Transcriptomic profiling during the post-harvest of heat-treated Dixiland Prunus persica fruits: common and distinct response to heat and cold

Abstract

Cold storage is extensively used to slow the rapid deterioration of peach (Prunus persica L. Batsch) fruit after harvest. However, peach fruit subjected to long periods of cold storage develop chilling injury (CI) symptoms. Post-harvest heat treatment (HT) of peach fruit prior to cold storage is effective in reducing some CI symptoms, maintaining fruit quality, preventing softening and controlling post-harvest diseases. To identify the molecular changes induced by HT, which may be associated to CI protection, the differential transcriptome of peach fruit subjected to HT was characterized by the differential display technique. A total of 127 differentially expressed unigenes (DEUs), with a presence-absence pattern, were identified comparing peach fruit ripening at 20°C with those exposed to a 39°C-HT for 3 days. The 127 DEUs were divided into four expression profile clusters, among which the heat-induced (47%) and heat-repressed (36%) groups resulted the most represented, including genes with unknown function, or involved in protein modification, transcription or RNA metabolism. Considering the CI-protection induced by HT, 23-heat-responsive genes were selected and analyzed during and after short-term cold storage of peach fruit. More than 90% of the genes selected resulted modified by cold, from which nearly 60% followed the same and nearly 40% opposite response to heat and cold. Moreover, by using available Arabidopsis microarray data, it was found that nearly 70% of the peach-heat responsive genes also respond to cold in Arabidopsis, either following the same trend or showing an opposite response. Overall, the high number of common responsive genes to heat and cold identified in the present work indicates that HT of peach fruit after harvest induces a cold response involving complex cellular processes; identifying genes that are involved in the better preparation of peach fruit for cold-storage and unraveling the basis for the CI protection induced by HT.

Figure 1. Treatments of peach fruits and clustering of the differential transcriptome. A. Schematic representation of Dixiland peach fruit samples employed for the transcriptomic analysis.

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 (Table 1) 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.

Figure 2. GO-term functional distribution of the heat-responsive unigenes.

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 (A) and molecular functions (B) vocabularies of their Arabidopsis thaliana orthologous.

Figure 3. Validation of differentially expressed unigenes by qRT-PCR.

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 Arabidopsis thaliana rad50 (gb|AF168748.1|AF168748). Bars with at least one equal letter mean no statistically significant difference (α = 0.05). The expression pattern code (Table 1) of each unigene is indicated on the bottom right corner of each graph.

Figure 4. Heat-induced unigenes in cold-treated peach fruit.

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 Arabidopsis thaliana rad50 (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.

Figure 5. Heat-repressed unigenes in cold treated peach fruit.

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 Arabidopsis thaliana rad50 (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.

Figure 6. Classification of peach-heat-differentially expressed unigenes regarding their response to cold in Arabidopsis.

Arabidopsis orthologs to the heat induced and repressed peach genes (Tables 2 and 3, respectively) were analyzed regarding their response to cold in Arabidopsis using the ColdArrayDB (http://cold.stanford.edu/cgi-bin/data.cgi). Within the induced and repressed peach genes, Arabidopsis orthologs with a fold-change higher than 1.5 in response to cold are classified as induced or repressed, respectively. Genes with an absolute fold-change less than 1.5 are indicated as not modified by cold.