activation(DHa)forkcatc

andKcinC4comparedtoC3plants [19,26,27]andlowerDHaforkcatc

inC3plantsfromcool habitats (C3cool

)relativeto C3plantsfromwarmhabitats (C3warm

) [18]. Rubisco-limited CO2 gross assimilation rate (ARubisco) modelled at varying temperatures and chloroplasticCO2concentrations(Cc)suggestedimproved photosynthetic performance of C3cool

plants at lower temperatures,and C3warm

plantsathigher temperatures, especially athigher CO2concentration[19]. However, thesepreviousanalyseswerebasedonalimitednumberof species;thus,more dataonthetemperatureresponseof Rubiscokineticsfromawiderrangeofspeciesareneeded toconfirmtheobservedtrends.

Thepresentreviewupdatesandsummarizestheinforma- tiononthethermalsensitivityof Rubiscocatalytictraits acrossspermatophytes,includingtherecentlargescreen- ing studies published during the last two years [16,25,26,28],andevaluatestheecologicalconstraints oftraitvariabilityandthepotentialcapacityforcropCO2

assimilation improvement under certain environmental conditionsbyintroducingcontrastingRubiscovariants.

Spanning the temperature response of Rubisco catalytic traits among

Spermatophyta: evidence of adaptation in relation to the photosynthetic mechanism but limited adaptation to the thermal environment

Atotal of138 Spermatophytaspecies,for whichatleast oneofthemainRubiscokineticcharacteristicshadbeen reportedatthreeormoredifferenttemperaturesinpre- vious studies, werecompiled,filtered, andstandardized accordingtoGalme´setal.[19]toaccountfortheeffects of study-to-study methodological differences (Supple- mentarymaterial).Fromthesedata,theenergyofactiva- tion (DHa) and thescaling constant (c) for the catalytic traitsofRubiscowereobtainedbyapplyinganArrhenius- type temperatureresponsefunction[19].

There was a large interspecific variability in DHa for Rubisco catalytic traits, especially for Kc, Ko, and kcat

c,

4 3

species, and C3cool

species (Figure 1a,b). Despite this ample variability, significant differences wereobserved whencomparinggroupaverages.Inparticular,Rubiscoin C4specieshaslowerSc/ovaluesthanthoseinC3speciesat therangeofstudiedtemperatures,andalsohigherther- malsensitivity(morenegativeDHa)forSc/othanRubisco inC3cool

(Figure1a,c),whileC4Rubiscohashighervalues than C3warm

Rubisco at temperatures25C and lower thermalsensitivity(lowerDHa)for kcat

cthanbothC3 cool

and C3warm

Rubiscos. The same differencesin Rubisco thermaldependencyresponses betweenC4andC3spe- cies were already observed in previous studies [19,26,27]. However, in contrast to these previous studies,nosignificantdifferenceswereobservedbetween C4and C3Rubiscos in DHafor Kc,althoughC4species possess Kc values higher than those reported for C3

species atphysiologically relevant temperatures.These resultsindicatethatasignificantincreasein thenumber ofspeciesincludedinthepresentanalysisevenedoutthe differences in Kc temperature responses among species withdifferentphotosynthetictype.

Overall, the present analysis strengthens the evidence thatC4andC3speciesevolvedtowardsdifferenttemper- aturesensitivitiesofRubiscokinetics,assuggestedbefore [16,27],withC4Rubiscoshavinga18%largerDHafor Sc/oand 20% smallerDHa for kcatc

(Figure 1a). Wealso found a positive relationship betweenDHa for Sc/oand DHaforkcatc

(P<0.001,r²=0.102,datanotshown)that hasnotbeen previouslyobservedinsmallersetsofdata [19].Thismayindicatethatthewidelyassumedtrade- offs betweentheseRubisco catalytictraitsat25C(Sc/o

decreaseslikethesquarerootofkcatc

)[2,3,16]varywith temperature,andthatcontrastingrelationshipsareprin- cipallypossible.Thisfactisacriticalcornerstonetofind naturalversionsofRubiscoofparticularinterestforfuture attempts to engineer this enzyme for contrasting envi- ronmentalconditions. For engineeringforeignRubiscos into crops, theoutliers of theserelationships as wellas those displaying extreme behavior (Figure 1b) are of particularinterest.

ThecomparisonofthetemperaturesensitivityofRubisco kinetics betweenC3cool

and C3warm

speciesrevealed no differencesinDHaforanykineticparameter(Figure1a, c),eventhoughsomesignificantdifferenceswerefound intheaveragevaluesatdiscretetemperatures(e.g.higher kcatc

inC3cool

speciesattemperatures25Candhigher Sc/oin C3cool

species attemperatures 35C, compared with C3warm

species). These results are in contrast to Galme´s et al. [18], who obtained a lower DHa for kcatc

in C3cool

compared with C3warm

species. Also, wefound non-significant relationship between the species opti- mum growthtemperature andDHafor all kinetictraits, except for Sc/o (P< 0.02, r²=0.12, data not shown) indicating a higher temperature sensitivity of this

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Figure1

(a) (b)

(c)

Group Group

S_c/o

Sc/o(mol mol–1) ΔHa Ko(kJ mol–1)ΔHa Sc/o(kJ mol–1) ΔHa Kc(kJ mol–1)ΔHa kcatc(kJ mol–1)

kcat(s–1) Kc(μM)

K_c

K_o k_cat^c

C₄ (n = 26) (n = 47) (n = 49) C₃^cool C₃^warm C₄ (n = 11) (n = 39) (n = 39) C₃^cool C₃^warm C₄

(n = 19) (n = 47) (n = 55) C₃^cool C₃^warm

C₄ (n = 5) (n = 30) (n = 28) C₃^cool C₃^warm

c ΔHa (kJ

mol^-1) c ΔHa (kJ

mol^-1)

c ΔH_a (kJ

mol^-1) c ΔH_a (kJ

mol^-1)

c

Group -3.9 ± 0.4 b -20.5 ± 0.9 b

-2.9 ± 0.2 a -18.3 ± 0.6 ab -2.2 ± 0.2 a -16.6 ± 0.5 a

21.9 ± 1.3 b 51.1 ± 3.2 b

27.8 ± 0.9 a 66.6 ± 2.3 a 25.2 ± 0.9 a 59.6 ± 2.1 a 19.1 ± 2.8 a 39.2 ± 7.0 a

-25

-100

150

100

50

50 40

10 20 15

0 0

5 15

1 2 5

70 80 60

5

0

0 30

10 20

60 90

15 25

Temperature (°C) Temperature (°C) Temperature (°C)

35

35 5 15

45 45

5 15 25 35 45 5 15 25 35 45

100

50 150

100

75

50

25 125

0 C4

E. furctus C. indicum

A. squarrosum

A. squarrosum E. tef

A. vulgaris

A. myriantha P. distans V. helioscopia

D. intortum

T. rhodesica C3

C4

C4 -15

-20 -10

21.2 ± 1.4 a 45.9 ± 3.5 a 19.3 ± 1.4 a 40.8 ± 3.4 a

-13.5 ± 9.4 a -49.6 ± 23.8 a

-2.0 ± 4.0 a -20.4 ± 10.0 a 1.3 ± 2.8 a -12.0 ± 7.0 a

cool C3

warm C4 C3

cool C3

warm

C4 C3

cool C3

C3 warm cool

C3cool C3

warm

C3warm

a b c

Current Opinion in Plant Biology

ThermaldependenciesofinvitroRubiscokinetictraitsfromSpermatophyta.Specieswereclassifiedaccordingtotheirphotosyntheticmechanism (C₃vs.C₄)andC₃specieswerefurtherclassifiedaswarm(T_growth25C),andcool-temperature(T_growth<25C)speciesaccordingtotheir optimumgrowthtemperature(Tgrowth).EstimatesofTgrowthwereobtainedfromliteratureorassignedaccordingtotheirclimateoforiginasin Galme´setal.[18,19].Thearbitrarythresholdof25CwasusedtoseparatecoolandwarmC3asinanalogousstudies[18,19,40].

RubiscokineticdatacompilationofGalme´setal.[18,19]wasextendedwithnewdatafromrecentstudies[16,25,26,28],overallreporting RubiscokinetictraitsatthreeormoredifferenttemperaturesforalargenumberofSpermatophytaspecies.Alloriginaldatawerecorrectedusing theformulationalreadydescribedbyGalme´setal.[18,19]toaccountfordifferencesintheassaybuffercompositionthataffectedtheionic strengthandtheacidityconstantofdissolvedCO₂(pK_a,CO₂),aswellasfordifferencesintheCO₂andO₂solubilitiesused,inordertoremovethe effectsofstudy-to-studydifferencesinRubiscokineticassays.

(A)Meanandstandarderroroftheactivationenergy(DHa)andthescalingconstant(c)foreachgroupofspeciesafterfittingthecorrected RubiscokinetictraitsobtainedfromtheoriginaldataatthedifferenttemperaturesassayedineachstudybytheArrhenius-typetemperature responsefunction,accordingtoGalme´setal.[19](Supplementarymaterial).Onlytheinitialrisingpartwasfittedinthecaseofk_cat^casthedata abovethethermaloptimumweremissinginmostcases.Poorfitswithr²<0.7wereeliminatedfromtheanalysis.Inthecaseofthespecies reportedbymorethanonestudy,anaverageforeachspeciesRubiscothermaldependencieswasobtainedbeforecalculatingthemeanandthe standarderrorforeachgroupofspecies.Differentlettersindicatestatisticallysignificantdifferences(P<0.05)betweenthethreegroupsof species.Afternormality(Anderson-Darlingtest)andhomogeneityofvariances(Levene’stest)wereconfirmed,meanswerecomparedbythe analysisofvariance(ANOVA)followedbyDuncan’stest.Forthosedatawhosenormalityorhomogeneityofvarianceswasnotconfirmed, Kruskal–WallistestwithBonferronicorrectionformultiplecomparisonwasdoneinsteadoftheANOVA.S_c/o,Rubiscospecificityfactor;K_c,K_o, semi-saturationconstantsforCO2andO2,respectively;kcatc

,themaximumcarboxylaseturnoverrate;n,numberofspeciesforeachRubisco catalyticconstantandgroup.

(B)BoxplotsdepictionofDH_aforRubiscokinetictraitsofeachgroupofspecies.Thehorizontallinesrepresentthemedian,andtheboxand whiskerrepresentthe25to75percentileandminimumtomaximumdistributionsofthedata,respectively.Singlepointsoutofthisrange representthevaluesconsideredoutlayers(Chrysanthellumindicum–DH_aforS_c/o;Agriophyllumsquarrosum,Artemisiavulgaris,Puccinelliadistans, EuphorbiahelioscopiaandDesmodiumintortum–DHaforKc;ElymusfarctusandTephrosiarhodesica–DHaforKo;A.squarrosum,Eragrostistef andArtemisiamyriantha-DHafork^ccat).

(C)Arrhenius-typetemperatureresponsefitsforS_c/o,K_candk_cat^c.Circles,squaresandtrianglesrepresentmeanvaluesforC₄,C₃^coolandC₃^warm speciesateachtemperature,respectively;barsrepresentstandarderrors,anddifferentlettersindicatestatisticallysignificantdifferences

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collectivelysuggestlimitedadaptivechangesinRubisco kinetictemperaturesensitivityinresponsetothespecies thermal environment. Despite the slight trend for a higher thermal sensitivity of Rubisco kinetic traits in C3warm

relativetoC3cool

species(Figure1a),thesediffer- encesarestatisticallynotsignificant,indicativethatfac- torsotherthantheenvironmentaltemperaturemayhave shaped thermal Rubisco adaptation. However, we also note that the recent large-scale screening studies have investigated Rubisco overavery limitedthermalrange, that is 20–30C in Orr et al. [25]. This limitation is particularlyrelevantfork^cat^cthathasathermaloptimum at 50–55C [18]. Furthermore, the current dataset is dominatedbycropswhoseoriginis,inmostcases,warm environments, while the current distribution of their cultivationhasbeenfrequentlyshiftedtocoolertemper- ateregions.Incrops,breedinghasalsoresultedinmulti- plehybridizationsandpolyploidizationandcombinations ofRubiscolargeandsmallsubunitsthatdonotnecessar- ilyoccurinNature.Giventhatsmallsubunitscanplaya criticalroleinRubiscocatalytictraits[29],cropbreeding historyitselfmighthaveresultedin thelossof adaptive signal in Rubisco traits. In fact, a significantly lower thermalsensitivityofkcatc

incropsrelativetowildspecies wasobtainedinthepresentdataset,andalsohigherSc/o

and lower Kc,Ko and/or kcatc

wereobtained at discrete temperatures for crop versus wild species (data not shown). Future studies mapping Rubisco component originandanalyzingtheeffectsofRubiscosmallsubunits on thetemperatureresponseof differentcatalytictraits are neededtotest thishypothesis.

Remarkable isthewidevariability ofdataregardingthe thermalsensitivityofKo,includingpositiveandnegative DHa values ranging from 124 to 136kJmol ¹ in the presentdataset(Figure1b).Althoughtheamountofdata of DHaforKohavesignificantlyincreasedincomparison to previous compilations of the thermal sensitivity of Rubisco kinetics [19], a large variability was also observed even for the same species across different studies,suggesting thepresenceofmethodological problems which preclude obtainingreliable data. Actually, compared to the rest of the kinetic traits, thefit of Ko

valuestoArrhenius-equationwasmuchpoorer(32outof 91 species with r²<0.7 for Ko and 17 out of these 32specieswithr²<0.2).MostKovaluesincludedinthis dataset were obtained indirectly from the inhibition of the carboxylase activity betweentwo differentO2con- centrationsusingtheradiolabelmethod[25,26].Only the values of Ko in Badger and Collatz [21] and in Lehnherretal.[23]werebasedontheoxygen-electrode

DHa

forKo,whichisalsothecaseofdatafrominvivoestima- tions [19]. Recently, Boyd et al. [30] used both radiolabel (indirect method for Ko determination) and membrane inlet mass spectrometry (MIMS; direct method forKodetermination)to studythetemperature responseofRubiscokineticsinArabidopsisandobtained positive and almost identical valuesfor theDHa for Ko

withbothmethods.Toelucidatewhetherthediscrepan- ciesinKotemperatureresponsesareduetotrueinterspe- cific variability or caused by methodological artefacts, moredirectmeasurementsaboutthethermalsensitivity oftheoxygenaseactivityofRubiscoincontrastingspecies are required. Under specific environmental conditions, likehightemperatureanddroughtstress,theoxygenase activityofRubiscoinC3speciesisenhanced,becominga majordeterminant fortheplant carbonbalance. There- fore,havingreliableinformationofthermalsensitivityof Ko is a critical factor for modelling the photosynthesis response tofutureclimaticconditions.

Modelling the assimilatory potential of contrasting Rubisco variants indicates that large improvements may be achieved in the photosynthetic capacity of crops under varying environmental conditions

The simulationanalysisbasedonthekinetictraitsfrom allspeciesincludedinthiscompilationdemonstratesthat Rubiscoadaptationtotemperaturecanleadtosignificant differences in the Rubisco-based assimilatory potential (ARubisco)amongdifferentgroups(Figure2).Forinstance, RubiscofromC3cool

speciesprovideshigherARubiscothan C₃^warmRubiscoatlowertemperatures,independentlyof thechloroplasticCO2concentration (Cc).But,contrarily to theresults of Galme´s et al. [19],ARubisco of C3warm

species was not significantly higher than that of C3cool

speciesatelevatedtemperatures.

Compared with Rubisco from C3 species, C4 Rubisco provided a significantly lower assimilatory potential at temperatures higher than 35C and at lower Cc (120–

200mmolmol ¹), and significantly higher assimilatory potential atlowertemperatures,irrespective ofCc(Fig- ure2).Theresultwasmaintainedwhenconsideringthe different C4 subtypes, although PCK subtype tend to present higher ARubisco than NAD-ME and NADP-ME subtypes attherangeof studiedtemperatures(datanot shown),in accordancewithpreviousreports[16].This surprising finding may be due to the higher thermal sensitivity of Sc/o and the lower thermal sensitivity of kcatc

in C4 Rubiscos (Figure 1a), and indicates a poor performance of C4 Rubisco at high temperatures when

(Figure1LegendContinued)(P<0.05accordingtoeitherone-wayANOVAfollowedbyDuncan’stestorKruskal–WallistestwithBonferroni correction.Thelatterstatisticaltestwasusedwhendatanormalityorhomogeneityofvarianceswasnotconfirmed)betweenthethreegroupsof speciesateachtemperature.SincethetemperaturesusedtomeasureRubiscocatalyticconstantsweredifferentacrossthecompiledstudies,the Arrhenius-typeequationswerefurtherusedtocalculatethediscretevaluesofeachtraitat5,15,25,35and45Cforeachspecies.

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operatesoutsidetherangeofchloroplasticCO2concen- trationswhereithasevolved,butageneralizedimprove- ment in theperformance over C3Rubisco at low temperatures.Thisisanimportantoutcome,suggestingthat

the improvement of the photosynthetic capacity in C3

speciesby transferring C4 Rubiscos will generally only succeed at temperatures 15C, contrary to what was previously suggested by Sharwood et al. [11,16]. The

Figure2

C_c = 120 μmol mol^–1 C_c = 200 μmol mol^–1

C_c = 400 μmol mol^–1

Temperature (°C) Temperature (°C) Group

C_c = 5000 μmol mol^–1

ARubisco (μmol m–2 s–1) ARubisco (μmol m–2 s–1)ARubisco (μmol m–2 s–1)

ARubisco (μmol m–2 s–1) 10

10

100 200 300

0

cool warm

C4

C₃ C₃

15 20

20 30 40 5

5

5 15 25 35 45 5 15 25 35 45

5 15 25 35 45

(a) (b)

(c) (d)

ModellingtheeffectofdifferenttemperatureresponsesofRubiscokinetictraitsinC₃^cool(bluesquares,n=32),C₃^warm(redtriangles,n=28)and C4(greencircles,n=13)ontheRubisco-limitedgrossassimilationrate(ARubisco)atchloroplasticCO2concentrations(Cc)of120(a),200(b), 400(c)and5000(d)mmolmol ¹.ThephotosynthesismodelofFarquharetal.[41]wasusedtomodelARubiscoatthedifferenttemperaturesand C_c,usingthevaluesforthetemperaturedependenceparametersofS_c/o,K_c,K_oandk_cat^cforallspeciesinthecompileddatabase(Supplementary material,onlyspecieswiththeexplainedvarianceoftraitvs.temperatureresponsecurvefits,r²,equalorgreaterthan0.7wereincludedinthe analysis)andaleafRubiscocontentof2gm ²(equivalenttoaconcentrationof29mmolcatalyticsitesm ²).ThespeciesreportedbySharwood etal.[16]weremodelledbyusingtheeffectiveMichaelis-MentenconstantforCO2under21%O2(Kcair

=Kc(1+[O2]/Ko)insteadofindividual valuesofKcandKointhephotosynthesismodel[41].Grossassimilationwassimulatedheretoavoidconfoundingeffectsofmitochondrial respiration.Althoughk_cat^chasathermaloptimum,typicallyat50Corhigher[18],inthisanalysisweonlyusetheactivationenergyfortheinitial risingpartduetolackofthehighertemperatureestimatesofk_cat^cforthebulkoftherecentscreeningstudies.A_Rubiscorepresentsthepotential estimateofphotosynthesisratesupportedbyagivensetofRubiscocharacteristicsunderRuBP-saturatedconditions.DifferentvaluesofC_cwere chosentorepresentatypicalCcofanon-stressedC3plant(200mmolmol ¹),adrought-stressedC3plant(120mmolmol ¹),anon-stressedC3

plantunderincreasedatmosphericCO2(400 mmolmol ¹)andapossibleCcforaC4plant(5000mmolmol ¹).Circles,squaresandtriangles representmeanvaluesforC₄,C₃^coolandC₃^warmspeciesateachtemperature,respectively;barsrepresentstandarderrors,anddifferentletters indicatestatisticallysignificantdifferences(P<0.05accordingtoeitherone-wayANOVAfollowedbyDuncan’stestorKruskal–Wallistestwith Bonferronicorrection.Thenon-parametrictestwasusedwhendatanormalityorhomogeneityofvarianceswasnotconfirmed)betweenthethree groupsofspeciesforeachtemperature.

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4

speciesmightbeofpotentialbenefittoC3-photosynthesis under current andfuture atmospheric CO2 pressuresat temperaturesequalorhigherthan25C.However,Shar- wood et al. [11,16] only compared the assimilatory potentialoftheseC4monocotRubiscoswiththeassimi- latory potential of tobacco and wheat Rubisco, and, as mentioned above,there isawidevariability inRubisco catalytictraitsamong C3species,evenalargestudy-to- study variability for the same species; therefore, it is necessary to be cautious when generalizing modeling responses based on a limited number of species. The effects of Rubiscoreplacementsvaryin dependenceon the native and alien Rubisco characteristics, and it is

photosynthesisimprovementoccursunderdifferentenvi- ronmentalconditionsoutoftheobservedgeneraltrends for C3 and C4 Rubiscos (see Figure 3). Overall, this simulation analysis clearly demonstrates that future attempts to increase the photosynthetic capacity by means of Rubisco design must consider the climatic conditionsinwhichthetargetspecieswillbecultivated, inparticular,theprevalentthermalenvironmentandthe existenceofprocesseslimitingorpromotingthetransfer of atmosphericCO2to thesites ofcarboxylation.

The high variability in the thermal dependency of Rubisco kinetics across Spermatophyta allows testing

Figure3

C_c (μmol mol^–1)

Puccinellia maritima Artemisia myriantha

Saccharum officinarum Panicum amarum

C_c (μmol mol^–1)

Temperature °C

Fold change in ARubisco for T.aestivumFold change in ARubisco for Z. mays Fold change in ARubisco for Z. maysFold change in ARubisco for T.aestivum

Temperature °C C_c (μmol mol^–1)

120200 400

12005000 1200

5000 120200 400

0

5 15 25 35 45

1 2 3 4

0 1 2 3 4

(a) (b)

(c) (d)

FoldchangeoftheRubisco-limitedgrossassimilationrate(A_Rubisco)ofTriticumaestivum(a,b)andZeamays(c,d)afterreplacementofthenative RubiscobyforeignRubiscosofPuccinelliamaritima(a),Artemisiamyriantha(b),Saccharumofficinarum(c)andPanicumamarum(d).The photosynthesismodelofFarquharetal.[41]wasusedtomodelA_Rubiscoatthedifferenttemperaturesandphysiologicalconcentrationsof chloroplasticCO2(Cc)fortheC3andtheC4species,consideringtheRubiscotemperatureparametersforeachspecies(Supplementarymaterial) andaconstantleafRubiscocontentof2gm ².

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howmuchARubiscowouldchangeintwoimportantcrops, wheatandmaize,iftheirnativeRubiscowerereplacedby Rubiscos exhibiting the most extreme temperature responses. As for wheat, Rubisco from Puccinellia maritima, a C3cool

species, would result in higher ARubisco

especially at elevated temperatures (Figure 3a). The replacement of wheat Rubisco by that from Artemisia myriantha, a C3warm

species, would result in a higher CO2 assimilation capacity at temperatures above 30C (Figure 3b). AmongC4plants, Rubisco fromSaccharum officinarum and from Panicum amarum would allow increasingARubiscoofmaizeatlowandhightemperatures, respectively (Figure 3c,d). These results demonstrate thatthere is place for CO2fixation improvement in C3

andC4crops byengineeringRubisco.

Current status of Rubisco transplantation in crops

Although the replacement of the gene coding for the Rubiscolargesubunit(rbcL)oftobaccowithforeignver- sionsfromphylogeneticallyclosespecieshasbeensuccess- ful,thescientificcommunityhasyetfailedtoengineera foreignRubiscosustaining,atleast,theinvivoCO2assimi- lationratesof thenativeRubisco[10,31].Thisismainly related to the inability to express sufficient amount of functional Rubisco, due to problems in the biogenesis, folding andassembly of foreign versions[32].Wefocus onspermatophytesbecausethevastmajorityofavailable data,inparticular,thoseprovidedbythescreeningstudies, correspond to this group. Although Rubisco from other domains of life might have unique characteristics [18,19,33–36], replacement and successful expression offunctionalRubiscotoresultinapositivenetcarbongain undercurrentatmosphericCO2conditionsiscurrentlyonly feasibleamonghigher plants[12,37].Recently, large advancesinunderstandingRubiscobiogenesisandrepair havebeenachieved[38]andfutureeffortswillcontinuein thisdirection[12].Moreover,forhavingsuccessin the increaseoftheinvivoCO2assimilationratesbytransplan- tomic plants withmoreefficient Rubisco versions at warmer temperatures,itmustbealsonecessarytotakeintoaccount thethermalstabilityofRubiscoactivase[18,39].

Conclusion

Thecomprehensive analysisof thedataavailablesofar demonstrates a high variability in the thermal depen- denceofRubiscocatalytictraitsamonghigherplants.Part ofthisvariabilityisrelatedtothephotosynthetictype,but only a limited adaptation to the climatic origin of the species has been observed due to large within group variationamongspeciesfromdifferentthermalenviron- ments.Nevertheless,theextensivevariabilityincludesa numberof uniquespecies whoseRubisco kinetictraits displayadistinctiveresponsetovaryingtemperatureand CO2 concentration. The simulation of the benefits of usingthesespecificRubisco versionsin futureattempts toimprovethephotosyntheticcapacityofimportantcrops

forglobalfoodsecurityoffershighlypromisingrevenues inchangingclimates.

Conflict of interest statement

Nothingdeclared.

Acknowledgements

JeroniGalme´shasbeensupportedbytheSpanishMinistryofEconomyand Competitiveness(grantsAGL2009-07999andAGL2013-42364-R),andthe EuropeanCommission(grant727929).Sebastia` Capo´ hasbeensupported byaFPUGrantfromtheSpanishMinistryofEducation.U¨ loNiinemetshas beensupportedbytheEstonianMinistryofScienceandEducation(team grantPRG537),andtheEuropeanCommissionthroughEuropeanRegional DevelopmentFund(CenterofExcellenceEcolChange).Concepcion In˜iguezhasbeensupportedbyapostdoctoralgrantfromtheGovernmentof theBalearicIslands.WethankTrinidadGarciafortechnicalhelpand organizationoftheradioisotopeinstallationattheServeisCientı´fico- Te`cnicsoftheUniversitatdelesIllesBalears(UIB).

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.

1016/j.pbi.2019.05.002.

References and recommended reading

Papersofparticularinterest,publishedwithintheperiodofreview, havebeenhighlightedas:

ofspecialinterest ofoutstandinginterest

1. MayaudonJ,BensonAA,CalvinM:Ribulose-1,5-diphosphate fromandCO2fixationbyTetragoniaexpansaleavesextract.

BiochimBiophysActa1957,23:342-351.

2. TcherkezGGB,FarquharGD,AndrewsTJ:Despiteslow catalysisandconfusedsubstratespecificity,allribulose bisphosphatecarboxylasesmaybenearlyperfectly optimized.ProcNatlAcadSciUSA2006,103:7246-7251.

3. SavirY,NoorE,MiloR,TlustyT:Cross-speciesanalysistraces adaptationofRubiscotowardoptimalityinalow-dimensional landscape.ProcNatlAcadSciUSA2010,107:3475-3480.

4. NisbetEG,FowlerCMR,NisbetRER:Theregulationoftheair:a hypothesis.SolidEarth2012,3:87-96.

5.

BathellierC,TcherkezG,LorimerGH,FarquharGD:Rubiscois notreallysobad.PlantCellEnviron2018,41:705-716.

Re-examinesRubisco’scatalyticperformancebycomparisonwithother chemicallyrelatedenzymes.

6. PeterhanselC,HorstI,NiessenM,BlumeC,KebeishR, Ku¨rkcu¨ogluS,KreuzalerF:Photorespiration.ArabidopsisBook 2010,8:e0130.

7. EvansJR:Photosynthesisandnitrogenrelationshipsinleaves ofC3plants.Oecologia1989,78:9-19.

8. LongSP,Marshall-ColonA,ZhuX:Meetingtheglobalfood demandofthefuturebyengineeringcropphotosynthesisand yieldpotential.Cell2015,161:56-66.

Reviewsthepotentialandemergingapproachestoimprovecropphoto- syntheticefficiency,andthenewtoolsneededtorealizethesechanges.

9.

OrtDR,MerchantSS,AlricJ,BarkanA,BlankenshipRE,BockR, CroceR,HansonMR,HibberdJM,LongSPetal.:Redesigning photosynthesistosustainablymeetglobalfoodandbioenergy demand.ProcNatlAcadSciUSA2015,112:8529-8536.

Exploresdifferent prospective redesignsof plantsystems atvarious scalesforincreasingcropyieldsthroughimprovedphotosyntheticeffi- ciencyandperformance.

10. FlexasJ,Dı´az-Espejo A,ConesaMA,CoopmanRE,DoutheC, GagoJ,Galle´ A,Galme´sJ,MedranoH,Ribas-CarboMetal.:

MesophyllconductancetoCO₂andRubiscoastargetsfor