Shoot organization genes regulate shoot apical meristem organization and the pattern of leaf primordium initiation in rice

Abstract

The mechanism regulating the pattern of leaf initiation was analyzed by using shoot organization (sho) mutants derived from three loci (SHO1, SHO2, and SHO3). In the early vegetative phase, sho mutants show an increased rate of leaf production with random phyllotaxy. The resulting leaves are malformed, threadlike, or short and narrow. Their shoot apical meristems are relatively low and wide, that is, flat shaped, although their shape and size are highly variable among plants of the same genotype. Statistical analysis reveals that the shape of the shoot meristem rather than its size is closely correlated with the variations of plastochron and phyllotaxy. Rapid and random leaf production in sho mutants is correlated with the frequent and disorganized cell divisions in the shoot meristem and with a reduction of expression domain of a rice homeobox gene, OSH1. These changes in the organization and behavior of the shoot apical meristems suggest that sho mutants have fewer indeterminate cells and more determinate cells than wild type, with many cells acting as leaf founder cells. Thus, the SHO genes have an important role in maintaining the proper organization of the shoot apical meristem, which is essential for the normal initiation pattern of leaf primordia.

Figure 1.

Developmental Course of a sho3 Embryo. (A) Wild-type embryo at 5 DAP differentiating scutellum, coleoptile, epiblast, and SAM (white arrowhead) with first leaf primordia (1l). (B) Mature wild-type embryo. SAM is indicated by the white arrowhead. (C) Shoot apex of a mature wild-type embryo with three leaf primordia (1l, 2l, and 3l) and the domelike SAM (white arrowhead). (D) sho3 embryo at 6 DAP differentiating scutellum, epiblast, and SAM (arrowhead) with first leaf primordia (1l) but no coleoptile. (E) Mature sho3 embryo with scutellum and abnormal shoot. SAM is indicated by the white arrowhead. (F) Shoot apex of a sho3 embryo with three abnormal leaf primordia (1l, 2l, and 3l) and flat SAM (white arrowhead). Co, coleoptile; Ep, epiblast; Sc, scutellum. ; .

Figure 2.

Seedlings and SAMs of Wild-Type Plants and sho Mutants at 1 Week after Germination. Each pair shows seedling and SAM, respectively. The SAM is indicated by arrowheads. In sho seedlings, many threadlike leaves are produced, and the SAMs are flat. (A) and (B) Wild-type. (C) and (D) sho1-1. (E) and (F) sho1-2. (G) and (H) sho1-3. (I) and (J) sho1-4. (K) and (L) sho2. (M) and (N) sho3. .

Figure 3.

Scanning Electron Microscopy of Shoot Apices and Leaves in sho Seedlings. (A) Side view of a wild-type shoot apex, showing foodlike P2 leaf primordium (arrow) and P1 primordium (*) in alternate phyllotaxy. (B) to (D) Top view of sho1-1 (B), sho2 (C), and sho3 (C) seedlings showing malformed leaf primordia and irregular phyllotaxy. In several leaf primordia, a pair of small bulges (*) are observed on both sides of the threadlike structure. SAMs are indicated by arrowheads and leaf primordia by arrows. (E) Threadlike leaf with a small ligule (arrow) on the adaxial side. (F) Adaxial surface of a wild-type leaf blade with stomatas (arrowheads) and hairs (arrows). (G) Adaxial surface of a threadlike leaf with stomatas (arrowheads) and hairs (arrows). (H) Trifurcated leaf in a 3-week-old seedling of sho2. In the apical region, the leaf blade is separated into a central region and two lateral regions. ; .

Figure 4.

Mature Plants. (A) Mature wild-type plants. (B) Mature sho1-2 plants with apparently normal but dwarf shoot. (C) Mature sho2 plants with apparently normal but dwarf shoot.

Figure 5.

Spikelets of Wild-Type Plants and sho2 Mutants. (A) Wild-type spikelet. (B) sho2 spikelet with awnlike lemma and degenerated palea. (C) Relatively normal sho2 spikelet with an elongated awn (arrowhead). L, lemma; P, palea.

Figure 6.

Schematic Representation of Phyllotactic Pattern in sho Mutants. Positions of the first to 10th leaf primordia are indicated by colored circles on concentric circles; the outermost circle corresponds to the first leaf, the innermost circle to the 10th leaf, and the center to the SAM. Different colors indicate different seedlings. In the wild type, leaves are formed in distichous phyllotaxis, as indicated by black circles. The positions of the first through third leaves in sho mutants are almost normal and are indicated by black circles.

Figure 7.

Definition of Entropy of Phyllotaxy. A top view of a shoot apex is shown. It was divided into four diagonally duplicated regions (A to D), and the entropy of phyllotaxy (H) was calculated from the number of leaf primordia in each region: H=−Pi 1n Pi. Pi is the probability of leaf primordium formed in region i and is expressed by the number of leaf primordia in region i divided by the total number of leaf primordia produced (n).

Figure 8.

Histone H4 Expression in Wild-Type and sho Meristems at 1 Week after Germination. Hybridization was conducted with the histone H4 antisense RNA probe. (A) Wild type. No signal is visible in the SAM. (B) sho1-1. (C) sho2. (D) sho3. In sho mutants, signals in the SAM are observed in several cells, including those in the central zone. SAM is indicated by arrowheads. .

Figure 9.

OSH1 Expression in Wild-Type and sho Meristems at 1 Week after Germination. The OSH1 antisense RNA probe was used for hybridization. (A) to (C) Wild type. (D) to (G) sho1-1. Four different shoot apices are shown. (H) to (K) sho2. Four different shoot apices are shown. (L) to (O) sho3. Four different shoot apices are shown. (C), (F), and (G) show cross-sections; the other panels show median longitudinal sections. In (C), the outline of each leaf primordium is traced. .