Journal of Experimental Botany, Vol. 56, No. 409, pp. 101–112, January 2005
doi:10.1093/jxb/eri015 Advance Access publication 1 November, 2004
RESEARCH PAPER
Developing pineapple fruit has a small transcriptome
dominated by metallothionein
Richard Moyle1, David J. Fairbairn1, Jonni Ripi1, Mark Crowe2 and Jose R. Botella1,*
1
Plant Genetic Engineering Laboratory, Department of Botany, University of Queensland, QLD 4072, Australia
2
Institute of Molecular Biosciences, University of Queensland, Australia
Received 5 April 2004; Accepted 20 August 2004
Abstract
Introduction
In a first step toward understanding the molecular basis
of pineapple fruit development, a sequencing project
was initiated to survey a range of expressed sequences
from green unripe and yellow ripe fruit tissue. A highly
abundant metallothionein transcript was identified during library construction, and was estimated to account
for up to 50% of all EST library clones. Library clones
with metallothionein subtracted were sequenced, and
408 unripe green and 1140 ripe yellow edited EST clone
sequences were retrieved. Clone redundancy was high,
with the combined 1548 clone sequences clustering
into just 634 contigs comprising 191 consensus sequences and 443 singletons. Half of the EST clone
sequences clustered within 13.5% and 9.3% of contigs
from green unripe and yellow ripe libraries, respectively, indicating that a small subset of genes dominate
the majority of the transcriptome. Furthermore, sequence cluster analysis, northern analysis, and functional classification revealed major differences
between genes expressed in the unripe green and ripe
yellow fruit tissues. Abundant genes identified from the
green fruit include a fruit bromelain and a bromelain
inhibitor. Abundant genes identified in the yellow fruit
library include a MADS box gene, and several genes
normally associated with protein synthesis, including
homologues of ribosomal L10 and the translation
factors SUI1 and eIF5A. Both the green unripe and
yellow ripe libraries contained high proportions of
clones associated with oxidative stress responses
and the detoxification of free radicals.
Pineapple (Ananas comosus L. Merrill) fruit cropping and
processing are important horticultural industries in many
countries with tropical climates. In terms of worldwide
production, pineapple is currently the third most important
tropical fruit after bananas and mangoes. Pineapple is
cultivated mainly for fresh or canned fruit and juice, but is
also the only source of bromelain, a complex proteolytic
enzyme used in the pharmaceutical market and as a meattenderizing agent.
Pineapples are diploid (2n=50) perennial monocotyledonous plants with a terminal inflorescence consisting
of 50–200 individual hermaphrodite flowers that cluster
together. As individual fruits develop from the flowers, they
join together forming a cone shaped, compound, juicy,
fleshy multiple fruit of approximately 30 cm or more in
height. Pineapple maturity is evaluated by the fruit eye
flatness, the extent of skin yellowing and by aroma. It takes
3–5 months for pineapple fruit to reach maturity and
pineapple fruit quality is highest if the fruit matures on
the plant. Pineapples harvested prematurely do not continue
to ripen or sweeten, as there are no starch reserves in the
fruit to be converted to sugar.
In general, fleshy fruits are divided in two large groups,
climacteric and non-climacteric, based upon the presence or
absence of an autocatalytic ethylene burst during ripening.
Pineapple is an example of a non-climacteric fruit. It does
not have an autocatalytic ethylene burst during ripening,
and exogenous application of ethylene does not rapidly
accelerate fruit ripening. In climacteric fruits, ethylene
plays a crucial role in the control of the ripening process, by
regulating the transcription of a large number of genes. The
ethylene biosynthetic pathway is well established (Yang and
Hoffman, 1984) and the expression patterns of the main ethylene biosynthetic genes, 1-aminocyclopropane-1-carboxylic
Key words: Ananas comosus, EST, metallothionein, nonclimacteric fruit, pineapple, fruit ripening.
* To whom correspondence should be addressed. Fax: +61 7 33651699. E-mail: [email protected]
Journal of Experimental Botany, Vol. 56, No. 409, ª Society for Experimental Biology 2004; all rights reserved
102 Moyle et al.
acid (ACC) synthase (ACS) and ACC oxidase (ACO),
have been well characterized (Giovannoni, 2001). Conversely, almost nothing is known of ripening processes
in non-climacteric fruit, although ripening-related genes
from non-climacteric fruits have been reported with grape,
Citrus unshiu, and strawberry among those most studied.
Davies and Robinson (2000) undertook the isolation of 17
ripening-associated cDNAs by differential screening of
a shiraz grape library. An EST sequencing project on grape
buds has also been reported, but the library clones were
isolated to investigate gene expression leading up to bud
release from dormancy (Pacey-Miller et al., 2003). A smallscale sequencing project from Citrus unshiu resulted in the
isolation of 297 EST sequences (Moriguchi et al., 1998). In
addition, an EST sequencing project has been reported from
strawberry, resulting in the isolation of 1100 clone sequences (Aharoni et al., 2002; Aharoni and O’Connell, 2002).
Microarray analysis of the strawberry clones identified
112 genes that were ripening-regulated (Aharoni and
O’Connell, 2002). A number of previously identified genes
involved in cell wall softening and pigmentation were
differentially expressed during strawberry fruit ripening.
Oxidative stress-related genes were also observed as upregulated in ripening strawberry fruits, and it has been
suggested that strawberry fruits may contain a transcriptional programme responsive to oxidative stress induced
during ripening (Aharoni and O’Connell, 2002). Despite
these efforts to isolate ripening-related clone sequences, the
relatively small number of genes available for study has
hampered the identification of genes controlling the ripening processes in non-climacteric fruits.
The development and automation of high-throughput
sequencing technology provides an opportunity to study
genomes at both the DNA and RNA level. Genomesequencing projects can lead to the identification of the
complete catalogue of genes within an organism, independent of gene expression levels. Alternatively, large-scale endsequencing of cDNA library clones generates expressed
sequence tags (ESTs) that can be used to obtain a snap-shot
of gene expression in a particular tissue and stage of
development at a fraction of the cost of genome sequencing
(Adams et al., 1993). EST sequencing has proved a popular
and cost-effective method of isolating vast collections of
coding sequences from a plethora of species. As at February
2004, the GenBank dbEST (database of expressed sequence
tags) contained 20 039 613 EST sequences from 611
organisms (Boguski et al., 1993). Substantial numbers
of EST sequences have been generated from a number
of plants, with plant species comprising 12 of the 30 most
abundant EST collections. In addition, comparing EST
clones generated from different tissues at different stages
of development provides a method for comparing relative
gene expression and identifying subsets of genes and
corresponding functions that probably play vital roles within
specific tissue types and stages of development.
Despite the economic importance of pineapple as a crop,
surprisingly little research has been undertaken to understand the molecular basis of pineapple fruit development.
A search of the GenBank nucleotide sequence database
(Benson et al., 2003) yielded only 51 sequence entries for
Ananas comosus (as of February 2004), the majority being
isolated from root and vegetative tissues. Almost half of
these sequence entries resulted from a study reporting 24
genes isolated from pineapple roots by differential display
(Neuteboom et al., 2002). This study reports the first EST
sequencing project from pineapple, resulting in the isolation
of 1548 fruit EST clone sequences. As far as is known, this is
the first report of EST sequencing on any pineapple tissue or
indeed on any bromeliad. The isolation of a range of pineapple gene sequences from green unripe and yellow ripe fruit
tissue is a first step toward building a valuable resource that
will help in understanding the molecular basis of fruit development and will aid pineapple improvement programmes.
Materials and methods
Plant material
Commercial field-grown pineapples (A. comosus L. cv. Smooth
Cayenne) were harvested early in the morning and graded according
to ripening appearance. Selected pineapple fruits were separated into
top, middle, and bottom thirds, with the skin and pith tissues removed. The remaining fruit flesh was snap frozen in liquid nitrogen,
pulverized, and stored at ÿ80 8C.
cDNA library construction and sequencing
PolyA RNA was isolated from the middle third of pineapple 4 (Fig.
1), a mature size but unripe green pineapple, and pineapple 9 (Fig. 1),
a mature ripe yellow pineapple, using the oligo dT dynabead kit
(Dynal). PolyA RNA was transcribed into cDNA and subsequently
amplified using the SMART cDNA synthesis kit (Clontech Laboratories, Palo Alto, CA). SMART cDNA synthesis utilizes a mechanism
to enhance the proportion of cDNA containing full-length coding
sequences, allows for PCR amplification of the synthesized cDNA,
and introduces SfiI restriction enzyme sites for directional cloning.
The resulting cDNA was size fractionated by electrophoresis through
0.8% agarose gels. cDNA fragments running higher than approximately 700 bp were recovered and purified using the GeneClean II kit
(Bio101). The resulting cDNA pools were digested with SfiI restriction enzyme and directionally cloned into pBS-SfiI. pBS-SfiI was
engineered by inserting a linker containing SfiI directional cloning
sites between the EcoRI and XhoI restriction enzyme sites of
pBLUESCRIPT (Stratagene). Transformed colonies were picked at
Fig. 1. Commercial field-grown pineapples harvested and sorted 1–9
according to visible ripening characteristics.
Gene expression in pineapple fruits
2
random and placed, using a grid pattern, on duplicate 25 cm LB
ampicillin plates, one of which was subsequently screened at high
stringency (0.13 SSC/0.1%SDS at 65 8C) with a radiolabelled metallothionein clone Ac180 using a colony lift procedure (Sambrook
et al., 1989). Colonies not hybridizing to the metallothionein Ac180
probe were picked from the duplicate plate for growth in 96 format
deep well plates. A robotic platform (Biomek 9600) was used to
replicate the 96 well clone plates into multiple glycerol stock bank
collections. Cultures were grown from glycerol stock plates and 59
end sequencing was conducted on purified plasmid preparations.
Bioinformatics
Raw sequences were manually edited for sequence quality, trimmed
of plasmid contaminant and polyA tail in the sequence viewer program Chromas v2.13 (Technelysium). Clones yielding less than 150
bp of insert sequence were eliminated from the data set. All EST sequences were submitted into the GenBank dbEST. Edited sequences
were compiled into contigs or singletons using SeqMan sequence
assembly software (DNASTAR Inc. Madison, USA), and key parameters of minimum 90% match over at least 45 bp overlap. Comparison analysis of the contig consensus sequences was conducted
with the advanced basic alignment search tool, BLASTX server
(Altschul et al., 1990) and the National Center for Biotechnological
Information (www.ncbi.nih.gov) non-redundant protein database and
resulting information parsed from nearest neighbour hits. Contig
consensus sequences returning hits better than the 10Eÿ20 E-value
cutoff were BLASTX searched against MATDB (mips.gsf.de/proj/
thal/db/index.html) to retrieve putative functional classifications.
Functional classes and subclasses retrieved were subjected to manual
curation.
Northern analysis
Total RNA was extracted from meristem, young leaves (shorter than
3 cm in length), mature-sized green leaves, the middle third of
pineapples 1–5 and 7–9 (Fig. 1) using a pineapple RNA extraction
protocol (Cazzonelli et al., 1998). Total RNA was isolated from root
tissue using TRIzol (Invitrogen). Five microgram aliquots of total
RNA from pineapple 1–5, 7–9, meristem, young leaf, old leaf, and
roots were size fractionated through two 1% agarose gels, and
transferred to HyBond N+ membranes (Amersham) by capillary
transfer (Sambrook et al., 1989). The duplicate membranes were
cross-linked by UV and prehybridized in Church and Gilbert buffer
(Church and Gilbert, 1984) at 50 8C. Radiolabelled probes were
synthesized using the Strip EZ DNA kit (Ambion) and hybridized to
membranes at 55–65 8C. Membranes were progressively washed to
a high stringency of 0.1% SSC/0.1% SDS at 60 8C prior to exposure
in phosphoimager cassettes. Membranes were stripped and reprobed
according to the Strip EZ DNA kit instructions (Ambion).
Real-time PCR
For real-time quantitative PCR experiments (RT-qPCR), 1 lg of total
RNA was reverse transcribed using Superscript III (Invitrogen)
following the manufacturer’s instructions. The resulting cDNA was
subsequently taken up in a volume of 250 ll and the fruit and
meristem samples further diluted 100-fold. Pineapple metallothionein
Ac180 and b-actin target genes were amplified using gene-specific
primers designed from the coding sequence, and in the case of Ac180
over an RNA splice junction, using the Primer Express 1.5 software
(Applied Biosystems, Foster City, CA). The primer pairs for Ac180
were 59-TTCTGCCTCATTTATTGGCTCAT-39 and 59-AGCCGTTTCCCTTCTTCACA-39 and for b-actin 59-ATGGAAGCTGCGGGTATTCA-39 and 59-CCACCACTGAGCACGATGTT-39. Both
primers pairs amplified 101 bp amplicons with similar melting temperatures of 81 8C. RT-qPCR was carried out using the ABI PRISM
7700 sequence detector and SYBR Green Master mix (Applied
103
Biosystems) using primers at a final concentration of 0.1 lM each and
2.5 ll (the equivalent of 10 or 0.1 ng total RNA) of cDNA as
template. PCR-cycling conditions comprised an initial polymerase
activation step at 95 8C for 10 min, followed by 45 cycles at 95 8C
for 15 s and 60 8C for 1 min. Real-time DNA amplification was
monitored and analysed using the Sequence Detector 1.7 program
(Applied Biosystems). Threshold cycle data were exported to
EXCEL and the quantification of Ac180 gene expression relative to
the b-actin reference gene was calculated using the Pfaffl equation
(Pfaffl, 2001). This equation takes into account differing primer
efficiencies. Cycle threshold values were corrected for genomic DNA
contamination in the RNA sample. The young leaf sample was used
as a calibrator to allow comparisons across samples to be made.
RT-PCR experiments were repeated on three separate occasions in
duplicate.
Results and discussion
cDNA library construction
PCR amplification of both unripe green and ripe yellow
fruit cDNA and subsequent fractionation through an agarose gel revealed an intense band within the green and
yellow fruit cDNA pools. Subsequent sequence analysis on
a test plate of 96 randomly picked library colonies resulted
in the identification of 40 clones as a member of the metallothionein gene family. Subsequent colonies were prescreened with radiolabelled Ac180 to reduce redundancy
due to the abundant metallothionein clone. One problem
traditionally associated with EST library construction is the
overabundance of short and truncated EST fragments due to
incomplete reverse-transcription and the ligation bias toward small inserts. This can result in a disproportionately
high number of false undiscovered ‘novel’ sequences due
to insufficient coding sequence to establish identity accurately by homology to existing coding sequences. To minimize the overabundance of very short inserts and enhance
the proportion of full-length and large EST fragments in the
libraries, the amplified cDNA were size-fractionated and
those running at less than approximately 700 bp were
discarded. Although the resulting cDNA pools still contain
fragments of less the 700 bp, it is possible that this sizefractionation strategy may subsequently result in the underrepresentation of very short genes in the cDNA libraries.
Restriction mapping of 24 randomly picked clones revealed
an estimated average EST insert size of approximately
1.2 kb for each library. A total of 480 unripe green pineapple and 1536 ripe yellow pineapple clones were subsequently picked for sequencing.
Sequence analysis
Raw sequence quality was consistently high, with over 80%
of clones returning an average Phred20 score of more than
700 bp. After editing raw sequences for quality and
discarding insert sequences of less than 150 bp, a total of
408 green unripe and 1140 yellow ripe pineapple EST sequences were retrieved at an average read length of 785 bp.
104 Moyle et al.
All edited EST sequences have been submitted to the
GenBank dbEST.
The large read lengths obtained compare favourably with
other recently reported EST sequence projects. For example,
Bhalerao et al. (2003) report average read lengths of 349 bp
and 355 bp from autumn leaf and young leaf libraries, while
Ronning et al. (2003) report an overall average read length
of 525 bp from various potato EST libraries. Maximum read
lengths are desirable, as short truncated EST clones containing 39 untranslated sequence with little or no coding
sequence cannot be accurately identified by homology to
protein coding sequences in public databases. The length
of the sequences generated also impacts on EST cluster
analysis, as the longer the EST sequences are, the more
likely that multiple EST clones encoded from a single gene
will overlap during contig assembly.
Cluster analysis reveals high redundancy within
the pineapple fruit EST libraries
All edited EST sequences were clustered using SeqMan
sequence assembly software (DNASTAR). The 1548 sequences clustered into 634 contigs. The unripe green and
ripe yellow sequences clustered with 62% and 54% redundancy, respectively. Further analysis of all contigs
containing two or more green and/or yellow fruit clones
revealed that 1105 ESTs (71% of all sequences) cluster
within the 191 contigs, whereas just 443 ESTs (29%)
represent singletons. To investigate the high level of
redundancy, contigs were listed by clone abundance—from
contigs containing the largest number of EST clones to
those containing a single EST clone from either unripe
green or ripe yellow libraries. The resulting data were
converted to percentages to enable a direct comparison
between the green unripe and yellow ripe cumulative
abundance of EST clones (Fig. 2). The results reveal 50%
of EST clone sequences cluster within just 13.5% and 9.3%
Fig. 2. The cumulative abundance of EST clone sequences from unripe
green and ripe yellow fruit libraries was calculated by sorting contigs
containing the most abundant clones to singletons. The cumulative
numbers of clones and contigs are displayed as a percentage of the unripe
green or ripe yellow clone and contig totals.
of contigs from green unripe and yellow ripe libraries,
respectively. Thus a small subset of genes appears to
dominate the majority of the transcriptome in both unripe
green and ripe yellow fruit. However, as library construction involved an amplification step, the possibility that
amplification bias may have contributed to the high
redundancies cannot be discounted.
Bioinformatics
Contig consensus sequences were exported in FASTA
format and batch BLASTX searches performed against
the GenBank non-redundant database. A stringent BlastX
E-value cut-off of 10Eÿ20 was chosen to ensure that the
annotations were based only on genes with a high degree of
similarity to this study’s cDNA clones (Hu et al., 2003;
Whitfield et al., 2003). A semi-automated process of
parsing BLAST hits and manually curating the putative
annotations resulted in a spreadsheet of information including cloneID, contig number, number of clones in each
contig, nearest BLASTX neighbour, accession number of
match, length of match, percentage similarity, and putative
annotation (supplementary data at JXB online).
Approximately 17% of all sequences did not have
significant homology to coding sequences in the GenBank
non-redundant database and therefore could not be annotated by similarity. This ‘undiscovered’ sequence subset
would probably contain some novel coding sequences that
have not previously been discovered. However, it is unrealistic to suggest that all of the undiscovered EST subset
encode for novel proteins with no significant homology to
those in public sequence databases. Instead, many clones
falling under the undiscovered sequence subset are likely to
contain insufficient coding sequence to accurately assign an
annotation based on homology. It is also possible that some
of these unidentified sequences may not encode proteins,
but instead may function as RNA molecules. Furthermore,
some of the undiscovered sequence ESTs may contain short
stretches of protein coding sequence homologous to proteins in the non-redundant database but, due to a small open
reading frame or short length of overlapping coding
sequence, do not produce an expected value better then
this cutoff limit of 10Eÿ20.
The majority (83%) of EST clones were putatively
annotated by similarity to coding sequences in the GenBank non-redundant database with over 55% of all EST
clones sharing 80% or higher similarity with existing
sequences. Approximately 17% of clones had similarity
of between 60–80%, and less than 5% shared lower than
60% similarity to known coding sequences. Contig consensus sequences returning a valid BLASTX hit from
GenBank were individually searched against the MIPS
MATDB database (Schoof et al., 2002). Manual curation
of MIPS MATDB functional classification resulted in
the assignment of major functional classes and subclasses
Gene expression in pineapple fruits
105
to 1184 EST clones generated from the green unripe and
yellow ripe pineapple libraries.
Metallothionein clone Ac180 is very highly
expressed in pineapple fruits
Fractionating an aliquot of the unripe green and ripe yellow
pineapple cDNA through an agarose gel revealed the
presence of a very highly abundant transcript of approximately 500–600 bp in length. Despite gel purification to
enrich for cDNA greater than 700 bp, sequence analysis of
an initial test plate of 96 clones picked resulted in the
identification of 40 clones as a member of the metallothionein gene family, subsequently named Ac180. Excluding the polyA tail, the metallothionein EST clones were
approximately 500 bp in length, matching the estimated
size of the abundant transcript in the amplified fruit cDNA
pools. When subsequent library colonies were screened
using radiolabelled Ac180 as a probe, up to 50% of the fruit
library colonies screened hybridized strongly. Reports of
highly abundant clones isolated in plant EST sequencing
projects include 14% of EST sequences from a poplar
young leaf library encoding the small subunit of Rubisco
(Bhalerao et al., 2003) and 20% of EST sequences from
a poplar cambium transition to dormancy library encoding
a major storage protein (Schrader et al., 2004). Furthermore, a small-scale EST sequencing project from a Citrus
unshiu fruit library identified 20% of clone sequences as
encoding for metallothionein (Moriguchi et al., 1998).
However, as far as is known, there have been no reports of
a single gene product dominating a transcriptome to the
extent that the metallothionein clone Ac180 appears to
dominate the unripe green and ripe yellow pineapple fruit
tissue. However, as the libraries were constructed from
PCR amplified cDNA, the possibility that the abundance of
the metallothionein clone Ac180 is exaggerated due to
amplification bias cannot be discounted.
The metallothionein Ac180 expression was further analysed by quantitative real-time PCR using first strand
cDNA synthesized from the middle third of the nine
pineapples harvested (Fig. 1). b-actin (Ac42) was used as
a normalization control (Fig. 3). The expression of the
metallothionein Ac180 gene increased during fruit development and was highest in mature fruit. Expression was
highest in fruit tissue, but high levels relative to b-actin
were also observed in root and leaf tissue, particularly in the
older leaf tissue. Metallothionein Ac180 expression was
calculated to be more than 50-fold higher than b-actin
(Ac42) in the ripening fruit samples 7–9, confirming
metallothionein transcripts are highly abundant in ripe
pineapple fruit.
A second metallothionein gene, Ac181, was also discovered in the yellow fruit library with 17 copies isolated.
No copies of Ac181 were isolated from the green fruit
library. The two metallothionein sequences share approximately 80% identity at the amino acid and nucleotide level.
Fig. 3. Quantitative real-time PCR analysis of metallothionein clone
Ac180 gene expression in various pineapple tissues.
Although Ac180 appears to be considerably more abundant
than Ac181, Northern analysis revealed both genes exhibit
a similar expression pattern across the range of ripening
pineapple fruits sampled, and are similarly up-regulated in
older leaves (Fig. 4).
Metallothioneins are small cysteine-rich proteins required for heavy metal tolerance in animals and fungi. In
plants, metallothionein genes are up-regulated in response
to heavy metal stress (van Hoof et al., 2001), natural and
induced leaf senescence (Buchanan-Wollaston, 1994; Chen
et al., 2003), ethylene-induced abscission (Coupe et al.,
1995), drought and salt stress (Oztur et al., 2002), light
stress (Dunaeva and Adamska, 2001), nematode infection
(Potenza et al., 2001), pathogen-induced necrosis (Butt
et al., 1998), abscisic acid (Chatthai et al., 1997; Reynolds
and Crawford, 1996), wounding (Choi et al., 1996),
sucrose starvation and heat shock (Hsieh et al., 1996),
and cold stress (Reid and Ross, 1997). Metallothionein
genes up-regulated during fruit development have been
isolated from climacteric fruits such as banana (Clendennen
and May, 1997), apple (Reid and Ross, 1997), and kiwifruit
(Ledger and Gardner, 1994) and non-climacteric fruit such
as grape (Davies and Robinson, 2000), Citrus unshiu
(Moriguchi et al., 1998), and strawberry (Nam et al., 1999).
Two kiwifruit metallothionein-like clones were differentially expressed during fruit development. pKIWI4 was
up-regulated early while pKIWI3 was up-regulated late in
fruit development. Similarly, two banana metallothioneinlike clones were found to be differentially expressed during
fruit ripening, pBAN3-23 expression decreased during fruit
ripening while pBAN3-6 expression peaked during a later
stage of fruit ripening. For Citrus unshiu, 62 metallothionein clones were isolated from the 297 clones analysed
from a mature fruit library. A comparison of the expression
106 Moyle et al.
Fig. 4. Northern hybridization analysis of total RNA with abundant
pineapple fruit clone probes. RNA was extracted from the middle third of
pineapples 1–9 (Fig. 1), fruit meristem (M), young leaf (YL), old leaf
(OL), and root tissues. Duplicate membranes were prepared and probed
with EST clones from the contig clusters Ac124 (fruit bromelain), Ac122
(bromelain inhibitor), Ac180 (metallothionein), Ac181 (metallothionein),
Ac146 (MADS box protein), Ac3085 (S-like ribonuclease RNS2), Ac183
(mitochondrial ADP/ATP carrier protein), Ac75 (protein translation
factor SUI1), and Ac8 (60S ribosomal protein L10). The northern was
also probed with pineapple actin as a control for observing loading
differences.
of the mature fruit abundant metallothionein clone,
citMT45, and a metallothionein clone previously isolated
from developing fruit, citMT36, again revealed a differential
expression pattern. CitMT36 was detected in all organs
tested whereas citMT45 was strongly up-regulated in fruit
from 70 d after fertilization.
It is not yet clear why metallothioneins are up-regulated
during fruit development, or how they function in plants.
One potential link between the reported up-regulation of
metallothionein during various stress responses and the
abundance during pineapple fruit ripening and in old leaves
could be oxidative stress. Reactive oxygen species accumulate during stress conditions such as wounding, abscission, necrosis, pathogen infection, drought and salt stress,
and multiple genes involved in oxidative stress responses
and the detoxification of free radicals have been isolated
from these fruit libraries. As northern analysis revealed that
a number of other abundant genes share a similar pattern of
expression to the Ac180 and Ac181 metallothionein genes
(Fig. 4), it is therefore possible that a suite of genes are upregulated during fruit ripening and in ageing leaves by
common causal and regulatory mechanisms.
Unripe green and ripe yellow pineapple fruits express
a high proportion of oxidative stress-related genes
Functional classifications were assigned to contig consensus sequences that had an E-value better than 10Eÿ20,
using a manually curated system based on MIPS MATDB
functional classification. Results obtained from contig consensus sequences were subsequently assigned to the individual EST sequences clustering within each contig. The
highly abundant metallothionein gene Ac180 was excluded
from the functional classification analysis.
Functional class distribution indicated that 10% of unripe
green and 6% of ripe yellow clones encode proteins falling
within the cell rescue, defence, and virulence major class
(Fig. 5). This represents an unusually high proportion of
clones, although similar percentages of cell rescue, defence,
and virulence clones have been found in senescing leaves
(Bhalerao et al., 2003). The majority of the clones clustered
within the subclasses representing detoxification and stress
responses, particularly oxidative stress and the detoxification of reactive oxygen species and free radicals. Indices of
oxidative processes have been measured in tomato fruits
(Jimenez et al., 2002). Hydrogen peroxide content, lipid
peroxidation, and protein oxidation were all found to increase at the breaker stage (fully developed green fruit just
prior to turning red). Six EST copies of a (S)-2-hydroxy-acid
gene (Ac3388) were identified from the green pineapple
fruit. (S)-2-hydroxy-acid oxidase (glycolate oxidase) catalyses a reaction resulting in the production of H2O2. It is
a known source of peroxide produced during photorespiration and is enhanced during abiotic stress responses (Mittler,
2002). As (S)-2-hydroxy-acid oxidase appears to be abundant in the green unripe fruit, it is speculated that it is
a significant source of peroxide, contributing to a state of
oxidative stress within the fruit. Peroxiredoxin and glutathione transferase are two genes involved in the detoxification
of H2O2. Peroxiredoxin (Ac21) and glutathione transferase
(Ac135) were among the most abundant gene sequences
from the green fruit library, with six EST copies isolated
each (Table 1). Three copies of peroxiredoxin (Ac21) were
also isolated from the yellow fruit library. Clones encoding
additional glutathione transferase genes were isolated from
the green fruit library (three copies of Ac134 and two copies
of Ac54), and the yellow fruit library (three copies of
Ac55, three copies of Ac2038, and two copies of Ac2248).
Glutathione transferases are up-regulated in many plants in
response to a range of stress conditions (Conklin and Last,
1995; Marrs and Walbot, 1997; Mittova et al., 2003; Moons,
2003; Ruzsa et al., 1999; Skipsey et al., 1997; Thom et al.,
2002) and have been reported as up-regulated during fruit
ripening in strawberry (Aharoni et al., 2002; Aharoni and
O’Connell, 2002). In plants, the functions of glutathione
transferase include detoxification of xenobiotics and the
stabilization of flavanoids (Dixon et al., 2002). In addition,
stress-inducible glutathione transferases have been shown to
protect plants from oxidative injury by functioning as
glutathione peroxidases (Cummins et al., 1999; Roxas
et al., 1997). It is probable that some glutathione transferase
genes are up-regulated by the general oxidative stress caused
during a range of stress conditions (Dixon et al., 2002), and
that oxidative stress also causes up-regulation of glutathione
transferases in fruits. Similarly, plant peroxiredoxins are upregulated during stress. They function as antioxidant proteins, detoxifying various peroxide substrates (Dietz, 2003;
Gene expression in pineapple fruits
107
Fig. 5. The distribution of EST clones by modified MIPS based functional classification for ripe yellow (A) and unripe green (B) fruit.
Hofmann et al., 2002). In addition, two ascorbate peroxidase
genes, Ac50 (two copies) and Ac3096 (one copy), one
monodehydroascorbate reductase gene (Ac3333), and a glutaredoxin gene (Ac37) were isolated from the yellow fruit
library. Ascorbate peroxidase and monodehydroascorbate
reductase also function to scavenge H2O2 (Mittler, 2002)
while glutaredoxin is described as an electron donor to
peroxiredoxin (Rouhier et al., 2003). Three superoxide
dismutase genes, Ac119 (one copy), Ac3200 (one copy)
and Ac3219 (three copies) were isolated from the yellow
fruit library. One copy of Ac3219 was also isolated from
the green fruit library. Superoxide dismutase functions as
a scavenger of Oÿ
2 (Alscher et al., 2002). The relative
abundance of antioxidant proteins and enzymes of the ROS
scavenging pathways in pineapple fruits supports the view
that ripening is an oxidative phenomenon requiring a balance
between the production of ROS and their removal by
antioxidant systems.
The green unripe and yellow ripe pineapple fruit
express different subsets of abundant genes
Unripe green and ripe yellow fruit clones were sorted by
contig abundance for direct comparison. The fruit libraries
shared few abundant genes in common, with most abundant
108 Moyle et al.
Table 1. The 20 most abundant EST clones isolated from green unripe fruit
Putative annotation
Pineapple clone
cluster ID
ESTs from unripe
green fruit
ESTs from ripe
yellow fruit
Fold enrichment in
green unripe fruit
Bromelain inhibitor
Mitochondrial ADP/ATP carrier protein
Unknown protein
S-like ribonuclease RNS2
Undiscovered sequence
Similar to transcription factor IIB
Fruit bromelain
PHD finger transcription factor, putative
GAST-like gene product
Glutathione transferase
Putative cinnamyl-alcohol dehydrogenase
Nuclear transport factor 2, putative
Peroxiredoxin
Putative dehydration-responsive protein
RD22 precursor
Putative (S)-2-hydroxy-acid oxidase
Ubiquitin conjugating enzyme
Unknown protein
Unknown protein
Glyceraldehyde 3-phosphate dehydrogenase
Putative fructose-bisphosphate aldolase
Ac124
Ac183
Ac101
Ac3085
Ac1885
Ac115
Ac122
Ac3400
Ac35
Ac135
Ac176
Ac187
Ac21
Ac3144
52
23
15
15
9
8
8
7
7
6
6
6
6
6
3
28
1
39
27
22
1
0
0
0
4
0
3
1
–
Ac3388
Ac153
Ac187
Ac105
Ac136
Ac3389
6
5
5
4
4
4
0
3
0
0
1
0
gene species being preferentially isolated from either the
unripe green or ripe yellow fruit (Tables 1, 2). Abundant
clones preferentially isolated from green fruit included a
bromelain inhibitor (Ac124) and a fruit bromelain (Ac122).
The bromelain inhibitor was the most abundant clone type
from the green fruit library with 52 clones isolated. Northern
analysis confirmed these two genes are down-regulated
during fruit ripening (Fig. 4). Why bromelain and a bromelain inhibitor are simultaneously expressed in developing fruits is unclear.
Cinnamyl alcohol dehydrogenase (CAD) (Ac176) clones
were isolated from both the green and yellow fruit libraries,
but were more abundant in the green than the yellow fruit
library A CAD gene was reported to be up-regulated during
fruit ripening in strawberry (Aharoni et al., 2002; BlancoPortales et al., 2002). CAD up-regulation in strawberry
fruits has been suggested to play a role in the interconversion
of aldehydes and alcohols implicated in flavour as well as
the lignification of vascular elements (Aharoni et al., 2002).
Eight copies of a putative PHD transcription factor
(Ac3400) were isolated from the green fruit library, while
none were identified among the yellow fruit library clones.
It is possible that this putative transcription factor directs
changes in gene expression during the course of fruit
ripening. Conversely, a MADS box transcription factor
homologue (Ac146) was the most abundant clone type
isolated from the subtracted yellow fruit library, but was not
isolated from the green fruit library. MADS-box genes
encode for a large family of transcription factor proteins
involved in regulating various aspects of plant development, particularly flowering and organ or meristem identity.
Analysis of the Arabidopsis genome revealed 107 genes
encode MADS box proteins, although the function of most
remain unknown (Parenicova et al., 2003). Northern
2
42
1
1
1
22
–
–
–
4
–
6
17
–
5
–
–
11
–
analysis of developing pineapple fruit confirmed that
Ac146 expression increases during ripening and is most
abundant in yellow ripe fruit tissue (Fig. 4). No expression
was detected in meristem, leaf or root tissue. It is somewhat
unusual for a transcription factor to be among the most
highly expressed genes, and, therefore, it is hypothesized
that the differential abundance of this MADS box gene
probably plays a significant role in regulating differential
gene expression during fruit ripening.
Interestingly, a protein translation factor SUI1, a eukaryotic translation initiation factor 5A isoform (eIF 5A), a 60S
ribosomal protein L10, and a putative nascent polypeptideassociated complex alpha chain were all abundant among the
yellow fruit library clones yet absent in green fruit library
clones. Why these translation-related genes are strongly upregulated in the ripe yellow fruit is not immediately obvious
as one would expect protein synthesis genes to be relatively
evenly expressed throughout the plant. However, recent
research in non-plant systems has determined that L10,
SUI1, and eRF5A perform alternative functions in addition
to protein synthesis. The pineapple 60S ribosomal L10 gene
is a homologue of the human QM ribosomal protein (Dowdy
et al., 1991) and the Jun interaction factor Jif-1 from chicken
(Monteclaro and Vogt, 1993). Both QM ribosomal protein
and Jif-1 function as tumour suppressor genes (Dowdy et al.,
1991; Monteclaro and Vogt, 1993), and Jif-1 has been demonstrated specifically to bind to c-Jun, causing the inhibition
of a certain AP-1 transcription factor function. SUI1 translation initiation factors have been implicated in the nonsensemediated decay pathway (NMD) in addition to their role in
recognition of the AUG codon during translation initiation
(Cui et al., 1999). The NMD pathway is an mRNA surveillance pathway, responsible for recognizing aberrant mRNA
transcripts with premature stop codons and accelerating their
Gene expression in pineapple fruits
109
Table 2. The 20 most abundant EST clones isolated from yellow ripened fruit
Putative Annotation
Pineapple clone
cluster ID
ESTs in ripe
yellow fruit
ESTs in unripe
green fruit
Fold enrichment in
yellow ripe fruit
MADS box protein
Undiscovered sequence
S-like ribonuclease RNS2
Mitochondrial ADP/ATP carrier protein
Undiscovered sequence
Similar to transcription factor IIB
Protein translation factor SUI1
Metallothionein
60S ribosomal protein L10
Undiscovered sequence
Glycine decarboxylase complex H-protein
Eukaryotic translation initiation factor 5A isoform 1
Putative uricase
Putative carboxymethylenebutenolidase
Submergence induced protein 2A
Putative auxin-repressed protein
Putative nascent polypeptide-associated complex alpha chain
Similar to protein disulphide isomerase
Calmodulin 4
Cytochrome b5
Ac146
Ac81
Ac3085
Ac183
Ac1885
Ac115
Ac75
Ac181
Ac8
Ac157
Ac138
Ac32
Ac109
Ac175
Ac117
Ac172
Ac185
Ac23
Ac127
Ac131
45
44
39
28
27
22
20
17
17
14
12
11
9
8
8
8
8
7
7
7
0
1
15
23
9
8
0
0
0
0
1
0
1
1
0
0
0
4
1
0
–
16
1
0.4
1
1
–
–
–
–
4
–
3
3
–
–
–
0.6
3
–
subsequent decay (Hillman et al., 2004; Wilusz et al., 2001).
Hypusine modified eIF5A is the active form of the protein
and is thought to act as a nucleo-cytoplasmic transporter of
mRNA from the nucleus to the cytoplasm, in addition to
translation initiation factor activity (Bevec and Hauber,
1997; Rosorius et al., 1999). The eIF5A mediated translocation of mRNA may occur for only certain RNA species,
as hypusine-modified eIF5A was found to bind RNA in
a sequence-specific manner (Xu and Chen, 2001). Yeast
cells, in which DHS has been inactivated are incapable of
dividing, prompted the proposal that hypusine-containing
eIF-5A facilitates translation of a subset of mRNAs required
for cell division. In Arabidopsis, eIF5A expression peaks
in rosette leaves at days 14 and 35 after emergence, coincident with the onset of bolting and the later stages of leaf
senescence (Wang et al., 2003). In tomato, eIF-5A mRNA
show a parallel increase in abundance in senescing flowers,
senescing fruit, and osmotically stressed tomato leaves
exhibiting programmed cell death (Wang et al., 2001).
Northern analysis of the pineapple eIF5A shows an upregulation of the eIF5A during fruit ripening and in older
leaves, consistent with the expression data reported for
Arabidopsis and tomato eIF5A (Wang et al., 2001, 2003).
Two highly abundant undiscovered sequences were
present in the ripe yellow fruit library, but absent in green
fruit library. The undiscovered sequence Ac81 represented
by 44 ripe yellow fruit clones appeared to have a very short
open reading frame encoding 64 amino acids. Although
annotated as an undiscovered sequence, there are two
hypothetical genes from rice that share approximately
64% similarity. The short open reading frame of only 64
amino acids resulted in an expected value falling below the
10Eÿ20 threshold, leading to the annotation as an undiscovered sequence. The undiscovered sequence Ac157
represented by 14 ripe yellow fruit clones is just 283 bp
in length and contains no significant open reading frame,
the longest starting at the fifth ATG and being just 16 amino
acids in length. As 13 of the 14 ESTs for this gene begin at
the same base and there were nine variations in the polyA
addition base, it is believed that the full-length clone
sequence for this transcript has been isolated rather than
partial sequence clones containing only a 39 untranslated
sequence. Together with the lack of a reasonable openreading frame, it is hypothesized that Ac157 may function
as an RNA molecule rather than encode for a protein.
Abundant genes found in common and in relatively
proportionate numbers between the unripe green and ripe
yellow libraries included an S-like ribonuclease, mitochondrial ADP/ATP carrier protein, and a putative transcription
factor IIB. However, northern analysis revealed that the
S-like ribonuclease and mitochondrial ADP/ATP carrier
protein genes are up-regulated during ripening, with highest
expression in the ripe yellow fruit. S-like ribonucleases
have been isolated from many plant species and characterized as being up-regulated during processes including
senescence, phosphate starvation, wounding, and drought
stress (Liang et al., 2002; Ma and Oliveira, 2000; Salekdeh
et al., 2002; Taylor et al., 1993). It has been suggested that
S-like ribonucleases are up-regulated during senescence
and in response to phosphate starvation to play a role in the
remobilization of phosphate.
Comparing clone distribution by functional class
reveals major differences between the unripe
green and ripe yellow fruit libraries
A number of differences between the unripe green and ripe
yellow fruit libraries were observed after clustering clones
by assigned functional classification (Fig. 5). The largest
difference was in the protein synthesis category, where
10% of ripe yellow fruit clones and 2% of unripe green fruit
110 Moyle et al.
clones clustered. The difference is largely attributable to
three abundant gene products, a 60S ribosomal protein L10,
an abundant SUI1 translation factor, and isoforms of
a eukaryotic translation initiation factor 5A.
Nine per cent of unripe green fruit and 4% of ripe yellow
fruit clones fell within the cellular transport functional
class. A single abundant gene encoding a mitochondrial
ADP/ATP carrier protein was largely responsible for the
difference, being twice as abundant in green fruit than in
yellow fruit. Conversely, the transport facilitation class
represented 2% of ripe yellow fruit clones and 1% of unripe
green fruit clones. The difference is largely due to the eight
clones isolated from ripe yellow fruit encoding various
subunits of H+ transporting vacuolar ATPase, compared
with just one clone isolated from unripe green fruit.
The transcription class contained 9% of clones from ripe
yellow fruit and 5% of unripe green fruit clones. The
percentage difference can be accounted for by the presence
of the abundant MADS box gene AC146 from the ripe
yellow library that was not found in the green fruit library.
Interestingly, a closer inspection revealed no transcription
factor genes were isolated in common from both the unripe
green and ripe yellow libraries. The yellow fruit library contained three different MADS box genes, a scarecrow-like protein clone, a bHLH transcription factor, and two different zinc
finger proteins. The green unripe library contained seven
copies of a putative PHD finger transcription factor protein, an
APETALA2 homologue, and a G-box binding factor.
The cellular communication and signal transduction
functional class contained approximately 3–4% of clones
from both the unripe green and ripe yellow libraries.
However, closer inspection revealed major differences in
the composition of the subclasses. The green unripe library
contained many clones that fell within the plant hormonal
regulation subclass whereas the yellow unripe fruit predominantly contained clones involved in Ca2+-mediated
and G-protein-mediated signal transduction.
Approximately 3% of clones from each library fell
within the energy functional class. However, the composition of energy-related clones differs between each library.
The ripe yellow fruit library produced 18 clones related to
electron transport and membrane-associated energy conservation that were not present in the unripe green fruit
library clones. These clones encode for photosystem II
protein K (seven clones), cytochrome b5 (seven clones),
and cytochrome b5 reductase (four clones). The green
unripe fruit produced six clones encoding a putative (S)-2hydroxy-acid oxidase assigned to the respiration subclass
that were absent from the yellow fruit library clones.
Differences were also observed between various metabolism subclasses. The ripe yellow library contained many
clones associated with cysteine biosynthesis that were
absent from the unripe green library clones. In addition,
12 clones of the subclass glycine degradation encoding for
glycine decarboxylase were isolated from the ripe yellow
fruit library, whereas just one clone was isolated from the
green fruit library. The nitrogen and sulphur utilization and
metabolism subclasses contained 11 clones from the yellow
fruit library and only one from the green fruit library.
Approximately 37–40% of the unripe green and ripe
yellow fruit sequences analysed shared homology to unclassified MATDB sequences, and as such were assigned as
of unknown or not clear-cut function.
Summary
A pineapple EST sequencing project was initiated to study
gene expression during fruit ripening and to develop a valuable molecular resource for pineapple biotechnology.
Significant differences have been identified in the most
abundant genes expressed in the green unripe tissue compared with the yellow ripe fruit tissue. The differential expression patterns of the most abundant EST species isolated
were validated by northern analysis of fruit tissue spanning
a range of developmental and ripening stages, as well as
apical meristem, leaf, and root tissues (Fig. 4). Northern
analysis confirmed the fruit bromelain and bromelain inhibitor genes found to be abundant in the green fruit library
are down-regulated during fruit ripening. Two metallothionein genes (Ac180 and Ac181), a MADS box gene (Ac146),
a protein translation factor SUI1 (Ac75), and a 60S ribosomal protein L10 (Ac8) were all abundant in the yellow
fruit library relative to the green fruit library. All were shown
to be strongly up-regulated during fruit ripening by northern
analysis. With the exception of MADS box protein Ac146,
the abundant clones isolated from the yellow fruit library
exhibit a similar expression pattern of up-regulation during
fruit ripening and up-regulation in older leaves. These results
suggest that a suite of genes up-regulated during ripening in
fruits is similarly up-regulated during maturation or senescence in leaves, perhaps by common regulatory processes.
The metallothionein clone Ac180 was extremely abundant in the green unripe and yellow ripened libraries. Over
40% of all library colonies hybridized strongly to a radiolabelled Ac180 probe. The metallothionein Ac180 expression level was further analysed by quantitative real-time
PCR. Expression of metallothionein Ac180 was calculated
at over 50-fold higher than the b-actin control in ripening
fruit tissues. Why metallothionein is so strongly expressed
in ripening pineapple fruit tissue remains unclear.
The present study provides a valuable resource for the
future study of fruit ripening in pineapple, and in nonclimacteric fruits in general. A number of genes likely to
play vital roles in fruit ripening and senescence have been
identified, providing many candidate genes for future
analysis. In addition, the isolation of the EST clone set
will allow microarrays to be constructed for the large-scale
examination of gene expression over a range of fruit development stages. It is hoped to investigate the similarities and
differences in genes expressed in this non-climacteric fruit
Gene expression in pineapple fruits
model compared with climacteric fruit systems. It is also
possible that key gene sequences identified could prove useful for future molecular breeding programmes, as markers
to aid selection of desirable traits in commercial pineapple
lines. The identification of pineapple fruit-specific genes
will also facilitate the isolation of promoter sequences
conferring tight gene control during fruit ripening.
Supplementary data
For supplementary data, please refer to JXB online.
Acknowledgements
This work was supported by the Australian Research Council (ARC)
and Golden Circle Limited.
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