Os04g0396500

The rice gene Os03g0230500 was reported as LAX2 in 2011.

Annotated Information

Function

• LAX2 encodes a nuclear protein with a plant-specific conserved domain and that LAX2 physically interacts with LAX1 through a region containing this conserved domain. Thus, the rsearchers propose that LAX2 is a novel genetic component that is required for the process of AM formation and acts together with LAX1 in rice.

• Although LAX1 and LAX2 have a unique role in the maintenance of the AM, it is possible that LAX2 modulates the activity of LAX1 at the posttranscriptional level.

Mutation

• lax2 mutations were isolated while screening for mutants with abnormal panicle development. The researchers found three independent mutant alleles, all of which exhibited a sparse-panicle phenotype. These alleles, recovered from ethyl methanesulfonate, tissue culture, and g-ray irradiation mutagenesis populations, were designated as lax2-1, lax2-2, and lax2-3, respectively. To characterize the nature of the sparse appearance of the lax2-1 panicle, the researchers counted the number of inflorescence branches in the wild type and mutant. The lax2-1 mutant panicle produced fewer spikelets compared with the wild-type panicle (Figures 1A and 1E). Quantitative analysis of the lax2-1 mutant panicle revealed that the number of PBs was not affected in the mutant (Figure 1F). However, the number of total lateral branches, which was the sum of the number of PBs, SBs, and spikelets in a panicle, was reduced in lax2-1 (Figure 1G). This indicates that the sparse panicle appearance and the reduction in spikelet number in the lax2-1 mutant are attributable both to reduced higher order branching, such as the number of SBs, and to a reduced number of spikelets borne directly on branches (Figure 1B). Because a spikelet other than a terminal spikelet is produced as a lateral branch from an inflorescence meristem, lax2-1 mutants seem to have a defect in producing all the lateral branches other than PBs in panicles. lax2-1 mutants also had defects in vegetative development. In lax2-1 mutants, the number of tillers produced during vegetative growth was reduced (Figures 1C and 1H). Because there was no trace of tiller buds at the base of the leaves in lax2-1 mutants (Figure 1D), the reduction in the number of tillers is caused by a defect in AM formation and not by enhanced apical dominance or growth arrest of tiller buds. As a result of the reduction in the number of tillers in lax2-1, mature lax2-1 plants had fewer panicles than wild-type plants. Thus, the lax2-1 mutant failed to develop lateral branches during both vegetative and reproductive development, although the number of PBs in the panicle was not affected.

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. .

• The other lax2 mutant alleles, lax2-2 and lax2-3, had similar branching defects, as observed in lax2-1 mutants in panicles, except that the number of PBs in lax2-2 and lax2-3 was slightly increased (Table 1).

Table 1. Phenotypic Characterization of lax2 Mutants and Their Respective Parental Wild Types in Rice .

• To clarify the genetic interactions of lax2 with other rice mutants that exhibit a defect in AM formation, the researchers made double mutants containing lax2 and either lax1 or moc1. lax1 had a sparse panicle with reduced SBs and spikelets, which was similar to the lax2 panicle, and lax1 had tillers with slightly reduced numbers at the vegetative stage (Figures 2A to 2C and 2G to 2I). The researchers made lax1 lax2 double mutants using null alleles of lax1, lax1-1, and lax1-6. lax1-1 is a deletion allele of lax1 , and lax1-6 has a single nucleotide deletion at the 59 end of the region encoding the conserved bHLH domain. The phenotype of the lax1 lax2 double mutant was more extreme than that of the single mutants. In the double mutant panicle, all SBs and spikelets except terminal spikelets were absent, whereas the number of PBs was not affected compared with the single mutants (Figure 2K, Table 2). At the vegetative stage, both single mutants shared the similar phenotype of a slightly reduced number of tillers (Figures 2B and 2C); however, there was a strong reduction in tiller branches in the double mutant (Figure 2E, Table 2). The allele of moc1 used in our analysis, moc1-4, also has a sparse panicle; however, the branching pattern of the moc1-4 panicle is different from that of the wild type, lax1, or lax2(Figure 2J,2G). The researchers used lax2-2 and moc1-4, both of which were derived from Nipponbare. The phenotype of the lax2-2 moc1-4 double mutant was more extreme than that of either single mutant. In the double mutant panicle, all PBs, SBs, and spikelets were absent (Figure 2L), and there were no tiller branches at the vegetative stage (Figure 2F).

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. .

Table 2. Quantification of the Double Mutants of lax1 and lax2 .

• The researchers observed the expression of the OSH1 protein (a marker of the meristem) and the morphology of the AM in single and double mutants of lax2 and moc1. At the vegetative stage in a wild-type plant, a population of cells that expresses OSH1 can be detected at the axil of the second and third youngest leaf primordia (P2-P3) as a bulge , depending on the sampling stage within a plastochron (Figures 3A and 3E). Similar structures were observed in both lax2-2 and moc1-4 single and double mutants (Figures 3B to 3D). These bulges possibly include cells that give rise to an incipient AM. At the P4 leaf axil, the AM is formed and grows in wild-type plants and in lax2 and moc1 single mutants (Figures 3F to 3H and 3J). At the P5 leaf axil, the AM grows continuously and is covered by prophyll in wild-type plants and in lax2 and moc1 single mutants (Figure 3K to 3M and 3O). However, in the lax2 moc1 double mutant, the bulge with OSH1-expressing cells, although seen initially at the P2-P3 leaf axil, did not proliferate at the P4 leaf axil (Figure 3I) and was absent at the P5 leaf axil (Figure 3N). The lack of the AM in the lax2 moc1 double mutant was also observed by scanning electron microscopy analysis (Figures 3R and 3S). This phenotype is quite consistent because there is never a tiller in the lax2 moc1 double mutant. This observation negates the possibility that the initiation of the SAM is stochastically arrested in the mutant and indicates that the double mutant is defective in the maintenance of the AM. Thus, at the vegetative stage, both LAX2 and MOC1 are involved in the maintenance of the AM. At the reproductive stage, AM primordia that give rise to PBs formed normally in the lax2 mutant as in the wild type (Figures 3P and 3Q).

Figure 3. Anti-OSH1 Immunostaining of the Wild Type, lax2, moc1, and lax2 moc1 DoubleMutant and Scanning ElectronMicroscopy Image of theWild 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 mm and 1 mm, respectively. .

Expression

• In the　vegetative shoots, LAX2 expression was observed at the axil of the P3 leaf, at the location where the AM is formed, as well as in　young leaves (Figures 5A, 5B, and 5G). In the reproductive stage, LAX2 expressionwas observed at all the AMsthat give rise toPBs, SBs, and spikelets (Figures 5C and 5D).

Figure 5. In Situ mRNA Accumulation Pattern of LAX2. .

Subcellular localization

• To identify the molecular function of LAX2, we investigated the cellular localization of LAX2 by expressing LAX2:green fluorescent protein (GFP) fusion proteins driven by a 35S constitutive promoter of Cauliflower mosaic virus in the root cells of stable rice transformants (Figures 4C to 4E). GFP fluorescence was localized exclusively in the nucleus, suggesting that LAX2 is a nuclear protein, although we could not find the typical nuclear localization signal in the predicted LAX2 amino acid sequence.

Figure 4.(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. .

Evolution

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Labs working on this gene

• Crop Development Division, National Agriculture and Food Research Organization Agricultural Research Center, Niigata 943-0193, Japan
• National Center for Plant Gene Research, Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China
• Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
• Photosynthesis and Photobiology Research Unit, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan
• State Key Laboratory of Rice Biology, China National Rice Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310006, People’s Republic of China
• Institute of Radiation Breeding, National Institute of Agrobiological Sciences, Hitachi-ohmiya 319-2293, Japan
• Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan
• Rice Research Division, National Institute of Crop Science, National Agriculture and Food Research Organization, Tsukuba 305-8518, Japan
• Department of Agricultural and Environmental biology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan
• Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan