Arabidopsis JINGUBANG Is a Negative Regulator of Pollen Germination That Prevents Pollination in Moist Environments

Abstract

The molecular mechanism of pollen germination and pollen tube growth has been revealed in detail during the last decade, while the mechanism that suspends pollen grains in a dormant state is largely unclear. Here, we identified the JINGUBANG (JGB) gene by screening pollen-specific genes for those that are unnecessary for pollen germination. We showed that the pollen of the jgb loss-of-function mutant exhibited hyperactive germination in sucrose-only medium and inside the anther, while this phenotype was rescued by the transgenic expression of JGB in jgb plants. JGB contains seven WD40 repeats and is highly conserved in flowering plants. Overexpression of JGB inhibits pollen germination. These results indicate that JGB is a novel negative regulator of pollen germination. In addition, we found that jasmonic acid (JA) abundance was significantly elevated in jgb pollen, while exogenous application of methyl jasmonate rescued the inhibition of pollen germination in plants overexpressing JGB Based on the molecular features of JGB and on the finding that it interacts with a known JA biosynthesis-related transcription factor, TCP4, we propose that JGB, together with TCP4, forms a regulatory complex that controls pollen JA synthesis, ensuring pollination in moist environments.

Figure 1.

JGB Is an Unknown Hydrophilic Protein Containing Seven WD40 Repeats and Inhibits Improper Pollen Germination in Vivo. (A) Diagrams of JGB, the jgb insertional mutant, and JGB protein containing seven WD40 repeats. (B) The hydrophilic feature of JGB analyzed with ProtParam and ProtScale tools of ExPASy. (C) JGB transcript was absent in the mature pollen grains of jgb plants (left panel, RT-PCR with GAPDH as a loading control). JGB was undetectable in jgb pollen using immunoblot analysis (right panel, with an anti-JGB antibody produced in this study and tubulin as a loading control). (D) to (G) Improper germination of jgb pollen occurred when the flowering plants were periodically sprayed with water. (D) Hyperactive germination of jgb pollen observed from the outside of the anther. Bar = 200 μm. (E) Pollen tubes inside the anther visualized with aniline blue staining. Bar = 20 μm. (F) Pollen tubes per anther in the flowers of (E). Error bars represent sd (n = 5). (G) Scanning electron microscopy showing the occurrence of hyperactive germination from the lower anther (pollen tubes highlighted in yellow). Panels in the right are magnifications of the boxed regions in the left panels. Bars = 200 μm.

Figure 2.

JGB Inhibits Pollen Germination in Vitro. (A) jgb pollen germinated on sucrose-only medium. Images (left panel) were taken 3 h after imbibition on sucrose-only medium; germination rates of Col-0 pollen with partial and full components of the standard medium (containing H₃BO₃, KCl, CaCl₂, and MgSO₄) 3 h after imbibition (right panel) are shown for comparison. Error bars represent sd (n = 3). Bar = 20 μm. (B) Hyperactive germination of jgb pollen on standard medium in vitro. Images (upper panel) were taken 45 min after imbibition and germination rates were compared at three time points (lower panel). Error bars represent sd (n = 3). Bar = 20 μm.

Figure 3.

JGB Is a Negative Regulator of Pollen Germination. (A) Construction of the transgenic vectors. (B) Immunoblot showing the native level of JGB in Col-0, the undetectable level of JGB in jgb, and the native JGB and JGB-GFP levels in JGB OX pollen with tubulin as a loading control. (C) to (E) Inhibition of pollen germination by excessive JGB in vitro. (C) Images of Col-0, jgb, and JGB OX pollen 1 and 2 h after imbibition. Bar = 20 μm. (D) Germination rates of Col-0, jgb, and JGB OX pollen 1, 2, 4, 6, and 10 h after imbibition. Error bars represent sd. (E) JGB OX pollen exhibiting weaker JGB-GFP fluorescence (indicated by an asterisk) usually germinated. (F) to (H) Inhibition of pollen germination by excessive JGB in vivo. (F) Pollen grains of Col-0 and jgb 20 min after being pollinated to Col-0 stigmas (Alexander staining). Bar = 20 μm. (G) Pollen grains of JGB OX 40 min after being pollinated to Col-0 stigmas (upper panel, JGB-GFP fluorescence; lower panel, bright field). Bar = 20 μm. (H) The germination rates of Col-0, jgb, and JGB OX pollen when pollinated to Col-0 stigmas. Error bars represent sd.

Figure 4.

Pollen Tubes of jgb Plants Are Defective during Later Periods of Growth in Vivo. (A) and (B) The slowdown of jgb pollen tube growth since 6 h after pollination. (A) Fluorescence images of pollen tubes in vivo (aniline blue staining). Arrows indicate the position of leading pollen tubes. Bar = 500 μm. (B) Pollen tube efficiency (length of the leading pollen tube/length of style) of pollen tubes in vivo. Error bars represent sd (Student’s t test: **P < 0.01). (C) The percentages of jgb progeny in the middle and bottom part of the siliques produced by the Col-0 × jgb/JGB cross.

Figure 5.

JGB Is Localized in the Nucleus and Cytoplasm of Vegetative Cells. (A) Localization of JGB-GFP fluorescence in both the nucleus (Hoechst 33342 staining) and cytoplasm of vegetative cells. Bar = 20 μm. (B) Appearance of JGB-GFP fluorescence from bicellular pollen (BCP) during pollen development. We show this finding with a jgb/JGB qrt/qrt plant having two pollen grains in a tetrad as the negative control. UNM, uninucleate microspores; TCP, tricellular pollen. Bar = 20 μm. (C) Subcellular localization of JGB did not change during pollen germination and pollen tube growth. Panel in the upper right corner is the merged picture of the two original pictures. pt, pollen tube; sn, sperm nucleus; vn, vegetative nucleus. Bar = 20 μm.

Figure 6.

Enrichment of JGB in the Vegetative Nucleus Does Not Influence Pollen Germination and Tube Growth. (A) Diagram of point mutation in JGB. Red indicates the change of the amino acid. (B) and (C) Enrichment of JGB-GFP fluorescence in the nucleus by the point mutation. (B) Fluorescence images of jgb RES and jgb RES^AEV pollen. Bar = 10 μm. (C) Relative fluorescence intensity of the pollen. Nu, nucleus; Cy, cytoplasm. (D) Pollen of jgb RES and jgb RES^AEV showed equal rates of (inhibited) germination in vivo (on Col-0 stigmas), implying excessive expression of JGB (JGB^AEV) in these transgenic lines and indicating that translocation of cytoplasmic JGB to the nucleus did not affect the function of JGB in controlling pollen germination. Error bars represent sd. (E) and (F) The growth of pollen tubes showed no difference between jgb RES and jgb RES^AEV, indicating that translocation of cytoplasmic JGB to the nucleus did not affect the function of JGB in stabilizing pollen tube growth. (E) Fluorescence images of pollen tubes 4 and 6 h after pollination. Arrows indicate the leading pollen tubes. Bar = 500 μm. (F) Pollen tube efficiency of jgb RES and jgb RES^AEV pollen tubes. Error bars represent sd.

Figure 7.

MeJA Activates Arabidopsis Pollen Germination in Vitro. (A) to (D) Hyperactivated pollen germination by supplying MeJA in vitro. (A) Images of Col-0 pollen germinated with MeJA (1 h after imbibition). Bar = 10 μm. (B) The germination rates show clear dose dependency, with the most germination on the medium supplemented with 500 μM MeJA. Error bars represent sd. (C) Effects of MeJA and DIECA on germination of Col-0, jgb, and JGB OX pollen. Error bars represent sd (n = 3). (D) Images of Col-0 and jgb pollen germinated with DIECA (1 h after imbibition). Bar = 10 μm. (E) jgb pollen has significantly higher transcript levels of genes (LOX2, AOC4, OPCL1, ACX1, ACX5, AIM1, KAT2, and JMT) encoding proteins in the JA synthesis pathway. Values were normalized to ACTIN11 with the transcript abundance of Col-0 taken as 1. Error bars represent sd (n = 3). (F) jgb pollen contains a significantly higher amount of JA. FW, fresh weight. Error bars represent sd (n = 3).

Figure 8.

JGB Interacts with TCP4 and a Schematic Model Showing That JGB Inhibits Pollen Germination by Inhibiting JA Synthesis. (A) Yeast were cotransformed with the plasmids as indicated on the left. Yeast were grown on SD/-2 (SD/-Leu-Trp) dropout media (left panel) and SD/-3 (SD/-Leu-Trp-His) dropout media (right panel). Cells grown on SD/-3 dropout media are indicative of the physical interaction between JGB and TCP4. (B) In vitro pull-down assays for the interactions between JGB and TCP4. The fusion proteins were expressed in E. coli and used for GST pull-down assays, and then immunoblotted with anti-His or anti-GST antibody. GST protein was used as a negative control. The arrows indicate GST-JGB fusion protein (upper) and GST protein (lower). (C) Working model of inhibition of pollen germination by JGB. TCP4 activates the JA biosynthesis gene LOX2. TCP4 and JGB regulate LOX2, affecting JA production and pollen germination.