Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics, Disrupts Actin Filaments and Affects Cell Wall Architecture

Abstract

A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca2+ influx, thereby elevating the cytosolic Ca2+ concentration [Ca2+]c and disrupting the [Ca2+]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca2+]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components.

Fig 1. Morphology of pollen tubes.

(A) Control pollen tubes. (B) Pollen tubes treated with 0.5 mM PBA. (C) PBA treated abnormal pollen tube showing a swollen tip (arrow). Scale bars = 50μm.

Fig 2. Influx of calcium at the apex of the pollen tube and [Ca²⁺]c.

(A) Influx of calcium at the apex of the pollen tube at different time points. The blue line represents the control, while the green line represents pollen tube treated with PBA. (B) The [Ca²⁺]c gradient at the apex of the control pollen tube. (C) Strong fluorescence was detected at the apex of the pollen tube treated with PBA, indicating the disappearance of the [Ca²⁺]c gradient. (D) Bright field image of C. Scale bars = 25μm

Fig 3. Actin filaments in pollen tubes grown in different media.

(A) Parallel actin filaments in a pollen tube in normal culture medium. Scale bars = 25μm. (B) Disrupted actin filament distribution in the subapical region of the pollen tube treated with 0.5 mM PBA. Scale bar = 25μm.

Fig 4. Influence of PBA on distribution of de-esterified (JIM5) and esterified pectins (JIM7).

(A) More de-esterified pectins indicated by JIM5 fluorescence were present at the basal part of the pollen tube in normal medium, whereas less in the apical region. (B) The signal intensity of JIM5 labeling indicated that there was more de-esterified pectin present in the apex of the PBA treated pollen tubes. (C) Quantitative analysis of the fluorescence signal obtained after JIM5 labeling of the wall of control pollen tubes (control, pink line) and PBA-treated tubes (0.5 mM PBA, blue line), indicating that PBA promoted the accumulation of de-esterified pectin at the apex of the tubes. (D) Higher levels of esterified pectins were present in the apical region of pollen tubes cultured in normal medium, indicated by fluorescence signal of JIM7. (E) Fluorescence was observed mainly at the tube tip and some at the base after JIM7 labeling of pollen tubes in the presence of PBA. (F) Quantitative analysis of the fluorescence signal obtained after labeling the wall of control pollen tubes (control, pink line) and PBA-treated tubes (0.5 mM PBA, blue line) with JIM7, showing that PBA reduced the accumulation of esterified pectin at the tube. Scale bars = 25μm.

Fig 5. Influence of PBA on the distribution of arabinogalactan proteins (AGPs).

(A) Fluorescence after labeling pollen tubes cultured in normal medium with the LM2 antibody, indicating a greater abundance of AGPs in the basal region and decreased levels from the base towards the pollen tip. (B) Pollen tubes treated with PBA, showing fluorescence of LM2 throughout the tube, with a higher signal at the tip and adjacent to the basal region near the grain. (C) Quantitative analysis of the fluorescent signal obtained after labeling the wall of control pollen tubes (control, pink line) and PBA-treated tubes (0.5 mM PBA, blue line) with LM2, indicating that PBA induced accumulation of AGPs at the pollen apex. Scale bars = 25μm.

Fig 6. Effect of PBA on the distribution of cellulose and callose in pollen tube.

Scale bars = 25μm. (A) Fluorescence of calcofluor white showed that cellulose distributed along the whole pollen tube. (B) PBA treated pollen tube were labeled by Calcofluor, showing that cellulose was present on the whole tube. (C) Quantitative analysis of the fluorescent signal from Calcofluor white staining of cellulose in the wall of control pollen tubes (control, pink line) and PBA treated tubes (0.5 mM PBA, blue line). (D) Callose distribution along the control pollen tube except for at the tip indicated by fluorescent signal from aniline blue. (E) Distribution of callose along the whole PBA treated pollen tube, with weak signal at the tip. (F) Quantitative analysis of the fluorescent signal from aniline blue staining of callose in the wall of control pollen tubes (control, pink line) and PBA treated tubes (0.5 mM PBA, blue line).

Fig 7. FTIR spectra gained from analysis of the tip regions of normal pollen tubes (control, blue line), pollen tubes treated with 0.5 mM phenylboronic acid (PBA, pink line), and the FTIR differential spectrum generated by digital subtraction of the control spectra from the spectra of the PBA treated samples (PBA-control, red line).