Group Title: BMC Plant Biology
Title: Dominance induction of fruitlet shedding in Malus x— domestica (L. Borkh): molecular changes associated with polar auxin transport
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Title: Dominance induction of fruitlet shedding in Malus x— domestica (L. Borkh): molecular changes associated with polar auxin transport
Physical Description: Book
Language: English
Creator: Dal Cin, Valeriano
Velasco, Riccardo
Ramina, Angelo
Publisher: BMC Plant Biology
Publication Date: 2009
Abstract: BACKGROUND:Apple fruitlet abscission is induced by dominance, a process in which hormones such as auxin, cytokinins and strigolactone play a pivotal role. The response to these hormones is controlled by transcription regulators such as Aux/IAA and ARR, whereas auxin transport is controlled by influx and efflux carriers.RESULTS:Seven partial clones encoding auxin efflux carriers (MdPIN1_A, MdPIN1_B, MdPIN10_A, MdPIN10_B, MdPIN4, MdPIN7_A and MdPIN7_B), three encoding auxin influx carriers (MdLAX1, MdLAX2 and MdLAX3) and three encoding type A ARR cytokinin response regulators (MdARR3, MdARR4 and MdARR6) were isolated by the use of degenerate primers. The organization of the PIN multigene family in apple is closer to Medicago truncatula than to Arabidopsis thaliana. The genes are differentially expressed in diverse plant organs and at different developmental stages. MdPIN1 and MdPIN7 are largely more expressed than MdPIN10 and MdPIN4. During abscission, the transcription of these genes increased in the cortex whereas in the seed a sharp fall was observed. The expression of these genes was found to be at least partially controlled by ethylene and auxin.CONCLUSION:The ethylene burst preceding abscission of fruitlets may be responsible for the decrease in transcript level of MDPIN1, MDARR5 and MDIAA3 in seed. This situation modulates the status of the fruitlet and its fate by hampering the PAT from the seeds down through the abscission zone (AZ) and this brings about the shedding of the fruitlet.
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BMC Plant Biology

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Research article

Dominance induction of fruitlet shedding in Malus x domestic
(L. Borkh): molecular changes associated with polar auxin transport
Valeriano Dal Cin*1,3, Riccardo Velasco2 and Angelo Raminal

Address: 'Department of Environmental Agronomy and Crop Science, University of Padova, Viale dell'Universita 16, 35020 Legnaro (Padova),
Italy, 2Experimental Institute for Agriculture, via Mach 2 San Michele all'Adige, 38010 Trento, Italy and 3Horticultural Sciences, University of
Florida, Gainesville, PO Box 116090, USA
Email: Valeriano Dal Cin*; Riccardo Velasco; Angelo Ramina
* Corresponding author

Published: 26 November 2009 Received: 17 June 2009
BMC Plant Biology 2009, 9:139 doi:10.1186/1471-2229-9-139 Accepted: 26 November 2009
This article is available from:
2009 Dal Cin et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.ore/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Apple fruitlet abscission is induced by dominance, a process in which hormones
such as auxin, cytokinins and strigolactone play a pivotal role. The response to these hormones is
controlled by transcription regulators such as Aux/IAA and ARR, whereas auxin transport is
controlled by influx and efflux carriers.
Results: Seven partial clones encoding auxin efflux carriers (MdPINI_A, MdPINI_B, MdPINIO_A,
MdPINIO_B, MdPIN4, MdPIN7_A and MdPIN7_B), three encoding auxin influx carriers (MdLAXI,
MdLAX2 and MdLAX3) and three encoding type A ARR cytokinin response regulators (MdARR3,
MdARR4 and MdARR6) were isolated by the use of degenerate primers. The organization of the PIN
multigene family in apple is closer to Medicago truncatula than to Arabidopsis thaliana. The genes are
differentially expressed in diverse plant organs and at different developmental stages. MdPINI and
MdPIN7 are largely more expressed than MdPINIO and MdPIN4. During abscission, the
transcription of these genes increased in the cortex whereas in the seed a sharp fall was observed.
The expression of these genes was found to be at least partially controlled by ethylene and auxin.
Conclusion: The ethylene burst preceding abscission of fruitlets may be responsible for the
decrease in transcript level of MDPINI, MDARR5 and MDIAA3 in seed. This situation modulates the
status of the fruitlet and its fate by hampering the PAT from the seeds down through the abscission
zone (AZ) and this brings about the shedding of the fruitlet.

Abscission is a coordinated process tightly regulated by
the interplay of several factors, among which auxin and
ethylene play a pivotal role [1,2]. Leaf deblading and eth-
ylene application lead to premature abscission of the
organ due to the disruption of the auxin flux and activa-
tion of the abscission zone (AZ) at the base of the petiole

In the commonly accepted model, as demonstrated in the
Arabidopsis etrl-1, ethylene coordinates abscission. In
this mutant, flower abscission is significantly delayed
because the ethylene receptor (ETR1) is hampered in eth-
ylene binding activity, leading to partial ethylene insensi-
tivity. Although ethylene accelerates abscission, it is
strictly not necessary for shedding, indicating that a very
complex interplay of events control the process [5-7].

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Indeed, it has been shown that ethylene-dependent and -
independent pathways converge in determining flower
abscission [8].

It has also been postulated that prevention of abscission
requires a continuous and constant auxin transport
through the AZ [1]. Besides preventing abscission, auxin
regulates a tremendous number of processes, for instance
root meristem activity, organogenesis, and vascular tissue
differentiation [9-11]. Only recently the outstanding com-
plex mode of action of auxin has been partially unraveled
[12]. The most common auxin in plant, indol-3-acetic
acid (IAA), binds and is perceived by TIR1, an F-box pro-
tein [ 13]. TIR1 interacts in the SCF complex to bring about
the degradation of Aux/IAA transcriptional regulators
[14]. These proteins are active repressors of auxin respon-
sive genes and are encoded by a large multigene family
[15]. Auxin applications enhance the transcript amount of
most of the Aux/IAAs in several species [16-19]. Another
enthralling field concerns auxin transport [20], which can
be classified as either polar (PAT) or non polar. However,
the PAT is acquiring ever-growing interest and may be the
most important means of auxin relocation [21]. IAA is
taken up into the cell by a combination of lipophilic dif-
fusion, symport via AUX and LAX (LIKE-AUXi) per-
meases, and ATP-dependent transport by a P-glycoprotein
[22-25]. Auxin export is mediated by PIN-FORMED (PIN)
facilitators and by ATP activated PGPs (Phosphoglycopro-
teins) [26-30]. PINs and PGPs were shown to characterize
coordinated and independent auxin transport mecha-
nisms, and function interactively in a tissue-specific man-
ner [31]. Nevertheless, the function of the PGPs is non-
specific and mainly applies to auxin excess [32]. As a mat-
ter of fact, it is the asymmetric cellular localization of PIN
proteins that determines the direction of the auxin flow
[20]. Although different PINs are implicated in specific
developmental processes, there seems to be redundancy
as indicated by the ectopic expression of PIN proteins in
some mutant combinations [20,33,34].

The modes of action of auxin and ethylene elucidated in
A. thaliana have been extended to other model species
such as tomato [35,36]. Yet, little is known about the
interactions between these two hormones during abscis-
sion induction of organs other than debladed leaves or
senescing flowers. In particular, the apple cluster during
the immature fruit drop represents an ideal system to
study the shedding of actively growing organs [37]. At this
developmental stage, the shedding process involves
almost exclusively lateral fruitlets in which abscission is
preceded by an increase in ethylene biosynthesis and sen-
sitivity [38-40]. According to the correlative basis
reknown model the central fruitlet exerts a dominant
effect over lateral fruitlets because it is at a more advanced
stage of development [37,41]. As assessed by the canaliza-
tion theory the strong auxin flow coming from the central

fruitlet, down to the peduncle through the AZ into the
twig, depolarizes the weak auxin flows from the lateral
fruitlets causing their abscission [42].

Apical dominance is a complex physiological process
largely controlled by auxin and its interaction with two
additional hormones: cytokinins and MAX (more axillary
branching [43-45]. Cytokinins produced in the roots are
directed to organs (shoot apical meristems, fruits, etc)
whose sink strength is related to their ability in producing
and exporting auxin [46]. This process directs more cyto-
kinins which stimulate growth [47,48]. In the case of api-
cal dominance of shoot meristems, lateral bud outgrowth
occurs when the auxin flow from the apex is hampered,
dominance is weakened, and cytokinins are redirected to
axillary meristems [43,44]. Besides the main cytokinin
stream coming from the roots, the hormone can also be
produced in other tissues. For instance, following decapi-
tation, a prompt increase in transcripts for the key enzyme
in cytokinin biosynthesis, adenosine phosphate-isopente-
nyltransferase, occurs in the stem xylem [49]. The cytoki-
nins produced here may then be translocated into the
axillary meristems where they stimulate the lateral bud
outgrowth. The cytokinin signaling relies on a two-com-
ponent signal transduction system and upon activation it
determines changes in transcript level of genes encoding
proteins, such as the same cytokinin response regulators
(ARRs type A) which are involved in various processes

The second hormone which interacts with auxin is MAX,
a carotenoid derived compound which has been only
recently characterized as strigolactone, but its existence
had been proven before by the discovery of the pea rms,
petunia dad and A. thaliana max mutants [52-55]. These
plants display excessive branching, indicating that strigol-
actone is a positive regulator of apical dominance. Among
the several genes related to this hormone, AtMAX2
encodes an F-box protein [55]. Although the F-box pro-
tein is not required for the synthesis of MAX it is involved
in the transduction of the signal at the level of the node

The isolation, characterization and expression of some
genes encoding elements involved in auxin transport, as
well as of type A MdARRs and MdMAX2, were pursued to
elucidate at the molecular level the interactions among
ethylene, PAT and cytokinin in relation to the immature
apple fruit abscission,

Identification of elements involved in PAT in
During a differential display study between abscising and
not abscising fruitlet populations, several clones related to
auxin were isolated. However, only two transporters were

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BMC Plant Biology 2009, 9:139


found: with one encoding an auxin hydrogen symporter
whereas the other one was too short and located in a
highly conserved region which made further studies com-
plicated [58]. We then pursued the isolation of other ele-
ments involved in auxin transport by the amplification of
fruitlet cDNAs with degenerate primers and the following
3' race. This approach allowed the identification of seven
PIN (Additional file 1) and three LAX (Additional file 2)
partial clones. The name was chosen according to the
highest level of similarity with the PIN of A. thaliana and
M. truncatula, two dicotyledonous species whose mem-
bers have all been well characterized at the genomic level.
MdPIN1_A (EF406255) MdPIN1_B (EF406256),
MdPIN10_A (EF406260), and MdPIN10_B (EF406261)
are likely to be orthologous to AtPIN1, whereas
MdPIN7_A (EF406258), MdPIN7_B (EF406259), and
MdPIN4 (EF406257) may be orthologous to AtPIN3,
AtPIN4 orAtPIN7 (Figure 1 and Additional file 3). Never-
theless, this association has to be definitely proven by
functional and synteny studies. The isolation of different
alleles (presented here with a letter) and the fact that
MdPIN1 and MdPIN1 0 are different genes and not allelic
forms was ascertained by the isolation and comparison of
the genomic clones (MdPIN1: EF406268; MdPIN10_A:
EF406269; MdPIN10_B: EF406270). The amplification of
the genomic clones allowed the identification of the third,
fourth and fifth intron as indicated in the work by [59].
The comparison of these regions indicated that the third
intron was identical among the three clones whereas the
fourth one was identical only between MdPIN1 0_A and
MdPIN10 B. The fifth intron was the most divergent, but
the identity between the two alleles of MdPIN1 0 was still
97.1% (Additional file 4). Concerning the LAX genes,
MdLAX1 (EF406263) may be orthologous to AtAUX1,
whereas MdLAX2 (EF406264) and MdLAX3 (EF406262)
are more divergent and closer to AtLAX (Figure 2 and
Additional file 5).

Since fruitlets are actively growing organs and auxins
install apical dominance by directing the cytokinin stream
derived from the root, some elements involved in cytoki-
nin signal transduction were also isolated. These clones
showed a high level of similarity to type A response regu-
lators (ARRs, A. thaliana Response Regulators) of several
species (results not shown) and were named according to
the most closely related AtARR: MdARR6 (EF406267),
MdARR3 (EF406265) and MdARR4 (EF406266).

Expression analysis in the different tissues
The RT- PCR expression analysis performed with 33P
labeled degenerate primers visualized the relative level of
expression among the different PIN genes. The high inten-
sity of the band of MdPIN1 and MdPIN7 indicates that the
transcripts of these genes are largely predominant on
MdPINIO0 and MdPIN4 (Additional file 6). This result was

AtPIN7 NP 564189 pro
AtPIN3 NP 177250 pro
- ,x AIPIN4 NP 565261 pro
1000 MdPIN7 A
100 0' MdPIN7 BEF406259 .pro
8 MtPIN1
1000 MdPIN1 A
9'4 MdPIN10 A EF406260 pr,
89 1 MdPIN10 B EF406261 pn
93 / ~ PttPINI
--Q__-. MtPIN10
94 .7 MtPIN2
M-- tPIN8_DAA05219 pro
------- AtPIN8_NP 197014 pro
AtPIN5 NP 197157 pro

90 80 70 60 50 40 30 20 10 0
Amino Acid Substitutions (xl00)
Bootstrap Trials 1000, seed 500
Figure I
PIN Phylogenetic tree. Phylogenetic study of the PIN iso-
lated in this study (Malus x domestic (Md)) with those of Ara-
bidopsis thaliana (At), Medicago truncatula (Mt), and Populus
tremula x Populus tremuloides (Ptt). (MdPINI_A, EF406255;
MdPINI_B, EF406256; MdPIN4, EF406257; MdPIN7 A,
EF406258; MdPIN7 B, EF406259; MdPINIO_A, EF406260;
MdPINIO_B, EF406261; AtPINI, NP_177500; AF089085;
AtPIN2, NP 568848; AtPIN3, NP I77250; AtPIN4,
NP_565261, NP 849923; AtPIN5, NP _197157; AtPIN6,
NP_177836; AtPIN7, NP_564189, NP 849700,
NP_001077584; AtPIN8, NP _197014; MtPINI, AAM55297;
MtPIN2, AAM55298; MtPIN3, AAM55299; MtPIN4,
AAM55300; MtPIN5, AAM5530 I; MtPIN6, AAT48627;
MtPIN7, AAT48628; MtPIN8, DAA05219; MtPIN9,
AAT48629; MtPIN 10, AAT48630; PttPINI, AAG 17172;
PttPIN2, AAM54033; PttPIN3, AAM54034). Bootstrap values
are reported.

also confirmed by the number of cycles used in the RT-
PCR: on average, MdPIN1 expression was studied at 33
cycles, MdPIN7 at 32 cycles, MdPINI0 at 35 cycles and
MdPIN4 at 37 cycles (Table 1). Results showed a differen-
tial expression of the orthologs (Figure 3). MdLAX1 tran-
scripts accumulated in all the tissues, although the signal
was barely detectable in fruit seed and senescing leaf.
MdLAX2 and MdLAX3 transcripts showed a similar pat-
tern: in the seedlings the transcripts accumulated mainly
in root and hypocotyl, whereas in fruit mainly in cortex.
The transcripts were also abundant in the flower. Tran-
scripts decreased during leaf ageing (from shoot to senesc-
ing leaf). Transcripts were also detected in shoot, whereas
in fruitlet, the highest amount was observed in peduncle.
MdPIN1 transcripts displayed a similar pattern to

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BMC Plant Biology 2009, 9:139


NA MLAX1 EF406263 pro
Mt1_AX2 O9FEL7 pro
52 PttLAX AAF21982 pro
MtLAX4 Q8L884 pro
376 1000 MdLAX2 -EF403264 pro
831 MdPLAX3 F02 AG771p!o
MiLAXI1974 pro LB
0A IIAX I I 3 S4
100 7X NP 177892 pro

Mt1_AX5_Q8L883 pro
PtILAX3 AAK(58522 pro

20 15 10
Ami~no Acid Substihtutins (xlOO)
Bootstrap Trias =l GO, seed =500

5 0

Figure 2
AUX Phylogenetic tree. Phylogenetic study of the AUX
and AUX-Like isolated in this study (Malus x domestic (Md))
with those of Arabidopsis thaliana (At), Medicago truncatula
(Mt), and Populus tremula x Populus tremuloides (Ptt).
(MdLAXI, EF406263; MdLAX2, EF406264; MdLAX3,
EF406262; AtAUXI, NP_565882; AtLAXI, NP_195744,
NP_974719; AtLAX2, NP_179701; AtLAX3, NP_177892;
MtLAX4, Q8L884; MtLAX5, Q8L883; PttLAXI, AAF21982;
PttLAX2, AAG 17171; PttLAX3, AAK58522). Bootstrap values
are reported.

MdLAX1, with the signal slightly decreasing during flower
and leaf senescence. MdPINI0 transcripts were mainly
detected in seedlings grown in dark, above all in
hypocotyl and peduncle; the transcripts slightly decreased
along senescence of both flower and leaf. MdPIN4 dis-
played a pattern similar to MdPIN1 0, except in the flower
where the signal was strong and apparently not affected by
ageing. MdPIN7 transcripts were detected in all organs,
with a slight increase in the seedlings grown in dark com-
pared to those grown in light, and during flower senes-
cence, whereas a decrease was observed in senescing leaf.
MdIAA3 transcripts were present in all tissues at similar
level, whereas MdIAA7 transcripts displayed a decrease
during both flower and leaf senescence, and a weaker sig-
nal in seed than in peduncles and cortex. The strongest
MdMAX2 signal was observed in cotyledons of light
grown seedlings and in seeds of ripe and immature fruits.
Furthermore, the transcripts accumulated preferentially in
shoots rather than in fully expanded and senescing leaves.
Besides fruit cortex and leaf, MdARR6 transcript accumu-
lation pattern was the opposite to that of MdMAX2, with
the signal decreasing in senescing flower and leaf. The
MdARR3 signal, although weak in all tissues, was mainly
detected in the root of light grown seedlings. In the other
tissues, transcripts mainly accumulated in leaf. MdARR4
transcripts accumulated preferentially in dark grown seed-
ling and, opposite to MdARR3, transcript amount
decreased along leaf ageing.

Expression analysis during abscission induction
MdPIN1 transcript amount displayed a dramatic decrease
in seeds whereas not much difference was observed in the
other tissues (Figure 4). MdPINIO 0 transcripts increased in

cortex by day 7 with a concurrent slight decrease in pedun-
cle, whereas in AZ the decrease started already at day 5.
MdPIN4 transcripts increased throughout the experiment
in cortex, whereas in peduncle the increase was moderate
and started later, concurrently with a decrease in AZ.
MdPIN7 expression showed only a late increase in cortex.
MdLAX1 transcripts slightly declined in seeds and
increased in both cortex and peduncles. In AZ a transient
decrease was observed at day 5. Since MdLAX2 and
MdLAX3 showed the same pattern of expression, only
results related to MdLAX2 are presented in Figure 2.
Besides a slight transient decrease at day 3 in peduncle
and a late surge in cortex, transcript levels remained
unchanged along the experiment. MdlAA3 transcripts
decreased by day 7 in seeds and increased by day 5 and 7
in peduncles and cortex, respectively. MdIAA7 transcript
amount significantly increased in cortex at day 7 along
with a dramatic decrease in peduncles. MdARR6 mRNA
amount steadily increased in cortex, whereas in seeds and
peduncles a decrease was observed at day 7. Analogously,
MdARR3 transcripts increased in cortex and decreased in
seeds and slightly in peduncles. Concerning MdARR4,
mRNA amount already increased in cortex at day 3, then
remained constant. MdMAX2 transcripts decreased late in
seeds and increased at day 3 in cortex. The up-regulation
was maintained in the following dates: in peduncle a tran-
sient decreased at day 3 and 5 was observed whereas in AZ
mRNA gradually declined along the experiment.

Peduncle development
Peduncle growth was monitored along abscission up to
14 days after BA application (Figure 5). The length of the
NAF peduncles was significantly shorter than the AF along
the whole experiment. The initial length of the peduncles,
according to the value of the intercepts calculated by a lin-
ear regression, was 2.63 cm and 3.04 cm for NAF and AF,
respectively. On the other hand, according to the line
equations (a regression value of 0.92 in NAF and 0.99 in
AF), the growth rate of the fruitlet peduncle was similar
with values of 0.027 and 0.034, respectively. Concerning
diameter, the NAF was characterized by a thicker peduncle
(0.15 cm) than the AF (0.12 cm), but no relevant changes
were observed along the experiment.

Auxin effect on PIN transcripts
Auxin application determined a dose-dependent response
in PIN transcripts. Nevertheless, there was a clear increase
only in MdPIN4 and MdPINI0, whereas in the case of
MdPIN1 and MdPIN7 auxin application seemed only to
counteract the natural decrease in transcripts as observed
in the mock control (Additional file 7).

Ethylene effect on transcript accumulation
Fruitlet clusters were flushed with propylene or treated
with 1-MCP for 24 hours and expression analysis was car-
ried out in peduncles (Figure 6). Compared to the control,

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BMC Plant Biology 2009, 9:139


Table I: List of primers utilized in the expression analysis

Gene Tm Ncycles

Primer forward

Primer reverse

Accession number













































Specific primers utilized for the expression analysis. Tm indicates the annealing temperature used in the PCR reaction. The number (N) of cycles is
that utilized in the experiments reported. The accession number is also reported in the right column.

MdARR3 transcript amount was decreased by propylene
and increased by 1-MCP, especially in the case ofMdPIN1.
As far as MdPIN4, MdPIN7 and MdMAX2 are concerned,
the transcripts accumulated throughout the experiment in
an ethylene-independent manner, whereas no chemical
effect was observed on MdARR4 transcripts.

Which are the orthologs?
The isolation of various PIN and LAX clones in Malus x
domestic indicates the presence of multigene families.
Because of the identical expression profile and the high
identity values, MdLAX2 and MdLAX3 are likely to be alle-
les. On the contrary, the alignment of the PINs presented
here with the PINs ofA. thaliana and M. truncatula, the iso-
lation of the two genomic alleles of MdPIN1 0 and the dif-
ferences at the level of the introns, and expression pattern
of MdPIN1 and MdPIN1 0 indicate that they may be paral-

ogous, as it has been reported for MtPIN4 and MtPIN5
[59]. The same organization was found in Vitis vinifera,
Populus trichocarpa and Oryza sativa. Interestingly, A. thal-
iana presents a transcript variation of the PIN1 gene
(Additional file 8). Another interesting point is that
MdPIN7 and MdPIN4 are closely related to each other as
found for MtPIN1 and MtPIN3, whereas in A. thaliana,
besides AtPIN3 and AtPIN7 (showing a high level of sim-
ilarity) a third member, AtPIN4, is present in the same
cluster. The overall data indicate a different organization
of the PIN members in the species discussed here. Similar
organization for this cluster was found in Populus and vitis
but not in Oryza (Additional file 8). This may be due to
duplication and specialization of the members along
plant speciation after P. patens. The organization of the
auxin influx carriers was not completely conserved either
(Additional file 9) This observation also highlights the
difficulty in identifying the physiological orthologs of
Arabidopsis genes.

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BMC Plant Biology 2009, 9:139


light dark Fr Fri
MdLAX2 -

MdPIN 10 -


, 1 % um.

MdMAX2 -
MdARR3 -

Figure 3
Expression study in different organs of MalusXdomes-
tica. Description: Expression analysis in different tissues and
at different developmental stages performed by RT-PCR.
Root (R), cotyledon (C) and hypocotyl (Hy) were collected
from seedlings after 4 days of de-etiolation or left in the dark.
Seed (S) and cortex (K) excised from fruit (Fr) at commercial
harvest. Flowers (Fl) collected at full bloom and senescing
un-pollinated flowers (SFI) were those not pollinated. Leaves
were divided in mature fully expanded leaf (L), senescing leaf
(SL) and shoot (leaflet and stem) (Sh). Fruitlets (FrI) were
collected at 7 days APF and seed (S), peduncle (P) and cortex
(K) dissected.

The expression of the PAT elements is driven by external
and internal cues
During germination when a young plant has to grow
through the soil particles and reach the surface, the coty-
ledons remain upright and the hypocotyl keeps on elon-
gating until it reaches the light. The difference between
hypocotyl length of seedlings grown in dark and seedlings
during de-etiolation was also observed in our experiment
(data not shown). Expression analysis performed on the
seedlings indicated that light mainly affects transcript
amount of the auxin efflux carriers (MdPINIO 0 and
MdPIN4) in hypocotyl as reported in pea, whereas the
influx carrier transcript quantity was barely changed
(MdLAX1) [60]. According to the results, light negatively
regulates PIN expression during germination. Consider-
ing the expression domains of the Arabidopsis PINs at this
stage the expression level at different developmental
stages and in different organs, and the sequence similarity

to known proteins and genes, MdPIN1 and MdPIN10O
analogously to AtPIN1, would be involved in PAT
directed to the root apical meristems[31]. On the other
hand, MdPIN4 and MdPIN7, similarly to AtPIN3 and
AtPIN7, may have an important role in hypocotyl elonga-
tion because they are expressed in hypocotyl epidermis
and bundle sheath. However, immunolocalization and
GFP studies are necessary to further investigate which ele-
ments in apple are the physiological orthologs to those of

Moreover, a general decrease in the transcript amount of
MdLAX1, MdLAX2, MdLAX3, MdPIN1 and MdPIN10 was
observed during senescence and ageing of different
organs/tissues such as seed, leaf, flower and cortex. As dis-
cussed above, it is well known that a drop in auxin level
occurs during leaf senescence preceding abscission. It has
also been extensively demonstrated that the majority of
Aux/IAA transcripts increase after auxin application in sev-
eral systems, making them good molecular markers for
the auxin endogenous level [18,27,61-63]. Furthermore,
analogously to the PIN clones herein isolated, transcripts
of some PINs were found to increase following auxin
application in poplar, pea and A. thaliana [60,64,65].
Therefore, it is conceivable that the change in MdIAA7 and
PIN transcripts occurring at certain developmental stages
is at least partially auxin-related. On the other hand, the
clone MdIAA3 may represent a gene that is highly
expressed and only partially controlled by auxin. In addi-
tion, a slight increase in MdLAX1, MdPIN4 and MdPIN7
transcripts was monitored in senescing flowers, as
reported during daffodil flower senescence [66]. These
results indicate that the expression of the PAT elements,
besides being under auxin control, is developmentally
and tissue specifically regulated, as previously reported for
hybrid aspen and A. thaliana, respectively [64,65].

PAT transcripts are differentially regulated during
abscission induction
The expression analysis performed along abscission
induction pointed out a general increase in the transcrip-
tion of PAT elements in cortex, whereas a predominant
decrease was observed in seeds. Furthermore, considering
that MdPIN1 and MdPIN7 are the most abundant tran-
scripts (Figure 3, Figure 4, and Table 1) and that MdPIN1 0
and MdPIN4 were not detected in seed, it appears that
during the fruitlet abscission a decrease in PAT at the level
of the seed occurs. On the other hand, the low growth rate
[61] and the high level of PAT transcripts, such as MdIAA3
and MdIAA7 (this work) observed in the cortex of AF,
indicate a delay in development. This hypothesis is
strengthened by the up-regulation of MdARR6, MdARR3
and MdARR4 occurring in the cortex of AF. In fact, the type
A ARR is the best indicator of the level of the cytokinin
(the hormone that stimulates cell division [67]), in which

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- -------- -

BMC Plant Biology 2009, 9:139

BMC Plant Biology 2009, 9:139




0 1 2 3 4 5 6 7



0 2 4



0 1 2 3 4 5 6 7


0 1 2 3 4 5 6 7

6 MdLAX1

-3 .m


0 1 2 3 4 5 6 7


6 MdLAX2
2 -
1 -

0.2 2 4 5 6
o 1 2 3 4 5 6 7

6 MdlAA3
4 Ul .....
2 1

-2 4
-3 "
0 1 2 3 4 5 6 7


S 1 2 3 4 5 6 7




0 1 2 3 4 5 6 7



0 1 2 3 4 5 6 7


0 1 2 3 4 5 6 7


-A --.- -

'-. .. -m

o 1 2 3 4 5 6 7

Figure 4
Expression study during abscission induction. Expression analysis in seed (solid triangle, continuous line), cortex (open
triangle, dashed line), peduncle (solid square, dotted line) and AZ (open square, dashed-dotted line) performed by RT-PCR
during abscission induction from 15 to 22 days APF. Samples were collected after triggering abscission with BA 15 days APF
(time 0) and 3, 5 and 7 days later. Results are presented as folds induction compared to the TO of the ratio between the
expression level in the abscising fruitlets and in the persistent ones, corrected for the internal control (ubiquitin).

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6 8 10 12 14

0.20 -







0 2 4 6 8 10 12 14

Figure 5
Peduncle growth. Peduncle length and diameter of the abscising fruitlets (open squares) and persistent fruitlets (solid
squares) during abscission induction from 15 to 29 days APF. Samples were collected 3, 5, 7, 9 and 13 days after triggering
abscission with BA at TO.

this phenomenon occurs in fruit exclusively at early devel-

The cellular concentration of auxin is due to both trans-
port rate and homeostasis, but in the wood forming tis-
sues, the latter is largely predominant on synthesis,
catabolism and conjugation due to the scarce capacity of
the cells [64]. The peduncle and the AZ are mainly made
up of this type of cells. Different patterns were found in
transcripts accumulation: MdPIN1 0 decreased in pedun-
cles and in the AZ, whereas MdPIN4 increased in peduncle
and decreased in AZ, indicating a different nature of the
tissues. We may hypothesize that during abscission induc-
tion the decrease in auxin flow coming from the seeds
determines a change in the peduncle both in terms of
auxin level, as assessed by the MdIAA7 transcript accumu-
lation and in terms of PAT, because peduncle is the fruitlet
organ in which MdPIN4 and MdPIN1 0 are mostly
expressed and that the diverse PIN mutants display par-
tially different phenotypes (as previously discussed). The
vertical flux may be hampered due to an increase in the
apolar distributed efflux carriers. In this scheme, the auxin
flow through the AZ will progressively decrease, leading to
AZ activation. Nevertheless, auxin at this stage may play a
pivotal role in tissue differentiation leading to wood for-
mation [68], as demonstrated by the isolation of auxin
related clones putatively involved in the process [69,70].
In this case, the auxin flow may be directed, according to
the signal flow canalization hypothesis [71], through a
lateral transport mediated by specifically localized AECs
such as MdPIN4. The difference in development between

AF and NAF is further confirmed by the higher level of
MdLAX1 transcripts and by the reduced diameter of the
lateral fruitlet peduncle. In agreement to what was
observed for the hypocotyls during de-etiolation, the dif-
ferential expression may also be related to a higher elon-
gation rate. Nevertheless, despite the fact that AF
peduncles are longer than NAF, there was no difference in
the growth rate, thus indicating that the differential
expression is unrelated to cell elongation but mainly
related to tissue formation. Concerning AZ, the transient
decrease in MdLAX1 and the continuous decrease in
MdPIN1 0 and MdPIN4 transcripts may indicate a redistri-
bution of auxin.

Abscission induction and dominance
Abscission in apple is proposed to be due to competition
between auxin flows coming from the different fruitlets in
a dominance-like fashion [37]. The data herein presented
indicate that during abscission induction there is a clear
decrease in PAT-related transcripts in the seed, the key ele-
ment in fruitlet dominance. It is then likely that the drop
in auxin export capacity of AF leads to a weak auxin flow
which cannot compete with the strong flux from the cen-
tral fruitlet. As previously discussed, dominance is related
to the capacity of the apex to attract cytokinins, which are
positive regulators of type A ARR expression. Indeed, a
decrease in MdARR6 and MdARR3 transcripts was
observed in seeds during abscission induction, thus indi-
cating a shortage in cytokinin. Nevertheless, the low level
of cytokinin is likely due to a low biosynthetic rate rather
than to a reduced import. It has been shown that there are

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E 3.0-



0 2 4

BMC Plant Biology 2009, 9:139


MdLAX2 u
MdPIN1 eo



MdMAX2 pmmo
MdARR3 i _


Figure 6
Ethylene effect on transcript accumulation. Expression
analysis in peduncle of fruitlet clusters after 24 hours of pro-
pylene treatment (P) or I -MCP (M) application. The control
(C) represents untreated fruitlets after 12 hours from the
beginning of the experiment (TO).

several IPT genes expressed at specific stages of seed devel-
opment [72] and there is a specific developmental win-
dow during seed formation in which a large increase in
seed cytokinin is observed in cereals and beans [73-76]. At
this stage, cytokinins are believed to strengthen sink activ-
ity and to induce endosperm proliferation. Furthermore,
the chalazal endosperm is thought to be involved in
importing assimilates into the developing seeds [77].
Since cytokinins in seed are not exported, it is likely that
they play a role as local mediators rather than systemic sig-
nals [78,79]. Considering MAX2, an additional element
involved in apical dominance, its expression pattern in
the different tissues is opposite to that of MdLAX2/3,
MdARR6 and MDPIN10, especially in seedlings. This
divergence may indicate some common regulatory mech-
anisms in terms of transcription. Nevertheless, such rela-
tion is lacking in the tissues undergoing abscission,
indicating that MdMAX2 may not play an important func-
tion in PIN regulation as reported in other systems but
instead act in other processes such as senescence [80,81].

It appears that the expression level of genes related to
auxin and the PAT elements are tightly correlated to
abscission induction driven by dominance in apple. It is
feasible that the strong auxin flow characterizing the cen-
tral fruitlet dominates the weak ones [41]. Nevertheless, it

is not clear what is the cue determining the fall in the seed
PAT that triggers abscission. The best candidate is devel-
opment, which is also affected by carbohydrate availabil-
ity [82]. The developmental stage acts through the same
auxin flow (small seeds produce less auxin and cytoki-
nins) and with the likely involvement of ethylene. Indeed,
ethylene has already been proven to affect auxin transport
[83]. In this study, ethylene was also clearly shown to
repress transcript accumulation of MdLAX1, MdLAX2,
MdPIN1, MdPIN10, MdARR6 and MdARR3, indicating
that the increase in its evolution at this stage may modu-
late seed development, leading to a reduced auxin export
and the induction of abscission.

Plant material
The experiments were performed on Malus x domestic.
Eight-year old apple trees (cv Golden Delicious/M9) were
grown interspaced by pollinator trees (cv Stark Red). In
order to investigate the expression profile of the identified
clones in different tissues and in different physiological
conditions, seed and cortex from ripe fruits at harvest,
flowers at full bloom and un-pollinated senescing flowers,
shoot, leaf and senescing leaf and seed, peduncle and cor-
tex of fruitlets at 7 days after petal fall (APF) were col-
lected. Furthermore, since seedlings are the usual system
to study elements involved in PAT, seeds were sown in
half MS media in the dark. Five days after germination,
seedlings were either left for a further 4 days in the dark or
moved to light. Root, cotyledon and hypocotyl were col-
lected from seedlings at 5 days after germination and after
4 days of de-etiolation or darkness.

Fruitlets differing in the abscission potential were
obtained as previously described with some minor modi-
fications [39]. Abscising fruitlets (AF) were obtained from
lateral fruitlets of clusters borne on trees sprayed with ben-
zyladenine (BA) at 200 |g L-1 when the average fruit
diameter was around 10 mm (15 days APF). The non-
abscising fruitlets (NAF) were comprised of central fruit-
lets of clusters in which all the laterals had been removed
7 days APF. Seed, cortex, peduncle and abscission zone
(AZ) were collected from NAF and AF at 0, 3, 5 and 7 days
after the BA treatment of the AF population. The northern
blot analysis (Additional file 10) was performed as previ-
ously described with the MdAC01 probe [40,84]. Results
confirmed the validity of the populations obtained
because the increase in MdAC01 transcript amount
showed to be a reliable abscission marker [38,40,84].
Peduncle length and diameter of NAF and AF were meas-
ured with a calibre at 0, 3, 5, 7, 9 and 11 days after BA
treatment. Statistical analysis (T-test) was performed with
the excel package. The equation and the regression coeffi-
cient were also calculated.

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BMC Plant Biology 2009, 9:139


Table 2: List of degenerate primers utilized in the isolation of genes







ARR type S











Degenerate primers used for the isolation of partial clones of LAX, PIN, and ARR type A identified in this study. S indicates the oligonucleotide
sequence while M the conserved protein sequence chosen to design the primers. The amplification length of the product (L) expressed in bp and
the annealing temperature are also reported.

Hormone treatments
Auxin treatment was performed only on the peduncle,
because the peduncle is the tissue most respondent to
auxin [61]. IAA was dissolved in a constant amount of
alcohol at a final concentration of 0.1, 1, 5 and 10 mM.
The mock control consisted of peduncle treated with
water and the quantity of alcohol used for auxin solubili-
sation. The entire experiment was performed in the dark.
The central part of the peduncle (1.5 cm) was isolated
from lateral fruitlets at 15 days after petal fall (DAPF)
(TO), immersed in the auxin solution and a vacuum
applied for 10 min, and then was left for an additional
120 min under normal conditions and sampled. Entire
apple fruitlet clusters at 15 DAPF were treated with propyl-
ene (1000 [iL- L-1) or 1-MCP (1 [LL- L-1) or left untreated
(control) as described in [61]. Tissue was collected at the
beginning of the experiment (TO) and after 24 hours for
molecular analysis.

Clone isolation and expression analysis
Total RNA extraction and single strand cDNA synthesis
were performed as previously reported [85,39]. The isola-
tion of the partial clones was performed with degenerate
primers designed on conserved areas of the ORFs (Table
2) [86]. The reaction was performed in lx PCR Buffer
(Amersham Pharmacia), 0.25 mM dNTPs, 1 jiM primer
forward, 1 jiM primer reverse, and 0.05 U/jil of Taq
(Amersham Pharmacia). The PCR profile was as follows:
5 min hold at 95C and 40 cycles composed by 60 s at
94 C, 60 s at the primer annealing temperature (Tm) and
90 s of extension at 72C. Tm was as reported in Table 1.
The cycles were followed by a final step of 7 min at 72 C.

The 3'UTR of the clones was subsequently obtained with
specific primers (Table 3) as previously described [40].
Briefly the 3'RACE was performed with a gene specific for-

ward primer and the anchored oligo dT as the reverse
primer. Annealing temperature was as reported in Table 2.

The genomic sequences of MdPIN1 and MdPIN 0 were
obtained from the amplification of DNA extracted from
leaves of Golden Delicious apples with the Qiagen DNA
easy according to manufacturer instructions. The template
(100 ng) was amplified in lx PCR buffer (Amersham),
0.03 U tL-1 Taq (Amersham Pharmacia), 0.3 jiM of primer
forward (5'-CGGGATCCATTGTCTCCA-3') and 0.3 jiM of
primer reverse (5'-GGAAACTCCATTGCAGCT-3'). The
amplification profile was as follows: an initial denatura-
tion for 5 min at 95C, 40 cycles composed by 60 s at
94C, 90 s at 62C and 90 s at 72C, and a final step of 7
min at 72 C

Amplification fragments were electrophoresed in agarose
gel, purified and cloned into pGEM-T Easy vector

Table 3: List of primers utilized in the isolation of the 3' part of
the genes isolated in this study









Specific primers used in the 3' RACE for the isolation of the 3' part of
LAX, PIN, and ARR type A. The annealing temperature is also

Page 10 of 14
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L Tm

bp C

840 58

1400 57

200 60

BMC Plant Biology 2009, 9:139


(Promega). Positive clones were grown and plasmids were
isolated with the Miniprep Kit (Qiagen) and sequenced
using the ABIPRISM BigDye Terminator v3.1 kit (Applied

Contigs were assembled by the SeqMan software (DNAs-
tar package) while sequence comparisons were performed
using BlastX and BlastN algorithms (NCBI, National Cen-
tre for Biotechnology Information). Sequence alignment
between the deduced proteins of the clones identified
here and those from LAX and PIN members of Arabidopsis
thaliana (AtAUX1, NP_565882; AtLAX1, NP_195744,
NP_974719; AtLAX2, NP_179701; AtLAX3, NP_177892;
AtPIN1, NP_177500, AF089085; AtPIN2, NP_568848;
AtPIN3, NP_177250; AtPIN4, NP_565261, NP_849923;
AtPIN5, NP_197157; AtPIN6, NP_177836; AtPIN7,
NP_564189, NP_849700, NP_001077584; AtPIN8,
NP_197014), Medicago truncatula (MtLAX1, Q9FEL8;
MtLAX2, Q9FEL7; MtLAX3, Q9FEL6; MtLAX4, Q8L884;
MtLAX5, Q8L883; MtPIN1, AAM55297; MtPIN2,
AAM55298; MtPIN3, AAM55299; MtPIN4, AAM55300;
MtPIN5, AAM55301; MtPIN6, AAT48627; MtPIN7,
AAT48628; MtPIN8, DAA05219; MtPIN9, AAT48629;
MtPINI 0, AAT48630) and Populus tremula x Populus trem-
uloides (PttLAX1, AAF21982; PttLAX2, AAG17171;
PttLAX3, AAK58522; PttPIN1, AAG17172; PttPIN2,
AAM54033; PttPIN3, AAM54034) were performed with
the aid of the Clustal W algorithm using default settings
(MegAlign software, DNAstar package).

Expression analysis of the clones was performed by the
semi-quantitative PCR implemented with 33P labeled
primers as previously described [40]. Specific conditions
of this experiment are reported in Table 3. Data concern-
ing the abscission experiment are expressed as fold induc-
tion of the ratio between the level of expression in AF and
NAF populations, corrected for the transcript amount of
the constitutive gene (MdUBI). Data referring to auxin
application are presented as fold induction after correc-
tion for the level of the constitutive gene (MdUBI).
Changes of at least 0.5 fold magnitudes are considered sig-

AF: Abscising Fruitlet; ARR: Arabidopsis Response Regula-
tor; At: Arabidopsis thaliana; Aux/IAA: Aux/IAA; AUX:
AUXin; AZ: abscission zone; DAPF: days after petal fall;
dad: Decreased Apical Dominance; GFP: green fluorescent
protein; IAA: indol acetic acid; LAX: Like AUX; MAX: Max-
imum Axillary; Md: Malus x domestic; Mt: Medicago trun-
catula; NAF: Non Abscising Fruitlet; PAT: Polar Auxin
Transport; PIN: PIN FORMED; rms: ramosus; TO: time 0;
UBI: UBIquitin.

Authors' contributions
VDC conceived and designed the experimental study, per-
formed the experiments, and wrote the manuscript. RV
gave intellectual guidance and financially supported the
research. AR conceived and designed the experimental
study, gave intellectual guidance, financially supported
the research and wrote the manuscript. All authors read
and approved the final manuscript.

Additional material

Additional file 1
Scheme of the PINs isolated in this study. The scheme illustrates the par-
tial clones of PIN isolated in this study. Rectangles represent the CDS
whereas the line indicates the UTR. The blank rectangle and the dotted
line represent the missing sequence. The stop codon is reported in capital
letter between the CDS and the 3' UTR. The string of four A, where
present, indicates the polyA tail. The length of the parts is reported in base
pairs (bp) at the bottom.
Click here for file

Additional file 2
Scheme of the LAX isolated in this study. The scheme illustrates the par-
tial clones of LAX isolated in this study. Rectangles represent the CDS
whereas the lines indicate the UTR. The blank rectangle and the dotted
line represent the missing sequence. The stop codon is reported in capital
letter between the CDS and the 3' UTR. The string of four A, where
present, indicates the polyA. The length of the parts is reported in base
pairs (bp) at the bottom.
Click here for file

Additional file 3
Identity values among the PIN isolated in this study with those of sev-
eral species. Identity values among the protein sequences of PIN isolated
in this study from Malus x domestic (Md) and those of Arabidopsis
thaliana (At), Medicago truncatula (Mt), and Populus tremula x
Populus tremuloides (Ptt). (MdPINI_A, EF406255; MdPINI_B,
EF406256; MdPIN4, EF406257; MdPIN7_A, EF406258;
MdPIN7_B, EF406259; MdPIN10_A, EF406260; MdPIN10_B,
EF406261; AtPIN1, NP 177500; AF089085; AtPIN2, NP 568848;
AtPIN3, NP 177250; AtPIN4, NP 565261, NP 849923; AtPIN5,
NP 197157; AtPIN6, NP 177836; AtPIN7, NP 564189,
NP 849700, NP 001077584; AtPIN8, NP 197014; MtPIN1,
AAM55297; MtPIN2, AAM55298; MtPIN3, AAM55299; MtPIN4,
AAM55300; MtPIN5, AAM55301; MtPIN6, AAT48627; MtPIN7,
AAT48628; MtPIN8, DAA05219; MtPIN9, AAT48629; MtPIN10,
AAT48630; PttPINI, AAG17172; PttPIN2, AAM54033; PttPIN3,
Click here for file
2229-9-1 * i' --]

Page 11 of 14
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BMC Plant Biology 2009, 9:139

BMC Plant Biology 2009, 9:139

Additional file 4
Identity values of the intronic regions. The values are the identity per-
centages of the intronic nucleotide sequences of MdPINl (EF4062 68),
MdPIN10_A (EF406269) and MdPIN10_B (EF4062 70) obtained
with the clastalW alignment. Length is represented on the left and
expressed as base pairs (bp).
Click here for file

Additional file 5
Identity values among the LAX isolated in this study and those of sev-
eral species. Identity values among the protein sequencesLAX isolated in
this study from Malus x domestic (Md) with those of Arabidopsis
thaliana (At), Medicago truncatula (Mt), and Populus tremula x
Populus tremuloides (Ptt). (MdLAX1, EF406263; MdLAX2,
EF406264; MdLAX3, EF406262; AtAUXI, NP 565882; AtLAX1,
NP 195744, NP 974719; AtLAX2, NP 179701; AtLAX3,
NP 177892; MtLAXI, Q9FEL8; MtLAX2, Q9FEL7; MtLAX3,
Q9FEL6; MtLAX4, Q8L884; MtLAX5, Q8L883; PttLAX1, AAF21982;
PttLAX2, AAG17171; PttLAX3, AAK58522).
Click here for file
2229-9-1 1 *- h-..--]

Additional file 6
Expression analysis with degenerative primers. The expression analysis
was performed with PIN degenerate primers on cDNA of peduncle.
Click here for file

Additional file 7
Expression analysis of PINs following auxin application. The expres-
sion analysis of PIN was preformed on CDNA from peduncle tissue
treated with auxin .1,t ... ,1 ...... 11 ,... 0.1, 1, 5, 10 mM and the
mock control (M) for 90 mmin. Expression data are corrected for the con-
stitutive gene and presented as fold induction compared to the beginning
of the experiment (TO).
Click here for file

Additional file 8
PIN Phylogenetic tree. Phylogenetic tree of the PINs isolated in this study
from MalusXdomestica (Md) and those ofArabidopsis thaliana (At),
Medicago truncatula (Mt), Oryza sativa (Os)cultivar indica (ind)
and japonica (jap), Populus tremula x Populus tremuloides (Ptt),
Populus balsamifera subsp. trichocarpa (Ptric), Physcomitrella pat-
ens subsp patens (Pp) and several varieties of Vitis vinifera (Vv) from
t, 1. ,1 varieties. Bootstrap values are indicated. Var means transcript
variation. The accession number is reported at the end of the sequence
Click here for file
2229-9-1 *'- ir..--]


Additional file 9
LAX phylogenetic tree. Phylogenetic tree of the LAX isolated in this study
from MalusXdomestica (Md) and those of Arabidopsis thaliana (At),
Medicago truncatula (Mt), Oryza sativa (Os)cultivar indica (ind)
and japonica (jap), Populus tremula x Populus tremuloides (Ptt),
Populus balsamifera subsp. trichocarpa (Ptric), Physcomitrella pat-
ens subsp patens (Pp) and several varieties ofVitis vinifera (Vv) from
t, 11. 11 varieties. Bootstrap values are indicated. The accession number
is reported after the sequence. The accession number is reported at the end
of the sequence name.
Click here for file

Additional file 10
MdACO1 expression analysis during abscission. The expression analy-
sis was performed by northern blot on the sample utilized in this study: AF
(abscising fruitlets) and NAF (non-abscising fruitlets) at 0, 3 5 and 7
days during abscission induction. The control is represented by 18S.
Click here for file

We are grateful to Giulio Galla for technical assistance in the expression
analysis of the PIN genes

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