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MFTis an important regulator of seed dormancy in flowering plants. A natural mutation in the A-box motif in the promoter of wheat (Triticum aestivum) MFT2on chromosome 3A (TaMFT2-3A) has been used to prevent pre-harvest sprouting (PHS) in wheat cultivars in East Asia. Previous research usingin situhybridization showed thatTaMFT2-3Ais primarily expressed in the seed scutellum. In this study, we analyzed the localization of transcripts and encoded proteins for theTaMFT2homologs from barley (Hordeum vulgare) and rice (Oryza sativa), HvMFT2andOsMFT2, respectively. RNAin situhybridization showed that, like the wheat genes, the rice and barley homologs are primarily expressed in the scutellum, indicating that these threeMFT2genes have a common expression pattern during seed development. Analysis of thecis-acting regulatory elements of their promoter sequences showed that the threeMFT2promoters share eight seed-specificcis-acting RY motifs, which are binding sites for B3-domain transcription factors of the AFL-B3 and VIVIPAROUS1/ABI3-LIKE (VAL) families. In addition, we detected tandemly repeated and partially overlapping A-box motifs in the promoters ofHvMFT2, TaMFT2-3B, andTaMFT2-3D, possibly explaining why the natural allele ofTaMFT2-3Ahas been employed in breeding. We generated transgenic rice plants expressing nuclear-localized green fluorescent protein (NLS-2xGFP) and OsMFT2-GFP under the control of a 3-kb fragment of theOsMFT2promoter. Immunohistochemical staining using anti-GFP antibodies mainly detected GFP in the scutellum and scutellar epithelium, which is an important tissue for initiating germination upon seed hydration. We confirmed these results by confocal microscopy of GFP fluorescence in seeds. Our results suggest thatMFT2expression in rice, barley, and wheat might be regulated by a similar network of transcription factors through multi-RY motifs in theMFT2promoters, with possible roles in scutellar epithelium development.

To clarify the genetic mechanisms of fertility restoration in sorghum F1 hybrids produced in Japan ('Ryokuryu', 'Hazuki', 'Haretaka', 'Natsuibuki', 'Hanaaoba', 'Akidachi' and 'Kazetachi'), we analyzed QTLs for fertility restoration using seven F2 populations derived from those hybrids. By QTL mapping with a series of SSR markers, we detected three major QTLs for fertility restoration. These data and the results of haplotype analysis of known fertility restorer (Rf) genes showed that qRf5, corresponding to the Rf5 locus, was the most widely used Rf gene for fertility restoration of sorghum F1 hybrids among the lines tested. Other major Rf genes detected were qRf8, corresponding to Rf1, and qRf2, corresponding to Rf2. QTLs for grain weight also corresponded to these Rf loci. A minor QTL, qRf3, may also affect restoration of fertility. Our data show that three major Rfs-Rf1, Rf2, and Rf5-were used in F1 hybrid sorghum production in Japan. This knowledge can be used to improve the efficiency of the F1 sorghum breeding program.

Pith parenchyma cells store water in various plant organs. These cells are especially important for producing sugar and ethanol from the sugar juice of grass stems. In many plants, the death of pith parenchyma cells reduces their stem water content. Previous studies proposed that a hypothetical D gene might be responsible for the death of stem pith parenchyma cells in Sorghum bicolor, a promising energy grass, although its identity and molecular function are unknown. Here, we identify the D gene and note that it is located on chromosome 6 in agreement with previous predictions. Sorghum varieties with a functional D allele had stems enriched with dry, dead pith parenchyma cells, whereas those with each of six independent nonfunctional D alleles had stems enriched with juicy, living pith parenchyma cells. D expression was spatiotemporally coupled with the appearance of dead, air-filled pith parenchyma cells in sorghum stems. Among D homologs that are present in flowering plants, Arabidopsis ANAC074 also is required for the death of stem pith parenchyma cells. D and ANAC074 encode previously uncharacterized NAC transcription factors and are sufficient to ectopically induce programmed death of Arabidopsis culture cells via the activation of autolytic enzymes. Taken together, these results indicate that D and its Arabidopsis ortholog, ANAC074, are master transcriptional switches that induce programmed death of stem pith parenchyma cells. Thus, targeting the D gene will provide an approach to breeding crops for sugar and ethanol production.

To clarify the genetic mechanisms of fertility restoration in sorghum F1 hybrids produced in Japan (‘Ryokuryu’, ‘Hazuki’, ‘Haretaka’, ‘Natsuibuki’, ‘Hanaaoba’, ‘Akidachi’ and ‘Kazetachi’), we analyzed QTLs for fertility restoration using seven F2 populations derived from those hybrids. By QTL mapping with a series of SSR markers, we detected three major QTLs for fertility restoration. These data and the results of haplotype analysis of known fertility restorer (Rf) genes showed that qRf5, corresponding to the Rf5 locus, was the most widely used Rf gene for fertility restoration of sorghum F1 hybrids among the lines tested. Other major Rf genes detected were qRf8, corresponding to Rf1, and qRf2, corresponding to Rf2. QTLs for grain weight also corresponded to these Rf loci. A minor QTL, qRf3, may also affect restoration of fertility. Our data show that three major Rfs—Rf1, Rf2, and Rf5—were used in F1 hybrid sorghum production in Japan. This knowledge can be used to improve the efficiency of the F1 sorghum breeding program.

Sorghum [Sorghum bicolor (L.) Moench] pigments are associated with resistance to leaf diseases and grain deterioration. As a defence response, tan sorghum produces flavones (apigenin and luteolin), whereas wild-type sorghum produces additional 3-deoxyanthocyanidins (apigeninidin and luteolinidin). The dominant leaf colour gene P (Sb06g029550) controls 3-deoxyanthocyanidin production; the loss of its function results in tan leaves. We investigated the polymorphism of the P gene alleles and found that 11 of 327 lines from sorghum core collections had tan phenotype due to the loss of Sb06g029550 function. Six alleles (with insertions 1–4, a deletion and a point mutation) were found in tan sorghum, most of them in African landraces. This distribution suggests that the tan phenotype arose independently in different geographical locations and was selected several times during sorghum domestication. As plants with white seed without tannin often have tan leaves, the polymorphism of the transcription factor genes Y1 (for white seeds) and Tan1 (for seed tannin) was analysed. The grain coloration was affected by these transcription factors as previously reported but the leaf coloration by wounding was not affected by them.

Upon wounding or pathogen invasion, leaves of sorghum [Sorghum bicolor (L.) Moench] plants with the P gene turn purple, whereas leaves with the recessive allele turn brown or tan. This purple phenotype is determined by the production of two 3-deoxyanthocyanidins, apigeninidin and luteolinidin, which are not produced by the tan-phenotype plants. Using map-based cloning in progeny from a cross between purple Nakei-MS3B (PP) and tan Greenleaf (pp) cultivars, we isolated this gene, which was located in a 27-kb genomic region around the 58.1 Mb position on chromosome 6. Four candidate genes identified in this region were similar to the maize leucoanthocyanidin reductase gene. None of them was expressed before wounding, and only the Sb06g029550 gene was induced in both cultivars after wounding. The Sb06g029550 protein was detected in Nakei-MS3B, but only slightly in Greenleaf, in which it may be unstable because of a Cys252Tyr substitution. A recombinant Sb06g029550 protein had a specific flavanone 4-reductase activity, and converted flavanones (naringenin or eriodictyol) to flavan-4-ols (apiforol or luteoforol) in vitro. Our data indicate that the Sb06g029550 gene is involved in the 3-deoxyanthocyanidin synthesis pathway.

A loss of granule-bound starch synthase I (GBSS I) activity results in starch granules that contain mostly amylopectin and little or no amylose, a phenotype described as waxy. Previously, two phenotypic classes of waxy alleles, wxa, associated with no detectable GBSS I, and wxb, associated with apparently inactive GBSS I in the endosperm, were reported in sorghum (Sorghum bicolor (L.) Moench). In this study, the waxy alleles in a sorghum core collection were investigated using DNA markers. Of the 337 sorghum accessions examined, 17 accessions that were confirmed to be waxy by a negative iodine staining result and 16 were found to be wxa. A novel waxy allele, wxc, was found in a Taiwanese landrace. This allele consists of a +1G to C mutation in the 5′ splice site at the intron 10–exon 11 boundary, a mutation that most likely resulted in the suppression of GBSS I gene expression. A DNA marker specific for wxc was produced to distinguish the wxc allele from other alleles, allowing the identification of heterozygous non-waxy plants.

Sorghum accumulates sugars (sucrose, glucose and fructose) in the stem after anthesis. Brix is commonly used to indicate total sugar content; however, the relationship between Brix and specific sugar components has not been sufficiently investigated in sorghum juice in Japan. In this study, we measured the sugar components of sorghum juices from 109 varieties using capillary electrophoresis, which can quantify each sugar component in crude juice without further purification. The results indicated that the Brix of sorghum juice was proportional to the total sugar and sucrose concentrations (r = 0.900 and r = 0.894, P

We have developed a high-frequency method for Agrobacterium-mediated gene targeting by combining an efficient transformation system using rice suspension-cultured calli and a positive/negative selection system. Compared with the conventional transformation system using calli on solid medium, transformation using suspension-cultured calli resulted in a 5- to 10-fold increase in the number of resistant calli per weight of starting material after positive/negative selection. Homologous recombination occurred in about 1.5% of the positive/negative selected calli. To evaluate the efficacy of our method, we show in this report that knockout rice plants containing either a disrupted Waxy (granule-bound starch synthase) or a disrupted Xyl (β1,2-xylosyltransferase) gene can be easily obtained by homologous recombination. Study of gene function using homologous recombination in higher plants can now be considered routine work as a direct result of this technical advance.

HvCO9 was characterized to elucidate the barley flowering control mechanisms and to investigate the functional diversification of the barley CONSTANS-like (CO-like) genes in flowering. HvCO9 was located on the same chromosome, 1HL, as Ppd-H2 (HvFT3), which is a positive regulator of short-day (SD) flowering. A phylogenetic analysis showed that HvCO9 was located on the same branch of the CO-like gene tree as rice Ghd7 and the barley and wheat VRN2 genes, which are all negative regulators of flowering. High level HvCO9 expressions were observed under SD conditions, whereas its expression levels were quite low under long-day (LD) conditions. HvCO9 expression correlated with HvFT1 and HvFT2 expression under SD conditions, although no clear effect of HvCO9 on HvFT3 expression, or vice versa, under SD conditions was observed. The over-expression of HvCO9 in rice plants produced a remarkable delay in flowering. In transgenic rice, the expression levels of the flowering-related Ehd1 gene, which is a target gene of Ghd7, and its downstream genes were suppressed, causing a delay in flowering. These results suggest that HvCO9 may act as a negative regulator of flowering under non-inductive SD conditions in barley; this activity is similar to that of rice Ghd7 under non-inductive LD conditions, but the functional targets of these genes may be different. Our results indicate that barley has developed its own pathways to control flowering by using homologous genes with modifications for the timing of expression. Further, it is hypothesized that each pathway may target different genes after gene duplication or species diversification.

Recently we cloned and characterized the gene for the wheat transcription factor TaWRKY45 and showed that TaWRKY45 was upregulated in response to benzothiadiazole (BTH) and Fusarium head blight (FHB) and that its overexpression conferred enhanced resistance against F. graminearum. To characterize the functional role of TaWRKY45 in the disease resistance of wheat, in the present study we conducted expression analyses of TaWRKY45 with inoculations of powdery mildew and leaf rust and evaluated TaWRKY45-overexpressing wheat plants for resistance to these diseases. TaWRKY45 was upregulated in response to infections with Blumeria graminis, a causal fungus for powdery mildew, and Puccinia triticina, a causal fungus for leaf rust. Constitutive overexpression of the TaWRKY45 transgene conferred enhanced resistance against these two fungi on transgenic wheat plants grown under greenhouse conditions. However, the expression of two resistance-related genes, Pm3 and Lr34, was not induced by the inoculation with powdery mildew in TaWRKY45-overexpressing wheat plants. These results suggest that TaWRKY45 is involved in the defense responses for multiple fungal diseases in wheat but that resistance involving TaWRKY45 differs from at least Pm3 and/or Lr34-related resistance. Our present and previous studies indicate that TaWRKY45 may be potentially utilized to improve a wide range of disease resistance in wheat.

The WRKY transcription factors belong to a large protein family characterized by the conserved WRKY domain. These factors have been identified to play biological functions in various plant developmental processes. WRKY proteins are also known to be involved in regulating plant responses to pathogen attack and stress-related hormones. In this study, we report the isolation and characterization of the gene (TaWRKY45) for the wheat WRKY45 transcription factor. Amino acid sequence alignment and phylogenetic analyses demonstrated that the TaWRKY45 protein is orthologous to rice OsWRKY45. Our analysis of its expression in wheat indicated that TaWRKY45 was constitutively expressed in various organs and throughout the lifetime of the plant. We observed that TaWRKY45 was upregulated in response to benzothiadiazole (BTH), a plant immune system strengthner, and Fusarium graminearum, which is a causal fungus for Fusarium head blight (FHB). The constitutive overexpression of the TaWRKY45 transgene conferred an enhanced resistance against F. graminearum to transgenic wheat plants grown under greenhouse conditions. These results indicate that TaWRKY45 is involved in the defense systems for the biotic stressors in wheat and that it may be potentially utilized to improve the disease resistance of wheat.

Background SWEET is a newly identified family of sugar transporters. Although SWEET transporters have been characterized by using Arabidopsis and rice, very little knowledge of sucrose accumulation in the stem region is available, as these model plants accumulate little sucrose in their stems. To elucidate the expression of key SWEET genes involved in sucrose accumulation of sorghum, we performed transcriptome profiling by RNA-seq, categorization using phylogenetic trees, analysis of chromosomal synteny, and comparison of amino acid sequences between SIL-05 (a sweet sorghum) and BTx623 (a grain sorghum). Results We identified 23 SWEET genes in the sorghum genome. In the leaf, SbSWEET8-1 was highly expressed and was grouped in the same clade as AtSWEET11 and AtSWEET12 that play a role in the efflux of photosynthesized sucrose. The key genes in sucrose synthesis (SPS3) and that in another step of sugar transport (SbSUT1 and SbSUT2) were also highly expressed, suggesting that sucrose is newly synthesized and actively exported from the leaf. In the stem, SbSWEET4-3 was uniquely highly expressed. SbSWEET4-1, SbSWEET4-2, and SbSWEET4-3 were categorized into the same clade, but their tissue specificities were different, suggesting that SbSWEET4-3 is a sugar transporter with specific roles in the stem. We found a putative SWEET4-3 ortholog in the corresponding region of the maize chromosome, but not the rice chromosome, suggesting that SbSWEET4-3 was copied after the branching of sorghum and maize from rice. In the panicle from the heading through to 36 days afterward, SbSWEET2-1 and SbSWEET7-1 were expressed and grouped in the same clade as rice OsSWEET11/Xa13 that is essential for seed development. SbSWEET9-3 was highly expressed in the panicle only just after heading and was grouped into the same clade as AtSWEET8/RPG1 that is essential for pollen viability. Five of 23 SWEET genes had SNPs that caused nonsynonymous amino acid substitutions between SIL-05 and BTx623. Conclusions We determined the key SWEET genes for technological improvement of sorghum in the production of biofuels: SbSWEET8-1 for efflux of sucrose from the leaf; SbSWEET4-3 for unloading sucrose from the phloem in the stem; SbSWEET2-1 and SbSWEET7-1 for seed development; SbSWEET9-3 for pollen nutrition. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0546-6) contains supplementary material, which is available to authorized users.

The Rice Annotation Project has integrated the whole rice genomic sequence and full-length cDNA (FL-cDNA) sequences. However, 2,159 FL-cDNAs have not been mapped to the genome sequence. Here, these unmapped FL-cDNAs were characterized. Of the 2,159 sequences, 359 were mapped separately to multiple loci, either on different chromosomes or in distant regions, suggesting the generation of chimeric FL-cDNAs. Chimeric transcripts with traits for trans-splicing were screened for, and expressed sequence tags in public databases with highly similar sequences were identified as chimeric. However, in vivo generation of chimeric transcripts was not confirmed by RT-PCR. Thus, there was biologically reasonable evidence for the generation of a few chimeric junctions in the independent cDNA libraries, even though their formation was an extremely rare event. Of the unmapped FL-cDNAs, 271 had similarity to rice blast fungus. Therefore, the FL-cDNA library potentially contained accidentally formed chimeric transcripts and transcripts from contaminating organisms. The descriptions of these unmapped FL-cDNA sequences in the public databases were revised.

To elucidate the genetic mechanism of flowering in wheat, we performed expression, mutant and transgenic studies of flowering-time genes. A diurnal expression analysis revealed that a flowering activator VRN1, an APETALA1/FRUITFULL homolog in wheat, was expressed in a rhythmic manner in leaves under both long-day (LD) and short-day (SD) conditions. Under LD conditions, the upregulation of VRN1 during the light period was followed by the accumulation of FLOWERING LOCUS T (FT) transcripts. Furthermore, FT was not expressed in a maintained vegetative phase (mvp) mutant of einkorn wheat (Triticum monococcum), which has null alleles of VRN1, and never transits from the vegetative to the reproductive phase. These results suggest that VRN1 is upstream of FT and upregulates the FT expression under LD conditions. The overexpression of FT in a transgenic bread wheat (Triticum aestivum) caused extremely early heading with the upregulation of VRN1 and the downregulation of VRN2, a putative repressor gene of VRN1. These results suggest that in the transgenic plant, FT suppresses VRN2 expression, leading to an increase in VRN1 expression. Based on these results, we present a model for a genetic network of flowering-time genes in wheat leaves, in which VRN1 is upstream of FT with a positive feedback loop through VRN2. The mvp mutant has a null allele of VRN2, as well as of VRN1, because it was obtained from a spring einkorn wheat strain lacking VRN2. The fact that FT is not expressed in the mvp mutant supports the present model.

Herbicides are economically important, but the non-point pollution that they cause may disrupt the surrounding environment. Phytoremediation of herbicides has been well studied using conventional plants. Transgenic plants produced for metabolizing herbicides and long-persisting pollutants can be used for phytoremediation of foreign chemicals in contaminated soil and water. The genes involved in the metabolism of chemical compounds can be isolated from various organisms, including bacteria, fungi, plants, and animals, and these genes are then introduced into candidate plants. Transgenic plants expressing mammalian P450s and the other enzymes showed tolerance and phytoremediation activity toward target herbicides. Transgenic plants can also enhance the absorption and detoxification of pollutants, thereby aiding the phytoremediation of contaminated environments.

Phytoremediation is the use of plants to remove xenobiotic compounds from the environment. Plants have the inherent ability to detoxify xenobiotic pollutants, but they are generally poor at degrading them. The introduction of genes involved in xenobiotic degradation is aimed at enhancing plants’ potential further. Rice (Oryza sativa) is a good candidate for this purpose and has been transformed with genes encoding cytochrome P450 monooxygenases CYP1A1, CYP2B6, and CYP2C19. The transgenic plants were more tolerant to various herbicides than nontransgenic Nipponbare rice plants, owing to enhanced metabolism by the introduced P450 enzymes. Transgenic plants were able to remove atrazine and metolachlor from soil. Field testing and risk assessment are very important for developing transgenic plants for phytoremediation. Transgenic rice plants should become useful as herbicide-tolerant crops and for phytoremediation of xenobiotic pollutants in future.

Cytochrome monooxygenase P450s (CYPs) comprise one of the largest enzyme families in plants. Some P450s are involved in xenobiotic metabolism: they confer herbicide tolerance and are induced by chemical treatments. We isolated a novel P450 cDNA, CYP72A21 (accession number, AB237166), from rice (Oryza sativa L. cv. Nipponbare) seedlings treated with a mixture of 2,4-dichlorophenoxyacetic acid (2,4-D), chlorotoluron, phenobarbital, salicylic acid, and naphthalic anhydride (each 100 μM). We also isolated the gene’s promoter region. Endogenous CYP72A21 expression in rice seedlings treated with 2,4-D, herbicides esprocarb, or trifluralin was increased in the aerial part of seedlings. An expression plasmid, pI21pg, containing the GUS gene under the control of the CYP72A21 promoter was introduced into rice plants. GUS was expressed constitutively in roots, but this expression was suppressed by 2,4-D treatment. 2,4-D and other auxins induced GUS expression effectively in the stem and leaves. Histological observation revealed that GUS was expressed mainly in the base of the stem. Treatment with the herbicides acetochlor, esprocarb, and propyzamide induced GUS expression in the aerial parts of the seedlings. The CYP72A21 promoter was highly responsive to treatments with various chemicals, and thus might be useful for producing transgenic plants for biomonitoring of environmental chemicals.

Rice varieties vary in their capacity for callus induction, growth, and regeneration. We identified the locus and candidate gene which conferred good callus growth and regenerative ability in the rice variety Koshihikari, a notorious poor rice line for genetic transformation. In addition, we succeeded in establishment of a new selectable marker system using the NiR gene for Agrobacterium-mediated transformation of rice, c.v. “Koshihikari.” The locus was mapped onto chromosome 1, and the nearest RFLP marker was C0178. A total of 500 segregating individuals (BC6F2 seeds) were screened for recombination by PCR-based screening and its location narrowed to a 540-kb region that had been sequenced by the International Rice Genome Sequencing Project. One ORF encoded a putative ferredoxin-nitrite reductase (NiR), which has been suggested to be required for callus induction and growth. The growth and regeneration ability of the calli initiated from Koshihikari was improved through integration of the NiR gene from Konansou. Analysis of ORFs and the promoter region of NiR indicated that the promoter region of NiR gene is responsible for growth and regeneration ability of calli in rice, c.v. Koshihikari. We established a NiR selection system for Agrobacterium-mediated transformation in rice, c.v. Koshihikari, by integration of the NiR gene isolated from rice c.v. Konansou. Transgenic rice plants regenerated from selected calli exhibited β-glucuronidase (GUS) activity. A transformation frequency of 9% was obtained. The results indicated that the NiR selection system is devoid of the disadvantages and concerns of using foreign genes (antibiotics and herbicide resistant) for selection.

The human gene for CYP2B6, a cytochrome P450 monooxygenase that inactivates xenobiotic chemicals, was introduced into Oryza sativa cv. Nipponbare by Agrobacterium-mediated transformation. At germination, R(1) seeds of transgenic rice plants expressing CYP2B6 (CYP2B6 rice) showed a high tolerance to 5 microM metolachlor, a preemergence herbicide that is degraded by CYP2B6. Thin-layer chromatography after culture with (14)C-labeled metolachlor revealed that the amounts of residual metolachlor decreased in plant tissues and the medium of CYP2B6 rice faster than those of untransformed Nipponbare. CYP2B6 rice plants were able to grow in the presence of 13 out of 17 herbicides: five chloroacetamides and mefenacet, pyributicarb, amiprofos-methyl, trifluralin, pendimethalin, norflurazon, and chlorotoluron. These herbicides differ in their modes of action and chemical structures. Transgenic rice expressing a xenobiotic-degrading human CYP2B6, which has broad substrate specificity, should be good not only for developing herbicide tolerant rice but also for reducing the environmental impact of agrochemicals.

We introduced an expression plasmid, pIKBACH, co-expressing the human cytochrome P450 genes CYP1A1, CYP2B6, and CYP2C19, into rice plants (Oryza sativa cv. ‘Nipponbare’). The transgenic rice plants (pIKBACH rice) were screened by a combination of hygromycin resistance, PCR, and Western blot analysis. The pIKBACH rice plants expressed all three P450 species and exhibited tolerance towards various herbicides with different chemical structures and different modes of action, including a photosynthesis inhibitor (chlortoluron), very long-chain fatty acids (VLCFAs) inhibitors (acetochlor, metolachlor, and thenylchlor), a carotenoid biosynthesis inhibitor (norflurazon), and a root-elongation inhibitor (pyributicarb). In addition, the pIKBACH rice plants showed high tolerance to two mixtures of three herbicides [quizalofop-ethyl (0.15 μM), metolachlor (2 μM), and norflurazon (0.4 μM); and mefenacet (2.5 μM), thenylchlor (2 μM), and pyributicarb (1.5 μM)]. Thin-layer chromatography analysis revealed that the pIKBACH transgenic rice plants exhibited high metabolic activity towards chlortoluron, metolachlor, and norflurazon. The metabolism of herbicides by the pIKBACH rice plants was enhanced additively by the introduced P450 species. Assuming that public and commercial acceptance is forthcoming, pIKBACH rice plants may become useful tools for the breeding of herbicide-tolerant crops and for phytoremediation of environmental pollution by organic chemicals.

We compared three transgenic rice plants transformed with pIJ2C9, pIES2C9, or pIJU2C9, which express the human CYP2C9 gene under the control of the CaMV 35S, chimeric CaMV 35S, or maize polyubiquitin 1 promoter, respectively. The plants, especially those transformed with pIJU2C9, showed high tolerance to the sulfonylurea herbicides chlorsulfuron, imazosulfuron, and triasulfuron, owing to metabolic detoxification promoted by the introduced CYP2C9. The levels of expression of CYP2C9 were highly related to the tolerance to sulfonylurea herbicides. In this study, we achieved to produce transgenic rice plants with high expression of CYP2C9 by use of a maize polyubiquitin 1 promoter. Imazosulfuron is used in rice, but chlorsulfuron and triasulfuron are not, because of the sensitivity of rice plants to them. Transgenic rice plants expressing CYP2C9 will be useful for introducing tolerance to chlorsulfuron and triasulfuron into rice lines.

We compared the transcription patterns of calli initiated from the cultivars Koshihikari, Sasanishiki and Konansou and from isogenic lines with the aim of identifying genes correlated with good regeneration ability of the callus in rice by mRNA differential display. Koshihikari and Sasanishiki isogenic lines exhibited the same good regeneration ability as that of Konansou, and Koshihikari and Sasanishiki exhibited poor regeneration ability compared with that of Konansou. One full-length cDNA ( Os22A ) whose expression was elevated in the rice calli that exhibited vigorous plant regeneration was isolated. Sequence analysis revealed that Os22A is identical to the EST clones expressed in Oryza sativa in the endosperm 10 days after pollination (accession number BI797481 ) and in the panicle at the ripening stage (accession number AU172698 ). Os22A is highly homologous to a barley gene coding for glucose dehydrogenase (accession number S72926 ) that is specifically expressed in developing embryos. Os22A encodes a transcript of 1391 nucleotides and is specifically expressed in calli with good regeneration ability in rice. Expression of Os22A mRNA decreased or disappeared with decrease in regeneration ability. In order to characterize the function of Os22A , we integrated Os22A into rice calli ( O. sativa cv . Koshihikari). Calli initiated from Koshihikari exhibited poor regeneration ability but the transformed calli showed good regeneration ability in the regeneration medium. The regeneration ability of transformed calli clearly improved by integration of Os22A . Our results suggest that Os22A is involved in plant regeneration ability of the callus.

We introduced a human cytochrome P450 CYP1A1 gene into rice plants ( Oryza sativa cv. Nipponbare) to confer herbicide tolerance. In germination tests, the R 1 seeds showed tolerance to various herbicides with different modes of action, including quizalofop-ethyl (0.2 μM), norflurazon (0.5 μM), mefenacet (2.5 μM), and chlortoluron (100 μM). We used 14 C-labeled atrazine, chlortoluron, and norflurazon to confirm the metabolism of herbicides by the action of the introduced CYP1A1. Although both CYP1A1 plants and nontransgenic control plants metabolized these herbicides into the same set of chemical compounds, the herbicides were metabolized more rapidly in the CYP1A1 plants. We were surprised to find that the levels of the intermediate metabolites were higher in the culture medium of the CYP1A1 plants than in the plants themselves, because it is commonly accepted that herbicides are taken up, metabolized, and stored in plants. The metabolites of herbicides seemed to be exuded into the medium from the roots of the CYP1A1 plants. The introduced P450 enhanced the metabolism of the herbicides in plants. Therefore, the CYP1A1 plants became more tolerant to various herbicides than the control plants.

Transgenic rice plants expressing human CYP1A1 , CYP2B6 , or CYP2C19 show strong cross-tolerance to various herbicides. However, these plants showed susceptibility to the herbicides ethofumesate and benfuresate. Both herbicides inhibited germination of transgenic rice plants at a concentration of 2.0 μM in the culture medium, whereas control Nipponbare plants showed normal growth in their presence. The CYP2B6 rice plants metabolized ethofumesate to produce the de-ethylated metabolite DHDBM (2,3-dihydro-2-hydroxy-3,3-dimethyl-5-benzofuranyl methanesulfonate), which was accumulated to levels up to 60 times higher than in control plants. Germination of both control and CYP2B6 rice plants was inhibited completely with 0.75 μM DHDBM in the culture medium. The phytotoxicity of DHDBM to rice plants was at least four times greater than that of ethofumesate. Because both ethofumesate and benfuresate are metabolized to give DHDBM or analogous metabolites, we consider that DHDBM was the major phytotoxic metabolite in these rice plants.

Background Sorghum (Sorghum bicolor L. Moench) accumulates 3-deoxyanthocyanidins and exhibits orange to purple coloration on parts of the leaf in response to infection with the fungus Bipolaris sorghicola. We aimed to identify the key genes determining this color variation. Results Sorghum populations derived from Nakei-MS3B and M36001 accumulated apigeninidin, or both apigeninidin and luteolinidin, in different proportions in lesions caused by B. sorghicola infection, suggesting that the relative proportions of the two 3-deoxyanthocyanidins determine color variation. QTL analysis and genomic sequencing indicated that two closely linked loci on chromosome 4, containing the flavonoid 3′-hydroxylase (F3′H) and Tannin1 (Tan1) genes, were responsible for the lesion color variation. The F3′H locus in Nakei-MS3B had a genomic deletion resulting in the fusion of two tandemly arrayed F3′H genes. The recessive allele at the Tan1 locus derived from M36001 had a genomic insertion and encoded a non-functional WD40 repeat transcription factor. Whole-mRNA sequencing revealed that expression of the fused F3′H gene was conspicuously induced in purple sorghum lines. The levels of expression of F3′H matched the relative proportions of apigeninidin and luteolinidin. Conclusions Expression of F3′H is responsible for the synthesis of luteolinidin; the expression level of this gene is therefore critical in determining color variation in sorghum leaves infected with B. sorghicola. Electronic supplementary material The online version of this article (doi:10.1186/1756-0500-7-761) contains supplementary material, which is available to authorized users.

The cDNAs of human drug-metabolizing cytochrome P450 species CYP2C9 and CYP2C19 were each expressed in rice ( Oryza sativa L.) plants under the control of CaMV35S promoter and Nos terminator. Western blot analysis of the selected transgenic rice plants revealed that CYP2C9 and CYP2C19 were produced in the transgenic rice plants 2C9-57 and 2C19-12, respectively. [ 14 C]Chlorsulfuron and [ 14 C]imazosulfuron were found to be more rapidly metabolized in R 2 seedlings from 2C9-57R 1 than in the control plants to yield polar metabolites which were mainly glucose conjugates of hydroxylated metabolites formed by the function of CYP2C9. The R 2 transgenic rice seeds from 2C9-57R 1 showed tolerance toward the sulfonylurea herbicide chlorsulfuron in a germination test. On the other hand, [ 14 [C]pyributicarb was also rapidly metabolized in R 1 seedlings from 2C19-12R 0 to give polar metabolites, but not in the control and PCR-negative transgenic plants. The R 1 transgenic rice seeds of 2C19-12R 0 showed cross-tolerance toward the herbicides mefenacet, metolachlor, norflurazon, and pyributicarb with different chemical structures and modes of herbicide action in a germination test. It was suggested that the herbicide-tolerant rice plant 2C9-57R 2 seems to be practically useful for evasion of the sensitivity of rice plants toward the sulfonylurea herbicide chlorsulfuron. In addition, the herbicide-tolerant 2C19-12R 1 appears to be potentially useful for the control of sulfonylurea-resistant weeds in a paddy field by the combined use of the herbicides mefenacet, metolachlor, norflurazon, and pyributicarb.

The pre-mRNA encoding the neural cell adhesion molecule (NCAM) is spliced to generate NCAM isoforms containing the muscle-specific domain (MSD) during myogenesis. Utilizing chimeric NCAM minigenes, we searched for cis-acting elements that contribute to the alternative selection of exon MSDb, one of the four exons encoding MSD, and identified an intronic cis-element located downstream of exon MSDb. The cis-element acted as a negative regulator for the selection of exon MSDb in nonmuscle fibroblasts but not in myoblasts, that are already destined to differentiate into muscle cells. The suppressive effect of this cis-element on the selection of exon MSDb was released in the process of myogenesis. When MyoD was co-expressed with a minigene containing this element in fibroblasts, the suppressive effect of the cis-element was released as the cells underwent differentiation. We propose that this cis-element contributes at least as one of the regulatory elements in the differentiation state-dependent selection of MSD exons in vivo.

The spring-type near isogenic line (NIL) of the winter-type barley (Hordeum vulgare ssp. vulgare) var. Hayakiso 2 (HK2) was developed by introducing VERNALIZATION-H1 (Vrn-H1) for spring growth habit from the spring-type var. Indo Omugi. Contrary to expectations, the spring-type NIL flowered later than winter-type HK2. This phenotypic difference was controlled by a single gene, which cosegregated only with phytochrome C (HvPhyC) among three candidates around the Vrn-H1 region (Vrn-H1, HvPhyC, and CASEIN KINASE IIα), indicating that HvPhyC was the most likely candidate gene. Compared with the late-flowering allele HvPhyC-l from the NIL, the early-flowering allele HvPhyC-e from HK2 had a single nucleotide polymorphism T1139C in exon 1, which caused a nonsynonymous amino acid substitution of phenylalanine at position 380 by serine in the functionally essential GAF (3′, 5′-cyclic-GMP phosphodiesterase, adenylate cyclase, formate hydrogen lyase activator protein) domain. Functional assay using a rice (Oryza sativa) phyA phyC double mutant line showed that both of the HvPhyC alleles are functional, but HvPhyC-e may have a hyperfunction. Expression analysis using NILs carrying HvPhyC-e and HvPhyC-l (NIL [HvPhyC-e] and NIL [HvPhyC-l], respectively) showed that HvPhyC-e up-regulated only the flowering promoter FLOWERING LOCUS T1 by bypassing the circadian clock genes and flowering integrator CONSTANS1 under a long photoperiod. Consistent with the up-regulation, NIL (HvPhyC-e) flowered earlier than NIL (HvPhyC-l) under long photoperiods. These results implied that HvPhyC is a key factor to control long-day flowering directly.

Background Sorghum (Sorghum bicolor L. Moench) is a rich source of natural phytochemicals. We performed massive parallel sequencing of mRNA to identify differentially expressed genes after sorghum BTx623 had been infected with Bipolaris sorghicola, a necrotrophic fungus causing a sorghum disease called target leaf spot. Result Seventy-six-base-pair reads from mRNAs of mock- or pathogen-infected leaves were sequenced. Unannotated transcripts were predicted on the basis of the piling-up of mapped short reads. Differentially expressed genes were identified statistically; particular genes in tandemly duplicated putative paralogs were highly upregulated. Pathogen infection activated the glyoxylate shunt in the TCA cycle; this changes the role of the TCA cycle from energy production to synthesis of cell components. The secondary metabolic pathways of phytoalexin synthesis and of sulfur-dependent detoxification were activated by upregulation of the genes encoding amino acid metabolizing enzymes located at the branch point between primary and secondary metabolism. Coordinated gene expression could guide the metabolic pathway for accumulation of the sorghum-specific phytochemicals 3-deoxyanthocyanidin and dhurrin. Key enzymes for synthesizing these sorghum-specific phytochemicals were not found in the corresponding region of the rice genome. Conclusion Pathogen infection dramatically changed the expression of particular paralogs that putatively encode enzymes involved in the sorghum-specific metabolic network.

Gene duplication occurs by either DNA- or RNA-based processes; the latter duplicates single genes via retroposition of messenger RNA. The expression of a retroposed gene copy (retrocopy) is expected to be uncorrelated with its source gene because upstream promoter regions are usually not part of the retroposition process. In contrast, DNA-based duplication often encompasses both the coding and the intergenic (promoter) regions; hence, expression is often correlated, at least initially, between DNA-based duplicates. In this study, we identified 150 retrocopies in rice (Oryza sativa L. ssp japonica), most of which represent ancient retroposition events. We measured their expression from high-throughput RNA sequencing (RNAseq) data generated from seven tissues. At least 66% of the retrocopies were expressed but at lower levels than their source genes. However, the tissue specificity of retrogenes was similar to their source genes, and expression between retrocopies and source genes was correlated across tissues. The level of correlation was similar between RNA- and DNA-based duplicates, and they decreased over time at statistically indistinguishable rates. We extended these observations to previously identified retrocopies in Arabidopsis thaliana, suggesting they may be general features of the process of retention of plant retrogenes.

Target leaf spot is one of the major sorghum diseases in southern Japan and caused by a necrotrophic fungus, Bipolaris sorghicola. Sorghum resistance to target leaf spot is controlled by a single recessive gene (ds1). A high-density genetic map of the ds1 locus was constructed with simple sequence repeat markers using progeny from crosses between a sensitive variety, bmr-6, and a resistant one, SIL-05, which allowed the ds1 gene to be genetically located within a 26-kb region on the short arm of sorghum chromosome 5. The sorghum genome annotation database for BTx623, for which the whole genome sequence was recently published, indicated a candidate gene from the Leucine-Rich Repeat Receptor Kinase family in this region. The candidate protein kinase gene was expressed in susceptible plants but was not expressed or was severely reduced in resistant plants. The expression patterns of ds1 gene and the phenotype of target leaf spot resistance were clearly correlated. Genomic sequences of this region in parental varieties showed a deletion in the promoter region of SIL-05 that could cause reduction of gene expression. We also found two ds1 alleles for resistant phenotypes with a stop codon in the coding region. The results shown here strongly suggest that the loss of function or suppression of the ds1 protein kinase gene leads to resistance to target leaf spot in sorghum.

Five barley (Hordeum vulgare) PEBP (for phosphatidylethanolamine-binding protein) genes were analyzed to clarify their functional roles in flowering using transgenic, expression, and quantitative trait locus analyses. Introduction of HvTFL1 and HvMFT1 into rice (Oryza sativa) plants did not result in any changes in flowering, suggesting that these two genes have functions distinct from flowering. Overexpression of HvFT1, HvFT2, and HvFT3 in rice resulted in early heading, indicating that these FT-like genes can act as promoters of the floral transition. HvFT1 transgenic plants showed the most robust flowering initiation. In barley, HvFT1 was expressed at the time of shoot meristem phase transition. These results suggest that HvFT1 is the key gene responsible for flowering in the barley FT-like gene family. HvFT2 transgenic plants also showed robust flowering initiation, but HvFT2 was expressed only under short-day (SD) conditions during the phase transition, suggesting that its role is limited to specific photoperiodic conditions in barley. Flowering activity in HvFT3 transgenic rice was not as strong and was modulated by the photoperiod. These results suggest that HvFT3 functions in flowering promotion but that its effect is indirect. HvFT3 expression was observed in Morex, a barley cultivar carrying a dominant allele of Ppd-H2, a major quantitative trait locus for flowering under SD conditions, although no expression was detected in Steptoe, a cultivar carrying ppd-H2. HvFT3 was expressed in Morex under both long-day and SD conditions, although its expression was increased under SD conditions. HvFT3 was mapped to chromosome 1HL, the same chromosome that carries Ppd-H2. Genomic sequence analyses revealed that Morex possesses an intact HvFT3 gene, whereas most of this gene has been lost in Steptoe. These data strongly suggest that HvFT3 may be identical to Ppd-H2.

CYP72A21, a rice cytochrome P450 gene, is induced by chloroacetamide herbicides. OSB2, a rice myc-type transcription factor, induces anthocyanin accumulation in rice leaves. To produce plants for biomonitoring by color change, we combined the CYP72A21 promoter and the OSB2 gene and introduced them into the rice isogenic line Taichung-65 CB A (T65), which contains loci CB and A from the rice cultivar Murasakiine. Leaves of the transgenic plants turned red upon treatment with the chloroacetamide herbicides acetochlor, alachlor, and metolachlor. Seedling shoots reddened upon treatment with alachlor or metolachlor at 10 microM, a concentration slightly higher than that used in the field. Anthocyanin content was increased approximately 200% by the treatment. The color changes were consistent with increased shoot expression of OSB2 and the anthocyanidin synthase gene (ANS). This system promises easy detection of rice plant gene expression. Transgenic plants could be used in the future to biomonitor accumulated herbicides.

Acetolactate synthase (ALS) is a target enzyme for many herbicides, including sulfonylurea and imidazolinone. We investigated the usefulness of a mutated ALS gene of rice, which had double point mutations and encoded an herbicide-resistant form of the enzyme, as a selectable marker for wheat transformation. After the genomic DNA fragment from rice containing the mutated ALS gene was introduced into immature embryos by means of particle bombardment, transgenic plants were efficiently selected with the herbicide bispyribac sodium (BS). Southern blot analysis confirmed that transgenic plants had one to more than ten copies of the transgene in their chromosomes. Adjustment of the BS concentration combined with repeated selection effectively prevented nontransgenic plants from escaping herbicide selection. Measurement of ALS activity indicated that transgenic plants produced an herbicide-resistant form of ALS and therefore had acquired the resistance to BS. This report is the first to describe a selection system for wheat transformation that uses a selectable marker gene of plant origin.

Anthocyanin pigmentation provides an excellent system with which to study the regulation of gene expression in higher plants. In this study, OsPR1.1 promoter was isolated and the promoter activity was monitored using a reporter gene OSB2, which encodes a transcription factor for anthocyanin synthesis in rice plants. We introduced PR::OSB2 plasmid into an isogenic Taichung 65, no. 99-962 T-65 CBA B9F5 (T65 CBA), rice line (Oryza sativa L.) and found that the transgenic rice plants exhibited anthocyanin accumulation by the induced expression of OSB2 after chemical treatments with methyl jasmonate (MeJA) and 2,6-dichloroisonicotinic acid (DCINA). The shoots of the PR::OSB2 transgenic rice plants changed color to red after application of the chemicals accompanying with the increased anthocyanin content to approximately 5-fold by MeJA and 2-fold by DCINA, respectively. The anthocyanin accumulation was consistent with the increase of the expression of OSB2 and anthocyanidin synthase (ANS). This color change system could provide a useful and easy way to produce transgenic plants for monitoring of chemicals in the environment.

Cytochrome P450 monooxygenases (P450s) metabolize herbicides to produce mainly non-phytotoxic metabolites. Although rice plants endogenously express multiple P450 enzymes, transgenic plants expressing other P450 isoforms might show improved herbicide resistance or reduce herbicide residues. Mammalian P450s metabolizing xenobiotics are reported to show a broad and overlapping substrate specificity towards lipophilic foreign chemicals, including herbicides. These P450s are ideal for enhancing xenobiotic metabolism in plants. A human P450, CYP1A1, metabolizes various herbicides with different structures and modes of herbicide action. We introduced human CYP1A1 into rice plants, and the transgenic rice plants showed broad cross-resistance towards various herbicides and metabolized them. The introduced CYP1A1 enhanced the metabolism of chlorotoluron and norflurazon. The herbicides were metabolized more rapidly in the transgenic rice plants than in non-transgenic controls. Transgenic rice plants expressing P450 might be useful for reducing concentrations of various chemicals in the environment.

This study evaluated the expression of human cytochrome P450 genes CYP1A1, CYP2B6, and CYP2C19 in rice plants (Oryza sativa cv. Nipponbare) introduced using the plasmid pIKBACH. The transgenic rice plants (pIKBACH rice plants) became more tolerant toward various herbicides than nontransgenic Nipponbare rice plants. Rice plants expressing pIKBACH grown in soil showed tolerance to the herbicides atrazine, metolachlor, and norflurazon and to a mixture of the three herbicides. The degradation of atrazine and metolachlor by pIKBACH rice plants was evaluated to confirm the metabolic activity of the introduced P450s. Although both pIKBACH and nontransgenic Nipponbare rice plants could decrease the amounts of the herbicides in plant tissue and culture medium, pIKBACH rice plants removed greater amounts in greenhouse experiments. The ability of pIKBACH rice plants to remove atrazine and metolachlor from soil was confirmed in large-scale experiments. The metabolism of herbicides by pIKBACH rice plants was enhanced by the introduced P450 species. Assuming that public and commercial acceptance is forthcoming, pIKBACH rice plants may become useful tools for the breeding of herbicide-tolerant crops and for phytoremediation of environmental pollution by organic chemicals.