the operation of two decarboxylases, transamination, and ... · the operation of two...

The Operation of Two Decarboxylases, Transamination,and Partitioning of C4 Metabolic Processes betweenMesophyll and Bundle Sheath Cells Allows LightCapture To Be Balanced for the Maize C4 Pathway1[W]

Chandra Bellasio* and Howard Griffiths

Physiological Ecology Group, Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA,United Kingdom

ORCID ID: 0000-0002-3865-7521 (C.B.).

The C4 photosynthesis carbon-concentrating mechanism in maize (Zea mays) has two CO2 delivery pathways to the bundle sheath(BS; viamalate or aspartate), and rates of phosphoglyceric acid reduction, starch synthesis, and phosphoenolpyruvate regenerationalso vary between BS andmesophyll (M) cells. The theoretical partitioning of ATP supply betweenM and BS cells was derived forthesemetabolic activities fromsimulatedprofiles of light penetration across a leaf,with apotential 3-folddifference in the fraction ofATP produced in the BS relative to M (from 0.29 to 0.96). A steady-state metabolic model was tested using varying light quality todifferentially stimulate M or BS photosystems. CO2 uptake, ATP production rate (JATP; derived with a low oxygen/chlorophyllfluorescencemethod), and carbon isotope discriminationweremeasured on plants under a low light intensity, which is consideredto affect C4 operating efficiency. The light quality treatments did not change the empirical ATP cost of gross CO2 assimilation (JATP/GA).Using themetabolic model, measured JATP/GAwas comparedwith the predicted ATP demand asmetabolic functions were variedbetweenMandBS.Transaminationandthe twodecarboxylasesystems(NADP-malic enzymeandphosphoenolpyruvate carboxykinase)were critical for matching ATP and reduced NADP demand in BS and M when light capture was varied under contrasting lightqualities.

Interest in the C4 pathway has been increased by thepotential for enhancing crop productivity and main-taining yield stability in the face of global warming andpopulation pressure (Friso et al., 2010; Zhu et al., 2010;Covshoff and Hibberd, 2012). Maize (Zea mays), a C4plant of the NADP-malic enzyme (ME) subtype, is aleading grain production cereal (www.fao.org). C4 photo-synthesis is a shared activity between mesophyll (M; ab-breviations are listed in Table I) and bundle sheath (BS)cells, coupled to allow the operation of a biochemicalcarbon-concentrating mechanism (CCM). The CCM effec-tively minimizes photorespiration by increasing the CO2concentration in the bundle sheath (CBS), where Rubisco isexclusively expressed. Since BS and M are connected byplasmodesmata, some CO2 retrodiffuses. The refixation ofthat escaping CO2 by the CCM increases the activity of theCCMand the total ATPdemand (ATPBS +ATPM) for grossCO2 assimilation (GA; [ATPBS + ATPM]/GA), from atheoreticalminimumoffiveATPs (Furbank et al., 1990).

Leakiness (F), the amount of CO2 retrodiffusing relativeto phosphoenolpyruvate (PEP) carboxylation rate, istherefore a proxy for the coordination between theCCMand assimilatory activity (Henderson et al., 1992; Tazoeet al., 2008; Kromdijk et al., 2010; Ubierna et al., 2011;Bellasio and Griffiths, 2013).

Recently, the maize C4 subgroup has been shown tobe complicated by the presence of two BS decarboxyl-ation enzyme systems (NADP-ME and phosphoenol-pyruvate carboxykinase [PEPCK]), presumably bothacting as CO2 delivery pathways (viamalate [MAL] andAsp, respectively; Furumoto et al., 1999, 2000; Wingleret al., 1999; Eprintsev et al., 2011; Furbank, 2011; Picket al., 2011). There is also an extensive overlap betweenBS andM functions, since both cell types can synthesizestarch (Spilatro and Preiss, 1987; Kanai and Edwards,1999) and reduce phosphoglyceric acid (PGA; Majeranand van Wijk, 2009; see the overall scheme in Fig. 1).Additionally, energetic partitioning can also varybetween cell types, since the total ATP produced (JATP)per CO2 fixed in GA (JATP/GA) may be produced in BS(mainly through cyclic electron flow [CEF] around PSI)or in M (mainly through linear electron flow [LEF]),depending on the light locally available in BS or M(Kramer and Evans, 2011; Yin and Struik, 2012). Fur-thermore, although all NADPH is produced in M, theonly compartment operating linear electron transportand oxidizing water, some NADPH is exported to BSthrough MAL diffusion, to meet the reducing powerdemand therein (NADPHBS). To capture the complex C4physiology, several models of C4 photosynthesis have

1 This work was supported by the European Union SeventhFramework Programme Marie Curie Initial Training Network Har-vest program (grant no. 238017).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Chandra Bellasio ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.113.228221

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http://orcid.org/0000-0002-3865-7521

http://www.fao.org

mailto:[email protected]

http://www.plantphysiol.org

mailto:[email protected]

http://www.plantphysiol.org/cgi/doi/10.1104/pp.113.228221


been developed (Berry and Farquhar, 1978; Laisk andEdwards, 2000, 2009; vonCaemmerer, 2000). The earlierapproaches were developed into the von Caemmerer(2000) C4 model. In particular, the associated light-limited equations (referred to subsequently as the “C4model”) are used to estimate the parameters needed toresolve the isotopic discrimination (D) model, widelyemployed to study F under low-light conditions (forreview, seeUbierna et al., 2011). TheC4model partitionsJATP into two fractions: (1) the ATP consumed by PEPcarboxylase, and (2) the ATP consumed by the C3 ac-tivity (glyoxylate recycling, PGA reduction [PR], andribulose 1,5-bisphosphate [RuBP] regeneration). Theseactivities are located in M, BS, or both compartments(see the overall scheme in Fig. 1).However, theC4modelsimplifies the spatial compartmentalization between BSandM, and in this paper, we now develop the energeticimplications of the differential contribution ofM and BSto C4 photosynthesis under different light regimes.

Because of these anatomical, metabolic, and energeticcomplexities, C4 metabolism is highly sensitive to lim-iting light intensity (Bellasio and Griffiths, 2013) and,potentially, light quality (Evans et al., 2007). Lightquality has a greater influence on C4 photosynthesisthan on C3. Leaf pigments preferentially absorb the blueand red region of the spectra, and some wavelengthspenetrate deeper into leaves. It was shown in C3 leavesthat exposure to different wavelengths results in char-acteristic light penetration profiles, which, translatedinto different gradients in PSII yield, rates of ATP pro-duction, and assimilation (A) within the leaf (Terashimaet al., 2009). In C4 leaves, because of the concentric anat-omy, light reaches M cells before the deeper BS (Evanset al., 2007) and could alter the balance between lightharvesting and energetic partitioning betweenBS andM.

In this paper, we model the likely profiles of lightpenetration for specific wavelengths associated withred, green, and blue light within a maize M and BS leafcross section and calculate the impact on potential ATPproduction for each cell type. We calculate the pro-portion of absorbed light (AB) for each wavelength,expressed asABBS/M, the fraction of photons absorbedin BS relative to the photons absorbed inM, fromwhichwe derive JATPBS/JATPM, the fraction of ATP produced inBS relative to the ATP produced in M. Second, we de-veloped a steady-statemetabolicmodel (Fig. 1; Table II),which augments the conventional C4 model (vonCaemmerer 2000), to capture the spatial separationbetween BS and M and partitions the ATP demand be-tween BS and M cells in terms of PR, starch synthesis(SS), and PEP regeneration, so as to meet the ATPavailability in each cell type (Evans et al., 2007). Third,photosynthetic characteristics (leaf-level ATP produc-tion rate, CO2 assimilation, stomatal conductance, andF derived fromonline carbon isotope discrimination [D])were measured under red, green, and blue light, andred, green, and blue light in combination (RGB), using adecreasingphotonfluxdensity (from500 to 50mEm22 s21)to investigate the importance of metabolic plasticity un-der limiting light intensities.

Table I. Variables and acronyms described in the text

Abbreviation Definition Unit

A Net assimilation mmol m22 s21

AB Absorbed lightAB BS/M Partitioning of absorbed light DimensionlessATPBS ATP demand in BS mmol m22 s21

ATPM ATP demand in M mmol m22 s21

BS Bundle sheathCBS CO2 concentration in BS mmol mol21

CCM Carbon-concentrating mechanismCEF Cyclic electron flowDHAP Dihydroxyacetone phosphateETR Electron transport rate mmol m22 s21

GA Gross assimilation (A + RLIGHT) mmol m22 s21

gBS Bundle sheath conductance to CO2,calculated by fitting JMOD to JATP

mol m2 s21

IRGA Infrared gas analyzerJATP Total ATP production rate mmol m22 s21

JATPBS ATP production rate in BS mmol m22 s21

JATPM ATP production rate in M mmol m22 s21

JMOD Modeled ATP production rate mmol m22 s21

LEF Linear electron flowLCP Light compensation pointM MesophyllMAL MalateMDH Malate dehydrogenaseMDHBS Malate dehydrogenase reaction

rate in BSmmol m22 s21

MDHM Malate dehydrogenase reactionrate in M

mmol m22 s21

ME Malic enzymeME Malic enzyme reaction rate mmol m22 s21

NADPHBS NADPH demand in BS mmol m22 s21

NADPHTOT Total NADPH demand mmol m22 s21

OAA Oxaloacetic acidPAR Photosynthetically active radiation mE m22 s21

PEP PhosphoenolpyruvatePEPCK Phosphoenolpyruvate carboxykinasePEPCK PEPCK reaction rate mmol m22 s21

PGA 3-Phosphoglyceric acidPPDK Pyruvate phosphate dikinasePPDK PPDK reaction rate mmol m22 s21

PR PGA reductionPRBS PR rate in BS mmol m22 s21

PRM PR rate in M mmol m22 s21

RBS Respiration in the light in BS mmol m22 s21

RLIGHT Respiration in the light mmol m22 s21

RPP Reductive pentose phosphateRuBP Ribulose-1,5-bisphosphateRuP Ribulose-5-phosphateSS Starch synthesisSSBS Starch synthesis rate in BS mmol m22 s21

SSM Starch synthesis rate in M mmol m22 s21

SSTOT Total starch synthesis rate mmol m22 s21

T Transamination rate mmol m22 s21

VC Rubisco carboxylation rate mmol m22 s21

VO Rubisco oxygenation rate mmol m22 s21

VP PEP carboxylation rate mmol m22 s21

Y(II) Yield of PSIID 13C isotopic discrimination ‰

d13C 13C isotopic composition relativeto Pee Dee Belemnite

‰

F Leakiness Dimensionless

Plant Physiol. Vol. 164, 2014 467

Plasticity of C4 Biochemistry

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For instance, AB BS/M and JATPBS/JATPM were bothlower under the blue light (wavelength 460 nm), whichis rapidly extinguished within the M leaf profile, thanunderwhite light, confirming that light quality perturbsC4 energetics. In spite of this shift, when maize plantswere exposed to different light qualities, there was nochange in F, indicating that, at steady state, the coordi-nation between CCM activity and Rubisco assimilationwas retained (Ubierna et al., 2011; Sun et al., 2012). Themodeled metabolic plasticity projected a window forATP demand partitioning (ATPBS/ATPM), whichmatched the values for JATPBS/JATPM supply estimatedunder red, green, and blue light. We show that theplasticity of C4 metabolism, and in particular the possi-bility of shifting between MAL and Asp as a primarycarboxylase product, was of pivotal importance in

allowing the plasticity of ATP and NADPH demand. Inconclusion, our study explains the extensive overlap be-tween BS and M functions and the requirement for atleast two decarboxylase systems in NADP-ME subtypeplants such as maize, providing an explanation for em-pirical observations on the diversity of decarboxylaseactivities and PEP regeneration pathways (Rathnam,1978; Chapman and Hatch, 1981; Wingler et al., 1999;Eprintsev et al., 2011; Furbank, 2011; Pick et al., 2011).

RESULTS

Metabolic Modeling of Partitioning between BS and M

The complexity of C4 biochemistry (Furbank, 2011)was first integrated in a comprehensive steady-state

Figure 1. Metabolic model of C4 assimilation, rates of reaction, and net fluxes between BS and M. The overall scheme reportsthe reactions of the CCM (Furbank, 2011), Rubisco carboxylation, the reactions of the RPP pathway, the synthesis of starch,respiration, and glyoxylate recycling reactions. The tables, with the corresponding enzyme names, show the actual reactionrates, expressed relative to GA (5.13 mmol m22 s21), per unit of substrate transformed. Rates were estimated by parameterizing themodel equations (Table II) with data measured under PAR = 125 mE m22 s21 (A = 3.96 mmol m22 s21; RLIGHT = 1.17 mmol m22 s21;JATP = 28.6 mmol m22 s21), the output of the C4 model (VC = 5.35 mmol m22 s21; VP = 5.89 mmol m22 s21; VO = 0.44 mmol m22 s21),and the output of the D model (F = 0.23) under three characteristic ratios of ATP partitionings. These were numbered 1, 2, and 3.Condition 1 corresponds to the lowest ATP available in BS (ATP partitioning similar to that under blue light; Fig. 4B),condition 2 corresponds to an intermediate ATP availability in BS (ATP partitioning equal to that under red light; Fig. 4B), andcondition 3 corresponds to the highest ATP available in BS (ATP partitioning equal to that under green light; Fig. 4B). The insetshows net metabolite fluxes between M and BS in multiples of GA. The ATP demand in BS (ATPBS) and M (ATPM), the totalNADPH demand (NADPHTOT), and the NADPHBS were also calculated in the same three relevant conditions. PYR, Pyruvic acid.

468 Plant Physiol. Vol. 164, 2014

Bellasio and Griffiths



Tab

leII.Steady-stateeq

uationsforthemetab

olicmodel

ofC4assimilation

Proce

sses

described

byEq

uations4to

10ca

nbeca

lculateddirec

tlyfrom

themea

sureddataforA,RLIGHT,an

dtheoutputofthevo

nCaemmerer

C4model

(VO,VP,an

dVC),whileEq

uations11to

21requireprioralloca

tionofSS

,PR,an

dPEP

CK.Fo

rsimplicity,en

zymenam

esin

italicsrepresenttheen

zymereac

tionrate.Fo

rstoichiometricco

nsisten

cy,reac

tionratesareca

lculatedas

rates

ofsubstrate

tran

sform

ation.

Process

Symbol

ReactionRate

Equation

Localization

Notes

Gross

assimilation

GA

AþRLIGHT

(4)

GAan

dRLIGHTratesareex

pressed

per

CO

2.

RuP phosphorylation

–V

CþV

O(5)

BS

RuPphosphorylationsupplies

Rubisco

carboxy

latingac

tivity

(VC)toge

ther

with

oxy

genatingac

tivity

(VO).

TotalPR

PRTOT

2VCþ

3 2V

O2

1 3RLIGHT

(6)

BSan

dM

Thiseq

uationca

lculatesthetotalrate

ofPRonthebasisofthePGAproduce

dby

Rubisco

carboxylatio

n(2VC),Rubisco

oxygenation(V

O),andglyoxylaterecycling(0.5VO)

andconsidersthePGAconsumed

byrespiration;1/3

isthestoichiometricconversion

betweenrespiration(expressed

per

CO

2)andPR(expressed

per

triose).

TotalNADPH

dem

and

NADPH

TOT

PRTOTþ

1 2V

O(7)

BSan

dM

PRco

nsumes

oneNADPH

per

PGA;thetotalrate

ofPRisPRTOT(see

note

toEq

.6);in

glyo

xylate

rege

neration(per

glyo

xylate),0.5

NADH

isproduce

dbyGly

dec

arboxy

lase,0.5

NADH

isco

nsumed

byhyd

roxy

pyruvate

reductase,

andone

ferred

oxin(equivalen

tto

0.5

NADPH)isco

nsumed

byGln

synthetase;

intotal,0.5

NADPH

isco

nsumed

per

glyo

xylate

(equivalen

tto

VOrate;Su

pplemen

talTable

S1;

Yoshim

ura

etal.,2004).

DHAPen

tering

RPP

–PRTOT2

SSTOT

(8)

BS

TheDHAPen

teringtheRPPpathway

correspondsto

thetotalPRrate

minusthe

DHAPusedforstarch

synthesis,whichin

thiswork

isexpressed

per

triose.

TotalSS

SSTOT

1 3A

(9)

BSan

dM

Inthismodel,assimilationisen

tirely

converted

tostarch

;thisassumptiondoes

not

influen

ceen

erge

tics,as

starch

synthesishas

thesameATPdem

andas

phloem

-load

edSu

c;in

Equation9,1/3

convertsthestoichiometry

ofA(exp

ressed

per

CO

2)

tothestoichiometry

ofSS

(exp

ressed

per

triose).

TotalPEP

rege

neration

–V

P(10)

BSan

dM

PEP

rege

nerationrate

equalsPEP

consumptionrate

VPat

steadystate;

PEP

canbe

regenerated

either

byPPDK(m

ainly

inM

butactive

also

inBS)

orbyPEP

CKin

BS;

inthisstudy,

PPDKactivity

was

assumed

tobezero

inBS.

TotalATPdem

and

ATPBS+

ATPM

PRTOTþV

Cþ2V

Oþ

1 2SS

TOTþPEPCKþ2P

PDK

(11)

BSan

dM

Equation11ca

lculatesthetotalATPdem

andas

thesum

ofATPdem

andforPR(one

ATPper

PGA,co

rrespondingto

PR),RuBPrege

neration(oneATPper

RuP,

correspondingto

VC+VO),glyo

xylate

recycling(oneATPper

glyo

xylate,

correspondingto

VO),starch

synthesis(0.5

ATPper

triose,co

rrespondingto

1 2SS

),an

dPEP

rege

neration(oneATPper

PEP

CKca

talyticeven

tortw

oATPper

PPDK

catalyticeven

t);co

mpared

withtheoriginal

form

ulationoftheC4model,Eq

uation

11separates

theATPdem

andforPEP

CKan

dPPDK,includes

theATPdem

andfor

SS,an

dco

nsidersthePGAutilizedbyrespiration,whichdoes

notnee

dto

be

reduce

d(see

Eq.6).

ATPdem

andin

BS

ATPBS

PRBSþV

Cþ2V

Oþ

1 2SS

BSþPEPCK

(12)

BS

TheATPdem

andin

BSisbrough

tab

outbyPR(attherate

ofPRBS),RuBPrege

neration

(attherate

ofVC+VO),glyo

xylate

recycling(attherate

ofVO),starch

synthesis(0.5

ATPper

triose),an

dPEP

CKac

tivity

(oneATPper

OAA;seenote

toEq

.11).

ATPdem

andin

MATPM

2PPDKþ

1 2SS

MþPRM

(13)

MTh

eATPdem

andin

Misbrough

tab

outbyPR(attherate

ofPRM),SS

,an

dPPDK(two

ATPsper

pyruvicac

id;seenote

toEq

.11).

NADPH

dem

and

inBS

NADPH

BS

PRBSþ

1 2V

O(14)

BS

TheNADPH

dem

andin

BSisbrough

tab

outbyPR(oneNADPH

per

PGA)an

dglyo

xylate

recycling,

whichco

nsumes

0.5

NADPH

per

glyo

xylate

(corresponding

toVO;seeSu

pplemen

talTable

S2).

(Tab

leco

ntinues

onfollowingpage.)




http://www.plantphysiol.org/cgi/content/full/pp.113.228221/DC1



Tab

leII.(Continued

from

previouspage.)

Process

Symbol

ReactionRate

Equation

Localization

Notes

NADPH

supply

toBS

–MDH

M(15)

BS

AllNADPH

available

inBSisproduce

din

Man

dex

ported

through

theMALshuttle

bec

ause

wehaveassumed

that

nolinearelec

trontran

sport(i.e.water

oxidation)

occ

urred

inBS;

forthisreason,theNADPH

supply

toBSco

rrespondsto

the

NADPH

consumed

toreduce

OAAto

MALin

M,theproce

ssresponsible

for

NADPH

export,an

dnotto

therate

ofMALdecarboxy

lationin

BS,

whichdep

ends

onT,PEP

CK,an

dMDH

BS(Eq.19).

MDH

activity

inM

MDH

MPRBSþ

1 2V

O(16)

MMDH

activity

supplies

theNADPH

dem

andin

BS;

Equation16was

derived

from

Equations14an

d15.

Tran

samination

TV

P2MDH

M(17)

BSan

dM

Equation17ex

presses

that,at

stea

dystate,

allOAAiseither

tran

saminated

or

reduce

d;since

Tbyp

assestheMDH

Mreac

tion,whichisthereac

tionresponsible

forNADPH

exportto

BS(see

note

toEq

.15),Thas

thefunctionofbalan

cing

NADPH

supply

anddem

and,whichbec

omes

apparen

twhen

Equations15an

d17

areco

mbined

.MDH

MDH

BS

T2

PEP

CK

(18)

BS

MDH

isassumed

tooperateafastco

nversionat

equilibrium;therefore,itispassively

regu

latedbythesubstrate

availability:

theOAAthat

isnotusedbyPEP

CKis

reduce

dto

MALbyMDH;MDH

may

use

NADH,since

noNADPH-dep

enden

treductionofOAAhas

bee

nobserved

inmaize

(Kan

aian

dEd

wards,1999)an

ditis

like

lymitoch

ondrial

(Rathnam

,1978;Chap

man

andHatch

,1981);theNADH

rege

nerationmay

beca

rriedoutbych

loroplastic

ME,

whichisreported

toreac

tboth

withNADPan

dNAD

(Chap

man

andHatch

,1981);however,theproce

ssmay

bemore

complicated(Eprintsev

etal.,2011,an

drefs.therein);note

that

inthis

study,

weassumed

that

cellsaredecompartm

entalize

dwhilePEP

CKrate

was

man

ipulatedto

increa

sebetwee

nze

roan

damax

imum

rate

inresponse

toATP

availability(see

“Minim

um

andMax

imum

BSAllocation”fordetails).

ME

ME

MDH

M+MDH

BS

19

BS

Equation19ex

presses

that

therate

ofMALoxidationbyMEco

rrespondsto

therate

of

MALproduce

dbyMDH

activity

inM

plustherate

ofMALproduce

dbyMDH

activity

inBS.

PPDK

PPDK

VP2

PEP

CK

20

MTh

ePEP

rege

nerated

byPEP

CKin

BSdiffusesto

Man

dreduce

stherequirem

entofPEP

rege

nerated

byPPDKin

M.

PRin

MPRM

PRTOT2

PRBS

21

MPRisashared

proce

ssbetwee

nBSan

dM.





model (Fig. 1), with key processes described by rateequations (Fig. 1, box; Table II) and associated ATPdemand (Eqs. 11–13 in Table II). Thismodel captures thespatial separation between BS and M, the differentpathways of the CCM (through Asp and MAL), thedifferent carboxylating enzymes, and the process of SS,as a means to develop the traditional C4 model (vonCaemmerer 2000). The metabolic model (Fig. 1) wasgenerated on the assumption that ATP does not freelydiffuse betweenBS andMandany light-induced JATPBS/JATPM fluctuations have to be countered by changing thepartitioning of ATP demand (for a list of abbreviations,see Table I). Figure 1 depicts the reactions that arelocalized in BS, inM, or in both compartments. Ribulose-5-phosphate (RuP)phosphorylation isuniquely localizedinBS to supplyRuBPdirectly inproximity toRubiscoandfacilitate the substrate saturation of the enzyme. Gly-oxylate recycling is also a BS-exclusive reaction (Fig. 1;Yoshimura et al., 2004). This feature contributes to theCCM (the so-called C2 cycle), and in an evolutionaryperspective, it was acquired at an early stage (Sage et al.,2012; Schulze et al., 2013). PEP regeneration throughPEPCK is located uniquely in BS (Wingler et al., 1999),while PEP regeneration through pyruvate phosphatedikinase (PPDK) is located primarily in M (Fig. 1; Frisoet al., 2010; Majeran et al., 2010), and any PPDK activityin BS is generally neglected (von Caemmerer, 2000; see“Discussion”). PR, respiration, and SS are processes lo-cated both in BS andM (Spilatro and Preiss, 1987; Kanaiand Edwards, 1999; Majeran and van Wijk, 2009; Frisoet al., 2010).These processes are described in detail below, after an

initial comparison of modeled light profiles and mea-sured photosynthetic characteristics under red, green,and bluewavelengths, to give a quantitative descriptionof the biochemical mechanisms underpinning acclima-tion, fluxes and reaction rates, the dynamics of F, andthe effects on the total and relative ATP demand forassimilation.

Effect of Light Quality on ATP Production in BS and M

To study the influence of light quality on JATPBS/JATPM,we first modeled C4 anatomy (Fig. 2, left). Light pene-tration was modeled in two characteristic profiles usingthe absorption-scattering theory (Fig. 2, right). Profileswere calibrated with leaf transmittance and reflectanceat different wavelengths. Blue light was strongly ab-sorbed (steep profile), green light was weakly absorbed(gradual profile), while red light had an intermediateprofile of light penetration. These three profiles wereintegrated to estimate the contribution of absorbed lightwithin adaxial + abaxialM, interveinalM, andBS (Fig. 2)and calculated the partitioning of AB BS/M under fiverelevant conditions (Table III). AB BS/M was used inturn to estimate JATPBS/JATPM (Table III) under theassumption that photochemical yield did not varythrough the leaf profile (see “Materials and Methods”).At 400 nm, AB BS/M was lowest, representing JATPBS/

JATPM of 0.29; at 540 nm, AB BS/Mwas highest togetherwith JATPBS/JATPM (0.96). Under blue light, JATPBS/JATPMwas close to the lowest value (0.31), increasingunder redlight (0.68), natural white light (0.76), and green light(0.80). These valueswere derived independently of lightintensity, so they can be considered to represent theactual ATP availability in BS andM under a wide rangeof light intensities. Since ATP does not diffuse betweenBS andMand has a relatively small pool, at steady state,JATPBS/JATPM can be directly compared with ATPBS/ATPM. For this comparison, the values for JATPBS/JATPMunder blue, red, white, and green light were used sub-sequently to plot Figure 4B below. The model predictsthat light quality will unbalance the partitioning of ATPproduction. A comprehensive ecophysiological investi-gation was undertaken to study how assimilation andconversion efficiency would be affected.

Effect of Light Quality on Assimilatory Traits

Figure 3 shows the responses of maize to differentlight qualities (red, green, blue, or RGB) measured un-der decreasing irradiance. NetA, measured through gasexchange (Fig. 3A), and JATP, measured with the lowoxygen-electron transport rate (ETR) method using asaturating light pulse (Fig. 3B),were significantly higherunder red light and decreased under RGB, green, andblue. Light quality had no significant effect on stomatalconductance (Supplemental Fig. S1A), but Ci/Ca (for sub-stomatal CO2 concentration/cuvette CO2 concentration)was lower under red light (Supplemental Fig. S1B) as aconsequence of the higher A. The CBS, estimated by fit-ting a C4 photosynthesis model to JATP, was higher

Figure 2. Light penetration in a maize leaf. At left is the modeledmaize anatomy. A square BS is surrounded by three portions of M:interveinal M (MI), adaxial M (MAD), and abaxial M (MAB). Epidermiswas approximated as a flat reflecting surface. Light penetration wasstudied through profiles P1 and P2. At right are P1 light profiles(thick lines) and P2 light profiles (thin lines) calculated with theKubelka-Munk (absorption + scattering) theory and calibrated withspectroscopic data (Table III). Radiation is expressed as the sum ofdownward + upward photon flux, as a fraction of incident photonflux (dimensionless), and plotted against the depth in the absorbingpath of the leaf.







under red and green light (Supplemental Fig. S1C) be-cause of the higher A. The light compensation point(LCP), BS conductance (gBS), and respiration in the light(RLIGHT)were not significantly influencedby light quality(Table IV).

With the precise estimate of RLIGHT and JATP, we cal-culated JATP/GA, which represents the experimentallydetermined ATP cost for gross assimilation. JATP/GAwas not influenced by light intensity but varied betweenlight qualities from 5.73 to 5.37 under blue, red, RGB,and green light. JATP/GA was then used in Figure 4B,plotted against the JATPBS/JATPM found above. The rela-tively minor increase in JATP/GA (approximately 0.4ATP per CO2) observed experimentally contrasts withthe metabolic disruption theoretically predicted underthese conditions (Evans et al., 2007). Our data weresupported by real-time D (Fig. 3C) and F (Fig. 3D),which were not influenced by light quality, showingthat the plant coped with a 2.53 shift in JATPBS/JATPMwithout any imbalance in the coordination of CCM ac-tivity and assimilation. Obtaining F data under suchlow light intensities represents a technical challenge,and the experimental protocol was carefully optimizedfor low light intensity (Bellasio andGriffiths, 2013). Dataobtained under low light are subject to a potential errormagnification due to the small difference in CO2 con-centration between the air entering and leaving thecuvette (Supplemental Fig. S2; Evans et al., 1986), asclearly shown by the large error bars in Figure 3D, andtherefore should be interpreted with care. F, JATP/GA,and the window of JATPBS/JATPM formed the basis of ametabolic model used to describe these biochemicalresponses.

Influence of BS Activity on Assimilatory Metabolism andATP Demand (Total and Relative)

The comprehensive metabolic model (Fig. 1) wasdeveloped to describe the biochemical reactions directlyand indirectly involved in C4 assimilation by rateequations (Table II). The proportions of three assimila-toryATP-consumingprocesses (SS, PR, andPEPCK)weremanipulated to increase within BS (allocated; Fig. 4A).These processes overlap between BS and M and couldbe allocated to BS without influencing the overall

assimilation rate. By means of this progressive alloca-tion, we predicted the following: (1) the minimum andmaximum ATPBS/ATPM (Eqs. 12 and 13 in Table II);(2) the reaction rates and metabolite fluxes at a givenATPBS/ATPM, including the rate of PEPCK and PPDK,the relative CO2 flux through Asp and MAL, and thepartitioning of PR between BS and M; and (3) the dy-namics of (ATPBS +ATPM)/GA at variableATPBS/ATPM.

When theBS allocation rate of PR, PEPCK, andSSwaszero (referred as condition 1; no. 1 in Figs. 1 and 4B), thepredicted ATPBS/ATPM was lowest (0.27). This valuewas comparable to JATPBS/JATPM resolved from the op-tical model under blue wavelengths (400 and 460 nm;Table III), showing that metabolism could reduce ATPdemand to match even the lowest ATP supply in BS. Inthis condition, the ATP demand in BS was broughtabout by RuP phosphorylation and glyoxylate recy-cling, two processes that are known to be exclusive toBS. The predicted (ATPBS +ATPM)/GAwas 5.74 (Fig. 4B,dashed line), in agreement with JATP/GA measuredunder blue light (5.73; Fig. 4B, blue square). Since pho-tosynthetic PGA production is exclusive to BS (fromRubisco carboxylase or oxygenase activity, and glyox-ylate recycling), when there is no ATP available for PR(e.g. in condition 1), the PGAdiffuses toMand isreducedto dihydroxyacetone phosphate (DHAP) therein. The pre-dicted NADPHBS, therefore, was the lowest (Eq. 14 inTable II; Fig. 4A, dotted line), corresponding to theNADPH demand for glyoxylate recycling in BS. DHAPsupplies SS inM and diffuses back to BSwhere it is usedto regenerate RuBP. The activity of malate dehydrog-enase (MDH) in M (MDHM), a process responsible forexporting NADPH, was reduced by diverting the sub-strate oxaloacetate (OAA) to transamination to Asp.Hence, in condition 1, MDHM had the lowest activity(Fig. 1; Eq. 16 in Table II), while transamination hadthe highest rate (Figs. 1 and 4A, dashed line; Eq. 17 inTable II). Once Asp diffused to BS, it underwent a futilereduction-oxidative decarboxylation that resulted in netCO2fluxwithout a conjointNADPH translocation (Fig. 1).

When PR and SS were progressively allocated in BS,the predicted ATPBS/ATPM (Figs. 1 and 4) progres-sively increased. The activation of PEPCK in BS notonly contributed to the predicted increasing ATP de-mand in BS but also lowered the predicted ATP cost of

Table III. Energy partitioning between BS and M at different wavelengths

Measured leaf reflectance and transmittance (Woolley, 1971) was used to parameterize the opticalmodel (Fig. 2) to calculate the likely profiles of light penetration at different wavelengths. AB BS/M wascalculated integrating such light absorption profiles (Fig. 2, right) over the corresponding BS and M areas(Fig. 2 left) and used to calculate JATPBS/JATPM (Eq. 3).

Wavelength Description Reflectance Transmittance AB BS/M JATPBS/JATPM

nm %

400 Lowest AB BS/M 4 0.1 0.15 0.29460 Blue light-emitting diode used 5 1 0.16 0.31635 Red light-emitting diode used 6 7 0.34 0.68400–700 Natural white light 8 9 0.38 0.76522 Green light-emitting diode used 8 11 0.40 0.80540 Highest AB BS/M 13 23 0.48 0.96







assimilation (ATPBS + ATPM)/GA, because PEPCK re-generates PEP with half the ATP demand of PPDK.Condition 2 (no. 2 in Figs. 1 and 4) represents a statewhere ATPBS/ATPM equals JATPBS/JATPM resolved fromthe optical model under red light (0.68; Table III). Thepredicted (ATPBS + ATPM)/GA (5.45; Fig. 4B, dashedline) agreed with JATP/GA measured under red light(5.47; Fig. 4B, red square).

When the projected allocation of PR, PEPCK, and SSto BS was highest (referred to as condition 3; no. 3 inFigs. 1 and4A), thepredictedATPBS/ATPMwas 0.8. Thispartitioning equals JATPBS/JATPM estimatedby the opticalmodel under green light and is similar to JATPBS/JATPMestimated under natural white light (0.76; Table III).Because PEPCK was activated at the highest rate, only70%of PEPwas regenerated throughPPDKand (ATPBS +ATPM)/GA was lowest (5.39; Fig. 4B, dashed line), pre-dicting well JATP/GA values measured under RGB (5.38;Fig. 4B, RGB square) and green light (5.37; Fig. 4B, greensquare). In condition 3, PR in BS was highest, de-termining the highestNADPHBS (Figs. 1 and 4A, dottedline; Eq. 14 in Table II) and the highest MDHM activity(Fig. 1; Eq. 16 in Table II). Because most of the OAAproduced by PEP carboxylase was reduced by MDHM,the transamination rate was lowest, just enough tosupply PEPCK activity in BS.

Estimate of Actual Reaction and Diffusion Rates

Since allC4 reactionsweredescribedby rate equations(Table II), we could estimate actual reaction rates. Al-though the predictions shown so far (including the op-tical part) are largely independent of light intensity, inthis step, rates had to be calculated at a specific lightintensity. However, in order to compare these values inawider range of light intensities, rateswere expressed asrelative to GA. This also had the advantage of avoidingany computational distortion caused by respiration inthe vicinity of the light compensation point. The irra-diance of 125 mE m22 s21, characteristic of illuminationin the shade, was chosen because ongoing studies onlow light and light quality are relevant to the physiologyof shading (see “Discussion”). Furthermore, since lowlight provides a means to directly manipulate C4 me-tabolism, a great deal of comparable work has beenundertaken both in this laboratory (Kromdijk et al.,2008, 2010; Bellasio and Griffiths, 2013) and by otherinvestigators (Ubierna et al., 2011, 2013) under low ir-radiances. Reaction rates, shown in the boxes withinFigure 1, were obtained by parameterizing the modelwith the data obtained during the experiment and withthe output of the C4model (all equations are reported inSupplemental Table S1; see also Bellasio and Griffiths[2013] and refs. therein). Rates were calculated for thethree relevant conditions mentioned above (i.e. condi-tion 1 corresponds to the lowest ATP demand in BS,condition 2 corresponds to an intermediate ATP de-mand [matching ATP supply under red light], andcondition 3 correspond to thehighestATPdemand inBS[matching ATP supply under green light]). Conditions1, 2, and 3 are numbered 1, 2, and 3 in Figures 1 and 4.

DISCUSSION

The implications of the metabolic model for parti-tioning ATP demand are first considered in terms ofprevious studies of C4 decarboxylases in NADP-ME

Figure 3. Maize responses to decreasing light intensity under differentlight qualities. A, Net A. The curves were fitted in order to calculate thelight compensation point (Table IV). The inset shows a magnification.B, JATP, measured with the low oxygen-ETR method. C, Online D duringphotosynthesis. D, F resolved from D. Error bars represent SE; n = 4.B, Blue light; G, green light; R, red light.






systems. The resultant ATP partitioning and metabolicplasticity provided by these processes are then consid-ered in terms of overall C4 energetic limitations (Evanset al., 2007). Finally, we consider the implications formultiple decarboxylase origins and function in terms ofC4 pathway evolution as well as light use and energypartitioning within a C4 crop canopy.

Modeling ATP Demand: Decarboxylase Diversity inC4 Systems

Recent developments in C4 research have highlightedthe complexity of C4 metabolism in terms of extensiveoverlapping of BS and M functions (Friso et al., 2010;Majeran et al., 2010), the presence of transamination andtwo distinct decarboxylating pathways (Meister et al.,1996;Wingler et al., 1999; Pick et al., 2011), andplasticityin MAL metabolism (Eprintsev et al., 2011, and refs.therein). Although an involvement in balancing theenergetic capacity in response to environmental condi-tions has been proposed (Furbank, 2011), empirical ev-idence and an associated metabolic model were neededto validate this suggestion. In this study, we tested thecapability of metabolism to respond to different ATPallocations between BS and M by means of a compre-hensive metabolic model. To overcome uncertainties inthe causal relationship between ATP availability andenzyme kinetics, we deduced the highest and lowestpossible BS reaction rates from physiological consider-ations and studied how the predicted ATPBS/ATPMwould vary in response to incremental activation. Wefound thatATPBS/ATPMcouldvary between 0.27 to 0.80if SS, PR, and PEP regeneration were freely allocatedbetween BS and M. In particular, the rate of PEP re-generation in BS was modulated by manipulating theengagement of PEPCK. The availability of PEPCK in BSand the possibility to engage PEPCK at a variable ratehad a twofold importance. Firstly, it expanded thewindow of ATPBS/ATPM to match the predicted win-dow of JATPBS/JATPM. Secondly, it allowed to closelypredict the decreasing ATP cost of GA at increasingJATPBS/JATPM, observed experimentally. In other words,metabolism could take advantage of the increased ATPavailability in BS under penetrating light quality byactivating PEPCK, which regenerates PEP with half theATP cost of PPDK in M. In addition, we suggest thathigher ATP consumption in BS than the predictedmaximum could result from the transient activation ofPPDK in BS (Aoyagi and Nakamoto, 1985; Friso et al.,

2010) or in a long-term response from the de novo syn-thesis of PR enzymes. This shows the importance of thepresence of PPDK in BS and the possibility of metab-olism to regulate the maximum BS rate of PR in re-sponse to contrasting environmental conditions. Theseprocesses could take advantage of the increased ATPavailability in BS under 540-nm green light (Table III)or under high irradiances when ATP production in M isreduced (because of PSII yield quenching; SupplementalFig. S3).

The extensive overlap between BS and M functionswas important for preserving the overall assimilationrate, and for any process activated in BS, a comple-mentary decrease in M could rebalance overall me-tabolism so that the total rate of assimilation and ATPdemand remained constant in spite of contrasting BS/Mengagement. In addition, we showed the importanceof transamination rate (T) in balancing the reducingpower needs in BS. When the ATP availability in BSwas low (e.g. condition 1), PR in BS was down-regulated; therefore, there was a lower NADPHdemand in BS (Fig. 4A, dotted line). Under theseconditions, CO2 was delivered to BS through Asp, apathway that bypasses MAL reduction in M and,hence, NADPH export to BS. This shift between Asp-and MAL-mediated CCM indicates the importance ofmaintaining both pathways in NADP-ME subtype C4plants. The predicted T varied in response to envi-ronmental conditions from a minimum PEP carboxyl-ation rate (VP) of 0.35 to the entirety of the CO2delivered to BS, in line with the observation that Aspcan support physiological rates of photosynthesis(Rathnam, 1978; Chapman and Hatch, 1981; Meisteret al., 1996; Pick et al., 2011). Under white light, themodel predicted a 33% T/VP, which is in line withradiolabeling and biochemical observations (Downton,1970; Hatch, 1971; Chapman and Hatch, 1981). Thesepredictions are not influenced by whether transami-nation is mediated by Glu aminotransferases (Fig. 1) orby the more recently documented Asp aminotransfer-ases (Pick et al., 2011), because, in the model, trans-amination is simply assumed to be a fast, passivelyregulated process at equilibrium. In addition, havingtwo independent pathways of CO2 delivery (throughMAL and Asp) decreases the MAL concentrationgradient required to sustain a physiological assimila-tion rate (Pick et al., 2011).

Although a mechanistic explanation goes beyond thescope of this study, it is worth noting that the fine-tuning

Table IV. Physiological responses of maize plants to different light qualities

The LCP was determined by fitting light curves with dedicated software; RLIGHT was determined by linear regression of A against PAR 3 Y(II)/3; gBSwas determined by fitting a modeled JMOD to the measured JATP (Fig. 3). Differences were not significant for P , 0.05. Values shown are means 6 SE;n = 4.

Parameter Unit Mean RGB Red Green Blue

LCP mE m22 s21 29.04 28.05 6 1.8 21.24 6 3.1 33.10 6 4.1 33.75 6 5.7RLIGHT mmol oxygen m22 s21 1.169 1.202 6 0.090 1.231 6 0.11 1.148 6 0.11 1.095 6 0.12gBS mol m22 s21 0.00104 0.00127 6 3.1 3 1024 0.00117 6 3.1 3 1024 0.00075 6 3.1 3 1024 0.00247 6 3.1 3 1024







between contrasting scenarios may be relativelystraightforward at the metabolic level. In fact, boththe CCM and the reductive pentose phosphate (RPP)pathways share diffusible metabolites between BS andM cells and are mediated by fast reactions, which, inphysiological conditions, are close to the thermody-namic equilibrium (e.g. transamination reactions orsugar phosphate conversions). The regulation of thefluxes, therefore, may be regulated in just a few keysteps, for instance, at the level of PR or MAL decar-boxylation. These have long been known to be regu-lated by the stromal pH, by feedback from metabolitepools, and by feed forward from light reactions

(Johnson and Hatch, 1970; Drincovich and Andreo,1994; Detarsio et al., 2003; Murmu et al., 2003; Trostet al., 2006; Eprintsev et al., 2011). The adjustment ofthe other reaction and diffusion rates may then fol-low passively, mediated by the feedback provided bychanging relative metabolite concentrations in one orthe other compartment.

Modeling ATP Supply as a Function of Light Quality

Previously, fluorescence microimaging had shownthat, because of the characteristic C4 concentric leafanatomy, strongly absorbed blue wavelengths wouldresult in preferential absorption in M cells as comparedwith wavelengths of light that could penetrate deeperinto the leaf profile (Evans et al., 2007). However, diffi-culties in the interpretation of fluorescence imaging,which are dependent on the different fluorescenceyields of PSI-rich BS and PSII-rich M, have preventedinvestigators from predicting the relative light harvest-ing in BS and M (Evans et al., 2007). To overcome thesedifficulties, we characterized the profiles of light pene-tration in a maize leaf by means of an absorption-scattering model, which represents, to our knowledge,a first attempt to calculate the extent of light absorptionimbalance caused by light quality. Because both BS andM produce ATP in light reactions, light harvesting im-balances would alter ATP partitioning. These ATPproduction imbalances were estimated using the rela-tive stoichiometry of the electron transport operating inBS and M. The effect was very different from the re-sponse to changing light intensity; in fact, light qualityonly marginally influenced the total ATP available atthe leaf level but resulted in a 3-fold difference in thefraction of ATP produced in the BS, from 0.29 to 0.96(Table III). These imbalances are potentially indepen-dent of the light intensity; however, photochemicalyield (i.e. quenching) was assumed to be independentof the position within the leaf (see description of Eq. 3below), an assumption that may not hold under highlight intensity. Regulation ofphotosystemyield throughthe leaf profile (Terashima et al., 2009) might also occurunder changing light quality. Differential quenchingwould represent an additional point of plasticity in re-sponse to changing light quality, which would have theeffect of narrowing the window of possible ATPBS/ATPM. Since we were interested in biochemical plastic-ity in response toATP imbalance rather than in detailingthe mechanics of electron transport processes, we didnot include the possible plasticity at the thylakoid leveland, hence, maximized the operating range of possibleATPBS/ATPM variation.

This spatial partitioning of ATP production (JATPBS/JATPM) is different from the functional partitioning ofATP consumption of the C4 model (von Caemmerer,2000; Ubierna et al., 2011; Bellasio and Griffiths, 2013)and from the theoretical ATPBS/ATPM of the metabolicmodel presented here. In theC4model, the JATP is simplyassumed to be produced by an undivided electron

Figure 4. Partitioning of metabolic activities in BS cells and associatedshifts in ATP and NADPH demand. A, The output of the metabolicmodel, shown as a function of increasing theoretical ATPBS/ATPM: theincreasing contribution of BS (solid lines) to the total PR (relative to thetotal), SS (relative to the total), and PEPCK (relative to the highest rate);the predicted NADPH demand in BS (relative to the total [NADPHBS/NADPHTOT]; dotted line); and the predicted T (relative to VP [T/VP];dashed line). B, The output of the metabolic model is compared withthe empirical data. Model output is shown by the dashed line: thepredicted (ATPBS + ATPM)/GA is plotted as a function of the predictedATPBS/ATPM. Empirical data are shown as diamonds: the measuredJATP/GA, under blue, red, RGB, and green light (Table I), is plottedagainst the estimated ATP production partitioning JATPBS/JATPM at 460 nm,635 nm, white light, and 522 nm (estimated through the optical model;Table III). The lowest ATPBS/ATPM was named condition 1 (left arrow),the partitioning corresponding to red light was named condition 2(middle arrow), and the highest ATPBS/ATPM was named condition 3(right arrow).





transport chain (Yin and Struik, 2012) and then parti-tioned to PEP regeneration activity or C3 activity by aparameter known as x (Supplemental Table S1). Thisoperation does not involve spatial separations betweenBS and M. In this study, we followed this conventionalapproach, which has been widely validated, and thencaptured the partitioning between BS and M with theequations of themetabolicmodel (Table II). On the basisof this division of work, we calculated the theoreticalATPBS/ATPM. Values for ATPBS/ATPM, therefore, werederived independently from JATPBS/JATPM (JATPBS/JATPMwas not used in model parameterization). These inde-pendently derived JATPBS/JATPM andATPBS/ATPM valueswere compared in Figure 4.

Implications for Electron Transport Processes

To allow these ATP partitioning rearrangements,there must be a high degree of flexibility at the electrontransport chain level. In fact, although LEF activity in BSis often neglected (because of negligible expression ofthe oxygen-evolving complex [Majeran and van Wijk,2009; Friso et al., 2010] and nonappreciable oxygen-evolving activity [Meierhoff and Westhoff, 1993]), evi-dence that appreciable linear flow can be supported bystromal reductants such as glutathione and ascorbatehas been presented (Walker and Izawa, 1979; Ivanovet al., 2001, 2005, 2007). These reductants are likely to beproduced from NADPH, supplied by MAL importedfromM (Kanai and Edwards, 1999; Laisk and Edwards,2000). These processes couple reductant pools at thethylakoid and stromal levels andare likely to function asplasticity mechanisms, playing a pivotal role in accli-mation to changing light conditions.

Most of the LEF activity is localized inM chloroplasts,which evolve oxygen and supply all reducing powerrequirements. For this reason, many reactions requiringNADPH (such as nitrogen reduction) are localized inMto benefit from NADPH availability (Majeran et al.,2005). Even ifM chloroplasts are specialized inNADPHproduction, the ratio of ATP to NADPH demand ishighly sensitive to BS/Massimilation partitioning (Fig. 1).Meeting this variable requirement will involve differ-ential engagement of LEF versus CEF. The particularfeatures of the CEF operating in maize (Ivanov et al.,2007; Laisk et al., 2010; Munekage et al., 2010; Hertleet al., 2013) may reflect, beyond the heterogeneity be-tween BS and M specialization, this characteristic needfor plasticity in CEF/LEF engagement.

Regardless of this electron transport plasticity, theATP production deficits induced by changing lightquality cannot be rebalanced within the individual BSchloroplast. In fact, increasing ATP production at theelectron transport chain level would require light(Takabayashi et al., 2005; Kramer and Evans, 2011),whose availability is not undermetabolic control. At thesame time, the electron transport-mediated dark pro-duction of ATP (Morstadt et al., 2002; Bukhov andCarpentier, 2004; Egorova and Bukhov, 2004; Kuntz,2004) has low conversion efficiency (Kramer and Evans,

2011); hence, an engagement of ATP chemiosynthesiswould be incompatible with the observed pattern ofJATP/GA. ATP itself is not a suitable shuttle to rebalanceATP deficits because the ATPmolecule is relatively big,it has a relatively small pool, and homeostasis is critical;therefore, every chloroplast has an independent ATPpool. Maintaining balanced ATP consumption in spiteof local ATP deficits therefore requires rearranging thelocalization of ATP demand, the fluxes, and the parti-tioning of metabolic work between the mutually inter-dependent BS and M cells.

Metabolic Plasticity Is Effective in Maintaining OverallAssimilation Efficiency

Previously, on the basis of theoretical considerations,it had been predicted that an unbalanced ATP supplywould result in a disruption of the delicate equilibriumbetween BS and M functions with consequent loss inassimilatory efficiency (Henderson et al., 1992; Evanset al., 2007; Tazoe et al., 2008). This prediction arosebecause metabolic rigidity would be expected undersome of the common simplifications used for C4 bio-chemistry,whereby transamination is neglected, PGA isreduced at afixed rate in BS, andNADPHdelivery to BSis equimolar to CO2 delivery (Laisk and Edwards, 2000,2009). In the updated description of C4 metabolismprovided in this paper, reaction rates are variable andtunable. When the ATP availability in BS was low (e.g.condition 1), PR in BS was down-regulated, leaving allavailable ATP for RuBP regeneration, resulting in un-altered Rubisco efficiency. Because the Asp-mediatedCCM delivers solely CO2 while the MAL-mediatedCCM delivers both NADPH and CO2, the variable en-gagement of the two pathways allows the activity of theCCM to be regulated independently of NADPH de-mand in BS; hence, the optimal CBS could bemaintainedunder all light qualities.

These predictions are supported by further modeloutputs, where we found no significant effect of lightquality onF and on CBS, confirming the response foundin a similar experiment where plants were acclimatedunder high light (Sun et al., 2012). This observation,together with the relatively minor change in JATP/GA(Fig. 4B), show that C4 metabolic balance was adjustingto the shifts in ATP supply without the potential majordisruptions mentioned above.

Implications for Light Use at the Leaf and Canopy Levels

Photosynthesis in shaded conditions has critical im-portance inC4 canopies, as itmay represent up to 50%ofCO2 uptake (Baker and Long, 1988; Long, 1993). Shadelight has a reduced intensity (typically one-twentiethof full sunlight; Shirley, 1929) and differs in spectralquality from red-rich sunlight: diffuse sky radiation is en-riched in blue, whereas canopy-filtered light is enrichedin green (Smith, 1982). Under low-light conditions, ithas been shown that F may increase both at the leaf






(Kromdijk et al., 2010; Bellasio and Griffiths, 2013) andcanopy (Kromdijk et al., 2008) levels. Theoretical consid-erationshaveassociated thisF increasewithdecreasedC4efficiency and a potential loss of photosynthetic carbonuptake (Furbank et al., 1990; von Caemmerer, 2000;Kromdijk et al., 2008; Tazoe et al., 2008). Although otherstudies have compared the effect of light quality on Funder low irradiance or under different light qualities(Kromdijk et al., 2010; Sun et al., 2012; Bellasio andGriffiths, 2013; Ubierna et al., 2013), the novelty of theapproach presented here has been to couple the mea-sured and predicted ATP supply during assimilationunder these conditions. In this experiment, which wasspecifically optimized to acquire data under low light(Bellasio and Griffiths, 2013), we showed that the totalATP cost of gross assimilation was not significantlyinfluenced by light intensity and underwent a littlevariation under different light quality (Fig. 4B). Thisshowed that metabolism at steady state under low lightintensities maintained efficiency in spite of changes inlight quality or intensity. This implies that the hyper-bolic increase of F observed under decreasing lightintensities (Fig. 3D), which underpins the predictedphotosynthetic efficiency loss, actually did not cost ad-ditional ATP but resulted instead from mitochondrialdecarboxylation in BS (Bellasio and Griffiths, 2013).Similar conclusions were highlighted by Ubierna et al.(2013). This observation is consistent with VP/VC (forRubisco carboxylation rate) and the optimal x beinglargely independent of light intensity (von Caemmerer,2000; Kromdijk et al., 2010), indicating a constant degreeof engagement of the CCM even under an apparent Fincrease. Therefore, care should be taken when the ATPcost (and quantum yield) of C4 photosynthesis is de-rived from F, measured either at the leaf or canopyscale, particularly in the vicinity of the light compensa-tion point (Furbank et al., 1990; von Caemmerer, 2000;Tazoe et al., 2008). In these conditions, we propose thatthe ATP cost should be calculated by summing the ATPcost of all the active biochemical processes (see Eq. 11 inTable II) instead of usingF as a proxy forC4 biochemicalefficiency. The actual impact of F on canopy-level car-bon uptake may depend upon the extent of steady-statephotosynthesis under low light or altered light qualityconditions (e.g. green enriched), and shorter term, moretransient conditions, when F may be more variable.

CONCLUSION

In this study, we set out to investigate whether themaize C4 system could respond to changing environ-mental conditions by adjusting the C4 (amino) acid(MAL or Asp) delivered fromM to BS as well as the pro-portions of other metabolic reactions shared betweenboth cell types, such as SS, PR, and PEP regeneration(Walker et al., 1986; Spilatro and Preiss, 1987; Wingleret al., 1999; Friso et al., 2010;Majeran et al., 2010; Furbank,2011). Using contrasting light qualities and their pro-jected extinctions within the leaf profile, we could then

estimate the rate of ATP synthesis in M and BS com-partments as compared with the overall leaf-level oper-ating efficiencymeasured by gas exchange and real-timecarbon isotope discrimination. We depicted a scenariowhereby metabolism, although subject to the generalconstraints imposed by C4 physiology, was able to takemaximum advantage of environmental conditions bychanging the relative engagement of BS andM functions,whichwere ultimately under environmental control. Theoutputs, based on metabolic modeling and empiricalmeasurements, providedefinitive evidence for the role ofcomplementarity between BS andM functions, allowingATP demand to be regulated in response to contrastingenvironmental conditions. The two decarboxylase sys-tems in BS of maize, with a variable rate of transamina-tion, allow the regulation of NADPH supply to matchdemand in BS independently of the delivery of CO2.

The findings of this study highlight the importance ofC4 metabolic models in helping to explain acclimationand adaptation to changing light intensity for all C4subgroups. The emerging complexity of the NADP-ME/PEPCK interactions certainly demands some re-finement to the widespread simplifications used todescribe C4 systems and to the assumptions regardingthe relatively fixed energetic partitioning in maize. Fur-thermore, we have clearly linked metabolic plasticity tothe capacity to maintain high photosynthetic efficiencyunder changing environmental conditions, which couldwell be related to the original functions of BS decarboxy-lases and the evolution of the C4 syndrome (Hibberd andQuick, 2002; Griffiths et al., 2013). Finally, the extent towhich such steady-state conditions of low light and al-tered light quality affect carbon uptake within an intactcrop canopy remains to be determined, as comparedwithmore transient responses, whichmaywell increaseF andreduce carbon assimilation under low-light conditions.

MATERIALS AND METHODS

Metabolic Model

The processes contributing to assimilatory metabolism in maize (Zea mays;Furbank, 2011) were integrated in a comprehensive steady-state model (Fig. 1).Some functional simplifications were made. Cells were decompartmentalized.NADPH and NADH were considered equivalent or convertible. Starch wasassumed tobe theonlyfinal product of photosynthesis (SShas the sameATPcostper hexose than phloem-loaded Suc, considering the stoichiometry of one ATPper H+ of the membrane H+-ATPase and one H+ per Suc of the Suc symporter).The ATP and NADH produced during respiration (assumed to be supplied byPGA) were neglected in calculations because they are likely to be consumedby basalmetabolism. Transaminationwas assumed to be passively regulated bysubstrate availability (all OAA not reduced by MDH was transaminated), astransamination reactions are rapid conversions at equilibrium (see “Discussion”).

The specialization of BS andMwas captured by assigningprocesses to BS,M,or the conjoint work of both compartments (allocatable processes). PEPCK(Walker et al., 1986; Furumoto et al., 1999, 2000; Wingler et al., 1999) andglyoxylate recycling (Yoshimura et al., 2004) were allocated to BS; LEF(Meierhoff andWesthoff, 1993; Romanowska et al., 2006;Majeran and vanWijk,2009; Friso etal., 2010;KramerandEvans, 2011)andPPDK(see“Discussion”)wereallocated toM;RLIGHTwas split equally betweenMandBS (vonCaemmerer, 2000;Kromdijk et al., 2010; Ubierna et al., 2011); PR and SS were variably allocated. Twas equal in BS and M, as the pool of Asp and Ala is shared. The model wasdescribed by steady-state rate equations (Table II).





Minimum and Maximum BS Allocation

The rate of variably allocated processes ranged between a minimum rateand a maximum rate, deduced from physiological considerations (for theenvironmental influence on maximum rates, see “Discussion”). Both BS and Mexpress SS enzymes (Spilatro and Preiss, 1987; Majeran and van Wijk, 2009)and synthesize starch (Rascio et al., 1980; Kanai and Edwards, 1999), so SS wasallocated to BS between zero and total SS rate. PR is not essential to BS, sominimum PR was set at zero. PR is mainly an M process (Majeran et al., 2005;Majeran and van Wijk, 2009), so maximum PR rate in BS was limited at0.353 total PR rate. PEPCK is not essential to BS, so minimum PEPCK was setat zero. Maximum PEPCK was set at 0.3 3 VP, identified by fitting the totalATP demand of assimilation to JATP/GA (Fig. 4B).

Parameterization

Equations describing overall assimilation (Eqs. 4–10 in Table II) were pa-rameterized with the measured data A, RLIGHT, the output of the von Caem-merer C4 model (Rubisco oxygenation rate [VO], VP, and VC; SupplementalTable S1; Bellasio and Griffiths, 2013), and F calculated from D under pho-tosynthetically active radiation (PAR) = 125 mE m22 s21 (see below). Then,reaction rates (Eqs. 4–21 in Table II) were calculated under the minimum(condition 1) and the maximum (condition 3) BS allocation (Figs. 1 and 4B).Finally, intermediate states (e.g. condition 2) were calculated by allocatingreactions to BS in linear increments (see continuous lines in Fig. 4A).

The ATP demand in BS was calculated by adding the ATP demand of BSprocesses (Eq. 12 inTable II; Supplemental Table S2).Analogously,ATPdemandin M was calculated by summing the ATP demand of M processes (Eq. 13 inTable II; Supplemental Table S2), and ATPBS/ATPM was calculated by dividingEquation 12 by Equation 13. Similarly, NADPHBS was calculated by summingtheNADPHdemand of BS processes (Eq. 14 in Table II; Supplemental Table S2).Rate equations for other processes are listed in Table II.

Estimated AB BS/M

A maize leaf cross section was simulated by rectangular units, enclosing asquare BS (Fig. 2, left). Interveinal distance (106mm),M thickness (100mm), BS/Marea (0.26), and the resulting BS side (46 mm) were averaged from the literature(Hattersley, 1984; Usuda, 1985; Bongard-Pierce et al., 1996; Moreno-Sotomayoret al., 2002; Kromdijk et al., 2010). Because of the square anatomy, the leaf lightenvironment could be described by two light profiles: P1 and P2. These werecalculated applying the Kubelka-Munk absorption-scattering theory with themethod ofAllen andRichardson (1968) andothers (Gates, 1980; Terashima et al.,2009), modified to describe the simulated C4 anatomy. Briefly, each profile wasconsidered to be made of a number n of light-absorbing and light-scatteringelements; the total number of elements in the profile was N. The element n = 0was the illuminated, or adaxial, point of the profile, which included the upperepidermis, approximated to a single reflecting element. The element Nwas theabaxial point of the profile, which included the lower epidermis, approximatedto a single reflecting element. Radiant flux directed downward was I, and thatupwardwas J. Incidentfluxwas I0 andwas taken tobe theunity. For eachprofile,the flux reflected by the first element was equivalent to the point reflectance andthe flux transmitted by the last element was equivalent to the point transmit-tance: J(0) = point reflectance and I(N) = point transmittance.

Incremental absorption and scattering were calculated as (Gates, 1980):

dI ¼ 2 ðk þ sÞIdnþ sJdn ð1Þ

dJ ¼ ðk þ sÞJdn2 sIdn ð2Þwhere k is an absorption coefficient and s is a scattering coefficient. In P1, kwas constant throughout the profile. In P2, k was three times higher in theelements corresponding to BS, because chlorophyll concentration is threetimes higher than in the surrounding M (BS/M chlorophyll content = 0.74[Kanai and Edwards, 1973] multiplied by M/BS area = 4 [Fig. 2, left]). I and Jwere solved for all elements of P1 and P2 according to the integration ofEquations 1 and 2 proposed by Gates (1980). Modeled leaf reflectance resultedfrom averaging J(0) contributions to the total leaf reflectance from P1 and P2(56% P1 and 44% P2). Similarly, modeled leaf transmittance was calculated asa weighted average of I(N) from P1 and P2. Modeled leaf reflectance andtransmittance were calibrated with measured leaf reflectance and transmit-tance at different wavelengths (Table III; Woolley, 1971) by varying k and s.This procedure allowed calculating P1 and P2 at different wavelengths (Fig. 2,right). AB in BS resulted from integrating P2 over BS area. AB in M resulted

from integrating P2 over adaxial and abaxial M area (MAD + MAB) plus theintegral of P1 over the two interveinal M areas (MI).

Estimated JATPBS/JATPM

M chloroplasts are engaged in NADPH and ATP production while BSchloroplasts only produce ATP; therefore, for a given number of light quanta,M chloroplasts produce approximately half the ATP produced in BS chloro-plast. JATPBS/JATPM was calculated as:

JATPBSJATPM

¼ 2$ABBSM

ð3Þ

The coefficient 2 (in Eq. 3) is based on awidely accepted assumption that light isequally shared between photosystems and on the simplification that thephotochemical yield is independent of the position within the leaf profile. Indetail, we assumed (1) exclusive linear electron transport in M with equalPSI/PSII absorption partitioning (Meierhoff and Westhoff, 1993; Kramer andEvans, 2011); (2) exclusive cyclic electron transport in BS with no PSII ab-sorption (Romanowska et al., 2006; Majeran and van Wijk, 2009); (3) equalyield of PSI and PSII (Miyake et al., 2005; Supplemental Fig. S3C); (4) equalH+/ATP stoichiometry of the ATP synthase in BS and M (the enzyme complexis the same; Majeran and van Wijk, 2009; Friso et al., 2010); and (5) twice theH+ per photochemical event pumped in BS versus M: in M, 1.5 H+ are extrudedper photochemical event (Kramer and Evans, 2011), while in BS, we assumedthree H+ extruded per photochemical event (average between four H+ of theNDH-mediated electron flow [Kramer and Evans, 2011; Peng et al., 2011] and twoH+ of the PGR5/PGRL1-mediated electron flow [Kramer and Evans, 2011; Hertleet al., 2013], both operating in maize BS [Ivanov et al., 2007]).

Plants

Maize (F1 hybrid PR31N27; Pioneer Hi-Bred) plants were grown in 1.5-L potsfilled with Levington pro M3 (Scotts) in growth rooms (Conviron) set at 16-hdaylength, temperature of 25°C/23°C (day/night), 40% relative humidity, andPAR = 600 mE m22 s21 and manually watered daily with particular care to avoidoverwatering. After 3 weeks, plants were measured once and then discarded.

Gas-Exchange Measurements with Concurrent PSI/PSIIYield and Online Carbon D

The experimental setup was described previously (Bellasio and Griffiths,2013). Briefly, an infrared gas analyzer (IRGA; LI6400XT; Li-Cor), was fittedwith a 6400-06 PAM2000 adapter and with a Li-Cor 6400-18 RGB light source.The IRGAwas fedwith CO2 (d

13C =28.3‰; Isi Soda) and either amixture of 2%oxygen/nitrogen or ambient air. PSI yield and PSII yield [Y(II)] were measuredwith aDual Pam-F (HeinzWalz). The IRGAwas connected to a cryogenicwater-and CO2-trapping purification line. To determine the relationship between Y(II)and JATP, a light-response curve was measured at 2% oxygen and Ca = 600 mmolmol21. Under the same light quality, 21% oxygen, and reference CO2 set at400mmolmol21, a second light-response curvewasmeasured, duringwhich Y(II)was determined and exhaust gas was trapped to determine D. In 1 d, a total of12 CO2 samples and six CO2 references from each individual plant were ana-lyzed directly with a VG SIRA dual-inlet isotope ratio mass spectrometer(modified andmaintained by Pro-Vac Services). Dwas calculated as reported inSupplemental Figure S2, using Equation 22. RLIGHT was calculated as the y in-tercept of the linear regression of A against PAR 3 Y(II)/3. JATP was calculatedfrom chlorophyllfluorescence data, by calibrating the relationship between ETRand Y(II) under low oxygen, and then using such a calibration to determine thesmall fraction of ATP consumed by photorespiration. The correction was min-imal because of the low oxygen sensitivity of maize (Supplemental Table S1;Bellasio and Griffiths, 2013); therefore, the potential errors caused by usingfluorescence measurements under contrasting light qualities were negligible.Light responses were treated with dedicated software (Photosyn assistant 1.2;Dundee Scientific) to calculate the light compensation point and by repeated-measures ANOVA (Genstat); point estimates were subject to ANOVA andTukey’s multiple comparison (Genstat).

F from D

F was resolved from D by use of the full Farquhar model (Farquhar, 1983;Farquhar and Cernusak, 2012), parameterized with a C4 photosynthesis model













(von Caemmerer, 2000), using equations and a fitting approach that weredescribed previously (Supplemental Table S1; Bellasio and Griffiths, 2013).Briefly, F was resolved from D by calculating the weighted individual frac-tionations of the discriminating processes operating in C4 photosynthesis. TheCO2 concentration in the cellular compartments was calculated by means of aC4 model, parameterized with the light-response data (A, Ci, Ca, and JATP) andRLIGHT. The C4 model was rearranged to express a modeled ATP productionrate JMOD and fit to the JATP (Supplemental Table S1), to yield a value for BSconductance for each individual plant, independently from D.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Ecophysiological responses to decreasing lightintensity under different light qualities.

Supplemental Figure S2. j values for the calculation of D.

Supplemental Figure S3. Photochemical responses to decreasing light in-tensity under different light qualities.

Supplemental Table S1. Definitions, equations, and variables used in themodels.

Supplemental Table S2. ATP and NADPH demand for key C4 processes.

ACKNOWLEDGMENTS

We are deeply grateful to Bernard Genty, Gerald Edwards, and Joe Berryfor discussion, to Julian Hibberd and Jessica Royles for review, to thereviewers for the constructive feedback, and to Davide Gusberti for providingseeds.

Received September 9, 2013; accepted November 14, 2013; published November19, 2013.

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