Int. J. Mol. Sci. 2012, 13, 9460-9477; doi:10.3390/ijms13089460
International Journal of
Expression Patterns, Activities and Carbohydrate-Metabolizing
Regulation of Sucrose Phosphate Synthase, Sucrose Synthase
and Neutral Invertase in Pineapple Fruit during Development
, Wei Wang
, Li-Qing Du
, Jiang-Hui Xie
, Yan-Li Yao
Key Laboratory of Tropical Fruit Biology, Ministry of Agriculture, South Subtropical Crop
Research Institute, Chinese Academy of Tropical Agricultural Science (CATAS), Zhanjiang
524091, Guangdong, China; E-Mails: email@example.com (X.-M.Z.);
firstname.lastname@example.org (L.-Q.D.); email@example.com (J.-H.X.); firstname.lastname@example.org (Y.-L.Y.)
Department of Food Science, Lousiana State University, Baton Rouge, LA 70803, USA
Laboratory of Plant Genetic & Breeding, Anhui Agricultural University School of Life Science,
130 Changjiang West Road, Hefei 230036, Anhui, China; E-Mail: email@example.com
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: firstname.lastname@example.org;
Tel.: +86-759-2859205; Fax: +86-759-2858124.
Received: 12 June 2012; in revised form: 14 July 2012 / Accepted: 19 July 2012 /
Published: 26 July 2012
Abstract: Differences in carbohydrate contents and metabolizing-enzyme activities were
monitored in apical, medial, basal and core sections of pineapple (Ananas comosus cv.
Comte de paris) during fruit development and ripening. Fructose and glucose of various
sections in nearly equal amounts were the predominant sugars in the fruitlets, and had
obvious differences until the fruit matured. The large rise of sucrose/hexose was
accompanied by dramatic changes in sucrose phosphate synthase (SPS) and sucrose
synthase (SuSy) activities. By contrast, neutral invertase (NI) activity may provide a
mechanism to increase fruit sink strength by increasing hexose concentrations.
Furthermore, two cDNAs of Ac-sps (accession no. GQ996582) and Ac-ni (accession no.
GQ996581) were first isolated from pineapple fruits utilizing conserved amino-acid
sequences. Homology alignment reveals that the amino acid sequences contain some
conserved function domains. Transcription expression analysis of Ac-sps, Ac-susy and
Int. J. Mol. Sci. 2012, 13 9461
Ac-ni also indicated distinct patterns related to sugar accumulation and composition of
pineapple fruits. It suggests that differential expressions of multiple gene families are
necessary for sugar metabolism in various parts and developmental stages of pineapple
fruit. A cycle of sucrose breakdown in the cytosol of sink tissues could be mediated
through both Ac-SuSy and Ac-NI, and Ac-NI could be involved in regulating crucial steps
by generating sugar signals to the cells in a temporally and spatially restricted fashion.
Keywords: Ananas comosusr; carbohydrate metabolism; enzyme activties; gene cloning;
Fruit taste and quality in pineapple (Ananas comosusr) mainly depend upon such factors as sugars,
organic acids, ﬁrmness, amino acids and aromatic compounds. Sucrose and its components, glucose
and fructose synthesized in source tissues are one of the most important sugars, which are transported
into sink tissues such as fruit, shoots and other tissues . Sucrose serves an integral role as a source of
carbon and energy for non-photosynthetic tissues, which is central to plant metabolism and the most
dominant metabolite involved in fruit growth and development . Until now, attempts to elucidate
the changes in metabolism that lead to accumulation of sucrose had focused on sucrose-metabolizing
enzymes like sucrose phosphate synthase (SPS, EC 184.108.40.206), sucrose synthase (SuSy, EC 220.127.116.11)
and neutral invertase (NI, EC 18.104.22.168) [3,4].
With the full-length or fragment SPS cDNAs isolated from fewer than 20 plant species from
GenBank database, some functional characteristics of SPS were studied both developmentally and in
response to specific stimuli [5–7]. SPS had also been shown to be regulated posttranslationally by
protein phosphorylation, binding to 14-3-3 proteins, and allosteric regulation . Although there is a
direct correlation between sucrose accumulation and SPS enzymatic activity, the mechanism of its
regulation is unknown during fruit ripening .
Some researchers had reported that SuSy activity was correlated with the direction of reversible
sucrose cleavage in plant sink tissues supplied with ample sucrose and with high demand from carbon
biosynthetic and respiratory pathways, cellulose and callose synthesis and sink strength . It plays a
key role in sucrose breakdown for the generation of uridie diphosphate glucose (UDPG) and possible
preservation of energy via uridine diphosphate (UDP) activation of the hexose moiety . In many
species, SuSy had also been identified repeatedly as playing a central role in modulating sink strength
of plants .
To keep the balance of sucrose-sink during fruit growth and development, invertases catalyse the
irreversible hydrolysis of sucrose to glucose and fructose. In plant cells, there are three invertase
isoforms each having different biochemical properties and subcellular localisation. Soluble neutral
invertase (NI) and acid invertase (AI) are located in the cytosol and vacuole, respectively, and
insoluble extracellular invertase is bound to the cell wall. By contrast, the physiological roles of plant
NIs have largely remained elusive until recently, and only limited and fragmentary information is
available, mainly because of low enzyme activity and gene expression levels .
Int. J. Mol. Sci. 2012, 13 9462
In our previous study, a Ac-SPS gene coding pineapple SPS protein was isolated and might play a
key role in sucrose accumulation during the fruit development . Although the importance of sucrose
metabolism in plant development through the generation of sugar signals had been reviewed ,
analysis of function and expression of three enzymes had been limited, especially in sucrose- or
hexose-storing fruits such as pineapple. In this study, differences in carbohydrate contents and
activities of the related enzymes were monitored to evaluate changes in sugar metabolism of different
pineapple parts throughout fruit development. Moreover, three cDNAs coding Ac-susy, Ac-ni and
Ac-sps were isolated from the mRNA of pineapple fruit. A comparison of their molecular structures
and phylogenic origin was presented here, and the relationship between sugar accumulation and gene
differential expressions was also investigated in apical, medial, basal and core sections of pineapple fruits.
2. Results and Discussion
2.1. Soluble sugar Contents
Though fruit flavor is related to both taste (e.g., sugar and acid content) and aroma components, the
main component dictating pineapple fruit eating quality is influenced by total sugar content as well as
composition . In this study, three major sugar (fructose, sucrose and glucose) contents of pineapple
fruits were determined throughout development stages.
In the early stages of fruit growth, hexose (fructose and glucose) was the predominant sugar in
nearly equal amounts except for medial section, accounting for ca. 80% of total soluble sugars
(Figure 1a,b). A parallel gradual accumulation of glucose and fructose contents was detected in the
apical and basal sections from 50 DAA to 70 DAA with the exception of core section, and then
decreased in mature fruits. In medial sections, the fructose and glucose of medial sections also
increased dramatically from 50 to 70 DAA and reached the highest levels at 70 DAA
FW) and 60 DAA (36.37503 mg·g
FW), respectively. Diametrically, the dynamic
activities of two sugars are slightly decreased during fruit growth and maturation in core sections,
which may be related to metabolizing-enzyme activities [13–15].
As reported by many researchers, sucrose is the chief contributor to sweetness, and is transported
through sieve elements and can enter into sink organs directly by the plasmodesmata or the apoplastic
space [15–16]. In our study, gradual increases of sucrose contents were detected starting from 20 DAA
up to the end of fruit growth regardless of fruit sections, and reached 7 fold of initial content in basal
section (Figure 1c,d). This may explain why sweet has a certain difference in the various portions
However, the variation tendency of sucrose was moderate in different parts of fruit between 50 and
60 DAA, accordingly, hexose (fructose and glucose) contents were sharply increased (Figure 1a,b). It
indicated that the conversion of sucrose to hexoses was indispensable and may have other benefits. For
example, hexose accumulation may function to increase the osmotic potential of the tissues and allow
improved nutrient uptake . Hexoses also play a role in regulating gene expression and therefore,
accumulation in the vacuole would permit hexose storage without adverse effects on gene expression
since the hexoses would be effectively excluded from the cytosol .
Int. J. Mol. Sci. 2012, 13 9463
Figure 1. (a) Changes of fructose; (b) glucose; (c) sucrose contents; (d) sucrose/hexose
ratio in the different fruit sections during various development stages. Each point is the
mean of three determinations, and vertical bars are representative of ± S.E. (n = 3).
2.2. Isolation of Ac-Sps, Ac-Ni and Ac-Susy Genes and Sequence Analysis
2.2.1. Multiple Alignments of Three Genes
The present study firstly isolated two cDNAs of Ac-sps and Ac-ni from pineapple fruits utilizing
two pairs of degenerate primers according to the conserved domain of SPS and NI amino acid
sequences from other plant species. Two 1131 bp and 1036 bp cDNA fragments were amplified by
RT-PCR, and named Ac-sps (GenBank ID: GQ996582) and Ac-ni (GenBank ID: GQ996581),
respectively. By alignment, the deduced SPS and NI polypeptides present high-level identities with
corresponding regions of glucosyltransferases and neutral invertase from other plants (data not shown).
In SPS amino acid sequences, the alignment analysis showed that Ac-SPS shared with
amino acid identities of 85%, 84%, 82% and 81% to rice (Oryza sativa, AAQ56529) and
Int. J. Mol. Sci. 2012, 13 9464
green bamboo (Bambusa oldhamii, AAR16190), broomcorn (Sorghum bicolor, XP_002441522),
wheat (Triticum aestivum, AAQ15106) and kiwifruit (Actinidia deliciosa, AAC39434), respectively.
In addition, the encoded NI polypeptide shared 94% identity with rice (O. sativa, EAY86114),
93% identity with broomcorn (S. bicolor, XP002452195) and grape (Vitis vinifera, XP002264960),
88% identity with Arabidopsis (Arabidopsis thaliana, NP197643), respectively. Interestingly, the
amino acid homology of NI is more significant in the carboxyl terminal domain than in the amino
terminal due to the fact that the catalytic domain is concentrated in the carboxyl terminal region within
about 200 residues .
As expected, Ac-SuSy reported (GenBank ID: DQ438976) also showed a high degree of similarity
to those of sucrose synthases from other species, and was thus classified among the large group of
dicot susy genes (data not shown). The carboxyl terminal domain was more conserved than that of
amino terminal. By contrast, the deduced amino acid has 87% homolog with rice (O. sativa, ABF95854)
and maize (Zea mays, ACF78506), 86% homolog with broomcorn (S. bicolor, XP002465303) and
84% homology with soybean (Glycine max, XP003521575).
2.2.2. Domain Characteristics and Phylogenetic Analysis of Ac-SPS
By alignment of the SPS amino acid sequences, some functional domains are highly conserved,
which are mostly located in the N-terminal half of the protein including the glucosyltransferase and
SPP-like domains (Figure 2a). The Fru6P binding region (I) is identical in all sequences with the
exception of an isoleucine substitution for valine in SPS2 of Nicotiana tabacum, The regulatory
phosphoserine (SPS-229) involved in 14-3-3 protein and UDPG binding sites have not been conserved (II),
but the suggested recognition elements for the protein kinase are also nearly identical in all sequences.
SPS is modulated by multisite protein phosphorylation in response to light, osmotic stress
(SolSer-424), nitrogen supply, and temperature and also binds 14-3-3 proteins (SolSer-229) .
A phylogenetic analysis of plant sps genes had been reported previously within the broader A, B,
and C families, according to the classification of Langenkämper et al. (2002) . Although only one
member (AtSPS4) of the C family was represented in the full-length sequences, A and B-families had
obvious internal splits between the dicot and the monocot in our study. Up to now, none of the known
monocot SPS genes belongs to the C family because of the relative scarcity of SPS sequences from
monocots. Family A has 16 members with well-supported internal splits between the 11
dicotyledonous sequences (subfamily a1) and the six monocotyledonous sequences (subfamily a2).
B family has four members containing two monocotyledons and two dicotyledons, respectively.
Though the Ac-SPS from A. comosus is seemed to be allied with the A family, it is somewhat more
divergent than the rest (Figure 2b). It seemed clear that these three gene families must have arisen
before the separation of the monocots and dicots, which was thought to have occurred about
200 million years ago . Furthermore, Lunn (2002) also found that the conservation of the
exon-intron splice sites within the A, B and C family genes in Arabidopsis, rice and maize were
consistent with all three families having a common origin .
Int. J. Mol. Sci. 2012, 13 9465
Figure 2. (a) Conserved regions and phylogenetic analysis of sucrose phosphate synthase
(SPS) protein sequences. (I) some considered function domains were uncovered as the
putative Fru6P binding site; (II) the 14-3-3 regulated phosphoserine and uridie diphosphate
glucose (UDPG) binding domain; (III) the osmotically regulated phosphoserine; (IV, V)
various aspartate-proline pairs determined in either spinach or maize or both . Each
active site of the various motifs was highlighted in capital letters under the alignment.
Numbering of amino acids is according to S. oleracea; (b) the amino acid sequences of the
ORFs were initially aligned and tree was calculated by Neighbor-Joing algorithms and
rooted with Synechocystis. The GenBank accession numbers and name of the sequences
Int. J. Mol. Sci. 2012, 13 9466
2.2.3. Domain Characteristics and Phylogenetic Analysis of Ac-NI
The eight conserved regions of the seven NIs identified from different plants were shown including
the A. comosus in our study (Figure 3a). Two residues (Asp
) attributed to the enzyme
active sites were proposed in І and II conserved regions of NI amino acids. Interestingly, this VII
peptide domain was lack of Val
and changed Ser
with Thr in the conserved peptide sequence.
However, whether the presence of high conserved domains plays a key role in regulating the sugar
metabolism needs to await further analysis.
Thirteen full protein sequences of NI family from 12 organisms were identified from Genbank.
Analysis of the evolution revealed that the NIs of higher plants were classified into the three clades by
comparing the conserved function domains (Figure 3b). The Ac-NI belongs to the clade from Ⅰ
monocots, and is conserved among them sharing about 68%–93% identity between each other. The
other two clades (II, III) are composed of dicots from A. thaliana, Beta vulgaris and V. vinifera, etc.
Ac-NI protein is predicted to be localized in mitochondria, because all proteins from the common
organelles are located in the same cluster divided into two subclusters, plastid- and mitochondria-localized
types . Vargas et al. (2003) suggested that NI homologues were restricted to cyanobacterial species
and plants, and higher plant NIs might have originated from an orthologous ancestral gene after the
endosymbiotic origin of chloroplasts . Though this hypothesis could well explain the presence of
plastid-localized form of neutral invertase, it was difficult to imagine that mitochondrial form in higher
plants occurred from plastid-form by exchanging of targeting sequences to plastids and mitochondria.
2.2.4. Phylogenetic Tree Analysis of Ac-SuSy
In order to explore a comprehensive analysis of evolutionary relationships among SuSy gene
families between pineapple and other plant species, including the twelve isoforms of rice and
Arabidopsis SuSy, a total of 31 plant and one bacteria SuSy amino acid sequences, representing 18
species, were aligned (Figure 4). The phylogenetic tree analysis revealed both relatively deep
evolutionary root and the existence of more recent duplications for the SuSy genes. Using one SuSy
from bacteria as the outgroup, the plant SuSy genes could be subdivided into three clearly distinct
groups, including genes from angiosperms and gymnosperms. These groups were designated class I,
class II and class III, respectively. For pineapple SuSy, it was fallen into this monocot-specific group II,
and clusters together with other monocot genes by forming an independent clade to the exclusion of
dicots genes. As shown in Figure 4, SuSys from dicot and monocotyledonous plants were found in all
the three groups, especially, SuSy proteins in class I can be well classified into two distinct subclades.
In addition, six rice SuSys, six Arabidopsis SuSys and four pea SuSys were also distributed in the
dicot branch of classes I, II, and III (Figure 3). Those results suggested that SuSy evolutionary
divergence originated before the common ancestor of dicots and monocots, and most second
duplication events occurred before the monocot/dicot divergence.
Int. J. Mol. Sci. 2012, 13 9467
Figure 3. Conserved regions and phylogenetic analysis of, neutral invertase (NI) protein
sequences. (a) Large regions of homology are indicated in boxes І-VIII. Putative catalytic
) are highlighted in І and II conserved regions, respectively. The
numbers above the alignment represent the neutral invertases sequence of L. temulentum;
(b) Phylogenetic dendrogram was generated by the multi-alignment using Molecular
Evolutionary Genetics Analysis (MEGA) 4.0 based on identity and rooted with Cyanothece.
Sequences are designated by accession numbers and name of organisms from GenBank
Int. J. Mol. Sci. 2012, 13 9468
Figure 4. Phylogenetic dendrogram of the deduced amino acid sequences of pineapple
SuSy gene constructed using the Neighbor-Joining method with MEGA software 4.0.
Sequences were designated by accession numbers and name of organisms from
2.3. Transcript Expression Analysis of Ac-sps, Ac-susy and Ac-ni genes
In order to evaluate the importance of carbohydrate-metabolizing enzymes throughout pineapple
fruit development, the spatial and temporal expression patterns revealed that three genes were
differentially expressed in immature and mature fruits. 18S rRNA expression levels were chosen as
standards for constitutive expression and gave closely similar results. Expression levels of Ac-sps
mRNA revealed some differences in all developmental stages and different parts of pineapple fruits.
Recent evidence suggested that SPS may play a more important role in sucrose synthesis and
accumulation [7,15,17,19], as supported by the transcript characteristics of Ac-sps in sucrose
accumulation (Figure 5a). In contrast to mRNA levels during the initial phase of anthesis, Ac-sps
transcript levels of fruit different sections were nearly devoid in 20 DAA except for medial parts,
which was associated with low levels of sucrose in immature fruit sections, as also observed by
Botha & Black (2000) . They gradually reached two peaks of accumulation with maxima at 30 and
70 DAA in basal and medial sections, respectively, and then were down-regulated up to 80 DAA.
However, a transcript peak of 30 DAA was lost in apical and core sections. This reason may be caused
by the differences of organic acid and secondary metabolites in various fruit sections .
Experiments with banana had consistently demonstrated SPS activity increase with accumulation of
sucrose in agreement with our results . Strand et al. also provided the evidence that decreased
Int. J. Mol. Sci. 2012, 13 9469
expression of sps can inhibit sucrose synthesis in transgenic A. thaliana . Though a remarkable rise
is showed along with fruit maturation, there is a significant difference between the core and apical
sections and the basal and medial sections (p < 0.05, n = 3) throughout the ripening process of
pineapple fruits. Zhang et al. (2010) also found that different harvest seasons had serious influence on
the activities of carbohydrate-metabolizing enzymes and sugar component in the different stages of
fruit development .
The other most significant contributions of our study are that not only SPS but also SuSy and NI
activities are positively correlated with sugar accumulation. Though SPS activity had also been
implicated in sugar metabolism, the roles of other two enzymes had been less clear . In our
experiments, Ac-susy transcription levels were gradually up-regulated, and exhibited obvious profiles
in the different sections with fruit development (Figure 5b). Thereafter, Ac-susy transcripts in four
different sections showed a marked decrease to basal levels after 70 DAA until harvest. In medial and
basal sections, there are more obvious than the other parts in Ac-susy transcript levels. However, some
studies had attributed sucrose breakdown roles to these two latter enzymes , but our study clearly
indicated that activities of SuSy were increased with sucrose accumulation in different sections of
pineapple fruits, rather than the opposite, similar with results detected in watermelon  as well as
other fruit . These results indicated Ac-susy transcript was bound up with sucrose increase.
Ciereszko et al. also demonstrated that the sucrose/glucose-dependent SuSy expression was strongly
induced in transgenic Arabidopsis hexokinase-overexpressing plants . Although details of this
regulation are unclear, SuSy activity and expression of the corresponding gene(s) were more or less
related with hexokinase.
Due to low enzyme activity and gene transcription levels, NI was scarcely studied .
Nevertheless, higher levels of the Ac-ni transcripts were detected between 30 DAA and 50 DAA with a
transient increase of hexose contents in our study (Figure 5c). On the other hand, though the abundance
of Ac-ni mRNA also increased and had a single peak around 40 DAA regardless of different sections
in the early development of the fruits, Ac-ni expression levels decrease come up more early than that of
Ac-susy and Ac-sps. From 40 to 80 DAA, Ac-ni transcription was down-regulated to a negligible level,
especially in the core and basal sections. These data together suggest the putative role of hexose in
regulating Ac-ni transcription through a negative feedback control had been evaluated. This hypothesis
may be further supported by the Ac-ni transcript levels of different fruit sections, because hexose
accumulation of the basal section analogous to Ac-ni transcript levels precedes the other parts.
Nonis et al. (2007) also reported Ac-ni transcription levels can be improved using the hexokinase
inhibitor NAG . While previous studies on NI revealed small changes in gene expression among
different tissues and during organ development , the pineapple NI studied in this article displayed a
highly modulated pattern of expression in response to developmental clues. The major function of the
high and constant transcription activity of Ac-ni in immature pineapple fruit is to maintain the cellular
hexose concentrations. A decreased invertase activity would lead to increases in sucrose/hexose
ratios [30,31]. This emphasizes not only the continuous hydrolysis of sucrose catalysed by invertase,
but also the possibility of continuous sugar exchange between cytosol and vacuole (sucrose influx and
Int. J. Mol. Sci. 2012, 13 9470
Figure 5. Transcript expression analysis of (a) Ac-sps; (b) Ac-susy; (c) Ac-ni genes during
fruit development using quantitative PCR.
2.4. Detection of Ac-SPS, Ac-SuSy and Ac-NI Activities
As expected, analogous results had been obtained in a comparison of enzyme activities and gene
transcript levels. Those clearly indicated that sucrose accumulation occurs only in the presence of
threshold activities of the three enzymes directly involved in sucrose metabolism. However, the
dynamic patterns of three enzyme activities and transcripts during fruit development could not
compare directly with each other, because multiple gene families could be differentially expressed in
various parts and developmental stages of plants . SPS, SuSy and NI had been reported in different
flowering plants and database searches supported the existence of many isoforms of these
enzymes [10,19,30]. The enzyme activity is synthetically performed by translations of many isozymes,
but the mRNA transcript is one of the isozymes. Moreover, the protein contents of enzyme increased,
and the increase of transcripts may push up the activity of enzymes.
In some fruits, sucrose is translocated from the source to the fruit and is either apoplastically
hydrolysed to fructose and glucose by invertase, or is cleaved symplastically to fructose and
UDP-glucose by sucrose synthase . In the present work, the Ac-NI activities were very low in
basal and core sections at 20 DAA, but rapidly increased and reached maxima (14-fold and 10-fold,
respectively) at 40 DAA. A pronounced increase was also detected in apical and medial activities from
30 to 40 DAA (Figure 6a). Thereafter, activities of various sections showed similar patterns with a
slight peak at 70 DAA, followed by a gradual decrease towards becoming undetectable at 80 DAA. A
positive correlation between sugar accumulation (Figure 1) and levels of SuSy and SPS activities
(Figure 6b,c) were indicated in fruit different sections during development, which is paralleled with the
transcript characteristics of two genes. These results suggest that sucrose synthase can only cleave the
Int. J. Mol. Sci. 2012, 13 9471
sucrose translocated to fruit tissue at a mature stage, and that NI activity is closely related to the
accumulation of fructose and glucose in immature pineapple fruit. This is consistent with the
suggestion that the two steps of sucrose degradation by SuSy and NI and the re-synthesis by SPS in
fruit could generate a locally increased sucrose concentration gradient in the zone of phloem
unloading, thus favoring sucrose import . The potential capacity for re-synthesis of sucrose by the
action of SPS in fruit is also supported with the presence of SPS activity and transcript of Ac-sps,
which increase until the mature stage in fruit different sections (Figure 6c). A series of actions by SuSy
and SPS during maturation could be important to sink strength in citrus fruit at this stage . Thus, NI
may have a role in supplying materials for growth and cell wall construction, whereas SuSy may
supply SPS with substrates (UDP-glucose and fructose) for the re-synthesis of sucrose . Substantial
progress has been also made in studies on SuSy synthesizing UDPG which is essential for sucrose and
cell wall biosynthesis . Since pineapple fruits have a high capacity to cleave sucrose to glucose and
fructose in immature stages (Figure 1), it seems likely that it is glucose rather than sucrose that serves
as a signal in upregulating Ac-susy expression. Though the physiological role of NI and SuSy in
sucrose metabolism is not completely clear in fruit development, the positive correlation with sucrose
accumulation in our study suggests the need to rethink this step of sucrose metabolism in the context of
sink sugar accumulation.
Figure 6. Activity profiles of (a) NI; (b) SuSy; (c) SPS in crude protein extracts of
different fruit sections during pineapple development. Activities were expressed as μmol
product synthesized g
FW. Each point is the mean of three determinations, and
vertical bars are representative of ± S.E. (n = 3).
Int. J. Mol. Sci. 2012, 13 9472
Figure 6. Cont.
3. Experimental Section
3.1. Plant Materials
Field-grown pineapples (A. comosus cv. Comte de paris) were cultivated in the pineapple resource
bank of South Subtropical Crops Research Institute. The experimental design used in this study was
under the same management conditions such as irrigation, fertilization, soil management, disease
control and pruning. Ninety fruit samples had been selected after the full florescence period from April
to June in 2007. The fruits were randomly sampled every 10 days from the 20th day after anthesis
(DAA) and cut transversely into apical, medial, basal and core sections, after the size and weight of
crowns and fruits were measured. And then, the tissues were immediately frozen in liquid nitrogen and
stored at −80 °C before being analyzed. These sliced fleshes of 30 fruits were pooled together as one
of three replications at each harvesting time.
3.2. Plant Materials Determination of Sugar Content and Composition
For glucose, fructose and sucrose content, the sugars were extracted by grinding flesh tissues (10 g)
in 80% ethanol, adjusted to pH 7.0 with 0.1 M NaOH, and heated for 5 min at 80 °C. The extract
corresponding to 0.5 g fresh weight (FW) was dried in vacuum, redissolved in water and the solution
was passed through an ion-exchange column (Dowex 50W-X8 and Dowex 1-X8). The eluate (10 μL)
was then analyzed by high-performance liquid chromatography (HPLC)  (Shimadzu LC-6A;
Kyoto, Japan) equipped with a RI detector and a SP1010 column (Showa Denko K. K., Tokyo, Japan)
at a flow rate of 0.5 mL·min
Int. J. Mol. Sci. 2012, 13 9473
3.3. Cloning and Expression Analysis of Ac-sps, Ac-susy and Ac-ni
3.3.1. Isolation of Total RNA and Sequence Amplification
Total RNA was extracted with Trizol Reagent (Invitrogen, Carlsbed, CA, USA) from different parts
of pineapple fruits. The purity and integrity of total RNA were verified with a spectrophotometer at
230 nm, 260 nm and 280 nm (NanoDrop, Technologies Inc.) and 1.2% denaturing agarose gels
(Qiagen, RNeasy Mini Handbook). For cloning of the genes related to sugar-metabolizing, SMAR™
PCR cDNA Synthesis Kit (BD Biosciences Clontech, United States) was used for the synthesis of
cDNA starting from 0.5–1 μg of poly (A)
RNA according to the instructions of the manufacturer.
Based on conserved amino acids from the available Genbank (http://www.ncbi.nlm.nih.gov/), two
sets of degenerate primers and a pair of specific primers were designed for cloning Ac-sps, Ac-ni and
Ac-susy genes, respectively (Table 1). PCR was carried out in a 25 μL reaction mixture containing
2.5 μL of reaction mixture 10× PCR Buffer II (Takara, Japan), 1.25 mM MgCl
, 4 mM dNTP/analog
Mixture, 1 U ExTaq, 2 μL cDNA, 0.8 μM forward and reverse primers. The reaction mixture was
overlaid with a drop of mineral oil and was allowed to run for 30 cycles (95 °C for 30 s, 52 °C, 54 °C
and 55 °C for 30 s, 72 °C for 1.5 min for Ac-sps, Ac-ni, Ac-susy, respectively). PCR products were
visualized on a 1.2 % agarose gel (w/v) including ethidium bromide (1 μg·mL
), and then cloned into
the pGEM-T Easy Vector System І (Promega, Madison, WI, USA) with T4 DNA ligase (Takara,
Japan) according to the manufacturer’s instructions. Electro-transformation was used for Escherichia coli
(JM109) with the Gene PulserⅡsystem (Bio-Rad Laboratories Inc., Hercules, CA, USA) at 1500 V.
Table 1. Primer sets used for PCR amplification and expression analysis.
Primer name Forward and reverse primes (5'-3') Tm (°C)
Ac-sps degenerate primers
Ac-sps specific primers
Ac-susy specific primers
Ac-ni degenerate primers
Ac-ni specific primers
18S rRNA specific primers
Oligonucleotides designed based on the Ac-susy sequence from pineapple fruit (GenBank ID: DQ438976);
Oligonucleotides designed based on the 18S rRNA sequence from pineapple (GenBank ID: D29786).
3.3.2. Sequence Comparisons
All similarity searches were executed using blastn or blastx algorithms . Multiple alignment and
phylogenetic analysis were carried out using the Clustal W  and viewed by the Megalign (DNAstar
Int. J. Mol. Sci. 2012, 13 9474
Inc.). A phylogenetic tree was constructed by the Neighbor-Joining (NJ) method using the NJ algorithm
implemented in the Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0.
3.3.3. Real-Time PCR Analysis of Gene Expressions
Quantitative PCR was performed by using the first strand cDNA as templates on a Lightcycler
(Roche Diagnostics), with the Light Cycler Fast Start Reaction Mix MasterPLUS SYBR Green
according to the manufacturer’s recommendations. Cycling conditions were as follow: 95 °C for
5 min, 40 cycles at 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 30 s. All amplifications were
normalized with an 18S rRNA gene to confirm equal amounts of RNA template. PCR parameters were
analogous to above descriptions using specific primers (Table 1). The data shown represent means of
values obtained from three independent biological replicates.
3.4. The Extraction and Activity Analysis of NI, SPS and SuSy
Sugar-metabolizing enzymes were prepared essentially in frozen flesh tissue as described by
Zhang et al. . Fruit material was homogenised in 10 mL of ice-cold buffer containing 50 mmol·L
Hepes-NaOH (pH 7.5), 0.5 mmol·L
Na-ethylenediaminetetraacetic acid (EDTA), 2.5 mmol·L
diethyldithiocarbamic acid, 0.5% (w/v) bovine serum albumin (BSA) and 1% (w/v)
insoluble polyvinylpyrrolidone (PVP). After centrifugation at 12,000 g for 20 min at 4 °C,
supernatants were dialyzed for approximately 20 h against 25 mmol·L
Hepes-NaOH (pH 7.5) and
Na-EDTA and used as the crude soluble enzyme extract. The insoluble pellet was
washed twice in homogenising medium and then incubated with shaking for 4 h in ice-cold
homogenising medium containing 1 mol·L
NI activities were assayed in a final volume of 25 mL, containing 0.2 mL of dialyzed enzymatic
extraction, 0.8 mL of reaction solution (pH 4.8 or 7.2, 0.1 mol·L
, 0.1 mol·L
citrate, 0.1 mol·L
sucrose for neutral invertase). The activities were measured by the quantity of
reducing sugars released in the assay media with dinitrosalicylic acid .
Activities of SPS were assayed of using 0.15 mL of reaction medium and 0.2 mL of enzyme
sample. The reaction medium is composed of 50 mmol·L
Mops-NaOH (pH 7.5), 10 mM MgCl
glucose-6-phosphate, 10 mmol·L
fructose-6-phosphate and 5 mmol·L
UDPG. After the
mixture was incubated for 30 min at 37 °C, the reaction was stopped by adding 0.1 mL 30% (w/v)
NaOH and kept in boiling water for 5 min. When cooled to room temperature, the resorcinol solution
(12%, v/v) of 0.5 mL and HCl (12 mol·L
) of 0.5 mL were added into the mixture and held in an
80 °C water bath for 10 min. Blank controls were obtained by adding the sterile water to the reaction
medium containing resorcinol. The procedure for the SuSy assay was identical to that of SPS except
for the reaction mixture, where fructose 6-phosphate or glucose 6-phosphate was replaced by
contained 10 mmol·L
Int. J. Mol. Sci. 2012, 13 9475
3.5. Statistical Analysis
DPSv3.01 for the variance analysis and the correlation analysis by SAS 9.0 according to different
requirements was done. Quantitative analyses were presented as mean values and the reproducibility of
the results was expressed as standard error (S.E.).
In summary, Ac-SPS and Ac-SuSy activities are differentially responsive to sugar dynamic
characteristics and show contrasting expression patterns consistent with different functional roles. The
phenomena are expected to shed further light on the important horticultural phenomenon of sugar
accumulation that determines fruit quality. A cycle of sucrose breakdown in the cytosol of sink tissues
could be mediated to keep the balance of sugars through both Ac-SuSy and Ac-NI. In addition, we also
conclude that Ac-NI could be involved in regulating crucial steps of plant development or at least play
a supportive role to other sugar metabolism enzymes by providing substrates to the cells and by
generating sugar signals in a temporally and spatially restricted fashion.
Conflict of Interest
The authors declare no conflict of interest.
Financial support was provided by the Hainan Natural Science Fund of China (808182), Special
Fund for Agro-scientific Research in the Public Interest (3–41), and Basic Scientific Research Project
of Nonprofit Central Research Institutions (200701).
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