Temperature-dependent sugar accumulation in interspecific Capsicum F1 plants showing hybrid weakness.

In plants, F1 hybrids showing hybrid weakness exhibit weaker growth than their parents. The phenotypes of hybrid weakness are often suppressed at certain temperatures. However, it is unclear whether hybrid weakness in Capsicum annuum × C. chinense is temperature-dependent or not. Our study showed that Capsicum hybrid weakness was suppressed at 30 and 35 °C and was induced at 15, 20, and 25 °C. Moreover, we investigated the time course of hybrid weakness in cell death, metabolite content, and gene expression in leaves of plants transferred to 20 °C after growing at 30 °C for 21 days. The expression of pathogen defense-related genes was upregulated at 1 day after transfer to 20 °C (DAT). Cell death was detected at 7 DAT, plant growth had almost stopped since 14 DAT, and sugars were accumulated at 42 DAT in hybrid plants. The study revealed that some sugar transporter genes, which had been upregulated since 7 DAT, were involved in sugar accumulation in Capsicum hybrid weakness. Thus, our results demonstrated that gene expression changes occur first, followed by physiological and morphological changes after induction of hybrid weakness. These responses observed in this study in Capsicum hybrid weakness are likely to be owed to plant defense responses-like reactions.

A hypersensitive response-like reaction is involved in hybrid weakness in F1 plants of the cross Capsicum annuum × Capsicum chinense.

Hybrid weakness in Capsicum is characterized by the termination of leaf differentiation after the development of several leaves. F1 plants in some crosses between Capsicum annuum and Capsicum chinense show weakness; this phenomenon has not been investigated in detail since first reported. In the present study, we characterized morphologically and physiologically hybrid weakness in Capsicum. F1 plants did not show weaker growth than their parents 20 days after germination (DAG), but at 40 DAG, the hybrid weakness phenotype was evidenced by almost complete arrest of new leaf formation, delayed increase in plant height, and reduced upper internode length. The shoot apical meristem (SAM) of F1 plants exhibited delayed development and an abnormal structure characterized by a flat shape and the presence of fuzzy cell layers on the surface. These abnormal SAMs of F1 plants may lead to dwarfism. Dead cells and accumulation of H2O2 were visually detected in leaves of F1 plants, and cell death was considered to be programmed, as it was accompanied by internucleosomal fragmentation of DNA. The expression of immunity marker genes PR1 and PR2 was upregulated in leaves of F1 plants. These results suggest that a hypersensitive response-like reaction is involved in Capsicum hybrid weakness.

Morphological changes during juvenile-to-adult phase transition in sorghum.

MAIN CONCLUSION: Morphological and genetic markers indicate that in sorghum, the juvenile-to-adult phase transition occurs during the fourth and fifth leaf stages. This timing differs from those reported for other plants. The juvenile-to-adult (JA) phase transition is an important event for optimizing vegetative growth and reproductive success in plants. Among the Poaceae crops, which are a vital food source for humans, studies of the JA phase transition have been restricted to rice and maize. We studied the morphological and genetic changes that occur during the early development of sorghum and found that dramatic changes occur in shoot architecture during the early vegetative stages. Changes were observed in leaf size, leaf shape, numbers of trichomes, and size of the shoot apical meristem. In particular, the length/width ratios of the leaf blades in the fifth and upper leaves were completely different from those of the second to fourth leaves. The fifth and upper leaves have trichomes on their adaxial sides, which were absent on the lower leaves. We also analyzed expression of two microRNAs that are known to be molecular markers of the JA phase transition and found that expression of miR156 was highest in the second to fourth leaves and then was gradually down-regulated, whereas miR172 expression followed the opposite pattern. These results suggest that in sorghum, the second and third leaves represent the juvenile phase, the fourth and fifth leaves are in the transition stage, and the sixth and upper leaves are in the adult phase. Thus, the JA phase transition occurs during the fourth and fifth leaf stages. These findings are expected to be useful for understanding the early development of sorghum.

Nicotiana suaveolens accessions with different ploidy levels exhibit different reproductive isolation mechanisms in interspecific crosses with Nicotiana tabacum.

Reproductive isolation, including prezygotic and postzygotic barriers, is a mechanism that separates species. Many species in the Nicotiana section Suaveolentes exhibit reproductive isolation in crosses with Nicotiana tabacum. In this study, we investigated whether the chromosome numbers and ploidy levels of eight Nicotiana suaveolens accessions are related to the reproductive isolation after crosses with N. tabacum by flow cytometry and chromosome analyses. Additionally, the internal transcribed spacer (ITS) regions of the eight N. suaveolens accessions were sequenced and compared with the previously reported sequences of 22 Suaveolentes species to elucidate the phylogenetic relationships in the section Suaveolentes. We revealed that four N. suaveolens accessions comprised 64 chromosomes, while the other four accessions carried 32 chromosomes. Depending on the ploidy levels of N. suaveolens, several types of reproductive isolation were observed after crosses with N. tabacum, including decreases in the number of capsules and the germination rates of hybrid seeds, as well as hybrid lethality and abscission of enlarged ovaries at 12-17 days after pollination. A phylogenetic analysis involving ITS sequences divided the eight N. suaveolens accessions into three distinct clades. Based on the results, we confirmed that N. suaveolens accessions vary regarding ploidy levels and reproductive isolation mechanisms in crosses with N. tabacum. These accessions will be very useful for revealing and characterizing the reproductive isolation mechanisms in interspecific crosses and their relationships with ploidy levels.

QTL-seq analysis identifies two genomic regions determining the heading date of foxtail millet, Setaria italica (L.) P.Beauv.

Heading date is an important event to ensure successful seed production. Although foxtail millet (Setaria italica (L.) P.Beauv.) is an important foodstuff in semiarid regions around the world, the genetic basis determining heading date is unclear. To identify genomic regions regulating days to heading (DTH), we conducted a QTL-seq analysis based on combining whole-genome re-sequencing and bulked-segregant analysis of an F2 population derived from crosses between the middle-heading cultivar Shinanotsubuhime and the early-heading cultivar Yuikogane. Under field conditions, transgressive segregation of DTH toward late heading was observed in the F2 population. We made three types of bulk samples: Y-bulk (early-heading), S-bulk (late-heading) and L-bulk (extremely late-heading). By genome-wide comparison of SNPs in the Y-bulk vs. the S-bulk and the Y-bulk vs. the L-bulk, we identified two QTLs associated with DTH. The first QTL, qDTH2, was detected on chromosome 2 from the Y-bulk and S-bulk comparison. The second QTL, qDTH7, was detected on chromosome 7 from the Y-bulk and L-bulk comparison. The Shinanotsubuhime allele for qDTH2 caused late heading in the F2 population, whereas the Yuikogane allele for qDTH7 led to extremely late heading. These results suggest that allelic differences in both qDTH2 and qDTH7 determine regional adaptability in S. italica.

Combining mapping of physiological quantitative trait loci and transcriptome for cold tolerance for counteracting male sterility induced by low temperatures during reproductive stage in rice.

Male sterility induced by low temperatures (LTs) during the reproductive stage is a major constraint for temperate zone rice. To detect physiological quantitative trait loci (QTLs), we modeled genotypic variation in the physiological processes involved in low temperature spikelet sterility on the basis of anther length (AL), a proxy for microspore and pollen grain number per anther. The model accounted for 83% of the genotypic variation in potential AL at normal temperature and the ability to maintain AL at LT. We tested the model on 208 recombinant inbred lines of cold-tolerant 'Tohoku-PL3' (PL3) × cold-sensitive 'Akihikari' (AH) for 2 years. QTLs for spikelet fertility (FRT) at LT were detected on chromosomes 5 (QTL for Cold Tolerance at Reproductive stage, qCTR5) and 12 (qCTR12). qCTR12 was annotated with the ability to maintain AL under LTs. qCTR5 was in a region shared with QTLs for culm length and heading date. Genome-wide expression analysis showed 798 genes differentially expressed in the spikelets between the parents at LTs. Of these, 12 were near qCTR5 and 23 were near qCTR12. Gene expression analysis confirmed two candidate genes for qCTR5 (O-methyltransferase ZRP4, Os05g0515600; beta-1,3-glucanase-like protein, Os05g0535100) and one for qCTR12 (conserved hypothetical protein, Os12g0550600). Nucleotide polymorphisms (21 deletions, 2 insertions and 10 single nucleotide polymorphisms) in PL3 were found near the candidate conserved hypothetical protein (Os12g0550600) and upstream in PL3, but not in AH. Haplotype analysis revealed that this gene came from 'Kuchum'. The combination of mapping physiological QTLs with gene expression analysis can be extended to identify other genes for abiotic stress response in cereals.

Identification of loci associated with embryo yield in microspore culture of Brassica rapa by segregation distortion analysis.

KEY MESSAGE: We identified three physical positions associated with embryo yield in microspore culture of Brassica rapa by segregation distortion analysis. We also confirmed their genetic effects on the embryo yield. Isolated microspore culture is well utilized for the production of haploid or doubled-haploid plants in Brassica crops. Brassica rapa cv. 'Ho Mei' is one of the most excellent cultivars in embryo yield of microspore culture. To identify the loci associated with microspore embryogenesis, segregation analysis of 154 DNA markers anchored to B. rapa chromosomes (A01-A10) was performed using a population of microspore-derived embryos obtained from an F1 hybrid between 'CR-Seiga', a low yield cultivar in microspore-derived embryos, and 'Ho Mei'. Three regions showing significant segregation distortion with increasing 'Ho Mei' alleles were detected on A05, A08 and A09, although these regions showed the expected Mendelian segregation ratio in an F2 population. The additive effect of alleles in these regions on embryo yield was confirmed in a BC3F1 population. One region on A08 containing Br071-5c had a higher effect than the other regions. Polymorphism of nucleotide sequences around the Br071-5c locus was investigated to find the gene possibly responsible for efficient embryogenesis from microspores.

Change of shoot architecture during juvenile-to-adult phase transition in soybean.

Juvenile-to-adult phase change is an indispensable event which guarantees a successful life cycle. Phase change has been studied in maize, Arabidopsis and rice, but is mostly unknown in other species. Soybean/Fabaceae plants undergo drastic changes of shoot architecture at the early vegetative stage including phyllotactic change and leaf type alteration from simple to compound. These characteristics make soybean/Fabaceae plants an interesting taxon for investigating vegetative phase change. Following the expansion of two cotyledons, two simple leaves simultaneously emerge in opposite phyllotaxy. The phyllotaxy of the third and fourth leaves is not fixed; both opposite and distichous phyllotaxis are observed within the same population. Leaves were compound from the third leaf. But the third leaf was rarely simple. Morphological and quantitative changes in early vegetative phase were recognized in leaf size, leaf shape, number of trichomes, stipule size and shape, and shoot meristem shape. Two microRNA genes, miR156 and miR172, are known to be associated with vegetative phase change. Examination of the expression level revealed that miR156 expression was high in the first two leaves and subsequently down-regulated, and that of miR172 showed the inverse expression pattern. These expression patterns coincided with the case of other species. Taken all data together, the first and second leaves represent juvenile phase, the fifth and upper leaves adult phase, and the third and fourth leaves intermediate stage. Further investigation of soybean phase change would give fruitful understandings on plant development.

Efficient haploid and doubled haploid production from unfertilized ovule culture of gentians (Gentiana spp.).

Factors affecting reliable plant regeneration from unfertilized ovule culture of gentians (Gentiana spp.) were examined. Cold pretreatment (4°C) of flower buds enhanced or maintained production of embryo-like structure (ELS). When 43 genotypes were surveyed in two different labs, 40 of them produced ELSs ranging from 0.01 to 26.5 ELSs per flower bud. No ELSs could be obtained in three genotypes. A significant correlation (r = 0.64) was observed between the number of ELS per flower and the frequency of responding flower buds. Eight genotypes of G. triflora, which were used as common materials in two different labs, produced ELSs in both labs. The ploidy levels of a total of 1,515 regenerated plantlets were determined, revealing that the majority of these plants consisted of haploids (57.9%) and diploids (34.3%). However, the frequency of haploids and diploids was different between G. triflora and G. scabra, and G. triflora showed higher frequencies of haploids than G. scabra. When haploids were treated with oryzalin for chromosome doubling, diploids and tetraploids were obtained. These results demonstrate that the unfertilized ovule culture technique of gentians is a powerful tool for obtaining haploids and DHs because of its reproducible and reliable nature and application to a wide range of genotypes.

A gene network for long-day flowering activates RFT1 encoding a mobile flowering signal in rice.

Although some genes that encode sensory or regulatory elements for photoperiodic flowering are conserved between the long-day (LD) plant Arabidopsis thaliana and the short-day (SD) plant rice, the gene networks that control rice flowering, and particularly flowering under LD conditions, are not well understood. We show here that RICE FLOWERING LOCUS T 1 (RFT1), the closest homolog to Heading date 3a (Hd3a), is a major floral activator under LD conditions. An RFT1:GFP fusion protein localized in the shoot apical meristem (SAM) under LD conditions, suggesting that RFT1 is a florigen gene in rice. Furthermore, mutants in OsMADS50, a rice ortholog of Arabidopsis SUPPRESOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) did not flower up to 300 days after sowing under LD conditions, indicating that OsMADS50, which acts upstream of RFT1, promotes flowering under LD conditions. We propose that both positive (OsMADS50 and Ehd1) and negative (Hd1, phyB and Ghd7) regulators of RFT1 form a gene network that regulates LD flowering in rice. Among these regulators, Ehd1, a rice-specific floral inducer, integrates multiple pathways to regulate RFT1, leading to flowering under appropriate photoperiod conditions. A rice ortholog of Arabidopsis APETALA1, OsMADS14, was expressed in the floral meristem in wild-type but not in RFT1 RNAi plants, suggesting that OsMADS14 is activated by RFT1 protein in the SAM after the transition to flowering. We have thus exposed a network of genes that regulate LD flowering in rice.

Variations in Hd1 proteins, Hd3a promoters, and Ehd1 expression levels contribute to diversity of flowering time in cultivated rice.

Rice is a facultative short-day plant, and molecular genetic studies have identified the major genes involved in short-day flowering. However, the molecular mechanisms promoting the diversity of flowering time in cultivated rice are not known. We used a core collection of 64 rice cultivars that represent the genetic diversity of 332 accessions from around the world and studied the expression levels and polymorphisms of 6 genes in the short-day flowering pathway. The RNA levels of Heading date 3a (Hd3a), encoding a floral activator, are highly correlated with flowering time, and there is a high degree of polymorphism in the Heading date 1 (Hd1) protein, which is a major regulator of Hd3a expression. Functional and nonfunctional alleles of Hd1 are associated with early and late flowering, respectively, suggesting that Hd1 is a major determinant of variation in flowering time of cultivated rice. We also found that the type of Hd3a promoter and the level of Ehd1 expression contribute to the diversity in flowering time and Hd3a expression level. We evaluated the contributions of these 3 factors by a statistical analysis using a simple linear model, and the results supported our experimental observations.

Parental Genome Imbalance Causes Hybrid Seed Lethality as Well as Ovary Abscission in Interspecific and Interploidy Crosses in Nicotiana.

Enhanced ovary abscission after pollination and hybrid seed lethality result in post-zygotic reproductive isolation in plant interspecific crosses. However, the connection between these barriers remains unclear. Here, we report that an imbalance in parental genomes or endosperm balance number (EBN) causes hybrid seed lethality and ovary abscission in both interspecific and intraspecific-interploidy crosses in the genus Nicotiana. Auxin treatment suppressed ovary abscission, but not hybrid seed lethality, in an interspecific cross between Nicotiana suaveolens and N. tabacum, suggesting that ovary abscission-related genes are located downstream of those involved in hybrid seed lethality. We performed interploidy crosses among N. suaveolens tetraploids, octoploids, and neopolyploids and revealed hybrid seed lethality and ovary abscission in interploid crosses. Furthermore, a higher maternal EBN than paternal EBN caused these barriers, as previously observed in N. suaveolens × N. tabacum crosses. Altogether, these results suggest that maternal excess of EBN causes hybrid seed lethality, which in turn leads to ovary abscission through the same mechanism in both interspecific and interploidy crosses.

Capsicum annuum with causal allele of hybrid weakness is prevalent in Asia.

Reproductive isolation, including hybrid weakness, plays an important role in the formation of species. Hybrid weakness in Capsicum, the cessation of plant growth, is caused by two complementary dominant genes, A from C. chinense or C. frutescens and B from C. annuum. In the present study, we surveyed whether 94 C. annuum accessions had B or b alleles by crossing with C. chinense having the A allele. Of the 94 C. annuum accessions, five had the B allele, three of which were native to Latin America and two were native to Asia. When combined with previous studies, the percentage of B carriers was 41% in Japan, 13% in Asia excluding Japan, 6% in Latin America, and 0% in Europe and Africa. In addition, 48 accessions of C. annuum from various countries were subjected to SSR analysis. Clades with high percentages of B-carriers were formed in the phylogenetic trees. In the principal coordinate analysis, most B-carriers were localized in a single group, although the group also included b-carriers. Based on these results, we presumed that the B allele was acquired in some C. annuum lines in Latin America, and B-carriers were introduced to the world during the Age of Discovery, as along with the b-carriers.

Phytochrome B regulates Heading date 1 (Hd1)-mediated expression of rice florigen Hd3a and critical day length in rice.

Many plants require circadian clock and light information for the photoperiodic control of flowering. In Arabidopsis, a long-day plant (LDP), flowering is triggered by the circadian clock-controlled expression of CONSTANS (CO) and light stabilization of the CO protein to induce FT (FLOWERING LOCUS T). In rice, a short-day plant (SDP), the CO ortholog Heading date 1 (Hd1) regulates FT ortholog Hd3a, but regulation of Hd3a by Hd1 differs from that in Arabidopsis. Here, we report that phytochrome B (phyB)-mediated suppression of Hd3a is a primary cause of long-day suppression of flowering in rice, based on the three complementary discoveries. First, overexpression of Hd1 causes a delay in flowering under SD conditions and this effect requires phyB, suggesting that light modulates Hd1 control of Hd3a transcription. Second, a single extension of day length decreases Hd3a expression proportionately with the length of daylight. Third, Hd1 protein levels in Hd1-overexpressing plants are not altered in the presence of light. These results also suggest that phyB-mediated suppression of Hd3a expression is a component of the molecular mechanism for critical day length in rice.

Gynogenesis in gentians (Gentiana triflora, G. scabra): production of haploids and doubled haploids.

Gynogenesis was investigated on gentian (Gentiana triflora, G. scabra and their hybrids), which is an important ornamental flower. When unfertilized ovules were cultured in 1/2 NLN medium containing a high concentration of sucrose (100 g/l), embryo-like structures (ELS) were induced. Although genotypic variation was observed in ELS induction, all four genotypes produced ELSs ranging from 0.93 to 0.04 ELSs per flower bud. The ovules collected from flower buds of later stages (just before anthesis or flower anthesis) tended to exhibit higher response. The dark culture condition produced more than four times as many ELSs than in 16-h light condition. A significant number of plantlets were directly regenerated from ELSs on MS regeneration medium. The ploidy levels of 179 regenerated plants were determined by flow cytometry, revealing that the majority of them were diploid (55.9%) and haploid (31.3%). When a total of 54 diploid plants were examined by molecular genetic markers, 52 (96.3%) were considered as doubled haploids (DHs). This is the first report showing successful gynogenesis in gentian. The production of haploids and DHs by unfertilized ovule culture opens a novel prospect in gentian F1 hybrid breeding.

Genetic Mapping of the HLA1 Locus Causing Hybrid Lethality in Nicotiana Interspecific Hybrids.

Hybrid lethality, a postzygotic mechanism of reproductive isolation, is a phenomenon that causes the death of F1 hybrid seedlings. Hybrid lethality is generally caused by the epistatic interaction of two or more loci. In the genus Nicotiana, N. debneyi has the dominant allele Hla1-1 at the HLA1 locus that causes hybrid lethality in F1 hybrid seedlings by interaction with N. tabacum allele(s). Here, we mapped the HLA1 locus using the F2 population segregating for the Hla1-1 allele derived from the interspecific cross between N. debneyi and N. fragrans. To map HLA1, several DNA markers including random amplified polymorphic DNA, amplified fragment length polymorphism, and simple sequence repeat markers, were used. Additionally, DNA markers were developed based on disease resistance gene homologs identified from the genome sequence of N. benthamiana. Linkage analysis revealed that HLA1 was located between two cleaved amplified polymorphic sequence markers Nb14-CAPS and NbRGH1-CAPS at a distance of 10.8 and 10.9 cM, respectively. The distance between these markers was equivalent to a 682 kb interval in the genome sequence of N. benthamiana.

Two Nicotiana occidentalis accessions enable gene identification for Type II hybrid lethality by the cross to N. sylvestris.

Hybrid lethality, meaning the death of F1 hybrid seedlings, has been observed in many plant species, including Nicotiana. Previously, we have revealed that hybrids of the selected Nicotiana occidentalis accession and N. tabacum, an allotetraploid with S and T genomes, exhibited lethality characterized by the fading of shoot color. The lethality was suggested to be controlled by alleles of loci on the S and T genomes derived from N. sylvestris and N. tomentosiformis, respectively. Here, we extended the analysis of hybrid lethality using other two accessions of N. occidentalis identified from the five tested accessions. The two accessions were crossed with N. tabacum and its two progenitors, N. sylvestris and N. tomentosiformis. After crosses with N. tabacum, the two N. occidentalis accessions yielded inviable hybrid seedlings whose lethality was characterized by the fading of shoot color, but only the T genome of N. tabacum was responsible for hybrid lethality. Genetic analysis indicated that first-mentioned N. occidentalis accession carries a single gene causing hybrid lethality by allelic interaction with the S genome.

Arabidopsis EMBRYOMAKER encoding an AP2 domain transcription factor plays a key role in developmental change from vegetative to embryonic phase.

Although several types of plant cells retain the competence to enter into embryonic development without fertilization, the molecular mechanism(s) underlying ectopic embryogenesis is largely unknown. To gain insight into this mechanism, in a previous study we identified 136 ESTs specifically expressed in microspore embryogenesis of Brassica napus. Here, we describe the characterization of the Arabidopsis EMBRYOMAKER (EMK) gene, which is homologous to one of the identified Brassica ESTs (BnGemb-58) and encodes an AP2 domain transcription factor. The AtEMK was expressed in developing and mature embryos, but its rapid disappearance occurred during germination. After germination, the expression of AtEMK was found in the root apical meristem and the distal parts of cotyledons. Although a mutant lacking AtEMK exhibited no distinctive defects in the embryo, ectopic expression of AtEMK induced embryo-like structures from cotyledons. The embryo-like structures contained high concentration of lipids, expressed several embryo-specific genes, and could convert into independent plants, indicating that the structures are somatic embryos. In vitro culture, AtEMK enhanced the efficiency of somatic embryogenesis. Furthermore, ectopic expression of AtEMK caused the formation of trichomes on cotyledons, dedifferentiated several tissues into calli, and retarded root development, demonstrating that AtEMK is harmful for the normal development of plants after germination. From these results, we conclude that the AtEMK is a key player to maintain embryonic identity, and the rapid disappearance of AtEMK expression during germination is essential for the developmental transition between the embryonic and vegetative phases in plants.

A high maternal genome excess causes severe seed abortion leading to ovary abscission in Nicotiana interploidy-interspecific crosses.

Seed abortion and ovary abscission, two types of postzygotic reproductive barriers, are often observed in interspecific and/or interploidy crosses in plants. However, the mechanisms underlying these reproductive barriers remain unclear. Here, we show that the distinct types of seed developmental abnormalities (type I and type II seed abortion) occur in a phased manner as maternal to paternal genome dosage increases and that type II seed abortion is followed by ovary abscission. We revealed that these two types of seed developmental abnormalities are observed during seed development in the interploidy-interspecific crosses of Nicotiana suaveolens and N. tabacum. Moreover, in the cross showing type II seed abortion, several events, such as changes in abscission-related gene expression and lignin deposition, occurred in the ovary abscission zone, eventually leading to ovary abscission. Notably, successive increases in maternal ploidy using ploidy manipulated lines resulted in successive type I and type II seed abortions, and the latter was accompanied by ovary abscission. Conversely, both types of seed abortion and ovary abscission could be overcome with a ploidy manipulation technique that balances parental ploidy levels. We thus concluded that a high maternal genome excess cross may cause severe seed developmental defects and ovary abscission. Based on our findings, we propose a model explaining the abortion phenomena, where an interaction between the promotive and inhibitive effects of the parental genomes determines the developmental destiny of seeds. SIGNIFICANCE STATEMENT: We demonstrate that a stepwise increase in maternal ploidy results in a stepwise increase in seed abortion severity, leading to ovary abscission in plants. We propose a model explaining the abortion phenomena, where an interaction between the promotive and inhibitive effects of the parental genomes determines the developmental destiny of seeds.

Identification of dynamin as an interactor of rice GIGANTEA by tandem affinity purification (TAP).

GIGANTEA (GI), CONSTANS (CO) and FLOWERING LOCUS T (FT) regulate photoperiodic flowering in Arabidopsis. In rice, OsGI, Hd1 and Hd3a were identified as orthologs of GI, CO and FT, respectively, and are also important regulators of flowering. Although GI has roles in both flowering and the circadian clock, our understanding of its biochemical functions is still limited. In this study, we purified novel OsGI-interacting proteins by using the tandem affinity purification (TAP) method. The TAP method has been used effectively in a number of model species to isolate proteins that interact with proteins of interest. However, in plants, the TAP method has been used in only a few studies, and no novel proteins have previously been isolated by this method. We generated transgenic rice plants and cell cultures expressing a TAP-tagged version of OsGI. After a two-step purification procedure, the interacting proteins were analyzed by mass spectrometry. Seven proteins, including dynamin, were identified as OsGI-interacting proteins. The interaction of OsGI with dynamin was verified by co-immunoprecipitation using a myc-tagged version of OsGI. Moreover, an analysis of Arabidopsis dynamin mutants indicated that although the flowering times of the mutants were not different from those of wild-type plants, an aerial rosette phenotype was observed in the mutants. We also found that OsGI is present in both the nucleus and the cytosol by Western blot analysis and by transient assays. These results indicate that the TAP method is effective for the isolation of novel proteins that interact with target proteins in plants.