Generation of Superoxide by OeRbohH, a NADPH Oxidase Activity During Olive (Olea europaea L.) Pollen Development and Germination

Abstract

Reactive oxygen species (ROS) are produced in the olive reproductive organs as the result of intense metabolism. ROS production and pattern of distribution depend on the developmental stage, supposedly playing a broad panel of functions, which include defense and signaling between pollen and pistil. Among ROS-producing mechanisms, plasma membrane NADPH-oxidase activity is being highlighted in plant tissues, and two enzymes of this type have been characterized in Arabidopsis thaliana pollen (RbohH and RbohJ), playing important roles in pollen physiology. Besides, pollen from different species has shown distinct ROS production mechanism and patterns of distribution. In the olive reproductive tissues, a significant production of superoxide has been described. However, the enzymes responsible for such generation are unknown. Here, we have identified an Rboh-type gene (OeRbohH), mainly expressed in olive pollen. OeRbohH possesses a high degree of identity with RbohH and RbohJ from Arabidopsis, sharing most structural features and motifs. Immunohistochemistry experiments allowed us to localize OeRbohH throughout pollen ontogeny as well as during pollen tube elongation. Furthermore, the balanced activity of tip-localized OeRbohH during pollen tube growth has been shown to be important for normal pollen physiology. This was evidenced by the fact that overexpression caused abnormal phenotypes, whereas incubation with specific NADPH oxidase inhibitor or gene knockdown lead to impaired ROS production and subsequent inhibition of pollen germination and pollen tube growth.

Keywords: NADPH oxidase; NOX; Rboh; olive; pollen; sexual plant reproduction.

Figure 1

(A) Amino acid sequence alignment and predicted motifs of OeRbohH, AtRbohH, AtRbohJ, and NtNOX. Conserved residues are highlighted in red. The blue boxes indicate the predicted calcium-binding domains (2). Predicted transmembrane domains (6) are boxed in green, including the conserved heme-coordinating histidine residues (4) highlighted with black circles. The predicted FAD-binding sites are marked with purple boxes (2). Predicted sites for NADPH binding (4) are indicated with red boxes. (B) Analogous color coding was used to show domains/motifs in a simplified diagram of OeRbohH. (C) Phylogenetic relationships of the 10 NADPH oxidase proteins from Arabidopsis (AtRbohA-J), together with olive pollen OeRbohH and a partial tobacco pollen sequence Rboh NtNOX. The tree was constructed by the maximum likelihood (ML) from Clustal Omega multiple alignment and rooted with human NOX isoform HsNOX5. Numbers at nodes indicate bootstrap values. The circle marks the putative pollen-Rboh subgroup.

Figure 2

(A) OeRbohH expression analysis using semiquantitative PCR of seedling and tree tissues (upper panel), floral tissues (medium panel), and pollen ontogeny (lower panel). (B) mRNA levels of OeRboh determined by real-time PCR in pollen along in vitro germination, expressed as percentages compared to the mRNA level of the mature pollen (100%). Averages of three biological replicates ± SD are presented. No significant differences were found. ANOVA test (p < 0.05).

Figure 3

OeRbohH protein expression along pollen ontogeny (A) and in vitro pollen germination (B). Western blot using the anti-OeRbohH antibody (lower panel) was performed after native-PAGE (upper panel).

Figure 4

Fluorescence microscopy localization of OeRbohH in the olive anther and in vitro germinated pollen grains. Sections from olive anthers at the following stages: pollen mother cells prior to meiosis (A), tetrads (B), young microspores (C), vacuolated microspores (D), and mature pollen (E) were incubated with an anti-OeRbohH Ab, followed by an anti-chicken IgG-Alexa Fluor 488–conjugated secondary Ab. In insets, detailed view of gametophytic tissue is shown in B-E. Right insets in D and E show a detailed view of the sporophytic tissues of the anther. Negative control sections (anthers at the tetrad stage) were treated with the preimmune serum (F). In vitro germinated pollen grains were also used for fluorescence microscopy localization of OeRbohH. Recently emerged pollen tube showed intense labeling at the pollen tube tip (G, arrow). Elongated pollen tubes also showed intense labeling at the tip (H, arrow). High magnification of the pollen tube apex after immunolocalization of OeRbohH (I). The protein accumulates at the very tip, although it can be also weakly localized at the plasma membrane (arrowheads). Negative control (using the preimmune serum as the primary antibody) did not show labeling (J). Note the autofluorescence of the exine. En, endothecium; Ep, epidermis; IC, intermediate cells; Mi, microspore; MP, mature pollen grain; T, tapetum; Te, tetrad; YP, young pollen. Bar = 20µm. Boundaries of the anther layers and the pollen grain/pollen tube contour are shown for reference in several pictures (A, F, H, J).

Figure 5

Transient expression of OeRbohH in tobacco pollen tubes as YFP fusions, observed by CLSM. (A) YFP construction alone (control) showed homogeneous yellow fluorescence throughout the cytoplasm of the pollen tube. (B) YFP : OeRbohH and (C–F) OeRbohH : YFP transformants showed labeling in the plasma membrane (arrowheads) as well as in the cytoplasm without significant differences among both constructs. (D) Fluorescence was also observed at the edge of callose plugs in their proximal side (referred to the pollen grain). The majority of transformed pollen tubes showed accumulation spots (C, E, F) and abnormal phenotypes (i.e., swelling) at the tip (E–F), and their growth was significantly inhibited after 4h in vitro culture (G). Insets at pictures B, C, and D show bright field images of the pollen tubes for reference. CP, callose plug; Cy, cytoplasm. *indicates that the mean is significantly different from the control at P < 0.05. n = 100 from three independent experiments. Bars = 20 µm.

Figure 6

Detection of O₂^•− putatively generated by NADPH oxidase activity in pollen tubes. (A) Control sample incubated in growth medium in the absence of NBT. (B) NBT precipitate is observed in the pollen tube near the apex and in the proximity of callose plugs. (C) NBT stain remains even in the presence of sodium azide, an inhibitor of NADPH peroxidase. (D) Addition of NBT in the presence of DPI, an inhibitor of NADPH oxidases, did not lead to staining. (E) Quantification of the precipitate intensity along 50 µm of the apical region of the pollen tubes. Data represent means ± SEM. *indicates that the mean value is significantly different from the control at P < 0.001. n = 100 from three independent experiments. Bars = 10 µm. NBT, NBT-treated samples; DPI+NBT, samples treated with NBT and DPI; AZIDE+NBT, samples treated with NBT and sodium azide; A.U., arbitrary units.

Figure 7

DPI inhibits germination and pollen tube elongation. Control sample (A). When the NADPH oxidase inhibitor was added at the beginning of the process, the in vitro germination percentage was affected (B). When the inhibitor was added during the germination, the length of the pollen tube was affected (C) in both cases when compared to the control. Representative pictures from three independent experiments are shown. Quantification of the germination rate (D) and pollen tube lengths (E). Data represent means ± SEM. n = 100. *indicates that the mean is significantly different from the control at P < 0.05. Bars = 100 µm.

Figure 8

(A) In gel NADPH oxidase activity of mature (MP), hydrated (H), and germinated pollen extracts at different times (1, 4, 8, and 12 h) fractionated by native PAGE (50 µg/lane). (B) In mature pollen, the activity revealed by NBT was challenged by the addition of sodium azide or DPI during the incubation. (C, D) Densitometry quantification of NADPH oxidase activity as in (A) and (B), respectively. *indicates that the mean from three independent experiments is significantly different from the control (mature pollen) at P < 0.05.

Figure 9

Transfection of olive pollen tubes with OeRbohH-specific antisense oligodeoxynucleotides (ODNs). The OeRbohH sequence obtained was used to design antisense ODNs. Control samples (A, E). Transfection of olive pollen tubes with such antisense ODNs resulted in pollen tube growth inhibition (B) as well as in a reduction of the production of tip-localized O₂^•− (F) when compared with the control samples or the sense ODNs (C, G). Quantification of pollen tube lengths (D) and precipitate intensity (H). Data represent means ± SEM; n = 300. *indicates that the mean value is significantly different from the control at P < 0.05. Bars in upper panel = 100 µm. Bars in bottom panel = 10 µm.