BACKGROUND
european pears , including ‘bartlett’, ‘d’anjou’, and ‘comice’, are economically significant fruit in the united states, with a production value of $ <dig> million in  <dig>  <cit> .
as a climacteric fruit, pears ripen in association with a substantial increase in rates of respiration and ethylene biosynthesis  <cit> .
unlike many climacteric fruit such as apple and mango, european pears develop poor texture and flavor if left to ripen on the tree  <cit> .
therefore, most european pears are harvested at the mature-green stage and then usually exposed to ethylene or cold temperatures  prior to ripening to enhance their ability to produce ethylene and ripen at 20 °c  <cit> .
hansen found that early maturity ‘bartlett’ and ‘d’anjou’ pear might not respond to ethylene or cold treatment while late maturity fruit could ripen without any conditioning treatment  <cit> .
however, the underlying molecular mechanisms governing this developmental shift are still not well understood.
furthermore, as a climacteric fruit, pear fruit ripening includes the transition from auto-inhibitory ethylene  to autocatalytic ethylene  that regulates the numerous metabolic processes associated with fruit ripening  <cit> .
the intrinsic developmental factors that regulate the transition from system  <dig> to system  <dig> remain mostly unknown  <cit> .
ripening is postulated to be initiated by activation of specific transcriptional regulators, such as colorless non ripening  and ripening-inhibitor , as first identified in tomato, a model organism to study fruit ripening.
these regulators lead to signal transduction pathways that include ethylene as an essential signaling molecule  <cit> .
these signaling pathways control many ripening-related biochemical events such as chlorophyll degradation, starch degradation to sugars, decreases in organic acids, and production of aroma compounds  <cit> .
several studies designed to elucidate the molecular pathways of fruit ripening have focused on genes associated with hormone and cell wall metabolism, as well as transcriptional regulation  <cit> .
some of the molecular aspects of european pear ripening have been investigated  <cit> .
several studies reported an increase in ethylene biosynthesis enzymes, 1-aminocyclopropane-1-carboxylate  synthase and acc oxidase, following ethylene treatment and cold storage .
increases in transcript abundance of pear fruit ethylene biosynthesis genes   <cit>  and ethylene perception genes including pc-etr1a and pc-ers1a  <cit>  during fruit ripening were also reported.
low transcript abundance of genes encoding cell wall modifying proteins such as β-galactosidases and expansins were detected during fruit development in ‘rocha’ pear  <cit> .
in addition, large-scale expression profiles of ‘rocha’ and ‘la france’ pear during fruit growth and ripening have been generated  <cit> .
however, these two studies utilized microarrays with a limited number of fruit-specific sequences.
to our knowledge, genes associated with hormones other than ethylene and transcription factors have not been characterized during pear fruit development.
in the last 5 years, next generation sequencing  technologies accompanied by sophisticated bioinformatics tools have been developed and provide a powerful approach to examine the transcriptomes of non-model plants  <cit> .
accordingly, these tools have been utilized to determine transcriptional changes during fruit growth and development in a variety of species including chinese bayberry   <cit> , orange   <cit> , and korean black raspberry   <cit> .
in the present study, ngs technology was used to characterize the molecular mechanisms regulating the development of ripening capacity in ‘bartlett’ pear fruit.
the specific objectives were to 1) develop a better understanding of the acquisition of pear ripening capacity and 2) define the molecular regulation of pear fruit ripening, focusing on genes associated with cell wall metabolism, hormone biosynthesis and signaling, and transcription factors.
methods
plant materials and physico-chemical analysis
‘bartlett’ pear fruit were produced at a commercial orchard in sacramento county, california, usa.
fruit were harvested at 7-day intervals for 4 weeks, from  <dig> to  <dig> dafb; the fourth harvest time was equivalent to the first commercial harvest.
sixty fruit were collected at each harvest time from a total of five trees.
immediately after harvest, fruit were randomized and divided into five groups of  <dig>  each group was composed of three biological replications with four fruit each.
group  <dig> fruit were analyzed within 24 h of harvest for ethylene production rate, respiration rate, weight, diameter, skin color, flesh firmness, and ssc.
group  <dig> fruit were used to measure the internal ethylene concentration.
peel tissues for molecular analysis were collected from fruit in group  <dig>  fruit from groups  <dig> and  <dig> were enclosed in separate 20 l glass jars and treated with  <dig> or  <dig> μll− <dig> ethylene in flowing air streams of  <dig> mlmin− <dig> for 24 h at 20 °c.
these fruit were then held at 20 °c and 90 % relative humidity for 14 days to allow for ripening.
after 14 days , fruit were evaluated for skin color, flesh firmness, and ssc.
rates of ethylene production and respiration were assessed for each replication by sealing four fruit inside a  <dig>  l glass jar and using the method described by villalobos et al.
<cit> .
headspace samples were collected with 10 ml syringes and injected into a gas chromatograph for ethylene quantification  or a pir-2000r infrared analyzer for co <dig> analysis .
fruit diameter was measured across the widest point of each fruit with a caliper.
pear skin color was determined on two diametrically opposite sides of each fruit using a chroma meter cr- <dig> .
the color data were captured using the cie  <dig>  color space and expressed as the hue angle , where 90° represents full yellow and 180° corresponds to full green.
flesh firmness was quantified as the resistance to 9 mm penetration with an 8 mm-diameter probe using a fruit texture analyzer  on two opposite sides of the fruit after the peel was removed.
ssc was measured in juice samples extracted by squeezing cortical wedges cut from two opposite sides of each of four fruit in two layers of cheesecloth, with a reichert ar <dig> series refractometer .
the internal ethylene concentration was determined according to coombe and hale  <cit>  and chervin et al.
<cit> .
briefly, pre-weighed fruit were placed individually in a chamber containing a saturated solution of nacl.
each fruit was submerged in the solution under an inverted funnel with the narrow end capped with a rubber septum.
the air trapped in the narrow end of the funnel was withdrawn with a syringe.
the chamber was sealed and a partial vacuum of −700 mm hg was applied for 5 min.
after returning to atmospheric pressure, 1 ml of the fruit internal atmosphere trapped in the narrow end of the funnel was sampled by syringe and the ethylene concentration was determined by gas chromatography as described above.
statistical analysis was performed on each variable by means of analysis of variance using the sas statistical package .
the mean values of three replications were compared using tukey’s test .
rna extraction
total rna was isolated from  <dig>  g tissues ground in liquid n <dig>  which contained both skin and flesh tissues peeled from two opposite sides of  <dig> fruit , using the qiagen rneasy plant mini kit  according to the manufacturer’s instructions.
the total rna was then treated with dnase i recombinant, rnase-free  to remove dna contamination.
the total rna concentration was quantified using a nanodrop spectrophotometer , with absorbance at 260 nm.
the quality of total rna was verified by examining the ratio od260/od <dig> and formaldehyde agarose gel electrophoresis.
rna sequencing
illumina library preparation and sequencing of  <dig> samples  were completed following standard protocols at the uc davis dna technologies core .
the integrity and quantity of total rna was examined using an agilent  <dig> bioanalyzer rna  <dig> kit and invitrogen’s qubit.
mrna was isolated from total rna using dynabeads oligo-d <dig> .
the rna-seq library was constructed by following the truseq protocol .
individual libraries were prepared with barcodes and pooled for sequencing on one lane of the illumina hiseq  <dig> platform.
paired-end reads of 100 cycles were collected and fastq files were generated using the illumina pipeline.
de novo assembly and count estimation
given that inclusion of a greater number of reads in de novo assembly produces a greater contiguity of sequences  <cit> , illumina reads obtained from this experiment  and a second ‘bartlett’ pear ripening capacity experiment  were combined for the assembly.
the raw reads were trimmed to remove truseq adapters and low quality bases, using trimmomatic   <cit> .
surviving paired reads were used as input for de novo transcript assembly.
the assembly was carried out using trinity   <cit>  with default parameters except --min_kmer_cov was set to  <dig>  to minimize redundancy in the set of putative transcripts, the contigs were clustered using cd-hit  <cit>  and then with tgicl  <cit> .
stringent similarity parameters were selected to minimize the likelihood of merging paralogous transcripts.
this reduced the number of contigs in the original output by trinity.
as these contigs may still represent multiple isoforms of the same gene, contigs that shared a common trinity component and sub-component were naively grouped into unigenes by rsem   <cit> .
estimated read counts associated with the assembled contigs were determined with rsem, which utilizes bowtie to map reads to a reference database composed of the assembled contigs  <cit> .
in preparing this database the unigene to contig mapping described above was provided to permit rsem to estimate read counts at both the individual contig  and unigene level.
the rsem output represented the estimated counts of reads associated with each isoform or unigene, recognizing the uncertainty inherent in assigning reads to isoforms that may share one or more exons.
sequence identity validation and quantitative pcr validation
sequences in the de novo transcriptome were mapped to the reference genome of asian pear   <cit>  and european pear   <cit>  using gmap   <cit>  to check for possible chimeric and non-match sequences, using k-mer  <dig>  orfs were examined using orfpredictor  <cit>  with an orf cut-off length of  <dig> base pairs.
the blastx program   <cit>  was used to perform similarity searches of the contigs against the tair v <dig> and refseq  protein databases with an e-value threshold of 1e− <dig>  the contigs were annotated with the description inherited from the best hits in each database.
cdna was synthesized from 1 μg dnase - treated total rna, using superscript™ iii first strand synthesis systems for rt-pcr systems .
before qpcr validation, sequences of interest were aligned against the available sequences of asian and european pears on the ncbi est database and their published genome to confirm sequence identity, using the local tblastn function in bioedit   <cit> .
the gene expression was examined using sybr green pcr master mix and a  <dig> real time pcr system .
ef1alpha was chosen as the housekeeping gene after testing with 18 s, 26 s, β-actin, and tubulin <dig>  primers for sequences of interest were designed using primer  <dig>  <cit>  and passed the primer efficiency check for qpcr.
in the regression analysis, the fc of qpcr was ΔΔct  <cit>  and the fc of rna-seq was the base- <dig> logarithm of the ratio rsem count in treatment 2/rsem count in treatment  <dig>
mapman functional annotation analysis
mapman functional annotation analysis was utilized to gain an understanding about the general function of genes expressed during fruit growth and to identify gene families that may play essential roles in regulating the development of pear ripening capacity.
contigs were classified into specific functional groups, using mercator  <cit>  with a blast cut-off of  <dig>  because one unigene might have multiple contigs, a functional term of a unigene was derived from its representative contig that had the highest bit score.
enrichment analysis was completed through fisher’s test using mefisto  with bonferroni correction.
gene expression changes were viewed in mapman  <dig> .1r <dig>  <cit> .
differential expression analysis
the unigene counts were subjected to both pairwise and multi-condition analysis to detect de sequences between two harvest times and among four harvest times of pear fruit, respectively, using the ebseq package  with false discovery rate of  <dig>   <cit> .
the method employed by ebseq manages the varying uncertainty in counts across isoform groups.
in convergence checking, the maximum round of each comparison was chosen based on a difference less than  <dig>  between the two last iterations of ebout$alpha, and of ebout$beta .
for pairwise analysis, unigenes with a posterior probability of being differentially expressed  of ≥  <dig>  were identified as differentially expressed between two harvest times.
for multi-condition analysis, unigenes with p1 ≤  <dig>   were identified as differentially expressed across the four harvest times.
normalized counts of unigenes for calculating gene fold changes were obtained from the multi-condition analysis.
k-means cluster analysis
k-means clustering was utilized to determine particular patterns in gene expression throughout the four harvest times, using the base- <dig> logarithm of the average normalized counts of three biological replicates.
the number of clusters was identified using the figures of merit application embedded in mev  <cit> .
unigenes in each cluster were then identified using the r package amap  with pearson correlation, in which  <dig> random sets were applied to generate reproducible clusters.
a heatmap of the number of unigenes in mapman categories in each cluster was built on the r package gplots .
accession code
the clean reads produced in this study have been deposited at ddbj/embl/genbank short read archive:  <dig> biosample numbers samn <dig> - samn <dig>   <dig> accession codes srr <dig> - srr <dig>  and under project number prjna <dig>  this transcriptome shotgun assembly project has been deposited at ddbj/embl/genbank under the accession gbxl <dig>  the version described in this paper is the first version, gbxl <dig>  the gene id, contig id, and their putative function can be accessed through additional file  <dig>
computer system
except for the de novo transcriptome assembly and mapping to a reference genome, all data analyses were completed with a dell optiplex  <dig> 4gb ram, 32-bit, intel core i5- <dig> cpu with windows  <dig> enterprise, microsoft office  <dig>  and r  <dig> .
<dig> , rstudio i386-pc-mingw32/i <dig> platform.
RESULTS
physico-chemical changes during fruit growth and development
to characterize the relationship between pear fruit maturation and the development of ripening capacity, ‘bartlett’ pear fruit were harvested at weekly intervals commencing 3 weeks before commercial harvest to capture four progressive stages of maturity: s1: 100 days after full bloom , s2:  <dig> dafb, s3:  <dig> dafb, and s4:  <dig> dafb  .
fruit growth and maturity were monitored by physico-chemical measurements .
fruit weight and diameter at harvest increased considerably with these advancing stages of maturity .
in contrast, the flesh firmness at harvest steadily decreased as fruit maturity increased .
rates of respiration and the internal ethylene concentration were highest in fruit harvested at s <dig> and relatively low in s <dig>  s <dig>  and s <dig> .
rates of ethylene production were also relatively higher in s <dig> than s <dig> and s <dig> before increasing again at s <dig> .
despite the higher ethylene production rate at s <dig> , the level was substantially lower than typical rates produced during climacteric ripening of ‘bartlett’ pear, which can be as high as  <dig> μlkg−1h− <dig>  <cit> .
there were no significant differences in fruit soluble solids content  and skin color among four harvest maturity stages examined in this study .fig.
<dig> pear fruit at four harvest times.s1:  <dig> dafb, s2:  <dig> dafb, s3:  <dig> dafb, and s4:  <dig> dafb
amean values with different letters are significantly different according to tukey’s test
after harvest, fruit at each of the four maturity stages were treated with  <dig> or  <dig> μll− <dig> ethylene for 24 h and then evaluated for their ripening capacity based on softening after being held at 20 °c for 14 days.
the ability of fruit to soften in the presence or absence of ethylene increased with advancing harvest maturity .
when treated with ethylene, s <dig> fruit failed to soften, s <dig> fruit displayed partial softening , and s <dig> and s <dig> fruit softened to a firmness of <5 n. in the absence of ethylene treatment, fruit harvested at stages s <dig> and s <dig> failed to soften, while s <dig> and s <dig> fruit softened to 60 n and  <dig>  n, respectively.
therefore, it appears that the slightly higher rate of ethylene production during the preclimacteric phase in s <dig> had no positive effect on the ability of fruit to ripen when harvested at this stage.
the slight increase in firmness observed for s <dig> fruit at day  <dig> shelf life presumably reflected water loss during storage of fruit harvested at an immature stage; this agrees with what has been found in apple and bell pepper  <cit> .
the general fruit softening response was also accompanied by similar changes in peel color, as evidenced by the hue angle  .
there was no significant effect of harvest maturity and ethylene treatment on fruit ssc at the completion of a 14-day shelf life .fig.
<dig> firmness changes of pears harvested at four maturity stages at harvest and after air/ethylene treatment.
s <dig>  s <dig>  s <dig>  and s <dig> were harvested a week apart; s <dig> coincident with commercial harvest .
d14: 14 days at 20 °c following treatment of pears with air or  <dig> μll− <dig> ethylene  for 24 h. bars indicate standard errors; letters indicate significant differences among the three firmness values within a stage according to tukey’s test
in other species, standard stages of fruit development have been well established.
for instance, these stages in tomato include green, mature green, breaker, pink, and red ripe  <cit> , while peach and plum development is described as an s <dig> to s <dig> double sigmoid pattern  <cit> .
for ‘bartlett’ pear, we are unaware of defined standard stages of fruit development, except those utilizing firmness as a ripeness indicator: 85–98 n, when the fruit are ready to harvest  <cit>  and 20 n, when the fruit are ready to consume  <cit> , with a consideration of ssc  and size   <cit> .
in the current study, because of the low ripening capacity of pear fruit at early maturity stages, we considered full ripening capacity was achieved when fruit firmness reached 20 n after 14 days at 20 °c; this was named “rc14” for “ripening capacity at 14 days” after harvest.
given this definition of ripening capacity, we observed the following response of the four harvest maturity stages: s <dig> and s <dig> did not achieve rc14; s <dig> achieved rc <dig> with ethylene treatment; and s <dig> fruit achieved rc <dig> without ethylene treatment.
rna-seq and de novo assembly
rna sequencing of the peel tissue of the four maturity stages  generated  <dig>  million  2x <dig> bp paired-end reads.
of the  <dig>  mil paired-end reads from both experiments,  <dig>  % were retained after the quality check, in which the unqualified read was mostly due to bottom middle swath in the sequencing system.
a trinity assembly on  <dig> mil qualified paired-end reads generated  <dig>  contigs that were clustered into  <dig>  unigenes.
the contig length ranged from  <dig> to  <dig>  bp, with a median length of 502 bp and a mean length of 911 bp.
validation of the transcriptome in sequence identity and expression levels
sequence identity of the de novo transcriptome was first validated through putative function determination.
blastx of the contigs against the ncbi refseq  and arabidopsis  protein databases identified similar proteins  in these reference sets for  <dig>  % and  <dig>  %, respectively, of the  <dig>  unigenes.
this indicates that the functions of a large portion of the genes of p. communis have not yet been identified.
using the ncbi non-redundant database with a threshold of 1e− <dig>  68 %, 80 %, and 93 % of unigenes of chinese bayberry, korean black raspberry, and ‘suli’ pear transcriptomes, respectively, were annotated  <cit> .
in the general functional description of the transcriptome examined using mapman,  <dig>  % of the unigenes were assigned to  <dig> meaningful bincodes of mapman, with the highest numbers of unigenes classified into protein , rna , signaling , stress , and transport  categories .fig.
<dig> mapman functional annotations of the transcriptome.
2nd: secondary, aa: amino acid, ps: photosynthesis, met.
: metabolism, syn: synthesis, other 1: nucleotide met., minor cho met., major cho met., mitochondrial electron transport/atp and glycolysis; other 2: co-factor and vitamin met., tca/org transformation, metal handling, tetrapyrrole syn., c1-met., opp, n-met., biodegradation of xenobiotics, fermentation, polyamine met., gluconeogenesis/glyoxylate cycle, s-assimilation, micro rna and natural antisense
open reading frame  finders evaluate the degree to which full coding sequence are assembled  <cit> .
this analysis determined  <dig>   contigs had an orf of length ≥ 200 bp, with an average length of 724 bp.
moreover, the high percentages of mapped contigs when mapping to reference genomes indicated good sequence identity of our de novo transcriptome.
mapping all contigs of the de novo transcriptome to the asian pear  genome  <cit>  revealed that  <dig>   were mapped to the reference genome, in which  <dig>   contigs were possibly chimera sequences, and  <dig>   contigs were non-matched sequences.
additionally, mapping to the recently published european pear  genome  <cit>  showed  <dig>   mapped contigs, in which  <dig>   were possible chimeras, and  <dig>   were non-matched sequences.
to validate gene expression values obtained from rna-seq data, we examined the correlation between fold changes  calculated on rsem  counts  <cit>  and the equivalent values measured by quantitative pcr .
the validation on eleven transcripts associated with cell wall metabolism, hormone biosynthesis and signaling, and transcriptional regulation  yielded an r <dig> of  <dig>   .
the correlation was stronger than those recently published for ‘suli’ pear   <cit>  and for chinese bayberry   <cit> .
this analysis confirmed the reliability of the gene expression values generated from rna-seq.fig.
<dig> regression analysis of gene expression fold changes  obtained from quatitative pcr and rna-seq
multi-condition and pairwise differential expression analysis
differential expression analysis was conducted comparing multiple treatments or two treatments   <cit> .
the analysis on all four harvest maturity stages generated  <dig>  unigenes that were significantly different across these stages.
the results of the pairwise analysis on two maturity stages are presented in table  <dig>  the increased number of significant differentially expressed  unigenes from  <dig>  between s <dig> and s <dig>  to  <dig>  between s <dig> and s <dig>  and to  <dig>  between s <dig> and s <dig> suggests there were fewer transcriptional differences between closer stages.
regarding the transition between two adjacent stages, fewer gene expression changes occurred during the s2-s <dig> transition, when fruit gained the ability to soften to 20 n after ethylene treatment, than during the earlier s1-s <dig> transition, when fruit failed to ripen, and the later s3-s <dig> transition, when fruit developed the capacity to soften without ethylene treatment.
moreover, the highest number of de unigenes in the s3-s <dig> transition  suggests sophisticated molecular mechanisms occurred during this transition.
further analysis identified which de unigenes between two adjacent maturity stages were unique or shared across the three transitions .
a total of  <dig> de unigenes were shared across all three transitions.
the function of selected shared de genes, along with unique de unigenes, as related to fruit maturity and ripening capacity development is discussed later in this paper.table  <dig> number of differentially expressed unigenes between two maturity stages
na: not applicable
fig.
<dig> unique and shared differentially expressed unigenes in s1-s <dig>  s2-s <dig>  and s3-s <dig> pairwise analysis
k-means clusters and functional annotation analysis of the clusters
k-means clustering revealed representative patterns of gene expression over the four harvest maturity stages.
because we considered these patterns across four maturity stages, k-means clustering was processed on the  <dig>  de unigenes generated from multi-condition differential expression analysis, instead of the de unigenes from pairwise comparison.
using mapman classification,  <dig>  % of  <dig>  de unigenes were assigned to the  <dig> functional groups, while only  <dig>  % of total unigenes were assigned to these groups .
this indicates that a large portion of de unigenes associated with the four maturity stages had their putative functions identified and they could be visualized using mapman.
twelve clusters containing between  <dig> and  <dig> unigenes were determined .
of the considered unigenes,  <dig>  % fell into four clusters  that increased in transcript abundance from s <dig> to s <dig> while  <dig>  % belonged to four clusters  that decreased in expression.
clusters k <dig> and k <dig> contained unigenes that were strongly expressed at the s <dig> and s <dig> stages, respectively.
however, no enriched categories were identified in k <dig> and the enriched categories in k <dig> were not associated with our functional groups of interest, including cell wall metabolism, hormone biosynthesis and signaling, and transcription factors .
clusters k <dig> and k <dig> showed high expression of unigenes at s <dig>  the aux/iaa transcription factor family was enriched in both of these clusters, and the auxin-associated functional group was enriched in k <dig>  .
this suggests that the auxin-associated transcripts may play an important role in the s2-s <dig> transition.fig.
<dig> k-means clustering for differentially expressed unigenes across four stages  and functional classification within clusters .
a the y-axis is the base- <dig> logarithm of the mean of normalized counts of three biological replicates.
the number of unigenes in each cluster is indicated.
b clusters from left to right: from the most abundant k <dig> to the least abundant k <dig>  * indicates enriched categories according to fisher’s test
expression change in genes associated with cell walls, hormones, and transcription factors
given the importance of cell wall metabolism, hormone biosynthesis and signaling, and transcriptional regulation in overall fruit development processes, de transcripts putatively encoding proteins of these functions in the three transitions s1-s <dig>  s2-s <dig>  and s3-s <dig> were further investigated with mapman .
herein we mainly discuss transcripts with fc ≥  <dig>  with fc defined as the base- <dig> logarithm of the ratio rsem count in treatment 2/rsem count in treatment  <dig> .fig.
<dig> expression change of unigenes associated with cell wall metabolism in three transitions in mapman.
a <dig>  modification, a <dig>  pectin esterases, b. cellulose syn., c. degradation, d. precursor synthesis, e. cell wall proteins
fig.
<dig> expression change of unigenes associated with hormone metabolism in three transitions in mapman.
iaa: auxin/indole-3-acetic acid, aba: abscisic acid, ba: brassinosteroid, sa: salicylic acid, ga: gibberellin
fig.
<dig> number of differentially expressed unigenes putatively encoding transcription factors in three transitions
fig.
<dig> expression change of unigenes associated with transcriptional regulation in three transitions in mapman
cell wall metabolism
the identities of various cell wall metabolism-associated genes that are expressed during fruit development and ripening have been established for a range of species  <cit> .
our de novo pear fruit transcriptome contained  <dig> transcripts annotated to be associated with cell wall metabolism; of these transcripts,  <dig>  % were de across the four advancing stages of fruit maturity examined in this study.
the enrichment of the cell wall category in all three transitions   supports the idea that cell wall metabolism is critical during pear fruit growth and development.
more de transcripts putatively encoding different cell wall proteins were identified in the s1-s <dig> and s3-s <dig> transitions than in the s2-s <dig> transition .
the numbers of the transcripts with fc ≥  <dig> were  <dig> in s1-s <dig> ,  <dig> in s3-s <dig> , and  <dig> in s2-s <dig> .
furthermore, the numbers of de transcripts encoding proteins of the same cell wall groups such as degradation and modification were larger in the s1-s <dig> and s3-s <dig> transitions than in the s2-s <dig> transition .
these results suggest that transcripts of cell wall-associated genes experienced a more stable period during the s2-s <dig> transition, as compared to the s1-s <dig> and s3-s <dig> transitions, even though the fruit weight and diameter continually increased from s <dig> to s <dig> .table  <dig> unigenes associated with cell wall exhibiting a fold change ≥ <dig> in at least one transition
*the unigene is differentially expressed in the correspondent pairwise analysis
in the studies of fruit development in several species such as tomato, apple, grapevine, and orange, genes associated with cell wall synthesis, modification, and degradation received a large amount of attention .
the de transcripts in these functional categories were considered through the three transitions .
cellulose synthase contributes to building the cellulose backbone of the plant cell wall  <cit> .
in ‘la france’ pear, the expression of a cellulose synthase a catalytic subunit  was up-regulated from − <dig>  to  <dig> dafb, but there were no significant changes in its abundance during later fruit development stages  <cit> .
in our study, the putative cellulose synthase gene group was enriched only in the s2-s <dig> transition .
moreover, in the s3-s <dig> transition, three annotated cellulose synthase genes  were down-regulated.
ahmed found that cellulose content did not change during ripening of ‘bartlett’ pear  <cit> .
our results therefore suggest that accumulation of putative cellulose synthase transcripts and likely more cellulose construction occurred before the s <dig> stage in ‘bartlett’ pear.
furthermore, it is interesting to note that a down regulation of putative cellulose synthase genes coincided with the fruit's reaching s <dig> .
whether this down-regulation could be a prerequisite for, or a consequence of, attainment of ripening capacity requires further investigation.
xyloglucan endotransglucosylases/hydrolases  are cell wall modification enzymes that are thought to be involved in disassembly of the cellulose-xyloglucan matrix by cleaving the xyloglucan β-d-glucan backbones and then linking xyloglucan segments into them to loosen the cross-links between cellulose  <cit> .
this cell wall-modifying action may contribute to the relaxation of cell wall structures and fruit softening as ripening proceeds.
transcript abundance of most annotated xthsincreased from s <dig> to s <dig> and thereafter decreased from s <dig> to s <dig> .
our expression results for an xth transcript pcm_ <dig> contrast with earlier findings by fonseca et al., where a ‘rocha’ pear homolog  had low expression during fruit growth  and only exhibited an increase in abundance during fruit ripening   <cit> .
however, consistent with our data, several xth genes were upregulated during tomato fruit growth  <cit> .
miedes and lorences also reported an increase of the overall xth enzyme activity coincident with these xth gene expression changes, suggesting the contribution of xth to cell wall formation during fruit growth  <cit> .
therefore, we suggest that xth genes and their enzyme activity play an important structural role in cell wall during the s1-s <dig> transition.
in addition to xth, the cell wall modification group contains exp proteins that have been identified to be involved in polysaccharide association leading to cell wall loosening  <cit> .
the expression of six out of the eight putative exp genes increased from s <dig> to s <dig>  while the transcript abundance of all eight exps decreased from s <dig> to s <dig> .
these results agree with and complement the report by hiwasa et al., in which transcripts of pcexp <dig> and pcexp <dig>  were more abundant in young growing fruit than in mature fruit of ‘la france’ pear  <cit> .
this highlighted the important function of some exps in cell wall modification during fruit development.
any loosening of the cell wall caused by exp proteins also may enhance the abilities of other cell wall-targeting enzymes to move within the apoplast  and, consequently, facilitate fruit softening.
genes encoding pectin degradation enzymes involved in fruit softening, including pectin lyases/pectate lyases/polygalacturonases , have been well characterized in several species, such as tomato, banana, and strawberry  <cit> .
in the present study, this gene group became more enriched in the s1-s <dig> transition than in the s3-s <dig> transition , suggesting that pectin degradation processes become more active once pear fruit approach the mature stage.
consistent with the results found for pg transcripts in ‘rocha’ pear  <cit> , we detected a slight increase in transcript abundance of pc-pg <dig>  during the s1-s <dig> transition  and pc-pg <dig>  during the s2-s <dig> transition .
the results confirm that the high accumulation of pg transcripts does not start until pear fruit near the climacteric onset.
however, in contrast to the expression of these pgs, several other pectin degradation-related transcripts presented more significant changes: an increase of three out of four de pt/pg-annotated transcripts in the s1-s <dig> transition and a decrease of all de pt/pg-annotated transcripts in the s3-s <dig> transition.
it was shown that pectin-derived oligomers  induced an increase in ethylene biosynthesis in cultured pear fruit cells  <cit>  and that the pdos that accumulated when tomato fruit started to ripen could stimulate the ripening of tomato pericarp discs cut from mature-green fruit  <cit> .
therefore, we suspect that pectic oligomers could be produced by the pectolytic enzymes encoded by the genes with high transcript abundance during the s1-s <dig> and s2-s <dig> transitions, and these events may contribute to the increased ethylene production at s <dig> that subsequently lead to the softening of these fruit during 20 °c storage without the need for ethylene treatment.
our results showing high expression of three pg genes prior to fruit ripening is the first evidence at the transcript level of possible increases in cell wall degradation enzymes that could generate signal molecules from cell wall fragments to stimulate the development of ripening capacity in european pears.
additionally, we found similar gene expression patterns of different cell wall functional groups including exps and pts/pgs, which had high levels at s <dig>  s <dig> and a lower level at s <dig> .
the co-expression of these genes may imply the collaboration of these proteins in modifications of complex cell wall polysaccharide networks that are required for fruit cell growth.
this finding is similar to what was reported in tomato, in which, compared to the wild type, a significantly greater fruit firmness and reduction in cell wall pectin solubilization and depolymerization was shown in the double suppression line of leexp <dig> and lepg but not in the single mutant lines that were tested  <cit> .
hormone biosynthesis and signaling
hormone-associated genes play important roles in the regulation of ripening capacity  <cit> .
in our de novo pear fruit transcriptome,  <dig> unigenes were annotated as hormone-associated; of these,  <dig>  % were de among the four maturity stages.
in the hormone functional group, the highest number of de unigenes was associated with auxin .
the greatest changes in expression across stage transitions were observed for these unigenes , highlighting the potential role of auxin in regulating developmental processes that lead to the attainment of ripening capacity.
the transcript abundance of annotated tir <dig> , which is considered to be a key hormone receptor component in the auxin transduction pathway  <cit> , increased from s <dig> to s <dig> and decreased in s <dig>  additionally, most putative auxin-associated transcripts included saurs  and gh3s, which have been identified as auxin-responsive genes in a wide range of plants  <cit> .
furthermore, various gh <dig> genes were reported to be involved in iaa conjugation in many plant species  <cit> .
our data showed that the gene expression of several annotated saurs significantly increased during the s1-s <dig> transition, and putative auxin-responsive gh <dig> transcripts were up-regulated in the s2-s <dig> transition and then down-regulated in the s3-s <dig> transition .
our k-means cluster analysis had also determined that clusters k <dig> and k <dig>  representing unigenes most highly expressed in s <dig>  were enriched in auxin-associated unigenes .
auxin is considered a senescence retardant in fruit, and the breakdown of endogenous auxin has been reported to initiate ‘bartlett’ pear ripening  <cit> .
moreover, iaa levels declined prior to ripening in tomato, grape, and strawberry fruit  <cit> .
the changes in abundance of auxin-associated transcripts in our data suggest an important function of auxin in the s2-s <dig> transition in particular, where pear fruit developed a capacity to respond to ethylene and ripen.
we postulate that a decrease in auxin levels regulated the pear fruit’s responsiveness to ethylene and that this process occurred prior to autocatalytic ethylene biosynthesis.table  <dig> unigenes associated with hormone metabolism exhibiting a fc ≥ <dig> in at least one transition
*the unigene is differentially expressed in the correspondent pairwise analysis
ethylene is well known to be the main hormone regulating climacteric fruit ripening  <cit> .
our data showed that while the expression of both de acs genes increased from s <dig> to s <dig> and decreased from s <dig> to s <dig> , their overall expression was low throughout the four stages considered .
therefore, we suggest that the high fcs of the acs transcripts were probably biased due to their low rsem counts  <cit> .
the abundance of the aco transcript was slightly decreased in the s1-s <dig> transition , but did not significantly change during the later s2-s <dig> and s3-s <dig> transitions.
this behavior of the aco gene may explain the physiological data, where a higher internal ethylene concentration and ethylene production rate were detected in the s <dig> fruit that failed to ripen after 14 days at 20 °c.
fonseca et al.
reported aco activity was below detectable levels in ‘rocha’ pear during fruit growth  <cit> .
hence, we conclude that neither the expression changes of aco at the s1-s <dig> transition nor the ethylene produced at s <dig> had a significant effect on the ripening capacity of ‘bartlett’ pear.
moreover, our results on the expression of acs and aco genes agree with an ethylene biosynthesis and action model proposed in tomato, in which autocatalytic ethylene production is initiated by induction of an acs  <cit> .
ethylene is perceived by protein receptors in plant tissues and this binding inactivates kinase activity of ctr <dig> , allowing ein <dig>  and ein <dig> to transduce ethylene signaling  <cit> .
in the present study, transcript abundance of the ethylene receptor pc-ers1a decreased from s <dig> to s <dig> and had a maximum fc of − <dig>  in the s3-s <dig> transition .
the gene expression of a pc-ctr <dig>  pcm_ <dig>  increased from s <dig> to s <dig>  then stayed at a similar level in s <dig> .
previous studies have shown that a decrease in gene expression of the ethylene receptors leetr <dig> and leetr <dig> increased ethylene sensitivity in tomato  <cit> .
in the present study, s <dig> fruit were capable of ripening after 14 days without ethylene treatment.
therefore, it appears that the ethylene receptor pc-ers1a and ethylene signaling protein pc-ctr <dig> are involved in signal transduction of ethylene that consequently activated autocatalytic ethylene production in s <dig> fruit.
gibberellin  has been reported to stimulate pericarp growth of pea fruit  <cit>  and silique growth of arabidopsis  <cit> .
in the present study, ga-associated gene subcategories were enriched at the s1-s <dig> and s2-s <dig> transitions .
ga-stimulated transcripts  are known as targets of ga regulation  <cit> .
two annotated gasts and three putative ga-regulated transcripts were up-regulated from s <dig> to s <dig> .
this may indicate that gas play a role in fruit growth during the s1-s <dig> transition.
jasmonic acid  was suggested to regulate fruit growth in apple  <cit> .
our data showed that similar to ga-associated transcripts, ja-associated transcripts were enriched at the s1-s <dig> and s2-s <dig> transitions .
however, in contrast to the expression patterns of ga-associated genes, transcript abundance of the majority of de ja-associated transcripts decreased in the s1-s <dig> transition and increased in the s2-s <dig> transition .
these transcripts included three putative allene oxide synthases  and an annotated lipoxygenase, which encode enzymes involved in jasmonic acid biosynthesis.
as aos is considered to be a rate-limiting step in ja biosynthesis  <cit> , the expression patterns of our ja-associated transcripts complement findings by kondo et al.
in growing apple fruit  <cit> ; there, ja was at a high concentration early in fruit development, decreased, and then increased again.
therefore, ja may be involved in the regulation of pear growth and development through stages s <dig> to s <dig>
a key enzyme in aba biosynthesis, 9-cis-epoxycarotenoid dioxygenase , has been reported to be associated with ripening of several fruit such as ‘gold nijisseiki’ pear and strawberry  <cit> .
in our data, one nced showed a high fc at s2-s <dig>  the transition to ethylene responsiveness .
however, similar to genes related to ethylene biosynthesis, the expression level was very low in all four stages  , suggesting a possible bias of high fc.
we also found genes associated with aba such as genes encoding gram domain proteins  <cit>  and hva <dig>  <cit> .
however, we have not seen clear evidence of the importance of aba genes in pear growth or in association with the development of ripening capacity.
our results seem to agree with those of an earlier study, where the increase in aba concentration was merely coincident with ethylene evolution during ripening in ‘jingbaili’ and ‘gold nijisseiki’, asian pears  <cit> .
transcription factors
analyzing the expression of genes encoding transcription factors  help to identify key factors that regulate fruit growth and development, particularly those factors that control the fruit’s response to exogenous ethylene and/or its capacity to soften without ethylene treatment.
in the pear fruit, a large number of unigenes in the de novo transcriptome  were annotated as tfs.
of these unigenes,  <dig>  % were de across the four maturity stages.
among all de transcripts putatively identified as tfs, the ap2/erebp family members were the most abundant .
similar to the results obtained from microarray analyses of tomato and peach  <cit> , the gene expression of various putative erebps was either up- or down-regulated across stage transitions .
annotated bhlh transcripts were also highly represented and enriched in all transitions with various expression patterns.
given the broad range of processes affected by ap2/erebps- or bhlh-mediated regulation, which includes plant development, primary and secondary metabolism, hormone signaling, and response to biotic and abiotic stresses, as well as the intricate target specificity of each member of these tf families  <cit> , it is not surprising to find such diversity in transcriptional activation or repression across the fruit maturity stages considered in this work.table  <dig> unigenes associated with transcriptional regulation exhibiting a fc ≥ <dig> in at least one transition
*the unigene is differentially expressed in the correspondent pairwise analysis
in contrast to the unsystematic behaviors of ap2/erebp or bhlh genes, the gene expression of members of bzip , wrky, arf, and aux/iaa families showed a more consistent response; i.e., either up or down-regulation, with an enrichment  in a specific transition .
in particular, high up-regulation of several putative bzip genes occurred at the s1-s <dig> transition, compliments the previous results that showed one annotated bzip gene with higher expression in early maturity fruit compared to mature and ripening fruit of ‘rocha’ pear .
while bzip tfs have been implicated in the regulation of a wide range of processes including biotic and abiotic stress responses, hormone signaling, and development  <cit> , the concerted expression of a subset of bzip members in this study may point to a common functional feature in fruit development that warrants further investigation.
strikingly, expression of a majority of genes putatively encoding the tf aux/iaa, arf, and wrky were up-regulated in the s2-s <dig> transition and down-regulated in the s3-s <dig> transition .
these concerted aux/iaa and arf gene expression patterns paralleled the transcript abundance changes in auxin-associated genes, which peaked in the s2-s <dig> transition .
these results further underscore the important role of auxin in the development of ripening capacity in response to ethylene; i.e., transition from s <dig>  to s <dig> .
CONCLUSIONS
in this study, we characterized the physico-chemical features and transcriptional profiles associated with the development of ripening capacity in ‘bartlett’ pear across four maturity stages .
our analysis, which focused on the differential expression of genes associated with cell wall metabolism, hormone signaling, and transcriptional regulation, suggested a role for specific transcripts, as well as the coordination of members in the same gene family or among gene families, in the attainment of ripening capacity .
we postulate that pectin degradation enzymes may produce early signal molecules  that stimulate ethylene biosynthesis associated with the development of fruit ripening capacity.
additionally, auxin-associated genes appear to play an important role in regulating the ability of ethylene-treated fruit to ripen at 20 °c.
the transcription factor family bzip appears to regulate the s1-s <dig> transition and aux/iaa, arf and wrky may regulate the s2-s <dig> and s3-s <dig> transitions.
our results represent a resource for further investigation of some candidate genes or gene groups that regulate the responsiveness of pear, and perhaps other fruit, to ethylene and other plant hormones.
in addition, the candidate genes could be examined as molecular markers to indicate the status of ripening capacity, as well as determining appropriate postharvest treatments.fig.
<dig> proposed mechanisms regulating ripening capacity development during the final stages of pear fruit growth.
transition 1: fruit develop ripening capacity responsive to ethylene treatment; transition 2: fruit develop ripening capacity without the need of ethylene treatment.
ap2/erebp: apetala 2/ethylene response element binding protein, bhlh: basic helix-loop-helix, bzip: basic region/leucine zipper, arf: auxin response factors, hb: homeobox, c2h2: cys2his <dig> zinc finger; ga: gibberellin, ja: jasmonic acid, iaa: auxin/indole-3-acetic acid; xth :xyloglucan endotransglucosylase/hydrolase , pt/pg: pectin lyase/pectate lyase/polygalacturonase, exp: expansin
additional files
additional file 1:
color changes of pears harvested at four harvest times at harvest and after air/ethylene treatment.
s <dig>  s <dig>  s <dig>  and s <dig> were harvested a week apart; s <dig> coincident with commercial harvest.
d14: 14 days at 20 °c following treatment of pears with air or  <dig> μll− <dig> ethylene  for 24 h. rna extracted from peel tissues ah of s <dig> to s <dig> were used for rna-sequencing.
.
additional file 2:
soluble solids content  and skin color of ‘bartlett’ pears after 14 days at 20 °c.
pears were treated with either air or 100μll− <dig> ethylene  for 24 h at 20 °c.
* mean values with different uppercase letters between air and et-treated fruit and different lowercase letters among maturity stages are statistically different according to tukey’s test .
additional file 3:
primers for quantitative pcr validation.
additional file 4:
mapman classification of gene in the
de novo
transcriptome.
additional file 5:
enriched functional groups in each cluster and enrichment analysis.
* fisher’s test with bonferroni correction .
additional file 6:
enrichment of groups of cell wall, hormone metabolism, and transcription factors in the three transitions: s1-s <dig>  s2-s <dig>  and s3-s <dig>  ns: no significant difference, * fisher’s test with bonferroni correction.
additional file 7:
number of unigenes associated with a. cell wall and b. hormones according to mapman classifications in three transitions: s1-s <dig>  s2-s <dig>  and s3-s <dig>
additional file 8:
sequences of de no vo transcriptome and their putative functions.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
sf and am designed the experiment, collected fruit, and analyzed physiological characteristics.
sf extracted and purified the rna for sequencing and analyzed physio-chemical data.
kc carried out the quality check for the illumina reads, de novo assembly, and blastx.
nn conducted transcriptome validation of sequence identity and qpcr, differential expression, and functional annotation analysis.
tk and ag participated in primer design and qpcr.
cj provided advice for the experiment design.
fz and em assisted with data interpretation and provided extensive editing of the manuscript.
em coordinated all of the work.
nn led the writing of the manuscript draft, with sections provided by sf and kc.
all authors contributed to editing the manuscript.
authors’ information
not applicable
availability of data and materials
not applicable

 european pears, including ‘bartlett’, ‘d’anjou’, and ‘comice’, are economically significant fruit in the united states, with a production value of $ million in. as a climacteric fruit, pears ripen in association with a substantial increase in rates of respiration and ethylene biosynthesis. unlike many climacteric fruit such as apple and mango, european pears develop poor texture and flavor if left to ripen on the tree. therefore, most european pears are harvested at the mature-green stage and then usually exposed to ethylene or cold temperatures prior to ripening to enhance their ability to produce ethylene and ripen at 20 °c. hansen found that early maturity ‘bartlett’ and ‘d’anjou’ pear might not respond to ethylene or cold treatment while late maturity fruit could ripen without any conditioning treatment. however, the underlying molecular mechanisms governing this developmental shift are still not well understood. furthermore, as a climacteric fruit, pear fruit ripening includes the transition from auto-inhibitory ethylene to autocatalytic ethylene that regulates the numerous metabolic processes associated with fruit ripening. the intrinsic developmental factors that regulate the transition from system to system remain mostly unknown. ripening is postulated to be initiated by activation of specific transcriptional regulators, such as colorless non ripening and ripening-inhibitor, as first identified in tomato, a model organism to study fruit ripening. these regulators lead to signal transduction pathways that include ethylene as an essential signaling molecule. these signaling pathways control many ripening-related biochemical events such as chlorophyll degradation, starch degradation to sugars, decreases in organic acids, and production of aroma compounds. several studies designed to elucidate the molecular pathways of fruit ripening have focused on genes associated with hormone and cell wall metabolism, as well as transcriptional regulation. some of the molecular aspects of european pear ripening have been investigated. several studies reported an increase in ethylene biosynthesis enzymes, 1-aminocyclopropane-1-carboxylate synthase and acc oxidase, following ethylene treatment and cold storage. increases in transcript abundance of pear fruit ethylene biosynthesis genes and ethylene perception genes including pc-etr1a and pc-ers1a during fruit ripening were also reported. low transcript abundance of genes encoding cell wall modifying proteins such as β-galactosidases and expansins were detected during fruit development in ‘rocha’ pear. in addition, large-scale expression profiles of ‘rocha’ and ‘la france’ pear during fruit growth and ripening have been generated. however, these two studies utilized microarrays with a limited number of fruit-specific sequences. to our knowledge, genes associated with hormones other than ethylene and transcription factors have not been characterized during pear fruit development. in the last 5 years, next generation sequencing technologies accompanied by sophisticated bioinformatics tools have been developed and provide a powerful approach to examine the transcriptomes of non-model plants. accordingly, these tools have been utilized to determine transcriptional changes during fruit growth and development in a variety of species including chinese bayberry, orange, and korean black raspberry. in the present study, ngs technology was used to characterize the molecular mechanisms regulating the development of ripening capacity in ‘bartlett’ pear fruit. the specific objectives were to 1) develop a better understanding of the acquisition of pear ripening capacity and 2) define the molecular regulation of pear fruit ripening, focusing on genes associated with cell wall metabolism, hormone biosynthesis and signaling, and transcription factors. methods plant materials and physico-chemical analysis ‘bartlett’ pear fruit were produced at a commercial orchard in sacramento county, california, usa. fruit were harvested at 7-day intervals for 4 weeks, from to dafb; the fourth harvest time was equivalent to the first commercial harvest. sixty fruit were collected at each harvest time from a total of five trees. immediately after harvest, fruit were randomized and divided into five groups of each group was composed of three biological replications with four fruit each. group fruit were analyzed within 24 h of harvest for ethylene production rate, respiration rate, weight, diameter, skin color, flesh firmness, and ssc. group fruit were used to measure the internal ethylene concentration. peel tissues for molecular analysis were collected from fruit in group fruit from groups and were enclosed in separate 20 l glass jars and treated with or μll− ethylene in flowing air streams of mlmin− for 24 h at 20 °c. these fruit were then held at 20 °c and 90 % relative humidity for 14 days to allow for ripening. after 14 days, fruit were evaluated for skin color, flesh firmness, and ssc. rates of ethylene production and respiration were assessed for each replication by sealing four fruit inside a  l glass jar and using the method described by villalobos et al.. headspace samples were collected with 10 ml syringes and injected into a gas chromatograph for ethylene quantification or a pir-2000r infrared analyzer for co analysis. fruit diameter was measured across the widest point of each fruit with a caliper. pear skin color was determined on two diametrically opposite sides of each fruit using a chroma meter cr-. the color data were captured using the cie color space and expressed as the hue angle, where 90° represents full yellow and 180° corresponds to full green. flesh firmness was quantified as the resistance to 9 mm penetration with an 8 mm-diameter probe using a fruit texture analyzer on two opposite sides of the fruit after the peel was removed. ssc was measured in juice samples extracted by squeezing cortical wedges cut from two opposite sides of each of four fruit in two layers of cheesecloth, with a reichert ar series refractometer. the internal ethylene concentration was determined according to coombe and hale and chervin et al.. briefly, pre-weighed fruit were placed individually in a chamber containing a saturated solution of nacl. each fruit was submerged in the solution under an inverted funnel with the narrow end capped with a rubber septum. the air trapped in the narrow end of the funnel was withdrawn with a syringe. the chamber was sealed and a partial vacuum of −700 mm hg was applied for 5 min. after returning to atmospheric pressure, 1 ml of the fruit internal atmosphere trapped in the narrow end of the funnel was sampled by syringe and the ethylene concentration was determined by gas chromatography as described above. statistical analysis was performed on each variable by means of analysis of variance using the sas statistical package. the mean values of three replications were compared using tukey’s test. rna extraction total rna was isolated from  g tissues ground in liquid n which contained both skin and flesh tissues peeled from two opposite sides of fruit, using the qiagen rneasy plant mini kit according to the manufacturer’s instructions. the total rna was then treated with dnase i recombinant, rnase-free to remove dna contamination. the total rna concentration was quantified using a nanodrop spectrophotometer, with absorbance at 260 nm. the quality of total rna was verified by examining the ratio od260/od and formaldehyde agarose gel electrophoresis. rna sequencing illumina library preparation and sequencing of samples were completed following standard protocols at the uc davis dna technologies core. the integrity and quantity of total rna was examined using an agilent bioanalyzer rna kit and invitrogen’s qubit. mrna was isolated from total rna using dynabeads oligo-d. the rna-seq library was constructed by following the truseq protocol. individual libraries were prepared with barcodes and pooled for sequencing on one lane of the illumina hiseq platform. paired-end reads of 100 cycles were collected and fastq files were generated using the illumina pipeline. de novo assembly and count estimation given that inclusion of a greater number of reads in de novo assembly produces a greater contiguity of sequences, illumina reads obtained from this experiment and a second ‘bartlett’ pear ripening capacity experiment were combined for the assembly. the raw reads were trimmed to remove truseq adapters and low quality bases, using trimmomatic. surviving paired reads were used as input for de novo transcript assembly. the assembly was carried out using trinity with default parameters except --min_kmer_cov was set to to minimize redundancy in the set of putative transcripts, the contigs were clustered using cd-hit and then with tgicl. stringent similarity parameters were selected to minimize the likelihood of merging paralogous transcripts. this reduced the number of contigs in the original output by trinity. as these contigs may still represent multiple isoforms of the same gene, contigs that shared a common trinity component and sub-component were naively grouped into unigenes by rsem. estimated read counts associated with the assembled contigs were determined with rsem, which utilizes bowtie to map reads to a reference database composed of the assembled contigs. in preparing this database the unigene to contig mapping described above was provided to permit rsem to estimate read counts at both the individual contig and unigene level. the rsem output represented the estimated counts of reads associated with each isoform or unigene, recognizing the uncertainty inherent in assigning reads to isoforms that may share one or more exons. sequence identity validation and quantitative pcr validation sequences in the de novo transcriptome were mapped to the reference genome of asian pear and european pear using gmap to check for possible chimeric and non-match sequences, using k-mer orfs were examined using orfpredictor with an orf cut-off length of base pairs. the blastx program was used to perform similarity searches of the contigs against the tair v and refseq protein databases with an e-value threshold of 1e− the contigs were annotated with the description inherited from the best hits in each database. cdna was synthesized from 1 μg dnase - treated total rna, using superscript™ iii first strand synthesis systems for rt-pcr systems. before qpcr validation, sequences of interest were aligned against the available sequences of asian and european pears on the ncbi est database and their published genome to confirm sequence identity, using the local tblastn function in bioedit. the gene expression was examined using sybr green pcr master mix and a real time pcr system. ef1alpha was chosen as the housekeeping gene after testing with 18 s, 26 s, β-actin, and tubulin primers for sequences of interest were designed using primer and passed the primer efficiency check for qpcr. in the regression analysis, the fc of qpcr was ΔΔct and the fc of rna-seq was the base- logarithm of the ratio rsem count in treatment 2/rsem count in treatment mapman functional annotation analysis mapman functional annotation analysis was utilized to gain an understanding about the general function of genes expressed during fruit growth and to identify gene families that may play essential roles in regulating the development of pear ripening capacity. contigs were classified into specific functional groups, using mercator with a blast cut-off of because one unigene might have multiple contigs, a functional term of a unigene was derived from its representative contig that had the highest bit score. enrichment analysis was completed through fisher’s test using mefisto with bonferroni correction. gene expression changes were viewed in mapman.1r. differential expression analysis the unigene counts were subjected to both pairwise and multi-condition analysis to detect de sequences between two harvest times and among four harvest times of pear fruit, respectively, using the ebseq package with false discovery rate of. the method employed by ebseq manages the varying uncertainty in counts across isoform groups. in convergence checking, the maximum round of each comparison was chosen based on a difference less than between the two last iterations of ebout$alpha, and of ebout$beta. for pairwise analysis, unigenes with a posterior probability of being differentially expressed of ≥  were identified as differentially expressed between two harvest times. for multi-condition analysis, unigenes with p1 ≤  were identified as differentially expressed across the four harvest times. normalized counts of unigenes for calculating gene fold changes were obtained from the multi-condition analysis. k-means cluster analysis k-means clustering was utilized to determine particular patterns in gene expression throughout the four harvest times, using the base- logarithm of the average normalized counts of three biological replicates. the number of clusters was identified using the figures of merit application embedded in mev. unigenes in each cluster were then identified using the r package amap with pearson correlation, in which random sets were applied to generate reproducible clusters. a heatmap of the number of unigenes in mapman categories in each cluster was built on the r package gplots. accession code the clean reads produced in this study have been deposited at ddbj/embl/genbank short read archive: biosample numbers samn - samn accession codes srr - srr and under project number prjna this transcriptome shotgun assembly project has been deposited at ddbj/embl/genbank under the accession gbxl the version described in this paper is the first version, gbxl the gene id, contig id, and their putative function can be accessed through additional file computer system except for the de novo transcriptome assembly and mapping to a reference genome, all data analyses were completed with a dell optiplex 4gb ram, 32-bit, intel core i5- cpu with windows enterprise, microsoft office and r., rstudio i386-pc-mingw32/i platform.  physico-chemical changes during fruit growth and development to characterize the relationship between pear fruit maturation and the development of ripening capacity, ‘bartlett’ pear fruit were harvested at weekly intervals commencing 3 weeks before commercial harvest to capture four progressive stages of maturity: s1: 100 days after full bloom, s2: dafb, s3: dafb, and s4: dafb. fruit growth and maturity were monitored by physico-chemical measurements. fruit weight and diameter at harvest increased considerably with these advancing stages of maturity. in contrast, the flesh firmness at harvest steadily decreased as fruit maturity increased. rates of respiration and the internal ethylene concentration were highest in fruit harvested at s and relatively low in s s and s. rates of ethylene production were also relatively higher in s than s and s before increasing again at s. despite the higher ethylene production rate at s, the level was substantially lower than typical rates produced during climacteric ripening of ‘bartlett’ pear, which can be as high as μlkg−1h−. there were no significant differences in fruit soluble solids content and skin color among four harvest maturity stages examined in this study.fig. pear fruit at four harvest times.s1: dafb, s2: dafb, s3: dafb, and s4: dafb amean values with different letters are significantly different according to tukey’s test after harvest, fruit at each of the four maturity stages were treated with or μll− ethylene for 24 h and then evaluated for their ripening capacity based on softening after being held at 20 °c for 14 days. the ability of fruit to soften in the presence or absence of ethylene increased with advancing harvest maturity. when treated with ethylene, s fruit failed to soften, s fruit displayed partial softening, and s and s fruit softened to a firmness of <5 n. in the absence of ethylene treatment, fruit harvested at stages s and s failed to soften, while s and s fruit softened to 60 n and  n, respectively. therefore, it appears that the slightly higher rate of ethylene production during the preclimacteric phase in s had no positive effect on the ability of fruit to ripen when harvested at this stage. the slight increase in firmness observed for s fruit at day shelf life presumably reflected water loss during storage of fruit harvested at an immature stage; this agrees with what has been found in apple and bell pepper. the general fruit softening response was also accompanied by similar changes in peel color, as evidenced by the hue angle. there was no significant effect of harvest maturity and ethylene treatment on fruit ssc at the completion of a 14-day shelf life.fig. firmness changes of pears harvested at four maturity stages at harvest and after air/ethylene treatment. s s s and s were harvested a week apart; s coincident with commercial harvest. d14: 14 days at 20 °c following treatment of pears with air or μll− ethylene for 24 h. bars indicate standard errors; letters indicate significant differences among the three firmness values within a stage according to tukey’s test in other species, standard stages of fruit development have been well established. for instance, these stages in tomato include green, mature green, breaker, pink, and red ripe, while peach and plum development is described as an s to s double sigmoid pattern. for ‘bartlett’ pear, we are unaware of defined standard stages of fruit development, except those utilizing firmness as a ripeness indicator: 85–98 n, when the fruit are ready to harvest and 20 n, when the fruit are ready to consume, with a consideration of ssc and size. in the current study, because of the low ripening capacity of pear fruit at early maturity stages, we considered full ripening capacity was achieved when fruit firmness reached 20 n after 14 days at 20 °c; this was named “rc14” for “ripening capacity at 14 days” after harvest. given this definition of ripening capacity, we observed the following response of the four harvest maturity stages: s and s did not achieve rc14; s achieved rc with ethylene treatment; and s fruit achieved rc without ethylene treatment. rna-seq and de novo assembly rna sequencing of the peel tissue of the four maturity stages generated million 2x bp paired-end reads. of the mil paired-end reads from both experiments,  % were retained after the quality check, in which the unqualified read was mostly due to bottom middle swath in the sequencing system. a trinity assembly on mil qualified paired-end reads generated contigs that were clustered into unigenes. the contig length ranged from to  bp, with a median length of 502 bp and a mean length of 911 bp. validation of the transcriptome in sequence identity and expression levels sequence identity of the de novo transcriptome was first validated through putative function determination. blastx of the contigs against the ncbi refseq and arabidopsis protein databases identified similar proteins in these reference sets for  % and  %, respectively, of the unigenes. this indicates that the functions of a large portion of the genes of p. communis have not yet been identified. using the ncbi non-redundant database with a threshold of 1e− 68 %, 80 %, and 93 % of unigenes of chinese bayberry, korean black raspberry, and ‘suli’ pear transcriptomes, respectively, were annotated. in the general functional description of the transcriptome examined using mapman,  % of the unigenes were assigned to meaningful bincodes of mapman, with the highest numbers of unigenes classified into protein, rna, signaling, stress, and transport categories.fig. mapman functional annotations of the transcriptome. 2nd: secondary, aa: amino acid, ps: photosynthesis, met.: metabolism, syn: synthesis, other 1: nucleotide met., minor cho met., major cho met., mitochondrial electron transport/atp and glycolysis; other 2: co-factor and vitamin met., tca/org transformation, metal handling, tetrapyrrole syn., c1-met., opp, n-met., biodegradation of xenobiotics, fermentation, polyamine met., gluconeogenesis/glyoxylate cycle, s-assimilation, micro rna and natural antisense open reading frame finders evaluate the degree to which full coding sequence are assembled. this analysis determined contigs had an orf of length ≥ 200 bp, with an average length of 724 bp. moreover, the high percentages of mapped contigs when mapping to reference genomes indicated good sequence identity of our de novo transcriptome. mapping all contigs of the de novo transcriptome to the asian pear genome revealed that were mapped to the reference genome, in which contigs were possibly chimera sequences, and contigs were non-matched sequences. additionally, mapping to the recently published european pear genome showed mapped contigs, in which were possible chimeras, and were non-matched sequences. to validate gene expression values obtained from rna-seq data, we examined the correlation between fold changes calculated on rsem counts and the equivalent values measured by quantitative pcr. the validation on eleven transcripts associated with cell wall metabolism, hormone biosynthesis and signaling, and transcriptional regulation yielded an r of. the correlation was stronger than those recently published for ‘suli’ pear and for chinese bayberry. this analysis confirmed the reliability of the gene expression values generated from rna-seq.fig. regression analysis of gene expression fold changes obtained from quatitative pcr and rna-seq multi-condition and pairwise differential expression analysis differential expression analysis was conducted comparing multiple treatments or two treatments. the analysis on all four harvest maturity stages generated unigenes that were significantly different across these stages. the results of the pairwise analysis on two maturity stages are presented in table  the increased number of significant differentially expressed unigenes from between s and s to between s and s and to between s and s suggests there were fewer transcriptional differences between closer stages. regarding the transition between two adjacent stages, fewer gene expression changes occurred during the s2-s transition, when fruit gained the ability to soften to 20 n after ethylene treatment, than during the earlier s1-s transition, when fruit failed to ripen, and the later s3-s transition, when fruit developed the capacity to soften without ethylene treatment. moreover, the highest number of de unigenes in the s3-s transition suggests sophisticated molecular mechanisms occurred during this transition. further analysis identified which de unigenes between two adjacent maturity stages were unique or shared across the three transitions. a total of de unigenes were shared across all three transitions. the function of selected shared de genes, along with unique de unigenes, as related to fruit maturity and ripening capacity development is discussed later in this paper.table number of differentially expressed unigenes between two maturity stages na: not applicable fig. unique and shared differentially expressed unigenes in s1-s s2-s and s3-s pairwise analysis k-means clusters and functional annotation analysis of the clusters k-means clustering revealed representative patterns of gene expression over the four harvest maturity stages. because we considered these patterns across four maturity stages, k-means clustering was processed on the de unigenes generated from multi-condition differential expression analysis, instead of the de unigenes from pairwise comparison. using mapman classification,  % of de unigenes were assigned to the functional groups, while only  % of total unigenes were assigned to these groups. this indicates that a large portion of de unigenes associated with the four maturity stages had their putative functions identified and they could be visualized using mapman. twelve clusters containing between and unigenes were determined. of the considered unigenes,  % fell into four clusters that increased in transcript abundance from s to s while  % belonged to four clusters that decreased in expression. clusters k and k contained unigenes that were strongly expressed at the s and s stages, respectively. however, no enriched categories were identified in k and the enriched categories in k were not associated with our functional groups of interest, including cell wall metabolism, hormone biosynthesis and signaling, and transcription factors. clusters k and k showed high expression of unigenes at s the aux/iaa transcription factor family was enriched in both of these clusters, and the auxin-associated functional group was enriched in k. this suggests that the auxin-associated transcripts may play an important role in the s2-s transition.fig. k-means clustering for differentially expressed unigenes across four stages and functional classification within clusters. a the y-axis is the base- logarithm of the mean of normalized counts of three biological replicates. the number of unigenes in each cluster is indicated. b clusters from left to right: from the most abundant k to the least abundant k * indicates enriched categories according to fisher’s test expression change in genes associated with cell walls, hormones, and transcription factors given the importance of cell wall metabolism, hormone biosynthesis and signaling, and transcriptional regulation in overall fruit development processes, de transcripts putatively encoding proteins of these functions in the three transitions s1-s s2-s and s3-s were further investigated with mapman. herein we mainly discuss transcripts with fc ≥  with fc defined as the base- logarithm of the ratio rsem count in treatment 2/rsem count in treatment.fig. expression change of unigenes associated with cell wall metabolism in three transitions in mapman. a modification, a pectin esterases, b. cellulose syn., c. degradation, d. precursor synthesis, e. cell wall proteins fig. expression change of unigenes associated with hormone metabolism in three transitions in mapman. iaa: auxin/indole-3-acetic acid, aba: abscisic acid, ba: brassinosteroid, sa: salicylic acid, ga: gibberellin fig. number of differentially expressed unigenes putatively encoding transcription factors in three transitions fig. expression change of unigenes associated with transcriptional regulation in three transitions in mapman cell wall metabolism the identities of various cell wall metabolism-associated genes that are expressed during fruit development and ripening have been established for a range of species. our de novo pear fruit transcriptome contained transcripts annotated to be associated with cell wall metabolism; of these transcripts,  % were de across the four advancing stages of fruit maturity examined in this study. the enrichment of the cell wall category in all three transitions supports the idea that cell wall metabolism is critical during pear fruit growth and development. more de transcripts putatively encoding different cell wall proteins were identified in the s1-s and s3-s transitions than in the s2-s transition. the numbers of the transcripts with fc ≥  were in s1-s, in s3-s, and in s2-s. furthermore, the numbers of de transcripts encoding proteins of the same cell wall groups such as degradation and modification were larger in the s1-s and s3-s transitions than in the s2-s transition. these results suggest that transcripts of cell wall-associated genes experienced a more stable period during the s2-s transition, as compared to the s1-s and s3-s transitions, even though the fruit weight and diameter continually increased from s to s.table unigenes associated with cell wall exhibiting a fold change ≥ in at least one transition *the unigene is differentially expressed in the correspondent pairwise analysis in the studies of fruit development in several species such as tomato, apple, grapevine, and orange, genes associated with cell wall synthesis, modification, and degradation received a large amount of attention. the de transcripts in these functional categories were considered through the three transitions. cellulose synthase contributes to building the cellulose backbone of the plant cell wall. in ‘la france’ pear, the expression of a cellulose synthase a catalytic subunit was up-regulated from − to dafb, but there were no significant changes in its abundance during later fruit development stages. in our study, the putative cellulose synthase gene group was enriched only in the s2-s transition. moreover, in the s3-s transition, three annotated cellulose synthase genes were down-regulated. ahmed found that cellulose content did not change during ripening of ‘bartlett’ pear. our results therefore suggest that accumulation of putative cellulose synthase transcripts and likely more cellulose construction occurred before the s stage in ‘bartlett’ pear. furthermore, it is interesting to note that a down regulation of putative cellulose synthase genes coincided with the fruit's reaching s. whether this down-regulation could be a prerequisite for, or a consequence of, attainment of ripening capacity requires further investigation. xyloglucan endotransglucosylases/hydrolases are cell wall modification enzymes that are thought to be involved in disassembly of the cellulose-xyloglucan matrix by cleaving the xyloglucan β-d-glucan backbones and then linking xyloglucan segments into them to loosen the cross-links between cellulose. this cell wall-modifying action may contribute to the relaxation of cell wall structures and fruit softening as ripening proceeds. transcript abundance of most annotated xthsincreased from s to s and thereafter decreased from s to s. our expression results for an xth transcript pcm_ contrast with earlier findings by fonseca et al., where a ‘rocha’ pear homolog had low expression during fruit growth and only exhibited an increase in abundance during fruit ripening. however, consistent with our data, several xth genes were upregulated during tomato fruit growth. miedes and lorences also reported an increase of the overall xth enzyme activity coincident with these xth gene expression changes, suggesting the contribution of xth to cell wall formation during fruit growth. therefore, we suggest that xth genes and their enzyme activity play an important structural role in cell wall during the s1-s transition. in addition to xth, the cell wall modification group contains exp proteins that have been identified to be involved in polysaccharide association leading to cell wall loosening. the expression of six out of the eight putative exp genes increased from s to s while the transcript abundance of all eight exps decreased from s to s. these results agree with and complement the report by hiwasa et al., in which transcripts of pcexp and pcexp were more abundant in young growing fruit than in mature fruit of ‘la france’ pear. this highlighted the important function of some exps in cell wall modification during fruit development. any loosening of the cell wall caused by exp proteins also may enhance the abilities of other cell wall-targeting enzymes to move within the apoplast and, consequently, facilitate fruit softening. genes encoding pectin degradation enzymes involved in fruit softening, including pectin lyases/pectate lyases/polygalacturonases, have been well characterized in several species, such as tomato, banana, and strawberry. in the present study, this gene group became more enriched in the s1-s transition than in the s3-s transition, suggesting that pectin degradation processes become more active once pear fruit approach the mature stage. consistent with the results found for pg transcripts in ‘rocha’ pear, we detected a slight increase in transcript abundance of pc-pg during the s1-s transition and pc-pg during the s2-s transition. the results confirm that the high accumulation of pg transcripts does not start until pear fruit near the climacteric onset. however, in contrast to the expression of these pgs, several other pectin degradation-related transcripts presented more significant changes: an increase of three out of four de pt/pg-annotated transcripts in the s1-s transition and a decrease of all de pt/pg-annotated transcripts in the s3-s transition. it was shown that pectin-derived oligomers induced an increase in ethylene biosynthesis in cultured pear fruit cells and that the pdos that accumulated when tomato fruit started to ripen could stimulate the ripening of tomato pericarp discs cut from mature-green fruit. therefore, we suspect that pectic oligomers could be produced by the pectolytic enzymes encoded by the genes with high transcript abundance during the s1-s and s2-s transitions, and these events may contribute to the increased ethylene production at s that subsequently lead to the softening of these fruit during 20 °c storage without the need for ethylene treatment. our results showing high expression of three pg genes prior to fruit ripening is the first evidence at the transcript level of possible increases in cell wall degradation enzymes that could generate signal molecules from cell wall fragments to stimulate the development of ripening capacity in european pears. additionally, we found similar gene expression patterns of different cell wall functional groups including exps and pts/pgs, which had high levels at s s and a lower level at s. the co-expression of these genes may imply the collaboration of these proteins in modifications of complex cell wall polysaccharide networks that are required for fruit cell growth. this finding is similar to what was reported in tomato, in which, compared to the wild type, a significantly greater fruit firmness and reduction in cell wall pectin solubilization and depolymerization was shown in the double suppression line of leexp and lepg but not in the single mutant lines that were tested. hormone biosynthesis and signaling hormone-associated genes play important roles in the regulation of ripening capacity. in our de novo pear fruit transcriptome, unigenes were annotated as hormone-associated; of these,  % were de among the four maturity stages. in the hormone functional group, the highest number of de unigenes was associated with auxin. the greatest changes in expression across stage transitions were observed for these unigenes, highlighting the potential role of auxin in regulating developmental processes that lead to the attainment of ripening capacity. the transcript abundance of annotated tir, which is considered to be a key hormone receptor component in the auxin transduction pathway, increased from s to s and decreased in s additionally, most putative auxin-associated transcripts included saurs and gh3s, which have been identified as auxin-responsive genes in a wide range of plants. furthermore, various gh genes were reported to be involved in iaa conjugation in many plant species. our data showed that the gene expression of several annotated saurs significantly increased during the s1-s transition, and putative auxin-responsive gh transcripts were up-regulated in the s2-s transition and then down-regulated in the s3-s transition. our k-means cluster analysis had also determined that clusters k and k representing unigenes most highly expressed in s were enriched in auxin-associated unigenes. auxin is considered a senescence retardant in fruit, and the breakdown of endogenous auxin has been reported to initiate ‘bartlett’ pear ripening. moreover, iaa levels declined prior to ripening in tomato, grape, and strawberry fruit. the changes in abundance of auxin-associated transcripts in our data suggest an important function of auxin in the s2-s transition in particular, where pear fruit developed a capacity to respond to ethylene and ripen. we postulate that a decrease in auxin levels regulated the pear fruit’s responsiveness to ethylene and that this process occurred prior to autocatalytic ethylene biosynthesis.table unigenes associated with hormone metabolism exhibiting a fc ≥ in at least one transition *the unigene is differentially expressed in the correspondent pairwise analysis ethylene is well known to be the main hormone regulating climacteric fruit ripening. our data showed that while the expression of both de acs genes increased from s to s and decreased from s to s, their overall expression was low throughout the four stages considered. therefore, we suggest that the high fcs of the acs transcripts were probably biased due to their low rsem counts. the abundance of the aco transcript was slightly decreased in the s1-s transition, but did not significantly change during the later s2-s and s3-s transitions. this behavior of the aco gene may explain the physiological data, where a higher internal ethylene concentration and ethylene production rate were detected in the s fruit that failed to ripen after 14 days at 20 °c. fonseca et al. reported aco activity was below detectable levels in ‘rocha’ pear during fruit growth. hence, we conclude that neither the expression changes of aco at the s1-s transition nor the ethylene produced at s had a significant effect on the ripening capacity of ‘bartlett’ pear. moreover, our results on the expression of acs and aco genes agree with an ethylene biosynthesis and action model proposed in tomato, in which autocatalytic ethylene production is initiated by induction of an acs. ethylene is perceived by protein receptors in plant tissues and this binding inactivates kinase activity of ctr, allowing ein and ein to transduce ethylene signaling. in the present study, transcript abundance of the ethylene receptor pc-ers1a decreased from s to s and had a maximum fc of − in the s3-s transition. the gene expression of a pc-ctr pcm_ increased from s to s then stayed at a similar level in s. previous studies have shown that a decrease in gene expression of the ethylene receptors leetr and leetr increased ethylene sensitivity in tomato. in the present study, s fruit were capable of ripening after 14 days without ethylene treatment. therefore, it appears that the ethylene receptor pc-ers1a and ethylene signaling protein pc-ctr are involved in signal transduction of ethylene that consequently activated autocatalytic ethylene production in s fruit. gibberellin has been reported to stimulate pericarp growth of pea fruit and silique growth of arabidopsis. in the present study, ga-associated gene subcategories were enriched at the s1-s and s2-s transitions. ga-stimulated transcripts are known as targets of ga regulation. two annotated gasts and three putative ga-regulated transcripts were up-regulated from s to s. this may indicate that gas play a role in fruit growth during the s1-s transition. jasmonic acid was suggested to regulate fruit growth in apple. our data showed that similar to ga-associated transcripts, ja-associated transcripts were enriched at the s1-s and s2-s transitions. however, in contrast to the expression patterns of ga-associated genes, transcript abundance of the majority of de ja-associated transcripts decreased in the s1-s transition and increased in the s2-s transition. these transcripts included three putative allene oxide synthases and an annotated lipoxygenase, which encode enzymes involved in jasmonic acid biosynthesis. as aos is considered to be a rate-limiting step in ja biosynthesis, the expression patterns of our ja-associated transcripts complement findings by kondo et al. in growing apple fruit; there, ja was at a high concentration early in fruit development, decreased, and then increased again. therefore, ja may be involved in the regulation of pear growth and development through stages s to s a key enzyme in aba biosynthesis, 9-cis-epoxycarotenoid dioxygenase, has been reported to be associated with ripening of several fruit such as ‘gold nijisseiki’ pear and strawberry. in our data, one nced showed a high fc at s2-s the transition to ethylene responsiveness. however, similar to genes related to ethylene biosynthesis, the expression level was very low in all four stages, suggesting a possible bias of high fc. we also found genes associated with aba such as genes encoding gram domain proteins and hva. however, we have not seen clear evidence of the importance of aba genes in pear growth or in association with the development of ripening capacity. our results seem to agree with those of an earlier study, where the increase in aba concentration was merely coincident with ethylene evolution during ripening in ‘jingbaili’ and ‘gold nijisseiki’, asian pears. transcription factors analyzing the expression of genes encoding transcription factors help to identify key factors that regulate fruit growth and development, particularly those factors that control the fruit’s response to exogenous ethylene and/or its capacity to soften without ethylene treatment. in the pear fruit, a large number of unigenes in the de novo transcriptome were annotated as tfs. of these unigenes,  % were de across the four maturity stages. among all de transcripts putatively identified as tfs, the ap2/erebp family members were the most abundant. similar to the results obtained from microarray analyses of tomato and peach, the gene expression of various putative erebps was either up- or down-regulated across stage transitions. annotated bhlh transcripts were also highly represented and enriched in all transitions with various expression patterns. given the broad range of processes affected by ap2/erebps- or bhlh-mediated regulation, which includes plant development, primary and secondary metabolism, hormone signaling, and response to biotic and abiotic stresses, as well as the intricate target specificity of each member of these tf families, it is not surprising to find such diversity in transcriptional activation or repression across the fruit maturity stages considered in this work.table unigenes associated with transcriptional regulation exhibiting a fc ≥ in at least one transition *the unigene is differentially expressed in the correspondent pairwise analysis in contrast to the unsystematic behaviors of ap2/erebp or bhlh genes, the gene expression of members of bzip, wrky, arf, and aux/iaa families showed a more consistent response; i.e., either up or down-regulation, with an enrichment in a specific transition. in particular, high up-regulation of several putative bzip genes occurred at the s1-s transition, compliments the previous results that showed one annotated bzip gene with higher expression in early maturity fruit compared to mature and ripening fruit of ‘rocha’ pear. while bzip tfs have been implicated in the regulation of a wide range of processes including biotic and abiotic stress responses, hormone signaling, and development, the concerted expression of a subset of bzip members in this study may point to a common functional feature in fruit development that warrants further investigation. strikingly, expression of a majority of genes putatively encoding the tf aux/iaa, arf, and wrky were up-regulated in the s2-s transition and down-regulated in the s3-s transition. these concerted aux/iaa and arf gene expression patterns paralleled the transcript abundance changes in auxin-associated genes, which peaked in the s2-s transition. these results further underscore the important role of auxin in the development of ripening capacity in response to ethylene; i.e., transition from s to s.  in this study, we characterized the physico-chemical features and transcriptional profiles associated with the development of ripening capacity in ‘bartlett’ pear across four maturity stages. our analysis, which focused on the differential expression of genes associated with cell wall metabolism, hormone signaling, and transcriptional regulation, suggested a role for specific transcripts, as well as the coordination of members in the same gene family or among gene families, in the attainment of ripening capacity. we postulate that pectin degradation enzymes may produce early signal molecules that stimulate ethylene biosynthesis associated with the development of fruit ripening capacity. additionally, auxin-associated genes appear to play an important role in regulating the ability of ethylene-treated fruit to ripen at 20 °c. the transcription factor family bzip appears to regulate the s1-s transition and aux/iaa, arf and wrky may regulate the s2-s and s3-s transitions. our results represent a resource for further investigation of some candidate genes or gene groups that regulate the responsiveness of pear, and perhaps other fruit, to ethylene and other plant hormones. in addition, the candidate genes could be examined as molecular markers to indicate the status of ripening capacity, as well as determining appropriate postharvest treatments.fig. proposed mechanisms regulating ripening capacity development during the final stages of pear fruit growth. transition 1: fruit develop ripening capacity responsive to ethylene treatment; transition 2: fruit develop ripening capacity without the need of ethylene treatment. ap2/erebp: apetala 2/ethylene response element binding protein, bhlh: basic helix-loop-helix, bzip: basic region/leucine zipper, arf: auxin response factors, hb: homeobox, c2h2: cys2his zinc finger; ga: gibberellin, ja: jasmonic acid, iaa: auxin/indole-3-acetic acid; xth:xyloglucan endotransglucosylase/hydrolase, pt/pg: pectin lyase/pectate lyase/polygalacturonase, exp: expansin additional files additional file 1: color changes of pears harvested at four harvest times at harvest and after air/ethylene treatment. s s s and s were harvested a week apart; s coincident with commercial harvest. d14: 14 days at 20 °c following treatment of pears with air or μll− ethylene for 24 h. rna extracted from peel tissues ah of s to s were used for rna-sequencing.. additional file 2: soluble solids content and skin color of ‘bartlett’ pears after 14 days at 20 °c. pears were treated with either air or 100μll− ethylene for 24 h at 20 °c. * mean values with different uppercase letters between air and et-treated fruit and different lowercase letters among maturity stages are statistically different according to tukey’s test. additional file 3: primers for quantitative pcr validation. additional file 4: mapman classification of gene in the de novo transcriptome. additional file 5: enriched functional groups in each cluster and enrichment analysis. * fisher’s test with bonferroni correction. additional file 6: enrichment of groups of cell wall, hormone metabolism, and transcription factors in the three transitions: s1-s s2-s and s3-s ns: no significant difference, * fisher’s test with bonferroni correction. additional file 7: number of unigenes associated with a. cell wall and b. hormones according to mapman classifications in three transitions: s1-s s2-s and s3-s additional file 8: sequences of de no vo transcriptome and their putative functions. competing interests the authors declare that they have no competing interests. authors’ contributions sf and am designed the experiment, collected fruit, and analyzed physiological characteristics. sf extracted and purified the rna for sequencing and analyzed physio-chemical data. kc carried out the quality check for the illumina reads, de novo assembly, and blastx. nn conducted transcriptome validation of sequence identity and qpcr, differential expression, and functional annotation analysis. tk and ag participated in primer design and qpcr. cj provided advice for the experiment design. fz and em assisted with data interpretation and provided extensive editing of the manuscript. em coordinated all of the work. nn led the writing of the manuscript draft, with sections provided by sf and kc. all authors contributed to editing the manuscript. authors’ information not applicable availability of data and materials not applicable 
k-means cluster analysis revealed that auxin signaling associated transcripts were enriched in cluster k that showed the highest gene expression at s ap2/erebp and bhlh transcripts were enriched in all three transition s1-s s2-s and s3-s several members of aux/iaa, arf, and wrky appeared to play an important role in orchestrating the s2-s transition. illumina sequencing and trinity assembly generated unigenes, of which  % were annotated to the refseq plant database. the capacity of european pear fruit to ripen after harvest develops during the final stages of growth on the tree. the objective of this study was to characterize changes in ‘bartlett’ pear fruit physico-chemical properties and transcription profiles during fruit maturation leading to attainment of ripening capacity. the softening response of pear fruit held for 14 days at 20 °c after harvest depended on their maturity. high expression of genes putatively encoding pectin degradation enzymes in the s1-s transition suggests pectic oligomers may be involved as early signals triggering the transition to responsiveness to ethylene in pear fruit. our findings suggest that auxin is essential in regulating the transition of pear fruit from being ethylene-unresponsive to ethylene-responsive, resulting in fruit softening. we identified four maturity stages: s1-failed to soften and s2- displayed partial softening; s - able to soften following et; and s - able to soften without et. we identified maturity stages associated with the development of ripening capacity in ‘bartlett’ pear, and described the transcription profile of fruit at these stages. moreover, the co-expression of these genes with exps suggests their collaboration in modifying cell wall polysaccharide networks that are required for fruit growth.
 the capacity of european pear fruit to ripen after harvest develops during the final stages of growth on the tree. the objective of this study was to characterize changes in ‘bartlett’ pear fruit physico-chemical properties and transcription profiles during fruit maturation leading to attainment of ripening capacity.  the softening response of pear fruit held for 14 days at 20 °c after harvest depended on their maturity. we identified four maturity stages: s1-failed to soften and s2- displayed partial softening; s - able to soften following et; and s - able to soften without et. illumina sequencing and trinity assembly generated unigenes, of which  % were annotated to the refseq plant database. higher numbers of differentially expressed transcripts were recorded in the s3-s and s1-s transitions than in the s2-s transition. high expression of genes putatively encoding pectin degradation enzymes in the s1-s transition suggests pectic oligomers may be involved as early signals triggering the transition to responsiveness to ethylene in pear fruit. moreover, the co-expression of these genes with exps suggests their collaboration in modifying cell wall polysaccharide networks that are required for fruit growth. k-means cluster analysis revealed that auxin signaling associated transcripts were enriched in cluster k that showed the highest gene expression at s ap2/erebp and bhlh transcripts were enriched in all three transition s1-s s2-s and s3-s several members of aux/iaa, arf, and wrky appeared to play an important role in orchestrating the s2-s transition.  we identified maturity stages associated with the development of ripening capacity in ‘bartlett’ pear, and described the transcription profile of fruit at these stages. our findings suggest that auxin is essential in regulating the transition of pear fruit from being ethylene-unresponsive to ethylene-responsive, resulting in fruit softening. the transcriptome will be helpful for future studies about specific developmental pathways regulating the transition to ripening. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords rna-seqebseqcell wallauxinethylenebzipap2/erebpbhlhwrkyaux/iaaissue-copyright-statement© the author 2015 