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UGP gene expression and UDP-glucose

pyrophosphorylase enzymatic activity in
grafting annonaceous plant

Article in Acta Physiologiae Plantarum · March 2016

Impact Factor: 1.58 · DOI: 10.1007/s11738-016-2097-7

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Acta Physiol Plant (2016) 38:79
DOI 10.1007/s11738-016-2097-7

ORIGINAL ARTICLE

UGP gene expression and UDP-glucose pyrophosphorylase

enzymatic activity in grafting annonaceous plants
Daniel Baron1 • Juliana P. Bravo2 • Ivan G. Maia2 • Ana Pina3 • Gisela Ferreira1

Received: 15 August 2015 / Revised: 16 December 2015 / Accepted: 8 February 2016

Ó Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2016

Abstract Grafting is commonly used to propagate com- levels of gene expression during the early stages of grafting
mercial fruit species to ensure that the genetic character- development. However, no significant differences were
istics of selected clones are maintained. However, the detected in UGPase enzyme activity between the graft
biochemical and molecular mechanisms involved in the combinations. In addition, SDS-PAGE and MALDI-TOF
graft incompatibility of woody trees are not well under- analyses detected similar UGPase amino acid sequences in
stood. We investigated the effect of grafting in vegetative ungrafted atemoya samples to cherimoya (Annona cheri-
growth, UDP-glucose pyrophosphorylase expression and mola Mill.), a female parent of the atemoya hybrid. These
activity of Annonaceous grafted plants: atemoya (Annona findings suggest that expression of the UGPase protein is
cherimola Mill. x Annona squamosa L.) ‘Thompson’ related to graft compatibility in grafted Annona plants.
grafted onto different rootstocks, araticum-de-terra-fria
(Annona emarginata Schltdl. H. Rainer ‘‘var. terra-fria’’), Keywords Annonaceae  Enzymatic activity  Graft
araticum-mirim (Annona emarginata Schltdl. H. Rainer incompatibility  Plant propagation  UGP
‘‘var. mirim’’) and biribá (Annona mucosa Schltdl.
H. Rainer) at different post-grafting times. The growth of Abbreviations
atemoya grafted onto araticum-mirim was lower than that CTAB Hexadecyl trimethyl ammonium bromide
of the rootstocks araticum-de terra-fria and biribá. The DAG Days after grafting
results also indicated that grafting alters UGPase gene MALDI-TOF Matrix-assisted laser desorption/
expression; showing the combination atemoya grafted onto ionisation—time of flight
araticum-de-terra-fria (a compatible union) the higher UGPase UDP-glucose pyrophosphorylase
SDS-PAGE Sodium dodecyl sulfate polyacrylamide
Communicated by LA Kleczkowski. gel electrophoresis

& Daniel Baron

[email protected]; [email protected]
1 Introduction
Laboratório de Fisiologia Vegetal, Departamento de
Botânica, Instituto de Biociências, Universidade Estadual
Paulista, UNESP, campus de Botucatu, District of Rubião The production areas for Annonaceae, such as atemoya
Júnior, S/N8, 18618-970 Botucatu, São Paulo, Brazil (Annona cherimola Mill. x Annona squamosa L.) and
2
Laboratório de Biotecnologia e Genética Molecular, soursop (Annona muricata L.) have dramatically increased
Departamento de Genética, Instituto de Biociências, and many issues of incompatibility have been observed in
Universidade Estadual Paulista, UNESP, campus de the field. In orchards, Annonaceae species of economic
Botucatu, District of Rubião Júnior, S/N8,
18618-970 Botucatu, São Paulo, Brazil importance are grafted to ensure that the genetic charac-
3
teristics of productive scions are maintained (Almeida et al.
Unidad de Hortofruticultura, Centro de Investigación y
Tecnologı́a Agroalimentaria (CITA de Aragón), Avda. 2010; Encina et al. 2014), however, graft incompatibility is
Montanãna, 930, 500059 Zaragoza, Spain frequently observed. Graft incompatibility is generally

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defined as the interruption in cambial and vascular conti- been studied during graft union formation in the genus
nuity between rootstock and scion (Hartmann et al. 2011; Annona. Therefore, considering the need to understand the
Kostopoulou and Therios 2014; Li et al. 2012). Further underlying mechanisms involved in Annona graft incom-
understanding of the mechanisms related to graft incom- patibility, the aim of this study was to determine the effect
patibility in Annonaceae is valuable, especially for com- of grafting in UGP gene expression and enzymatic activity
mercial species, such as sweetsop and atemoya. in various graft combinations of atemoya scion grafted
The rootstocks most often used to graft atemoya, a hybrid onto three different rootstocks: araticum-de-terra-fria,
fruit with high organoleptic quality, are araticum-de-terra- araticum-mirim and biribá plants.
fria (Annona emarginata Schltdl. H. Rainer ‘‘var. terra-fria’’)
and araticum-mirim (Annona emarginata H. Rainer ‘‘var.
mirim’’). Atemoya grafted onto araticum-de-terra-fria Materials and methods
rootstock results in further development of the scion and
tolerance to cave nematodes [Radopholus similis (Cobb) Plant material and treatments
Thorne], stem borers (Cratosomus bombina F.) and water
stress (Tokunaga 2005). On the other hand, araticum-mirim The experiment was conducted in a greenhouse at the
rootstock causes dwarfism, which is considered beneficial Instituto de Biociências (IB), Universidade Estadual Pau-
because it facilitates the management of commercial orch- lista (UNESP), Botany Department, Botucatu, São Paulo,
ards (Prassinos et al. 2009). Despite research indicating graft Brazil (altitude 850 m). Seeds of three rootstocks arati-
incompatibility between atemoya and biribá (Almeida et al. cum-de-terra-fria, araticum-mirim, biribá and atemoya
2010), this authors suggested that wild Annonaceae species, were sown in polystyrene trays containing vermiculite,
such as ‘biribás’ and ‘araticuns’, are potential rootstocks in according to Baron et al. (2011). When the seedlings
breeding and in selection of new fruits. developed fully expanded leaves, they were transplanted to
It is widely known that the more closely related root- plastic pots (approximately 17 dm3) containing a mixture
stock-scion are, the better the chances for the graft to be substrate with fertile soil, textured vermiculite and coconut
successful facilitating new tissue formation and re-estab- fibre. The whip and tongue graft technique was performed
lishment of vascular connection (Hartmann et al. 2011; according to Tokunaga (2005). The rootstocks were pre-
Pina and Errea 2005). Several factors are attributed to graft pared 18 months after sowing, when the plants had stem
incompatibility including physiological intolerance at the diameters ranging from 8 to 10 mm and 15 cm in height.
cellular level between rootstock and scion (Andrew and The atemoya ‘Thompson’ was prepared using stem seg-
Marquez 1993; Nocito et al. 2010; Pina and Errea 2005; ments (12 cm in length, 8–10 mm in diameter) from the
Pina et al. 2012). Currently, molecular and proteomics same plant. Experiments were performed independently for
approaches are contributing in the study of graft union each graft combination (atemoya scions grafted onto ara-
formation and incompatibility, highlighting that it involves ticum-de-terra-fria, araticum-mirim and biribá rootstocks).
a network of different metabolic pathways (Cookson et al. Ungrafted genotypes were used as controls. The vegetative
2013; 2014; Irisarri et al. 2015; Liu et al. 2013; Pina and growth of grafted and ungrafted plants was evaluated using
Errea 2008; Prinsi et al. 2015; Zhang et al. 2015). different parameters: stem diameter, plant height, number
The enzyme UDP-glucose pyrophosporylase (UGPase) of fully expanded leaves and number of shoots 60 and
has been suggested as a candidate marker for re-estab- 90 days after grafting (DAG).
lishment during plant post-grafting in fruit trees (Pina and
Errea 2008). This enzyme (UGPase; EC 2.7.7.9), catalyses Real time qRT-PCR expression analyses
the interconversion of starch and sucrose being responsible
for the synthesis of UDP-glucose, which is an important Total RNA was isolated from stem bark tissue containing
precursor in the biosynthesis and regulation of plant cell 5 cm of the different graft interfaces and ungrafted plants
wall components, including cellulose, hemicellulose and 30 and 60 DAG using the CTAB protocol described by
pectin (Ciereszko et al. 2001; Kleczkowski 1994, 2004; Korimbocus et al. (2002). RNA integrity was confirmed
Kleczkowski and Decker 2015). using denaturing agarose gel electrophoresis and concen-
A decrease in the accumulation and activity of the UDP- tration was measured using a NanoDrop ND1000 Spec-
glucose pyrophosphorylase was associated to graft trophotometer (NanoDropÒ Technologies).
incompatibility in apricot/plum combinations (Pina and Total RNA (2 lg) was treated with RNase-free DNase I
Errea 2008). Likewise, Prinsi et al. (2015) suggested that (FermentasÒ) and then used for cDNA synthesis using the
the pear/quince incompatibility is somehow associated High Capacity RNA-to-cDNA Master Mix kit (Invitro-
with an alteration of the carbohydrate metabolism in pear genÒ) and the SuperScript III Reverse Transcriptase Kit
tissues. However, UGPase expression and activity have not (InvitrogenÒ) according to the manufacturer’s instructions.

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Acta Physiol Plant (2016) 38:79 Page 3 of 8 79

Gene-specific primer pair (Forward) 50 -AGAACTA approximately 55 kDa) were excised from the gel, washed
ATCCATCAAACCCT-30 and (Reverse) 50 -CACCAAAC several times with sterilised water and subjected to
CATACATCACCA-30 were used for real time amplifica- MALDI-TOF analysis essentially as described in Pina and
tion. Each reaction was performed in a total volume of Errea (2008).
10 ll containing 60 ng of cDNA and 0.2 lM of each
primer, using the following cycle conditions: 5 min at Experimental design and statistical analysis
95 °C, followed by 45 cycles of 15 s at 95 °C and 60 s at
60 °C and a final dissociation curve in a StepOnePlus The experiment was conducted using a randomised complete
Real Time PCR System (Applied BiosystemsÒ). Relative block design with four blocks of scion/rootstock unions for
expression levels were normalised using the 18S rRNA vegetative growth, gene expression and enzyme activity.
gene from cherimoya plants using the forward primer There were at least three replicates per group for grafted
(F) 50 -CGCAAATTACCCAATCTTGA-30 and reverse 50 - plants at 30 and 60 DAG, and four replicates were evaluated
ACTCATTCCAATTACCAGACTC-30 (R) (González- for ungrafted plants. The data were analysed using the soft-
Agüero et al. 2011). Relative quantification was deter- ware SAS 9.2. Variance homogeneity of the treatment
mined using the 2-DDCt method as described previously groups was analysed using Levene’s Test. The data were
by Livak and Schmittgen (2001). Amplification efficien- then analysed using ANOVA, and the group means were
cies were calculated from the amplification plots using the compared using Tukey’s Test at 5 % probability (p B 0.05).
program LinRegPCR (Ramakers et al. 2003). The quan-
titative data were analysed using the Relative Expression
Software Tool (Rest2009ÒQiagen), and differences were Results
considered significant compared to the ungrafted and
homograft (atemoya grafted onto atemoya) control at Plant growth
p B 0.05.
At the time of grafting, the ungrafted plants were of uni-
Protein extraction, UGPase activity assays form diameter and height, which facilitated grafting.
and MALDI-TOF analysis However, ungrafted atemoya and araticum-mirim plants
differed in leaf number (Table 1). Atemoya had fewer
Total protein was extracted from each graft combination in leaves than araticum-mirim but did not differ from arati-
four replicates. The extraction was performed in 100 mM cum-de terra- fria or biribá, even though all species were
Tris–HCl (pH 7.5), 5 mM MgCl2, 1 mM EDTA, 2 mM grown under the same environmental conditions.
DTT, 0.05 % (v/v) Triton-X-100 and 1 % (w/v) PVPP. The At 60 DAG, the stem diameter, number of leaves and
samples were centrifuged at 9000g for 20 min at 4 °C, and shoots did not differ between the grafted plant combina-
the supernatants were collected as described previously tions. There were significant differences (p B 0.05) in
(Ciereszko et al. 2001). To ensure equal loading of the plant height between the graft combinations atemoya onto
samples, aliquots of the crude protein extracts were used to araticum-mirim and atemoya onto biribá (Table 1). At 90
determine the protein concentrations using the Bio-Rad DAG, there were significant differences (p B 0.05)
protein assay using bovine serum album (BSA) as the between atemoya onto araticum-de-terra-fria and atemoya
standard. UGPase enzymatic activity was examined fol- onto biribá with respect to plant height with other graft
lowing the methodology described by Ciereszko et al. combinations atemoya onto atemoya and atemoya onto
(2001) with minor modifications. Protein extracts (2 ll) araticum-mirim. A significant increase in plant height of
were mixed in extraction buffer containing 100 mM of grafted atemoya onto the rootstocks araticum-de-terra-fria
Tris–HCl, 5.0 mM of MgCl2, 0.8 mM of NAD, 0.8 mM of and biribá was obtained with longer graft length for these
UDP-glucose as substrate, glucose 1,6-bisphosphate, 4 grafted plants. Regarding number of leaves, the combina-
phosphoglucomutase units and dehydrogenaseglucose-6- tion atemoya onto biribá had the higher number of leaves
phosphate (SigmaÒ). Finally, 2 mM of pyrophosphate than the other graft combinations, but the number of shoots
(PPi) was added to initiate the reaction. UGPase activity did not differ between them (Table 1).
was measured based on the absorbance at 340 nm, and
reported in units of lmol min-1 (g FW)-1. Activity levels UGP gene expression in grafted and ungrafted
were indicated for the media (n = 4) and ± standard error Annonaceae plants
(SE).
Proteins from ungrafted stem tissues of atemoya, ara- To examine the effects of grafting on UGP expression,
ticum-de-terra-fria, araticum-mirim and biribá were sepa- total RNA from grafted plants (at 30 and 60 DAG) and
rated by SDS-PAGE and putative UGPase bands (of ungrafted control plants was subjected to RT-qPCR. The

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Table 1 Growth differences, as

Moment before grafting Stem diameter (cm) Plant height (cm) Number of leaves (unit)
measured by stem diameter,
plant height, number of leaves Atemoya ungrafted 0.9 ± 0.3a 159.3 ± 15.7a 45.3 ± 19.5b
and number of shoots, before
grafting and at 60 and 90 days araticum-de-terra-fria ungrafted 1.2 ± 0.2a 121.0 ± 11.3a 107.3 ± 18.7ab
after grafting araticum-mirim ungrafted 1.4 ± 0.3a 148.3 ± 5.8a 136.5 ± 18.6a
biribá ungrafted 1.1 ± 0.2a 121.3 ± 12.6a 74.3 ± 17.8ab
F value 1.06ns 2.65ns 4.51*
LSD 0.9567 49.966 78.302
60 days after grafting Stem diameter Plant height Number of leaves Number of shoots
(cm) (cm) (unit) (unit)

Atemoya ungrafted 1.4 ± 0.2a 24.8 ± 1.4b 14.4 ± 3.62a 2.2 ± 0.42a
araticum-de-terra-fria 1.5 ± 0.2a 29.4 ± 1.4ab 15.8 ± 2.7a 2.2 ± 0.22a
ungrafted
araticum-mirim ungrafted 1.5 ± 0.1a 25.6 ± 4.9b 13.8 ± 1.1a 2.6 ± 0.27a
biribá ungrafted 1.4 ± 0.1a 37.2 ± 2.1a 17.8 ± 2.6a 2.8 ± 0.22a
F value 0.02ns 4.96* 0.6ns 1.29ns
LSD 0.5754 10.296 9.2176 1.0705
90 days after grafting Stem diameter Plant height Number of leaves Number of shoots
(cm) (cm) (unit) (unit)

Atemoya ungrafted 1.5 ± 0.14a 29.6 ± 4.72b 17.2 ± 3.3b 2.0 ± 0.0a
araticum-de-terra-fria 1.6 ± 0.12a 66 ± 5.65a 21 ± 2.32ab 2 ± 0.0a
ungrafted
araticum-mirim ungrafted 1.5 ± 0.11a 33.6 ± 7.29b 14.2 ± 2.68b 1.2 ± 0.22a
biribá ungrafted 1.7 ± 0.08a 70.6 ± 6.65a 27.6 ± 3.47a 2.0 ± 0.0a
F value 0.91ns 15.02* 5.38* 16ns
LSD 0.4211 22.27 11.11 0.4046
Means (±standard error, SE) followed by the same letter in the column do not differ according to Tukey’s
test at 5 % probability, n = 5
LSD least significant difference

results indicated that the expression of the gene coding for these results indicated that UGP expression differs between
the UGPase was detected in all ungrafted genotypes the investigated graft combinations at the transcriptional
(Fig. 1a). The constitutive UGPase transcription level level.
depends on the genotype, ranging from high levels in the
rootstock biribá to lower levels in atemoya, araticum-de- UGPase activity and protein identification
terra-fria and araticum-mirim (Fig. 1a). As shown in
Fig. 1, significant differences in relative UGP expression To determine whether UGPase activity is affected by
were observed between ungrafted and grafted plants and grafting, we measured enzyme activity in ungrafted and
the mRNA expression levels followed the same trend grafted plants immediately after grafting and at 30 and 60
between graft combinations at 30 and 60 DAG (Fig. 1b, c). DAG (Table 2). Among the ungrafted plants, atemoya
UGP expression levels were significantly different between exhibited the greatest UGPase activity compared to the
the tested homograft atemoya onto atemoya and the other genotypes. In contrast, the homograft atemoya onto
heterografts atemoya onto araticum-de-terra-fria and ate- atemoya exhibited lower UGPase activity compared to
moya onto araticum-mirim. The graft combination ate- ungrafted atemoya at 60 DAG, but not 30 DAG (Table 2).
moya onto araticum-de-terra-fria exhibited higher UGP The graft combination atemoya onto araticum-de-terra-fria
expression levels than atemoya onto araticum-mirim which exhibited greater UGPase activity than the ungrafted
showed lower UGP expression than the homograft and rootstock araticum-de-terra-fria; this difference was of
ungrafted atemoya throughout graft union development equal magnitude to the difference in UGPase activity
(Fig. 1b, c). UGP expression levels in ungrafted biribá observed between the ungrafted rootstocks araticum-de-
were higher than those determined for atemoya onto biribá terra-fria and atemoya. The graft combination atemoya
and the atemoya reference plant, respectively. Overall, onto araticum-mirim showed the same trend as atemoya

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Acta Physiol Plant (2016) 38:79 Page 5 of 8 79

Fig. 1 Relative UGP gene

expression in Annona plants.
a Ungrafted plants (0 DAG);
b Grafted plants at 30 DAG;
c Grafted plants at 60 DAG. The
data represent averages and
standard errors obtained from
three biological replicates
(*p B 0.05). Ungrafted atemoya
was used as the reference
(expression arbitrarily set to 1)

Table 2 UGPase activity assessed in ungrafted and grafted Annonaceous plants

UGPase activity [lmol min-1 (g FW)-1]
DAG (days after Atemoya onto Atemoya onto Atemoya onto Atemoya
grafting) atemoya araticum-de-terra-fria araticum-mirim onto biribá

0 17.90 ± 0.16Aa 14.80 ± 0.19Bb 8.1 ± 0.80Dc 10.5 ± 0.18Cc

30 15.63 ± 1.35BCab 18.80 ± 0.43Ba 12.7 ± 0.50Cb 22.1 ± 0.42Aa
60 13.17 ± 0.28Bb 19.90 ± 1.44Aa 18.58 ± 0.47Aa 13.95 ± 0.74Bb
Means followed by the same capital letter (lines) and lower case letter (columns) do not differ according to Tukey’s Test at 5 % probability
UGPase activity levels are reported for ungrafted atemoya, araticum-de-terra-fria, araticum-mirim and biribá immediately before grafting (0
DAG), and for atemoya onto atemoya (atemoya onto araticum-de-terra-fria, atemoya onto araticum-mirim and atemoya grafted onto biribá at 30
and 60 DAG
a = \0.0001* (treatments); b = \ 0.0001* (time); a 9 b = \0.0001* (treatments 9 time)

onto araticum-de-terra-fria during graft union develop- ungrafted atemoya and araticum-de-terra-fria. However,
ment, reaching values similar to ungrafted atemoya at 60 when grafted atemoya onto biribá, this union exhibited
DAG. These data showed that grafting plants with atemoya greater UGPase activity at 30 DAG compared to ungrafted
increases UGPase activity (Table 2). The ungrafted root- biribá, which turned to be similar to that of ungrafted
stock biribá exhibited lower UGPase activity than atemoya. At 60 DAG, UGPase activity in this graft

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seedling formation in commercial orchards (Kavati 1992,

2013; Tokunaga 2005).
In contrast, no increase in UGP expression was detected
when atemoya was grafted as scion onto araticum-mirim
rootstock, suggesting that no significant interaction occur-
red between these species, as observed with the araticum-
de-terra-fria plants. Intriguing, the observed UGPase
expression pattern clearly did not impair the post-graft
restoration of the plants, especially because the corre-
Fig. 2 The UGPase amino acid sequence obtained using peptide
maps generated from in-gel trypsin digests. UGPase of the atemoya sponding graft combination survived the entire experi-
rootstock is approximately 55 kDa in mass. The protein was excised mental observation period (60 DAG). Nevertheless, a
from a 15 % polyacrylamide gel, eluted, and analysed using mass previous report suggests that araticum-mirim rootstock
spectrometry. Trypsinised peptides matching UGPase from cheri- induces plant dwarfing, and as consequence, the resulting
moya are shown in bold. Peptide mass fingerprints were searched
using the Mascot search engine plants do not survive longer than 4 years in orchards due to
growth restrictions in the grafted stem tissue (Tokunaga
2005). This response is supposed to reflect incompatibility
combination decreased to similar levels to those observed in the induction or repression of genes between plants
in atemoya onto atemoya at 60 DAG (Table 2). (Prassinos et al. 2009).
In addition, we evaluated the presence of UGPase pro- In addition, previous research describes biribá species as
tein in ungrafted atemoya, araticum-de-terra-fria, arati- being immediately incompatible after grafting. It has been
cum-mirim and biribá immediately before grafting. reported that they lack a stem tissue union during the
UGPase identification was performed using SDS-PAGE in nursery phase after grafting, and consequently cannot sur-
combination with MALDI-TOF. Protein bands around vive (Almeida et al. 2010; Santos et al. 2005). However,
55 kDa were subjected to trypsin digestion and MALDI- biribá plants can be restored during the initial period post-
TOF analysis to rapidly calculate the peptide masses. The grafting (until 60 DAG). Here, we show that UGPase gene
MALDI-TOF spectrum identified 470 peptides comprising expression in the graft combination atemoya onto biribá
the trypsin fragments from atemoya genotype, and these was similar to that in the homograft atemoya onto atemoya
fragments were used as queries against the Mascot data- and ungrafted atemoya but lower than that in ungrafted
base (Fig. 2). Of these peptides, 31 % (145) matched biribá. Several studies have found incompatibility symp-
cherimoya, UDP-glucose pyrophosphorylase (accession toms when atemoya was grafted on the rootstock biribá
number NCBI: ACN50183.1). However, UGPase derived 1 year of orchard growth that include: shoot death, drying
peptides were not identified in the ungrafted rootstocks out of scion branches (Kavati 2013) and blackening at the
araticum-de-terra-fria, araticum-mirim and biribá. graft interface due to the presence of phenolic compounds
(Almeida et al. 2010).
It is evident from the present study that the atemoya
Discussion scion affected UGPase activity depending on the rootstock/
scion combination. In this context, enzymatic activity in
UDP-glucose pyrophosphorylase (UGPase) plays an ungrafted plants was lower than in ungrafted atemoya. All
important role in the production/metabolism of UDP-glu- tested graft combinations exhibited higher UGPase activity
cose, a key metabolite for sucrose and cell wall biosyn- than the rootstock and ungrafted atemoya; however, none
thesis (Lerouxel et al. 2006). Graft incompatibility has also of the grafts exhibited symptoms of incompatibility. Sur-
strong effects on regulation of UGPase gene in apri- prisingly, only the atemoya onto araticum-de-terra-fria
cot/plum combinations (Pina and Errea 2008). In this showed a positive correlation between UGP gene expres-
context, early increased expression of this gene is indica- sion and UGPase activity, while this correlation was
tive of a rapid union of plant tissues after grafting. The moderated in atemoya grafts with both araticum-mirim and
UGP expression results described here corroborate the biribá at 30 and 60 DAG, respectively. A previous study
existence of a significant interaction between scion and found little or no correlation between UGP gene expression
rootstock after grafting. According to our results, UGP and activity between ungrafted and grafted combinations of
expression levels were higher in grafts of atemoya onto plum and nectarine 2 weeks after grafting (Pina and Errea
araticum-de-terra-fria than in ungrafted atemoya species, 2008). In this case, the suggestion that UGPase is involved
which exhibited naturally lower UGP expression. These in the success of the graft is supported by the observation
findings are consistent with the literature indicating that that UGPase transcripts were diminished in the rootstock
atemoya grafts with araticum-de-terra-fria are suitable for from incompatible combinations belonging to the genus

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Acta Physiol Plant (2016) 38:79 Page 7 of 8 79

Prunus (plum and apricot) compared with compatible ones contributed with manuscript preparation and approved the
(Pina and Errea 2008). The same study found that there was manuscript in its final form.
no correlation between the expression and activity of
UGPase in the apricot cultivar under different graft part- Acknowledgments The authors wish to thank the Foundation for
Research Support of the State of São Paulo (FAPESP). Daniel Baron
ners (ungrafted, homo- and heterografts), suggesting a was the recipient of FAPESP Grant n° 2011/00853-8 and FAPESP
posttranscriptional/translational regulation of the enzyme. B.E.P.E. Grant n° 2013/22036-7.
Regarding the MALDI-TOF results, we found that only
ungrafted atemoya presented UGPase-derived amino acid
sequences that were similar to that of the UGPase descri- References
bed in cherimoya. One possible explanation is that atemoya
is an interspecies cross between sweetsop and cherimoya Abe T, Niiyama H, Sasahara T (2002) Cloning of cDNA for UDP-
glucose pyrophosphorylase and the expression of mRNA in rice
(Sanewski 1991). On the other hand, the UGPase derived endosperm. Theor Appl Genet 105:216–221. doi:10.1007/
amino acid sequences from ungrafted araticum-de-terra- s00122-002-0927-z
fria, araticum-mirim and biribá could not be aligned to Almeida LFPd, Alencar CMd, Yamanishi OK (2010) Propagação por
known plant UGPases sequences. Although the entire enxertia de atemoia ‘Thompson’ sobre espécies de Rollinia. Rev
Bras Frutic 32:653–656. doi:10.1590/S0100-2945201000500
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The possible existence of other UGPase isoforms play- Baron D, Ferreira G, Boaro CSF, Mischan MM (2011) Evaluation of
substrates on the emergence of ‘‘araticum-de-terra-fria’’ (Annona
ing a role in the regeneration of the grafted plants is also emarginata (Schltdl.) H. Rainer) Seedlings. Rev Bras Frutic
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native splicing and post-translational modifications (such Phosphate status affects the gene expression, protein content
as phosphorylation, glycosylation and acetylations), have and enzymatic activity of UDP-glucose pyrophosphorylase in
been proposed as important for the regulation of UGPase wild-type and pho mutants of Arabidopsis. Planta 212:598–605.
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activity and the formation of UGPase variants (Chen et al. Cookson SJ, Moreno MJC, Hevin C, Mendome LZN, Delrot S,
2007; Meng et al. 2007, 2009). Recently, a UGPase from Trossat-Magnin C, Ollat N (2013) Graft union formation in
sugarcane has been found to be phosphorylated in vivo grapevine induces transcriptional changes related to cell wall
(Soares et al. 2014). Moreover, post-translational modifi- modification, wounding, hormone signalling, and secondary
metabolism. J Exp Bot 64:2997–3008. doi:10.1093/jxb/ert144
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Kleczkowski et al. 2004; Meng et al. 2009). in stress responses at the graft interface when compared with
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