A bulk segregant gene expression analysis of a peach population reveals components of the underlying mechanism of the fruit cold response

Abstract

Peach fruits subjected for long periods of cold storage are primed to develop chilling injury once fruits are shelf ripened at room temperature. Very little is known about the molecular changes occurring in fruits during cold exposure. To get some insight into this process a transcript profiling analyses was performed on fruits from a PopDG population segregating for chilling injury CI responses. A bulked segregant gene expression analysis based on groups of fruits showing extreme CI responses indicated that the transcriptome of peach fruits was modified already during cold storage consistently with eventual CI development. Most peach cold-responsive genes have orthologs in Arabidopsis that participate in cold acclimation and other stresses responses, while some of them showed expression patterns that differs in fruits according to their susceptibility to develop mealiness. Members of ICE1, CBF1/3 and HOS9 regulons seem to have a prominent role in differential cold responses between low and high sensitive fruits. In high sensitive fruits, an alternative cold response program is detected. This program is probably associated with dehydration/osmotic stress and regulated by ABA, auxins and ethylene. In addition, the observation that tolerant siblings showed a series of genes encoding for stress protective activities with higher expression both at harvest and during cold treatment, suggests that preprogrammed mechanisms could shape fruit ability to tolerate postharvest cold-induced stress. A number of genes differentially expressed were validated and extended to individual genotypes by medium-throughput RT-qPCR. Analyses presented here provide a global view of the responses of peach fruits to cold storage and highlights new peach genes that probably play important roles in the tolerance/sensitivity to cold storage. Our results provide a roadmap for further experiments and would help to develop new postharvest protocols and gene directed breeding strategies to better cope with chilling injury.

Figure 1. Mealiness index of pools of peach Pop-DG siblings and global gene expression analysis of Chillpeach transcripts in response to cold storage.

A) Average mealiness index (MI) of pools S and LS from fruits shelf life ripened for 2–3 days at 20°C after being stored for up to 3 weeks at 5°C; B) Principal Component Analysis (PCA) of the global expression profile showing the most variation of each treatment condition (averaged from three replicates). First principal component (PC1) is shown on x-axis while the second principal component (PC2) is shown on y-axis. C) Clusters resulting from the unsupervised two-dimensional hierarchical clustering (Fig. S2). Y-axes represent the normalized expression ratio (Log2 M) of three biological replicates in relation to a reference pool. Red represents qualitative differences, purple depicts the genes regulated in a similar manner and green refers to the genes showing quantitative differences between the LS and S pools. D) The functional categories overrepresented in each cluster (Fig. 1C) are shown as a heatmap obtained with matrix2png. Enriched functional categories with Fisher test p-values <0.05 are colored in grades of yellow. The number of genes in each cluster is indicated to the right of the heatmap. M = mature fruits, R = mature with 2–4 days ripening at 20°C, CS1 = M +1 week cold storage at 5°C, CS2 = M +2 weeks cold storage at 5°C, CS3 = M +3 weeks cold storage at 5°C.

Figure 2. Differential gene expression between the S and LS fruit across the cold storage CS series.

A) A Venn diagram depicting the differentially expressed genes (FDR<0.05 and q-value<0.05) between tolerant and sensitive fruit at each time of cold storage. B) The over-represented functional categories (p-value 0.05) corresponding to the differentially expressed genes between the LS and S pools at each time of cold storage. C) The functional categories enriched in the genes whose expression profiles correlated with the projected MI fruits should have when shelf life ripened. Pearson: 1

Figure 3. Preformed mechanisms and effect of ripening.

A) The hierarchical cluster of the 63 genes differentially expressed between fruits LS and S at the mature stage. The expression values for samples M, R and CS and the M-LS vs. M-S ratio are shown. B) Hierarchical clustering of the expression values for 862 ripening genes (up or down in fruits R respect to M) during cold storage. M = mature fruits, R = mature with 2–4 days ripening at 20°C, CS1 = M +1 week cold storage at 5°C, CS2 = M +2 weeks cold storage at 5°C, CS3 = M +3 weeks cold storage at 5°C.

Figure 4. Comparison of the chillpeach data with the available microarray public domain data.

A) The differentially expressed peach genes in the global analysis (Fig. 1) and reported as cold and/or Stress Response genes. B) The differentially expressed peach genes in the global analysis (Fig. 1) and reported as hormone responsive genes.

Figure 5. Degree of association between the genes validated by Fluidigm in a pre-defined expression pattern from the pools in the microarray and in individual Pop-DG siblings.

A) The differentially expressed genes at 1 week of cold storage; B) The differentially expressed genes in the M stage and at 1 week of cold storage; C) The differentially expressed genes in the M stage. The Heatmap values correspond to the Pearson correlation coefficients between pairs of genes. For each gene in a gene set, the expression profile from the microarray results was defined and the Pearson correlation coefficients were calculated for pairs of genes in the individual sibling lines.