Grafting with rootstocks induces extensive transcriptional re-programming in the shoot apical ...

14 Pages·2013·1.49 MB·English

CooksonandOllatBMCPlantBiology2013,13:147
http://www.biomedcentral.com/1471-2229/13/147
RESEARCH ARTICLE Open Access
Grafting with rootstocks induces extensive
transcriptional re-programming in the shoot
apical meristem of grapevine
Sarah Jane Cookson and Nathalie Ollat*
Abstract
Background: Grafting is widely used inthe agriculture of fruit-bearing crops; rootstocks are known to confer
differences inscion biomass in addition to improving other traits of agricultural interest. However,little is known
about theeffect ofrootstockson scion gene expression. The objective ofthis study was to determine whether
hetero-grafting thegrapevine variety Vitis viniferacv. ‘CabernetSauvignon N’with two different rootstocks alters
gene expression intheshoot apex incomparison to theauto-grafted control. CabernetSauvignon was
hetero-grafted with two commercial rootstock genotypes and auto-grafted with itself. Vigor was quantified by
measurements ofroot, stem, leaf and trunk biomass.Gene expression profiling was done using a whole genome
grapevine microarray; four pools of five shoot apex samples were harvested 4months after grafting for each
scion/rootstock combination.
Results: The rootstocks increased stem biomass or conferred increased vigor bythe end of thefirst growth cycle.
Globally hetero-grafting two different genotypestogether triggered anincreasein shoot apex gene expression;
however nogenes were differentially expressed between thetwo hetero-grafts.The functional categoriesrelated to
DNA, chromatin structure, histones, flavonoids and leucine rich repeat containingreceptor kinases were the most
enriched inthe up-regulated genes inthe shoot apex of hetero-grafted plants.
Conclusions: The choice ofrootstockgenotype had little effect on thegene expression inthe shoot apex; this
could suggest that auto- and hetero-grafting was the major factorregulatinggene expression.
Background Despite the widespread use of grafting, we know little
Grafting is widely used in the agriculture of fruit crops. of how grafting with rootstocks confers differences to
Rootstocksareselectedtoprovideresistancetosoil-borne the vigor of the scion. Rootstock conferred vigor in
pests and diseases, toincreasetolerance to environmental horticulture is generally described in terms of shoot bio-
stresses and to improve crop productivity and/or quality mass accumulation, i.e. stem biomass, yield or biomass
(e.g.asreviewedby[1,2]).Inviticulture,graftingisprimar- allocation within the plant (such as the ratio between
ilyusedtofacilitategrapevinegrowthinsoilsinfectedwith yield and stem biomass). Various hypotheses explaining
the phylloxera, a soil-dwelling insect pest introduced to rootstock conferred vigor have been proposed including
Europe from America (e.g. [3]). The use of rootstocks, in alterations in nutrient and water movement, hormone
which the entire root system of a plant is replaced, has a concentrations and the anatomy of the graft union (as
profound effect on scion development. Rootstocks are reviewed by [2,8]). In apple orchards, the rootstocks
knowntoaltervariousphysiologicalprocessesinthescion used have dramatic dwarfing effects on the scion, redu-
suchasvigororbiomassaccumulation[4],fruitquality[5] cing trunk diameter by up to 70% e.g. [9]. In cherry
and response to abiotic stresses (e.g. to water deficit [6] trees, rootstock-induced dwarfing (reducing stem length
andsalinity[7]). by 25%) is caused by differential cessation of terminal
meristem growth e.g. [10]. Commercial rootstocks used
in viticulture have much smaller effects on scion vigor.
*Correspondence:[email protected] They are not associated with dwarfing phenotypes
INRA,ISVV,EGFV,UMR1287,F-33140,Villenaved’Ornon,France
©2013CooksonandOllat;licenseeBioMedCentralLtd.Thisisanopenaccessarticledistributedunderthetermsofthe
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distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
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observed in fruit trees, but grapevine rootstocks are still in scion vigor. In viticulture, vigor is typically quantified
important for fruit quality and yield. Although vigor by stem pruning weight at the end of the growing cycle
control mechanisms of different rootstocks are not and, in this work, conferred vigor was also quantified by
necessarily the same for different scions, there is some using measurements of dry stem biomass. The objective
degree of consistency with most commercial rootstock was to associate differences in rootstock conferred vigor
genotypes, being defined by viticulturists as high, mode- (i.e. stem biomass) with differences in gene expression in
rate orlowvigore.g.[3]. the shoot apex. Shoot apex samples were harvested four
Gene expression studies have been used to shed light months after grafting when presumably the stress associ-
on the mechanisms behind rootstock conferred vigor in ated with grafting and the formation of a successful graft
fruit trees. Gene expression in the shoot tip of apple union has been overcome. Some of the genes differen-
trees was studied in response to a range of rootstock ge- tially expressed in the microarray data were confirmed
notypes to correlate shoot tip expression to plant stature by qPCR (Additional file 1).
[11]. The Gene Ontology (GO) terms enriched in the
differentially expressed genes were response to stimulus Rootstockconferredvigoreffectsonbiomass
and response to abiotic and biotic stress as well as accumulationingraftedgrapevine
unknown biological processes and other biological pro- The rootstocks 1103P and RG increased stem biomass
cesses. Jensen et al. [11] identified 116 genes whose compared to the auto-grafted control at the end of the
expression levels were correlated with plant size; the growth cycle (Figure 1C), i.e. the rootstock genotypes
genesmoststronglycorrelatedwithtrunkcrosssectional conferred increased vigor to the scion. In addition, the
diameter were a sorbitol dehydrogenase, a homeobox- rootstock 1103P increased leaf biomass relative to the
leucine zipper protein and a hevein-like protein. Simi- auto-graftedcontrol(Figure1D),whereasthetrunkbio-
larly, Prassinos et al. [10]used cDNA amplified fragment mass was decreased by the grafting with the rootstock
length polymorphism to compare the effect of a dwar- RG (Figure 1B). Root biomass was not significantly dif-
fing and a semi-vigorous rootstocks on gene expression ferent between the three scion/rootstock combinations
in the scion and identified the differential regulation of a at the end of the growth cycle (Figure 1A). However,
number of transcription factors and genes involved in there was a difference in biomass allocation between
signalingprocesses. However,auto-grafted control plants the shoot and the root (Figure 2). The shoot/root ratio
were absent from the studies of Jenson et al. [11] and of hetero-grafts was significantlyhigher than that of the
Prassinos et al. [10], therefore the effect of grafting with auto-grafted control (Kruskal-Wilk ANOVA on ranks
a non-self rootstock on the gene expression of the scion followedbyTukeymultiple comparisonprocedure,data
remains tobedetermined. notshown).
Our objective was to study the effects of grafting on The shoot apex samples were harvested four months
gene expression in the shoot apical meristem of grape- aftergrafting,beforedifferencesinscion vigor(stembio-
vine Vitis vinifera cv. ‘Cabernet Sauvignon N’ (CS) het- mass) became apparent (Figure 3C). Leaf biomass was
erografts (with two different commercial rootstock also unaffected by grafting with rootstocks at the time of
genotypes originating from American Vitis spp.) and the apex harvest (Figure 3D). However, the root biomass of
auto-grafted control (CS grafted with CS). The rootstock the auto-grafted plants was significantly higher than that
genotypes chosen were ‘Riparia Gloire de Montpellier’ of the hetero-grafted plants (Figure 3A) and the trunk
(RG) and ‘1103 Paulsen’ (1103P), these genotypes are biomass was significantly lower with the rootstock RG
known to confer low and high scion vigor respectively. thanwiththeauto-grafted plantsandplantsgraftedonto
Our sampling strategy was designed to minimize the 1103P (Figure 3B).
secondary effects related to rootstock physiology, such
as, differences in plant water status, gas exchange and Rootstockeffectsongeneexpressionintheshootapex
transpiration. Therefore, shoot apical meristems were Hetero-grafting CS with American rootstock varieties,
harvested at the end of the night when presumably RG and 1103P, resulted in the differential expression of
plants are in a stable environment to reduce confusion a large number of genes in the shoot apex when com-
of the direct and indirect effects of grafting with root- pared to the auto-grafted control CS/CS (Figure 4). In
stocks.Genesfrom thefunctionalcategoriesDNA,chro- general, the differential expression of most genes was
matin structure, histones, flavonoids and leucine rich similar in both heterograft combinations: 1203 and 837
repeats(LRR) were enriched intheup-regulatedgenes. genes were up- and down-regulated in both CS/RG and
CS/1103P, whereas only 108 and 168 genes were specif-
Results ically up- and down-regulated in CS/1103P, and only
Inhorticulture, rootstocksare oftenselected for their abi- 164 and 92 genes were specifically up- and down-
litytoalterthegrowthofthescion,ortoconferdifferences regulatedin CS/RG. In fact, no genes were differentially
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A C
35 25
30
W) W) 20
D 25 D
mass (g 20 mass (g 15
ot bio 1105 nk bio 10
o u
R Tr 5
5
B D
0 400
50
35
ass (g DW) 3400 ass (gDW) 223050
m m
o o
bi 20 bi 15
m af
e e 10
St 10 L
5
0 0
400 600 800 1000 1200 1400 400 600 800 1000 1200 1400
Thermal time after transfer to pots (°Cd) Thermal time after transfer to pots (°Cd)
Figure1Rootstockeffectsonbiomassaccumulationingrapevineduringthefirstgrowthcycle.Biomassaccumulationwasfittedto
curvesand95%confidenceintervalswerecalculated,forpanelsA,CandDthecurvefittedwasy=eaxandforBitwasy=mx+C.Biomassin
theroot(A),trunk(B),stem(C)andleaves(D)ofCabernetSauvignongraftedwiththerootstocks1103Paulsen(filledcircles,greenlines)and
RipariaGloiredeMontpellier(filledtriangles,pinklines)andtheauto-graftedcontrol(opencircles,bluelines).
expressed between the two hetero-graft combinations. MapMan functional categories (BINs) enriched in the
The normalized expression values, gene expression genes differentially expressed between the hetero-
comparisons, p values, adjusted p values and the grafts and the auto-graft control also highlight the
gomapmanannotationforallsignificantlydifferentially similarity of response of the two hetero-graft combina-
expressed genes are given in Additional file 2. The tions(Figure5,[12-14]).
Figure2Hetero-graftingincreasedshoottorootratioincomparisontotheauto-graftedcontrol.Biomassallocationbetweentheshoot
andtherootofCabernetSauvignon(CS)hetero-graftedwiththerootstocks1103Paulsen(closedcircles)andRipariaGloiredeMontpellier
(closedtriangles)andtheauto-graftedcontrol(opencircles)duringthefirstyearofgrowth.
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A B
7 14
6 b 12
W) W) a a
D 5 D 10
g g
ass ( 4 a ass ( 8 b
m m
o 3 a o 6
bi bi
ot 2 nk 4
o u
R r
T
1 2
0 0
C D
8 8
W) 6 W) 6
D D
g g
ass ( 4 ass ( 4
m m
o o
bi bi
m af
e 2 e 2
St L
0 0
1103P RG CS 1103P RG CS
Rootstock genotype
Figure3Rootstockeffectsonbiomassaccumulation4monthsaftergrafting.BiomassofCabernetSauvignon(CS)graftedwiththe
rootstocks1103Paulsen(1103P)andRipariaGloiredeMontpellier(RG)andtheauto-graftedcontrol(CS)fourmonthsaftergrafting:root(A),
trunk(B),stem(C)andleaf(D)biomass.Meansandstandarddeviationsshown,n=10,resultsofANOVAfollowedbyHolm-Sidakmultiple
comparisonprocedureindicatedbyletterswheresignificantdifferenceswereobserved.
Genesup-regulatedintheshootapicalmeristemby associated with DNA and chromatin modification, such
hetero-graftingwithrootstocks as, genes from the category chromatin structure (e.g.
The MapMan functional categories [12,13] over- VIT_01s0150g00390, VIT_00s0184g00040, VIT_07s0005g
representedinthegenesup-regulatedintheshootapexof 01430 and VIT_11s0149g00130) and genes annotated as
hetero- compared to auto-grafts belonged to the func- being involved in replication control (e.g. VIT_06s0
tional categories cell wall, cell, cell organization, DNA, 004g06300) as well as one SET-domain transcription fac-
chromatin structure, histones, LRR receptor kinases, ter- tor (VIT_16s0098g01510) and two chromatin remodeling
penoids and isoflavonols (Table 1 and Figure 5). The GO factors (VIT_05s0049g00150 and VIT_04s0023g01610)
biological processes DNA replication, DNA metabolic (Additional file 3 and Figure 5). In addition to the up-
processesandothercellularmetabolicprocesseswerealso regulationofmanygenesinvolvedinDNAandchromatin
enriched in this gene list, along with the functions regulation, numerous transcription factors (Figure 6), two
helicase, motor, hydrolase and pyrophosphatase activity sugar transporters (VIT_14s0030g00240 and VIT_14s00
and the compartments chromosome, organelle, nucleus 30g00230), a developmental gene (TERMINAL FLOWER-
andextracellular(Table2). LIKEPROTIEN 1(TFL1),VIT_06s0080g00290),andgenes
Examination of the most strongly up-regulated genes involvedinaminoacidmetabolismandcellwallmodifica-
(Additional file 3) highlights the up-regulation of genes tionwereup-regulated(Additionalfile3).
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Figure4Venndiagramsshowingthegenesdifferentiallyexpressedbetweenauto-andheterograftsofgrapevine.Genesdifferentially
expressedintheshootapexbetweenCabernetSauvignonauto-grafted(CS/CS)andgraftedwithdifferentrootstocks(CS/RGandCS/1103P)are
presentedinaVenndiagram.Textthicknessindicatesthedirectionofgeneexpressionchange,thenumbersofgenessignificantlyup-
anddown-regulatedarerepresentedbyboldandnormaltextrespectively(differentialexpressiondefinedasameanlogfoldchangeof1
andpvalue<0.05adjustedwithBenjamini-Hochberg).
Genesdown-regulatedintheshootapicalmeristemby proteinsand DNA methyltransferases). Thehistonegenes
hetero-graftingwithrootstocks inducedbytherootstockswerecorehistonesgenes(H2A,
The MapMan functional categories [12,13] over- H2B, H3 and H4 (Hist1h4h)) except for one centromeric
represented in the genes down-regulated in the hetero- histone(VIT_15s0046g01110),whereasaH1variant,alin-
compared to auto-grafts belonged to the functional kerhistone,wasdown-regulated.Twonucleosomeassem-
categoriesreceptorkinases,hormonemetabolism(particu- bly proteins were also up-regulated (Additional file 5 and
larlyethylene),secondarymetabolism,pathogenesisrelated Figure 6); these proteins are chaperones of the core his-
(PR) proteins, redox and various miscellaneous genes tones H2A andH2B.Five SETdomain proteinswerealso
(Table 3 and Figure 4). The GO biological processes re- inducedbytherootstocks(Additionalfile5andFigure6);
sponse to stimulus, cell communication and signaling and theSETdomainisknowntohavemethyltransferaseactiv-
the functions transferase and oxidoreductase activity was itytargetedtospecificlysineresiduesofhistoneH3orH4
also over-represented (Table 4). [16]. Three DNA methyltransferases were induced by the
Examination of the most strongly down-regulated rootstocks (Additional file 5 and Figure 6); these genes
genes (Additional file 4)highlights the importanceof the were all belonging to the plant-specific chromomethylase
down-regulation of many classes of transcription factors, group responsible for asymmetric cytosine methylation in
particularly those belonging to the WRKY class (e.g. Arabidopsis [17]. Three Werner Syndrome-like exonucle-
VIT_17s0000g01280, VIT_08s0058g01390 and VIT_04s0 asegenesweredown-regulatedinresponsetotherootstock
008g05760) (Figure 6). WRKY transcription factors have genotypes.In Arabidopsis oneWernerSyndrome-likeexo-
widerolesintranscriptionalactivationandrepressingplant nuclease is known to be involved in post-transcriptional
developmentandstressresponses(asreviewedby[15]). gene silencing [18]. One gene involved in RNA processing
(VIT_12s0059g01120, a Ribonuclease 3-like/dicer protein)
Graftingwithrootstockstriggersthedifferentialexpression wasalsodown-regulated,thisgeneisknowntobeinvolved
ofmanygenesinvolvedinDNAandchromatinmodification in the 24 nt small interfering RNA (siRNA) pathway that
Grafting with non-self rootstocks induced the transcrip- modifieschromatinanddirectsDNAmethylation[19].
tional reprogramming of many genes associated with
chromatin modification. Seventy genes assigned to the Differentialexpressionofhormonerelatedgenes
functional categories associated with DNA were differen- Grafting CS with the rootstocks RG and 1103P triggered
tially expressed in the shoot apex in response to grafting thedifferentialregulationofanumberofgenesassociated
withtherootstocks1103PorRG(Figure6andAdditional with hormone metabolism: many genes from the func-
file5)alongwithothergenespotentiallyinvolvedinDNA tional categories IAA/auxin and gibberellins were both
or histone modification (such as SET domain containing up-anddown-regulated,whereasgenesfromthecategory
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Figure5PageMandisplayofgenecategoriesenrichedinthegenesdifferentiallyexpressedbetweenauto-andheterografts.Gene
categoryenrichmentofgenesdifferentiallyexpressedintheshootapexofCabernetSauvignonafterhetero-graftingwiththerootstocksRiparia
GloiredeMontpellier(RG)and1103Paulsen(1103P)incomparisontotheauto-graftedcontrol,A)genesup-regulatedby1103P,B)genes
up-regulatedbyRG,C)genesdown-regulatedby1103PandD)genesdown-regulatedbyRG.Fisher’sexacttest(withBonferronicorrection)was
usedtotestwhethersignificantlymoregenesinagivencategorywereoverorunder-represented(Colorscaleis:blue,significant
over-representationofgenes;red,significantunder-representationofgenes).Inthedisplay,theoverrepresentedMapManfunctionalcategories
aregivenbycollapsingnon-significantcategories.
brassinosteroids were generally up-regulated and genes 8s0040g01230) and one AUX1-like (VIT_03s0038g02
from the categories ABA, salicylic acid and ethylene were 140), and one protein ligase involved in auxin signal
generally down-regulated (Additional file 6). Most of the transduction (VIT_01s0127g00910) was down-regulated
differentially expressed genes from the category auxin (Additional file 7). Six genes from the functional ca-
were auxin responsive genes, however the expression of tegorybrassinosteroidswereup-regulatedincludingtwo
three auxin transporters were up-regulated, (three PIN1- brassinosteroid receptor genes (VIT_05s0062g01100
like (VIT_18s0001g15420,VIT_14s0108g00020 and VIT_0 andVIT_16s0100g00710)(Additionalfile8).
Table1MapManBINsenrichedinthegenesup-regulatedintheshootapexofhetero-grafts
Binnumber BinName Contingency pvalue Adustedp-value
31 Cell 61,605,1082,26089 7.43E-09 2.40E-06
10 Cellwall 47,509,1096,26185 3.75E-06 1.21E-03
31.1 Cellorganisation 41,374,1102,26320 2.53E-07 8.16E-05
28 DNA 43,304,1100,26390 1.26E-10 4.09E-08
28.1 DNAsynthesis,chromatinstructure 34,190,1109,26504 4.81E-11 1.55E-08
28.1.3 DNAsynthesis,chromatinstructure,histone 15,35,1128,26659 8.46E-10 2.73E-07
16.8.5 Secondarymetabolism,flavonoids,isoflavonols 6,15,1137,26679 1.51E-04 4.87E-02
16.1.5 Secondarymetabolism,isoprenoids,terpenoids 23,145,1120,26549 4.27E-07 1.38E-04
30.2.3 Signalling,receptorkinases,leucinerichrepeatIII 8,28,1135,26666 8.55E-05 2.76E-02
MapManBINsenrichedinthegenesup-regulatedbygraftingCSwiththerootstocksRGand1103Pincomparisontotheauto-graftedcontrol(logfoldchangeof1,
pvalue<0.05adjustedwithHolm).ContingencygivesthenumberofgenesininputlistfromtheBIN,thenumberofgenesonmicroarrayfromtheBIN,the
numberofgenesfrominputlistnotintheBINandnumberofgenesonmicroarraynotintheBIN.
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Table2GOtermenrichmentanalysisofgenesup-regulatedintheshootapexofhetero-grafts
GOterm Ontologya Description Contingency p-value Adusted
p-value
GO:0006260 P DNAreplication 6,9,763,18735 3.70E-07 1.20E-05
GO:0006259 P DNAmetabolicprocess 6,9,763,18735 3.70E-07 1.20E-05
GO:0044249 P Cellularbiosyntheticprocess 6,16,763,18728 2.70E-05 0.00088
GO:0034645 P Cellularmacromoleculebiosyntheticprocess 6,16,763,18728 2.70E-05 0.00088
GO:0009059 P Macromoleculebiosyntheticprocess 6,16,763,18728 2.70E-05 0.00088
GO:0044260 P Cellularmacromoleculemetabolicprocess 6,23,763,18721 0.00027 0.0086
GO:0044237 P Cellularmetabolicprocess 207,3914,562,14830 0.00068 0.022
GO:0044238 P Primarymetabolicprocess 207,3915,562,14829 0.00068 0.022
GO:0003774 F Motoractivity 26,119,743,18625 3.40E-12 1.10E-10
GO:0017111 F Nucleoside-triphosphataseactivity 45,326,724,18418 3.40E-12 1.10E-10
GO:0016818 F Hydrolaseactivity,actingonacidanhydrides,in 45,326,724,18418 3.40E-12 1.10E-10
phosphorus-containinganhydrides
GO:0016817 F Hydrolaseactivity,actingonacidanhydrides 45,326,724,18418 3.40E-12 1.10E-10
GO:0016462 F Pyrophosphataseactivity 45,326,724,18418 3.40E-12 1.10E-10
GO:0004386 F Helicaseactivity 19,207,750,18537 0.0011 0.033
GO:0005694 C Chromosome 67,277,702,18467 3.30E-31 5.90E-30
GO:0043228 C Non-membrane-boundedorganelle 67,277,702,18467 3.30E-31 5.90E-30
GO:0043232 C Intracellularnon-membrane-boundedorganelle 67,277,702,18467 3.30E-31 5.90E-30
GO:0043229 C Intracellularorganelle 243,3945,526,14799 2.90E-08 5.20E-07
GO:0043226 C Organelle 243,3945,526,14799 2.90E-08 5.20E-07
GO:0043227 C Membrane-boundedorganelle 242,3930,527,14814 3.10E-08 5.60E-07
GO:0043231 C Intracellularmembrane-boundedorganelle 242,3930,527,14814 3.10E-08 5.60E-07
GO:0005634 C Nucleus 241,3922,528,14822 3.80E-08 6.90E-07
GO:0005576 C Extracellularregion 86,1245,683,17499 7.00E-06 0.00013
GO:0030312 C Externalencapsulatingstructure 39,511,730,18233 0.00025 0.0044
GOtermenrichmentofgenessignificantlyup-regulated(logfoldchangeof1,pvalue<0.05adjustedwithHolm)intheshootapexinresponsetograftingwith
therootstocksRGand1103Pincomparisontotheauto-graftedcontrol.ContingencygivesthenumberofgenesininputlistfromtheGOterm,thenumberof
genesonmicroarrayfromtheGOterm,thenumberofgenesfrominputlistnotfromtheGOtermandnumberofgenesonmicroarraynotfromtheGOterm.
aKey:P,biologicalprocess;F,molecularfunctionandC,cellularcompartment.
Differentialregulationofreceptorkinases inthisstudywasdesignedtoreducethesecondaryeffects
Hetero-graftingwithrootstocksRGand1103Presultedin of rootstocks on gene expression (such as the indirect ef-
the up-regulation of many LRR containing receptor ki- fects associated with differences in scion transpiration,
nases and the down-regulation of many other classes of water status, photosynthesis, temperature, etc.). Shoot
receptorkinasessuchasS-locusglycoproteinlike,Domain apical meristems were harvested 1 hour before sunrise
of Unknown Function (DUF) 26, wall-associated kinase (harvested under green light) from plants growing
(WAK)andwheatleafrustresistanceLRK10-likereceptor outside in well watered pots. The shoot apices were
kinases(Additionalfiles8and9).Globally,37receptorki- harvested 4 months after grafting when presumably
nases were up- and 63 receptor kinases were down- the scions were in a ‘steady-state’; the plants had
regulated in the hetero-grafts compared to the auto-graft formed successful graft unions, had recovered from
control(Additionalfile8). the grafting process, had presumably adapted to the
presence of a rootstock but had not yet shown significant
Discussion modificationsofscionvigor.
Samplingstrategy
Rootstocks are widely used in agriculture and rootstocks Rootstockeffectsonplantdevelopment
can have a profound influence on many aspects of plant Rootstocks are known to have a wide range of effects on
development and plant responses to the environment scion development; the rootstocks used in this work are
(e.g.asreviewedby[1,2,8]).Thesamplingstrategyselected known to confer differences in scion vigor in the
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Figure6(Seelegendonnextpage.)
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(Seefigureonpreviouspage.)
Figure6MapMantranscriptionoverviewmapsshowingdifferencesintranscriptlevelsbetweenauto-andhetero-grafts.Comparisons
madebetweentheshootapexesofCabernetSauvignonauto-graftedandgraftedontotherootstocks1103Paulsen(A)andRipariaGloirede
Montpellier(B).Up-anddown-regulatedgenesaregiveninshadesofblueandredrespectively.Thecompletesetofgenes,derivedfunctional
categories,normalizedexpressionvaluesandcalculatedratiosaregiveninAdditionalfile2.
vineyard. The rootstock 1103P increases yields in com- this work on grapevine in which grafting with vigor in-
parison to the auto-grafted control when grafted with creasing rootstocks triggered the up-regulation, rather
the scion Shiraz, similarly RG increases the yield of the than the down-regulation, of gene expression. The tran-
scion Muscat Gordo Blanco in comparison to the auto- scriptthatbestcorrelatedwithplant sizeingraftedapple
grafted control (as reviewed by [3]). In the work trees was a sorbitol dehydrogenase [11]; sorbitol is the
presented here on young plants grown in pots, the root- primary transport form of carbon in apple and Jensen
stocks RG and 1103P also affected scion biomass accu- etal.[11]suggestedthattheshootapexof vigoroustrees
mulation and root/shootbiomass partitioning during the were more effective carbon sinks and that this facilitated
first growth cycle. Previous work has shown that these more vigorous growth. Similarly grafting with rootstocks
rootstocks alter secondary (stem thickness) rather than in grapevine (which increased scion vigor) resulted in
primary(stem length)shoot growth[20]. the differential regulation of many genes from the func-
In addition to affecting scion development, the root- tional categories major and minor carbohydrate meta-
stocks RG and 1103P affected the partitioning of biomass bolism and sugar transporters, suggesting that a similar
between the shoot and the root, this relationship was mechanism mayexistingrapevine.
stable throughout plant development. The intrinsic diffe- The up-regulation of genes from the functional ca-
rencesinshoot/rootbiomassallocationarereminiscentof tegories cell, cell wall and cell organization could also be
plants responses to a number of environmental factors, associated with the differences in scion growth observed
such as, nutrient availability, water availability and in the hetero-grafted grapevines. The up-regulation of a
temperature [21]. TFL 1gene(VIT_06s0080g00290)could berelated to the
non-self rootstock mediated increase in scion vegetative
Graftingwithrootstocksinducedchangesinthe growth. In apple trees, the silencing of MdTFL1 is asso-
expressionofgrowthrelatedtranscripts ciated with decreased vegetative growth and reduced
Increased rootstock conferred vigor in apple trees has generation time [22]. The grapevine homologue of TFL1
been associated with an increase in the number of genes is also known to delay flowering when over-expressed in
up-regulated in the shoot [11]; this was also observed in Arabidopsisandtobacco [23].
Table3MapManBINsenrichedinthegenesdown-regulatedintheshootapexofhetero-grafts
Binnumber BinName Contingency pvalue Adusted
p-value
17 Hormonemetabolism 34,549,747,26507 6.46E-05 1.54E-02
17.5.1 Hormonemetabolism,ethylene,synthesis-degradation 10,78,771,26978 1.79E-04 4.26E-02
26 Miscellaneous 106,1621,675,25435 2.49E-14 5.92E-12
26.1 MiscellaneouscytochromeP450 25,312,756,26744 1.15E-05 2.74E-03
26.8 Miscellaneousnitrilases,nitrilelyases,berberinebridgeenzymes, 16,76,765,26980 5.48E-09 1.30E-06
reticulineoxidases,troponinereductases
26.2 MiscellaneousUDPglucosylandglucoronyltransferases 22,263,759,26793 2.07E-05 4.92E-03
21.3 Redoxheme 5,6,776,27050 6.89E-06 1.64E-03
27.3.3 RNA,regulationoftranscription,AP2/EREBP,APETALA2/Ethylene-responsive 14,104,767,26952 5.88E-06 1.40E-03
elementbindingproteinfamily
16.8 Secondarymetabolism,flavonoids 25,239,756,26817 1.35E-07 3.22E-05
16.8.2 Secondarymetabolismflavonoids,chalcones 17,40,764,27016 1.70E-13 4.06E-11
16.8.2.3 Secondarymetabolismflavonoids,chalcones,stilbenesynthase 17,26,764,27030 7.42E-16 1.77E-13
30.2 Signallingreceptorkinases 38,637,743,26419 7.35E-05 1.75E-02
20.1.7 Stressbiotic,PR-proteins 18,125,763,26931 1.15E-07 2.74E-05
MapManBINsenrichedinthegenesdown-regulatedbygraftingCSwiththerootstocksRGand1103Pincomparisontotheauto-graftedcontrol(logfoldchange
of1,pvalue<0.05adjustedwithHolm).ContingencyasdescribedinTable1.
CooksonandOllatBMCPlantBiology2013,13:147 Page10of14
http://www.biomedcentral.com/1471-2229/13/147
Table4GOtermenrichmentanalysisofgenesdown-regulatedintheshootapexofhetero-grafts
GOterm Ontologya Description Contingency p-value Adustedp-value
GO:0050896 P Responsetostimulus 154,3959,375,14785 0.00032 0.0076
GO:0007154 P Cellcommunication 64,1511,465,17233 0.0018 0.044
GO:0023052 P Signaling 64,1512,465,17232 0.0018 0.044
GO:0016740 F Transferaseactivity 147,3766,382,14978 0.00035 0.0088
GO:0016491 F Oxidoreductaseactivity 81,1986,448,16758 0.0016 0.039
GOtermenrichmentofgenessignificantlydown-regulated(logfoldchangeof1,pvalue<0.05adjustedwithHolm)intheshootapexinresponsetografting
withtherootstocksRGand1103Pincomparisontotheauto-graftedcontrol.ContingencyasdescribedinTable2.
aKey:P,biologicalprocessandF,molecularfunction.
Graftingwithrootstockstriggersthedifferentialexpression Thedifferentialexpressionofreceptorkinasesin
ofmanygenesinvolvedinDNAandchromatinmodification responsetohetero-graftingwithanon-selfrootstock
In eukaryotes epigenetic mechanisms can regulation Plant genomes contain large numbers of receptor ki-
chromatin structure and gene expression, for example nases with very divergent extracellular domains. The re-
by DNA methylation, histone modification and certain ceptor kinases that were differentially expressed in
aspects of the siRNA signaling pathway [24]. DNA response to grafting with non-self rootstocks included
methyltransferasesaddamethylgrouptoacytosinebase genes from the functional categories LRR, S-locus glyco-
to produce 5 methylcytosine; DNA methylation is protein like, DUF 26, WAK and LRK10-like. LRR pro-
thought to repress transposons activity and in some teins are the largest group of receptor kinases in plants
cases gene expression [25]. The up-regulation of genes andthemotifisthoughttobeinvolvedinsignaltransduc-
associated with DNA methylation could be associated tion and to mediate protein-protein interactions. In this
with the repression of transposon activity or gene ex- study, generally the receptor kinases from the LRR family
pression in hetero-grafted plants. The up-regulation of were up-regulated in the shoot apex of the hetero-grafts
many histones could also be related to alterations in the (14geneswereupandonly4genesweredown-regulated).
chromatin structure of hetero-grafted plants and could LRR domain containing proteins have been implicated in
be involved in some of the gene expression differences many developmental pathways and defense responses
observed between the hetero- and auto-grafted plants. (as reviewed by [28]); this could imply that grafting with
Both DNA methylation and histone modification can be rootstocks triggered a defense response throughout the
affected by siRNAs [24] and small RNAs are known to plant. Similarly, LRK10 receptor kinases were down-
be graft transmissible in plants (e.g. [26]). We propose regulated in the hetero-grafts and LRK10 was first identi-
the hypothesis that grafting with non-self rootstocks al- fiedasaleafrustresistancegeneinwheat.
tersthe small RNA population ofthescion andmediates S-locusglycoprotein-likereceptorkinaseswerefirstiden-
epigenetics changes in the recipient tissue. However, tified as being important in self-incompatibility responses
chromatin modification is known to be induced in re- in Brassica flowers and have since been shown to be in-
sponse to various stresses such as salt, drought and cold volved in plant defense responses (as reviewed by [29]).
stress as well as being involved in a number of hormone Surprisingly, the S-locus receptor kinases were also down-
signaling cascades (as reviewed by [27]). Therefore, the regulated, rather than up-regulated as we would have
differential expression of genes in response to hetero- expected. However, at least one S-locus receptor kinase in
grafting could be an indirect response of chromatin Arabidopsis is a negative regulator of plant defense re-
modification genes via grafting-related stress responses sponses [30] suggesting that the interpretation of differen-
and/or a direct consequence of siRNA trafficking be- tialexpressionofreceptorkinasesismorecomplicatedthat
tween the rootstock and scion. In addition to possible it may first appear. However, it is possible that the S-locus
epigenetic regulation induced by hetero-grafting, both receptor kinases differentially expressed in response to
post-transcriptional and post-translational modifications graftingwithanon-selfrootstockgenotypeinordertosup-
are also possible. Numerous genes involved in protein pressaself-incompatibilityresponsethroughouttheplant.
synthesis, degradation and targeting were differentially
expressed in the hetero-grafts. Furthermore, six genes Thedifferentialexpressionofhormonesignalingrelated
involved in RNA processing and three genes involved in genesinresponsetohetero-graftingwithanon-self
RNA binding were up-regulated in the hetero-grafts (the rootstock
onlydown-regulatedgenewastheribonuclease3-like/dicer The differential regulation of genes from the functional
proteinpreviouslymentioned). category hormones could suggest the involvement of