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. 2009 Apr;21(4):1095-108.
doi: 10.1105/tpc.108.065425. Epub 2009 Apr 3.

Two-Step Regulation of LAX PANICLE1 Protein Accumulation in Axillary Meristem Formation in Rice

Tetsuo Oikawa  1 , Junko Kyozuka
Affiliations

Two-Step Regulation of LAX PANICLE1 Protein Accumulation in Axillary Meristem Formation in Rice

Tetsuo Oikawa et al. Plant Cell. 2009 Apr.

Abstract

Axillary meristem (AM) formation is an important determinant of plant architecture. In rice (Oryza sativa), LAX PANICLE1 (LAX1) function is required for the generation of AM throughout the plant's lifespan. Here, we show a close relationship between AM initiation and leaf development; specifically, the plastochron 4 (P4) stage of leaf development is crucial for the proliferation of meristematic cells. Coincident with this, LAX1 expression starts in the axils of leaves at P4 stage. LAX1 mRNA accumulates in two to three layers of cells in the boundary region between the initiating AM and the shoot apical meristem. In lax1 mutants, the proliferation of meristematic cells is initiated but fails to progress into the formation of AM. The difference in sites of LAX1 mRNA expression and its action suggests non-cell-autonomous characteristics of LAX1 function. We found that LAX1 protein is trafficked to AM in a stage- and direction-specific manner. Furthermore, we present evidence that LAX1 protein movement is required for the full function of LAX1. Thus, we propose that LAX1 protein accumulates transiently in the initiating AM at P4 stage by a strict regulation of mRNA expression and a subsequent control of protein trafficking. This two-step regulation is crucial to the establishment of the new AM.

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Figures

Figure 1.
Figure 1.
Axillary Bud Formation during Vegetative Development of Rice. (A) Longitudinal section of shoot apex stained with safranin-orange G-ferrous tannic acid. The newest leaf is designated as P1, and other leaves are numbered accordingly. (B) to (F) Transverse sections of the shoot in (A). Arrowheads indicate small bulge that will become the AM. (G1) to (G17) Localization of OSH1 mRNA that serves as a marker for meristematic cells. The number indicates the distance from the top of the SAM in micrometers. Panels labeled “e” are close-up views of the region delineated by a rectangle in the previous panel. The SAM region is presented as a red circle in (G1e) to (G3e). Arrows in (G4) and (G5e) indicate the margins of P2 leaf. Arrows in (G6e) and (G7e) indicate OSH1 expression underneath the site where P2 leaf margins overlap. Arrowheads in (G7e) and (G8e) indicate the lateral bulge of cells. (H) A schematic of OSH1 expression in the shoot apex. The region of OSH1 mRNA expression is depicted in red. Bars = 50 μm.
Figure 2.
Figure 2.
Expression Analysis of LAX1 and Related Genes. In situ hybridization analysis of LAX1 and related genes mRNA expression in transverse sections of a developing meristem. Column labels indicate the stage of the leaf surrounding the meristem. (A1) to (A5) LAX mRNA expression. (B1) to (B5) OSH1 mRNA expression. (C1) to (C5) Histone H4 mRNA expression. (D1) to (D5) CDKB2;1 mRNA expression. (E1) to (E5) MOC1 mRNA expression. (F1) to (F5) Rice CUC3 mRNA expression. (G1) to (G5) Rice ra2 mRNA expression. Bars = 50 μm.
Figure 3.
Figure 3.
Defects of AM Formation in lax1 Mutants. (A) and (B) Tiller formation in the wild type and lax1-2 mutant at 30 d after sowing. The lax1-2 mutant has a reduced number of tillers. (C) and (D) Axillary bud formation in the wild type and lax1-2 mutant. Arrowheads indicate tillers and axillary buds. Yellow arrows indicate the absence of axillary bud in the axils of leaves. The numbers indicate the absolute number of leaves counted from germination. Leaves were removed to show axillary buds. (E) and (F) Axillary bud formation in the wild type and moc1-3 mutant. The numbers indicate the absolute number of leaves counted from germination. Leaves were removed to show axillary buds. (G) Tiller bud formation in five lax1 alleles and moc1-3 mutants. The frequency of tiller bud formation in each leaf axil is shown. Twelve plants were examined for each lax1 mutant allele. In this analysis, wild-type plants of all genetic background showed 100% tiller bud formation. The main culm of each plant was examined. Bars = 10 cm in (A), 4 cm in (B), and 500 μm (C) to (F).
Figure 4.
Figure 4.
Expression Pattern of Histone H4 and OSH1 in the Axils of the Fourth Leaf in the Wild Type and lax1-2. (A) to (D) Expression of Histone H4 ([A] and [B]) and OSH1 ([C] and [D]) in the developing AM in the axil of the forth leaf when the forth leaf corresponded to P3. (E) to (H) Expression of Histone H4 ([E] and [F]) and OSH1 ([G] and [H]) in the developing AM in the axil of the forth leaf when the forth leaf corresponded to P4. (I) to (L) Expression of Histone H4 ([I] and [J]) and OSH1 ([K] and [L]) in the developing AM in the axil of the forth leaf when the forth leaf corresponded to P5. (A), (C), (E), (G), (I), and (K) show the wild type, and (B), (D), (F), (H), (J), and (L) show lax1-2. Bars = 50 μm.
Figure 5.
Figure 5.
Trafficking of LAX1 Protein. (A) In situ localization of GFP:LAX1 mRNA in the developing secondary rachis branch of a transgenic plant. The expression of GFP:LAX1 mRNA was restricted to the boundary region (arrowhead). An antisense GFP probe was used to detect GFP:LAX1. (B) Confocal image of GFP:LAX1 protein distribution. GFP signals were observed in AM cells in a stage-specific manner (white arrowhead). Yellow arrows indicate the LAX1 protein in the boundary region where the protein was produced. The sample stage is identical to (A). GFP fluorescence is green, and FM4-64–stained plasma membrane is blue. (C) to (F) Confocal image of transverse sections in the developing axillary buds during vegetative phase. In the P4 stage, GFP:LAX1 protein was observed in the AM cells ([C] and [D]). By contrast, GFP signals were restricted to the boundary region at the early (E) and late (F) P5 stage. (G) and (H) Confocal image of 3xGFP:LAX1 (G) and GFP:LAX1 (H) fusion proteins in the developing primary rachis branch. GFP signals were limited to the boundary region resulting from the inhibition of cell-to-cell protein trafficking (G). By contrast, GFP:LAX1 protein was shown in AM cells (H). Bars = 50 μm in (A), (B), (G), and (H) and 100 μm in (C) to (F).
Figure 6.
Figure 6.
Complementation of lax1 Phenotype by GFP:LAX1 and 3xGFP:LAX1 Fusion Constructs. (A) and (B) Panicle morphology of representative transgenic plants harboring 1xGFP:LAX1 or 3xGFP:LAX1. These constructs were introduced into the lax1-3 mutant. (C) Frequency of lateral spikelet formation in the transgenic (1xGFP:LAX1 or 3xGFL:LAX1) and nontransgenic (WT) plants. The ratio of lateral spikelets relative to the number of bracts, modified leaves, in a panicle is shown. 1xGFP:LAX1 or 3xGFP:LAX1 genes under the control of the LAX1 regulatory region were introduced into lax1-3 mutants. The first regenerated T1 plants were used for the analysis.
Figure 7.
Figure 7.
Model of LAX1 Function in the AM Formation. LAX1 mRNA is specifically expressed in the boundary region when the subtending leaf is at P4 and P5 stages. At P4 stage, LAX1 protein synthesized in the boundary region moves toward the future AM and enhances cell proliferation. In the absence of LAX1 function, the cell proliferation does not take place. As a consequence, the AM is not established. LAX1 mRNA accumulation is shown in blue, and the LAX1 protein distribution is shown in green.
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