Over-expression of microRNA171 affects phase transitions and floral meristem determinancy in barley.

1.9 MB·English

by  Curaba, Julien

Curabaetal.BMCPlantBiology2013,13:6
http://www.biomedcentral.com/1471-2229/13/6
RESEARCH ARTICLE Open Access
Over-expression of microRNA171 affects phase
transitions and floral meristem determinancy
in barley
Julien Curaba1, Mark Talbot1, Zhongyi Li1 and Chris Helliwell2*
Abstract
Background: The transitions from juvenile to adult and adult to reproductive phases of growth are important
stages inthelife cycle of plants. The regulators of thesetransitionsinclude miRNAs, in particular miR156 and
miR172 which are partof a regulatory module conserved across theangiosperms. InArabidopsis miR171 represses
differentiation of axillary meristems by repressing expression of SCARECROW-LIKE(SCL) transcription factors, however
therole of miR171 has not been examined inother plants.
Results: Toinvestigate theroles of mir171 and itstarget genes ina monocot, theHvu pri-miR171a was
over-expressed inbarley (Hordeum vulgare L.cv. Golden promise) leading to reduced expression of atleast one
HvSCL gene. The resulting transgenic plants displayed a pleiotropic phenotype which included branching defects,
anincreased number ofshort vegetativephytomersand late flowering. These phenotypesappear to be the
consequence of changes in theorganisation of theshoot meristem. Inaddition, thedata show that miR171
over-expression alters thevegetativeto reproductive phase transition byactivatingthemiR156 pathway and
repressing the expression oftheTRD (THIRDOUTERGLUME) and HvPLA1 (Plastochron1) genes.
Conclusions: Our data suggest that some ofthe roles of miR171 and its target genes that have been determined
inArabidopsis are conserved in barley and that theyhave additional functionsin barley including activation of the
miR156 pathway.
Keywords: Barley, miR171, Scarecrow-like, Phase change, Meristems, Flowering time
Background The miRNAs with the best-defined roles in regulating
During their life cycle, flowering plants undergo three phase changes in the shoot meristem are miR156 and
developmental phases, the juvenile and adult vegetative miR172 which form a regulatory module that is widely
stages and a reproductive phase. During the last decade conserved in plants. In Arabidopsis, rice and maize,
it has become clear that microRNAs (miRNAs) are im- miR156 regulates shoot branching, leaf initiation and
portant regulators of transitions between these phases. juvenile-to-adult phase transition through the down-
miRNAsaresmallregulatoryRNAmoleculeswhichtrig- regulation of several SPL genes [2-9]. In grass plants,
ger the post-transcriptional repression of target genes over-expression of miR156 promotes vegetative branch-
through a base-pairing mechanism [1]. There are some ing producing an increased number of tillers but inhibits
20 miRNA families that are highly conserved in flo- inflorescence branching resulting in a reduced number
wering plants. Many of these conserved miRNA fam- of spikelets [2-5,9]. Detailed analysis of miR156 function
ilies control crucial developmental processes through in the maize inflorescence has shown that it has a role
the down-regulation of conserved transcription factor- in establishing axillary meristem boundaries by spatially
encoding genes. restricting expression of the SPL gene Tasselsheath4
(TSH4) to the bract. This leads to the allocation of
*Correspondence:[email protected] more cells to the outgrowth of spikelet meristems at the
2Currentaddress:SchoolofAgricultureandFoodSystems,Universityof
expense of leaf initiation [3,10]. Recent studies have de-
Melbourne,Parkville,Victoria3010,Australia
Fulllistofauthorinformationisavailableattheendofthearticle monstrated cross talk between the miR156 and miR172
©2013Curabaetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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pathways, which appear to have conserved roles in co- orIV[32],suggestingthatthethreetargetshavepartially
ordinating the timing of vegetative phase changes and redundant function. SCL6-IV, which is expressed at the
competency to flower in all angiosperms [11]. During boundary between the axillary meristem and the SAM,
vegetative shoot development in Arabidopsis and maize, seems to have adifferentfunctiontothe two otherSCL6
miR156 expression gradually decreases and is inversely genes, being linked to branching but not SAM main-
correlated with that of miR172 [2,12,13]. Over-expres- tenance [30,32]. A more detailed analysis of SCL6-II/III
sionofmiR156leadstoadecreaseinmiR172abundance showed that these genes are expressed in the peripheral
in young shoots [2,13]. In Arabidopsis miR156 down- zone and vascular tissues of the SAM and promote the
regulates AtSPL9/10 which in turn directly activates the maintenance of the pool of meristematic cells and the
transcription of MIR172b [13]. The AtSPL3/4/5 genes differentiationoftheaxillarymeristem[28,30].
are regulated both transcriptionally and post-transcrip- Although studies in Arabidopsis have revealed import-
tionally by miR172 [14] and miR156, [15], respectively. ant roles for miR171 and its SCL targets, their roles in
In maize, miR172 is thought to repress the maintenance monocot plants are unknown. This study investigated
of juvenile traits and promote the onset of reproductive the role of the miR171-SCL module in controlling plant
development by down-regulating Glossy15 (GL15) [16]. architecture in barley (Hordeum vulgare L. cv Golden
miR172 expression reaches a maximum in reproductive promise). Over-expression of miR171 affects expression
shootsandpromotesfloralinitiationandflowerdevelop- of meristem identity genes suggesting a conservation of
ment by repressing expression of AP2-Like (AP2L) genes the role identified in Arabidopsis. In addition a delay in
[12,13,17-23]. In maize and barley, loss of miR172 func- the transition to reproductive growth involving miR156
tion promotes the formation of ectopic undifferentiated was observed which suggests that there are monocot
branches in the place of spikelet meristems (SMs) which specific functions formiR171anditstarget genes.
in barley eventually lead to the formation of a nascent
spike [12,24]. In barley miR172 has also been shown to Results and discussion
be responsible for the cleistogamy phenotype by repres- ConservedmolecularfunctionsofmiR171inbarley
sing an AP2-like gene, Cleistogamy1 (Cly1), in the lodi- InbarleytwomaturemiR171sequences(hvu-miR171a/b)
cules, preventing the outgrowth and opening of the have been identified [34] which differ by one central nu-
floret [25]. cleotide. The only miRNA precursor identified is that for
miR171 is a well conserved miRNA family known to hvu-miR171a(Additionalfile1).Thereareninerice,four-
regulate members of the SCARECROW-LIKE (SCL) teen maize and four Brachypodium miR171 family mem-
transcription factor family. The SCLs belong to a sub- bers in miRBase, indicating that barley is likely to have
class of the highly conserved GRAS family composed of additionalmiR171genes.
homologs of GAI, RGA and SCR [26]. Among other The abundance of miR171 was examined in various
functions, GRAS proteins are known to be involved in tissues and it was found to be predominantly expres-
GAresponses controlling flowering andregulating apical sed in reproductive tissues (Figure 1A). These data
meristem development [26,27]. In Arabidopsis, there are correlatewithpreviousobservationsinArabidopsiswhere
three MIR171 genes (a, b and c) which are predicted to miR171 mostly accumulates in reproductive organs
regulate three SCL6 genes (SCLII, III, IV, also known as [29,31,33]. The psRNAtarget server (http://plantgrn.
HAIRY MERISTEM (HAM) and LOST MERISTEMS noble.org/psRNATarget) was used to search for poten-
(LOM), [28-30]. The expression domains of the miR171 tial targets of hvu-miR171a and b amongst a recently
family members and the SCL6-II/III mRNAs overlap, assembled full-length cDNA dataset from barley [35].
suggesting a redundant function for both miRNA and This analysis identified 11 potential target ESTs pre-
target mRNAs [30-32]. miR171a is most highly expres- dicted to be miR171-regulated by cleavage or trans-
sed in the inflorescence where it regulates SCL6-III and lational repression using a mismatch score of 4.0 or
SCL6-IV expression through mRNA cleavage [29,33]. less (Additional file 2). Of the target mRNAs with the
Arabidopsis plants over-expressing miR171c (OE171c) lowest mismatch scores (1.5 or less), three (AK368048,
and the triple scl6 mutant show similar pleiotropic phe- AK371946andAK364580)encodeSCL6-likeproteinsand
notypes, including altered shoot branching, plant height, are all predicted to be regulated by miR171 cleavage. The
chlorophyll accumulation, primary root elongation, flo- fourth potential miR171 target (AK362896) encodes a
wer structure, leaf shape and patterning, indicating that protein of unknown function and is predicted to be regu-
miR171 acts mainly by down-regulating SCL6 genes to lated by translational repression.We searched degradome
control a wide range of developmental processes during data generated from developing barley seed [36] for evi-
shoot development [32]. The reduced branching pheno- dence of cleavage of these four mRNAs and found evi-
type observed in OE171c plants can be rescued by the dence for cleavageofthe three SCL6-like mRNAs but not
expression of a miR171c-resistant version of SCL6-II, III, AK362896 (Additional file 3: AK364580 and AK368048
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phenotypes and expression of the mature miR171. T
1
A
OE171 plants were selected based on the presence of the
miR171 transgene and grown under long day (LD) and short
EtBr day (SD) conditions with 16 and 10 hour photoperiods,
respectively.
w) w) w) m) m) m) m) Under LD conditions the first wild-type (WT) stems
Shoot (1 Basal node (1 Stem (1 Young leaf (1 Sheath leaf (2 Green spike (2 Ovary (2 sDttohitfnoPegpOGspaEae(m1Ddj7uea1vpyeraspnoglPdiealueonc-scttotsionnG-gstatiedanlrreuumtaleetviddnetsarttatooianonspendixrt)oitosewdtnnuahdrcedtereeaedlfnaatseyetr.woOT7eEl5hel1oae7nDv1iegnPsaptG,teleia,rnnnawdottishdc3ieaal0es-t
WT plants were already flowering (Figure 3A, Additional
B
file 4). OE171 plants were extremely late flowering
L 1000 with the first spike starting to flower after 140 DPG
C
S (Figure 3B). At this stage the stems were fully elongated
Hvon 100 and OE171 plants were dwarfed compared to WTat the
Relative expressi 101 swpaaamcsteidousnteaogtfoetorhefedduelceveaedvleoispnmtweerrnnaptopd(Feedigleuanrrgeotuh3n,Adr,eBstuh).eltTinihngefliondrweasaccrefoinsmcme-
w) w) w) m) m) m) m)
1 1 1 1 2 2 2 (Figure3E, H, I). OE171 produced on average one or two
Shoot ( Basal node ( Stem ( Young leaf ( Sheath leaf ( Green spike ( Ovary ( aledndAgitthiontaolwavrisdisblethenotdoeps,owf itthheastedmecre(Fasiginugrein3tIe).rnTohdee
Figure1Expressionprofileofhvu-miR171andHvSCL.(A) 500
Northernblotusingahvu-miR171aantisenseprobe.Thetissues
wereharvestedfromdifferentstagesofdevelopmentandtheagein 1a 400
weeks(w)ormonths(m)isindicatedinbrackets.(B)RT-qPCR 17
R
showingtherelativeabundanceofHvSCLmRNAinthesametissues mi 300
ausserdefeforernthcee.northernblotin(A),usingthelevelinthesheathleaf e pri-sion 200
vs
areidenticalatthemiR171cleavagesitesocannotbedis- RelatiExpre 100
tinguished in the degradome data). The degradome data
0
for AK371946 had the greatest number of reads 2 bp up-
0 WT 1 3 5 6 7 9 10
stream of the predicted cleavage site however RLM-5
RACE showed that the predicted miR171 cleavage site OE171
wasthemostabundant(Additionalfile3).
B OE171
Mis-expressionofmiR171inbarleyleadstopleiotropic WT 1 3 5 6 7 9 10
phenotypes
miR171
To study the role of miR171 and its targets in barley,
0 1.0 1.7 0.9 1.0 1.0 0.6 0.9
pri-miR171a was over-expressed under control of the
miR156
maize ubiquitin promoter. Seven independent trans- 1.0 1.3 1.8 0.7 1.3 1.0 1.1 1.1
genic lines were obtained with at least one hundred fold miR172
over-expression of pri-miR171a compared to wild-type 1.0 0.2 0.1 0 0 0 0 0
miR168
(Figure 2A). A corresponding increase in the abundance
1.0 0.6 0.7 0.6 0.6 0.6 0.5 0.6
of mature miR171 was also detected in all the transgenic EtBr
lines (Figure 2B). The T miR171 over-expressing plants
o Figure2Expressionofpri-miR171aandmiR171inOE171
(OE171) showed a pleiotropic phenotype with altered transgeniclines.(A)RT-qPCRshowingtheabundanceofpri-
shoot architecture and delayed flowering. For lines where miR171amRNAinT leavesofOE171lines(numbered)relativeto
0
we were able to obtain seed, these phenotypes were WT(setas1.0).(B)Northernblotshowingthelevelofdifferent
maturemiRNAsequencesinleavesofT OE171linesandWT.
confirmed in the T generation. In our subsequent ana- 0
1 miR168wasusedasloadingcontrol.
lyses, T1 lines 1, 6 and 9 were used; these had similar
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A LD (75) B LD (140) E F G I OE
LD
WT (100)
LD
(60)
6 7
6
WT OE 5
5 4
WT OE OE H
3
C SD (75) D SD (75) 4 2
1
3
2
WT OE
WT OE OE LD (50) LD (100) 1
J
25
Short Day Long Day
s
er 20
Till
of 15
er
b
m
u 10
N
5
0
WT 1 5 6 7 9 WT 5 6 9
OE171 OE171
Figure3OE171affectsbarleyshootarchitecture.T OE171plants(OE)andWTgrownunderlongday(LD)orshortday(SD)conditions
1
(A-E).TheplantageisindicatedinDaysPostGermination(DPG).(F-G)Dryspikes.(I)WTandOE171culmswithouttheleavesshowingthe
nodesnumberedfromthecrown.(J)Numberofemergingtillerson2month-oldplantsgrownunderSDandLD.2to4plantswereobserved
foreachtransgenicline.
transgenic plants eventually developed short and partially spikelet meristems into floral organs was delayed, but
sterile spikes (Figure 3F, G). A similar phenotype was ob- their morphology and phyllotaxy on the inflorescence
served under SD conditions where OE171 showed an meristem appeared normal (Figure 4B to E). At the base
increasednumberofphytomers,reducedinternodelength of each OE171 inflorescence one to at least four indeter-
(Figure 3C, D) and a delay in the juvenile-to-adult transi- minate meristems which formed new inflorescence mer-
tion compared to the WTgrown under the same condi- istems were observed (Figure 4B, C, D). These ectopic
tions (Additional file 4). In addition, OE171 plants grown inflorescence meristems were sometimes preceded by
under SD produced fewer tillers (on average 4 tillers the formation of short aerial tillers producing additional
compared to 12) than the WT plants (Figure 3C, D, J, leaves in a condensed structure at the top of the culm
Additionalfile5)andneverdevelopedanyfertilespikes. (Figure 4F, G). The formation of a new branch appeared
A closer examination of the shoot apex of OE171 to be at the expense of the adjoining inflorescences
plants grown under LD showed an accumulation of phy- which stopped developing and dried out (Figure 4G). At
tomers with a progressive reduction of bending at the the base of the WT spike at flowering stage, the floral
internode. These eventually form a compact structure of structure on the first rachilla phytomer was restricted to
joined nodes with a series ofleavesthat wraparoundthe a bract (instead of a lemma-awn) and a pair of glumes
inflorescence (Figure 4A). The main inflorescences, as (Figure 4H). In comparison, OE171 spikes at the same
well as the resulting spikes (Figure 3G), were shorter developmental stage possess several phytomers with
with a reduced number of spikelet meristems compared intermediate culm/rachis characteristics at the base of
to the wild-type (Figure 4D, E). The development of the the inflorescence (Figure 4I, J). Internode lengths were
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A B OE C OE D OE E WT
eIM
eIM
eIM
OE
F G H I J
4
lem
3 glu
glu eIM
2
bra AT eIM
1 col
OE OE WT OE OE
K L M N O
WT WT OE OE OE
P eIM Q R S OE T OE
eIM
eIM
lateral
spike
glu
eIM
bra
OE OE WT
Figure4OE171affectsfloralmeristemdifferentiationandpollendevelopment.T1plantsofOE171(OE)andWTgrownunderLD
(A-Q)andSD(R-T)conditions.(A)OE171Shootwithleavesmanuallyopened.(B-C)YoungOE171inflorescenceat100DPGshowingectopic
inflorescencemeristems(eIM).(D-E)ScanningelectronmicroscopyofOE171andWTinflorescencesat100DPGand50DPGrespectively.
(F)CondensedleafstructureontheapicalshootofanOE171plant.(G)Aerialtillersproducingmultipleinflorescencemeristems(numbered1to
4)revealedafterremovingtheleavesfrom(F).(H-J)Baseofgreenspikesatfloweringtimeshowingthefirstrachisphytomers;glumes(glu),bract
(bra),collar(col),lemma-awn(lem),aerialtiller(AT)andectopicinflorescencemeristems.(K)WTfloret,(L)WTanthersandovary,(M)OE171
floret,(N-O)OE171anthersandovary.(P)EctopicinflorescencemeristemsemergingfromthetopofaOE171maturespike.(Q)OE171
inflorescencedriedoutat150DPG.(R)BaseofaWTgreenspikesgrownunderSDshowinganectopiclateralspike.(S-T)Younginflorescenceof
OE171at120DPGand150DPG(T).Scalebarsrepresent1mm.
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intermediate between those of culm and rachis phyto- plants grown under short day conditions to quantify the
mers and indeterminate inflorescence meristems emer- abundanceofmiR171anditstargets.Alltransgeniclines
ged from the axillary buds instead of spikelet meristems showed an over-accumulation of the pri-miR171a tran-
(Figure 4I). These secondary inflorescence meristems scripts and mature miR171 sequences (Figure 5A, B).
eventually produced lemma-awn structures but never We were unable to detect AK364580, AK362896 and
grew and formed a fully developed floret (Figure 4J). AK368048 in meristem tissues by RT-qPCR (data not
Meanwhile, the main inflorescence produced several shown). AK371946 was expressed in wildtype and sho-
WT-like florets in the middle of the spike but only a wed reduced levels in OE171 plants (Figure 5A) sugges-
fewwerefertile(Figure4K,M).OE171anthersweresmal- ting that this was the main target down-regulated by
lerandtranslucentincomparisontotheWT,suggestinga miR171 over-expression in the shoot apex. However it is
pollen development defect that could explain the partial possible that other target genes with higher mismatch
sterility phenotype (Figure 4L, N, O, 3G). In addition, the
terminalbranchstructureofthespikesometimesgaverise
A
to an ectopic inflorescence meristem (Figure 4P). In the
1000
strongestlines,thedevelopmentaldelaywassuchthatthe
OE171-6 OE171-9WT OE171
spike did not form any floral organs and dried out T
W
(Figure 4Q). The OE171-3 T0 plant had the highest o 100
miR171expressionandneverflowered.Itdevelopedtillers e t
v
wloittahxyandeefxetcrteomfetlhyedsipsoikreglaentimzeedriisntfelmorseswchenicchenweivtehratrpahnysli-- elati 10
tioned to floral meristems (Additional file 5). Under SD ge r
n
conditions WT plants sometimes developed one ectopic ha 1
inflorescence meristem at the base of the primary inflo- A c
rescence which eventually formed a fertile lateral spike N
R
(Figure 4R). In comparison, the inflorescences of OE171 m 0.1
d
plants developed several ectopic inflorescence meristems, ol
F
asobservedinlongdayconditions(Figure4S),butthede-
0.01
vbeeTlfoohpreemsreeenaotcbahsliendrgvelatathyioewnflsaoswsusegtrrgionengsgtsettrhagaaetnb(dFaitrghlueeyresmp4iTikRe)1.s7d1riceadnorue-t miR171a HvSCL HvSPL HvAP2L HvPLA1 TRD HvWUS HvKN1
gulate axillary meristem development and the timing of pri-
the juvenile to adult phase transtition. In Arabidopsis,
B
miR171 acts mainly through the down-regulation of the
OE171
SCL6-II/III/IV genes; over-expression of miR171 or loss
of SCL6 function represses axillary meristem differenti- WT WT 6 9 1
ation resulting in reduced shoot branching [30,32], and SD LD LD LD LD
eventually a complete developmental arrest under SD
miR171
conditions [30]. This phenotype correlates with the ob-
servations here and suggests that miR171 regulates shoot
branching through a conserved mechanism between miR156
monocots and dicots. However, in contrast to the OE171
phenotype inbarley, OE171 inArabidopsis alsopromotes miR172
stemelongation[32],repressesleafinitiation[30]andonly
slightly delays flowering [28], suggesting that the roles of EtBr-sRNAs
miR171 and its targets evolved differently for these pro-
cessesinbarleyandArabidopsis. Figure5Hvu-miR171-SCLmolecularpathway.(A)RT-qPCR
showingthemRNAabundanceofdifferentgenesofinterestin
younginflorescenceoftwoindependentOE171linesrelativeto
Mis-regulationofgeneexpressioninOE171barleyplants
WTatsimilardevelopmentalstage(asshownbytheSAMpictures
In both barley and Arabidopsis, OE171 affects shoot
onthetoprightcorner;scalebars=1mm),grownunderLD.
branching [32]. In Arabidopsis, the miR171 family acts (B)NorthernblotshowingthelevelofdifferentmaturemiRNA
mainlythroughthedown-regulationofthreeSCL6genes sequencesinyounginflorescenceofthreeindependentOE171lines
(SCL6-II/III/IV) [32]. As OE171 disrupts meristem func- growninLDandWTplantsgrowninSDandLD.Ethidiumbromide
stainingofthesmallRNAswasusedasloadingcontrol(SAM,shoot
tion we extracted RNA from young inflorescence me-
apicalmeristem;AM,axillarymeristem).
ristem tissues of T OE171 transgenic lines and WT
1
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scores are affected in OE171 plants and that the other phenotypic similarities suggest that miR171 is linked to
SCL6 genes are targeted by miR171 outside of the meri- themiR156-miR172pathwayinbarley.
stem tissues that we have focused on. For simplicity we TheeffectofOE171onmiR156andmiR172expression
refertoAK371946asHvSCLfrom here onwards. was examined using inflorescence tissue from T OE171
1
We determined the tissue specificity of HvSCL expres- and WT plants grown under LD conditions (Figure 5B).
sion by RT-qPCR (Figure 1B). HvSCL is expressed in the miR156 abundance was higher in OE171 lines but no
shoot, basal node, green spike and ovary. The strong ex- change in miR172 expression was detected. Using the
pression of HvSCL in green spike and ovary overlaps same tissues, we quantified the relative abundance of two
with thehighestmiR171abundance, at leastatthe tissue barley mRNAs, coding for an SPL gene predicted to be
level, suggesting that miR171 acts to dampen HvSCL ex- targeted by miR156, HvSPL (U21_18637) and a potential
pression rather than restrict it [37]. These data correlate target of miR172, HvAP2L (U21_18652) (Figure 5A). As
with previous observations in Arabidopsis where both expected, HvSPL was down-regulated in OE171, rein-
miR171 and SCL target mRNAs are predominantly de- forcing the hypothesis that miR171 acts at least par-
tected inthesame tissues [33]. tially through the up-regulation of miR156. Surprisingly,
RNA in situ hybridization of Arabidopsis shoot meri- HvAP2L was up-regulated in OE171, suggesting that
stem tissues shows that SCL6-II/III are mostly expressed miR171regulatesHvAP2L expression independently from
in the peripheral zone and the vascular strands of devel- miR172. This last observation correlates with previous
oping axillary buds where they promote cell differenti- work in Arabidopsis reporting that TOE3 (an AP2-like
ation and help maintain the polar organization of the gene containing a miR172 binding site) expression grad-
SAM [30]. The maintenance of the SAM requires the uallyincreasesalongwithmiR171abundanceinthedeve-
expression of WUSCHEL (WUS) and the knotted-like loping shoot [20]. Additionally, AP2 was recently shown
geneSHOOTMERISTEMLESS (STM)topreventthedif- to directly activate MIR156e expression in Arabidopsis
ferentiation of the meristematic cells located in the cen- [38], suggesting that the up-regulation of HvAP2L in
tral zone. In the double mutant scl6-II scl6-III grown OE171 could be responsible for the increasing abun-
under short day conditions, the spatial expression pat- dance of miR156.
terns of WUS and STM are altered [30]. A WUS-related The increased accumulation of miR156 in OE171 did
homeobox gene, HvWUS (U21_34817), and a class I not cause a reduction of miR172 abundance in contrast
Knotted-like gene, HvKN1 (TC279468) were identified in with previous observations in Arabidopsis and maize
barley EST databases. Quantification of their relative ex- [2,13]. However, observations by Jung et al. [14] showed
pressionintheinflorescencesofOE171plantsshowsare- that OE156 driven by an inducible promoter in Arabi-
duction for both genes compared to the WT (Figure 5A). dopsis did not noticeably affect miR172 abundance. The
This effect may reflect a disorganization of the SAM and authors proposed that miR156 principally affects miR172
loss of maintenance of meristem cells, which correlates during the early vegetative stage by repressing MIR172b
with miR171 function in Arabidopsis and could explain expression and that the increasing miR172 abundance
the reduction of shoot branching observed in barley duringthelate vegetativestageisdrivenbyotherMIR172
OE171. genesindependentofmiR156.Thiscorrelateswiththeob-
The OE171 phenotype resembles that of OE156 in servation that miR172 abundance continues to increase
maize [2,12]. In maize, miR156 controls plant architec- duringlater vegetativestageswhilethemiR156levelstays
ture by regulating at least 2 SPL genes, Teosinte Glume at a constant minimum level [13,14]. In agreement with
Architecture1 (TGA1) and Tasselsheath4 (TSH4) [10,12]. this hypothesis, a decrease in miR172 expression was de-
The dominant Corngrass1 (Cg1) mutant, in which zma- tectedintheyoungleavesofOE171T plantswhichover-
0
miR156b/c is over-expressed, shows an extended vegeta- accumulatedmiR156(Figure2B).MaizeOE156plantsdid
tive program characterized by an increased number of not show the same floweringdefect as for loss of miR172
leaves and the development of large bract leaves in the function [2,12], suggesting that the function of miR172 in
inflorescences [2]. Over-expression of zma-miR156b/c theinflorescenceisindependentofmiR156.
also causes the reduction of zma-miR172 abundance The phenotype of the barley OE171 plants was also
whichisknowntocontroljuvenile-to-adultphasetransi- reminiscent of mutants in maize, rice and barley genes
tion through the down-regulation of Glossy15 (GL15) that affect the rate of leaf initiation. We did not observe
[16]andspikelet meristemdeterminancythroughthere- any clear changes in the leaf initiation rate in OE171
pression of IDS1 and SID1 [12,19]. In barley, the pheno- (Additional file 6) but similarities in other phenotypes
type associated with loss of miR172 function is similar ledustoexaminethesegenesinOE171plants.InArabi-
to the OE171 plants, blocking spikelet meristem deter- dopsis the leaf initiation rate is likely to be controlled
minacy and exhibiting ectopic branch structures from by two independent pathways, one involving miR156-
thebaseandtheendofthespike[24](Figure 4A).These SPLs and the other involving two orthologs of the rice
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PLASTOCHRON1(PLA1)genewhichencodecytochrome may be monocotyledon specific. Thirdly, miR171 promo-
P450 enzymes [39,40]. In rice, loss of PLA1 function cau- tes vegetative traits in barley through a secondary path-
ses an extension of the vegetative program characterized way, independent from miR156 [40], that involves TRD
by an increased number of short vegetative phytomers and HvPLA1 [42,43]. These apparent additional roles for
and the production of vegetative shoots (with the emer- miR171 and its targets in barley shoot development may
gence of new leaves) instead of primary rachis branches represent an important evolutionary difference between
[41], a phenotype that closely resembles OE171 in barley. monocotanddicotplants.
More recently it has been shown that PLA1 expression is
under control of the transcription factor NECK LEAF 1 Methods
(NL1),whichwhenmutatedcausesasimilarphenotypeto Vectorconstruction
thatofthepla1mutant[42].NL1isorthologoustoThird The sequence corresponding to pri-miR171a was ampli-
Outer Glume (TRD) in barley and Tasselsheath-1 (TSH-1) fied using the primers MIR171-1F/2R (Additional file 7)
in maize. In both trd and tsh-1 mutants the repression of and cloned into the pENTR/D topo vector (Invitrogen).
bract growth is suppressed, producing leaves that sheath The construct was then recombined (according to the
theinflorescenceandareductioninthenumberofinflor- manufacturer’s instructions) into the Agrobacterium bin-
escence branches [43]. Therefore the regulation of TRD ary vector pWBVec8 [46] modified to contain a Gateway
andPLA1bymiR171inbarleywasinvestigated.TRDwas cassette down-stream of the Ubiquitin promoter. The
previously cloned using degenerate PCR (GU722206, [43] construct was then introduced into the Agrobacterium
andonebarleyESThomologtoPLA1wasfound,HvPLA1 strain AGL1 which was used to transform Golden Pro-
(U21_15786). The mRNA abundance for both genes was mise barleyplants.
significantlyreduced inthe inflorescences ofOE171com-
pared to the WT (Figure 5A). These results suggest that Planttransformationandgrowingconditions
miR171 coordinates vegetative phase changes in barley The transformation was performed as described [47]. In
through the regulation of two potentially distinct path- brief, barley plants were grown at 12°C / 16h light and
ways. It is proposed that miR171 acts up-stream of immature grains were harvested at about DPA20 (when
miR156andTRDwhichinturnmayregulateHvPLA1ex- the embryo turned from translucent to white and the
pressionaswasobservedfortheirorthologsinrice[44]. scutellum is about 2mm in diameter). After sterilization
ofthegrains,theembryoswereisolated andtheaxis was
Conclusion removed. Dissected scutella(approximately150)wereput
Overall, the data presented here suggest roles for miR171 in contact with a fresh culture of AGL1-MIR171a for
and its targets in regulating shoot development in barley. 3 days on antibiotic-free media. Transformed calli were
Over-expressionofmiR171resultsinanextendedvegeta- selected on hygromycin containing media. The resulting
tivephase characterised by anincreasednumberof leaves T plantsweretransferredintosoilandgrowninnaturally
0
and the initiation of indeterminate axillary meristems in- lit phytotron glasshouses. T plants were selected for the
1
stead of spikelet meristems. Additionally, OE171 plants presenceofthetransgeneandgrownwithairtemperature
have a reduced number of tillers emerging from the axil- set at 17°C/9°C day/night cycle under long day (LD) and
lary meristems of the crown (under SD conditions) and a short day (SD) conditions with 16 and 10 hours of day
delayinthedifferentiationofspikeletmeristemsintofloral light,respectively.
organs. A model is proposed in which miR171 is an up-
stream regulator that coordinates the timing of shoot de- QuantitativeRT-PCR
velopmentinbarleythroughthree independent pathways. RT-PCR reactions were performed as described [21]. In
First, the results, together with current knowledge from brief, total RNA was extracted using TRIzol reagent
Arabidopsis, suggest that miR171 affects meristem main- (Invitrogen) and 4μg was used for first-strand cDNA
tenance and axillary meristem differentiation in barley synthesis using Oligo(dT) primers and the Super-Script
through the down-regulation of HvSCL [28,30], and con- III RT kit (Invitrogen). PCRs were performed according
sequentlyaffectstheexpressionofmeristemspecificgenes to the manufacturer on an ABI 7900 HT Fast Real-Time
such as the homologs of WUS and KN1 analysed in this PCR System (Applied Biosystems). 1μl of 1:10 diluted
study. Secondly, miR171 could repress vegetative phase template cDNA was used in a 10μl reaction. The ampli-
transitions in barley through the upregulation of miR156, fication program was: 15″ at 95°C, (15″ at 95°C, 30″ at
a known regulator of the transition from juvenile to adult 60°C, 30″ at 72°C) for 35 cycles, followed by a thermal
phases across the angiosperms [2,6-9,13,45]. Interestingly, denaturing step. Relative transcript levels were calcu-
OE171 and OE156 in Arabidopsis show opposite effects lated with the ΔΔCt method (Applied Biosystems) using
on leaf initiation [30,32,40], suggesting that the possible the ACTIN gene as a reference. Each value presented
connection between the miR171 and miR156 pathways in the histograms represents an average from three
Curabaetal.BMCPlantBiology2013,13:6 Page9of10
http://www.biomedcentral.com/1471-2229/13/6
independent replicates plus/minus Standard Deviation.
Additionalfile4:Timingofthetransitionphasesduringshoot
Forward and reverse primer sequences are presented in development.Thediagramshowstheapproximatetimingofthe
Additionalfile7. transitionsfromjuveniletoadultphasesandadulttoreproductive
phases.Thefirsttransitionwasdeterminedbythemomentwhenthe
firststemoftheplantstartedtoelongate(jointing)andthesecond
RNAgelblotanalysis transition(flowering)whenthefirstspikereachedanthesis.UnderourSD
condition,OE171plantsdidnotflower.
40 μg of total RNA, extracted using TRIzol reagent
Additionalfile5:DevelopmentalarrestofOE171-3(T0).(A)Tillerof
(Invitrogen) and separated on a denaturing 15% polya-
theT plantOE171-3.ScanningelectronmicroscopyoftheSAMofa
0
cryamide gel containing 7M urea at 120V for 2hr. RNA OE171-3(B)andWT(C)plants.Ectopicinflorescencemeristem(eIM).
loading was based on spectroscopic measurement and Scalebarsrepresent1mm.
confirmed by ethidium bromide staining of the gel prior Additionalfile6:ComparisonofvegetativeWTandOE171plants.
RepresentativeWTandOE171plantsat4weeksoldinLD
to transfer. RNA was electrophoretically transferred to
conditions.
Zeta-probe GT membrane (BioRad) at 40V for 90 min
Additionalfile7:PrimerandProbesequencesusedinthisstudy.
and fixed by UVcrosslinking. The membrane was first in-
cubated in hybridization buffer [Na2PO4-pH7.2 125 mM,
Abbreviations
NaCl 250mM, SDS 7%, formamide 50%] for 4h at 42°C SCL:Scarecrow-like;miRNA:microRNA.
and then incubated in the presence of 32P-end-labeled
Competinginterests
oligonucleotide probes at 42°C overnight. The membrane
Theauthorsdeclarenocompetingfinancialinterests.
was washed in [2× SSC, 0.2% SDS] at 42°C and the radio-
activity was detected by phosphorimager. Oligonucleotide Authors’contributions
probesequencesarepresentedinAdditionalfile7. JCdesignedthestudy,carriedoutexperiments,analyseddataanddrafted
themanuscript.MTcarriedoutexperiments,ZLhelpeddesignthestudyand
draftthemanuscript.CHdesignedthestudycarriedoutexperiments,
RLM-50RACE analyseddataandhelpeddraftthemanuscript.Allauthorsreadand
approvedthefinalmanuscript.
0
RNAligase-mediated5 rapidamplificationofcDNAends
0
(RLM 5-RACE) was performed using the GeneRacer kit Acknowledgements
(Invitrogen). The manufacturer’s protocol was followed WewouldliketothankSueAllen,AnnaWielopolskaandKerrieRammfor
0 0 theirexcellenttechnicalassistanceandDrsTonyMillarandScottBodenfor
for 5 end analysis with exception of the 5 de-capping theircriticalreadingofthemanuscript.
step. In brief, total RNA was isolated from 2 week-old
0 Authordetails
shoot tissues and ligated to a 5 end RNA adaptor before
1CSIROPlantIndustry,GPOBox1600,CanberraACT2601,Australia.2Current
being reverse transcribed using an oligo(dT) primer. The address:SchoolofAgricultureandFoodSystems,UniversityofMelbourne,
PCRreactionswereperformedusinggenespecificreverse Parkville,Victoria3010,Australia.
primers (Additional file 7). The amplicons were gel pu-
Received:12November2012Accepted:2January2013
rified, cloned into the pGEM-T vector (Promega) and Published:7January2013
sequenced.
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