BACKGROUND
pineapple  is a tropical fruit crop of significant commercial value and is the most important economic plant in the bromeliaceae family.
however, surprisingly little research has been undertaken to understand the molecular basis of pineapple fruit development - an essential pre-requisite for future improvement by molecular breeding or genetic modification.
in terms of fruit ripening, fleshy fruits are classified as either climacteric or non-climacteric.
in climacteric fruits, such as apples and bananas, a burst of ethylene biosynthesis and an increase in respiration is observed at the onset of ripening.
conversely, non-climacteric fruits such as pineapple lack the autocatalytic ethylene burst and the increase in respiration.
although the physiological changes that occur during pineapple fruit development are well known, little is known about the molecular events that govern pineapple fruit ripening.
the advent of high throughput sequencing and microarray technologies has facilitated large-scale studies on gene expression changes during fruit development in a number of species, including climacteric fruits tomato and apple, and the non-climacteric fruit strawberry  <cit> .
in a previous study, we reported the construction of subtracted and normalized est cdna libraries, including the green mature and yellow mature ripening stages of pineapple fruits  <cit> .
more recently, short read next generation sequencing technology was applied to a ripe pineapple fruit gene discovery project  <cit> .
in this study, we report the development of an est-based pineapple microarray and its use to identify differentially expressed genes during fruit ripening.
online bioinformatics tools were used to assign putative identity and function to those pineapple genes displaying differential expression during fruit ripening.
we also applied visual mapping tools such as mapman , the kyoto encyclopedia of genes and genomes  online resource and heat maps generated through david to visualize biological processes and pathways of significance during pineapple fruit ripening.
quantitative real-time pcr analysis was used to validate a subset of the differentially expressed genes.
this study contributes to our understanding of the molecular basis of pineapple fruit ripening and non-climacteric ripening in general.
RESULTS
microarray analysis of pineapple fruit ripening identifies gene expression changes associated with important metabolic pathways and processes
<dig> cdnas isolated from several pineapple tissues including roots, green mature and yellow mature fruits were spotted in duplicate onto microarray slides .
hybridization was carried out using probes derived from mature green fruits and fully ripened yellow fruits.
in total, three replicate microarray hybridizations were performed and analysis of the results identified  <dig> differentially expressed ests .
of these,  <dig> were up-regulated and  <dig> down-regulated during pineapple fruit ripening.
among the  <dig> est sequences,  <dig> shared significant homology with known gene products,  <dig> shared homology with products of unknown function, and  <dig> had no significant homology with any known plant gene sequence in the genbank database.
est sequences with homologs were subjected to functional classification analysis and assigned an arabidopsis thaliana homolog id tag .
the microarray data generated in this study has been deposited in ncbi's gene expression omnibus  <cit>  and are accessible through geo series accession number gse <dig> .
the expression data was analyzed using the functional annotation cluster  tool contained in the database for annotation, visualization and integrated discovery   <cit> .
david fac analysis of the  <dig> up-regulated genes  produced a total of  <dig> enriched functional clusters under high stringency conditions.
redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin c biosynthesis and antioxidant-ros activity showed high enrichment scores with strong confidence levels .
the enrichment score gives an indication of the biological significance of the gene groups being analyzed, from which the top  <dig> were considered in our study .
david fac analysis of the  <dig> down-regulated genes .
redox activity
reduction-oxidation  activity showed the highest enrichment score in the fac analysis of genes up-regulated in mature yellow fruits .
david heat map analyses identified  <dig> genes with an expression range of  <dig>  to  <dig>  that functionally clustered into common go terms related to oxidation-reduction and oxidoreductase activity .
a number of genes identified in the heat map relate to dismutase, peroxiredoxin, glutaredoxin, ascorbate-glutathionine and thioredoxin activities.
the state of oxidation for a given organism is influenced by stress response-related enzymes such as dismutases, peroxidase, glutaredoxin, ascorbate-glutathionine and thioredoxin  <cit> .
the state of oxidation may influence the rate at which ethylene is produced at the onset of ripening in climacteric fruits  <cit> .
fruit maturation may be thought of as a catabolic process involving an array of redox enzymes causing degradation of fruit tissue and emanation of ethylene as a catabolic by-product that accelerates ripening.
possible differences between the oxidation state and modes of catabolism stress responses ought to be investigated between climacteric and non-climacteric fruit species.
the observed redox activity in pineapple might also be related to the large size of the fruit and the low partial pressure of oxygen.
organic acid metabolism
fac analysis identified organic acid metabolism as an important biological process during pineapple fruit ripening .
david heat map analyses identified  <dig> genes with an expression range of  <dig>  to  <dig>  that functionally clustered into common go terms related to organic acid metabolism and other associated processes such as cellular ketone metabolism, oxoacid metabolism and carboxylic acid metabolism .
organic acid content has been reported to increase during ripening of climacteric and non-climacteric fruits  <cit> .
in pineapple, acidity increases during development and starts declining once the fruit approaches maturity and ripens  <cit> .
citric and malic acid are the two main organic acids that contribute to the sourness and acidity of most climacteric and non-climacteric fruits such as peach  <cit> , apple  <cit> , kiwifruit  <cit> , grape  <cit> , orange  <cit> , cherry  <cit> , strawberry  <cit>  and pineapple  <cit> .
furthermore, citric acid was shown to undergo the greatest increase in developing pineapple, reaching a peak prior to fruit ripening, whereas malic acid showed little change during development  <cit> .
metalloenzyme activity
fac analysis identified responses to inorganic compounds, metal ion cofactors and cadmium as significant biological processes during pineapple fruit ripening .
a total of  <dig> genes ranging in expression from  <dig>  to  <dig>  fold, were identified in the david heat map .
a number of metalloenzymes activated in response to inorganic substances, metal ion cofactors and cadmium were identified in the heat map analysis .
these include superoxide dismutase , metallothionein  and phytochrome a  homologs.
metalloenzymes are known to regulate the redox state of many plant crops and are involved in many important biological processes such as oxidative stress, metal ion homeostasis, pathogenicity, metal-mediated catalysis and cell death.
pineapple waste  has been used in the bioremediation removal of toxic heavy metals from wastewater and sewage plants  <cit> .
this is possibly due to an abundance of metalloenzymes such as met, sod and phyt in ripe fruit, where these enzymes have the ability to act as ligands and chelate excess metal ions in wastewater treatment procedures.
indeed, met has been implicated in cadmium detoxification and metal ion homeostasis  <cit> .
previous studies confirm that yellow pineapple fruit contains a small transcriptome that is dominated by high levels of metallothionein transcript  <cit> .
glycolysis and sugar catabolism
fac and david heat map analysis identified genes involved in glycolysis and sugar catabolism .
kegg pathway visual analysis identified six genes in the glycolysis pathway map that were up-regulated in yellow ripe fruits by  <dig>  to  <dig>  fold .
these findings indicate the importance of glycolysis and sugar catabolism during pineapple fruit development.
most of the genes identified on the kegg map were also identified in the corresponding heat map matrix.
interestingly, gene expression profiling of non-climacteric grape, also identified a significant cluster of up-regulated genes involved in glycolysis and sugar catabolic activity  <cit> .
taken together, the results in grape and pineapple underlay the significance of glycolytic activity during non-climacteric ripening.
it is possible that ripening fruits generate increased atp energy reserves and reducing power by increasing glycolysis that could be later used by enzymes involved in important fruit metabolic processes.
the up-regulation of glycolysis related genes might also be related to phloem unloading of sugars and their storage in the vacuole.
anti-oxidant activity and reactive oxygen species  metabolism
fac analysis shows a significant enrichment of l-ascorbate  biosynthesis genes, anti-oxidant activity and ros metabolism during pineapple fruit ripening .
david heat map analysis revealed a total of five pineapple genes that functionally cluster with go terms related to vitamin biosynthesis, polyol metabolism, inositol metabolism and l-ascorbic acid biosynthesis .
to name a few, myo-inositol oxygenase  <dig> , myo-inositol oxygenase  <dig>  and l-galactose-1-phosphate phosphatase , were identified with expression levels ranging from  <dig>  to  <dig> -fold increase.
radiotracer evidence showed that grape gpp  is a key enzyme involved in l-ascorbate biosynthesis as non-climacteric grape fruits develop  <cit> .
our pineapple gpp expression profile also correlates with the peach  <cit>  and tomato  <cit>  gpp orthologs during fruit ripening.
the pineapple miox <dig> and miox <dig> genes correlate with miox orthologous genes found in grape  <cit>  and tomato  <cit>  and are involved in the conversion of myo-inositol to d-glucoronic acid during l-ascorbic acid biosynthesis.
david heat map analyses also identified a cluster of six pineapple genes found to be involved in anti-oxidant activity, response to ros, oxygen and ros metabolism, cellular response to oxidative stress and cellular response to ros activity .
l-ascorbate  <cit>  and glutathione  <cit>  have been linked to plant antioxidant activity and fruit ripening.
in fact, fruit ripening is considered to be an oxidative process, where l-ascorbate and glutathione are linked by a cycle of enzymes whose biological role is to detoxify hydrogen peroxide  <cit> .
kegg pathway mapping  identified a cluster of genes involved in l-cysteine biosynthesis, an important precursor for glutathione.
aside from ripening, l-ascorbate production is influenced by various environmental cues such as light  <cit> , temperature  <cit>  and ambient ozone concentrations  <cit> .
these stresses induce the formation of ros, which are removed by the plant’s antioxidant system involving catalase, superoxide dismutase, peroxidases and enzymes involved in the ascorbate-glutathione cycle  <cit> .
non-climacteric grape orthologs of pineapple gpp, apox <dig>  pxdn-b <dig>  sod- <dig> and sod- <dig> were also reported to be involved in antioxidant activity  <cit> .
hydrogen peroxide  is the most stable ros species and plays a crucial role as a signaling molecule in various physiological processes  <cit> .
h2o <dig> is also involved in cell membrane and wall degradation of fleshy tomato fruit tissue, thereby causing fruit softening  <cit> .
this may also be the case in developing pineapple fruit tissue.
proteolysis and ubiquitin-type housekeeping processes
david fac analysis indicated that proteolysis and ubiquitin protein catabolism are active in both green and yellow pineapple fruit .
the enrichment score for proteolysis and ubiquitin protein catabolism was comparable between both fac analyses , however it was significantly lower than other up-regulated biological processes .
david heat map analysis identified six down-regulated genes  and  <dig> up-regulated genes  involved in proteolysis and ubiquitin protein catabolism.
kegg pathway analysis visually created an ubiquitin mediated proteolysis map , showing genes which are active in both yellow  and green  mature pineapple fruit.
mapman  pathway analysis of a biotic-abiotic overview  also identified  <dig> down-regulated genes and  <dig> up-regulated genes involved in proteolysis and ubiquitin-type activity during pineapple fruit ripening.
proteolysis is essential for many aspects of plant development.
it is responsible for cellular housekeeping and stress responses by removing abnormal and miss-folded proteins, supplying amino acids needed to make new proteins, controlling homeostasis by reducing the abundance of key enzymes and regulatory proteins, and controlling gene function and programmed cell death of specific plant organs or cells  <cit> .
our bioinformatics analysis indicates that protein degradation during pineapple fruit development is a complex process involving a multitude of proteolytic pathways.
selective proteolysis of damaged proteins is mainly controlled through ubiquitin-mediated processes involving ubiquitin and several ubiquitin conjugating enzymes  <cit> .
pineapple fruit bromelain
david fac analysis of the  <dig> down-regulated genes , suggested that cysteine type peptidase activity was the most significant process in green mature fruit .
this is mainly due to two pineapple fruit bromelains, ac- <dig>  and ac- <dig>  that were identified on a david heat map  and found to be strongly down-regulated as pineapple ripens from mature green to mature yellow fruit.
bromelain is a cysteine-type protease unique to pineapple.
cysteine-type proteases are also found in other fruits, such as kiwifruit  <cit> , papaya and fig  <cit> .
pineapple est library analyses identified an abundance of a bromelain inhibitor  and a fruit bromelain  from green mature fruit  <cit> .
the bromelain inhibitor ac <dig> was the most abundant est in the green fruit library with  <dig> clones isolated.
northern analyses further confirmed decreased expression of ac <dig> and ac <dig> as pineapple fruit develops  <cit> .
the ac <dig> bromelain inhibitor corresponds to a non-cystatin bromelain inhibitor precursor homolog in pineapple  <cit> , that does not have an inhibitory effect on pineapple fruit bromelain.
it is possible that ac- <dig> and ac- <dig> are more abundant in green mature pineapple fruit due to the role that cysteine-type proteases play in plant defense mechanisms  <cit> , particularly before fruit maturation.
validation of microarray data by real-time rt-pcr
in order to validate our microarray results we performed quantitative real time pcr  to determine the expression levels of ten pineapple genes randomly selected from the list of genes differentially expressed across mature green and mature yellow fruits.
to avoid bias, the tested genes were chosen from an array of different processes including redox activity, organic acid metabolism, metalloenzyme activity, vitamin c biosynthesis, antioxidant activity, cysteine-type peptidase activity and ubiquitin proteolysis.
the qrt-pcr expression results correlated with the microarray expression data for  <dig> out of the  <dig> genes tested .
qrt-pcr determination of mgs and g3pd- <dig> mrna levels showed a  <dig> -fold and  <dig> -fold increase respectively in mature yellow fruits over mature green fruits and these results compare favorably to the  <dig> -fold and  <dig> -fold increase in expression determined by the microarray analysis.
both of these genes are involved in organic acid metabolism.
asp, gpp and csd <dig> are involved in vitamin c biosynthesis, redox and antioxidant activity.
qrt-pcr analysis showed an increase of  <dig> -fold,  <dig> -fold and  <dig> -fold in the levels of asp, gpp and csd <dig> respectively.
these results are in good agreement with our microarray data that showed  <dig> -fold ,  <dig> -fold  and  <dig> -fold  increases in expression during fruit ripening .
expression levels of genes involved in cysteine-type peptidase  and ubiquitin proteolysis  were also quantitatively analyzed.
real-time data confirmed that ac- <dig> is significantly down-regulated  and ube2a up-regulated  as pineapple fruit ripens.
our microarray data correlated with the qrt-pcr data, showing a  <dig> -fold down-regulation of ac- <dig> and  <dig> -fold up-regulation of ube2a.
expression analysis of otu by qrt-pcr revealed a  <dig> -fold up-regulation, in contrast with the microarray data which showed otu to be down-regulated by  <dig> -fold.
otu belongs to the cysteine proteinase superfamily  <cit> , a large multigene family that is likely to contain genes with highly similar sequences.
while qrt-pcr results are usually accurate and gene specific, the microarray used in this study was constructed spotting cdna clones and it is therefore possible that the hybridization results for genes encoded by multigene families could be adulterated by cross-hybridization of homologous probes.
qrt-pcr analysis further confirmed up-regulated expression of met  and down-regulated expression of gast , involved in metalloenzyme activity and stress response pathways respectively.
the microarray data correlated with qrt-pcr showing a  <dig> -fold  increase and  <dig> -fold  decrease, respectively.
met cdna levels were found to be highly abundant in yellow pineapple fruit tissue and shown to be significantly up-regulated during pineapple fruit development  <cit> .
CONCLUSIONS
in this study we have analyzed the changes in expression levels during pineapple fruit ripening using a purpose built pineapple microarray containing  <dig>  est elements.
we identified  <dig> unique pineapple clones with differential expression of at least  <dig> -fold from mature green to mature yellow stages of fruit ripening.
of these  <dig> ests,  <dig> displayed significant homology to plant sequences encoding proteins of known or unknown function.
of these,  <dig> ests were up-regulated and  <dig> ests down-regulated during pineapple fruit ripening.
furthermore, quantitative real-time pcr analysis validated the microarray results for nine out of ten genes randomly chosen from different cellular processes.
bioinformatics analyses of the microarray results suggest that redox activity is a prominent biological process during pineapple fruit ripening.
organic acid metabolism of citric and malic acid and metalloenzyme activity were also up-regulated during pineapple fruit ripening.
the significant increases observed in metalloenzyme encoding genes, may contribute to the bioremediation properties of pineapple waste  <cit> .
glycolysis related genes were also significantly up-regulated during pineapple fruit ripening, possibly to generate atp energy reserves for important ripening processes or in response to the influx of sugars and their storage in the vacuole.
furthermore, l-ascorbate biosynthesis, antioxidant activity and ros metabolism were determined to be up-regulated during pineapple fruit development.
the antioxidant properties of l-ascorbate and glutathionine may be involved in detoxifying h2o <dig> as pineapple fruit ripens.
h2o <dig> may have a biological role activating an up-regulated response of vitamin c accumulation and antioxidant activity during pineapple fruit development.
our studies also identified two pineapple bromelains, ac- <dig> and ac- <dig>  which were highly abundant in green mature pineapple tissues and may possibly be involved in plant defense mechanisms against pathogenic attack prior to fruit maturation.
applying a microarray based approach to studying large scale gene expression changes during fruit development has contributed to our understanding of the molecular basis of pineapple fruit ripening and non-climacteric fruit ripening in general.
the development of the pineapple microarray will also enable the future large scale analysis of gene expression changes in other pineapple developmental processes, conditions and treatments.

 pineapple is a tropical fruit crop of significant commercial value and is the most important economic plant in the bromeliaceae family. however, surprisingly little research has been undertaken to understand the molecular basis of pineapple fruit development - an essential pre-requisite for future improvement by molecular breeding or genetic modification. in terms of fruit ripening, fleshy fruits are classified as either climacteric or non-climacteric. in climacteric fruits, such as apples and bananas, a burst of ethylene biosynthesis and an increase in respiration is observed at the onset of ripening. conversely, non-climacteric fruits such as pineapple lack the autocatalytic ethylene burst and the increase in respiration. although the physiological changes that occur during pineapple fruit development are well known, little is known about the molecular events that govern pineapple fruit ripening. the advent of high throughput sequencing and microarray technologies has facilitated large-scale studies on gene expression changes during fruit development in a number of species, including climacteric fruits tomato and apple, and the non-climacteric fruit strawberry. in a previous study, we reported the construction of subtracted and normalized est cdna libraries, including the green mature and yellow mature ripening stages of pineapple fruits. more recently, short read next generation sequencing technology was applied to a ripe pineapple fruit gene discovery project. in this study, we report the development of an est-based pineapple microarray and its use to identify differentially expressed genes during fruit ripening. online bioinformatics tools were used to assign putative identity and function to those pineapple genes displaying differential expression during fruit ripening. we also applied visual mapping tools such as mapman, the kyoto encyclopedia of genes and genomes online resource and heat maps generated through david to visualize biological processes and pathways of significance during pineapple fruit ripening. quantitative real-time pcr analysis was used to validate a subset of the differentially expressed genes. this study contributes to our understanding of the molecular basis of pineapple fruit ripening and non-climacteric ripening in general.  microarray analysis of pineapple fruit ripening identifies gene expression changes associated with important metabolic pathways and processes cdnas isolated from several pineapple tissues including roots, green mature and yellow mature fruits were spotted in duplicate onto microarray slides. hybridization was carried out using probes derived from mature green fruits and fully ripened yellow fruits. in total, three replicate microarray hybridizations were performed and analysis of the results identified differentially expressed ests. of these, were up-regulated and down-regulated during pineapple fruit ripening. among the est sequences, shared significant homology with known gene products, shared homology with products of unknown function, and had no significant homology with any known plant gene sequence in the genbank database. est sequences with homologs were subjected to functional classification analysis and assigned an arabidopsis thaliana homolog id tag. the microarray data generated in this study has been deposited in ncbi's gene expression omnibus and are accessible through geo series accession number gse. the expression data was analyzed using the functional annotation cluster tool contained in the database for annotation, visualization and integrated discovery. david fac analysis of the up-regulated genes produced a total of enriched functional clusters under high stringency conditions. redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin c biosynthesis and antioxidant-ros activity showed high enrichment scores with strong confidence levels. the enrichment score gives an indication of the biological significance of the gene groups being analyzed, from which the top were considered in our study. david fac analysis of the down-regulated genes. redox activity reduction-oxidation activity showed the highest enrichment score in the fac analysis of genes up-regulated in mature yellow fruits. david heat map analyses identified genes with an expression range of to that functionally clustered into common go terms related to oxidation-reduction and oxidoreductase activity. a number of genes identified in the heat map relate to dismutase, peroxiredoxin, glutaredoxin, ascorbate-glutathionine and thioredoxin activities. the state of oxidation for a given organism is influenced by stress response-related enzymes such as dismutases, peroxidase, glutaredoxin, ascorbate-glutathionine and thioredoxin. the state of oxidation may influence the rate at which ethylene is produced at the onset of ripening in climacteric fruits. fruit maturation may be thought of as a catabolic process involving an array of redox enzymes causing degradation of fruit tissue and emanation of ethylene as a catabolic by-product that accelerates ripening. possible differences between the oxidation state and modes of catabolism stress responses ought to be investigated between climacteric and non-climacteric fruit species. the observed redox activity in pineapple might also be related to the large size of the fruit and the low partial pressure of oxygen. organic acid metabolism fac analysis identified organic acid metabolism as an important biological process during pineapple fruit ripening. david heat map analyses identified genes with an expression range of to that functionally clustered into common go terms related to organic acid metabolism and other associated processes such as cellular ketone metabolism, oxoacid metabolism and carboxylic acid metabolism. organic acid content has been reported to increase during ripening of climacteric and non-climacteric fruits. in pineapple, acidity increases during development and starts declining once the fruit approaches maturity and ripens. citric and malic acid are the two main organic acids that contribute to the sourness and acidity of most climacteric and non-climacteric fruits such as peach, apple, kiwifruit, grape, orange, cherry, strawberry and pineapple. furthermore, citric acid was shown to undergo the greatest increase in developing pineapple, reaching a peak prior to fruit ripening, whereas malic acid showed little change during development. metalloenzyme activity fac analysis identified responses to inorganic compounds, metal ion cofactors and cadmium as significant biological processes during pineapple fruit ripening. a total of genes ranging in expression from to fold, were identified in the david heat map. a number of metalloenzymes activated in response to inorganic substances, metal ion cofactors and cadmium were identified in the heat map analysis. these include superoxide dismutase, metallothionein and phytochrome a homologs. metalloenzymes are known to regulate the redox state of many plant crops and are involved in many important biological processes such as oxidative stress, metal ion homeostasis, pathogenicity, metal-mediated catalysis and cell death. pineapple waste has been used in the bioremediation removal of toxic heavy metals from wastewater and sewage plants. this is possibly due to an abundance of metalloenzymes such as met, sod and phyt in ripe fruit, where these enzymes have the ability to act as ligands and chelate excess metal ions in wastewater treatment procedures. indeed, met has been implicated in cadmium detoxification and metal ion homeostasis. previous studies confirm that yellow pineapple fruit contains a small transcriptome that is dominated by high levels of metallothionein transcript. glycolysis and sugar catabolism fac and david heat map analysis identified genes involved in glycolysis and sugar catabolism. kegg pathway visual analysis identified six genes in the glycolysis pathway map that were up-regulated in yellow ripe fruits by to fold. these findings indicate the importance of glycolysis and sugar catabolism during pineapple fruit development. most of the genes identified on the kegg map were also identified in the corresponding heat map matrix. interestingly, gene expression profiling of non-climacteric grape, also identified a significant cluster of up-regulated genes involved in glycolysis and sugar catabolic activity. taken together, the results in grape and pineapple underlay the significance of glycolytic activity during non-climacteric ripening. it is possible that ripening fruits generate increased atp energy reserves and reducing power by increasing glycolysis that could be later used by enzymes involved in important fruit metabolic processes. the up-regulation of glycolysis related genes might also be related to phloem unloading of sugars and their storage in the vacuole. anti-oxidant activity and reactive oxygen species metabolism fac analysis shows a significant enrichment of l-ascorbate biosynthesis genes, anti-oxidant activity and ros metabolism during pineapple fruit ripening. david heat map analysis revealed a total of five pineapple genes that functionally cluster with go terms related to vitamin biosynthesis, polyol metabolism, inositol metabolism and l-ascorbic acid biosynthesis. to name a few, myo-inositol oxygenase, myo-inositol oxygenase and l-galactose-1-phosphate phosphatase, were identified with expression levels ranging from to -fold increase. radiotracer evidence showed that grape gpp is a key enzyme involved in l-ascorbate biosynthesis as non-climacteric grape fruits develop. our pineapple gpp expression profile also correlates with the peach and tomato gpp orthologs during fruit ripening. the pineapple miox and miox genes correlate with miox orthologous genes found in grape and tomato and are involved in the conversion of myo-inositol to d-glucoronic acid during l-ascorbic acid biosynthesis. david heat map analyses also identified a cluster of six pineapple genes found to be involved in anti-oxidant activity, response to ros, oxygen and ros metabolism, cellular response to oxidative stress and cellular response to ros activity. l-ascorbate and glutathione have been linked to plant antioxidant activity and fruit ripening. in fact, fruit ripening is considered to be an oxidative process, where l-ascorbate and glutathione are linked by a cycle of enzymes whose biological role is to detoxify hydrogen peroxide. kegg pathway mapping identified a cluster of genes involved in l-cysteine biosynthesis, an important precursor for glutathione. aside from ripening, l-ascorbate production is influenced by various environmental cues such as light, temperature and ambient ozone concentrations. these stresses induce the formation of ros, which are removed by the plant’s antioxidant system involving catalase, superoxide dismutase, peroxidases and enzymes involved in the ascorbate-glutathione cycle. non-climacteric grape orthologs of pineapple gpp, apox pxdn-b sod- and sod- were also reported to be involved in antioxidant activity. hydrogen peroxide is the most stable ros species and plays a crucial role as a signaling molecule in various physiological processes. h2o is also involved in cell membrane and wall degradation of fleshy tomato fruit tissue, thereby causing fruit softening. this may also be the case in developing pineapple fruit tissue. proteolysis and ubiquitin-type housekeeping processes david fac analysis indicated that proteolysis and ubiquitin protein catabolism are active in both green and yellow pineapple fruit. the enrichment score for proteolysis and ubiquitin protein catabolism was comparable between both fac analyses, however it was significantly lower than other up-regulated biological processes. david heat map analysis identified six down-regulated genes and up-regulated genes involved in proteolysis and ubiquitin protein catabolism. kegg pathway analysis visually created an ubiquitin mediated proteolysis map, showing genes which are active in both yellow and green mature pineapple fruit. mapman pathway analysis of a biotic-abiotic overview also identified down-regulated genes and up-regulated genes involved in proteolysis and ubiquitin-type activity during pineapple fruit ripening. proteolysis is essential for many aspects of plant development. it is responsible for cellular housekeeping and stress responses by removing abnormal and miss-folded proteins, supplying amino acids needed to make new proteins, controlling homeostasis by reducing the abundance of key enzymes and regulatory proteins, and controlling gene function and programmed cell death of specific plant organs or cells. our bioinformatics analysis indicates that protein degradation during pineapple fruit development is a complex process involving a multitude of proteolytic pathways. selective proteolysis of damaged proteins is mainly controlled through ubiquitin-mediated processes involving ubiquitin and several ubiquitin conjugating enzymes. pineapple fruit bromelain david fac analysis of the down-regulated genes, suggested that cysteine type peptidase activity was the most significant process in green mature fruit. this is mainly due to two pineapple fruit bromelains, ac- and ac- that were identified on a david heat map and found to be strongly down-regulated as pineapple ripens from mature green to mature yellow fruit. bromelain is a cysteine-type protease unique to pineapple. cysteine-type proteases are also found in other fruits, such as kiwifruit, papaya and fig. pineapple est library analyses identified an abundance of a bromelain inhibitor and a fruit bromelain from green mature fruit. the bromelain inhibitor ac was the most abundant est in the green fruit library with clones isolated. northern analyses further confirmed decreased expression of ac and ac as pineapple fruit develops. the ac bromelain inhibitor corresponds to a non-cystatin bromelain inhibitor precursor homolog in pineapple, that does not have an inhibitory effect on pineapple fruit bromelain. it is possible that ac- and ac- are more abundant in green mature pineapple fruit due to the role that cysteine-type proteases play in plant defense mechanisms, particularly before fruit maturation. validation of microarray data by real-time rt-pcr in order to validate our microarray results we performed quantitative real time pcr to determine the expression levels of ten pineapple genes randomly selected from the list of genes differentially expressed across mature green and mature yellow fruits. to avoid bias, the tested genes were chosen from an array of different processes including redox activity, organic acid metabolism, metalloenzyme activity, vitamin c biosynthesis, antioxidant activity, cysteine-type peptidase activity and ubiquitin proteolysis. the qrt-pcr expression results correlated with the microarray expression data for out of the genes tested. qrt-pcr determination of mgs and g3pd- mrna levels showed a -fold and -fold increase respectively in mature yellow fruits over mature green fruits and these results compare favorably to the -fold and -fold increase in expression determined by the microarray analysis. both of these genes are involved in organic acid metabolism. asp, gpp and csd are involved in vitamin c biosynthesis, redox and antioxidant activity. qrt-pcr analysis showed an increase of -fold, -fold and -fold in the levels of asp, gpp and csd respectively. these results are in good agreement with our microarray data that showed -fold, -fold and -fold increases in expression during fruit ripening. expression levels of genes involved in cysteine-type peptidase and ubiquitin proteolysis were also quantitatively analyzed. real-time data confirmed that ac- is significantly down-regulated and ube2a up-regulated as pineapple fruit ripens. our microarray data correlated with the qrt-pcr data, showing a -fold down-regulation of ac- and -fold up-regulation of ube2a. expression analysis of otu by qrt-pcr revealed a -fold up-regulation, in contrast with the microarray data which showed otu to be down-regulated by -fold. otu belongs to the cysteine proteinase superfamily, a large multigene family that is likely to contain genes with highly similar sequences. while qrt-pcr results are usually accurate and gene specific, the microarray used in this study was constructed spotting cdna clones and it is therefore possible that the hybridization results for genes encoded by multigene families could be adulterated by cross-hybridization of homologous probes. qrt-pcr analysis further confirmed up-regulated expression of met and down-regulated expression of gast, involved in metalloenzyme activity and stress response pathways respectively. the microarray data correlated with qrt-pcr showing a -fold increase and -fold decrease, respectively. met cdna levels were found to be highly abundant in yellow pineapple fruit tissue and shown to be significantly up-regulated during pineapple fruit development.  in this study we have analyzed the changes in expression levels during pineapple fruit ripening using a purpose built pineapple microarray containing est elements. we identified unique pineapple clones with differential expression of at least -fold from mature green to mature yellow stages of fruit ripening. of these ests, displayed significant homology to plant sequences encoding proteins of known or unknown function. of these, ests were up-regulated and ests down-regulated during pineapple fruit ripening. furthermore, quantitative real-time pcr analysis validated the microarray results for nine out of ten genes randomly chosen from different cellular processes. bioinformatics analyses of the microarray results suggest that redox activity is a prominent biological process during pineapple fruit ripening. organic acid metabolism of citric and malic acid and metalloenzyme activity were also up-regulated during pineapple fruit ripening. the significant increases observed in metalloenzyme encoding genes, may contribute to the bioremediation properties of pineapple waste. glycolysis related genes were also significantly up-regulated during pineapple fruit ripening, possibly to generate atp energy reserves for important ripening processes or in response to the influx of sugars and their storage in the vacuole. furthermore, l-ascorbate biosynthesis, antioxidant activity and ros metabolism were determined to be up-regulated during pineapple fruit development. the antioxidant properties of l-ascorbate and glutathionine may be involved in detoxifying h2o as pineapple fruit ripens. h2o may have a biological role activating an up-regulated response of vitamin c accumulation and antioxidant activity during pineapple fruit development. our studies also identified two pineapple bromelains, ac- and ac- which were highly abundant in green mature pineapple tissues and may possibly be involved in plant defense mechanisms against pathogenic attack prior to fruit maturation. applying a microarray based approach to studying large scale gene expression changes during fruit development has contributed to our understanding of the molecular basis of pineapple fruit ripening and non-climacteric fruit ripening in general. the development of the pineapple microarray will also enable the future large scale analysis of gene expression changes in other pineapple developmental processes, conditions and treatments. 
quantitative real-time pcr analysis validated the microarray expression results for nine out of ten genes tested. among these sequences, share significant homology with genes encoding proteins of known function, share homology with genes encoding proteins of unknown function and share no significant homology with any database accession. although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. in this study we developed a element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening. microarray analyses identified unique cdnas differentially expressed at least -fold between the mature green and mature yellow stages of pineapple fruit ripening. of the pineapple sequences with homologs, were up-regulated and were down-regulated during pineapple fruit ripening. david functional annotation cluster analysis of all sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin c biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. this study extends our knowledge of the molecular basis of pineapple fruit ripening and non-climacteric fruit ripening in general. pineapple is a tropical fruit crop of significant commercial importance.
 pineapple is a tropical fruit crop of significant commercial importance. although the physiological changes that occur during pineapple fruit development have been well characterized, little is known about the molecular events that occur during the fruit ripening process. understanding the molecular basis of pineapple fruit ripening will aid the development of new varieties via molecular breeding or genetic modification. in this study we developed a element pineapple microarray and used it to profile gene expression changes that occur during pineapple fruit ripening.  microarray analyses identified unique cdnas differentially expressed at least -fold between the mature green and mature yellow stages of pineapple fruit ripening. among these sequences, share significant homology with genes encoding proteins of known function, share homology with genes encoding proteins of unknown function and share no significant homology with any database accession. of the pineapple sequences with homologs, were up-regulated and were down-regulated during pineapple fruit ripening. david functional annotation cluster analysis of all sequences with homologs revealed confident enrichment scores for redox activity, organic acid metabolism, metalloenzyme activity, glycolysis, vitamin c biosynthesis, antioxidant activity and cysteine peptidase activity, indicating the functional significance and importance of these processes and pathways during pineapple fruit development. quantitative real-time pcr analysis validated the microarray expression results for nine out of ten genes tested.  this is the first report of a microarray based gene expression study undertaken in pineapple. our bioinformatic analyses of the transcript profiles have identified a number of genes, processes and pathways with putative involvement in the pineapple fruit ripening process. this study extends our knowledge of the molecular basis of pineapple fruit ripening and non-climacteric fruit ripening in general. pineapplenon-climactericfruit ripeningmicroarray 