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, 23 (9), 3276-87

LAX PANICLE2 of Rice Encodes a Novel Nuclear Protein and Regulates the Formation of Axillary Meristems

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LAX PANICLE2 of Rice Encodes a Novel Nuclear Protein and Regulates the Formation of Axillary Meristems

Hiroaki Tabuchi et al. Plant Cell.

Abstract

Aerial architecture in higher plants is dependent on the activity of the shoot apical meristem (SAM) and axillary meristems (AMs). The SAM produces a main shoot and leaf primordia, while AMs are generated at the axils of leaf primordia and give rise to branches and flowers. Therefore, the formation of AMs is a critical step in the construction of plant architecture. Here, we characterized the rice (Oryza sativa) lax panicle2 (lax2) mutant, which has altered AM formation. LAX2 regulates the branching of the aboveground parts of a rice plant throughout plant development, except for the primary branch in the panicle. The lax2 mutant is similar to lax panicle1 (lax1) in that it lacks an AM in most of the lateral branching of the panicle and has a reduced number of AMs at the vegetative stage. The lax1 lax2 double mutant synergistically enhances the reduced-branching phenotype, indicating the presence of multiple pathways for branching. LAX2 encodes a nuclear protein that contains a plant-specific conserved domain and physically interacts with LAX1. We propose that LAX2 is a novel factor that acts together with LAX1 in rice to regulate the process of AM formation.

Figures

Figure 1.
Figure 1.
Plant Morphology of the lax2-1 Mutant. (A) Mature panicles. The lax2-1 mutant has a sparse appearance due to the production of fewer branches and spikelets. WT, wild type. (B) Enlarged view of boxes in (A). (C) Whole plants at the vegetative growth stage. lax2-1 mutant has fewer tillers than the wild type. (D) Culms of the wild type and lax2-1 after removal of the surrounding leaves. White arrowheads indicate the prophyll, which encloses the AM. (E) Quantification of the number of spikelets in a panicle. (F) Quantification of the number of PBs per panicle. (G) Quantification of the number of lateral branches, which is the sum of the number of PBs, SBs, and spikelets in a panicle. (H) Quantification of the number of tillers per plant. Error bars in (E) to (H) represent sd. The sample size for (E) to (H) is n = 9.
Figure 2.
Figure 2.
Morphology of Double Mutants of lax2 with lax1 or moc1. (A) to (F) Mature plants. WT, wild type. (G) to (L) Panicle. (A) and (G) The wild type. (B) and (H) lax1-1. (C) and (I) lax2-1. (D) and (J) moc1-4. (E) and (K) lax1-1 lax2-1 double mutant. (F) and (L) lax2-2 moc1-4 double mutant. The inset in (L) is a higher magnification of the double mutant panicle. Bar = 2 cm. White and red arrowheads indicate the position of nodes in the panicle and terminal spikelets, respectively.
Figure 3.
Figure 3.
Anti-OSH1 Immunostaining of the Wild Type, lax2, moc1, and lax2 moc1 Double Mutant and Scanning Electron Microscopy Image of the Wild Type and lax2 moc1 Double Mutant. (A), (F), and (K) Vegetative shoot of wild-type (WT) Nipponbare. (B), (G), and (L) Vegetative shoot of lax2-2. (C), (H), and (M) Vegetative shoot of moc1-4. (D), (I), and (N) Vegetative shoot of the lax2-2 moc1-4 double mutant. (E), (J), and (O) Position of photograph in shoot in each row is shown in red boxes. (P) Developing wild-type Nipponbare inflorescence with SB primordia. (Q) Developing inflorescence of lax2 at the same stage as in (P). (R) and (S) Scanning electron microscopy images of the wild type (R) and double mutant (S) at the base of P4 leaves. P3, P4, and P5 represent the 3rd, 4th, and 5th youngest leaf primordia, respectively (Steeves and Sussex, 1989). Red arrows indicate the position of AMs marked by morphology and OSH1 expression. Red arrowheads indicate the position of incipient AMs identified by the condensed OSH1 signals. Black and white bars indicate 100 μm and 1 mm, respectively.
Figure 4.
Figure 4.
Molecular Cloning of LAX2. (A) Positional cloning of LAX2. Black horizontal bars represent chromosomal segments or BAC clones corresponding to the region of interest on chromosome 4. Numbers above the top bar represent the number of recombinants between markers and the lax2 mutation after screening an F2 population derived from crosses between the lax2 mutation and an indica cultivar. Blue lines represent the positions of predicted genes. The detailed gene structure of LAX2 is shown at the bottom. Boxes and lines connecting boxes represent exons and introns, respectively. The gene structure presented here is modified from that predicted in the Rice Annotation Project Database or The Institute for Genomic Research database based on our experiments. aa, amino acids. (B) Phylogenetic analysis of amino acid sequences of conserved domains shared with LAX2 from rice and Arabidopsis. The alignment used to construct the phylogenetic tree was made using ClustalX (Jeanmougin et al., 1998) and is shown in Supplemental Figure 2 online. The phylogenetic tree was constructed by the NJ method (Saito and Nei, 1987) using PAUP* 4.0 software. Proteins sharing the conserved domain with LAX2 are divided into two groups, one with a zinc finger motif at the N-terminal side of the conserved domain (red box) and one without a zinc finger (green box). Numbers adjacent to each branch point of the tree indicate bootstrap support of that branch after 1000 replicates. The bar in the figure represents the degree of amino acids changes. (C) to (E) Subcellular localization of LAX2:GFP fusion protein in transgenic rice plants. GFP fluorescence, differential interference contrast, and merged images are shown in (C), (D), and (E), respectively. Bars = 50 um.
Figure 5.
Figure 5.
In Situ mRNA Accumulation Pattern of LAX2. (A) In situ hybridization with a LAX2-specific probe on a longitudinal section of a shoot during vegetative development. The dashed line indicates the approximate position of the cross section shown in (B). (B) In situ hybridization with a LAX2-specific probe on a cross section of a shoot during vegetative development. (C) In situ hybridization with a LAX2-specific probe on a longitudinal section of an inflorescence meristem with PB primordia. (D) In situ hybridization with a LAX2-specific probe on a longitudinal section of an inflorescence meristem with secondary PB primordia. (E) In situ hybridization with a LAX1-specific probe on a longitudinal section of a primary branch meristem with SB primordia. (F) Two-color in situ hybridization with LAX1- and LAX2-specific probes on a longitudinal section of a primary branch meristem with SB primordia. LAX2 expression is detected by a purple color and is visualized on the same section in (E). LAX1 mRNA localization is visualized by red color. (G) In situ hybridization with a LAX2 sense probe on a longitudinal section of the vegetative shoot. Bars = 100 μm. Arrows indicate LAX2 expression at the vegetative AM.
Figure 6.
Figure 6.
Molecular Interaction between LAX1 and LAX2. (A) Interaction between LAX1 and LAX2 was tested by a yeast two-hybrid assay. The full-size LAX2 cDNA and a deletion series of LAX2 cDNAs were fused with a DNA binding domain construct (BD), and the full-size cDNA of LAX1 was fused with an activation domain construct (AD). Yeast cells were grown on −Leu, Trp, His medium, and the interactions were monitored by growth on the selective medium. Numbers in parentheses are the positions of amino acid residues included in each construct. Red boxes indicate the position of the conserved domain. (B) In vitro binding of LAX1 and LAX2. In vitro–synthesized HA-tagged T antigen and HA-tagged LAX1 along with LAX2 (1 to 394) and p53 proteins in the presence of 35S-Met were used as bait and prey, respectively. After incubation of bait and prey proteins, bait proteins were immunoprecipitated using anti-HA antibody. LAX2 and p53 proteins were coprecipitated with HA-LAX1 and HA-T antigen, respectively.
Figure 7.
Figure 7.
A Model for the Functions of LAX1, LAX2, and MOC1. LAX2 is a novel nuclear factor that is involved in branching events throughout rice development. There are multiple pathways for AM formation where LAX1 and LAX2 are involved. LAX2 acts in two pathways, LAX1 dependent and LAX1 independent. In the LAX1-dependent pathway, LAX1 and LAX2 form a dimer. Both LAX1 and LAX2 can also function independently. Factors X and Y could be redundant factors of LAX2 and LAX1, respectively, and they could be partners of LAX1 and LAX2, respectively. Alternatively, it is also possible that factor X and factor Y could correspond to MOC1 because the phenotype of lax1 moc1 and lax2 moc1 double mutants are similar. In this case, MOC1 may or may not form dimers with LAX1 or LAX2. Once the activity of two proteins from the group consisting of LAX1, LAX2, and MOC1 is removed by mutations in the corresponding genes, the formation of the AM is almost completely suppressed at all developmental stages, except PB formation. Thus, there is expected be another layer of regulation for the formation of PB.

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