Hitoshi Yoshida

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.

Production of Human Monoclonal and Polyclonal Antibodies in TransChromo Animals

We have developed TransChromo (TC) technology, which enables the introduction of megabase-sized segments of DNA into cells. We have used this approach to derive mice that carry megabases of human DNA by the use of a human chromosome fragment (HCF) as a vector. TC technology has been applied to the construction of the TC Mouse,trade mark which incorporates entire human immunoglobulin (hIg) loci. TC Mouse expresses a fully diverse repertoire of hIgs, including all the subclasses of IgGs (IgG1-G4). Immunization of the TC Mouse with various human antigens produced antibody responses comprised of human antibodies. Furthermore, it was possible to obtain hybridoma clones expressing fully human antibodies specific for the target human antigen. However, because of the instability of the Igkappa locus-bearing HCF2, the efficiency of hybridoma production was less than one-tenth of that observed in normal mice. An instant solution to this problem was to cross-breed the Kirin TC Mouse carrying the HCF14, which was stable in mouse cells, with the Medarex YAC-transgenic mouse carrying about 50% of the hIgVkappa gene segments as a region that is stably integrated into the mouse genome. The resulting mouse, dubbed the KM Mouse, performed as well as normal mice with regard to immune responsiveness and efficiency of hybridoma production. Another application of TC technology is the production of polyclonal antibodies in large animals such as chickens and cows. To test the efficacy of human polyclonal antibodies derived from TC animals, feasibility studies were performed using antisera and purified gamma-globulin from TC mice immunized with Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MRSA), or Japanese encephalitis virus (JEV). The TC mouse-derived antisera and gamma-globulin showed a much higher titer and efficacy in terms of the neutralizing activity of the pathogens in vitro and in vivo than either human serum or gamma-globulin prepared from human blood.

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

This work examines the reproductive phase transition in rice using laser microdissection and microarrays in addition to biochemical and genetic approaches. The results support that PAP2, a MADS box gene belonging to a grass-specific subclade of the SEP subfamily, and three AP1/FUL-like genes coordinately act in the meristem to specify the identity of the inflorescence meristem downstream of the florigen signal. 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.

Flower development in rice

The flower of rice diverged from those of model eudicot species such as Arabidopsis, Antirrhinum, or Petunia, and is thus of great interest in developmental and evolutionary biology. Specific to grass species, including rice, are the structural units of the inflorescence called the spikelet and floret, which comprise grass-specific peripheral organs and conserved sexual organs. Recent advances in molecular genetic studies have provided an understanding of the functions of rapidly increasing numbers of genes involved in rice flower development. The genetic framework of rice flower development is in part similar to that of model eudicots. However, rice also probably recruits specific genetic mechanisms, which probably contribute to the establishment of the specific floral architecture of rice. In this review, the molecular genetic mechanisms of rice flowering are outlined, focusing on recent information and in comparison with those of model eudicots.

Enhanced Apoptosis and Tumor Regression Induced by a Direct Agonist Antibody to Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Receptor 2

Purpose: Substantial evidence indicates that supraoligomerization of the death receptors for Fas ligand and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is necessary for efficient activation of the apoptotic pathway. Bivalent IgG antibodies can induce the efficient apoptosis by mimicking the natural ligands but only after these antibodies are further oligomerized by cross-linking. In this study, we generated a novel agonist antibody to TRAIL receptor 2 (TRAIL-R2) capable of inducing apoptosis without cross-linking and elucidated its mode of action and efficacy. Experimental Design: A fully human antibody to TRAIL-R2, KMTR2, was generated from KM Mouse immunized with TRAIL-R2 ectodomain. Apoptosis-inducing activities of unfractionated or purified monomeric IgG of KMTR2 was evaluated in the presence or absence of cross-linkers, secondary antibodies or Fc receptor–expressing effector cells, against human colorectal adenocarcinoma Colo205. Oligomerization of TRAIL-R2 was analyzed by size exclusion chromatography and confocal microscopy, and in vivo efficacy was examined in Colo205 xenograft model. Results: KMTR2 specifically recognized TRAIL-R2 and induced apoptosis with or without cross-linking. Size exclusion chromatography showed that the apoptosis activity coeluted with monomeric IgG and was effective independent of secondary antibody or Fc receptor–expressing effector cells. The antibody formed supracomplexes with soluble recombinant and membrane-anchored TRAIL-R2 and enhanced clustering of TRAIL-R2 on cell surface without cross-linking. KMTR2 was dramatically efficacious in reducing established human tumor. Conclusion: Our findings indicate that novel agonist antibody KMTR2 can direct antibody-dependent oligomerization of TRAIL-R2 and initiates efficient apoptotic signaling and tumor regression independent of host effector function. Thus, the direct agonist would be a lead candidate for cancer therapeutics.

Activation of a Rac GTPase by the NLR Family Disease Resistance Protein Pit Plays a Critical Role in Rice Innate Immunity

The nucleotide-binding domain and leucine-rich repeat-containing (NLR) family proteins recognize pathogen-derived molecules and trigger immune responses in both plants and animals. In plants, the direct or indirect recognition of specific pathogen effectors by NLRs culminates in a hypersensitive response (HR) and the production of reactive oxygen species (ROS), key components of the plant defense response. However, the molecules activated by NLRs and how they induce immune responses are largely unknown. We found that the rice GTPase OsRac1 at the plasma membrane interacts directly with Pit, an NLR protein that confers resistance to the rice blast fungus. OsRac1 contributes to Pit-mediated ROS production as well as the HR and is required for Pit-mediated disease resistance in rice. Furthermore, the active form of Pit induces the activation of OsRac1 at the plasma membrane. Thus, OsRac1 is activated by Pit during pathogen attack and plays a critical role in Pit-mediated immunity in rice.

LAX PANICLE2 of Rice Encodes a Novel Nuclear Protein and Regulates the Formation of Axillary Meristems

This study reports the identification of a novel regulator of axillary meristem formation in rice, showing that LAX PANICLE2 (LAX2) likely acts in the maintenance of the axillary meristem. In addition, it reveals that LAX2 localizes to the nucleus and appears to form a dimer with LAX1, which is a basic helix-loop-helix transcriptional factor. Aerial architecture in higher plants is dependent on the activity of the shoot apical meristem (SAM) and axillary meristems (AMs). The SAM produces a main shoot and leaf primordia, while AMs are generated at the axils of leaf primordia and give rise to branches and flowers. Therefore, the formation of AMs is a critical step in the construction of plant architecture. Here, we characterized the rice (Oryza sativa) lax panicle2 (lax2) mutant, which has altered AM formation. LAX2 regulates the branching of the aboveground parts of a rice plant throughout plant development, except for the primary branch in the panicle. The lax2 mutant is similar to lax panicle1 (lax1) in that it lacks an AM in most of the lateral branching of the panicle and has a reduced number of AMs at the vegetative stage. The lax1 lax2 double mutant synergistically enhances the reduced-branching phenotype, indicating the presence of multiple pathways for branching. LAX2 encodes a nuclear protein that contains a plant-specific conserved domain and physically interacts with LAX1. We propose that LAX2 is a novel factor that acts together with LAX1 in rice to regulate the process of AM formation.

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.

The ACC synthase TOE sequence is required for interaction with ETO1 family proteins and destabilization of target proteins

The Arabidopsis ETO1 protein is a negative regulator of ethylene biosynthesis. It specifically inhibits the enzyme activity of type 2 1-aminocyclopropane-1-carboxylate synthases (ACC synthases or ACS) and promotes their degradation by a proteasome-dependent pathway. To further understand the function of the ETO1 family in the plant kingdom, we cloned a cDNA of LeEOL1 (Lycopersicon esculentum ETO1-LIKE 1), an ETO1 homolog from tomato. LeEOL1 encodes a putative protein with domain architecture conserved in the Arabidopsis ETO1/EOL1/EOL2 proteins and in the predicted rice EOL proteins. LeEOL1 is expressed in leaf, stem, root, flower, and the full ripe stage of fruit, suggesting diverse regulatory roles in the development of tomato. Yeast two-hybrid analysis revealed specific interactions between LeEOL1 and type 2 ACC synthases. When the C-terminal 14 amino acids (TOE; target of ETO1) of LE-ACS3 specific to type 2 ACC synthases were fused to a type 1 ACS, LE-ACS2, at the corresponding position, it allowed LE-ACS2 to strongly interact with LeEOL1. A GFP-TOELE-ACS3 fusion protein expressed in rice calli and in the roots of wild-type Arabidopsis showed reduced stability compared to native GFP. However, the fluorescence of GFP-TOELE-ACS3 was comparable to that of the native GFP in Arabidopsiseto1-4 mutant. Furthermore, MG132 treatment significantly enhanced the fluorescence of GFP-TOELE-ACS3 in the roots of wild-type Arabidopsis. These results suggest that the ETO1-family-mediated ACS protein degradation pathway is conserved in both monocots and dicots, and that TOE acts as a protein destabilization signal recognized by the ETO1 protein family.

Is the lodicule a petal: Molecular evidence?

Lodicules are grass-specific floral organs with scale-like shapes that play an important role in flower opening. Because of their position just outside the stamens, lodicules have been suggested as analogous to eudicot petals. Previous molecular genetic studies in maize and rice revealed that identities of lodicule and stamen are specified by members of the AP3-lineage of B-class MADS-box genes, which specify petal and stamen identities in eudicots. This supported the hypothesis that lodicules may be equivalent to eudicot petals. Recent studies in rice, maize, and barley further demonstrated that the molecular genetic mechanism of lodicule development includes the PI-lineage of B-class, C-class, SEP-like, AGL6-like, and AP2-like genes. These findings consistently suggest that the genetic mechanisms behind lodicule and petal development are similar. Nevertheless, remarkable divergence in the appearances of lodicules and petals suggests that their developmental processes are very different. Critical mutations in cis-elements and coding sequences of the key regulatory genes may be major driving forces of the divergence between lodicules and petals.