Treatments of peach fruits and clustering of the differential transcriptome. A. Schematic representation of Dixiland peach fruit samples employed for the transcriptomic analysis.

<p>The differential transcriptome was obtained comparing fruits at six different stages: harvest time (H); after 3 and 7 days of harvest (H3 and H7, respectively) along the normal ripening process (light blue arrows) at 20°C; after the application of a heat treatment (upper red arrow) of 39°C during 3 days (HT) and after 3 and 7 days (HT3 and HT7, respectively) at 20°C after the heat treatment (lower red arrows) at 20°C. Both H3 and HT fruits have the same post-harvest lifetime. H7 and HT3 fruits have approximately the same post-harvest lifetime. However, HT7 fruits have no control counterparts of the same post-harvest lifetime. Crossed circles indicate the time-points of sample collection for the analysis. B. Pie chart representing the four clusters of unigenes of the differential transcriptome. Induced- and repressed-upon-heat-treatment unigenes were classified according to their expressional pattern (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051052#pone-0051052-t001" target="_blank">Table 1</a>) and gathered into the IG and RG, respectively. These clusters represent 83% of the differential post-harvest transcriptome. OG includes unigenes whose differential expression pattern can not be related to the applied treatment. HG includes unigenes expressed at harvest stage and that are repressed during the ripening of fruits.</p

Heat-repressed unigenes in cold treated peach fruit.

<p>The relative-to-harvest (H) level of accumulation of 9 selected heat-repressed transcripts was determined in the peach fruit subjected to 3 (R3) or 5 days at 0°C (R5) followed by 2 days at 20°C (R5+2). Gene expression levels were normalized against <i>Arabidopsis thaliana rad50</i> (gb|AF168748.1|AF168748). Bars with at least one equal letter mean no statistically significant difference (α = 0.05). Cold-repressed transcripts are grouped in red and cold-induced transcripts in green. The transcript that was not modified by the cold treatment (R14) is in grey; while transcript R2, which resulted induced in R5 but repressed in R5+2, is in yellow.</p

Validation of differentially expressed unigenes by qRT-PCR.

<p>The relative-to-harvest level of accumulation of the differentially expressed transcripts was determined in the different post-harvest conditions studied. Relative expression level indicated by black bars emphasized the presence-absent pattern of expression observed in the differential display experiments. Gene expression levels were normalized against <i>Arabidopsis thaliana rad50</i> (gb|AF168748.1|AF168748). Bars with at least one equal letter mean no statistically significant difference (α = 0.05). The expression pattern code (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051052#pone-0051052-t001" target="_blank">Table 1</a>) of each unigene is indicated on the bottom right corner of each graph.</p

Classification of peach-heat-differentially expressed unigenes regarding their response to cold in <i>Arabidopsis</i>.

<p><i>Arabidopsis</i> orthologs to the heat induced and repressed peach genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051052#pone-0051052-t002" target="_blank">Tables 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051052#pone-0051052-t003" target="_blank">3</a>, respectively) were analyzed regarding their response to cold in <i>Arabidopsis</i> using the ColdArrayDB (<a href="http://cold.stanford.edu/cgi-bin/data.cgi" target="_blank">http://cold.stanford.edu/cgi-bin/data.cgi</a>). Within the induced and repressed peach genes, <i>Arabidopsis</i> orthologs with a fold-change higher than 1.5 in response to cold <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051052#pone.0051052-Vogel1" target="_blank">[33]</a> are classified as induced or repressed, respectively. Genes with an absolute fold-change less than 1.5 are indicated as not modified by cold.</p

Clustering of the identified differentially expressed transcripts (DETs) and summary of the total differentially expressed unigenes (DEUs).

<p>The type of expression pattern consists of a six-number sequence which represents the absence (0) or presence (1) of a transcript in the control ripening conditions (H, H3 and H7, respectively) and after the application of HT and along the ripening after the treatment (HT, HT3 and HT7, respectively).</p

Heat-induced unigenes in cold-treated peach fruit.

<p>The relative-to-harvest (H) level of accumulation of 14 selected heat-induced transcripts was determined in peach fruit subjected to 3 (R3) or 5 days at 0°C (R5) followed by 2 days at 20°C (R5+2). Gene expression levels were normalized against <i>Arabidopsis thaliana rad50</i> (gb|AF168748.1|AF168748). Bars with at least one equal letter mean no statistically significant difference (α = 0.05). Cold-induced genes are grouped in green, cold-repressed transcripts in red and the transcript that was not modified by the cold treatment (I42) is in grey.</p

Functional identity and gene ontology description of the isolated unigenes corresponding to the “Harvest Genes” (HG) unigenes.

<p>Functional identity and gene ontology description of the isolated unigenes corresponding to the “Harvest Genes” (HG) unigenes.</p

Functional identity and gene ontology description of the isolated unigenes corresponding to the “Induced Group” (IG) unigenes.

<p>Functional identity and gene ontology description of the isolated unigenes corresponding to the “Induced Group” (IG) unigenes.</p

Functional identity and gene ontology description of the isolated unigenes corresponding to the “Other Group” (OG) unigenes.

<p>Functional identity and gene ontology description of the isolated unigenes corresponding to the “Other Group” (OG) unigenes.</p

GO-term functional distribution of the heat-responsive unigenes.

<p>The top BLAST hits of the 105 differential expressed unigenes from the IG and RG were classified according to the GO term in biological processes (<b>A</b>) and molecular functions (<b>B</b>) vocabularies of their <i>Arabidopsis thaliana</i> orthologous.</p