1040-2519/96/0601-0431$08.00 431

Annu.Rev. Plant Physiol. Plant Mol. Biol. 1996. 47:431–44Copyright© 1996by AnnualReviewsInc. All rightsreserved

ROLE AND REGULATION OFSUCROSE-PHOSPHATE SYNTHASEIN HIGHER PLANTS

Steven C.Huber1 andJoan L.Huber2

1United States Department of Agriculture/Agricultural Research Service, and Depart-ments of Crop Science andBotany, North Carolina State University, Raleigh, NorthCarolina27695-7631

2Departmentof Horticultural Science, North Carolina State University, Raleigh, NorthCarolina27695-7631

KEY WORDS: sucrosesynthesis, spinach(Spinacia oleraceaL.), maize(ZeamaysL.), regula-tory protein phosphorylation, protein kinase

ABSTRACT

Sucrose-phosphatesynthase(SPS;E.C.2.4.1.14)is theplantenzymethoughttoplay a major role in sucrosebiosynthesis. In photosynthetic and nonphotosyn-thetic tissues, SPSis regulated by metabolites and by reversible protein phos-phorylation. In leaves,phosphorylation modulatesSPS activity in response tolight/dark signals and end-product accumulation. SPS is phosphorylated onmultiple seryl residuesin vivo, and themajor regulatory phosphorylation siteinvolved is Ser158in spinach leavesand Ser162in maizeleaves.Regulation oftheenzymaticactivity of SPSappearsto involvecalcium, metabolites,andnovel“coarse” control of theprotein phosphatasethat activatesSPS.Activationof SPSalso occursduringosmotic stressof leaf tissuein darkness, which may functionto facil itatesucroseformationfor osmoregulation.Manipulation of SPSexpres-sion in vivo confirmstherole of this enzymein thecontrol of sucrose biosyn-thesis.

CONTENTSINTRODUCTION..................................................................................................................... 432BIOCHEMICAL AND MOLECULAR PROPERTIES........................................................... 433

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Physical and Regulatory Properties.................................................................................... 433Molecular Properties........................................................................................................... 434Variation AmongSpecies.................................................................................................... 436Possible Occurrenceof Enzyme Complexes....................................................................... 436

CONTROL BY REVERSIBLE PROTEIN PHOSPHORYLATION ...................................... 437Light/Dark Modulationof SPSActivity............................................................................... 437Osmotic StressActivation.................................................................................................... 439Nonregulatory PhosphorylationSites................................................................................. 440

SPSIN TRANSGENIC PLANTS............................................................................................. 440ROLEOFSPSIN VIVO........................................................................................................... 441

Sucrose Synthesisand Sugar Cycling.................................................................................. 441FactorsAffectingSPSExpression....................................................................................... 441

CONCLUDING REMARKS.................................................................................................... 442

INTRODUCTION

Sucroseplaysa pivotal role in plant growth anddevelopmentbecauseof itsfunction in translocation andstorage,andbecauseof the increasingevidencethatsucrose(or somemetabolite derivedfrom it) mayplay a nonnutritive roleasa regulatorof cellular metabolism, possiblyby actingat the level of geneexpression(19). It is the nonreducingnatureof the sucrosemolecule thatexplainsits wide distribution andutilization amonghigherplants.Trehalose,the only othernonreducingdisaccharidefound in nature,playsa comparablerole in insectsand fungi.

Sucrosesynthesis can becatalyzed bytwo distinct enzymes inhigherplants:sucrose-phosphatesynthase(SPS; EC2.4.1.14):

UDP-glucose+ Fru-6-P↔ sucrose-6′-P + UDP +H+,

and sucrose synthase(SuSy; EC2.4.1.13):

UDP-glucose+ fructose↔ sucrose + UDP + H+.

Although this review focuseson SPS,somecomparisonsare madewithSuSy.Both enzymesaresolublein the cytoplasmandcatalyzefreely revers-ible reactions.However,rapidremovalof sucrose-6′-Pby sucrosephosphatase(SPP;EC3.1.3.24)keepsthecytosolic[sucrose-P]low andtherebyrenderstheSPSreaction essentially irreversible.In fact,recentevidencesuggests thatSPSandSPPmayactuallyform a complexin vivo (seesectionon PossibleOccur-renceof EnzymeComplexes).Thus,sucrosesynthesisis generallyconsideredto becatalyzedby SPS(in conjunction with SPP),whereassucrosebreakdownis largely catalyzedby SuSy.Although a given tissuewill tend to haveanexcessof oneactivity overtheother(dependinguponwhetherit is engagedinnetsucrosesynthesisor breakdown),manytissueshaveboth enzymes,anditis clear thatsignificant“sugar cycling”can occur (discussedbelow).

A considerableamounthasbeenlearnedaboutSPSsinceits discovery,butstill muchremainsto belearned.Forageneraldiscussionof SPSandits role in

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sucrosebiosynthesisin leaves,seeStitt et al (36) andHuberet al (16). Sinceabout1990,severalnew aspectshavebeenelucidated.With respectto local-ization, it is now clearthat SPSis not confinedto photosynthetic tissuesbutalsooccursin nonphotosynthetictissuesthat areactivein sucrosebiosynthe-sis,e.g.ripeningfruits. With respectto mechanisms for control,it is now clearthat SPSis controlled (a) at the level of enzymeprotein (e.g. leaf develop-ment),(b) by allostericeffectors(Glc-6-PandPi), and(c) by reversibleserylphosphorylation. In addition, the geneencodingSPShasbeenclonedfromseveralspecies.As a result, the deducedsequenceis available,and cDNAprobescanbeused tomonitor changes inthe steady-statepoolof SPS mRNA.However,despitethe progressmadeto date,we arejust beginningto under-standthehierarchyof molecular mechanismsthat togethercontrolSPSenzy-maticactivity in vivo. This review focuseson recent developments withSPS.

BIOCHEMICAL AND MOLECULAR PROPERTIES

PhysicalandRegulatory Properties

SPSis a low-abundanceprotein (<0.1% of leaf solubleprotein) and is alsorelatively unstable.Consequently, progresson the purification and charac-terizationof the enzymehasbeenslow. In addition, therearesomeapparentdifferencesamongspeciesin someof thepropertiesof theenzyme;not all ofour information aboutSPShasbeenderivedfrom studiesof thesameenzymesource.Nonetheless,some generalstatementscanbemade. It is nowgenerallyacceptedthatsubstratesaturationprofilesfor UDP-Glc andFru-6-Pare hyper-bolic rather than sigmoidal and that the enzymefrom somespeciescan beallostericallyactivatedby Glc-6-Pandinhibited by Pi [seeStitt et al (36) for areview].Theseeffectorshavealargeeffectontheaffinitiesfor bothsubstrates,Fru-6-Pand UDP-Glc (29, 33). Alteration of the affinity for substratesandeffectorsis alsoinvolvedin thelight modulation of SPS thatoccursby revers-ible proteinphosphorylation in somespecies(38). In general,SPSfrom non-photosynthetic tissues(e.g.potatotubers)is regulatedby metabolitesandbyproteinphosphorylation in an analogousmannerto the enzymefrom photo-synthetictissues(29).

The nativeSPSmolecule is likely a dimer of 120–138-kDasubunits(15).Anomalousbehaviorof SPSon gel-filtration chromatographyprobably ac-countsfor the larger estimatesof molecular mass in some studies.Thespecificactivity of thenativespinachenzymeis about150IU/mg protein.In addition,the reactioncatalyzedby SPS is clearly reversible. Inthe mostcarefullyconductedstudy to date, Lunn & ap Rees(21) showedthat the apparentequilibrium constant(Kapp) of the pea seedenzymerangedfrom 5 to 65dependingupon[Mg2+] andpH. Underassumedin vivo conditions, theKapp

SUCROSE-PHOSPHATE SYNTHASE 433

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for SPShasbeenestimatedto beabout10.Thecalculatedmass-actionratioforthe SPSreactionin vivo indicatesthat the reactionis far from equilibrium,presumablybecause of rapidremovalof sucrose-P by SPP.

MolecularProperties

Cloning of the SPSgenewasaccomplishedfirst for the enzymefrom maize(42) andthenfor thespinach(20, 35), potato(34), sugarbeet(9), andrice (JJValdez-Alarco’n, B Jimenez-Moraila& LR Herrera-Estrella,submitted) en-zymes. Ingeneral, theN-terminalportions of the∼120-kDa subunitof SPSarehighly conserved,andtherearealsotwo regionsof strongsimilarity betweenSPSandSuSy(33). Oneof theregionsthat is highly conservedbetweenSPSand SuSy correspondsto residuesV176 to S214of spinach SPS;this region of39 aminoacidsexhibits an overall similarity of 64% betweenSPSandSuSy(Table1). Salvucciet al (33) notedthat within this highly conservedregionthere is a stretchof 11 amino acids (D197 to E206 of spinachSPS)thatresemblestheGly-rich motif of phosphate-bindingdomainsandthusmight beinvolved in bindingof Fru-6-P(to SPS)or UDP-Glc.This stretchcontains10residuesthatareidentical orsimilar; theonly exception isspinachresidue203,which is a conservedLys residuein SPSbut a conservedVal residueat theanalogousposition in SuSy.It hasbeensuggestedthatthefunctionof thebasicresiduein SPSmayinfluencetheselectivityfor thenegativelychargedFru-6-Prather than theneutralFrumolecule.

Theportionof theprimarysequencein thevicinity of theuridinemoiety ofthe substratemoleculeUDP-Glc hasbeendeterminedby photoaffinity label-ing of a recombinantspinachSPSfragmentusing[β-32P]5-N3UDP-Glc (32).It wasdeterminedthat the 5 position of the uridine ring wasproximal to theprimarysequenceQ227to E239. Notethatthissequenceis reasonablyclosetothe residuesthoughtto be involved in binding of theothersubstrate,Fru-6-P(D197 to E206). The uridine-binding region of the SPSmoleculeis highlyconserved amongspinach,maize, andpotato, but there is relatively littlehomologywith SuSy(33). This is perhapsnot surprisingbecausetherearenorecognizedconsensusbinding motifs for UDP-Glc orothernucleotidediphos-phate sugars.

The secondregion of strongsimilarity betweenSPSandSuSyis locatedtowardtheC-terminus of theSuSysequence(residuesD587 to P631of spin-ach SPS).Of the 44 residueswithin this region of the SPSmolecule,25residuesare identical and 8 are similar, for an overall similarity of 75%.However, the functionof thisportionof themoleculeis not known.

Another importantdomainof the SPSmoleculethat remainsto be identi-fied is the effector site involved in Glc-6-P and Pi binding. None of thespecificaminoacid residuesat theeffectorsite hasyet beenidentified. How-ever,the allostericsite containsessentialandaccessiblesulfhydryl group(s),

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Tab

le1

Par

tiala

min

oac

idse

quen

ceal

ignm

ento

fS

PSfr

omsp

inac

h(2

0,35

),m

aize

(42)

,pot

ato

(34)

,sug

arbe

et(9

),an

dri

ce(J

JV

alde

z-A

larc

o’n,

BJi

men

ez-M

orai

la&

LR

Her

rera

-Est

rella

,sub

mitt

ed)

with

aho

mol

ogou

sre

gion

ofpo

tato

SuS

y(3

0).a

Spec

ies

Res

idue

sSe

quen

ce

:*

::

**

*:

**

:-

**

-*

**

**

**

::

:*

:

Spin

ach

176–

212

VV

LI

SL

HG

LI

RG

EN

ME

LG

RD

SD

TG

GQ

VK

YV

VE

LA

RA

L

Mai

ze17

8–21

4I

..

..

V.

..

V.

..

..

..

..

..

..

..

..

..

..

..

..

.M

Pota

to16

8–20

4I

..

..

L.

..

I.

..

..

..

..

..

..

..

..

..

..

..

..

.L

Suga

rbee

t16

3–19

9L

..

..

L.

..

I.

..

..

..

..

..

..

..

..

..

..

..

..

.L

Ric

e17

9–21

5I

..

..

L.

..

VS

.D

..

..

..

..

..

..

..

..

..

..

..

.L

StSU

SY28

4–31

8V

.I

L.

P.

.Y

FA

Q.

.V

-.

.Y

P-

..

..

..

V.

IL

DQ

VP

AL

a With

inth

ese

quen

ces,

iden

tical

resi

dues

are

show

nas

dots

,and

gaps

are

show

nas

dash

es.R

esid

ues

iden

tical

inal

lsix

sequ

ence

sar

ein

dica

ted

byan

aste

risk

over

the

alig

nmen

t;co

nser

ved

resi

dues

are

indi

cate

dby

aco

lon.

Itha

sbe

ensu

gges

ted

(33)

that

this

high

lyco

nser

ved

regi

onm

aybe

inth

evi

cini

tyof

the

site

that

bind

sFr

u-6-

P(i

nSP

S)

orF

ru(i

nS

uSy)

.

SUCROSE-PHOSPHATE SYNTHASE 435

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whereasthe catalytic site doesnot (2). Comparisonof the SPSsequencesavailableto dateindicatesthat thereare10 conservedcysteineresidues.Pre-sumably,oneor moreof theseis at theeffectorsite.It is notknownwhetherallthe sulfhydryl groupsin the moleculearereduced;however,it is known thatthere are no intersubunit disulfide bonds(J Huber, unpublished data,1995).

Variation AmongSpecies

Thereappearto be significant quantitative differencesamongspeciesin theregulatorypropertiesof SPSin vitro, i.e. theextentof Glc-6-Pactivation andPi inhibition (1, 13). Therearealsodifferencesin the modulation of SPSinvivo. Somespeciesexhibit a markedlight activationof SPS(designatedasclassI andclassII species)(13),whereasothersdo not (classIII species).Thedistinctions among thethree classesof plants arequantitative rather thanqualitative in nature.For example,our original studiesof soybean(a classIIIspecies)involved cultivars of maturity group VII, e.g. “Ransom.” AlthoughsoybeanRansomplantsexhibitedlittl e, if any,light activation of SPSin vivo,we haverecentlyobservedthatsoybeancultivarsof maturitygroupOOO,e.g.“Maple Presto,”showsignificantlight activation(S Huber,unpublisheddata,1994).Similarly, we haveobserveddifferencesamongNicotiana speciesandamongcultivarsof Nicotianatabacum(S Huber,unpublisheddata).However,with N. tabacum,evenwhen light activationoccurs,it is subtle relative tospeciesof classesI andII. Nonetheless,theseresultssuggestthattherequisiteinterconversionenzymes[SPS-kinase(SPSk)and SPS-proteinphosphatase(SPS-PP)] may be presentat somelevel in all species.

Furthersupportfor this notion hasrecentlybeenobtainedfrom studiesoftransgenictobaccoplants expressingthe maize SPS gene (18). In controlplants,therewasa smallbut significant(∼30%increasein thelight comparedwith dark) activation of SPS assayedunder selectiveconditions (limitingsubstratesplusallostericinhibitor, Pi). In transgenictobaccoplantsexpressingthe maizeSPSgene,Vmax activity of SPSwasincreasedabout2.5-fold asaresultof expression ofthetransgene,andthemaizeenzymewaslight activatedin a very pronouncedmanner(∼150%increasein the light). TheseresultsarenoteworthybecausemaizeSPSexpressedin transgenictomatoplantsshowsrelativelylitt le light modulation (4, 18).Thebasisfor thelackof modulationinthis caseis not clearbut may involve slight differencesin quaternarystructure,which results in the phosphorylation site becomingless accessibleto theendogenousproteinkinases.

Possible Occurrenceof EnzymeComplexes

Thereis increasingevidencefrom other systemsthat solubleenzymesoftenoccurascomplexeswith other relatedenzymes.In the caseof SPS,thereisevidencefor anassociationwith SPSk (12),which mayfacilitatethephospho-

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rylation of this low-abundanceprotein.Recentevidencealsoshowsthat SPSandSPPmay form a complexin vitro. The primary observationis that SPSactivity in vitro is reversiblyreducedby removalof SPPduring purification(GL Salerno,E Echeverria,HG Pontis,submitted);efficient removalof inhibi-tory sucrose-6′-P via an enzymecomplex is speculatedto be necessaryformaximalSPSactivity. It will beinteresting todeterminewhetherphosphoryla-tion of SPS(at eitherregulatoryor nonregulatorysites)affectsthe interactionwith SPP.

CONTROLBY REVERSIBLE PROTEINPHOSPHORYLATION

Light/Dark Modulationof SPSActivity

REGULATORY PHOSPHORYLATION SITE The major (if not sole) regulatoryphosphorylation siteof spinachSPShasbeenidentified(23) asSer158(Table2). Phosphorylationof Ser158is both necessaryand sufficientfor theinactiva-tion of SPSin vitro. Additional linesof evidenceconsistentwith theassignmentinclude: (a) labeling of the tryptic phosphopeptidecontainingSer158[pre-viouslydesignatedphosphoprotein7 (11)] in situcorrelateswith inactivationofSPS;(b) asynthetic peptidebasedonthephosphorylationsitesequenceisagoodsubstratefor SPSkin vitro and competeswith native SPSfor phosphoryla-tion/inactivation(22); (c) labelingof Ser158in situ occursmorerapidly thanother(nonregulatory)phosphorylation siteson SPS andreflectsdifferentturn-overrates(17);and(d) polyclonal antibodiesgeneratedagainstthephosphory-lation site sequencepreferentially recognizeand immunoprecipitatehighlyactivateddephospho-SPS as opposedto inactivatedphospho-SPS (40).

Although the regulatoryphosphorylation sequenceof spinachSPSis notconservedexactly,all sequencesavailableto datecontaina homologousseryl

Table 2 Amino acid sequencessurrounding the putative regulatory phosphorylationsiteof SPS.a

Species Residues Sequence

: : : * * :

Spinach 150–162 K G R M R R I S S V E M M

Potato 142–154 R G R L P R I S S V E T M

Sugarbeet 137–149 R P R L P R I N S L D A M

Maize 154–166 K K K F Q R N F S D V T L

Rice 154–166 K K K F Q R N F S E L T V

−8 −6 −3 0 4aResiduesidenticalin all fivesequencesareindicatedbyanasteriskoverthealignment;

conservedresidues are indicatedby a colon. Residuesare numberedrelative to thephosphorylatedSerat position 0. Spinach(20, 35), maize(42), potato (34), sugarbeet(9), andrice(JJValdez-Alarco’n, B Jimenez-Moraila& LR Herrera-Estrella,submitted).

SUCROSE-PHOSPHATE SYNTHASE 437

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residue (Table2). Evidenceconsistent with phosphorylation of Ser162inmaizeSPShasbeenobtainedin studiesof maizeleavesaswell astransgenictobaccoexpressingthe maize SPSgene(18). It remainsto be determinedwhetherthehomologousSerresiduein theotherspeciesis phosphorylatedandis of regulatorysignificance,but it seemsquite likely. It is importantto notethatseveralof theresiduessurrounding the(putative) phosphorylation sitearealso conservedamongthe five species. Inparticular, therearebasic residues atP−3, P−6, andP−8 (numberingrelative totheSerat position 0)and hydropho-bic residuesat P−5 and P+4 (Table 2). At leastseveralof theseconservedresidues appear tobe importantfor recognitionby proteinkinase.

SPS-KINASE PartiallypurifiedspinachleafSPScontains acopurifyingproteinkinasethatcanphosphorylateandinactivateSPSwith [γ-32P]ATP(12).In vitro,approximately75–85% of the 32P is incorporatedinto Ser158, themajorregulatoryphosphorylationsite.Using a syntheticpeptidebasedon thephos-phorylationsite sequence,two protein kinaseswith apparentmolecularmassesof 45and150kDawereresolvedchromatographically fromspinachleaves(22).Thesmaller kinase(designatedpeakI) is mostlikely a monomer,whereasthelargerkinase(peakIII) hasasubunitmolecularmassof ∼65kDa.An importantdistinction betweenthe two kinasesis that thepeakI enzymeis strictly Ca2+

dependent,whereasthepeakIII enzyme,which tendsto copurify with SPS,isCa2+ independent. The substratespecificity of both kinaseshasbeencharac-terizedin vitro usingsyntheticpeptideanalogs.Themajorrecognitionelementsconsistof basicresiduesatP−6 andP−3 (24)andahydrophobic residueatP−5(S Huber & D Toroser,unpublished data, 1995). Theseresiduesare alsoconserved amongspecies(Table 2).

Studieswith maizeleaf SPSkhaveidentified a singleform of theenzyme,andthereis a clear requirementfor peptidesubstrateswith basicresiduesatP−3/P−6 anda hydrophobic residueat P−5 (R McMichael& S Huber,unpub-lished data, 1994). MaizeleafSPSkis also strictly Ca2+ dependent(18). Theseobservationsraisetheintriguing possibility thatcytosolic [Ca2+] mayregulatesucrosebiosynthesis,at leastin somespecies.Thereis evidencethatcytosolic[Ca2+] is reducedin the light relative to the dark (26). Thesechangesincytosolic[Ca2+] couldcontributeto thelight activation of SPS invivo (Figure1). Anotherfactorthatmaybeimportantin vivo is Glc-6-P,which is not onlyan allostericactivatorof SPSbutalsoan inhibitor of SPSkper se(24).

SPS-PROTEIN PHOSPHATASE Phospho-SPSis dephosphorylated/activatedby atype2A proteinphosphatase(SPS-PP)that is inhibitedby Pi (13). In spinach,thereis a distinct light activationof SPS-PPthat involvesan increasein totalextractableactivity aswell asadecreasein sensitivity to Pi inhibition (41).Thelight activation of SPS-PPcan be blocked by pretreatmentof leaveswith

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cycloheximide(CHX), whichsuggestsarolefor cytoplasmic proteinsynthesis.However,themolecularbasisfor thelight activation remainsunclear;it couldresultfrom eitheracovalentmodificationof existingproteinor thesynthesisofatarget/regulatory subunitor modifyingenzyme.Regardlessof themechanism,thelight modulation of SPS-PPandits regulationby Pi arethoughtto play animportantrolein theactivation of SPSafteradark-to-lighttransition(Figure1).Otherpotential effectorsof SPS-PPincludeavarietyof P-esters(41)andaminoacids(14).Theinhibitionbyaminoacidsmayplayanimportantrolein feedbackregulationof sucrosesynthesis.

OsmoticStressActivation

Activation of SPS, assayedunder selectiveconditions, occurs in spinachleaves(28) and potato tubers (29) incubatedin hyperosmotic solutions ofmannitolor sorbitol.Thesimplestexplanation, dephosphorylation of theregu-

Figure 1 Schematic representation of the regulation of spinach leaf SPSby reversible serylphosphorylation. Multisite phosphorylation andthe identifi cation of the major regulatory site asSer158 are indicated. An increasein Glc-6-P and a decreasein Pi, as might occur during adark-to-light transition, would favor dephosphorylation/activation of SPS andwould alsoincreasecatalyticactivity asaresultof allostericregulation.Anotherimportantfactor maybelight modulationof the regulatory propertiesof SPS-PP, andchangesin cytosolic [Ca2+]. Adaptedfrom Reference13.

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latorysitein responseto thestress,seemsnot to be the case. Rather,it appearsthat a uniquesite(s)on spinachSPSis phosphorylatedduring osmotic stressthat can partially antagonizethe inhibitory effect of phosphorylation of theregulatorysite (Ser158in spinach).Control of this processmay occurat thelevelof geneexpression(14).Indeed,stress-inducedproteinkinaseshavebeendemonstrated(10). Whether a stress-inducedkinaseand the identity of theputativenovelphosphorylation siteare involvedremainsto be established.

Nonregulatory PhosphorylationSites

Spinachleaf SPSappearsto be phosphorylatedon multiple seryl residuesinvivo. Apart from the regulatorysite (Ser158in spinach),thephosphorylationstatusof the other sitesremainsrelatively constantduring light/dark transi-tions, i.e. thesitesmaybeconstitutively phosphorylated.We havetentativelyidentified twoof thenonregulatorysites(17)andarein theprocessof identify-ing endogenousproteinkinasesthat might phosphorylate these residues.

SPS INTRANSGENIC PLANTS

Increasedactivity of SPSin leaveshasbeenachievedin severalspeciesbyoverexpressionof the geneencodingSPSin transgenicplants. Transgenictomatoplantsexpressingthe maizeSPSgenehadelevatedleaf SPS,andthemaizeenzymewasunregulatedwith respectto normal light/dark modulation(4, 5, 42). TheenhancedSPSactivity wasassociatedwith an increasedlight-andCO2-saturatedrateof photosynthesisand,underambientconditions,withincreasedratiosof sucroseto starchin leaves(4) andincreasedpartitioning offixed-C into sucrose(25). Overallgrowthof thetransgenictomatoplantswasnot increasedwhenthe transgeneexpressionwasleaf-specific,i.e. expressedfrom theRubisco smallsubunitpromoter. However,recent results suggest thatgrowth enhancementmay occurwhen SPS expressionis constitutive (i.e.35S-CaMV promoter)and occursin both photosynthetic and nonphotosyn-thetic tissues(3). Micallef et al (25) also found that vegetativegrowth oftransgenictomatoplants(expressingthe maizeSPSgenedriven by the Ru-biscoSSUpromoter) wasnot increasedbut notedthat reproductivedevelop-mentwasenhanced.Total fruit numberwasincreased,the fruit maturedear-lier, andtherewasa substantial increasein total fruit dry weight (25). Morework needs to bedone onthe growth response itself andto determine thebasisfor the enhancementwhen it is observed,but it is speculatedthat SPS,byaffectingtissue[sucrose]might influenceflowering at leastin somespecies.Another intriguingobservation by Micallef et al(25) is that SPStransformantsdid not exhibit thenormalacclimationresponseof leaf photosynthesisto highCO2. Thus,whengrownandmeasuredathighCO2, theSPStransformantshad

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higher ratesof photosynthesisper unit leaf areacomparedwith the controlplants.

SpinachSPShas also beenexpressedin transgenictobaccoand potatoplants.However,despitean increasein SPSprotein, the additionalenzymewasdownregulated,apparentlyby phosphorylation,suchthatmetabolismwasnot affected(37). In order to effectively upregulateSPSactivity andsucrosebiosynthesis, it may be necessaryto produceplantsexpressinga geneticallymodifiedSPSprotein,e.g. withtheregulatory phosphorylationsiteremoved.

A substantial reductionin SPS activityin potatoleavesandtubershasbeenachievedwith an SPS-antisense constructunder control of the 35S-CaMVpromoter(7). As aresultof theantisenseinhibition, sucrosesynthesisin leaveswas reduced, and starch andamino acid synthesiswas increased. Afluxcontrolcoefficientfor SPSwasestimatedto be0.30–0.45 in potatoleaves.Intubers,theresynthesisof sucrosefrom starchwasreducedin theSPS-antisensetransformants.Overall, the resultsstrongly supportthenotion thatSPSis oneof the important controlpointsin sucrosebiosynthesis.

ROLE OFSPS INVIVO

SucroseSynthesisandSugarCycling

In addition to the well-recognizedrole of SPS in sucrosebiosynthesis insourceleaves,it is becomingclearthatsomesucrosesynthesisoccurseveninheterotrophiccells that are engagedin net sucrosedegradation.Significantturnoverof the endogenoussucrosepool hasbeenidentified in a variety oftissues,including potatotubers(7) and germinating Ricinuscotyledons(8).Turnoverof sucroseis thought to involve a futile cycle of simultaneoussyn-thesis (by SPSand SuSy) and cleavage(by SuSy). Thus, relatively smallchangesin unidirectionalfluxescanoccurandproducemuchlargerchangesinnet flux throughthe sucrosepool, without large changesin metabolites (7).Changesin the activation stateof SPS,presumablyas a result of proteinphosphorylation, havebeen shown to contribute to changesin net flux throughthe sucrosepool in Ricinuscotyledons(8) and potatotubers (7).

Factors AffectingSPSExpression

Expressionof SPSmRNA andenzymeproteinis controlleddevelopmentally,e.g.duringleafdevelopment(20),andin matureleaves bya variety offactors,including irradiance(20) andN-nutrition (J Huber,unpublisheddata,1994).The responsesof SPSto changesin irradianceillustratethe integration ofmechanismsfor control of SPSactivity. Transferof spinachplantsgrown atlow irradianceto highirradiance resultsin a rapidincrease innet photosynthe-sis andflux of C into sucrose.Within 3 h of transfer,SPSprotein(andVmax

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activity) remainconstant,but activationstateof the enzymeis increasedpre-sumablyby dephosphorylationof Ser158(15).After longerperiodsof time atthe higher irradiance,thereis a gradualincreasein SPSproteinandmRNA(20). Thus,regulationof enzyme activityby covalent modificationand controlof SPS geneexpressionfunctionin anintegratedmannerto provideshort-andlong-termcontrol,respectively.

SPSgeneexpressionalsorespondsto sugars.Provisionof Glc to excisedsugarbeetor potato leavesstrongly increasedthe steadystatelevel of SPSmRNA, whereasexogenoussucroseslightly repressedexpression[at leastinsugarbeet(9)]. A similar responseis seenin potatotuberswhenstarchsynthe-sis is inhibited by antisenserepressionof ADP-Glc pyrophosphorylase.Thetransgenicpotato tubersaccumulatedsoluble sugars(sucroseand glucose),andtherewasa tremendousincreasein the steadystatelevel of SPSmRNA(27). SPSactivity, measuredunderselectiveassayconditions, was also in-creasedrelativeto wild-typetubers(7), but thebasisfor theincreasedactivitywasnot determined.The resultssuggestthat hexosesugars,or somerelatedmetabolite(s), might beinvolvedin the controlof expressionof SPSas wellasothergenes.It is importantto note that the sugareffectson SPSexpressionhavebeenidentified both whenexogenoussugarsareprovided(9) andwhenendogenoussugars are manipulated genetically(27).

CONCLUDING REMARKS

With thecloningof theSPSgene from fivespecies, we are beginningto betterunderstandthe SPS molecule with preliminary identification of importantdomainssuchassubstratebindingsitesandphosphorylation sites.In additionto itsrole in sourceleaves, SPSis alsosignificantin sink tissueswherea futilecycle of simultaneousdegradationandresynthesisoccursin a wide rangeoftissues.Manipulation of SPSactivity is now possible andholdspromiseforimpactingonplantgrowth andresourceallocation.SPSis clearly animportantfactorregulatingsucrosebiosynthesis,but it is importantto recognizethat it isnot the only factor, and in addition, changesin SPSprotein level are oftencompensatedfor by adjustments in the activation stateof the enzymeas aresult of phosphorylation/dephosphorylation.Consequently, future transfor-mationstudiesneedto considerproductionof plantswith SPSproteinmodi-fied in termsof phosphorylation control.

ACKNOWLEDGMENTS

We thank colleaguesfor sharingunpublishedmanuscriptsand ideas,espe-cially Mike Salvucci,ChristineFoyer,Mark Stitt, Luis Herrera-Estrella,andLothar Wil lmitzer, and other membersof the laboratoryfor discussions. Fi-nancialsupportfrom theUS Departmentof Agriculture/AgriculturalResearch

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Service,the USDA NationalResearchInitiative Competitive GrantProgram,and theDepartmentof Energy isgratefullyacknowledged.

Any Annual Reviewchapter, aswell asany arti clecited in an Annual Reviewchapter,may bepurchased fromthe Annual ReviewsPreprints and Reprints service.

1-800-347-8007; 415-259-5017; email: arpr@class.org

LiteratureCited

1. Crafts-Brandner SJ, Salvucci ME. 1989.Speciesandenvironmentalvariationsin theeffect of inorganic phosphate on sucrose-phosphatesynthaseactivity. Plant Physiol.91:469–72

2. Doehlert DC, HuberSC.1985. Theroleofsulfhydryl groupsin theregulation of spin-achleaf sucrose-phosphatesynthase.Bio-chim.Biophys.Acta 830:267–73

3. FoyerCH,GaltierN, QuickP. 1994. Modi-fications in carbon assimilation, carbonpartitioningandtotal biomassasaresultofover-expressionof sucrose phosphatesyn-thase in transgenic tomato plants. PlantPhysiol. 105:S23

4. Galtier N, Foyer CH, Huber JLA, VoelkerTA, Huber SC. 1993. Effects of elevatedsucrose-phosphate synthase activity onphotosynthesis,assimilatepartitioningandgrowth in tomato (Lycopersicon esculen-tum var. UC 82B). Plant Physiol. 101:535–43

5. Galtier N, Foyer CH, Murchie E, Alred R,Quick P, et al. 1995. Effects of light andatmosphericcarbon dioxideenrichment onphotosynthesisandcarbon partitioning inthe leaves of tomato (Lycopersiconescu-lentum L.) plants over-expressingsucrose-phosphate synthase. J. Exp. Bot. 46:1335–44

6. Deletedin proof7. Geigenberger P, KrauseK-P, Hill LM, Re-

imholzR,MacRaeE,etal.1995. Theregu-lation of sucrose synthesis in leaves andtuberof potato plants. In SucroseMetabo-lism, Biochemistry, Physiology and Mo-lecular Biology, ed.H Pontis,G Salerno, EEcheverria.Rockville,MD: Am.Soc.PlantPhysiol.

8. Geigenberger P, Stitt M. 1991. A “fut ile”cycle ofsucrose synthesisanddegradationis involved in regulating partitioning be-tween sucrose,starch and respiration incotyledonsof germinatingRicinuscommu-nis L. seedlings whenphloemtransport isinhibited. Planta 185:81–90

9. Hesse H, Sonnewald U, Wil lmitzer L.1995. Cloning andexpression analysis ofsucrose-phosphate synthase from sugar

beet(Betavulgaris). Mol. Gen.Genet. 247:515–20

10. HolappaLD, Walker-SimmonsMK. 1995.Thewheatabscisicacid–responsiveproteinkinasemRNA, PKABA1, is up-regulatedby dehydration, cold temperature, andos-motic stress.Plant Physiol. 108:1203–10

11. Huber JLA, Huber SC.1992. Site specificserinephosphorylation of spinachleaf su-crose-phosphate synthase. Biochem. J.283:877–82

12. Huber SC,Huber JL. 1991. In vitro phos-phorylationandinactivationof spinachleafsucrose-phosphate synthase by an endo-genous protein kinase.Biochim. Biophys.Acta1091:393–400

13. Huber SC,Huber JLA. 1992. Role of su-crose-phosphate synthase insucrosemeta-bolism in leaves. Plant Physiol. 99:1275–78

14. Huber SC,HuberJLA, McMichaelRW Jr.1993. The regulation of sucrosesynthesisin leaves. In Carbon Partitioning WithinandBetweenOrganisms,ed.CJPollock,JFFarrar,AJGordon,pp.1–26.Oxford:BIOSSci.

15. Huber SC, Huber JL, McMichaelRW Jr.1994. Control of plantenzymeactivity byreversible protein phosphorylation. Int.Rev. Cytol. 149:47–98

16. Huber SC, Huber JL, Pharr DM. 1993.Assimilate partitioning and util ization insource and sink tissues.In InternationalCrop Science,ed. DR Buxton, I:761–77.Madison, WI: Crop Sci.Soc.Am.

17. Huber SC,McMichaelRW Jr, BachmannM, Huber JL, Shannon JC, et al. 1995.Regulation of leaf sucrose-phosphatesyn-thaseand nitrate reductaseby reversibleprotein phosphorylation. In Protein Phos-phorylation in Plants,ed.PRShewry. Ox-ford: Oxford Univ. Press.In press

18. Huber SC, McMichaelRW Jr, Huber JL,Bachmann M, Yamamoto YT, ConklingMA. 1995. Light regulationof sucrosesyn-thesis: role of protein phosphorylation andpossible involvementof cytosolic [Ca2+].In Carbon Partitioning and Source-SinkInteractions in Plants,ed.MA Madore,W

SUCROSE-PHOSPHATE SYNTHASE 443

Ann

u. R

ev. P

lant

. Phy

siol

. Pla

nt. M

ol. B

iol.

1996

.47:

431-

444.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

gby

Uni

vers

ity o

f Q

ueen

slan

d on

10/

06/1

3. F

or p

erso

nal u

se o

nly.

Lucas, pp. 35–44. Rockville, MD: Am.Soc.Plant Physiol.

19. JangJ-C,SheenJ. 1994. Sugar sensing inhigherplants.Plant Cell 6:1665–79

20. Klein RR, Crafts-Brandner SJ, SalvucciME. 1993. Cloninganddevelopmental ex-pression of the sucrose-phosphate-syn-thase gene from spinach. Planta 190:498–510

21. Lunn JE,apReesT. 1990. Apparent equi-librium constant andmass-action ratio forsucrose-phosphate synthase in seedsofPisumsativum.Biochem.J. 267:739–43

22. McMichaelRW Jr, Bachmann M, HuberSC.1995. Spinachleaf sucrose-phosphatesynthaseandnitratereductasearephospho-rylated/inactivatedby multiple protein ki-nases invitro. Plant Physiol. 108:1077–82

23. McMichaelRWJr,KleinRR,SalvucciME,HuberSC.1993. Identificationof themajorregulatory phosphorylationsite in sucrose-phosphatesynthase.Arch. Biochem. Bio-phys.307:248–52

24. McMichaelRW Jr,KochanskyJ,KleinRR,Huber SC. 1995. Characterization of thesubstratespecificity of sucrose-phosphatesynthase protein kinase.Arch. Biochem.Biophys.321:71–75

25. Micallef BJ, Haskin KA, VanderveerPJ,Roth K-S, Shewmaker CK, Sharkey TD.1995. Altered photosynthesis, floweringandfruitingin transgenic tomatoplantsthathaveanincreasedcapacity for sucrosesyn-thesis.Planta 196:327–34

26. Mi ller AJ, SandersD. 1987. Depletion ofcytosolic free calcium induced by photo-synthesis.Nature326:397–400

27. Müller-Röber BT, Sonnewald U,Wil lmitzerL. 1992. Inhibitionof ADP-glu-cosepyrophosphorylaseleadstosugarstor-ing tubersandinfluencestuber formationand expressionof tuber storage proteingenes.EMBOJ. 11:1229–38

28. Quick P, Siegl G,NeuhausHE,FeilR,StittM. 1989. Short termwaterstressleads to astimulationof sucrosesynthesisbyactivat-ing sucrose phosphate-synthase. Planta177:536–46

29. Reimholz R,GeigenbergerP, Stitt M. 1994.Sucrose phosphate synthaseis regulated,via metabolites and protein phosphoryla-tionin potatotubers,in amanneranalogousto the enzyme in leaves. Planta 1992:480–88

30. SalanoubatM, BelliardG.1987. Molecularcloningandsequencingof sucrosesynthase

cDNA from potato (Solanum tuberosumL.): preliminarycharacterizationof sucrosesynthase mRNA distribution. Gene 60:47–56

31. Deletedin proof32. Salvucci ME, Klein RR. 1993. Identifica-

tion of the uridine-binding domain of su-crose-phosphate synthase:expression of aregion of the protein that photoaffinity la-belswith 5-azidouridine diphosphate-glu-cose.Plant Physiol. 102:529–36

33. Salvucci ME, van de Loo FJ, Klein RR.1995. The structure of sucrose-phosphatesynthase. In Sucrose Metabolism, Bio-chemistry, Physiology and Molecular Biol-ogy,ed.HGPontis,GLSalerno,EEchever-ria.Rockville,MD: Am.Soc.PlantPhysiol.

34. SonnewaldU,BasnerA. 1993.EMBL DataLibrary, AccessionNo.S34172

35. Sonnewald U, Quick WP, MacRae E,Krause KP, Stitt M. 1993. Purifi cation,cloning andexpressionof spinachleaf su-crose-phosphatesynthasein E.coli. Planta189:174–81

36. Stitt M, HuberSC,KerrP. 1987. Control ofphotosynthetic sucroseformation. In Bio-chemistry of Plants, ed. MD Hatch, NKBoardman, 8:327–409. New York: Aca-demic

37. Stitt M, Sonnewald U. 1995. Regulation ofmetabolismin transgenic plants.Annu.Rev.Plant Physiol. Plant Mol. Biol. 46:341–68

38. Stitt M, Wilke I, Feil R, Heldt HW. 1988.Coarsecontrol of sucrose-phosphate syn-thasein leaves: alterations of the kineticpropertiesin response tothe rateof photo-synthesisandtheaccumulation of sucrose.Planta 174:217–30

39. Deletedin proof40. Weiner H. 1995. Antibodies that distin-

guishbetweentheserine-158phospho-anddephospho-form of spinach leaf sucrose-phosphate synthase. Plant Physiol. 108:219–25

41. Weiner H, Weiner H, Stitt M. 1993. Su-crose-phosphate synthasephosphatase,atype 2A protein phosphatase,changes itssensitivity towardsinhibition by inorganicphosphate in spinach leaves.FEBS Lett.333:159–64

42. Worrell AC, BruneauJ-M, Summerfelt K,Boersig M, Voelker TA. 1991. Expressionof a maizesucrosephosphatesynthaseintomato alters leaf carbohydrate partition-ing. Plant Cell 3:1121–30

444 HUBER & HUBER

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