A Rice Ca2+ Binding Protein Is Required for Tapetum Function and Pollen Formation

Abstract

In flowering plants, successful male reproduction requires the sophisticated interaction between somatic anther wall layers and reproductive cells. Timely degradation of the innermost tissue of the anther wall layer, the tapetal layer, is critical for pollen development. Ca²⁺ is a well-known stimulus for plant development, but whether it plays a role in affecting male reproduction remains elusive. Here we report a role of Defective in Exine Formation 1 (OsDEX1) in rice (Oryza sativa), a Ca²⁺ binding protein, in regulating rice tapetal cell degradation and pollen formation. In osdex1 anthers, tapetal cell degeneration is delayed and degradation of the callose wall surrounding the microspores is compromised, leading to aborted pollen formation and complete male sterility. OsDEX1 is expressed in tapetal cells and microspores during early anther development. Recombinant OsDEX1 is able to bind Ca²⁺ and regulate Ca²⁺ homeostasis in vitro, and osdex1 exhibited disturbed Ca²⁺ homeostasis in tapetal cells. Phylogenetic analysis suggested that OsDEX1 may have a conserved function in binding Ca²⁺ in flowering plants, and genetic complementation of pollen wall defects of an Arabidopsis (Arabidopsis thaliana) dex1 mutant confirmed its evolutionary conservation in pollen development. Collectively, these findings suggest that OsDEX1 plays a fundamental role in the development of tapetal cells and pollen formation, possibly via modulating the Ca²⁺ homeostasis during pollen development.

Figure 1.

Phenotypic comparison between the wild type and the osdex1 mutant. A, Wild-type plant (left) and the osdex1 mutant plant (right) after heading. B, Part of the wild-type panicle showing the dehisced anther (left) and part of the osdex1 panicle (right) showing a smaller anther at the pollination stage. C, Wild-type (left) and osdex1 (right) flower organs after removal of the palea and lemma. D, Wild-type (left) and osdex1 (right) flowers before anthesis. E, Wild-type stained pollen at stage 13. Bars = 10 cm (A), 2 cm (B), 5 mm (C and D), and 100 μm (E).

Figure 2.

Bright field microscopy of transverse sections showing anther and microspore development in wild type and osdex1. Locules from the anther section of the wild type (A–D) and osdex1 (E–H) from stage 8 to stage 11. dT, degenerated tapetal layer; E, Epidermis; En, endothecium; M, middle layer; Msp, microspores; T, tapetal layer; Tds, tetrads. Bars = 15 µm. A and E, Stage 8b. B and F, Stage 9. C and G, Stage 10. D and I, Early stage 11.

Figure 3.

TEM images of the anthers from the wild type and osdex1. A to D, TEM observation showing tapetal cells of wild-type anthers at stage 8b (A), stage 9 (B), stage 10 (C), and stage11 (D). E to H, Wild-type tapetal cell wall (left) and microspore cell wall (right) at stage 8b (E), stage 9 (F), stage 10 (G), and stage11 (H). I to L, TEM observation showing tapetal cells of osdex1 anthers at stage 8b (I), stage 9 (J), stage 10 (K), and stage 11 (L). M, Higher magnification of the highlighted region in (I) showing details in tapetal cells. N, Vacuoles fusion in osdex1 tapetal cells at stage 9. Black arrows show the attachment of vacuoles. O and P, Higher magnification of the highlighted region in (J) showing details in tapetal cells. White arrows show the breakage of vacuoles. Black arrow shows the breakage of ER. Q to T, osdex1 tapetal cell wall (left) and microspore cell wall (right) at stage 8b (Q), stage 9 (R), stage 10 (S), and stage 11 (T). Ba, Bacula; CW, cell wall; eOr, early stage of orbicules; Pb, probacula; M, mitochondria; N, nucleus; Ne, nexine; Or, orbicules; T, tapetal cells; Te, tectum; V, vacuoles. Bars = 2 μm (A–C and I–K), 1 μm (D and N), 10 μm (L), and 0.5 μm (E–H, M, O–P, and Q–T).

Figure 4.

DNA fragmentation is initiated earlier and subsequently blocked in osdex1 mutant. A to H, DNA fragment signal at stage 7, stage 8a, stage 8b, and stage 9 in wild type (A–D) and osdex1 mutant (E–H). The red fluorescence shows the propidium iodide staining of anther cells using confocal laser scanning microscope; the yellow fluorescence shows the TUNEL-positive nuclei staining in confocal laser scanning microscope overlays of fluorescein staining and propidium iodide staining. Bars = 15 μm.

Figure 5.

Callose degradation is retarded in osdex1. A to D, TEM of extracellular materials from the wild type and osdex1 at stage 9 (A and B), and stage 10 (C and D). Black arrows show the site of callose deposition. E to L, Immunolabeling of wild-type (E–H) and osdex1 (I–L) anther sections from stage 7 to stage 10 observed by epifluorescence microscopy. M to P, Negative controls of immunolabeling. In (E) to (P), the green channel shows immunostaining with callose antibody; blue counterstaining shows 1,4- and 1,3;1,4-glucan polymers stained with 0.01% calcofluor white; and red staining shows background autofluorescence. Bars = 2 μm (A), 5 μm (B–D), and 15 μm (E–P).

Figure 6.

Phylogenetic analysis of OsDEX1 and its related proteins. Neighboring-joint analysis was performed using MEGA 6.1 (see “Materials and Methods”) based on the alignment given in Supplemental Data Set S1 online of OsDEX1 with the most similar OsDEX1 sequences from the species shown. The species were classified by evolutionary relationship.

Figure 7.

A to D, Transgenic lines containing Pro:DEX1:OsDEX1 in the Arabidopsis dex1 mutant display rescued male fertility. Insets show the pollen tested by Alexander staining using bright field microscopy. Ws, wild-type plant of Wassileskija ecotype. Bar = 10 mm.

Figure 8.

Recombinant OsDEX1 has Ca²⁺ binding activity. A, 3D structure of EF-hand motif of YesW. B, 3D structure of EF-hand motif of OsDEX1. C, In vitro Ca²⁺ binding assay shows that OsDEX1 has Ca²⁺ binding activity. D to G, Failure in complementation by OsDEX1 with the mutated Ca²⁺ binding sites in dex1. Insets show pollen tested by Alexander staining using bright field microscopy. Bar = 10 mm.

Figure 9.

The Ca²⁺ concentration calculated by emission ratios between YFP and CFP intensities using confocal laser scanning microscope in rice anthers and tobacco epidermal cells. A to H, Ca²⁺ concentration on plasma membrane in tapetal cells of wild type (A–D) and osdex1 (E and F) from stage 8 to stage 11. I to T, Comparison of cytosolic (I to N) and plasma membrane (O–T) Ca²⁺ concentration in tobacco epidermal cells overexpressed with OsDEX1 (J and P) or mOsDEX1 (M and S). I, L, O, and R, Control of YC3.6 overexpression. K, N, Q, and T, Statistical analysis of ratios between YFP and CFP intensities in the cells (K and N) and on the plasma membrane (Q and T).

Figure 10.

Model for OsDEX1 in anther development. Transcription factors such as TIP2, TDR, PTC1, and EAT1 function as master valves to switch cell death signaling on or off; OsDEX1 buffers the Ca²⁺ concentration in the cells to function as a component of cell death signaling.