A sweet orange mutant impaired in carotenoid biosynthesis and reduced ABA levels results in altered molecular responses along peel ripening

Abstract

Citrus fruit ripening is a complex process involving biochemical, physiological and molecular events that differ between the flesh and the peel of the fruit. We characterized sweet orange peel maturation by means of a comparative transcriptomic analysis between Navelate orange (Citrus sinensis L. Osbeck) and its mutant fruit Pinalate, which presents a severe blockage at early steps of the carotenoid biosynthetic pathway and consequently reduced ABA levels. Peel ripening involved the decrease of the photosynthetic activity and the transmembrane transport processes, as well as the buildup of starch and cuticular waxes and the cell wall modification. In addition, a number of biotic and abiotic stress responses, including the defense response, and the response to blue light, water deprivation and abscisic acid stimulus were modulated in a ripening-stage specific manner. The regulation of energy-related processes and secondary metabolism pathways was attenuated in Pinalate, while the molecular mechanisms underlying stress responses displayed dependency on ABA levels. These results indicate that ABA is a key signal inducing stress responses along orange peel ripening, which might determine the fruit postharvest performance.

Figure 1

Transcriptomic comparative analyses of Navelate and Pinalate fruit along ripening. (A) Venn diagrams showing the distribution of differentially expressed genes (DEG, SAM analysis, FDR < 0.01) for the comparisons between breaker (Bk) and full colored (FC) Navelate (N) and Pinalate (P) fruit respect to their mature green (MG) developmental stage. Inductions and repression are indicated in bold and italics, respectively. (B) Principal Component Analysis (PCA), (C) Hierarchical Cluster Analysis (HCA) and (D) Heatmap large-scale transcriptional profiles based on the 1116 DEG satisfying a P < 0.001 in an ANOVA test for all conditions represented in Venn diagrams (A). The colors in PCA for each condition are consistent with those in HCA. The three axes in PCA account for 82.1% of the total variance among genotypes and developmental stages. Heatmap colors vary from light blue (repression) to dark red (induction). Three biological replicates from each condition were used for the analyses.

Figure 2

Real-time qRT-PCR expression analysis for selected genes from microarray analysis. Relative transcript abundance for selected genes belonging to ‘Response to water deprivation’ (DREB2A, STZ) and ‘Response to abscisic acid stimulus’ (CYP707A1, RD26, SYP121, ERD14, ABF4, GBF3, ATAF1, ALDH3-H1) biological processes differentially regulated in Navelate (black) and Pinalate (white) fruit harvested at six ripening stages. Transcript levels for all conditions were expressed relative to MG Navelate fruit. Data are the mean values of three biological replicates ± SE. Asterisks indicate statistical differences between genotypes according to a t-test (pvalue < 0.05) for each ripening stage.

Figure 3

Effect of ABA treatment on the expression of stress-responsive genes. Relative transcript abundance for selected genes belonging to ‘Response to water deprivation’ (DREB2A, STZ) and ‘Response to abscisic acid stimulus’ (CYP707A1, RD26, SYP121, ERD14, ABF4, GBF3, ATAF1, ALDH3-H1) biological processes (Table 4) in Navelate and Pinalate fruit harvested at FC stage and treated or not with ABA. Transcript levels for all conditions were expressed relative to Navelate non-treated FC fruit. Data are the mean values of three biological replicates ± SE. Different letters indicate statistical differences among conditions according to an ANOVA test (pvalue < 0.05) for each gene.