The superwoman1-cleistogamy2 mutant is a novel resource for gene containment in rice.

Outcrossing between cultivated plants and their related wild species may result in the loss of favourable agricultural traits in the progeny or escape of transgenes in the environment. Outcrossing can be physically prevented by using cleistogamous (i.e. closed-flower) plants. In rice, flower opening is dependent on the mechanical action of fleshy organs called lodicules, which are generally regarded as the grass petal equivalents. Lodicule identity and development are specified by the action of protein complexes involving the SPW1 and OsMADS2 transcription factors. In the superwoman1-cleistogamy1 (spw1-cls1) mutant, SPW1 is impaired for heterodimerization with OsMADS2 and consequently spw1-cls1 shows thin, ineffective lodicules. However, low temperatures help stabilise the mutated SPW1/OsMADS2 heterodimer and lodicule development is restored when spw1-cls1 is grown in a cold environment, resulting in the loss of the cleistogamous phenotype. To identify a novel, temperature-stable cleistogamous allele of SPW1, targeted and random mutations were introduced into the SPW1 sequence and their effects over SPW1/OsMADS2 dimer formation were assessed in yeast two-hybrid experiments. In parallel, a novel cleistogamous allele of SPW1 called spw1-cls2 was isolated from a forward genetic screen. In spw1-cls2, a mutation leading to a change of an amino acid involved in DNA binding by the transcription factor was identified. Fertility of spw1-cls2 is somewhat decreased under low temperatures but unlike for spw1-cls1, the cleistogamous phenotype is maintained, making the line a safer and valuable genetic resource for gene containment.

Disulfide proteomics of rice cultured cells in response to OsRacl and probenazole-related immune signaling pathway in rice.

BACKGROUND: Reactive oxygen species (ROS) production is an early event in the immune response of plants. ROS production affects the redox-based modification of cysteine residues in redox proteins, which contribute to protein functions such as enzymatic activity, protein-protein interactions, oligomerization, and intracellular localization. Thus, the sensitivity of cysteine residues to changes in the cellular redox status is critical to the immune response of plants. METHODS: We used disulfide proteomics to identify immune response-related redox proteins. Total protein was extracted from rice cultured cells expressing constitutively active or dominant-negative OsRacl, which is a key regulator of the immune response in rice, and from rice cultured cells that were treated with probenazole, which is an activator of the plant immune response, in the presence of the thiol group-specific fluorescent probe monobromobimane (mBBr), which was a tag for reduced proteins in a differential display two-dimensional gel electrophoresis. The mBBr fluorescence was detected by using a charge-coupled device system, and total protein spots were detected using Coomassie brilliant blue staining. Both of the protein spots were analyzed by gel image software and identified using MS spectrometry. The possible disulfide bonds were identified using the disulfide bond prediction software. Subcellular localization and bimolecular fluorescence complementation analysis were performed in one of the identified proteins: Oryza sativa cold shock protein 2 (OsCSP2). RESULTS: We identified seven proteins carrying potential redox-sensitive cysteine residues. Two proteins of them were oxidized in cultured cells expressing DN-OsRac1, which indicates that these two proteins would be inactivated through the inhibition of OsRac1 signaling pathway. One of the two oxidized proteins, OsCSP2, contains 197 amino acid residues and six cysteine residues. Site-directed mutagenesis of these cysteine residues revealed that a Cys140 mutation causes mislocalization of a green fluorescent protein fusion protein in the root cells of rice. Bimolecular fluorescence complementation analysis revealed that OsCSP2 is localized in the nucleus as a homo dimer in rice root cells. CONCLUSIONS: The findings of the study indicate that redox-sensitive cysteine modification would contribute to the immune response in rice.

Inflorescence meristem identity in rice is specified by overlapping functions of three AP1/FUL-like MADS box genes and PAP2, a SEPALLATA MADS box gene.

In plants, the transition to reproductive growth is of particular importance for successful seed production. Transformation of the shoot apical meristem (SAM) to the inflorescence meristem (IM) is the crucial first step in this transition. Using laser microdissection and microarrays, we found that expression of PANICLE PHYTOMER2 (PAP2) and three APETALA1 (AP1)/FRUITFULL (FUL)-like genes (MADS14, MADS15, and MADS18) is induced in the SAM during meristem phase transition in rice (Oryza sativa). PAP2 is a MADS box gene belonging to a grass-specific subclade of the SEPALLATA subfamily. Suppression of these three AP1/FUL-like genes by RNA interference caused a slight delay in reproductive transition. Further depletion of PAP2 function from these triple knockdown plants inhibited the transition of the meristem to the IM. In the quadruple knockdown lines, the meristem continued to generate leaves, rather than becoming an IM. Consequently, multiple shoots were formed instead of an inflorescence. PAP2 physically interacts with MAD14 and MADS15 in vivo. Furthermore, the precocious flowering phenotype caused by the overexpression of Hd3a, a rice florigen gene, was weakened in pap2-1 mutants. Based on these results, we propose that PAP2 and the three AP1/FUL-like genes coordinately act in the meristem to specify the identity of the IM downstream of the florigen signal.

A simple set of plasmids for the production of transgenic plants.

A new set of plasmids for plant transgenic studies was developed. Its strong point is that independent gene cassettes are connected within one binary vector by the restriction endonuclease-based technique only. Using the set, two overexpressing cassettes and three RNA interference (RNAi) cassettes were successfully introduced into rice. Our plasmid set is useful for producing commercial transgenic plants, especially in the case of rice.

MOSAIC FLORAL ORGANS1, an AGL6-like MADS box gene, regulates floral organ identity and meristem fate in rice.

Floral organ identity and meristem determinacy in plants are controlled by combinations of activities mediated by MADS box genes. AGAMOUS-LIKE6 (AGL6)-like genes are MADS box genes expressed in floral tissues, but their biological functions are mostly unknown. Here, we describe an AGL6-like gene in rice (Oryza sativa), MOSAIC FLORAL ORGANS1 (MFO1/MADS6), that regulates floral organ identity and floral meristem determinacy. In the flower of mfo1 mutants, the identities of palea and lodicule are disturbed, and mosaic organs were observed. Furthermore, the determinacy of the floral meristem was lost, and extra carpels or spikelets developed in mfo1 florets. The expression patterns of floral MADS box genes were disturbed in the mutant florets. Suppression of another rice AGL6-like gene, MADS17, caused no morphological abnormalities in the wild-type background, but it enhanced the phenotype in the mfo1 background, indicating that MADS17 has a minor but redundant function with that of MFO1. Whereas single mutants in either MFO1 or the SEPALLATA-like gene LHS1 showed moderate phenotypes, the mfo1 lhs1 double mutant showed a severe phenotype, including the loss of spikelet meristem determinacy. We propose that rice AGL6-like genes help to control floral organ identity and the establishment and determinacy of the floral meristem redundantly with LHS1.

Unequal genetic redundancy of rice PISTILLATA orthologs, OsMADS2 and OsMADS4, in lodicule and stamen development.

Two homologs of PISTILLATA have been identified in rice: OsMADS2 and OsMADS4. However, their roles in floral organ development are controversial. Here, we demonstrate that the genes show unequal redundancy of class B function. Although OsMADS2 plays an important role in lodicule development, OsMADS4 also supports the specification of lodicule identity. In contrast, the genes are roughly equally important in stamen development. Consistent with their redundant functions, both OsMADS2 and OsMADS4 interact with the unique rice AP3 ortholog SPW1.

superwoman1-cleistogamy, a hopeful allele for gene containment in GM rice.

Cleistogamy is an efficient strategy for preventing gene flow from genetically modified (GM) crops. We identified a cleistogamous mutant of rice harbouring a missense mutation (the 45th residue isoleucine to threonine; I45T) in the class-B MADS-box gene SUPERWOMAN1 (SPW1), which specifies the identities of lodicules (equivalent to petals) and stamens. In the mutant, spw1-cls, the stamens are normal, but the lodicules are transformed homeotically to lodicule-glume mosaic organs, thereby engendering cleistogamy. Since this mutation does not affect other agronomic traits, it can be used in crosses to produce transgenic lines that do not cause environmental perturbation. Molecular analysis revealed that the reduced heterodimerization ability of SPW1(I45T) with its counterpart class-B proteins OsMADS2 and OsMADS4 caused altered lodicule identity. spw1-cls is the first useful mutant for practical gene containment in GM rice. Cleistogamy is possible in many cereals by engineering class-B floral homeotic genes and thereby inducing lodicule identity changes.