Ethephon induced oxidative stress in the olive leaf abscission zone enables development of a selective abscission compound

Abstract

Background: Table olives (Olea europaea L.), despite their widespread production, are still harvested manually. The low efficiency of manual harvesting and the rising costs of labor have reduced the profitability of this crop. A selective abscission treatment, inducing abscission of fruits but not leaves, is crucial for the adoption of mechanical harvesting of table olives. In the present work we studied the anatomical and molecular differences between the three abscission zones (AZs) of olive fruits and leaves.

Results: The fruit abscission zone 3 (FAZ3), located between the fruit and the pedicel, was found to be the active AZ in mature fruits and is sensitive to ethephon, whereas FAZ2, between the pedicel and the rachis, is the flower active AZ as well as functioning as the most ethephon induced fruit AZ. We found anatomical differences between the leaf AZ (LAZ) and the two FAZs. Unlike the FAZs, the LAZ is characterized by small cells with less pectin compared to neighboring cells. In an attempt to differentiate between the fruit and leaf AZs, we examined the effect of treating olive-bearing trees with ethephon, an ethylene-releasing compound, with or without antioxidants, on the detachment force (DF) of fruits and leaves 5 days after the treatment. Ethephon treatment enhanced pectinase activity and reduced DF in all the three olive AZs. A transcriptomic analysis of the three olive AZs after ethephon treatment revealed induction of several genes encoding for hormones (ethylene, auxin and ABA), as well as for several cell wall degrading enzymes. However, up-regulation of cellulase genes was found only in the LAZ. Many genes involved in oxidative stress were induced by the ethephon treatment in the LAZ alone. In addition, we found that reactive oxygen species (ROS) mediated abscission in response to ethephon only in leaves. Thus, adding antioxidants such as ascorbic acid or butyric acid to the ethephon inhibited leaf abscission but enhanced fruit abscission.

Conclusion: Our findings suggest that treating olive-bearing trees with a combination of ethephon and antioxidants reduces the detachment force (DF) of fruit without weakening that of the leaves. Hence, this selective abscission treatment may be used in turn to promote mechanized harvest of olives.

Keywords: Abscission; Antioxidant; Detachment force; Ethylene; Mechanical harvesting; Olive; Oxidative stress; ROS.

Fig. 1

Annual kinetics of changes in fruit and leaf detachment force (DF) after anthesis. The data are based on monthly DF measurements of 25 leaves and fruits taken from various trees. The samples were taken at the indicated Days Post Anthesis (DPA). The vertical dashed lines represent the time of harvest of table and oil olives. Below is an illustration of fruit development after anthesis

Fig. 2

Effect of ethephon treatment on changes in the detachment force of the leaf and fruit AZs (a), and on the percentage of abscised fruits from the two fruit AZs (b) during 7 days after treatment. The location of each AZ is represented by a rectangle in the picture on the left. Error bars in graph (a) represent confidence intervals (p < 0.95). b. The two colored columns represent the percentage of fruits abscised from FAZ2 (black) or FAZ3 (red) as a function of days after ethephon treatment

Fig. 3

Changes in the anatomy of leaf and fruit AZs during the 7 days after ethephon treatment. Images of longitudinal sections of the LAZ stained with ruthenium red (a) or alcian blue and neutral red (b), and of fruit FAZ2 (c) and FAZ3 (d) stained with ruthenium red at ×5 and ×40 magnitudes are presented. Separation of the LAZ 7 days after ethephon treatment is indicated by a white arrow. AZs are indicated by yellow arrows. The various regions are indicated as follow: LAZ – leaf abscission zone layer, PVT - petiole vascular tissue, AB - axillary bud, LP - leaf petiole, SVT - stem vascular tissue, FAZ2 - fruit abscission zone 2 layer, P – fruit pedicle, FAZ3 - fruit abscission zone 3 layer, F - fruit

Fig. 4

Differentially expressed contigs in the three different AZs 7 days after ethephon treatment. Venn diagrams of upregulated (a) and downregulated (b) contigs in response to ethephon treatment are presented. The number of contigs significantly regulated in each of the AZs is indicated inside the circles. Significant GO enriched biological processes are presented for the common contigs regulated in all three AZs (black), for contigs specifically regulated in the LAZ (green), and for contigs specifically regulated in the FAZ2 and FAZ3 (pink). Among the 109 common contigs upregulated in the FAZ2 and FAZ3, none of the GOs was significantly enriched

Fig. 5

Effect of ethephon treatment on the expression patterns of genes encoding three families of cell wall hydrolyzing enzymes in the three AZs. The expression pattern of all transcripts belonging to PG, cellulase and expansin gene families in the three AZs at 0 and 5 days after ethephon treatment (a), and the expression pattern of only the induced genes of PG (b), cellulase (c) and expansin (d) 5 days after the ethephon treatment, are presented

Fig. 6

Expression patterns of genes involved in the biosynthesis of and response to the plant hormones ethylene, auxin and ABA in the three AZs before and 5 days after ethephon treatment. Hierarchical clustering of the genes involved in the biosynthesis of (a) and the response to (b) the hormones in a green to red scale are presented

Fig. 7

Specific induction in the LAZ by ethephon of genes involved in oxidative stress (a) and in response to ROS (b). Gene expression patterns of 224 genes involved in oxidative stress (a) and of six genes involved in the cell response to ROS (b), which were significantly induced in in the LAZ in response to ethephon treatment. Expression levels are indicated on an abundance scale of green to red. b. The expression pattern of the genes Aldo-Keto Reductase family 4 member C9 (OeAKR4C9), Gibberellin 2-Oxidase 4 (OeGA2OX4), Cinnamyl-Alcohol Dehydrogenase 1 (OeCAD1), Cytochrome P450 81D11 (OeCYP81D11), Cinnamyl-Alcohol Dehydrogenase 8 (OeCAD8) and Flavodoxin-Like Quinone Reductase 1 (OeFQR1), all known to be involved in the cell response to stress is presented

Fig. 8

Effect of ethephon treatment on ROS formation in the leaf and fruit AZs before (a) and 3 days after (b) treatment. DCF fluorescence under a confocal scanning microscope of the LAZ and FAZ2 is presented. The location of the AZs is indicated by yellow arrows. P, proximal side; D, distal side

Fig. 9

Effect of a combined treatment of ethephon and antioxidants on the DF of leaves and fruits 3 days after treatment. Error bars represent confidence intervals based on 30 samples (p < 0.95) and different letters represent statistically different DF