Shinnosuke Ohmori

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.

Agronomic traits and gene containment capability of cleistogamous rice lines with the superwoman1-cleistogamy mutation.

Pollen-mediated transgene flow is a major concern for the production of genetically modified (GM) rice. Cleistogamy is a useful tool for preventing this form of gene flow. We previously identified the cleistogamous rice mutant superwoman1-cleistogamy (spw1-cls) and determined its molecular genetic mechanism. In the present study, we cultivated spw1-cls over five years to examine effects of cleistogamy on agronomic traits. Simultaneously, we cultivated cleistogamous backcross lines created by continuous backcrossing with “Yumeaoba” (a japonica cultivar) as the recurrent parent and by application of a DNA marker. In these experimental cultivations, spw1-cls and its backcross lines showed almost equal or slightly lower, but acceptable, agronomic traits compared with each control line. We also conducted natural crossing tests in paddy fields to assess the gene containment capability of spw1-cls. In a series of field experiments, there was no natural crossing between spw1-cls (pollen donor) and pollen recipient lines, but the wild-type donor and recipient lines were crossed. Thus, the cleistogamy of the spw1-cls mutation is able to inhibit natural crossing effectively, without significant loss of commercial benefits, such as yield. We conclude that spw1-cls cleistogamy is a practical tool for gene containment in GM rice cultivation.

Cleistogamy decreases the effect of high temperature stress at flowering in rice.

Abstract Rice sterility due to a high temperature at flowering is a serious agricultural problem that has been associated with global warming. The flowering stage in rice plants is most vulnerable to high temperature stress. Closed flowering rice plants may better withstand high temperature stress. The aim of this study was to determine the role of cleistogamy (closed flowering) in avoiding high temperature-induced sterility. Cleistogamy was induced by moderate heat treatment at 30°C during the panicle development stage. Both cleistogamous and chasmogamous (ordinary open flowering) rice plants, which possess the same genetic background, were subjected to 38°C or 36°C for 4 h just before flowering, and the percentage of fertility, number of pollen grains on a stigma, number of germinated pollen grains on a stigma, and temperatures inside and outside of the closed spikelets were examined. The cleistogamous rice plants showed a higher fertility percentage and a larger number of germinated pollen grains on a stigma than the chasmogamous rice plants. The temperature inside the closed flowering spikelets was 1.8°C lower than that outside the spikelets. The cleistogamous rice plants thus showed avoidance to high temperature stress at 38°C at flowering. On the basis of these results, we concluded that cleistogamy was advantageous to rice pollination and fertilization at high temperatures because of glume cooling.

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

Summary 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.

The cleistogamy of the superwoman1-cleistogamy1 mutation is sensitive to low temperatures during the lodicule-forming stage

We reported previously that the rice (Oryza sativa L.) cleistogamous mutation superwoman1-cleistogamy1 (spw1-cls1) was applicable to inhibit outcrossing between genetically modified varieties and their relatives, which causes pollen-mediated gene flow or disturbance of line purity. The cleistogamy of spw1-cls1 is caused by decreased protein-protein interactions between the mutant SPW1 and its partner proteins. Importantly, these interactions are restored under low-temperature conditions, but whether the cleistogamy of spw1-cls1 is affected by this phenomenon was unclear. In this study, we cultivated spw1-cls1 in various regions of Japan and confirmed that its flowers opened at low temperatures. Moreover, we compared the morphology of a series of lodicules generated at various temperatures. The results indicated that the cleistogamy of spw1-cls1 is thermosensitive and is gradually disturbed as the temperature decreases. This was correlated with the protein interaction pattern of the mutant SPW1 as reported previously. Then, we revealed the critical period for the low-temperature-induced instability of the phenotype of spw1-cls1 and examined the effect of daily temperature changes on cleistogamy. The results may facilitate simulation of the phenotype of spw1-cls1 at various temperatures and the prediction of regions where the cleistogamy of spw1-cls1 can be stably used to inhibit outcrossing.