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Molecular genetic studies using Arabidopsis thaliana as a model system have overwhelmingly revealed many important molecular mechanisms underlying the control of various biological events, including floral induction in plants. The major genetic pathways of flowering have been characterized in-depth, and include the photoperiod, vernalization, autonomous and gibberellin pathways. In recent years, novel flowering pathways are increasingly being identified. These include age, thermosensory, sugar, stress and hormonal signals to control floral transition. Among them, hormonal control of flowering except the gibberellin pathway is not formally considered a major flowering pathway per se, due to relatively weak and often pleiotropic genetic effects, complex phenotypic variations, including some controversial ones. However, a number of recent studies have suggested that various stress signals may be mediated by hormonal regulation of flowering. In view of molecular diversity in plant kingdoms, this review begins with an assessment of photoperiodic flowering, not in A. thaliana, but in rice (Oryza sativa); rice is a staple crop for human consumption worldwide, and is a model system of short-day plants, cereals and breeding crops. The rice flowering pathway is then compared with that of A. thaliana. This review then aims to update our knowledge on hormonal control of flowering, and integrate it into the entire flowering gene network.

The skin disease paracoccidioidomycosis ceti occurs in several dolphin species globally. Infection by the unculturable fungi Paracoccidioides brasilensis or other Paracoccidioides spp. results in chronic cutaneous and granulomatous lesions. In this study we used immunohistochemistry to investigate the seroprevalence of antibodies to Paracoccidioides spp. in captive dolphins from three aquaria in Japan. We had previously reported that there were serological cross-reactions for Paracoccidioides spp. with related species in the order Onygenales. We hypothesized that the degree of serological cross-reactions for Paracoccidioides spp. might be lower in areas, such as Japan, where the fungal diseases coccidiodomycosis and paracoccidiodomycosis are not endemic. Sera from 41 apparently healthy dolphins, including 20 Atlantic bottlenose dolphins (BD: Tursiops truncatus), 6 Indo-Pacific bottlenose dolphins (IPBD: Tursiops aduncus), 2 F1 generation of a cross between BD and IPBD (F1), 3 Pacific white-sided dolphins (PWD: Lagenorhynchus obliquidens), 2 pantropical spotted dolphins (PSD: Stenella attenuata), 6 false killer whales (FKW: Pseudorca crassidens), and 2 rough-toothed dolphins (RTD: Steno bredanensis) were investigated. Sera from three dolphins with paracoccidioidomycosis ceti were used as a positive control. The yeast-form cells of Paracoccidioides spp. in the cutaneous tissue sample derived from the first Japanese paracoccidioidomycosis ceti case were used as the antigen for the immunohistochemistry. Of the 41 dolphins tested, 61.0% had antibodies against Paracoccidioides spp. This indicates that dolphins of several species in Japanese aquaria have likely been exposed to the pathogen Paracoccidioides spp.

Androgen action generates sex-related differences that include changes in the gut microbiota composition. Hypoandrogenism and hyperandrogenism in males and females, respectively, are associated with the prevalence of metabolic disorders. Our recent work showed that male androgen receptor knockout (ARKO) mice developed high-fat diet (HFD)-dependent sarcopenic abdominal obesity, hyperglycemia, and hepatic steatosis, leading to early death. The ARKO mice also exhibited alterations in intestinal microbiota but did not experience metabolic abnormalities when administered with antibiotics. Here, we show that time-dependent changes in feed efficiency (ratio of body weight gain to food intake) and weight of dried feces-to-food ratio could be good markers for changes in gut microbiota. Turicibacter spp., Lactobacillus spp., and L. reuteri increased in the gut in both HFD-fed ARKO and castrated mice having metabolic abnormalities. HFD-fed ARKO mice showed increased plasma levels of aspartate, but not alanine, aminotransferase. Changes in the gut microbiome appear to provoke androgen deficiency-induced metabolic diseases, leading to early mortality.

The prominence of seafood in Japan motivates close monitoring of its seas and marine lives for potentially pathogenic fungi. During the treatments of the male Pacific white-sided dolphin (Lagenorhynchus obliquidens) for paracoccidioidomycosis ceti (PCM-C), 5 white and floccose colonies showing identical genotype and morphological characteristics were isolated from two skin biopsy samples of cutaneous granulomatous lesions in 2018. The isolates were identified as Parengyodontium album known as one of fungal species having abilities to produce industrially important proteases, and to become a causative agent for emerging mycosis based on morphological and molecular biological characteristics. These lesions consisted of non-malignant pearl-like structures of hyperplastic keratinocytes. Interestingly, although the isolates could grow at 35 °C, their DNA sequences were phylogenetically located in a cluster consisting of environmental and clinical isolates lacking the ability to grow at 35 °C, based on previous reports. The opportunistic infection we observed in the dolphin might be caused by immune disorder due to PCM-C. Notably, although P. album is recognized as non-harmful, and has significant industrial importance and antitumor activity, it has potential to cause not only superficial but also systemic infection, and presents difficulties in treatment because of its high resistance to antifungal compounds.

Two casein kinase genes, Hd16 and Hd6, modulate florigen genes within certain restricted ranges of photoperiod, thereby fine-tuning the critical day length in rice and potentially determining ecological niches within latitudinal clines., Many short-day plants have a critical day length that fixes the schedule for flowering time, limiting the range of natural growth habitats (or growth and cultivation areas). Thus, fine-tuning of the critical day-length setting in photoperiodic flowering determines ecological niches within latitudinal clines; however, little is known about the molecular mechanisms controlling the fine-tuning of the critical day-length setting in plants. Previously, we determined that florigen genes are regulated by day length, and identified several key genes involved in setting the critical day length in rice. Using a set of chromosomal segment substitution lines with the genetic background of an elite temperate japonica cultivar, we performed a series of expression analyses of flowering-time genes to identify those responsible for setting the critical day-length in rice. Here, we identified two casein kinase genes, Hd16 and Hd6, which modulate the expression of florigen genes within certain restricted ranges of photoperiod, thereby fine-tuning the critical day length. In addition, we determined that Hd16 functions as an enhancer of the bifunctional action of Hd1 (the Arabidopsis CONSTANS ortholog) in rice. Utilization of the natural variation in Hd16 and Hd6 was only found among temperate japonica cultivars adapted to northern areas. Therefore, this fine-tuning of the setting of the critical day length may contribute to the potential northward expansion of rice cultivation areas.

An 8-year-old female Magellanic penguin (Spheniscus magellanicus) started to show epilepsy-like seizures. Subsequent magnetic resonance imaging (MRI) examinations did not reveal any responsible lesions. The neurological symptoms worsened at the age of 10. This penguin became recumbent and died 6 months later after the apparition of the recumbency. At necropsy, only multiple yellowish necrotic lesions in the air sacs and lungs were found. Histopathological evaluation of the brain showed a marked loss of Purkinje cells and many hypertrophied parvalbumin-positive basket/stellate cells were seen in the cerebellar cortex. Calbindin immunohistochemistry demonstrated disrupted arrangement of dendrites in the Purkinje cells. This case was diagnosed as cerebellar cortical degeneration with a very late onset and a slow progression in a Magellanic penguin.

This study represents cases with spontaneous neuritis of peripheral nerves in electric eels. Two electric eels were presented with abnormal swimming behavior and loss of appetite. Electric eels had extensive histopathologic lesions in the splenic and cardiac nerves. The lesions were characterized by swelling of neuronal cells, central chromatolysis and marked inflammatory cell infiltration consisting mainly of lymphocytes around the affected nerves. To the best of our knowledge, this is the first case report of spontaneous neuritis of peripheral nerves in electric eels.

A white, lobular mass was found in the right ovary of a pregnant Risso's dolphin (Grampus griseus) at necropsy. The mass was unilateral and occupied most of the pre-existing ovarian tissue. Histologically, the mass was composed of diffuse sheets of polyhedral cells with abundant eosinophilic cytoplasm and oval nuclei, separated by fibrous connective tissue. Only a few ovarian follicles were observed at the periphery of the mass. Immunohistochemically, the large eosinophilic cells were positive for vimentin and negative for pan-cytokeratins. Based on the histopathological features, the present case was diagnosed as luteoma. In human medicine, luteoma of pregnancy, a tumor-like proliferative lesion occurring in pregnant women, is well described. In veterinary medicine, luteoma associated with pregnancy has never been described. The present study would provide useful information for understanding the characteristics of luteoma in animals.

Summary Flowering time is an important agronomic trait that affects crop yields. In cereals, several CCT-domain proteins unique to monocots, such as Grain number, plant height, and heading date 7 (Ghd7) gene, have been identified as key floral repressors, although the corresponding molecular mechanisms have been unknown until now. In rice, a short-day plant, Heading date 1 (Hd1) gene, a rice ortholog of Arabidopsis floral activator CONSTANS (CO), represses flowering under non-inductive long-day (LD) conditions and induces it under inductive short-day (SD) conditions. Here, we report biological interactions between Ghd7 and Hd1, which together repress Early heading date 1 (Ehd1), a key floral inducer under non-inductive LD conditions. In addition to this genetic interaction between them, Co-IP experiments further demonstrated that a Ghd7-Hd1 protein formed a complex in vivo and ChIP and luciferase reporter analyses suggested that this complex specifically binds to a cis-regulatory region in Ehd1 and represses its expression. These findings imply that Hd1, an evolutionally conserved transcriptional activator, can function as a strong transcriptional repressor within a monocot-specific flowering-time pathway through with Ghd7.

Organ-organ crosstalk is involved in homeostasis. Gastrointestinal symptoms are common in patients with renal failure. The aim of this study was to elucidate the relationship between gastrointestinal motility and gastrointestinal symptoms in chronic kidney disease. We performed studies in C57BL/6 mice with chronic kidney disease after 5/6 nephrectomy. Gastrointestinal motility was evaluated by assessing the ex vivo responses of ileum and distal colon strips to electrical field stimulation. Feces were collected from mice, and the composition of the gut microbiota was analyzed using 16S ribosomal RNA sequencing. Mice with chronic kidney disease after 5/6 nephrectomy showed a decreased amount of stool, and this constipation was correlated with a suppressed contraction response in ileum motility and decreased relaxation response in distal colon motility. Spermine, one of the uremic toxins, inhibited the contraction response in ileum motility, but four types of uremic toxins showed no effect on the relaxation response in distal colon motility. The 5/6 nephrectomy procedure disturbed the balance of the gut microbiota in the mice. The motility dysregulation and constipation were resolved by antibiotic treatments. The expression levels of interleukin 6, tumor necrosis factor-α, and iNOS in 5/6 nephrectomy mice were increased in the distal colon but not in the ileum. In addition, macrophage infiltration in 5/6 nephrectomy mice was increased in the distal colon but not in the ileum. We found that 5/6 nephrectomy altered gastrointestinal motility and caused constipation by changing the gut microbiota and causing colonic inflammation. These findings indicate that renal failure was remarkably associated with gastrointestinal dysregulation.

A Bengal tiger was presented for evaluation of weakness, ataxia and inappetance. Computed tomography (CT) and magnetic resonance imaging (MRI) revealed a mass extending from the T7-8 vertebral body to the left rib and compressing the spinal cord. On CT, the bone destruction and sequestrum were shown. On MRI, the multilobulated mass appeared hypo- to isointense in T1-weighted and hyperintense in T2-weighted images. The tiger died after imaging, most likely from renal failure. Chordoma without metastasis was diagnosed on necropsy. The imaging characteristics were similar to those found in chordoma in humans. This report describes the use of CT and MRI in an exotic species.

Stillbirth and neonatal mortality are significant problems in captive breeding of dolphins, however, the causes of these problems are not fully understood. Here, we report a case of meconium aspiration syndrome (MAS) in a male neonate of bottlenose dolphin (Tursiops truncates) who died immediately after birth. At necropsy, a true knot was found in the umbilical cord. The lungs showed diffuse intraalveolar edema, hyperemic congestion and atelectasis due to meconium aspiration with mild inflammatory cell infiltration. Although the exact cause of MAS in this case was unknown, fetal hypoxia due possibly to the umbilical knot might have been associated with MAS, which is the first report in dolphins. MAS due to perinatal asphyxia should be taken into account as a possible cause of neonatal mortality and stillbirth of dolphin calves.

A 9-year-old male Yellow-headed Amazon (Amazona oratrix) with a history of anorexia and vomiting died of a liver tumor. The tumor consisted of neoplastic cells with hepatocellular and cholangiocellular differentiations and their intermingled areas. Neoplastic hepatocytes showed islands or trabecular growth with vacuolated eosinophilic cytoplasm. Cells showing biliary differentiation formed ducts or tubules lined by cytokeratin AE1/AE3-positive epithelia, accompanied by desmoplasia consisting of myofibroblasts reacting to α-smooth muscle actin and desmin. The tumor was diagnosed as a combined hepatocellular-cholangiocarcinoma, which is very rare in the avian.

A 20-year-old Gentoo penguin was found dead with a clinical history of inappetence and dyspnoea. At necropsy, the lungs showed severe congestion/hemorrhage and atelectasis. Histopathologically, fibrosis was observed exclusively around parabronchi with severe collagen deposition. In fibrotic lesions, there were numerous depositions of crystalline structures accompanied by epithelioid cells and multinucleated giant cells (foreign body type). In addition to irregularly lamellar structures as the morphology, the crystals were demonstrated calcium oxalate (CaOx) by the Alizarin red S staining with and without polarized light and von Kossa's staining. Myocobacteria and fungi were not found by special and immuohistochemical stainings. Pulmonary dystrophic oxalosis is a very rare lesion in Gentoo penguin.

Low testosterone levels increase the risk for cardiovascular disease in men and lead to shorter life spans. Our recent study showed that androgen deprivation via castration altered fecal microbiota and exacerbated risk factors for cardiovascular disease, including obesity, impaired fasting glucose, excess hepatic triglyceride accumulation, and thigh muscle weight loss only in high-fat diet (HFD)-fed male mice. However, when mice were administered antibiotics that disrupted the gut microbiota, castration did not increase cardiovascular risks or decrease the ratio of dried feces to food intake. Here, we show that changes in cecal microbiota (e.g., an increased Firmicutes/Bacteroidetes ratio and number of Lactobacillus species) were consistent with changes in feces and that there was a decreased cecal content secondary to castration in HFD mice. Castration increased rectal body temperature and plasma adiponectin, irrespective of diet. Changes in the gut microbiome may provide novel insight into hypogonadism-induced cardiovascular diseases.

A 14-year-old female South African fur seal (Arctocephalus pusillus) was presented with a large skin mass on the right shoulder. At necropsy examination, multiple white nodules were found in the lungs, liver, spleen and right axillary lymph nodes. Histologically, the skin mass was composed of round to polygonal neoplastic cells with round to oval nuclei and variably sized cytoplasmic vacuoles. Cellular and nuclear atypia were prominent. Immunohistochemically, the neoplastic cells expressed vimentin, but not cytokeratins, S100 protein, adipophilin or desmin. The cytoplasmic lipid droplets stained positively with oil red O. Metastasis was seen in the lungs, liver, spleen and right axillary lymph nodes, with similar morphological features to the skin mass. Based on these findings, a diagnosis of a pleomorphic liposarcoma with systemic metastasis was made. No previous reports of metastatic liposarcomas have been published in marine mammals.

For many decades, researchers have focused on the self-sustainable oscillations of plant circadian clocks, which can only be observed under artificial constant environmental conditions. However, plants have evolved under natural diurnal conditions where several major environmental cues such as light, temperature and humidity are dramatically changing and interacting with each other. Therefore, little is known about the roles of the plant circadian clock in natural field conditions. Molecular genetic analyses in Arabidopsis thaliana have revealed that some core circadian clock genes are required for the establishment of robust circadian rhythms under artificial diurnal conditions, and that others function only as self-oscillators. However, it is largely unknown yet how those robust rhythms can be obtained under natural diurnal conditions. Recently, an extensive time-course transcriptome analysis of rice (Oryza sativa) leaves in natural field conditions revealed that OsGIGANTEA, the sole rice ortholog of the Arabidopsis GIGANTEA gene, governs the robust diurnal rhythm of rice leaf transcriptomes even under natural diurnal conditions; rice Osgi mutants exhibited severely defective transcriptome rhythms under strong diurnal changes in environmental cues. This review focuses on the physiological significance of the plant circadian clock in natural field conditions.

A 10-year-old female false killer whale (Pseudorca crassidens) developed skin lesions in the left breast fin. Histopathologically, the lesions consisted of multiple granulomas spread diffusely into the deep dermis and bone; characteristically, each granuloma had septate, branching fungal hyphae and chlamydospores surrounded by eosinophilic Splendore-Hoeppli materials. Macrophages, epithelioid cells and multinucleated giant cells in the granulomas reacted mainly to anti-SRA-E5 antibody against human macrophage scavenger receptor type I. Fusarium solani was isolated and its gene was detected from the skin samples. Mycotic skin lesions by Fusarium spp. reported so far in marine mammals were regarded as superficial dermatitis; therefore, the present case is very uncommon in that the lesions spread deeper into the skin.

A 19-year-old female Bengal tiger (Panthera tigris tigris) was presented with hind limb weakness, ataxia and respiratory distress. Computed tomography revealed a mass between the left side of the T7 vertebra and the base of the left 7th rib. The tiger then died, and necropsy was performed. Grossly, the vertebral mass was 6 × 5.7 × 3 cm, and invaded the adjacent vertebral bone and compressed the T7 spinal cord. Histologically, the mass was composed of large, clear, vacuolated and polygonal cells with osteochondral matrix. Cellular and nuclear atypia were moderate. The vacuolated cells stained positively for cytokeratin and vimentin and negatively for S-100. Based on these findings, the present case was diagnosed as a vertebral chordoma; the first report in a tiger.

Summary The temporal and spatial control of meristem identity is a key element in plant development. To better understand the molecular mechanisms that regulate inflorescence and flower architecture, we characterized the rice aberrant panicle organization 2 (apo2) mutant which exhibits small panicles with reduced number of primary branches due to the precocious formation of spikelet meristems. The apo2 mutants also display a shortened plastochron in the vegetative phase, late flowering, aberrant floral organ identities and loss of floral meristem determinacy. Map-based cloning revealed that 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).

Because plant reproductive development occurs only in adult plants, the juvenile-to-adult phase change is an indispensable part of the plant life cycle. We identified two allelic mutants, peter pan syndrome-1 (pps-1) and pps-2, that prolong the juvenile phase in rice (Oryza sativa) and showed that rice PPS is an ortholog of Arabidopsis thaliana CONSTITUTIVE PHOTOMORPHOGENIC1. The pps-1 mutant exhibits delayed expression of miR156 and miR172 and the suppression of GA biosynthetic genes, reducing the GA3 content in this mutant. In spite of its prolonged juvenile phase, the pps-1 mutant flowers early, and this is associated with derepression of RAP1B expression in pps-1 plants independently of the Hd1-Hd3a/RFT1 photoperiodic pathway. PPS is strongly expressed in the fourth and fifth leaves, suggesting that it regulates the onset of the adult phase downstream of MORI1 and upstream of miR156 and miR172. Its ability to regulate the vegetative phase change and the time of flowering suggests that rice PPS acquired novel functions during the evolution of rice/monocots.

The circadian clock controls physiological traits such as flowering time, photosynthesis, and growth in plants under laboratory conditions. Under natural field conditions, however, little is known about the significance of the circadian clock in plants. By time-course transcriptome analyses of rice (Oryza sativa) leaves, using a newly isolated rice circadian clock–related mutant carrying a null mutation in Os-GIGANTEA (Os-GI), we show here that Os-GI controlled 75% (false discovery rate = 0.05) of genes among 27,201 genes tested and was required for strong amplitudes and fine-tuning of the diurnal rhythm phases of global gene expression in the field. However, transcripts involved in primary metabolism were not greatly affected by osgi. Time-course metabolome analyses of leaves revealed no trends of change in primary metabolites in osgi plants, and net photosynthetic rates and grain yields were not affected. By contrast, some transcripts and metabolites in the phenylpropanoid metabolite pathway were consistently affected. Thus, net primary assimilation of rice was still robust in the face of such osgi mutation-related circadian clock defects in the field, unlike the case with defects caused by Arabidopsis thaliana toc1 and ztl mutations in the laboratory.

Flowering time is a major determinant for the local adaptation of crops. Hd1 is a key flowering-time gene in rice and is orthologous to the Arabidopsis CONSTANS gene. To elucidate the role of Hd1 in selection, we examined the Hd1 alleles of 60 landraces of Asian cultivated rice (Oryza sativa L.) originating from all regions of Asia, which comprised three cultivar groups, indica, japonica, and aus. The identified alleles were classified into four allele groups. The functional Hd1 alleles in allele groups I and II corresponded to indica and japonica, respectively. Non-functional alleles in these groups were not clearly associated with cultivar groups or locations. Allele groups III and IV corresponded to the aus cultivar group. The ancestry of each cultivar group was identified by the coalescent approach for Hd1 molecular evolution using the haplotype patterns of 14 regions over the 1.1 Mb chromosomal region surrounding Hd1 and the pSINE patterns of two loci, 1.4 and 4.4 Mb apart from Hd1. The haplotype patterns clearly revealed that Hd1 allele migration was caused by multiple and complex introgression events between cultivar groups. The Hd1 haplotypes among dozens of accessions of the wild species O. rufipogon were strongly divergent and only two of the haplotype clusters in O. rufipogon were closely related to those in cultivated rice. This strongly suggested that multiple introgression events have played an important role in the shaping and diversification of adaptation in addition to primary selection steps at the beginning of domestication.

A 10-year-old miniature sow was died, showing inappetence and weight loss. Grossly, neoplastic enlargement of the uterus was found. Histopathologically, the lesions consisted of acinar, ductular and cystic proliferations of mono- and multilayered epithelial cells; these cells reacted immunohistochemically strongly with three different cytokeratin antibodies, and occasionally to vimentin. Myofibroblastic desmoplastic cells, positive to alpha-smooth muscle actin, were present among neoplastic cells. Metastatic lesions were seen in the lungs and liver. Based on these findings, a diagnosis of uterine adenocarcinoma with marked desmoplasia was made. This case is the second report of uterine adenocarcinoma in the miniature pig.

Casein kinase II (CK2) is a protein kinase with an evolutionarily conserved function as a circadian clock component in several organisms, including the long-day plant Arabidopsis (Arabidopsis thaliana). The circadian clock component CIRCADIAN CLOCK ASSOCIATED1 (CCA1) is a CK2 target in Arabidopsis, where it influences photoperiodic flowering. In rice (Oryza sativa), a short-day plant, Heading date6 (Hd6) encodes a CK2α subunit that delays flowering time under long-day conditions. Here, we demonstrate that control of flowering time in rice by the Hd6 CK2α subunit requires a functional Hd1 gene (an Arabidopsis CONSTANS ortholog) and is independent of the circadian clock mechanism. Our findings from overexpressing the dominant-negative CK2 allele in rice support the independence of CK2 function from the circadian clock. This lack of control of the circadian clock by Hd6 CK2α might be due to the presence of glutamate in OsLHY (a CCA1 ortholog in rice) instead of the serine at the corresponding CK2 target site in CCA1. However, this glutamate is critical for the control of the OsPRR1 gene (a rice ortholog of the Arabidopsis TOC1/PRR1 gene) by OsLHY for regulation of the circadian clock. We also demonstrated that the other conserved CK2 target sites in OsLHY conferred robust rhythmic expression of OsLHY-LUC under diurnal conditions. These findings imply that the role of CK2 in flowering-time regulation in higher plants has diversified during evolution.

An adult female Aldabra giant tortoise (Geochelone gigantea) was found dead with a 2 month-history of decreased appetite. At necropsy, multiple ulcers were seen in the mucosa of stomach and colon. Histopathologically, the ulcers were characterized by extensive necrosis, hemorrhage, and marked edema, along with numerous chlamidospores and aseptate fungal hyphae. Fungal structures infiltrated into the lamina propria, submucosa and muscle layer; their invasion into blood vessels formed thrombosis. Immunohistochemically, the fungal structures were strongly positive with an antibody against Candida spp. This case is the first report on gastrointestinal candidiasis in an Aldabra giant tortoise.

Recent research into the flowering of rice (Oryza sativa) has revealed both unique and conserved genetic pathways in the photoperiodic control of flowering compared with those in Arabidopsis (Arabidopsis thaliana). We discovered an early heading date2 (ehd2) mutant that shows extremely late flowering under both short- and long-day conditions in line with a background deficient in Heading date1 (Hd1), a rice CONSTANS ortholog that belongs to the conserved pathway. This phenotype in the ehd2 mutants suggests that Ehd2 is pivotal for the floral transition in rice. Map-based cloning revealed that Ehd2 encodes a putative transcription factor with zinc finger motifs orthologous to the INDETERMINATE1 (ID1) gene, which promotes flowering in maize (Zea mays). Ehd2 mRNA in rice tissues accumulated most abundantly in developing leaves, but was present at very low levels around the shoot apex and in roots, patterns that are similar to those of ID1. To assign the position of Ehd2 within the flowering pathway of rice, we compared transcript levels of previously isolated flowering-time genes, such as Ehd1, a member of the unique pathway, Hd3a, and Rice FT-like1 (RFT1; rice florigens), between the wild-type plants and the ehd2 mutants. Severely reduced expression of these genes in ehd2 under both short- and long-day conditions suggests that Ehd2 acts as a flowering promoter mainly by up-regulating Ehd1 and by up-regulating the downstream Hd3a and RFT1 genes in the unique genetic network of photoperiodic flowering in rice.

Cypripedium macranthos var. rebunense is an endangered plant endemic to Rebun Island, Japan. A proper understanding of genetic diversity is needed when conducting conservation programs for rare and endangered species. We therefore examined the genetic diversity of C. macranthos var. rebunense using allozyme markers with a view to future conservation. Our study revealed that C. macranthos var. rebunense has relatively high genetic diversity (P was 0.62, na and ne were 1.85 and 1.28 respectively, and Ho and He were 0.163 and 0.187, respectively) when compared with other plant taxa. The natural habitats of C. macranthos var. rebunense are geographically separated into northern and the southern populations. Disappearance of alleles and increase in homozygosity expected as a result of the bottleneck effect were observed, particularly in the southern populations composed of a small number of plants. As additional negative effects (inbreeding depression and further genetic drift) due to fragmentation are predicted in these populations, the southern populations may show deterioration of genetic diversity in the near future.

The origin and spread of novel agronomic traits during crop domestication are complex events in plant evolution. Wild rice (Oryza rufipogon) has red grains due to the accumulation of proanthocyanidins, whereas most cultivated rice (Oryza sativa) varieties have white grains induced by a defective allele in the Rc basic helix-loop-helix (bHLH) gene. Although the events surrounding the origin and spread of black rice traits remain unknown, varieties with black grains due to anthocyanin accumulation are distributed in various locations throughout Asia. Here, we show that the black grain trait originated from ectopic expression of the Kala4 bHLH gene due to rearrangement in the promoter region. Both the Rc and Kala4 genes activate upstream flavonol biosynthesis genes, such as chalcone synthase and dihydroflavonol-4-reductase, and downstream genes, such as leucoanthocyanidin reductase and leucoanthocyanidin dioxygenase, to produce the respective specific pigments. Genome analysis of 21 black rice varieties as well as red- and white-grained landraces demonstrated that black rice arose in tropical japonica and its subsequent spread to the indica subspecies can be attributed to the causal alleles of Kala4. The relatively small size of genomic fragments of tropical japonica origin in some indica varieties indicates that refined introgression must have occurred by natural crossbreeding in the course of evolution of the black trait in rice.

Loss of seed shattering was a key event in the domestication of major cereals. We revealed that the qSH1 gene, a major quantitative trait locus of seed shattering in rice, encodes a BEL1-type homeobox gene and demonstrated that a single-nucleotide polymorphism (SNP) in the 5′ regulatory region of the qSH1 gene caused loss of seed shattering owing to the absence of abscission layer formation. Haplotype analysis and association analysis in various rice collections revealed that the SNP was highly associated with shattering among japonica subspecies of rice, implying that it was a target of artificial selection during rice domestication.

Huge advances in plant biology are possible now that we have the complete genome sequences of several flowering plants. Now, genomes can be comprehensively compared and map-based cloning can be performed more easily. Association study is emerging as a powerful method for the functional identification of genes and molecular genetics has begun to reveal the basis of plant diversity. Taking the flowering pathways as an example, we discuss the potential of several approaches to comparative biology.

To better understand the molecular mechanisms of the photoperiodic regulation of rice, a short-day plant, we isolated 27 cDNAs that were differentially expressed in the photoperiod-insensitive se5 mutant from approximately 8,400 independent mRNA species by the use of a fluorescent differential display (FDD). For this screening, we isolated mRNAs at five different time points during the night and compared their expression patterns between se5 and the wild type. Of 27 cDNAs isolated, 12 showed diurnal expression patterns often associated with genes involved in the determination of the flowering time. In se5, expression of nine cDNAs was increased. Five of these cDNAs were up-regulated under SD, suggesting that they may promote flowering under SD. They included genes encoding a cDNA containing a putative NAC domain, the fructose-bisphosphate aldolase, and a protease inhibitor. Expression of three cDNAs was decreased in se5 but not photoperiodically regulated. These cDNAs included a rice homolog of Arabidopsis GIGANTEA (GI), lir1, and a gene for myo-inositol 1-phosphate synthase, all of which were previously shown to be under the control of circadian clocks. The expression patterns of the rice homolog of GI, OsGI, were similar to those of the Arabidopsis GI, suggesting the conservation of some mechanisms for the photoperiodic regulation of flowering between these two species.

Plant circadian clocks that oscillate autonomously with a roughly 24-h period are entrained by fluctuating light and temperature and globally regulate downstream genes in the field. However, it remains unknown how punctual internal time produced by the circadian clock in the field is and how it is affected by environmental fluctuations due to weather or daylength. Using hundreds of samples of field-grown rice (Oryza sativa) leaves, we developed a statistical model for the expression of circadian clock-related genes integrating diurnally entrained circadian clock with phase setting by light, both responses to light and temperature gated by the circadian clock. We show that expression of individual genes was strongly affected by temperature. However, internal time estimated from expression of multiple genes, which may reflect transcriptional regulation of downstream genes, is punctual to 22 min and not affected by weather, daylength, or plant developmental age in the field. We also revealed perturbed progression of internal time under controlled environment or in a mutant of the circadian clock gene GIGANTEA. Thus, we demonstrated that the circadian clock is a regulatory network of multiple genes that retains accurate physical time of day by integrating the perturbations on individual genes under fluctuating environments in the field.

Summary The photoperiodic sensitivity 5 (se5) mutant of rice, a short-day plant, has a very early flowering phenotype and is completely deficient in photoperiodic response. We have cloned the SE5 gene by candidate cloning and demonstrated that it encodes a putative heme oxygenase. Lack of responses of coleoptile elongation by light pulses and photoreversible phytochromes in crude extracts of se5 indicate that SE5 may function in phytochrome chromophore biosynthesis. Ectopic expression of SE5 cDNA by the CaMV 35S promoter restored the photoperiodic response in the se5 mutant. Our results indicate that phytochromes confer the photoperiodic control of flowering in rice. Comparison of se5 with hy1, a counterpart mutant of Arabidopsis, suggests distinct roles of phytochromes in the photoperiodic control of flowering in these two species.

FLORICAULA ( FLO ) of Antirrhinum and LEAFY ( FLY ) of Arabidopsis regulate the formation of floral meristems. To examine whether same mechanisms control floral development in distantly related species such as grasses, we isolated RFL, FLO-LFY homolog of rice, and examined its expression and function. Northern analysis showed that RFL is expressed predominantly in very young panicle but not in mature florets, mature leaves, or roots. In situ hybridization revealed that RFL RNA was expressed in epidermal cells in young leaves at vegetative growth stage. After the transition to reproductive stage, RFL RNA was detected in all layers of very young panicle including the apical meristem, but absent in the incipient primary branches. As development of branches proceeds, RFL RNA accumulation localized in the developing branches except for the apical meristems of the branches and secondary branch primordia. Expression pattern of RFL raised a possibility that, unlike FLO and LFY, RFL might be involved in panicle branching. Transgenic Arabidopsis plants constitutively expressing RFL from the cauliflower mosaic virus 35S promoter were produced to test whether 35S- RFL would cause similar phenotype as observed in 35S- LFY plants. In 35S- RFL plants, transformation of inflorescence meristem to floral meristem was rarely observed. Instead, development of cotyledons, rosette leaves, petals, and stamens was severely affected, demonstrating that RFL function is distinct from that of LFY . Our results suggest that mechanisms controlling floral development in rice might be diverged from that of Arabidopsis and Antirrhinum .

The photoperiodic control of flowering time is essential for the adaptation of plants to variable environments and for successful reproduction. The identification of genes encoding florigens, which had been elusive but were supposedly synthesized in leaves and then transmitted to shoot apices to induce floral transitions, has greatly advanced our understanding of the photoperiodic regulation of flowering. Studies on the photoperiodism of Arabidopsis, a model long-day plant, revealed the molecular mechanisms regulating the expression of the Arabidopsis florigen gene FT, which is gradually induced in response to increase in day length. By contrast, in rice, a model short-day plant, the expression of the florigen gene Hd3a (an FT ortholog in rice) is regulated in an on/off fashion, with strong induction under short-day conditions and repression under long-day conditions. This critical day length dependence of Hd3a expression enables rice to recognize a slight change in the photoperiod as a trigger to initiate floral induction. Rice possesses a second florigen gene, RFT1, which can be expressed to induce floral transition under non-inductive long-day conditions. The complex transcriptional regulation of florigen genes and the resulting precise control over flowering time provides rice with the adaptability required for a crop species of increasing global importance.

Rice has been cultivated as a major crop for more than 7000 years, and it currently sustains more than half the world population. Rice and many other food plants are monocotyledons — such plants are of clear importance, and yet they are distinct from the dicotyledonous model plant Arabidopsis in many aspects of development. Recent advances in research on rice include efficient transformation, the creation of a highly saturated molecular map, and the large-scale analysis of expressed sequence tags. Indeed, the number of complementary DNAs analyzed in rice is approaching the number analyzed in Arabidopsis . Rice has reached the point where it can be usefully considered a model monocotyledonous plant.

Lacaziosis, formerly called lobomycosis, caused byLacazia loboi, is a zoonotic mycosis found in humans and dolphins and is endemic in the countries on the Atlantic Ocean. Although the Japanese coast is not considered an endemic area, photographic records of lacaziosis-like skin lesions were found in bottlenose dolphins (Tursiops truncatus) that were migrating in the Goto Islands (Nagasaki Prefecture, Japan). We diagnosed 2 cases of lacaziosis in bottlenose dolphins captured simultaneously at the same coast within Japanese territory on the basis of clinical characteristics, cytology, histopathology, immunological tests, and detection of partial sequences of a 43 kDa glycoprotein coding gene (gp43) with a nested-PCR system. The granulomatous skin lesions from the present cases were similar to those found in animals from endemic areas, containing multiple budding and chains of round yeast cells and positive in the immune-staining with anti-Paracoccidioides brasiliensisserum which is a fungal species related toL. loboi; however, thegp43gene sequences derived from the present cases showed 94.1% homology toP. brasiliensisand 84.1% toL. loboi. We confirmed that the causative agent at the present cases was different genotype ofL. loboifrom Amazon area.

We have recently isolated a rice circadian clock-related mutant carrying a null mutation in Os-GIGANTEA(GI) gene, the solo ortholog of Arabidopsis GI. Time-course global transcriptome analyses of leaves from wild-type and osgi mutant grown in the field have revealed that Os-GI affects gene expression of more than half of genes on rice 44k microarray. To better understand the biological significance of circadian clock function in growth and development of rice, we here investigated the gene expression involved in phytohormone biosynthesis. Here we found that mRNA levels of a few major genes encoding GA2-oxidase which can inactivate bioactive gibberellins (GAs) were remarkably increased in osgi-1 plants. This suggests that Os-GI functions to maintain bioactive GA level through the regulation of the GA-deactivating enzyme genes in rice. Consistently, osgi-1 plants showed semi-dwarf phenotype with reduced internode and leaf sheath elongation.

A case of cardiac hamartoma in a 2-month-old squirrel monkey is reported. The monkey showed a loss of appetite and died suddenly. Microscopically, an encapsulated nodular lesion was found at the right atrial wall. The lesion consisted of irregularly shaped, slender myocytes intermingled with a few fibroblasts and collagen fibers. Neither nuclear atypia nor inflammatory cell infiltrate was seen. The constituting cells had stratified striations in the cytoplasm and reacted immunohistochemically for desmin, indicating the nature of myocytes. Based on the above findings, a diagnosis of cardiac hamartoma was made. This is the first case of cardiac hamartoma in this species.

The adaptation of plants to natural environments depends on the adaptation of flowering-time control at the appropriate season to set seeds. Possible molecular mechanisms underlying this adaptation have recently been revealed. In Arabidopsis thaliana, a model long-day plant, control of floral transition by vernalization and long-day floral promotion pathways is a key regulator in adaptation to different regions. A floral repressor termed FLC and a floral promoter termed CONSTANS (CO), which control FT, a florigen gene, are key transcriptional regulators of these pathways. Recent analyses of haplotypes in accessions of A. thaliana revealed that FLC regulation by an activator termed FRIGIDA (FRI) had been a target for natural selection. By contrast, in rice (Oryza sativa), a model short-day plant, two independent floral pathways-Heading date 1 (Hd1, a CO orthologue)-dependent and Early heading date 1 (Ehd1)-dependent pathways-control Hd3a (an FT orthologue) and flowering time. Interestingly, there is an antagonistic action between Hd1 and Ehd1 in the control of flowering time under long-day conditions, because Hd1 represses floral transition whereas Ehd1 promotes it. A wild rice species, Oryza rufipogon, has common ancestry with cultivated rice and grows wild in the tropics, yet cultivated rice is grown even in the cold regions of northern latitudes. During domestication, the adaptation of O. sativa to northern regions by artificial selection may have become possible through interactions of the two pathways. These suggest that the domestication process of rice will provide novel insights into the adaptation of plants in evolution.

Flowering plants are largely categorized into short-day and long-day plants. In some species, no floral response to photoperiods is observed, which creates another group, day-neutral plants. In 1920, Garner and Allard reported that many flowering plants recognize the day-length to determine flowering-time and set seeds at appropriate seasons (see reviews by [Mouradov et al 2002], [Simpson and Dean 2002]). Recent studies in a short-day plant, rice, and a long-day plant, Ara- bidopsis thaliana, revealed that plants utilize an evolutionarily conserved flow- ering pathway to establish opposite photoperiodic responses (see reviews by [Izawa et al 2003], [Yanovsky and Kay 2003]). Here I summarize recent progress on molecular mechanisms of photoperiodic flowering to overlook genetic players at molecular levels to confer both short-day and long-day responses in photoperi- odic flowering.

Plants set seed at appropriate seasons. One major mechanism responsible for this adaptation involves photoperiodic flowering. Most plants are classified as either long-day plants, which flower under a longer photoperiod, or short-day plants, which flower under a shorter photoperiod. A third group, day-neutral plants, is not responsive to changes in photoperiod. During the past decade, molecular analysis has revealed at the molecular level how the long-day plant Arabidopsis thaliana measures daylength in photoperiodic flowering. In contrast, the molecular mechanisms underlying the responses of short-day plants are still under investigation. Progress in understanding photoperiodic flowering in rice (Oryza sativa), a short-day plant, revealed unique, evolutionarily conserved pathways involved in photoperiodic flowering at the molecular level. Furthermore, the conserved pathways promote flowering under short-day conditions and suppress flowering under long-day conditions in rice, but promote flowering under long-day conditions in Arabidopsis. In this chapter, we discuss the molecular mechanisms responsible for short-day flowering in rice in comparison with long-day flowering in Arabidopsis.

Two evolutionarily distant plant species, rice (Oryza sativa L.), a short-day (SD) plant, and Arabidopsis thaliana, a long-day plant, share a conserved genetic network controlling photoperiodic flowering. The orthologous floral regulators—rice Heading date 1 (Hd1) and Arabidopsis CONSTANS (CO)—integrate circadian clock and external light signals into mRNA expression of the FLOWERING LOCUS T (FT) group floral inducer. Here, we report that the rice Early heading date 1 (Ehd1) gene, which confers SD promotion of flowering in the absence of a functional allele of Hd1, encodes a B-type response regulator that might not have an ortholog in the Arabidopsis genome. Ehd1 mRNA was induced by 1-wk SD treatment, and Ehd1 may promote flowering by inducing FT-like gene expression only under SD conditions. Microarray analysis further revealed a few MADS box genes downstream of Ehd1. Our results indicate that a novel two-component signaling cascade is integrated into the conserved pathway in the photoperiodic control of flowering in rice.

Phytochromes confer the photoperiodic control of flowering in rice (Oryza sativa), a short-day plant. To better understand the molecular mechanisms of day-length recognition, we examined the interaction between phytochrome signals and circadian clocks in photoperiodic-flowering mutants of rice. Monitoring behaviors of circadian clocks revealed that phase setting of circadian clocks is not affected either under short-day (SD) or under long-day (LD) conditions in a phytochrome-deficient mutant that shows an early-flowering phenotype with no photoperiodic response. Non-24-hr-light/dark-cycle experiments revealed that a rice counterpart gene of Arabidopsis CONSTANS (CO), named PHOTOPERIOD SENSITIVITY 1(Heading date 1) [SE1 (Hd1)], functions as an output of circadian clocks. In addition, the phytochrome deficiency does not affect the diurnal mRNA expression of SE1 upon floral transition. Downstream floral switch genes were further identified with rice orthologs of Arabidopsis FLOWERING LOCUS T (FT). Our RT-PCR data indicate that phytochrome signals repress mRNA expression of FT orthologs, whereas SE1 can function to promote and suppress mRNA expression of the FT orthologs under SD and LD, respectively. This SE1 transcriptional activity may be posttranscriptionally regulated and may depend on the coincidence with Pfr phytochromes. We propose a model to explain how a short-day plant recognizes the day length in photoperiodic flowering.

To localize abscisic acid (ABA)-inducible gene expression of rab16 genes, rab16A promoter was linked to the gusA reporter gene encoding β-glucuronidase and introduced into rice (Oryza sativa L.) plants. The activity of rab16A promoter was induced by ABA and osmotic stresses in various tissues of vegetative and floral organs. In anthers and embryos, rab16A promoter was active in the absence of ABA. To elucidate cis-elements of the rab16 promoter that confer ABA-inducible expression, variously modified 40-bp fragments (-264 to -225) of the rab16B promoter were fused to a truncated (-46 bp) cauliflower mosaic virus 35S minimal promoter, and their activities in protoplasts were analyzed. The transient assays revealed that the 40-bp fragment consists of two separate ABA-responsive elements, motif I (AGTACGTGGC) and motif III (GCCGCGTGGC). Motif I and motif III are both required for ABA induction; however, each can substitute for the other. Further analyses of these motifs indicated that motif III has a distinct DNA sequence specificity as an ABA-responsive element from motif I, suggesting that the two motifs interact with different transcription factors in vivo.

The coat protein (CP) gene of rice stripe virus was introduced into two japonica varieties of rice by electroporation of protoplasts. The resultant transgenic plants expressed the CP at high levels (up to 0.5% of total soluble protein) and exhibited a significant level of resistance to virus infection. Plants derived from selfed progeny of the primary transformants also expressed the CP and showed viral resistance, indicating stable transmission of the CP gene and the trait of resistance to the next generation. Moreover, the virally encoded strip disease-specific protein was not detected in transgenic plants expressing CP 8 weeks after inoculation, indicating protection before viral multiplication. These studies demonstrated that CP-mediated resistance to virus infection can be extended to cereals and to the viruses transmitted by an insect vector (planthopper).

THE generation of transgenic plants using gene transfer techniques is important to both the investigation of gene regulation and the genetic engineering of crops1. The Ti plasmid of Agrobacterium tumefaciens is now routinely used to transform dicotyledonous plants2, and the transfer of foreign genes to unorganized tissue3–6and plants7,8 has been accomplished using direct DNA transfer methods9–11. A protocol for the easy and reproducible production of fertile transgenic cereals, however, has not yet been described. We report here the production of fertile transgenic rice plants obtained by introducing the bacterial hph gene, encoding hygromycin B resistance12 (Hmr), into protoplasts of Oryza sativa (L.) by electroporation. The non-selectable gene encoding β-glucuronidase was also transferred with the hph gene and its expression was detected in the progeny of the stable transformant.