The Role of ABA in the Interaction between Citrus Fruit and Penicillium digitatum

Abstract

Abscisic acid (ABA) protects citrus fruit against Penicillium digitatum infection. The global mechanisms involved in the role of ABA in the P. digitatum-citrus fruit interaction are unknown. Here, we determine the transcriptome differences between the Navelate (Citrus sinensis (L.) Osbeck) orange and its ABA-deficient mutant Pinalate, which is less resistant to infection. Low ABA levels may affect both the constitutive mechanisms that protect citrus fruit against P. digitatum and early responses to infection. The repression of terpenoid, phenylpropanoid and glutation metabolism; of oxidation-reduction processes; and of processes related to the defense response to fungus and plant hormone signal transduction may be one part of the constitutive defense reduced in the mutant against P. digitatum. Our results also provide potential targets for developing P. digitatum-citrus fruit-resistant varieties. Of those up-regulated by ABA, a thaumatin protein and a bifunctional inhibitor/LTP, which are relevant in plant immunity, were particularly remarkable. It is also worth highlighting chlorophyllase 1 (CLH1), induced by infection in Pinalate, and the OXS3 gene, which was down-regulated by ABA, because the absence of OXS3 activates ABA-responsive genes in plants.

Keywords: abscisic acid (ABA); fungal disease; green mold; postharvest rots; transcriptomic profiling.

Figure 1

Differences in the susceptibility of Pinalate and Navelate fruit to infection by P. digitatum. (A) Changes in the lesion area of the macerated zone of Pinalate (○) and Navelate fruit (●) and of the Pinalate fruit treated with ABA 1 mM (□); (B) Changes in the percentage of decay in the same samples; (C) Picture of Navelate, Pinalate, and the ABA-treated Pinalate fruit infected (8 dpi) with P. digitatum. The fruit were inoculated with P. digitatum (10⁴ conidia mL⁻¹) at a depth of 4 mm. The error interval indicates the standard error of the estimated mean value. * denotes no significant differences (p ≤ 0.05) between the Navelate and Pinalate fruit treated with ABA for the same storage period, but their mean values were significantly lower than those of the Pinalate fruit. ** represents significant differences (p ≤ 0.05) between the parental and both the mutant fruit treated or not with ABA.

Figure 2

ABA content in both the freshlyharvested (FH) fruit and wounded (1d W) and infected (1d I) samples taken at 1 dpi. Different letters mean significant differences (p ≤ 0.05) between Navelate fruit and its mutant Pinalate, treated or not with ABA, for the same storage period. (A) The Principal Component Analysis (PCA) (B) and the heatmap of the hierarchical cluster analysis (HCA) (C) of the expressed genes according to the RNA-Seq analysis. The DEGs meeting a cutoff of STEDV > 0.6 and |Log₂ FoldChange| ≥ 1 were considered in the HCA for all the conditions. The colors used in the PCA for each sample are the same as those employed for the same samples on the heatmap. FHN: freshly-harvested Navelate; WN: wounded Navelate at 1 dpi; IN: infected Navelate at 1 dpi; FHP: freshly-harvested Pinalate; WP: wounded Pinalate at 1 dpi; IP: infected Pinalate at 1 dpi; WPA: wounded Pinalate treated with ABA at 1 dpi; IPA: infected Pinalate treated with ABA at 1 dpi. The colors on the heatmap represent the median centered Log₂(RPM) expression values for each condition and change from dark red (induction) to light green (repression). Clusters 12 and 14 are highlighted in Figure 1C. The samples on the X-axis follow the same order as on the heatmap. Three biological replicates from each condition were used for all the analyses. The error interval indicates the standard error of the estimated mean value.

Figure 3

Venn diagrams of the up-(bold numbers) and down-regulated (regular numbers) genes in several comparisons (DEG, edgeR, BH p-value adjustment α ≤ 0.05). All the induced and repressed DEGs met a cutoff of |Log₂ FoldChange| ≥ 1. (A) The number of the up- and down-regulated genes of the infected Pinalate (IP) fruit and its respective wounded controls (WP), plus those DEGS of the infected Pinalate fruit treated with ABA (IPA) and their control (WPA), compared to the freshly-harvested (FH) Pinalate fruit; (B) The number of the up- and down-regulated genes in the wounded (control) (WN) and infected Navelate (IN) fruit compared to the FH Navelate fruit; (C) The number of the up- and down-regulated genes by infection in relation to wounding (control) in the Pinalate, Pinalate + ABA and Navelate samples. The numbers outside of the diagrams are the sum of all the induced and repressed genes under each condition. The infected fruit were inoculated with 10⁴ P. digitatum conidia mL⁻¹ and the control mock-wounded fruit with water. After wounding or infection, all the fruit were kept at 20 °C for 1 d (1 dpi).