Transcriptome and Hormone Analyses Revealed Insights into Hormonal and Vesicle Trafficking Regulation among Olea europaea Fruit Tissues in Late Development

Abstract

Fruit ripening and abscission are the results of the cell wall modification concerning different components of the signaling network. However, molecular-genetic information on the cross-talk between ripe fruit and their abscission zone (AZ) remains limited. In this study, we investigated transcriptional and hormonal changes in olive (Olea europaea L. cv Picual) pericarp and AZ tissues of fruit at the last stage of ripening, when fruit abscission occurs, to establish distinct tissue-specific expression patterns related to cell-wall modification, plant-hormone, and vesicle trafficking in combination with data on hormonal content. In this case, transcriptome profiling reveals that gene encoding members of the α-galactosidase and β-hexosaminidase families associated with up-regulation of RabB, RabD, and RabH classes of Rab-GTPases were exclusively transcribed in ripe fruit enriched in ABA, whereas genes of the arabinogalactan protein, laccase, lyase, endo-β-mannanase, ramnose synthase, and xyloglucan endotransglucosylase/hydrolase families associated with up-regulation of RabC, RabE, and RabG classes of Rab-GTPases were exclusively transcribed in AZ-enriched mainly in JA, which provide the first insights into the functional divergences among these protein families. The enrichment of these protein families in different tissues in combination with data on transcript abundance offer a tenable set of key genes of the regulatory network between olive fruit tissues in late development.

Keywords: cell wall; endomembrane trafficking; fruit ripening; olive; plant hormone; transcriptomic comparative.

Figure 1

Differentially expressed cell wall-related genes and classification of cell wall families between olive AZ and fruit (pericarp) at the last stage of ripening (217 DPA). (A) Overlap of overexpressed fruit genes, and overexpressed AZ genes. This figure shows the number of transcripts and families that were specific and common for each tissue. (B) Comparison of significantly overexpressed transcripts (p < 0.01) between olive AZ (black) and fruit (gray) at the last stage of ripening. Number of transcripts related to the cell wall in each cell wall family. (C) Distribution of cell wall-related families that were specific for each olive tissue. Additional information on the cell wall-related genes is presented in Table S1.

Figure 2

Profiles of IAA, GA₁, GA₄, ABA, JA, and SA levels measured from olive AZ and fruit (pericarp) at the last stage of ripening (217 DPA). Hormone levels not detected are indicated by a black dot (●). Data are the means ± SD of three biological replicates with three technical repeats each. Statistically significant differences based on unpaired Student’s t-test at p < 0.05 are denoted by an asterisk.

Figure 3

Differentially expressed hormone-related genes between olive abcission zone (AZ) and fruit (pericarp) at the last stage of ripening (217 DPA). (A) Overlap of overexpressed fruit genes, and overexpressed AZ genes. This figure shows the number of hormone-related transcripts that were specific and common for each tissue. (B) Comparison of significantly overexpressed transcripts (p < 0.01) between olive AZ (black) and fruit (gray) at the last stage of ripening. Number of transcripts related to the plant hormone in each tissue. (C) Heatmap of the expression of differentially expressed genes related to hormone metabolism and signaling in the indicated groups (IAA, GA₁ + GA₄, ABA, SA, JA levels) in olive AZ and fruit at the last stage of ripening. The gene expression of each sample was normalized using the mean expression for each condition. Then, the gene groups were defined in function of hormone relationship. Color codes for expression values are reported on the top. Additional information on the hormones-related genes is presented in Table S2 and Figure S1.

Figure 4

Simplified schematic representation of the hormone metabolism and signaling pathways in AZ and fruit at the last stage of ripening in olive (217 DPA). (A) Fruit-enriched genes encoding various hormone proteins at 217 DPA (p < 0.01). (B) AZ-enriched genes encoding various hormone proteins at 217 DPA (p < 0.01). Genes with elevated mRNA levels are in red typeface (ET = Ethylene). Additional information on the hormone-related genes is presented in Table S2.

Figure 5

Expression of OeERF3, OeERF4, OeSNRK2.4, OeNPR1, and OeJAR1 in AZ and fruit at the last stage of ripening (217 DPA) in olives. Data are the means ± SD of three biological replicates with three technical repeats each and were determined by qRT-PCR normalized against Olea europaea ubiquitine. Statistically significant differences based on unpaired Student’s t-test at p < 0.05 are denoted by an asterisk.

Figure 6

Simplified schematic representation of the trafficking pathways to and from the cell wall of olive fruit (A) and AZ (B) at the last stage of ripening. The Rab-GTPases likely involved at each step are indicated in red (up-regulated). 1. Synthesis of proteins in endoplasmic reticulum (ER). 2. Synthesis of matrix polysaccharides and assembly of proteins in Golgi and TGN/EE (the trans-Golgi network and early endosomal compartments). 3. Modification of wall elements by secreted enzymes. Pathways to and from the vacuole have been omitted for simplicity. Additional information on the vesicle-trafficking-related genes is presented in Table S3. (ECM: equivalent to the “cell wall” or “apoplast”. PM: plasma membrane).

Figure 7

Expression profiling of (A) fruit-enriched or (B) AZ-enriched genes related to transport as reconstructed from the pyrosequencing transcriptomes. Sequence transcripts showing significant variations (p < 0.01). p-values are visualized in a color-code scale. Additional information on the transport-related genes is presented in Table S4.