Distinct regulation of adaxial-abaxial polarity in anther patterning in rice

Abstract

Establishment of adaxial-abaxial polarity is essential for lateral organ development. The mechanisms underlying the polarity establishment in the stamen remain unclear, whereas those in the leaf are well understood. Here, we investigated a rod-like lemma (rol) mutant of rice (Oryza sativa), in which the development of the stamen and lemma is severely compromised. We found that the rod-like structure of the lemma and disturbed anther patterning resulted from defects in the regulation of adaxial-abaxial polarity. Gene isolation indicated that the rol phenotype was caused by a weak mutation in SHOOTLESS2 (SHL2), which encodes an RNA-dependent RNA polymerase and functions in trans-acting small interfering RNA (ta-siRNA) production. Thus, ta-siRNA likely plays an important role in regulating the adaxial-abaxial polarity of floral organs in rice. Furthermore, we found that the spatial expression patterns of marker genes for adaxial-abaxial polarity are rearranged during anther development in the wild type. After this rearrangement, a newly formed polarity is likely to be established in a new developmental unit, the theca primordium. This idea is supported by observations of abnormal stamen development in the shl2-rol mutant. By contrast, the stamen filament is likely formed by abaxialization. Thus, a unique regulatory mechanism may be involved in regulating adaxial-abaxial polarity in stamen development.

Figure 1.

Phenotypes of the rol Mutant. (A) A wild-type spikelet. le, lemma; pa, palea. (B) A rol spikelet with the rod-like lemma (arrowhead). (C) A rol spikelet without a lemma. Arrowhead and arrow indicate a rudimentary organ (arrested lemma) and curled anthers, respectively. (D) A rol spikelet with an elongated awn (arrow). (E) and (F) rol spikelets rescued by introducing an 8.6-kb genomic fragment of the SHL2 gene. In the rescued transgenic plant, the development of the lemma and stamens is similar to that in the wild type. (G) and (H) Adaxial (G) and abaxial (H) epidermal surface of the lemma in the wild type. (I) Epidermal surface of the rod-like lemma in rol. (J) A cross section of the lemma in the wild type. ab, abaxial side; ad, adaxial side. (K) A cross section of the rod-like lemma in rol. (L) and (M) In situ localization of ETT3 (L) and PHB3 (M) in the rod-like lemma of rol. Bars = 1 mm in (A) to (F), 100 μm (G) to (I), and 50 μm (J) to (M).

Figure 2.

Comparison of the Needle-Like Structure in the rol Mutant and the Awn in Kasalath (indica). (A) A spikelet with a needle-like structure in the rol mutant. The red bracket indicates the distal part, which has an awn identity. The blue bracket indicates the proximal part, which has lemma identity. (B) and (C) Scanning electron microscopy images of the distal (B) and proximal (C) part of the needle-like structure in the rol mutant. (D) to (F) A spikelet with a long awn in Kasalath. (E) and (F) Scanning electron microscopy images of the awn (E) and the lemma (F) of Kasalath. Bars = 2 mm (A) and (D) and 100 μm (B), (C), (E), and (F).

Figure 3.

Scanning Electron Microscopy Images of the Stamen in the Wild Type and rol. (A) and (E) Wild type. (B) and (F) Pin-like stamen. (C) and (G) OT-type stamen with only one theca. (D) and (H) ATT-type stamen showing four pollen sacs localized adaxially. (E) to (H) Close-up views of (A) to (D), respectively. Arrowheads and stars indicate the pollen sac and the connective, respectively. Brackets indicate a theca. an, anther; fi, filament. Bars = 200 μm in (A) to (D) and 100 μm in (E) to (H). [See online article for color version of this figure.]

Figure 4.

Spatial Expression Pattern of ETT1 and PHB3 during Stamen Development in the Wild Type. (A) Cross-section of an anther. Arrow and bracket indicate the connective and the thecae, respectively. (B) to (D) Spikelets at the early developmental stages in the wild type. Arrowheads indicate protrusions in the stamen primordia. (E) to (H) Spatial expression patterns of ETT1 in a longitudinal section (E) and cross sections ([F] to [H]) of the spikelet. (I) to (L) Spatial expression patterns of PHB3 in a longitudinal section (I) and cross sections ([J] to [L]) of the spikelet. Arrows indicate the PHB3 expression domain in the lateral region of the stamen primordium. (M) to (O) Two-color in situ hybridization in the anther. Purple, ETT1; pink, PHB3. Arrowheads indicate protrusions in the stamen primordia ([N] and [O]). (P) Cross section of a filament. (Q) and (R) Expression of ETT1 (Q) and PHB3 (R) in the filament. ca, carpel; fm, floral meristem; st, stamen; v, vascular tissue. Bars = 50 μm in (A) to (L) and 20 μm in (M) to (R).

Figure 5.

Scanning Electron Microscopy Images of Stamen Primordia and Spatial Expression Pattern of ETT1 and PHB3 during Stamen Development in rol. (A) and (B) Scanning electron microscopy images of stamen development. Red, blue, and yellow arrowheads indicate pin-like, OT-type, and ATT-type stamen, respectively. (C) An early stage of stamen development. (D) and (G) Pin-like stamen primordia. (E) and (H) Primordia of OT-type stamen. (F) and (I) Primordia of ATT-type stamen. Brackets indicate a theca ([E], [F], [H], and [I]) and arrows indicate the region where the stomium differentiates later ([H] and [I]). Black arrowheads indicate the procambium ([G], [H], and [I]). Solid circles indicate the direction of the center of the flower. pr, protrusion that differentiates later into the pollen sac; st, stamen. Bars = 50 μm in (A) to (C) and 20 μm in (D) to (I).

Figure 6.

Schematic Representation of the SHL2 (ROL) Gene and Its Encoded Protein, and Spatial Expression Pattern of SHL2. (A) The SHL2 gene. Boxes indicate coding regions. Arrowhead indicates the mutation site in the shl2-rol mutant. The red bar shows the 8.6-kb genomic fragment used for the complementation test. This fragment contains a promoter of ∼1.5 kb. (B) Schematic representation of the SHL2 protein. Arrowhead indicates the amino acid substitution in the shl2-rol mutant. The blue box indicates the conserved RdRP domain. (C) and (D) Spatial expression pattern of SHL2 in the spikelet. Bars = 50 μm. ca, carpel; le, lemma; pa, palea; st, stamen.

Figure 7.

Model of Anther Patterning in Rice. (A) Model of anther patterning in the wild type. (B) Adaxial-abaxial polarity in a theca. Arrowheads indicate the protrusions at the region between the domains of the adaxial and abaxial identities. (C) Model of anther patterning in rol. (a) Pin-like stamen, (b) OT-type stamen, and (c) ATT-type stamen. The domains with the adaxial and abaxial identities represented by the expression domains of ETT1 and PHB3 are shown in red and blue, respectively. Each bracket represents a unit of the adaxial-abaxial polarity. Double-headed arrows represent the axis of adaxial-abaxial polarity. ab, abaxial side; ad, adaxial side. Dashed arrow indicates developmental progression.