Ca2+-activated reactive oxygen species production by Arabidopsis RbohH and RbohJ is essential for proper pollen tube tip growth

Abstract

In flowering plants, pollen germinates on the stigma and pollen tubes grow through the style to fertilize the ovules. Enzymatic production of reactive oxygen species (ROS) has been suggested to be involved in pollen tube tip growth. Here, we characterized the function and regulation of the NADPH oxidases RbohH and RbohJ (Respiratory burst oxidase homolog H and J) in pollen tubes in Arabidopsis thaliana. In the rbohH and rbohJ single mutants, pollen tube tip growth was comparable to that of the wild type; however, tip growth was severely impaired in the double mutant. In vivo imaging showed that ROS accumulation in the pollen tube was impaired in the double mutant. Both RbohH and RbohJ, which contain Ca(2+) binding EF-hand motifs, possessed Ca(2+)-induced ROS-producing activity and localized at the plasma membrane of the pollen tube tip. Point mutations in the EF-hand motifs impaired Ca(2+)-induced ROS production and complementation of the double mutant phenotype. We also showed that a protein phosphatase inhibitor enhanced the Ca(2+)-induced ROS-producing activity of RbohH and RbohJ, suggesting their synergistic activation by protein phosphorylation and Ca(2+). Our results suggest that ROS production by RbohH and RbohJ is essential for proper pollen tube tip growth, and furthermore, that Ca(2+)-induced ROS positive feedback regulation is conserved in the polarized cell growth to shape the long tubular cell.

Figure 1.

RbohH and RbohJ Genes Were Expressed in Pollen Grains. (A) RT-PCR analysis of RbohH and RbohJ expression in different tissues. IM, inflorescence meristem. APETALA2 (AP2) was used as a control. Two biological and technical replicates were performed. (B) RT-PCR analysis of RbohH and RbohJ expression in pollen grains. Total RNA was extracted from pollen grains. ELONGATION FACTOR-1α (EF-1α) was used as a control. Two biological and technical replicates were performed. (C) and (D) GUS staining of an opened flower and pistil of RbohH_pro:GUS (C) or RbohJ_pro:GUS (D). Bars = 500 μm (left panels) and 200 μm (right panels).

Figure 2.

The rbohH rbohJ Double Mutant Was Defective in Pollen Tube Tip Growth. (A) Schematic representation of the T-DNA insertions in RbohH and RbohJ. Open triangles show the sites of T-DNA insertions in the mutant together with deleted nucleotide numbers. Closed boxes indicate the protein-coding exons. rbohH-2 and rbohJ-1 mutants were in the Ws background, and rbohH-3, rbohJ-2, and rbohJ-3 were in the Col-0 background. (B) RT-PCR analysis of the full-length transcripts of rbohH and RbohJ expressed in each mutant flower. AP2 was used as a control. Two biological and technical replicates were performed. (C) Dissected siliques harvested ∼7 d after hand pollination. White arrowheads indicate unfertilized ovules. ♂ indicates the genotype of the pollen grain, and ♀ indicates the genotype of the pistil. Bar = 1 mm. (D) Percentage of normal seeds, aborted seeds, and unfertilized ovules in siliques. Siliques were harvested 5 to 10 d after hand pollination. Closed bars indicate normal seeds; gray bars indicate aborted seeds; open bars indicate unfertilized ovules. Values are means ± se (n = 5 to 6 siliques); *P < 0.01 (Student’s t test). (E) Length of pollen tubes germinated in vitro. Values are means ± sd (n = 11–30); *P < 0.01 (Student’s t test). (F) Pollen tubes were visualized by aniline blue staining in vivo. Pistils were harvested 12 h after hand pollination. Bar = 500 μm.

Figure 3.

Detection of ROS on Stigmatic Papilla Cells. Detection of ROS is shown on stigmatic papilla cells with Oxyburst Green 20 min after pollination. (A) to (D) Intense fluorescence of Oxyburst Green around a wild-type pollen tube growing within the stigmatic papilla cell wall. (E) to (H) Faint fluorescence of Oxyburst Green around the rbohH-3 rbohJ-2 double mutant pollen tube growing within the stigmatic papilla cell wall. (A) and (E) show fluorescence images; (B) and (F) show pseudocolor fluorescence images; (C) and (G) show bright-field images; and (D) and (H) show overlay images. Bar = 10 μm.

Figure 4.

Ca²⁺ Activates the ROS-Producing Activity of RbohH and RbohJ via the EF-Hand Motif. (A) Amino acid sequences of wild-type and mutant EF-hand regions of RbohH and RbohJ. Two EF-hand motifs are indicated. The Ca²⁺ binding loops in the first and second EF-hand motifs are boxed. The consensus Ca²⁺ binding loop is shown: X, Y, Z, #, and –X = Ca²⁺ ligand; –Z = Ca²⁺ ligand, a bidentate Glu (E) or Asp (D); G = Gly; I = Ile or other aliphatic residue found at this position; * = any residue. (B) and (C) Ionomycin-induced ROS production of RbohH (B) and RbohJ (C) in HEK293T cells. FLAG-tagged proteins of the wild type and EF-hand mutants were transiently expressed in HEK293T cells. After 5 min of baseline measurement, 1 μM ionomycin was added to the medium. Expressed proteins were detected by immunoblotting with anti-FLAG antibody. As a loading control, β-actin was used. (D) and (E) Dose-dependent inhibition of ionomycin-induced ROS production by DPI. HEK293T cells transiently expressing FLAG-tagged RbohH (D) or RbohJ (E) were pretreated with different concentrations of DPI. After 5 min of baseline measurement, 1 μM ionomycin was added to the medium. For (B) to (E), all quantified data are means ± se (n = 3). Arrows indicate the time at which ionomycin was added. ROS production was measured by chemiluminescence and expressed as relative luminescence units per second (RLU/s). The maximum value of the luminescence activity was set at 1.0 unit.

Figure 5.

Synergistic Activation of RbohH and RbohJ by Calyculin A and Ionomycin. HEK293T cells were transiently transfected with FLAG-tagged RbohH (A) or RbohJ (B). After baseline measurement, either or both 0.1 μM calyculin A or 1 μM ionomycin was added to the medium at the time points indicated by the arrows. Insets show enlarged diagrams of calyculin A–induced ROS production for 15 min. All quantified data are means ± se (n = 3). ROS production was measured by chemiluminescence and expressed as relative luminescence units per second (RLU/s). The maximum value of ionomycin-induced luminescence activity was set at 1.0 unit.

Figure 6.

EF-Hand Mutations Affect Pollen Tube Tip Growth but Not Protein Subcellular Localization. (A) and (B) Complementation analysis of the rbohH-3 rbohJ-2 double mutant phenotype with GFP-tagged wild-type or EF-hand mutants (E219Q and E229Q). Pollen grains were from wild-type or homozygous transgenic plants. GFP:RbohH-WT and GFP:RbohH-E219Q were expressed under the control of the RbohH promoter in the rbohH-3 rbohJ-2 double mutant, while GFP:RbohJ-WT and GFP:RbohJ-E229Q were expressed under the control of the RbohJ promoter in the rbohH-2 rbohJ-2 double mutant. Pistils were wild type (Col-0). (A) Pistils were harvested 12 h after hand pollination and stained with aniline blue. Bar = 500 μm. (B) Percentage of normal seeds, aborted seeds, and unfertilized ovules in siliques. Siliques were harvested 5 to 10 d after hand pollination. Closed bars indicate normal seeds; gray bars indicate aborted seeds; open bars indicate unfertilized ovules. Values are means ± se (n = 6 to 11 siliques). (C) Subcellular localization of GFP-tagged wild-type and EF-hand mutants (E219Q and E229Q) in pollen tubes of the rbohH-3 rbohJ-2 double mutant. Pollen grains were germinated in vitro. GFP:RbohH-WT and GFP:RbohH-E219Q were expressed under the control of the RbohH promoter in the rbohH-3 rbohJ-2 double mutant, while GFP:RbohJ-WT and GFP:RbohJ-E229Q were expressed under the control of the RbohJ promoter in the rbohH-3 rbohJ-2 double mutant. The bottom right panel shows a cross section of a pollen tube expressing GFP:RbohJ-E229Q. Bars = 10 μm.