Os06g0665400

Function

The temporal and spatial control of meristem identity is a key element in plant development.Through certain research,we found out that the rice aberrant panicle organization 1 (apo1) mutants revealed that APO1 positively controls spikelet number by suppressing the precocious conversion of inflorescence meristems to spikelet meristems.Besides, the rice aberrant panicle organization 2 (apo2) mutant which exhibits small panicles reduces number of primary branches due to the precocious formation of spikelet meristems.

To better understand the molecular mechanisms that regulate inflorescence and flower architecture,To elucidate its molecular function, we isolated the APO1 and AP02 gene using a map-based strategy. And through analyses of phenotypes and gene expression,we revealed that phenotypic analyses of apo1 and floral homeotic double mutants demonstrate that APO1 positively regulates class-C floral homeotic genes, but not class-B genes. Molecular studies revealed that APO1 encodes an F-box protein, an ortholog of Arabidopsis UNUSUAL FLORAL ORGAN (UFO), which is a positive regulator of class-B genes. Over expression of APO1 caused an increase in inflorescence branches and spikelets. As the mutant inflorescences and flowers differed considerably between apo1 and ufo, the functions of APO1 and UFO appear to have diverged during evolution.

In addition,APO2 is identical to previously reported RFL gene, the rice ortholog of the Arabidopsis LEAFY(LFY) gene. Further analysis indicated that APO2/RFL and APO1, the rice ortholog of Arabidopsis UNUSUAL FLORAL ORGANS, act cooperatively to control inflorescence and flower development. The present study revealed functional differences between APO2/RFL and LFY. In particular, APO2/RFL and LFY act oppositely on inflorescence development. Therefore, the genetic mechanisms for controlling inflorescence architecture have evolutionarily diverged between rice (monocots) and Arabidopsis (eudicots).

Figure 1.Phenotypes of apo1.(a) Panicle structure.(b)Top view of the wild-type inflorescence.(c) Side view of the apo1-2inflorescence.(d) Wild-type flower comprising two lodicules (arrowhead), six stamens and one pistil(e) apo1-2 flower comprising two lodicules (arrowheads), two lodiculestamen mosaic organs (arrows), three stamens and three pistils.(f) SEM image of apo1-1 flower.(g) In situ expression of DL gene in wild-type (left)and apo1-1(right) flowers.DL is expressed in normal and extopic carpel primordia (arrowheads).(h), (i) Schematic representation of wild-type (f) and apo1 (g) flowers. (j), (k)A model of floral organ identity in wild type (h) and apo1 (i). Abbreviations: ca,carpel; cg, mosaic organ between carpel and glume; le, lemma; lo, lodicule; pa,palea; st, stamen. Scale bars = 10 cm in (a), and 50 lm in (b) and (c)..

Figure 2.Vegetative phenotypes of wild-type and apo2 plants. (a) Three-week-old seedlings of wild-type (left) and apo2-1 plants (right).Arrowheads indicate the fourth leaf. (b) Changing pattern in the number of leaves during development. (c) Size of second leaf in wild-type and apo2-1 plants. (d) Cell lengths of second and fourth leaf sheath in wild-type and apo2-1 plants. (e) Transverse sections of mature fourth leaf blades in wild-type (left) and apo2-1 plants (right). Vertical bars in (b), (c), and (d) indicate standard deviations. Asterisks in (c) and (d) indicate significant difference from wildtype at P < 0.01 (Student’s t-test). Scale bars, 5 cm in (a), 50 lm in (e), and (f)..

Molecular function of APO1

Using the F2 population ofAPO1/apo1-3 · cv. Kasalath (Oryza sativa spp. indica), APO1 was roughly mapped at around 106 cM between two sequence-tagged site (STS) markers, R3819 and C11635, of chromosome 6. This map position supports the close linkage with SPW1 located at around 120 cM. We also found a close homolog of UFO within this region. Occurrence of the mosaic floral organs observed in apo1 resembled the floral phenotypes of Arabidopsis ufo mutants, and prompted us to test the idea that the UFO homolog might be APO1. To test this idea, we examined the nucleotide sequence of this region in the three apo1 alleles. The UFO-like gene comprised two exons and one intron . Sequencing analysis detected one nucleotide substitution in the exon of each apo1 allele. Three substitutions caused non-sense mutations at positions 28, 211 and 378 of the deduced amino acid sequence. The mutant phenotype of apo1-3 was rescued when an 8.5-kb genomic fragment containing the UFO-like gene was introduced . Thus, we conclude that APO1 is the UFO-like gene. APO1 encodes a putative protein of 429 amino acids, with motifs that suggest a role as an F-box protein at the N-terminus. F-box proteins are characterized as components that bind substrates for ubiquitin-mediated proteolysis (Kipreos and Pagano, 2000). F-box proteins contain a protein–protein interaction motif in the C-terminal region that binds to the target protein (Kipreos and Pagano,2000).

APO1 encodes an F-box protein that is closely related to Antirrhinum FIM and Arabidopsis UFO. The F-box motif provides substrate specificity for large protein complexes called SCFs (Skp1-Culling-F-box protein complexes), which have E3 ubiquitin ligase activity and target proteins for degradation(Kipreos and Pagano, 2000). As genetic experiments and a binding assay revealed that UFO interacts with the Arabidopsis SKP1 homolog, ASK1 (Durfee et al., 2003; Zhao et al., 2001),UFOis thought to act as the receptor that recruits the substrate. F-box proteins generally contain a protein–protein interaction motif to bind the substrates in the C-terminal portion. However, such a motif and its target protein of FIM/UFO remain unknown. We also could not find a protein– protein interaction motif in the C-terminal portion of APO1..

Figure 3.Sequence analysis of the APO1 gene. (a) Exon/intron structure of APO1 and mutations in three alleles. Two open boxes indicate exons split by a small intron. Mutations in the three alleles are indicated. (b) Alignment of the deduced amino acid sequences encoded by APO1, STP, PFM, FIM, UFO, maize fimbriata A and maize fimbriata B. Residues that are>50% identical are shaded in black. Conservative substitutions at an amino acid position are shaded in gray. Dashes denote gaps that were introduced to optimize the alignment. The F-box domain is underlined. Asterisks indicate the completely conserved amino acid and dots indicate the similar residues. (c) An unrooted phyllogenetic tree generated by CLUSTALX using the neighbor-joining method from the predicted amino acid sequences of F-box genes from Arabidopsis and rice, and UFO homologs from other plants.Bootstrap values generated with 1000 replicates are indicated before the nodes. The bar represents the branch length equivalent to 0.1 amino acid changes per residue..

Molecular function of APO2

As for APO2,it was mapped to a location near the Rice FLORICAULA (RFL) gene (AB005620) on chromosome 4(Kyozuka et al., 1998). Sequencing of RFL in apo2 revealed that apo2-1 has a single base change (C fi A) in the middle of exon2 that causes a premature termination of translation(S210stop). Thus, the apo2-1 protein is presumed to lack theDNA binding domain in the C-terminal region. In contrast,apo2-2 has a T fi A transition in exon1 that causes an amino acid change (L102H) (Figure S3). To confirm that APO2 is identical to RFL, a genomic DNA fragment was introduced into apo2-2. The plants transformed with RFL showed panicles with an increased number of spikelets compared with plants harboring an empty vector, demonstrating that APO2 is RFL .

The spatial expression pattern of APO2/RFL was previously reported by Kyozuka et al. (1998) and Rao et al. (2008). We re-examined the detailed expression pattern by in situ hybridization. In the vegetative phase, APO2/RFL was expressed in shoot meristems and leaf primordia, in contrast to the result of Kyozuka et al. (1998) who did not detect the expression in the vegetative meristems.Because apo2 mutants exhibited several phenotypes in vegetative phase, the expression in shoot apical meristems would be reasonable. In the reproductive phase, APO2/RFL was expressed in the meristems of primary and secondary branches at primordial and elongating stages , but the expression transiently down regulated from the meristems at stage In5 (Figure 5d). When spikelets were differentiated,APO2/RFL expression was recovered in floral meristems and in primordia of all floral organs, including lodicules, stamens, carpels and ovules. This unique expression pattern during the reproductive phase has not been reported to date. Since the timing of the down-regulation coincided with that of the meristem identity change from inflorescence to spikelet and the mutant inflorescence phenotypes seemed to be independent from those in spikelets/flowers, APO2/RFL may independently function between inflorescence and the spikelet/flower stages.

Figure 4.Interaction between APO2/RFL and APO1 in rice Oc cells. (a) UBQ:APO1 panicle. Scale bar, 1 cm.(b) apo2-1 UBQ:APO1 panicle. Scale bar, 1 cm.(c) Co-immunoprecipitation of APO1 with APO2 in rice Oc cells. Immunoblot(IB) analysis was performed with anti-FLAG and anti-HA antibody. A band ofthe correct size for APO1-2HA was detected in the APO1-2HA APO2-3FLAG double transgenic line, but not in the single APO1-2HA transgenic line.(d) Co-immunoprecipitation of APO2-2HA with APO2-3FLAG indicates thatAPO2 forms a dimer in rice Oc cells.

Labs working on this gene

1 Kyoko Ikeda-Kawakatsu,Jun-Ichi Itoh1 and Yasuo Nagato,Graduate School of Agriculture and Life Sciences, University of Tokyo, Yayoi, Bunkyo, Tokyo 113-0032, Japan.

2 Masahiko Maekawa, National Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan.

3 Takeshi Izawa,National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.

4 Kyoko Ikeda,Momoyo Ito, Nobuhiro Nagasawa, Junko Kyozuka and Yasuo Nagato, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan