Transcriptomic profiling of citrus fruit peel tissues reveals fundamental effects of phenylpropanoids and ethylene on induced resistance

Abstract

Penicillium spp. are the major postharvest pathogens of citrus fruit in Mediterranean climatic regions. The induction of natural resistance constitutes one of the most promising alternatives to avoid the environmental contamination and health problems caused by chemical fungicides. To understand the bases of the induction of resistance in citrus fruit against Penicillium digitatum, we have used a 12k citrus cDNA microarray to study transcriptional changes in the outer and inner parts of the peel (flavedo and albedo, respectively) of elicited fruits. The elicitor treatment led to an over-representation of biological processes associated with secondary metabolism, mainly phenylpropanoids and cellular amino acid biosynthesis and methionine metabolism, and the down-regulation of genes related to biotic and abiotic stresses. Among phenylpropanoids, we detected the over-expression of a large subset of genes important for the synthesis of flavonoids, coumarins and lignin, especially in the internal tissue. Furthermore, these genes and those of ethylene biosynthesis showed the highest induction. The involvement of both phenylpropanoid and ethylene pathways was confirmed by examining changes in gene expression and ethylene production in elicited citrus fruit. Therefore, global results indicate that secondary metabolism, mainly phenylpropanoids, and ethylene play important roles in the induction of resistance in citrus fruit.

Figure 1

Summary of differentially expressed genes [significant analysis of microarrays (SAM), P < 0.01] in the flavedo (grey bars) and albedo (white bars) of fruits infected with Penicillium digitatum during 1 day (I1) and infected and cured fruits at 4 days (IC4), 5 days (IC5) and 7 days (IC7) after the beginning of the experiment, compared with nontreated (NT) fruits. Genes differentially expressed in both tissues are represented as striped bars.

Figure 2

Gene‐to‐gene correlation matrix of differentially expressed genes. (a) Main gene clusters are situated along the diagonal line (groups a–g). Correlations between genes are shown by the blue scale: the darker the blue colour, the higher the percentage of similarity between gene expression patterns. (b) Patterns of expression of flavedo (F) and albedo (A) genes included in each cluster. NT, nontreated fruits; I1, fruits infected with Penicillium digitatum during 1 day; IC, infected and cured fruits at 4, 5 and 7 days after the beginning of the experiment.

Figure 3

Multivariate analysis of differentially expressed genes in elicited citrus fruits. (a) Principal component analysis (PCA) showing the variation between fruits subjected or not to elicitor treatment in the first component, and the variation between tissues, flavedo (F) and albedo (A), in the second component. (b) PCA showing the distribution of genes. Genes with log₂ > 2 for induced and log₂ < –2 for repressed expression are indicated in different colours. Independent PCAs for the flavedo (c) and albedo (d) samples. See nomenclature in Fig. 1.

Figure 4

Hierarchical view of gene ontology (GO) biological categories significantly over‐represented in the elicited flavedo 5 days after the beginning of the experiment (FIC5) compared with nontreated flavedo (FNT), obtained with AgriGO. Significant categories (adjusted P < 0.05) are shown using grey scaling according to their significance level. Other categories required to complete the hierarchy are shown in white.

Figure 5

Graphical representation of the phenylpropanoid, free phenolic acids and suberin pathways showing gene expression values in the flavedo (F) and albedo (A) of elicited oranges. The first number in parentheses indicates the number of genes spotted onto the microarray that have an Arabidopsis thaliana homologue, and the second number indicates the total number of A. thaliana genes for each step in the pathway. Numbers in squares indicate the log₂ ratio of: a, I1/NT; b, IC4/NT; c, IC5/NT; d, IC7/NT. n.d., not detected. The symbol ‘+’ indicates no expression in NT and the symbol ‘–’ indicates expression in NT fruits but not in the compared treatment. NT, nontreated fruits. I1, fruits infected with Penicillium digitatum during 1 day; IC, infected and cured fruits at 4, 5 or 7 days after the beginning of the experiment.

Figure 6

Involvement of ethylene in the response of citrus fruits to elicitor treatment and to Penicillium digitatum infection. (a) Northern blot analysis of CsACO in the flavedo and albedo of oranges: NT, nontreated; I, infected; IC, infected and cured; X, nonwounded; C, cured; WC, wounded and cured. (b) CsACO mRNA accumulation in oranges infected with P. digitatum during 1, 2 and 3 days. Fruits were nontreated (NT), wounded and water inoculated (W) or wounded and inoculated with a suspension of 10⁶ conidia/mL of P. digitatum (I). In (a) and (b), relative accumulation (R.A.) values of CsACO mRNA in arbitrary units are shown at the bottom. Normalization was carried out with respect to the hybridization signal of the Citrus sinensis 26S rRNA using the flavedo infected with P. digitatum for 1 day (I1) as a reference. Hybridization with the P. digitatum 28S rDNA probe is shown at the bottom. (c) Ethylene production in discs of nontreated oranges (●), infected fruits (), cured fruits (), wounded and cured fruits (□) and infected and cured fruits (◊). Values represent the average of three replicates, including 10 discs in each replicate, ± SD.

Figure 7

mRNA relative accumulation of genes putatively involved in phenylpropanoid metabolism in elicited citrus fruits. The expression level was determined by Northern blot (●) or microarray (□) hybridizations. In general, a reference value of 1.0 was assigned to the nontreated flavedo, except for COMT1 and SAD genes as a result of a lack of expression in nontreated flavedo. Values from microarray hybridizations represent the average of three biological replicates ± SD. NT, nontreated fruits; I1, fruits infected with Penicillium digitatum during 1 day; IC, infected and cured fruits at 4, 5 and 7 days after the beginning of the experiment.