Apical dominance in saffron and the involvement of the branching enzymes CCD7 and CCD8 in ...

15 Pages·2014·0.95 MB·English

Rubio-Moragaetal.BMCPlantBiology2014,14:171
http://www.biomedcentral.com/1471-2229/14/171
RESEARCH ARTICLE Open Access
Apical dominance in saffron and the involvement
of the branching enzymes CCD7 and CCD8 in the
control of bud sprouting
Angela Rubio-Moraga1, Oussama Ahrazem1,2, Rosa M Pérez-Clemente3, Aurelio Gómez-Cadenas3,
Koichi Yoneyama4, Juan Antonio López-Ráez5, Rosa Victoria Molina6 and Lourdes Gómez-Gómez1*
Abstract
Background: Insaffron (Crocus sativus),new corms develop atthe base of every shoot developed from the
maternal corm, a globular underground storage stem. Since the degree of bud sprouts influences thenumberand
sizeofnewcorms,andstrigolactones(SLs)suppressgrowthofpre-formedaxillarybud,itwasconsideredappropriate
toinvestigateSLinvolvementinphysiologyandmolecularbiologyinsaffron.Wefocusedontwoofthegeneswithin
theSLpathway,CCD7andCCD8,encodingcarotenoidcleavageenzymesrequiredfortheproductionofSLs.
Results:TheCsCCD7andCsCCD8genesarethefirstonesisolatedandcharacterizedfromanon-grassmonocotyledonous
plant.CsCCD7andCsCCD8expressionshowedsomeoverlapping,althoughtheywerenotidentical.CsCCD8washighly
expressedinquiescentaxillarybudsanddecapitationdramaticallyreduceditsexpressionlevels,suggestingits
involvementinthesuppressionofaxillarybudoutgrowth.Furthermore,invitroexperimentsshowedalsothe
involvementofauxin,cytokininandjasmonicacidonthesproutingofaxillarybudsfromcormsinwhichtheapical
budwasremoved.Inaddition,CsCCD8expression,butnotCsCCD7,washigherinthenewlydevelopedvasculartissue
ofaxillarybudscomparedtothevasculartissueoftheapicalbud.
Conclusions:WeshowedthatproductionandtransportofauxininsaffroncormscouldactsynergisticallywithSLsto
arresttheoutgrowthoftheaxillarybuds,similartothecontrolofabove-groundshootbranching.Inaddition,jasmonic
acidseemstoplayaprominentroleinbuddormancyinsaffron.Whilecytokininsfromrootspromotebudoutgrowth.
InadditiontheexpressionresultsofCsCCD8suggestthatSLscouldpositivelyregulateprocambialactivityand
thedevelopmentofnewvasculartissuesconnectingleaveswiththemothercorm.
Keyword:Auxin,Buds,Carotenoidcleavageoxygenases,Corm,Saffron,Strigolactones
Background the beginning of the dry season (April-May), when the
C. sativus is an economically important monocotyledon- leaves senesce and wither, to the beginning of summer
ous crop producing saffron, the world’s highest priced (July), characterized by the formation of leaf primordia
spice [1]. In addition, the stigmas are used all over the [4].Shortlyafterwards,flower morphogenesis takesplace
world to treat different diseases [2]. Over the past and all the flower is already differentiated by the end of
5000 years, farmers have selected C. sativus for its stig- August [4]. With the onset of sprouting at the end of
mas characterized by the accumulation of apocarote- October, the corm turns into a source organ supporting
noids [3]. C. sativus is a triploid perennial sterile plant, growth of the developing corm. The importance of
adapted to overcome a dry dormant period in the form adequate corm production is self-evident in the sterile
of an underground corm. Corms remain dormant from taxon saffron, which has been reproduced vegetatively
for millennia by annually replacing corms. Since almost
*Correspondence:[email protected] every sprouting bud produces a corm, factors affecting
1DepartamentodeCienciayTecnologíaAgroforestalyGenética.Facultadde sprouting are highly important for corm and flower pro-
Farmacia,InstitutoBotánico.UniversidaddeCastilla-LaMancha,Campus
duction. Only one to three corms per mother corm are
Universitarios/n,02071Albacete,Spain
Fulllistofauthorinformationisavailableattheendofthearticle
©2014Rubio-Moragaetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse,
distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublic
DomainDedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthis
article,unlessotherwisestated.
Rubio-Moragaetal.BMCPlantBiology2014,14:171 Page2of15
http://www.biomedcentral.com/1471-2229/14/171
produced in one growing season through conventional pathway has been proposed [33]. Two carotenoid cleav-
methods [4]. It would take 9–10 years to produce corms age dioxygenases, CCD7 and CCD8, are involved in
required to sow one hectare from an initial corm [5]. SL biosynthesis. CCD7 catalyses the 9,10 cleavage of
Hence, low multiplication rates and fungal infestation of 9-cis-β-carotene to produce 10′-apo-β-carotenal and
corms reduce the productivity and quality, thereby β-ionone. Then, the 10′-apo-β-carotenal is cleaved by
restraining the availability of planting material. A corm CCD8 to produce C18-ketone β-apo-13-carotenone.
survivesforonlyoneseason,reproducingviadivisioninto This compound is immediately converted by CCD8 to
cormletsthateventuallygiverisetonewplants,andthere- carlactone, through a series of different reactions [33].
forecormsareindispensableforsaffronpropagation. Carlactone presumably serves as substrate for MAX1
Despite its importance, the sprouting process in saffron enzymes which catalyze the production of the different
hasnotbeencharacterizedprecisely.Asinotherplants,it formsofSLsfoundinnature[34,35].
is thought that this process should be orchestrated by a Orthologues of CCD7 and CCD8 have been character-
complex interplay of phytohormone and sugar signals [6]. ized in plants such as Arabidopsis, pea, petunia, rice,
Abscisic acid (ABA) has been associated with the onset chrysanthemum, kiwi, and tomato [14,25,32,36-40]. In
and maintenance of corm dormancy [7]. Gibberellins addition, a CCD8 orthologue has been isolated from the
(GAs) seem to be involved in apical sprout growth after moss Physcomitrellapatens[41].
dormancy cessation, but not in dormancy maintenance The saffron corm is a stem-derived organ formed by
[8]. So far, there is no data regarding the involvement of shortened nodes and internodes. Mature saffron corms
other hormones neither in the sprouting process nor on usually show one to three apical dominant buds which
apical dominance in saffron. In addition, it is not known will sprout in the following season plus many axillary
whether the corm behaves as the stem of other higher dormant buds (Figure 1). Each axillary bud has the same
plants and follows the same behaviour regarding apical developmental potential as the primary shoot apical
dominance. In higher plants not all of the axillary buds meristem in that it can produce a growing shoot axis.
develop, and each bud is subjected to a decision to con- However, axillary buds enter a dormant state after form-
tinue growth or to become dormant [9]. Plant hormones ing only a few leaves. The large number of axillary buds
aremajorplayersinthecontrolofaxillarybudoutgrowth. in the corm system allows the plant to recover quickly
It has been known for a long time that two hormones from damage and to adjust its growth according to
in particular, auxin and cytokinin, are involved in this environmental inputs. Apical dominance acts as a plant
control. Auxin, which is supplied from the apical bud, survival mechanism by providing a reservoir of meri-
indirectlysuppressesaxillarybudoutgrowth,whilecytoki- stems that can replace the damaged primary shoot.
nins directly induce branching [10]. During the past two This mechanism works when the primary shoot is
decades, genetic and physiological analyses in pea, Arabi- damaged or removed through disease, herbivore graz-
dopsis rice, and Petunia have predicted the involvement of ing, or pruning. In some plants, apical dominance can
an additional, novel hormone in the control of shoot also be released depending on developmental pro-
branching:theSLsthatactassecondmessengerstoinhibit grams. Dormant axillary buds start their outgrowth
axillary bud outgrowth [11]. The interactions between after the primary bud differentiates into the determin-
auxin and SLs in regulation of lateral branching are com- ateorgan,suchasaflower.Theseadditionalshootsare
plex. SLs may act by dampening auxin transport [12-14], important for increasing the total number of leaves or
act downstream of auxin [15], or be independent from the flowers to be more fruitful. However, this is not the
statusofstemauxin[16]toregulatelateralbranching. case of saffron, where no activation of additional axillary
SLs are long known for their role as germination stim- budsisinduced.
ulants for root parasitic plants [17] and pre-symbiotic In view of the importance of sprouting in saffron and
branching factors for arbuscular mycorrhizal fungi [18]. In the potential impact of apical dominance on the number
floweringplants,SLshavealsobeenimplicatedindevelop- ofcormsproduced,itwasconsideredtimelytoinvestigate
ment [19] as new hormonal players in the suppression of the role of SLs in this process, including the control of
the outgrowth of preformed axillary buds [20,21], in root axillary branching, which can be extended to other plants
systemarchitecture,adventitiousrooting,secondarygrowth propagatedbycorms.Inthisstudywehaveidentifiedtwo
andreproductivedevelopment[22-25].Theyhavebeenalso saffron genes required for SL biosynthesis as well as the
associated with plant responses to both abiotic and biotic involvementofkeygrowthregulatorsinthisprocess.
stresses [26-30]. However, it is on their effects on shoot
branching that some of the biosynthesis and responsive Results
genesintheSLpathwaywerefirstidentified[31,32]. Characterizationofaxillarybudsproutinginsaffron
Several lines of evidence demonstrate that SLs are Axillary buds are usually dormant, and are inhibited by
derived from apocarotenoids, and a putative biosynthetic auxin produced by the apical meristem. We checked
Rubio-Moragaetal.BMCPlantBiology2014,14:171 Page3of15
http://www.biomedcentral.com/1471-2229/14/171
A
apical bud
axillary bud
internode B
node
vascular
tissue
C
parenchyma
D
roots
basal
plate
Figure1Thecormofsaffronandthedifferentpartsusedinthisstudy.(A)Upperviewofthecormshowingtheapicalmeristemandthe
axillarybudslocatedinthedifferentnodes.(B)Transversalsectionofthecormthroughtheapicalmeristemshowingthevasculartissueandthe
parenchymaofthecorm.(C)Bottomviewofthecormshowingthebasalplatewhichconnectedthecormtothemothercorm,alongwiththe
roots.(D)Transversalsectionofthecormshowingtherootsandtheparenchymatissue.
whether the saffron corm exhibited classical stem-like buds; (iv) full removal of the basal complex with or
behaviour and also investigated the role of bud apical without apical bud removal; and (v) full removal of the
dominance in determining lateral bud dormancy release adventitious roots with or without apical bud removal.
and sprouting. Decapitation of the apical bud of the The sprouting was followed for 30 days. Decapitation
corm induced a loss of apical dominance and after of the apical bud induced accelerated loss of apical
15 days, all axillary buds were sprouted (Figure 2A). We dominance,affectingaxillarybudgrowth.After15days,
determinedthenumberofaxillarybudssproutedineach all axillary buds were sprouting (Figure 2C). The same
node and the number of axillary buds that remained pattern was observed when the apical complex (the
quiescent 35 days after decapitation of the main bud apical bud plus underlying tissues) was removed. As
(Figure 2B). The first node was set as the one that was expected, when auxin was applied to the decapitated
occupied by the basal plate. The total number of apex, axillary bud sprouting was suppressed (Figure 2C
axillary buds per node was increased from the bottom and Additional file 2: Figure S2). Interestingly, the
to the apical meristem, reaching the maximum at node removal of the basal plate inhibited the growth of the
6 (Figure 2B). In the first and second nodes the per- apical and axillary buds. The same results were
centage of sprouted buds was 13.5 and 38%, respect- obtained when the adventitious roots were removed
ively. The roots were formed in the third node in 98% (Figure 2C). However, in this case, when corms were
of the analysed corms, and in this node the percentage transferred to a humid surface allowing the regeneration
of sprouted axillary buds was 51.8%. From this node of the roots, the growth of the apical bud was restarted
onward, the pattern of sprouting changes, with an and secondary buds sprouted in the corms when the
increasing number of sprouted buds in comparison apical bud was removed. These experiments emphasize
withthebudsthatremainquiescentineachnode,from the importance of apical budpresence and viability in the
62% in the fourth node up to 92.7% in the eighth node control of axillary bud growth but also the involvement
(Figure 2B). Practically all these sprouted axillary buds of the roots on bud sprouting. Roots have been consid-
will form a new replacement corm, resulting in an ered to be the source of cytokinins, and cytokinins
increase in the number of corms (Additional file 1: induced the fast growth of axillaries buds in decapi-
Figure S1). To characterize this process in depth, the tatedcorms(Additionalfile2:FigureS2).
corms were subjected to the following treatments: (i) Several works have pointed out that after decapitation,
decapitation of the apical bud; (ii) full removal of the the axillary bud needed to form a vascular connection
apicalcomplex;(iii)fullremovalofsecondaryquiescent before it could grow. We investigated whether avascular
Rubio-Moragaetal.BMCPlantBiology2014,14:171 Page4of15
http://www.biomedcentral.com/1471-2229/14/171
A
a c
1cm
d
1cm
b
1cm 1cm
B
2,5 axillary buds sprouted
axillary buds quiescent
ds 2
u roots
b
y
yllar 1,5
x
a
of
er 1
b
m
u
N 0,5
0
node node node node node node node node
1 2 3 4 5 6 7 8
C
7
Serreiems1oval of the apical bud
m) 6 Sefruiells 2removal of apical complex
c
h ( 5 Sefruiells 3removal of basal plate
ngt Sefruiells 4removal of roots
ud le 4 rmeemriosvteaml a +pi cal
y b 3 NAA
ar
nd 2
o
c
Se 1
0
1 2 6 13 14 15 16 20 23 30
Time after capping
D
a 1 mm b 1 mm 1 mm c
d e f
100µm 100µm 100µm
Figure2(Seelegendonnextpage.)
Rubio-Moragaetal.BMCPlantBiology2014,14:171 Page5of15
http://www.biomedcentral.com/1471-2229/14/171
(Seefigureonpreviouspage.)
Figure2DominanceoftheapicalmeristeminnondormantCrocussativuscorms.(A)removaloftheapicalbudallowedthesproutingof
theaxillarybuds.a)cormswiththeapicalmeristemsremoved.b)intactcorms.c)cormswithsproutedsecondaryaxillarybud13daysafterapical
meristemremoval.d)intactcorms,ascontrol,13daysaftertheinitiationofthedecapitationexperiment.(B)numberofaxillarybudssproutedor
quiescentineachnodeafterdecapitationoftheapicalmeristem.Ineachtreatment,25cormsweredecapitated.ErrorbarsrepresentSDof25
replicates.(C)removaloftheapicalbudwithorwithoutothercormpartsandresultsinsproutingofaxillarybuds.Ineachtreatment,25corms
weredecapitated.ErrorbarsrepresentSDof25replicates.(D)vascularizationinthesproutedaxillarybudsofsaffroncorms(a-c).Absenceof
vasculatureinquiescentaxillarybuds(d-f).Budsweresectionedbyhandandsectionswerestainedforligninwithphloroglucinol-HCl.Lignin
stainingisred.Picturesweretakenunderadissectionmicroscope.
connection is already present in the axillary buds of buds, decapitated buds and axillary bud extracts were
saffron. Axillary buds in saffron are covered by a few applied to P. ramosa seeds, and germination was scored
leaves, thus these axillary buds might initiate a few after 7 d. The extracts of the main vascular tissue from
leaves and then become developmentally arrested or the apical buds induced 11.5% germination, whereas ger-
dormant because the terminal bud inhibits their growth. mination was not induced using extracts from the other
These dormant axillary buds need to develop a vascular tissues. The synthetic SL GR24 (10−9 to 10−10 M) was
connection previously to be able to grow, and this usedasapositive control andinduced 58%and34%ger-
process is relatively slow (Figure 2D) in comparison with mination, respectively, whereas water (negative control)
other systems studied [32]. Quiescent axillary buds in inducednogermination(Figure 4).
saffron have not developed the vascular connections
with mother corm (Figure 2D), but once the sprouting IdentificationofthefirstsaffronCCD7andCCD8genes
of these axillary buds is induced, the development of SLs are a new class of plant hormones that have been
thatvasculature begins (Figure 2D). shown to be involved in the regulation of the outgrowth
of preformed axillary buds. In order to study the rela-
Hormonesinsaffronbuds tionship between apical dominance and SLs in saffron,
Apical dominance was one of the first developmental the CCD7 and CCD8 genes were isolated using a com-
phenomenon shown to be regulated by plant hormones. bination of degenerate primer PCR and gene walking.
Auxin, derived from the apical bud, moves basipetally in Saffron CCD7,hereafterdesignatedasCsCCD7,contains
the stem through the polar auxin transport stream and five introns (Figure 5A), and the 1912 bp of the coding
inhibits the growth of axillary buds, whereas cytokinin sequence encodes a protein of 591 amino acids with a
derived mainly from the roots, promotes the outgrowth predicted pI of 6.6 and 66.17 kDa (GenBank accession
[42,43]. In order to characterize the hormonal regulation number KJ361477). The ChloroP 1.1 program predicted
of axillary bud sprouting in saffron corms, the level of a 50-amino acid, N-terminal transit peptide, consistent
auxin and several other hormones were measured in the with plastid localization of CCD7 [44]. CsCCD7 showed
differentpartsofthecorm(Figure3).Highlevelsofauxins the highest homology with Solanum lycopersicum CCD7
were detected in the apical buds, while auxins were not protein (67% identical). In the case of the CCD8, two
detectedintheothertissuestested,includingaxillarybuds .1pt?>different genes were isolated from saffron, desig-
(Figure 3A). Interestingly, a significant increase in auxin nated as CsCCD8a and CsCCD8b, which differ in the
content was detected in axillary buds after removal of the sequenceofthefirstexonandintron.CsCCD8awaspre-
apical meristem (Figure 3A), suggesting a reorganization dicted to have six exons (Figure 5B), 3151 nucleotides
ofthedominanceafterdecapitation.Bycontrast,thehigh- and a coding sequence of 1533 nucleotides encoding a
est level of jasmonic acid (JA) was detected in the quies- proteinof511aminoacidswithapredictedpIof6.6and
cent axillary buds, decreasing ten days after decapitation 57.17 kDa (GenBank accession number KJ361478).
oftheapicalbud(Figure3B).Thehighestlevelofsalycilic CsCCD8b was predicted to also have six exons, 3195
acid(SA)wasdetectedinapicalandaxillarybudsfollowed nucleotides and a coding sequence of 1671 nucleotides
bythebasalplate(Figure3C). encoding a protein of 557 amino acids with a predicted
pI of 6.1 and 62.03 kDa (GenBank accession number
SLsinsaffroncorms KJ361479). Both CsCCD8 proteins showed 83% identity
To assess the presence of SLs in saffron, several parts with DAD1 (CCD8 from Petunia hybrida) and 76% to
were dissected and tested for the induction of germin- D10 (CCD8 from Oryza sativa), and were predicted to
ation of Phelipanche ramosa seeds (Figure 4). Apical be localized in plastids using the ChloroP 1.1 program,
buds, axillary latent buds and sprouted axillary buds, consistentwithplastidlocalizationofCCD8[45].
externalcover (theexternalsurface ofthe corms without As part of the characterization of CsCCD7, CsCCD8a
buds), roots, basal plate, and vascular tissue from apical and CsCCD8b, amino acid sequence alignments were
Rubio-Moragaetal.BMCPlantBiology2014,14:171 Page6of15
http://www.biomedcentral.com/1471-2229/14/171
highest expression was detected in the axillary buds
A
24 h after removal of the apical bud, followed by the
60 levels of expression in the apical bud (Figure 6A). The
ht
g levels in the vascular tissue from the apical buds or from
ei 50
w
y the sprouted axillary buds were similar (Figure 6A).
gr dr 40 Interestingly, high expression levels were observed in
g/
nt (n 30 the orange stigma in contrast with the low levels de-
nte tectedinthesenescentstigma.Theexpressionlevelswere
o
n c 20 alsolow in leaves andadventitious roots (Figure 6A). The
uxi combined levels of both CsCCD8 transcripts in mRNA
A 10
extracted from different tissues were examined using
0 primersthatdonotdiscriminatebetweenthetwodifferent
Basal plate Epidermis Roots Apical bud Axillary bud Axillary bud
10 d. after copies/alleles. The highest levels were detected in the
B decapitation
quiescent axillary buds (Figure 6B). However, these levels
ght 2000 were drastically reduced in the axillary buds 24 h after
ei
gr dry w 1500 raepmicoalvabluodf wtheereapmicuaclhbruedd.uTcehde ienxpcroemsspioanrisloenvelwsitinh tthhee
ng/ 1000 expressionlevelsinthequiescentaxillarybuds,suggesting
ent ( that the levels of CsCCD8 are reduced during the sprout-
nt 500
co ingprocess(Figure6B).TheexpressionofCsCCD8inthe
A
J 0 vascular tissue from the apical buds was lower than
Basal plate Epidermis Roots Apical bud Axillary bud Axillary bud
10 d. after that in the newly developed vascular tissue of the
decapitation
C sproutedaxillarybuds,buthigherthanthatobservedin
300 adventitious roots (Figure 6B). Similarly to CsCCD7, a
eight 250 relatively high expression of CsCCD8 was detected in
y w orange stigma, while this expression was reduced in
dr 200
gr senescentstigma(Figure6B).
ng/ 150
ntent ( 100 Discussion
o
A c 50 Almost all bulbous plant species are monocots, includ-
S ing economically important plants such as saffron, tulip,
0 onion, garlic and lily. Their vegetative propagation
Basal plate Epidermis Roots Apical bud Axillary bud Axillary bud
10 d. after constitutes the most relevant process for agronomical
decapitation
Figure3Hormonalcontentsindifferentpartsofsaffroncorms. improvements to markedly increase the potential number
Eachcolumnrepresentsthemean±oftwotofourreplicatesof of bulblets, while the control of dormancy is crucial to
independentlyharvestedplantmaterial.(A)Auxincontentinthe solve many problems associated with the storage and
differentsamples.(B)Jasmonicacid(JA)contentinthedifferent
distribution of these crops. SLs play a key role in both
samples.(C)Salicylicacid(SA)contentintheanalyzedsamples.
processes, development of new buds and sprouting
inhibition by inhibition of bud outgrowth [20,21,46,47]
carried out, in order to build a phylogenetic tree using and several genes involved in the biosynthesis and
the CCD7 and CCD8 protein sequences from a variety signallingofSLshavebeenidentifiedfromadiverserange
of plant species (Figure 5A and B). This analysis showed of species [48], although excluding bulbous plants. In this
that CsCCD7 was closer to the eudicot sequences than paper, we describe and analyse the sprouting process in
to the grass sequences (Figure 5A) while CsCCD8a and saffron induced by decapitation, as well as the involve-
CsCCD8b were in a cluster separate from the eudicots ment of SLs in this process through the isolation and
(Figure 5B andAdditionalfile 3:FigureS3). characterization of two key genes in SL biosynthesis,
CsCCD7andCsCCD8.
GeneexpressionofCsCCD7andCsCCD8
TodeterminewhereCsCCD7andCsCCD8wereexpressed, IsolationofthesaffronCCD7andCCD8genes
the pattern of CsCCD7 and CsCCD8 transcript abundance The genes so far identified that control branching are
was determined in different tissues using quantitative frequently conserved between species. In particular,
real-time RT-PCR (qPCR). CsCCD7 expression was two carotenoid cleavage dioxygenase genes, CCD7
readily detected in all the analyzed tissues, but showing (MAX3/RMS5/DAD3/D17-HTD1) and CCD8 (MAX4/
different expression levels. At the level of buds, the RMS1/DAD1/D10), involved in SLs biosynthesis, appear
Rubio-Moragaetal.BMCPlantBiology2014,14:171 Page7of15
http://www.biomedcentral.com/1471-2229/14/171
70
%) 60
n (
o 50
ati
n
mi 40
r
e
g 30
a
s
mo 20
a
r
P. 10
0
P AB VT2 R Water 10-10M 10-9M
GR24
Figure4Analysisofroot-extractablestrigolactonesquantifiedbyagerminationbioassayindifferentindifferentcormtissues.
GerminationofP.ramosaseedsinducedbyextractsfrom:P,parenchyma;AB,axillarybud;VT2,vasculartissuefromaxillarybudsandR,roots.
GR24(10–9and10–10M)anddemineralizedwaterwereusedaspositiveandnegativecontrols,respectively.Barsrepresentthemeansoftwo
poolsfromfiveindependentsamples(±SE).
to be well conserved among the plant species studied. Apicaldominanceinsaffroninrelationwiththehormone
To characterize the SL pathway in saffron, the saffron contentandthepositionaleffectofaxillarybud
orthologues of CCD7 and CCD8 were isolated, and sprouting
theirorthologywasconfirmedbyphylogeneticanalysis. Apical dominance is thought to result from the develop-
Analyses of the genomes of several plants species mental arrest of lateral buds caused by auxin, which is
showed that CCD7 is a single copy gene, which also basipetally transported from the shoot apical meristem
seems to be the case in saffron, analysed in this study. [56]. This notion is supported by the fact that apical
However, in most of the analysed genomes CCD8 is dominance is maintained if an excised apex is replaced
present as a multicopy gene [49] (Additional file 3: by an exogenous source of auxin. Auxin derived from
Figure S3). In saffron, our results suggest that there are the shoot apex might control lateral bud outgrowth by
atleasttwolociencodingCCD8. the action of SLs, relaying the inhibitory signal from the
Despite the high functional conservation of the CCD7 main stem into the buds [15]. This process has not pre-
and CCD8 genes between species, there are interesting viously been studied in depth in saffron. As observed in
differences in the expression patterns. CsCCD7 and other plant systems, apical dominance in saffron was
CsCCD8wereexpressedinalltissuesandorgansexam- released by excision of the apical bud, which was the
ined, with very low expression levels in adventitious main source of auxin among the tested organs and
roots. In Arabidopsis [45], petunia [38], pea [50], kiwi tissues, and conversely, apical dominance was main-
[40] and tomato [25], root expression of CCD8 is at tained by the application of auxin to the cut surface of
least 10 times higher than that in the shoot. By contrast, thedecapitatedcorm.
inrice[51]andchrysanthemum,shootexpressionexceeds In addition, the importance of roots has been shown,
root expression [14], whereas in rose no expression is possibly as a source of cytokinin for sprouting of apical
detectedinroots[52].InArabidopsis,CCD7expression and secondary buds, in the latter, when apical domin-
was high in roots, [37] although recently, the highest ancehasbeen lost(Additional file 2:FigureS2).
expression has been detected in seeds and in the stem The buds more closely located to the apical bud were
vascular tissue [53]. In rice CCD7 is expressed in both more active, in terms of sprouting, than those distant
shootandroottissuesbeingmainlyexpressedinvascu- from the apical bud, suggesting that the outgrowth
larbundletissuesthroughouttheplant[54].Inpetunia, potential of each axillary bud is related to its position
its expression is higher in nodes and internodes [55] in the corm. The decision of which buds activate first
and in tomato, CCD7 expression in green tissue is far depends on the local bud competitiveness, which is
less than that detected in the stem and in the roots probably determined by the local environment and
[39]. These differences may reflect different contribu- developmental state of the bud [57]. On the other
tions of the root and shoot in the SL-regulation of hand, a perception of altered light quality, in particular
shootbranchingindifferentspecies. a decreased ratio of red light to far-red light (R/FR)
Rubio-Moragaetal.BMCPlantBiology2014,14:171 Page8of15
http://www.biomedcentral.com/1471-2229/14/171
A
PtCCD7
MtCCD7
AaCCD7
MAX3
SbCCD7
RMS1
RcCCD7
VvCCD7
DAD3
SlCCD7
CsCCD7
B
RMS1
MtCCD8
CcsCCD8
CcCCD8
RcCCD8
SlCCD8
SbCCD8
MAX4
OsCCD8
ZmCCD8
CsCCD8a
CsCCD8b
Figure5(Seelegendonnextpage.)
Rubio-Moragaetal.BMCPlantBiology2014,14:171 Page9of15
http://www.biomedcentral.com/1471-2229/14/171
(Seefigureonpreviouspage.)
Figure5GenestructuresofCsCCD7andCsCCD8andthephylogeneticrelationshipwithCsCCD7andCsCCD8homologuesfromother
plantspecies.(A)Thepostulatedintron/exonstructureforCsCCD7andpositionsoftheintronsinorthologuesofCsCCD7isshownintheleft
sideofthefigure.IntherightsidearepresentativephylogenetictreeofCCD7proteinsfromdifferentplantspeciesisshown.(B)Thepostulated
intron/exonstructureforCsCCD8andpositionsoftheintronsinorthologuesofCsCCD8isshownattheleftside.Intherightsidearepresentative
phylogenetictreeofCCD8proteinsfromdifferentplantspeciesisshown.Exonsandintronsareshownasboxesandlines,respectively.The
presenttreeswereobtainedafteralignmentoffull-lengthCCDssequencesusingClustalWandclusteringwiththeneighbour-joiningmethod.Accession
numbersareasfollow:SlCCD7(ACY39882.1),PhCCD7(ACY01408.1),AcCCD7(ADP37985.1),ZmCCD7(NP_001183928.1),RcCCD7(XP_002511629.1),
VvCCD7(XP_002274198.1),AaCCD7(ADB64459.1),PsCCD7(ABD67496.2),GmCCD7(ADK26570.1),AtCCD7(NP_182026.4),CcsCCD7(ADM18968.1),
SmCCD7(XP_002984696.1),OsCCD7(EAY95081.1),LjCCD7(ADM88552.1),MtCCD7(XP_003622555.1),BdCCD7(XP_003581501.1),PpCCD7
(ADK36680.1),PtCCD8a(XP002309543),PtCCD8b(XP002324797),AcCCD8(GU206812.1),AtCCD8(AT4G32810),BdCCD8(LOC100831734),
MtCCD8(Medtr3g127920),OsCCD8(Os01g0746400),Dad1(AY743219),RMS1(AY557342),SbCCD8(Sb03g034400),ZmCCD8(GRMZM2G446858),
RcCCD8,SlCCD8(NP_001266276.1),StCCD8(XP_006359761.1),CitCCD8a(KD079823),CitCCD8b(XP_006476130,CaCCD8b(XP_004501157),PpCCD8
(ADK36681.1),SmCCD8(XP_002972693.1),GmCCD8b(XP_003522713),GmCCD8a(XP_003522713.2).Branchsupportisunder5000Bootstrapreplicas.
perceived by the phytochrome B has a key role in this
process, inducing an inhibition of bud outgrowth
A
[58-60]. However, on our experimental system, consid-
16 eringthecormsasanundergroundorganitisnotclear
whether the buds nearer the apex are exposed to some
14
sunlight. However, this positional effect has been
12 observed in other plants such as pea, where only one
bud per axil is released at the upper nodes when
10
branching is promoted by decapitation [61,62], and
8 appears to be determined by a balance between several
hormones[63].
6
We also measured the levels of JA in several tissues of
4 saffron. JA and its derivatives have been implicated in
stress-induced responses and have also been shown to
2
inhibit plant growth and mitosis [64]. The highest levels
0 of JA were detected in quiescent axillary buds, but such
AM AB AB24 R VT VT2 SS OS L
levels were reduced after the decapitation of the apical
B bud and were undetectable in apical buds. These data
suggest that JA could play a prominent role in bud
20
dormancy in saffron. Similarly, JA has recently been
18
shown to be involved in bud dormancy in apricot [65]
16
and orchids [66]. The observed pattern of JA was oppos-
14 ite to the one observed for auxin. Cell elongation and
12 meristemactivityrequiredforplantgrowthareregulated
10 by auxins. Interestingly, JA shows extensive crosstalk
with auxin and down-regulates PIN1 and PIN2 protein
8
levels [67], suggesting a possible role of this hormone in
6
PIN protein trafficking and auxin transport, as suggested
4 for SLs [48]. In agreement with this observation, it was
2 shown that a gain in function mutation in IAA8 induced
0 more lateral branches and decreased shoot apical dom-
AM AB AB24 R VT VT2 SS OS L inance byreducingJAlevels [68].
Figure6CsCCD7andCsCCD8transcriptaccumulation Endogenous cytokinins (CKs) can enter axillary buds
(normalisedtothesaffronhouseholdgeneRSP18).(A)relative
and promote their outgrowth by promoting the cell
geneexpressionofCsCCD7indifferentplanttissues.(B)relative
cycle. CKs are synthesized throughout the plant, but the
geneexpressionofCsCCD8indifferentplanttissues.AM,apicalbud;
AB,axillarybud;AB24,axillarybud24hrafterdecapitation;R, origin ofCKs in bud regulationis still under debate [69].
adventitiousroot;VT,vasculartissuefromapicalmeristem;VT2, It has been shown that CKs produced in the roots are
vasculartissuefromsecondarybuds;SS,senescentstigma;OS,orange transported through the xylem [70,71] and exert their
stigma;L,leaf.Valuesaremeansofthreetechnicalreplicates±SE,
action on different tissues. Removal of roots in saffron
normalizedtotheinternalcontrolgene.
interrupts bud outgrowth and the development of new
Rubio-Moragaetal.BMCPlantBiology2014,14:171 Page10of15
http://www.biomedcentral.com/1471-2229/14/171
roots restart the sprouting process. Due to the involve- different from that found in xylem sap [81], suggesting
ment of CKs in outgrowth bud promotion (Additional that different SLs could be produced in different tissues
file 2: Figure S2), it is likely that CKs produced in the inwhich theyhavedifferentbiologicalfunctionalities.
rootsareresponsible for this process insaffron asshown Theexpression patternsofCsCCD7andCsCCD8were
inotherplant systems [72]. analysed in apical and in axillary buds at two different
SA levels have been shown to change between dor- developmental stages, quiescent and 24 h after elimin-
mant and waking saffron corms [73], suggesting its abil- ation of the main apical bud. For both genes expression
ity to break dormancy. Therefore, we determined the in apical buds was higher than in roots or leaves. How-
levels of SA in apical buds, axillary buds and in axillary ever, in axillary buds the expression patterns of both
buds 10 d after removal of the apical bud. However, we genes were clearly different. Although CsCCD8 showed
did not detect significant differences among the samples, the highest levels of expression in the quiescent axillary
suggesting that endogenous level of SA in buds is not buds, the expression levels of CsCCD7 were very low in
involvedinthe control ofparadormancy. this tissue. In Arabidopsis, expression of CCD8 has also
been reported to be relatively high in nodal tissue close
SLsinsaffroncormsandtherolesofCsCCD8and to the buds, while in rice, CCD7 was mainly found at
CsCCD7insproutingandvasculartissueformation the node of the stem where the axillary meristem initi-
Another major player in the control of shoot branching ates [54]. In fact, the CsCCD8 transcript levels in the
are the SLs [74], originally identified as germination axillary buds were rapidly down regulated by decapita-
stimulants for root parasitic plants [75]. SLs, together tion of the apical bud, although the expression of
with auxin, have been shown to have an inhibitory role CsCCD7 was up regulated, as has been observed in
on shoot branching [20]. The auxin transport auto- potato[82].PreviousdataonCCD7andCCD8expression
inhibition hypothesis [76] proposed that organs remain patterns in other plant systems revealed that decapitation
dormant because they are not able to export their own results in decreased expression of these genes in the
auxin into the stem polar auxin flow. Once axillary bud stem and in the axillary bud [14,15,32,51,83]. Even
dormancy is broken, bud outgrowth may depend on the though this was the case for CsCCD8,CsCCD7 showed
establishment of auxin transport from the bud via a the opposite behaviour. This result suggests that SL
process involving canalization, which is controlled by production related to bud dormancy is most probably
the strength of the polar auxin transport stream and or controlledatCsCCD8level.
may require an auxin-regulated second messenger [16]. Moreover, bud auxin export is also a prerequisite for
The SLs were proposed to act as regulators of auxin theformationof vascularconnectionstothestemvascu-
transport by reducing the expression and/or plasma lature in inhibited buds [16,84]. In the quiescent axillary
membranelocalizationofauxintransporters[77,78]. buds of saffron the vasculature is not well developed,
In the present work, the germination stimulatory ac- and the sprouting process is accompanied by the devel-
tivity of P. ramosa seeds of different extracts was tested, opment of this system. The leaf primordia of these
and the stimulatory activity, albeit low, was only de- quiescent axillary buds are not a source of auxins. How-
tected in the fractions from vascular tissue developed ever, once the bud start to grow, buds synthesize auxin,
from the apical bud indicating the presence of SLs in as observed in other plants [85], and its export may
this tissue. The absence of activity in the extracts from enhance vascular connections and nutrient flow to fur-
other tissues is most probably due to the presence of ther stimulate the growing bud. Interestingly, it is in the
SLs at extremely low levels. Grafting studies performed vasculature of the axillary buds where the CsCCD8
in several species showed that a wild-type rootstock expression levels were enhanced, compared with the
grafted to either accd7 or ccd8 mutant scion was able to vasculature of apical buds, although CsCCD7 levels
restore wild-type branching patterns, indicating that SL remained practically unchanged, but high. Recently [86],
was produced in the roots [31,79]. However, wild-type it has been provide evidence that SLs positively regulate
shoots on mutant roostocks also have near-wild-type cambial activity. The expression pattern of the studied
branching patterns [31,32,79]. In addition, wild-type epi- genes suggests that most probably SL or carlactone pro-
cotyl interstock grafts into rms1 and hypocotyl grafts duction in this vascular tissue is controlled at the level
into Arabidopsis max3 are also able to reduce branching of CsCCD8 but not CsCCD7. In agreement with this,
[80], indicating that biosynthesis of SLs is not limited to several reports point out to the involvement of CCD7 in
the root system. Further, the expression of CsCCD7 and the formation of other apocarotenoids [87,88]. Both
CsCCD8 in the vascular tissues connecting sprouting CCD4 and CCD7 are currently candidates to deliver C
27
buds with the mother corm, suggested that SLs are also intermediates for CCD1, which has been suggested to
synthesized in the stem vascular system in saffron. In act preferentially over apocarotenoids [89]. Consistent
addition, the SL profile found in tomato root exudates is with this additional role for CCD7, strongly elevated