The influence of the pollination compatibility type on the pistil S-RNase expression in European pear (Pyrus communis)

Abstract

The S-RNase gene plays an essential role in the gametophytic self-incompatibility (GSI) system of Pyrus. It codes for the stylar-expressed S-RNase protein which inhibits the growth of incompatible pollen tubes through cytotoxicity and the induction of programmed cell death in the pollen tube. While research on the Pyrus GSI system has primarily focused on the S-RNase gene, there is still a lack of insight into its spatiotemporal expression profile and the factors that regulate it. Previous studies have suggested that S-RNase expression in the style is influenced by pollination and is dependent on the compatibility type. We here continue on this basic hypothesis by analyzing the spatiotemporal expression of the S-RNase alleles in Pyrus communis "Conference" styles in response to different types of pollination; namely, upon full- and semi-compatible pollination and upon incompatible selfing. The results revealed that temporal dynamics of S-RNase expression are influenced by the pollen's compatibility type, indicating the presence of a signaling mechanism between pollen and style to control S-RNase production during pollen tube growth. In our experiment, S-RNase expression continuously decreased after cross-pollination and in the unpollinated control. However, after a fully incompatible pollination, S-RNase expression remained constant. Finally, semi-compatible pollination showed a initially constant S-RNase expression for both alleles followed by a strong decrease in expression. Based on these results and previous findings, we propose a regulatory mechanism to explain the effect of pollination and the associated compatibility type on S-RNase expression in the style. This proposed mechanism could be used as a starting point for future research.

Keywords: S-RNase; allele-specific expression; gametophytic self-incompatibility; pear (Pyrus communis); pollen-style interactions.

FIGURE 1

Schematic representation of the different parts of the pistil used for sampling (A), and a reference picture of the five pear styles of cultivar “Conference” to indicate the section sites used for tissue sampling (B). Representative images of the different phenological stages of flower development (C). a) Green bud stage: single flowers are visible but still closed. b-c) White/pink bud stage: petals start to appear and petal color is cultivar-dependent during this stage, the styles may still be very short. d) Start of the balloon stage: flowers are still closed and stamens are still tightly packed around the style. e) Balloon stage: petals are still closed, but can easily be moved aside. f) Anthesis: anthers are pink and closed but the petals have opened. In this stage, flowers can no longer be considered unpollinated since insects carrying pollen from nearby trees may have visited the flower; g) Open flower: anthers have dehisced.

FIGURE 2

Temporal dynamics of the PcS₁₀₈ (A) and PcS₁₂₁ (B) S-RNase expression levels denoted as -dCq values in both the upper and lower half of the style across five time points throughout development of an unpollinated style of the cultivar “Conference.” Data points represent mean values, error bars represent 95% confidence intervals. Asterisks mark the time points at which there is a significant difference between S-RNase allele expression in the upper versus the lower part of the style (p < 0.05) with *: p < 0.05, **: p < 0.01 and ***: p < 0.001.

FIGURE 3

Temporal dynamics of the expression of PcS₁₀₈ (A) and PcS₁₂₁ (B) S-RNase alleles in the styles of the cultivar “Conference” upon different types of pollination treatments, including unpollinated (UP), cross-pollination (Cross), selfing (Self) and semi-compatible pollination (Semi). Data points represent mean values, error bars represent 95% confidence intervals. Letters above the data points indicate significant differences between time points for specific pollination treatments as determined by the Tukey post-hoc test with a significance level of 5% (α = 0.05).

FIGURE 4

Temporal dynamics of the expression of PcS₁₀₈ (A) and PcS₁₂₁ (B) S-RNase alleles in the styles of the cultivar “Conference” upon different types of pollination treatments, including unpollinated (UP), cross-pollination (Cross), selfing (Self) and semi-compatible pollination (Semi). Data points represent mean values, error bars represent 95% confidence intervals. Letters above the data points indicate significant differences between treatments at a specific time point as determined by the Tukey post-hoc test with a significance level of 5% (α = 0.05).

FIGURE 5

Interaction plots for the significant interaction effect between pollination treatment and position for both PcS₁₀₈ (A) and PcS₁₂₁ (B) S-RNase expression levels in styles of the cultivar “Conference”. Data points represent mean values, error bars represent 95% confidence intervals. Letters above the data points indicate significant differences between treatments over all time points per style region as determined by the Tukey post-hoc test with a significance level of 5% (α = 0.05).

FIGURE 6

Illustration of the proposed mechanism underlying the effect of pollen tube (in)compatibility on S-RNase gene expression in the style of Pyrus. (A) The occurrence of an incompatibility reaction (red, multi-pointed star) in “self-type” pollen tubes sends a signal to the style to sense the stylar S-RNase protein concentration [S-RNase] and keep it at a minimum level required to maintain the self-incompatibility reaction. This minimum threshold amount is maintained by the de novo synthesis of extra S-RNase through a yet unknown system of transcriptional activation. (B) On the other hand, in the complete absence of an incompatibility reaction, such as upon cross-pollination, there is no activation of this [S-RNase] sensing mechanism to maintain S-RNase protein levels in the style, so that there is no feedback regulation on S-RNase expression and it thus follows the gradually decreasing “baseline” dynamics as occurs in the absence of pollination. Without active S-RNase replenishment from transcriptional activation, the continuous S-RNase degradation inside the compatible pollen tubes causes a general decrease in the stylar S-RNase protein level. (C) In the specific case of a semi-compatible pollination, the occurrence of an incompatibility reaction in the incompatible pollen tubes signals the style to sense the stylar S-RNase protein concentration. In the meantime, the progressive S-RNase degradation by the presence of compatible pollen tubes may strongly decrease S-RNase protein levels in the style therefore promoting S-RNase expression even further. The assumption that the depletion of non-self-S-RNase by incompatible pollen tubes is much lower than by compatible pollen tubes is schematically illustrated by dotted and full, colored arrows signifying the entrance of S-RNase protein into the pollen tubes.