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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.

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.

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.

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.

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.

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.

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.

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 .

Many plants flower in response to specific day lengths, which is crucial for successful reproduction, indicating that plants have developed sophisticated molecular mechanisms for day-length recognition. The external coincidence model proposes a crosstalk between the internal circadian clock and external light stimuli. This review focuses on recent progress in understanding the molecular mechanisms underlying the ‘gating effect’, by which any biological responses to environmental stimuli are restricted to certain phases of the circadian clock. For instance, in rice, two distinct predominant gating mechanisms recognise the critical day length. The gating mechanism for red light induces morning expression of the floral repressor Grain number, plant height and heading date7, but only under long-day conditions. The gating mechanism for blue light induces morning expression of the floral promoter Early heading date 1. Here, we summarise various gating mechanisms that recognise day length in several plant species, and also discuss the existence of nongating mechanisms involved in the photoperiodic control of flowering.

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.

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.

In order to analyze expression of the maize alcohol dehydrogenase 1 gene (Adh1), its promoter was fused with the gusA reporter gene and introduced into rice by protoplast transformation. Histochemical analysis of transgenic plants and their progeny showed that the maize Adh1 promoter is constitutively expressed in root caps, anthers, anther filaments, pollen, scutellum, endosperm and shoot and root meristem of the embryo. Induction of expression by the Adh1 promoter was examined using seedlings derived from selfed progeny of the transgenic plants. The results showed that expression of the Adh1 promoter was strongly induced (up to 81-fold) in roots of seedlings after 24 h of anaerobic treatment, concomitant with an increase in the level of gusA mRNA. 2,4-D also induced Adh1 promoter-directed expression of gusA to a similar extent. In contrast, little induction by anaerobic treatment was detected in transformed calli, leaves or roots of primary transformants or shoots of seedlings. A detailed examination of seedling roots during anaerobic treatment revealed that the induction started first at the meristem and after 3 h there was strong induction in the elongation zone which is located 1-2 mm above the meristem; the induction then progressed upward from this region. Our results suggest that transgenic rice plants carrying the gusA reporter gene fused with promoters are useful for the study of anaerobic regulation of genes derived from graminaceous species.

The domestication of crops involves a complex process of selection in plant evolution and is associated with changes in the DNA regulating agronomically important traits. Here we report the cloning of a newly identified QTL, qSW5 (QTL for seed width on chromosome 5), involved in the determination of grain width in rice. Through fine mapping, complementation testing and association analysis, we found that a deletion in qSW5 resulted in a significant increase in sink size owing to an increase in cell number in the outer glume of the rice flower; this trait might have been selected by ancient humans to increase yield of rice grains. In addition, we mapped two other defective functional nucleotide polymorphisms of rice domestication-related genes with genome-wide RFLP polymorphisms of various rice landraces. These analyses show that the qSW5 deletion had an important historical role in artificial selection, propagation of cultivation and natural crossings in rice domestication, and shed light on how the rice genome was domesticated.

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.

To develop a transposon tagging system in an important cereal plant, rice (Oryza sativa L.), the maize transposable element Ac (Activator) was introduced into rice protoplasts by electroporation. We employed a phenotypic assay for excision of Ac from the selectable hph gene encoding resistance to hygromycin B. Southern blot analysis of hygromycin B-resistant calli showed that the Ac element can transpose from the introduced hph gene into the rice chromosomes. Sequence analysis of several Ac excision sites in the hph genes revealed sequence alterations characteristic of the excision sites of this plant transposable element. The Ac element appears to be active during development of transgenic rice plants from calli. Moreover, hybridization patterns of different leaves from the same plant indicated that some Ac elements are stable whereas others are able to transpose further during development of leaves. The results indicate that the introduced Ac element can transpose efficiently in transgenic rice plants.

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.