Seediscussions,stats,andauthorprofilesforthispublicationat:http://www.researchgate.net/publication/282604076
ImplicationsofCandidatusPhytoplasmamali
infectiononphloemfunctionofappletrees
ARTICLEinENDOCYTOBIOSISANDCELLRESEARCH·OCTOBER2015
READS
11
6AUTHORS,INCLUDING:
AxelMithöfer
MichaelReichelt
FriedrichSchillerUniversityJena
MaxPlanckInstituteforChemicalEcology
110PUBLICATIONS3,390CITATIONS
101PUBLICATIONS3,114CITATIONS
SEEPROFILE
SEEPROFILE
AlexandraCUFurch
FriedrichSchillerUniversityJena
19PUBLICATIONS377CITATIONS
SEEPROFILE
Availablefrom:AlexandraCUFurch
Retrievedon:12October2015
Journal of Endocytobiosis and Cell Research (2015) 67-75 | International Society of Endocytobiology
zs.thulb.uni-jena.de/content/main/journals/ecb/info.xml
Journal of Endocytobiosis and
Cell Research
Implications of Candidatus Phytoplasma mali infection on
phloem function of apple trees
Matthias R. Zimmermann1+, Bernd Schneider2+, Axel Mithöfer3, Michael Reichelt4,
Erich Seemüller2 and Alexandra C.U. Furch1*
1InstituteofGeneralBotanyandPlantPhysiology,Friedrich‐
Schiller‐University Jena, Dornburger Str. 159, 07743 Jena,
Germany; 2 Julius Kuehn Institute (JKI), Federal Research
CentreforCultivatedPlants,InstituteforPlantProtectionin
Fruit Crops and Viticulture, Schwabenheimer Str. 101,
69221 Dossenheim, Germany; 3 Max Planck Institute for
Chemical Ecology, Department of Bioorganic Chemistry,
Hans‐Knöll‐Straße8,07745Jena,Germany;4MaxPlanckIn‐
stitute for Chemical Ecology, Department of Biochemistry,
Hans‐Knöll‐Straße8,D‐07745Jena,Germany;
+MatthiasR.ZimmermannandBerndSchneiderhavecon‐
tributed equallytothearticle. *correspondenceto:Alexandra.Furch@uni‐jena.de
Appleproliferation(AP)iscausedbyapsyllid‐transmit‐
tedphytoplasmaandisoneofthemosteconomicallyim‐
portant diseases on apple in Europe. AP was first re‐
portedinnorthernItalyandinthefollowingyearsfrom
manyEuropeancountries.Previousresearchonphyto‐
plasma‐induced diseases of rosaceous fruit trees
(mainlyAP,peardeclineandEuropeanstonefruityel‐
lows), exhibits strong evidence that phloem injuries
play a central and probably universal role in phyto‐
plasmapathogenicity.Theeffectofphytoplasmainfec‐
tionsonphloemfunctionandtheresultingdiseasesre‐
ceivedlittleattentioninthepast.Phytoplasmainfection
severelyimpairsassimilatetranslocationinhostplants
andmightberesponsibleformassivechangesinphloem
physiology including signalling components. As shown
for other phytoplasma species, infection brings about
Ca2+influxintosievetubes,leadingtosieve‐tubeocclu‐
sionbycallosedepositionorproteinplugging,whichis
assumedalsoforAPphytoplasma.Effectorsmaycause
gatingofsieve‐element Ca2+ channels leading tosieve‐
tube occlusion with presumptive dramatic effects on
phytoplasma spread, photoassimilate distribution and
thewholephloemphysiology.However,thereisindica‐
tionthatphloemloadingisaffectedbyphytoplasmain‐
fection.Assieveelementsneedapermanentinputofen‐
ergytoensuretheirviability,sugarmetabolismandthe
associate energy production of the companion cells
have a dramatic impact on the physiological fitness of
Journal of Endocytobiosis and Cell Research VOL 26 | 2015
phloemfunction.Itispresumptivethatsignalling sub‐
stances are produced prior sieve‐element occlusion to
ensurethespreadthroughouttheplantbody.Analyses
ofdiversephytohormonesinresponsetochallengewith
APphytoplasmashowastrongincreaseofsalicylicacid
accompaniedbyadecreaseofjasmonicacid.Thissup‐
portstheideathatphytohormoneshavearoleinplant
defencesignallingagainstphytoplasmainfection.Itisa
matter of debate whether mechanisms involved in
phloemimpairmentcoulddifferbetweenpathosystems
andvarywiththeplantsusceptibilitytoinfection.
JournalofEndocytobiosisandCellResearch(2015)67‐75
Category:Reviewarticle
Keywords:abscisicacid,appleproliferation,auxin,Candida‐
tusPhytoplasmamali,jasmonates,phloem,phytohormones,
plantdefence,plant‐phytoplasmainteraction,salicylicacid,
sieve‐elementocclusion
Accepted:14September2015
____________________________________________________________________
Introduction
Economical significance of phytoplasma‐induced
diseases
Phytoplasmascausediseasesinmorethan1000plantspe‐
cies worldwide (Seemüller et al. 2002). In Europe, mainly
perennialplantssuchasfruittreesandshrubs,grapevine,a
rangeofforestandlandscapetrees,andsomeornamentals
areknowntobeaffected.AmongthefruitcropsgrowninEu‐
rope,appleproliferation(AP),apricotchloroticleafroll,lep‐
tonecrosisofPrunussalicinaplums,peardeclineaswellas
phytoplasma diseases of grapevine are of particular im‐
portancebyimpairingsizeandqualityofthecropandvigor
and longevity of the trees. The economic losses of phyto‐
plasmadiseasesaredifficulttoassess.Forappleprolifera‐
tionalossof125mio€i.e.hasbeenestimatedforGermany
andItalyduetoproductionoftastelessfruits(Strauss2009).
Additional expenses accrue for plant protection measures
like insecticide applications or rouging. The diseases of
pome‐andstonefruitsarecausedbythecloselyrelatedspe‐
ciesCandidatusPhytoplasmamali,Ca.P.prunorumandCa.P.
pyri,respectively,thatformadistinctgroupwithinthephy‐
logenetic phytoplasma clade (Seemüller and Schneider
2004), while the grapevine diseases are caused by other
moredistantlyrelatedphytoplasmas.Duetotheireconomic
importance,thetemperatefruittreepathogensareamong
themostextensivelystudiedphytoplasmas.
67 Candidatus phytoplasma mali affects phloem function, Zimmermann MR et al.
Characteristicsofphytoplasmas
phloem tissue of new host plants (Figure 2). There is evi‐
dencethatsomephytoplasmasareverticallytransmittedto
theirprogeny(Hanboonsongetal.2002),ortransmittedvia
seedsasshownforcoconutembryos(Cordovaetal.2003),
however,thepredominantroutemeansofsurvivalofphyto‐
plasmasisthroughtransmissionbetweeninsectsandplants.
Theyappeartomanipulatetheirinsectandplanthoststoen‐
hancetheirowntransmissionefficiencies.Inplants,theyre‐
mainmainlyrestrictedtothephloemtissue(Doietal.1967;
WhitcombandTully1989),andspreadthroughouttheplant
bymovingthroughtheporesofthesieveplatesthatdivide
the phloem sieve tubes (Figure 2). As mature sieve tubes
usually contain the highest concentrationof phytoplasmas
(Christensenetal.2004),theywerealsofoundinsidethecy‐
toplasmofimmaturephloemelements.Nexttosievetubes,
phytoplasmas have been detected in the cytoplasm of
phloemparenchymacellsadjacenttosieveelements(Sears
andKlomparens1989;Silleretal.1987).
Phytoplasmasareplant‐pathogenicprokaryotesoftheclass
Mollicutes (mycoplasmas) characterized by the lack of a
rigid cell wall and a small (0.53 – 1.35 Mb), low‐GC (21 –
28%)genome(Kubeetal.2008;Marconeetal.1999).They
aremostcloselyrelatedtootherwall‐lessbacteriasuchas
the saprophytic acholeplasmas and, more distantly, to the
human‐ and animal‐pathogenic and commensal mycoplas‐
mas.Inplants,phytoplasmasinhabitalmostexclusivelythe
phloem sieve elements and are transmitted within their
planthostsbyphloemsap‐feedinghomopterousinsects,pri‐
marily leafhoppers (Cicadellidae) and, less frequently,
planthoppers(Fulgoroidea)andpsyllids(Psylloidea,Figure
1)(Seemülleretal.2002).
Figure1:AdultfemaleofCacopsyllapicta(MayerandGross,JKI).
Newgeneration(emigrant)onMalussp.
Phytoplasmainfectionsaresystemic,persistentanddifficult
tocontrol.Duetotheinabilitytocultivatephytoplasmasun‐
der axenic conditions, many biological aspects including
their phytopathogenic traits are poorly understood. Thus
far,themostimportantinformationonpathogenicandmet‐
abolictraitsofthesepathogenswasobtainedfromcomplete
genome sequences of six phytoplasmas that include Ca. P.
mali(Kubeetal.2008).Theremainingfivearerepresenta‐
tives of the aster yellows (Ca. P. asteris)/stolbur (Ca. P.
solani) phytoplasma complex. Facilitated by genomic se‐
quencedata,foureffectorproteinsrelatedtovirulencewere
identifiedingenomesoftwoCa.P.asterisstrainsthatinduce
witches’broom(WB)formationanddwarfism(Hoshietal.
2009)ortargetthenucleioftheplantandmayalterplant
cellphysiology(Sugioetal.2011).Noneoftheseeffectorsor
othervirulence‐relatedfactorsweredetectedinphytoplas‐
masinfectingrosaceousfruittrees,thatareonlydistantlyre‐
latedtoCa.P.asterisorCa.P.solani.However,phytoplasma
induced phloem injuries were observed in diseased fruit
treesleadingtoseveresymptomsandthereforemayplaya
centralandprobablyuniversalroleinphytoplasmapatho‐
genicity.
Lifecycle
Phytoplasmasarebacteriathatliveinandmanipulateplants
and insects (Hogenhout et al. 2008). They require replica‐
tion in both hosts for their survival. Phytoplasmas are in‐
gestedwiththeplant/phloemsapandmovethroughthein‐
sectstyletandinvadegut,epithelialandmuscletissues,he‐
molymph and salivary glands. They multiply in salivary
glandcellsandaretransportedwiththesalivabackintothe
68
Figure2:Phytoplasmalifecycle.Reddotsrepresentthephytoplas‐
masandredarrowsshowthespreaddirection.
Symptomsofappleproliferation
AP induces specific and nonspecific symptoms on shoots,
leaves, fruits, and roots. Specific symptoms of the vector‐
born disorder are WB (Figure 3B), rosettes, enlarged stip‐
ules, and non‐specific symptoms are foliar reddening, yel‐
lowing,undersizedfruitandgrowthsuppression.However,
symptomexpressionisoftensubjecttofluctuation.WBsand
undersizedfruitaretypicalfornewlyinfectedtreesandcan
beoftenobservedforonlyoneorafewyearsafterfirstap‐
pearance.Then,treesmayrecoverandshownooronlymild
symptomsforshorterorlongerperiods(Carraroetal.2004;
Seemülleretal.1984).Symptomsaremorepronouncedin
young trees where the root system is severely impaired
leadingtoalowvigorandoftenadeclineofthetree.Symp‐
tom severity also varies greatly within the genus Malus.
KartteandSeemüller(1988)founddifferentresponsesafter
experimental inoculation of more than 40 taxa. All trial
Journal of Endocytobiosis and Cell Research VOL 26 | 2015
Candidatus phytoplasma mali affects phloem function, Zimmermann MR et al. plants could be infected but some reacted hypersensitive
whereasotherswereastolerantascommercialrootstocks.
Symptomdevelopmentalsodependsonthevirulenceofthe
infectingAPphytoplasmastrainasshowninastudyof24
Ca. P. mali accessions of different origin (Seemüller and
Schneider2007).Virulent(Figure3B),moderatelyvirulent
andavirulent(Figure3AandD)accessionswerepresentin
almost equal numbers. The plant reaction due to the viru‐
lence of the pathogen is also evident in the experimental
phytoplasmahostCatharanthusroseusasexemplifiedwith
the avirulent and virulent apple proliferation strain 1/93
and12/93,respectively(Figure3D‐E).
Figure3:Malusxdomesticacv.GoldenDeliciousinfectedbydiffer‐
rentaccessionsoftheappleproliferationphytoplasma[(A)acces‐
sion1/93avirulent;(B)accession12/93virulent]andCatharanthus
roseusplants[(C)healthy;(D)accession1/93avirulent;(E)acces‐
sion12/93virulent].
TheplanthabitatofAPphytoplasmas
Theplantvascularsystemservesasmainrouteforthelong‐
and short‐distance transport of various compounds
throughout the plant (van Bel 1996; Hafke et al. 2005). It
consistsoftwomajortissuetypes—xylem,whichconducts
waterandnutrients,andphloem,whichtransportsmainly
organiccompounds.
Sieveelements(SEs),companioncells(CCs)andphloem
parenchymacells(PPCs)arethethreephloemelementsin‐
volved in long‐distance transport of photoassimilates (van
Bel1996;Hafkeetal.2005)andseverallong‐distancesig‐
nals, such as diverse RNA‐species, proteins, nitric oxide,
azelaic acid, SFD1/GLY1‐derived glycerol‐3‐phosphate, de‐
hydroabietinal, phytohormones (e.g. salicylic acid,
jasmonates),aswellaselectricalsignals(e.g.Davies2006;
Parketal.2007;Gaupelsetal.2008;Jungetal.2009;Demp‐
seyandKlessig2012;Furchetal.2014).Whiletheenucleate
SEs form the transport pathway, the CCs are engaged in
maintaining their viability. A high density of pore‐plasmo‐
desma units (PPUs) and tight endoplasmic reticulum (ER)
couplingSEandCCunderlinesanintimatesymplasmiccon‐
nectionacrossthisboundary(Kempersetal.1998;Martens
etal.2006).Permanentmetabolicandenergeticsupportfor
theSEsisrequiredsincetheircellularmachineryislargely
reducedduringontogenytomakewayforamoreefficient
massflow(vanBelandKnoblauch2000).
As phytoplasmas are obligate parasites, living in the
phloemsystemandspreadthroughouttheplantbymoving
throughtheporesofthesieveplates(Figure4),animpair‐
mentofthephloemisverylikely.
Phytoplasma‐induced injuries of the phloem were first
identifiedbySchneider(1973).Hefoundthatseverephyto‐
plasmainfectionsofpearareassociatedwiththepathologi‐
caldepositionofcalloseinsidethesieveelementsfollowed
bythecollapseoftheseSEsandtheirCCs.Thislossofcon‐
ductingtissuemayresultinahyperactivityofthecambium,
leading to the formation of a more or less pronounced re‐
placementphloem.Asthistissueusuallybecomessimilarly
affected, starch accumulates in the stem, especially in the
leaves, whereas the roots are starving. Depending on the
susceptibilityofthegenotype,diseasedtreesdeclineorsuf‐
fer more or less (Schneider 1976; Schaper and Seemüller
1982;KartteandSeemüller1991).Thephloemsystemofall
higherplantspervadestheplantfromrootstoleaftipsand
ensurescommunicationandnutrientsupplyamongallplant
organsandtissues.Apotentiallossofvascularsapduetoa
wound is prevented via efficient sealing mechanisms with
callose and proteins (Knoblauch et al. 2003; Furch et al.
Figure4:TransmissionelectronmicrographsofCa.P.maliinfectedCuscutaodorataplants.Theoverviewofphloemcells(A)showaninfection
ofsieveelements(SE),companioncells(CC)andphloemparenchymacells(PPC)withphytoplasmas.Highermagnification(B,C)illustrates
theaccumulationofphytoplasmasatthesieveplateanddenselypackedphytoplasmas,somearebudding(arrowheads)(D).Sieveporesare
markedwitharrows.SP,sieveplate;SER,sieveelementreticulum.
Journal of Endocytobiosis and Cell Research VOL 26 | 2015
69 Candidatus phytoplasma mali affects phloem function, Zimmermann MR et al.
2007). Thus, phloem sap cannot be usually collected by
wounding of the vascular system, as known for cucurbits
andRicinus(DinantandLemoine2010;Zimmermannetal.
2013). Interestingly, considerable volumes of phloem exu‐
date can be collected from severely affected trees (Figure
5A)butnotfrominfectednonsymptomaticorhealthytrees.
Thegreatvolumeof~300µlperdroplet,thenumerouspro‐
teins and the pH of 7.5‐8 indicates sieve‐tube content (Fi‐
gure5).Highproteinconcentrationsbetween0.07and110
mg ml‐1 with 150‐1000 different proteins were found in
sieve‐tubeexudatesofvariousplantspecies(e.g.Schobertet
al.1998;Rodriguez‐Medina2009;Zimmermannetal.2013).
ThepHvalueofthecytosol,respectivelysieve‐tubeexudate,
isacharacteristic,regulatedandconsistenttrait(Ojaetal.
1999;Felle2001,2005;Hafkeetal.2005)andisprobably
notorequallyinfluencedbytheinfluxofxylemwaterafter
wounding (Zimmermann et al. 2013). These findings indi‐
cate that the phytoplasma infection impairs the sealing
mechanismofthephloem(Kollaretal.1989).Ashasbeen
shownforseveralotherbacterialplantpathogens,itislikely
that phytoplasmas produce a series of virulence proteins
thatsuppressplantresponses(Hogenhoutetal.2008).
Figure5: AppletreeinfectedwithCa.P.malishowingwound‐inducedvascularexudation.Thegreatvolumeof~300µlperdroplet(A),the
numerousproteins(B)andthecytoplasmicpHof7.5‐8(C)indicatessieve‐tubecontent.Exudatewascollectedfromthestemofanappletree
aftercuttingbyapipetteandtransferredto4‐foldconcentratedreducingsamplebuffer(Roti‐Load1;Carl‐Roth,Karlsruhe,Germany)inthe
proportionof1:3.One‐dimensionalSDS–PAGEofexudatewascarriedoutaccordingtoLaemmli(1970)byusinga4%stackinggelanda12%
separationgelinaMiniProtean3ElectrophoresisSystem(Bio‐RadLaboratories,Hercules,CA,USA)withPrecisionPlusProteinStandard–All
Blue(Bio‐Rad)asaproteinsizemarker.Thegelswerestainedbysilver(HeukeshovenandDernick1988).Exudates(V=1–4µL)weredropped
onindicatorpaper(Macherey‐Nagel,Düren,Germany).MES/TRISbufferswithdifferentpHvalues(4–9.7)wereusedforcalibrationline.
Somephytoplasmasinduceseverephloemnecrosisintheir
hostplants,indicatingthattheseplantsreacttothephyto‐
plasma infection. However, many phytoplasmas, do not
seemtoinducephloemnecrosisbutstillaccumulatetohigh
densitiesinphloemelements.
Phloemlocatedplantdefence
Itisapermanentchallengeforplantstoprotectthemselves
againsttheattackandharmfuleffectsofpathogens.Inpar‐
ticular, the phloem is one preferred destination for plant
pathogens,asitmediatesthetranslocationofprimaryand
secondarymetabolites.Hence,thephloemreflectsaninter‐
estingpropagationpathwayandanimportantsourceofen‐
ergy and defence‐related information. Compromised
phloemfunctionalitymayhavedisastrousconsequencesfor
plant health and development. Focus on phloem research
overthepastyearshasbeenshiftedfromthetranslocation
ofnutrientstowardssignallinganddefence.Oneexampleis
theocclusionofSEs.
Sieve‐elementocclusion
The SE sealing prevents an invasion and manifestation of
pathogens and their released effectors/elicitors, redirects
nutrient flows, and enriches signal and defence molecules
70
andmaybeseenasageneraldefenceresponsetopathogen
attack.Sievetubesmaybeconstrictedbycallosepluggedby
proteins(e.g.Furchetal.2007).Callosedepositionisanuni‐
versal mode of sieve‐plate occlusion (Kudlicka and Brown
1997;Nakashimaetal.2003).Itisaß‐1.3‐glucanpolymer
thatisproducedenzymaticallyanddepositedextracellularly
aroundplasmodesmataandsieveporesintheformofcollars
(Blackmanetal.1998;Zabotinetal.2002)asareactionto
chemical or mechanical stress (Kudlicka and Brown 1997;
Nakashimaetal.2003;Levyetal.2007).Asforproteinplug‐
ging, the numerous aggregation forms of phloem‐specific
proteins(CronshawandSabnis1990)displayanimmense
variation within the plant kingdom. Spindle‐like protein
bodiesthatonlyoccurinFabaceae(PalevitzandNewcomb
1971)wereobservedinViciafabasievetubes.Triggeredby
osmoticshockorCa2+application,theseproteinbodiesdes‐
ignated as forisomes (Knoblauch et al. 2003) disperse di‐
rectly and recondense “spontaneously” into the original
stateinintactsievetubes(Knoblauchetal.2001,2003).Ma‐
nipulation of isolated forisomes showed a strong cal‐
cium‐dependentdispersion/recondensation.
TransientinfluxofCa2+constitutesanearlyelementof
signalling cascades triggering pathogen defence responses
Journal of Endocytobiosis and Cell Research VOL 26 | 2015
Candidatus phytoplasma mali affects phloem function, Zimmermann MR et al. inplantcells.Numerousstudieshaveprovidedevidencethat
Ca2+playsapivotalroleinactivatingtheplant’ssurveillance
system against attempted microbial invasion (Yang et al.
1997; Scheel 1998). Stimulus specific and spatio‐temporal
defined Ca2+ signatures of characteristic magnitude, fre‐
quency,anddurationareassumedtomaintainsignalspeci‐
ficityoftransductioncascades(Thuleauetal.1998;Trewa‐
vas1999).Subsequently,thebindingofcytosolicCa2+to,for
example,calmodulin,Ca2+‐dependentproteinkinases,phos‐
phatases and Ca2+‐gated ion channels facilitates down‐
stream signal transduction directed toward activation of a
signal‐specificcellularresponse(Blumwaldetal.1998).
V. faba sieve tubes infected by a Stolbur phytoplasma
containconsistentlydispersedforisomeshintingatCa2+lev‐
elsappreciablyhigherthaninhealthysievetubes(Musetti
etal.2013).Moreover,thesievetubesaresealedwiththick
depositsofcallose(Musettietal.2013).Undoubtedly,phy‐
toplasmasinduceCa2+influxwithinherentconsequencesfor
forisome dispersion and callose synthesis. As V. faba and
Stolbur phytoplasma is an exclusive artificial system, it
wouldbeofinteresttostudyhowapathogeninteractswith
anaturalhost(forexampleMalusandAPphytoplasma).
Theabovereportedeffectoftheimpairmentoftheseal‐
ingmechanismisunexpectedbecausebothocclusionmech‐
anismsandtheinducedphloemnecrosisreduceorinhibit
themassflow.Thefindingsindicateconditions,depending
onthephytoplasmatiterorthestrain(avirulentorvirulent)
where the sieve tubes are still functioning but where the
sealingmechanismisaffected.Sofar,ithasbeenunclearif
SEocclusion(maybereversible)ispartoftheplant’sstrat‐
egyagainstphytoplasmaspreadorifphytoplasmasinduce
andexploresymplasmicisolationforanundisturbedmulti‐
plication(vanBeletal.2014).Hence,onemajorfuturetask
should be to understand the physiological relationship of
phytoplasmainfection,sieve‐tubeocclusionandwoundin‐
ducedvascularexudation.
Stress‐relatedchangesofphytohormonesandproteins
TheformationofWB,thesmall‐fruitsymptom,andthere‐
ducedrootgrowthofdiseasedappletreescanbeexplained
withaninfection‐inducedhormonalimbalance,inparticular
byanalteredauxin(Indole‐3‐aceticacid;IAA)/cytokininra‐
tio(HegeleandBangerth1997).Thereisindicationthatsyn‐
thesis,metabolismand/ortransportofplanthormonesare
affectedindiseasedplants(Hare1977).Phytohormonedis‐
persioninleavesofCa.P.mali‐infected(virulentandaviru‐
lent) and healthy apple trees (Figure 6), show significant
phytoplasma‐triggered effects for all examined phytohor‐
mones(Figure6A:IAA‐auxin,ABA‐abscisicacid,SA‐salicylic
acid; Figure 6B: JA‐jasmonic acid, cis‐OPDA‐cis‐12‐oxo‐
phytodienoicacid,JA‐Ile‐(+)‐7‐iso‐jasmonoyl‐L‐isoleucine).
Thisisconsistentwitharoleinimmunesignalling(Dempsey
and Klessig 2012; Pieterse et al. 2012). It is generally ac‐
ceptedthatpathogenswithabiotrophiclifestylearemore
sensitive to SA‐mediated induced defence mechanisms
(Glazebrook2005),henceasignificantincreaseofSAisex‐
pected.TheincreaseofSAinplantsinfectedbythevirulent
andavirulentstrainbyeither100%andaparalleldecrease
ofJAandJA‐Ilepointstoacross‐talkandindicatethatanin‐
nate immunity response has been initiated (Kornneef and
Journal of Endocytobiosis and Cell Research VOL 26 | 2015
Pieterse2008).ThedownregulationoftheJA‐mediatedde‐
fencesmayhelptheplanttominimizeenergycostsandal‐
lowstheplanttofine‐tuneitsdefenceresponse(Pieterseet
al.2001;Bostock2005;KornneefandPieterse2008).ABA
andIAAhavealsobeenimplicatedinplantdefence,buttheir
significance is less well studied (Kornneef and Pieterse
2008).ABAisproducedinresponsetomicrobes(DeVleess‐
chauweretal.2010),transportedfromtheproducingroot
cellsintothexylemandreleasedtotheabove‐groundplant
parts(Zangetal.2008;Furchetal.2014).IAAhasprofound
effects on plant growth and development. Both plants and
some plant pathogens can produce IAA to modulate plant
growth(Zhao2010).Itisverylikelythatthesignificantlyin‐
creasedIAAlevelisresponsibleforformationofWB.Atpre‐
sentitisnotknown,iftheplantsproducemoreIAAdueto
thephytoplasmainfection,orifthephytoplasmasitselfpro‐
duceIAA.
Figure 6: Analysis of phytohormones in leaves of healthy apple
treesandtreesinfectedbyeitheranavirulent(a‐vir)oravirulent
(vir)Ca.P.malistrain.Eachbarrepresentsthemeanof13‐25repli‐
cates from 6 different plants ± standard deviation. The statistical
significanceistestedwithone‐wayANOVAbypairwisecomparison
with Students t‐test and marked with different letters (p = 0.05).
250mgofeitherleafmaterialwasgroundandextractedinthepres‐
enceofamixofinternalphytohormonestandards(Vadasseryetal.
2012).Subsequently,thereducedandresolvedsamplewasloaded
onto a reversed‐phase column (Oasis HLB 3cc; Waters, Eschborn,
Germany) that was first pre‐conditioned by the addition of 2 ml
methanol(MeOH)followedby2mlwater.Afterbeingwashedwith
2ml5%(v/v)MeOH/watersolutioncontaining0.5%(v/v)formic
acid,thecolumnwaselutedwith2mlMeOH.Theeluatewascom‐
pletely dried in a speed‐vac, resolved in 0.1 ml MeOH and subse‐
quently subjected to liquid chromatography–mass spectrometry
analysisaccordingtoVadasseryetal.(2012).
71 Candidatus phytoplasma mali affects phloem function, Zimmermann MR et al.
Supportedbythediversityofsymptoms,itislikelythatthe
natureofdiseasedevelopmentismorecomplex.Recentsur‐
veysrevealedthatthephloemisacentralactorinplantde‐
velopmentandnutrition,providingnutrientsandenergyto
sinkorgansandintegratinginterorgancommunication.Data
showthatthesieve‐tubesapcontainsatleast150different
water‐solubleproteins(Schobertetal.1995).Amajorpart
is engaged in sugar metabolism, transmembrane sugar
transport,proteindegradationanddetoxificationofreactive
oxygen species. Furthermore, long‐distance signalling is
supposedtobeanintegralpartofinduceddefenceresponse
ofplants(e.g.Davies2006;Furchetal.2014;Gaupelsetal.
2008; Jung et al. 2009; Park et al. 2007). In addition, the
phloemisbelievedtobeinvolvedintheproductionoram‐
plificationofsignalsthatarereleasedintothephloem‐sap
streaminresponsetostress,andintheproductionandstor‐
age of antimicrobial compounds acting in defence mecha‐
nisms. However, the interaction of phytoplasmas with
phloem constituents is still poorly understood (MacLean
andHogenhout2013).Thereisindicationthatphloemload‐
ingisaffectedbyphytoplasmainfection.AsSEsneedaper‐
manentinputofenergytoensuretheirviability,sugarme‐
tabolism and the associate energy production of the com‐
panioncellshaveadramaticimpactonthephysiologicalfit‐
nessofphloemfunction.Whentherequiredsmallpropor‐
tionsucrosebreakdowninthecompanioncellswasblocked
by insertion of a phosphorylase gene, sugar uptake or re‐
trievalwasinhibitedandphloemloadingwasseverelyim‐
paired, probably by the lack of proton pump fuelling. This
resulted in photoassimilate accumulation in source leaves,
chlorophyll loss, and reduced plant growth (Lerchl et al.
1995;vanBel2003),typicalsymptomsofphytoplasmaldis‐
eases.
PutativevirulencefactorsofCa.P.mali
The role of AAA+ proteins in phytoplasmal patho‐
genicity
Thereisnodirectmolecularorbiochemicalproofforviru‐
lenceproteinsoreffectorsofCa.P.mali.However,thereis
circumstantialevidencethatmembraneproteinsfromCa.P.
mali strains have a significant influence on pathogenicity.
Thesefindingsarebasedonmonitoringdiseasedataoflong‐
termtrialsfromAPaccessionsofdifferentoriginandmolec‐
ular data derived from the associated pathogen strains.
Basedonphenotypicsymptomstheaccessionswereclassi‐
fied as being strongly, moderately or mildly virulent
(SeemüllerandSchneider2007;Seemülleretal.2011).The
moleculardatacomefromagroupofgenes,encodingAAA+
proteinswhicharepresentinanunusuallyhighnumberin
phytoplasmas, namely the hflB‐ and AAA+ ATPase genes.
Analysisofnucleicacidsequencesorderivedaminoacidse‐
quences of some of the genes revealed virulence‐related
substitutions.Clusteranalysisclearlyseparatedthevirulent
fromnon‐virulentstrains.TheAAA+superfamilyisalarge,
functionallydiversegroupofproteinsandcomprehendvar‐
ious types of ATPases (e.g. ClpC, ClpV, p97) and proteases
(e.g. HflB, Clp group, Lon) possessing the ATPase module
(Langklotzetal.2012;Snideretal.2008).Bothareintegral
72
membraneproteinswiththelong,catalyticrelevantC‐termi‐
nal domain facing the cytosol. Topology prediction pro‐
grams however, indicated that the C‐tail of four AAA+
ATPasesandtwoofthreeHflBsofCa.P.malifacetotheout‐
side,hencethesievetubecytoplasm(Seemülleretal.2013).
AAA+proteinsareofcrucialimportanceforbacterialvir‐
ulence.TheClpCATPasefromStaphylococcusaureusforex‐
ample regulates transcription, which is a major virulence
factor(Luongetal.2011).Similarly,AAA+proteinsarees‐
sentialforthefunctionofsecretionsystems.Severalbacte‐
rial species such as Salmonella enterica, Agrobacterium tu‐
mefaciensand Pseudomonas syringaeusetypethreesecre‐
tionsystem(TTSS)toinjectvirulencefactorsintotheeukar‐
yotichost(VanMelderenandAertsen2009;AlixandBlanc‐
Potard2008).ThisunusualfindingonC‐tailorientationisa
newaspectinunderstandingphytoplasmapathogenicityat
thesievetubelevel.ItisconceivablethatthepowerfulAAA+
proteinsattackstructuresorcomponentsofsieveelements,
inparticularifthepathogensareattachedtothemembrane.
Adherencetohostmembranesiswellestablishedformost
mycoplasmaspathogenictohumansandanimalsandiscon‐
sidered to be a prerequisite for colonization and infection
evidencedbytheharmcausedtothemembranes(Razinet
al.1998).However,suchanattachment,whichisbasedon
protein/protein interaction, is not clearly established for
phytoplasmas.
Conclusion
The effect of phytoplasma infections on function of the
phloemandtheresultingdiseasereceivedlittleattentionin
the past. However, recent results in phytoplasma and
phloemresearch,likethecorrelationofphytoplasma‐AAA+
proteinswithvirulenceorchangesinphytohormonelevels
understresssituationsoffertheopportunitytomakesignif‐
icantprogressinunderstandinginductionanddevelopment
ofphytoplasmaldiseases.Forthefutureitwillbeimportant
tostudytheeffectofthesievetube‐borninhabitingpatho‐
gensonthephloem.Wesupposethatafterinfectionbydif‐
ferentphytoplasmastrainsandthereleaseofproposedef‐
fectors/proteases from plasma‐membrane proteins (e.g.
AAA+proteins),thefirstreactionisaCa2+increaseinsieve
elements ‐ resulting in filament formation of phloem‐pro‐
teins and, later on, callose deposition at sieve plates and
sievepores.Asubsequentreactionisthechangeofphyto‐
hormone dispersion, integrity of plasma membrane and
changeofmassflowdirection(Figure7).
Acknowledgements
The authors thank Andrea Lehr for technical assistance
concerning the phytohormone measurements and The
Deutsche Forschungsgemeinschaft in the frame of FU
969/2‐1 as well as the Max Planck Society for supporting
thiswork.
Journal of Endocytobiosis and Cell Research VOL 26 | 2015
Candidatus phytoplasma mali affects phloem function, Zimmermann MR et al. Figure7:Aproposedmodelaboutthe(sub‐)cellularlevelofplant‐phytoplasmainteraction.
References
AlixE,Blanc‐PotardAB.(2008)Peptide‐assisteddegradationofthe
SalmonellaMgtCvirulencefactor.EmboJ.27:546‐557.
BlackmanLM,GunningBES,OverallRL.(1998)A45kDaproteiniso‐
latedfromthenodalwallsofCharacorallinaislocalisedtoplas‐
modesmata.PlantJ.15:401‐411.
BlumwaldE,AharonGS,LamBCH.(1998)Earlysignaltransduction
pathwaysinplant–pathogeninteractions.TrendsPlantSci.3:342‐
346.
BostockRM.(2005)Signalcrosstalkandinducedresistance:strad‐
dling the line between cost and benefit. Ann Rev Phytopathol.
43:545–580.
CarraroL,ErmacoraP,LoiN,OslerR.(2004)Therecoveryphenom‐
enon in apple proliferation‐infected apple trees. J Plant Pathol.
86:141‐146.
ChristensenNM,NicolaisenM,HansenM,SchulzA.(2004)Distribu‐
tionofphytoplasmasininfectedplantsasrevealedbyreal‐time
PCRandbioimaging.MolPlant–MicrobeInteract.17:1175–1184.
CordovaI,JonesP,HarrisonNA,OropexaC.(2003)InsituPCRde‐
tectionofphytoplasmaDNAinembryosfromcoconutpalmswith
lethalyellowingdisease.MolPlantPathol.4:99–108.
Cronshaw J, Sabnis DD. (1990) Phloem proteins. In: Behnke HD,
SjolundRD.(eds.)SieveElements.ComparativeStructure,Induc‐
tion and Development. Berlin, Heidelberg, New York, Springer‐
Verlag,pp.257‐283.
DaviesE.(2006)Electricalsignalsinplants:factsandhypotheses.
In: Volkov AG. (ed.) Plant Electrophysiology. Berlin, Springer‐
Verlag,pp.407‐422.
DempseyMA,KlessigDF.(2012)SOS–toomanysignalsforsystemic
acquiredresistance?TrendsPlantSci.17:538–545.
DeVleesschauwerD,YangY,CruzCV,HöfteM.(2010)Abscisicacid‐
inducedresistanceagainstthebrownspotpathogenCochliobolus
miyabeanusinriceinvolves MAPkinase‐mediatedrepressionof
ethylenesignaling.PlantPhysiol.152:2036–2052.
DinantS,LemoineR.(2010)Thephloempathway:Newissuesand
olddebates.ComptesRendusBiol.333:307‐319.
DoiY,TeranakaM,YoraK,AsuyamaH.(1967)Mycoplasma‐orPLT
group‐like microorganisms found in the phloem elements of
plantsinfectedwithmulberrydwarf,potatowitches’broom,aster
yellows or paulownia witches’ broom. Ann Phytopathol Soc Jpn.
33:259–266.
Journal of Endocytobiosis and Cell Research VOL 26 | 2015
FelleHH.(2001)pH:signalandmessengersinplantcells.PlantBiol.
6:577–591.
FelleHH.(2005)pHregulationanoxicplants.AnnBot.96:519–532.
Furch ACU, Zimmermann MR, Kogel K‐H, Reichelt M, Mithöfer A.
(2014)Directandindividualanalysisofstress‐relatedphytohor‐
monedispersioninthevascularsystemofCucurbitamaximaafter
flagellin22treatment.NewPhytol.201:1176‐1182.
Furch ACU, Hafke JB, Schulz A, van Bel AJE. (2007) Ca2+‐mediated
remotecontrolofreversiblesievetubeocclusioninViciafaba.J
ExpBot.58:2827‐2838.
GaupelsF,FurchACU,WillT,MurLAJ,KogelK‐H,vanBelAJE.(2008)
NitricoxidegenerationinViciafabaphloemcellsrevealsthemto
besensitivedetectorsaswellaspossiblesystemictransducersof
stresssignals.NewPhytol.178:634‐646.
Glazebrook J. (2005) Contrasting mechanisms of defense against
biotrophic and necrotrophic pathogens. Annu Rev Phytopathol.
43:205–227.
HafkeJB,vanAmerongenJ‐K,KellingF,FurchACU,GaupelsF,van
Bel AJE. (2005) Thermodynamic battle for photosynthate
acquisition between sieve tubes and adjoining parenchyma in
transportphloem.PlantPhysiol.138:1527‐1537.
HanboonsongY,ChoosaiC,PanyimS,DamakS.(2002)Transovarial
transmission of sugarcane white leaf phytoplasma in the insect
vector Matsumuratettix hiroglyphicus (Matsumura). Insect Mol
Biol.11:97‐103.
HarePE.(1977)In:MethodsinEnzymology.47:3‐18.
HegeleM,BangerthF.(1997)ChangesinIAAandABAlevelsandIAA
transport of proliferation‐diseased apple trees. Acta Hortic.
463:97‐103.
HeukeshovenJ,DernickR.(1988)Improvedsilverstainprocedure
for fast staining in Phastsystem development unit; staining of
sodiumdodecylsulfategels.Electrophoresis.9:28‐32.
Hogenhout SA, Oshima K, Ammar el‐D, Kakizawa S, Kingdom HN,
NambaS.(2008)Phytoplasmas:bacteriathatmanipulateplants
andinsects.MolPlantPathol.9:403‐423.
Hoshi A, Oshima K, Kakizawa S, Ishii Y, Ozeki J, Hashimoto M,
Komatsu K, Kagiwada S, Yamaji Y, Namba S. (2009) A unique
virulencefactorforproliferationanddwarfisminplantsidentified
from a phytopathogenic bacterium. Proc Natl Acad Sci USA.
106:6416‐6421.
Jung HW, Tschaplinski TJ, Wang L, Glazebrook J, Greenberg JT.
(2009)Priminginsystemicplantimmunity.Science.324:89‐91.
73 Candidatus phytoplasma mali affects phloem function, Zimmermann MR et al.
Kartte S, Seemüller E. (1988) Variable response within the genus
Malustotheappleproliferationdisease.ZeitschrPflanzenkrankh
Pflanzenschutz.95:25‐34.
KartteS,SeemüllerE.(1991)Histopathologyofappleproliferation
inMalustaxaandhybridsofdifferentsusceptibility.JPhytopathol.
131:149‐160.
Kempers R, Ammerlaan A, van Bel AJE. (1998) Symplasmic con‐
striction and ultrastructural features of the sieve element/com‐
panion cell complex in the transport phloem of apoplasmically
and symplasmically phloem‐loading species. Plant Physiol.
116:271‐278.
KnoblauchM,PetersWS,EhlersK,vanBelAJE.(2001)Reversible
calcium‐regulated stopcocks in legume sieve tubes. Plant Cell.
13:1221‐1230.
KnoblauchM,NollGA,MüllerT,PrüferD,Schneider‐HütherI,Schar‐
nerD,vanBelAJE,PetersWS.(2003)ATPindependentcontractile
proteinsfromplants.NatMat.2:600‐603.
KollarA,SeemüllerE,KrczalG.(1989)Impairmentofthesievetube
sealing mechanism of trees infected by mycoplasmalike
organisms.JPlantDisProt.124:7‐12.
KoornneefA,PieterseCMJ.(2008)Crosstalkindefensesignaling.
PlantPhysiol.146:839–844.
KubeM,SchneiderB,KuhlH,DandekarT,HeitmannK,MigdollA,
ReinhardtR,SeemüllerE.(2008)Thelinearchromosomeofthe
plant‐pathogenic mycoplasma 'Candidatus Phytoplasma mali'.
BMCGenom.9:26.
KudlickaK,BrownRM.(1997)Celluloseandcallosebiosynthesisin
higherplants.PlantPhysiol.115:643‐656.
LangklotzS,BaumannU,NarberhausF.(2012)Structureandfunc‐
tion of the bacterial AAA protease FtsH. Biochim Biophys Acta
(BBA)‐MolCellRes.1823:40–48.
Laemmli UK. (1970) Cleavage of structural proteins during the
assemblyoftheheadofbacteriophageT4.Nature.227:680–685.
Lerchl J, Geigenberger P, Stitt M, Sonnewald U. (1995) Impaired
photoassimilatepartitioningcausedbyphloem‐specificremoval
of pyrophosphate can be complemented by a phloem‐specific
cytosolicyeast‐derivedinvertaseintransgenicplants.PlantCell.
7:259‐270.
Levy A, Guenoune‐Gelbart D, Epel BL. (2007) ß‐1,3‐glucanases.
Plasmodesmal gate keepers for intercellular communication.
PlantSignalBehav.5:288‐290.
Luong TT, Sau K, Roux C, Sau S, Dunman PM, Lee CY. (2011)
StaphylococcusaureusClpCdivergentlyregulatescapsuleviasae
and codY in strain Newman but activates capsule via codY in
strain UAMS‐1 and in strain Newman with repaired saeS. J
Bacteriol.193:686‐694.
MacLean AM, Hogenhout SA. (2013) Phytoplasmas and Spiroplas‐
mas:Thephytopathogenicmollicutesofthephloem.In:Phloem:
MolecularCellBiology,SystemicCommunication,BioticInterac‐
tions.Oxford,UK,JohnWiley&Sons,Inc.,pp.293‐309.
MarconeC,NeimarkH,RagozzinoA,LauerU,SeemüllerE.(1999)
Chromosome sizes of phytoplasmas composing major phyloge‐
neticgroupsandsubgroups.Phytopathology.89:805‐810.
MartensHJ,RobertsAG,OparkaKJ,SchulzA.(2006)Quantification
of plasmodesmatal endoplasmic reticulum coupling between
sieveelementsandcompanioncells using fluorescenceredistri‐
butionafterphotobleaching.PlantPhysiol.142:471‐480.
MusettiR,BuxaSV,DeMarcoF,LoschiA,PolizzottoR,KogelK‐H,
vanBelAJE.(2013)Phytoplasma‐triggeredCa2+influxisinvolved
insieve‐tubeblockage.MolPlant‐MicrobeInteract.26:379‐386.
NakashimaJ,LaosinchaiW,CuiX,BrownRMJr.(2003)Newinsight
intothemechanismofcelluloseandcallosebiosynthesis:prote‐
asesmayregulatecallosebiosynthesisuponwounding.Cellulose.
10:369‐389.
OjaV,SavchenkoG,JakobB,HeberU.(1999)pHandbuffercapaci‐
ties of apoplastic and cytoplasmic cell compartments in leaves.
Planta.209:239–249.
PalevitzBA,NewcombEH.(1971)Theultrastructureanddevelop‐
mentoftubularandcristallinePproteininthesieveelementsof
certainpapilionaceouslegumes.Protoplasma.72:399‐427.
ParkS‐W,KaiyomoE,KumarD,MosherSL,KlessigDF.(2007)Me‐
thyl salicylate is a critical mobile signal for plant systemic ac‐
quiredresistance.Science.318:113‐116.
74
PieterseCMJ,TonJ,VanLoonLC.(2001)Cross‐talkbetweenplant
defence signalling pathways: boost or burden? Ag Biotech Net.
3:ABN068.
PieterseCMJ,VanderDoesD,ZamioudisC,Leon‐ReyesA,VanWees
SCM.(2012)Hormonalmodulationofplantimmunity.AnnuRev
CellDevelopBiol.28:489–521.
Razin S, Yogev D, Naot Y. (1998) Molecular biology and
pathogenicity of mycoplasmas. Microbiol Mol Biol Rev. 62:1094‐
1156.
Rodriguez‐Medina C. (2009) Study of Macromolecules in Phloem
Exudates of Lupinus albus. Ph.D. dissertation, School of Plant
Biology,UniversityofWesternAustralia,Crawley.
Schaper U, Seemüller E. (1982) Condition of the phloem and
persistenceofmycoplasmalikeorganisms associatedwithapple
proliferationandpeardecline.Phytopathology.72:736‐742.
Scheel D. (1998) Resistance response physiology and signal
transduction.CurrOpinPlantBiol.1:305‐310.
SchneiderH.(1976)Indicatorhostforpeardecline:Symptomatol‐
ogy, histopathology and distribution of mycolpasmalike organ‐
ismsinleafveins.Phytopathology.67:592‐601.
Schneider H. (1973) Cytological and histological aberrations in
woody plants followinginfection with viruses, mycoplasmas,
rickettsiasandflagellates.AnnuRevPhytopathol.11:119‐146.
SchobertC,GroßmannP,GottschalkM,KomorE,PecsvaradiA,zur
Nieden U. (1995) Sieve‐tube exudate from Ricinus communis L
seedlings contains ubiquitin and chaperones. Planta. 196:205‐
210.
SchobertC,BakerL,SzederkényiJ,GroßmannP,KomorE,Hayashi
H, Chino M, Lucas WJ. (1998) Identification of immunologically
related proteins in sieve‐tube exudate collected from
monocotyledonousanddicotyledonousplants.Planta.206:245–
252.
Sears BB, Klomparens KL. (1989) Leaf tip cultures of the evening
primrose allow stable aseptic culture of mycoplasma‐like
organisms.CanJPlantPathol.11:343–348.
Seemüller E, Kunze L, Schaper U. (1984) Colonization behavior of
MLO, and symptom expression of proliferation‐diseased apple
trees and decline‐diseased pear trees over a period of several
years.ZeitschrPflanzenkrankhPflanzenchutz.91:525‐532.
SeemüllerE,GarnierM,SchneiderB.(2002)Mycoplasmasofplants
andinsects.In:RazinS,HerrmannR.(eds.)MolecularBiologyand
Pathology of Mycoplasmas. London, Kluwer Academic/Plenum
Publishers,pp.91‐116.
Seemüller E, Schneider B. (2004) 'Candidatus Phytoplasma mali',
'Candidatus Phytoplasma pyri' and 'Candidatus Phytoplasma
prunorum',thecausalagentsofappleproliferation,peardecline
andeuropeanstonefruityellows,respectively.IntJSystemEvol
Microbiol.54:1217‐1226.
Seemüller E, Schneider B. (2007) Differences in virulence and
genomicfeaturesofstrainsof'CandidatusPhytoplasmamali',the
appleproliferationagent.Phytopathology.97:964‐970.
SeemüllerE,KampmannM,KissE,SchneiderB.(2011)HfIBgene‐
basedphytopathogenicclassificationof'CandidatusPhytoplasma
mali'strainsandevidencethatstraincompositiondeterminesvir‐
ulenceinmultiplyinfectedappletrees.MolPlant‐MicrobeInteract.
24:1258‐1266.
SeemüllerE,SuleS,KubeM,JelkmannW,SchneiderB.(2013)The
AAA plus ATPases and HfIB/FtsH proteases of 'Candidatus
Phytoplasma mali': Phylogenetic diversity, membrane topology,
and relationship to strain virulence. Mol Plant‐Microbe Interact.
26:367‐376.
SillerW,KuhbandnerB,MarwitzR,PetzoldH,SeemüllerE.(1987)
Occurrenceofmycoplasma‐likeorganismsinparenchymacellsof
Cuscutaodorata(RuizetPav.).Phytopathology.119:147‐159.
Snider J, Thibault G, Houry WA. (2008) The AAA+ superfamily of
functionallydiverseproteins.GenBiol.9:216.
Strauss E. (2009) Microbiology. Phytoplasmas research begins to
bloom.Science.325:388‐390.
Sugio A, Kingdom HN, MacLean AM, Grieve VM, Hogenhout SA.
(2011)PhytoplasmaproteineffectorSAP11enhancesinsectvec‐
torreproductionbymanipulatingplantdevelopmentanddefense
hormonebiosynthesis.ProcNatlAcadSciUSA.108:E1254‐E1263.
ThuleauP,SchroederJI,RanjevaR.(1998)Recentadvancesinthe
regulationofplantcalciumchannels:Evidenceforregulationby
Journal of Endocytobiosis and Cell Research VOL 26 | 2015
Candidatus phytoplasma mali affects phloem function, Zimmermann MR et al. G‐proteins, the cytoskeleton and second messengers. Curr Opin
PlantBiol.1:424‐427.
TrewavasA.(1999)Lecalcium,c’estlavie:Calciummakeswaves.
PlantPhysiol.120:1‐6.
VadasseryJ,ReicheltM,HauseB,GershenzonJ,BolandW,Mithöfer
A. (2012) CML42‐mediated calcium signaling co‐ordinates re‐
sponsestoSpodopteraherbivoryandabioticstressesinArabidop‐
sis.PlantPhysiol.159:1159–1175.
vanBelAJE.(1996)Interactionbetweensieveelementandcompan‐
ion cell and the consequences for photoassimilate distribution.
Two structural hardware frames with associated physiological
softwarepackagesindicotyledons?JExpBot.47:1129‐1140.
vanBelAJE,KnoblauchM.(2000)Sieveelementandcompanioncell:
thestoryofthecomatosepatientandthehyperactivenurse.Aus‐
tralJPlantPhysiol.27:477‐487.
vanBelAJE.(2003)Thephloem,amiracleofingenuity.PlantCell
Environ.26:125‐149.
vanBelAJE,KnoblauchM,FurchACU,HafkeJB.(2011)(Questions)n
on phloem biology. 1. Electropotential waves, Ca2+ fluxes and
cellular cascades along the propagation pathway. Plant Sci.
181:210‐218.
vanBelAJE,FurchACU,WillT,BuxaSV,MusettiR,HafkeJB.(2014)
Spreadthenews:systemicdisseminationandlocalimpactofCa2+
signalsalongthephloempathway.JExpBot.65:761‐1787.
VanMelderenL,AertsenA.(2009)Regulationandqualitycontrolby
Lon‐dependentproteolysis.ResMicrobiol.160:645‐651.
WhitcombRF,TullyED.(1989)TheMycoplasmas.Vol.V,SanDiego,
AcademicPress,Inc.
Yang Y, Shah J, Klessig DF. (1997) Signal perception and
transduction in plant defense responses. Gen Develop. 11:1621‐
1639.
Zabotin AI, Barysheva TS, Trofimova OI, Lozovaya VV, Widholm J.
(2002)Regulationofcallosemetabolismin higher plantcellsin
vitro.RussJPlantPhysiol.49:792‐798.
ZhangYP,QiaoYX,ZhangYL,ZhouYH,YuJQ.(2008)Effectsofroot
temperature on leaf gas exchange and xylem sap abscisic acid
concentrations in six cucurbitaceae species. Photosynthetica.
46:356–362.
ZhaoY.(2010)Auxinbiosynthesisanditsroleinplantdevelopment.
AnnuRevPlantBiol.61:49‐64.
Zimmermann MR, Hafke JB, van Bel AJE, Furch ACU. (2013)
Interaction of xylem and phloem during exudation and wound
occlusioninCucurbitamaxima.PlantCellEnviron.36:237‐247.
Journal of Endocytobiosis and Cell Research VOL 26 | 2015
75