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1.
Theor Appl Genet ; 133(7): 2105-2115, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32200415

RESUMO

KEY MESSAGE: A frame shift invoked by a single-base deletion in the gene encoding a cytochrome P450 hydroxylase, CYP81E22, causes the loss of bentazon detoxification function in soybean. Bentazon is an effective herbicide in soybean cultivation applied at post-emergence stages for control of several broadleaf weeds. However, some soybean cultivars are highly sensitive to bentazon and are killed upon application. In this study, the gene related to the high sensitivity of soybean cultivars to bentazon was mapped to chromosome 16, and its location was narrowed down to a 257-kb region where three cytochrome P450 genes were located. In these genes, a single-base deletion of cytosine was detected in the coding region of Glyma.16G149300, CYP81E22, at + 1465 bp downstream from the translation start codon, leading to a frame shift in the open reading frame and creating a premature stop codon. This stop codon resulted in the loss of more than half of the P450, and consequently, the remaining molecule failed to form a functioning protein. This single-base deletion was common among the highly sensitive cultivars screened from the soybean mini-core collection and other previously reported highly sensitive cultivars. Furthermore, we screened plant lines from the targeting-induced local lesions in genomes library of the soybean cultivar Enrei based on a modelled 3D structure of CYP81E22. The lines with mutations in Glyma.16G149300 were highly sensitive to bentazon, which provides strong evidence that Glyma.16G149300 is the gene responsible for high sensitivity to bentazon.


Assuntos
Benzotiadiazinas , Sistema Enzimático do Citocromo P-450/genética , Glycine max/efeitos dos fármacos , Glycine max/genética , Herbicidas , Folhas de Planta/genética , Alelos , Mapeamento Cromossômico , Códon de Terminação , Cruzamentos Genéticos , Resistência à Doença/genética , Mutação da Fase de Leitura , Deleção de Genes , Genótipo , Folhas de Planta/enzimologia , Proteínas de Plantas/genética , Glycine max/enzimologia
2.
Plant Cell Physiol ; 60(11): 2496-2509, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31418782

RESUMO

Lotus japonicus is an important model legume plant in several fields of research, such as secondary (specialized) metabolism and symbiotic nodulation. This plant accumulates triterpenoids; however, less information regarding its composition, content and biosynthesis is available compared with Medicago truncatula and Glycine max. In this study, we analyzed the triterpenoid content and composition of L. japonicus. Lotus japonicus accumulated C-28-oxidized triterpenoids (ursolic, betulinic and oleanolic acids) and soyasapogenols (soyasapogenol B, A and E) in a tissue-dependent manner. We identified an oxidosqualene cyclase (OSC) and two cytochrome P450 enzymes (P450s) involved in triterpenoid biosynthesis using a yeast heterologous expression system. OSC9 was the first enzyme derived from L. japonicus that showed α-amyrin (a precursor of ursolic acid)-producing activity. CYP716A51 showed triterpenoid C-28 oxidation activity. LjCYP93E1 converted ß-amyrin into 24-hydroxy-ß-amyrin, a metabolic intermediate of soyasapogenols. The involvement of the identified genes in triterpenoid biosynthesis in L. japonicus plants was evaluated by quantitative real-time PCR analysis. Furthermore, gene loss-of-function analysis of CYP716A51 and LjCYP93E1 was conducted. The cyp716a51-mutant L. japonicus hairy roots generated by the genome-editing technique produced no C-28 oxidized triterpenoids. Likewise, the complete abolition of soyasapogenols and soyasaponin I was observed in mutant plants harboring Lotus retrotransposon 1 (LORE1) in LjCYP93E1. These results indicate that the activities of these P450 enzymes are essential for triterpenoid biosynthesis in L. japonicus. This study increases our understanding of triterpenoid biosynthesis in leguminous plants and provides information that will facilitate further studies of the physiological functions of triterpenoids using L. japonicus.


Assuntos
Lotus/metabolismo , Triterpenos/metabolismo , Regulação da Expressão Gênica de Plantas , Ácido Oleanólico/metabolismo , Proteínas de Plantas/metabolismo , Ácido Ursólico
3.
Plant Cell Physiol ; 60(5): 1082-1097, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30753604

RESUMO

Triterpenes (C30) constitute one of the diverse class of natural products with potential applications in food, cosmetic and pharmaceutical industries. Soyasaponins are oleanane-type triterpenoids widespread among legumes and particularly abundant in soybean seeds. They have associated with various pharmacological implications and undesirable taste properties of soybean-based food products. Uncovering the biosynthetic genes of soyasaponins will provide new opportunities to control the pathway for human benefits. However, the pathway of soyasaponin biosynthesis has not been fully elucidated in part because of a paucity of natural mutants. Here, we applied a structured high-density soybean mutant library for the forward genetic screening of triterpenoid biosynthesis. The seed soyasaponin polymorphism in the mutant library was evaluated using a high-throughput thin-layer chromatography and liquid chromatography tandem mass spectrometry analysis. This screening identified 35 mutants (3.85% of 909 mutant lines) with seven unusual soyasaponin phenotypes (Categories 1-7), which was greater than the number of natural mutants reported previously (22 mutants, 0.18% of ∼12,428 accessions). Nine unique intermediates of soyasaponin biosynthesis were identified and their chemical structures were estimated based on their MS/MS fragment patterns. Based on published information, 19 mutants could be associated with loss of function of four individual soyasaponin biosynthesis genes identified through expressed sequence tag mining or positional cloning, whereas the remaining 16 mutants were novel and may facilitate discovery of the unknown biosynthetic genes of soyasaponins. Our approach and library may help to identify new phenotype materials and causative genes associated with specialized metabolite production and other traits.


Assuntos
Glycine max/genética , Triterpenos/metabolismo , Mutação/genética , Saponinas/metabolismo , Espectrometria de Massas em Tandem
4.
Plant Cell Physiol ; 59(4): 792-805, 2018 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-29401289

RESUMO

Soyasaponins are specialized metabolites present in soybean seeds that affect the taste and quality of soy-based foods. The composition of the sugar chains attached to the aglycone moiety of soyasaponins is regulated by genetic loci such as sg-1, sg-3 and sg-4. Here, we report the cloning and characterization of the Sg-3 gene, which is responsible for conjugating the terminal (third) glucose (Glc) at the C-3 sugar chain of soyasaponins. The gene Glyma.10G104700 is disabled in the sg-3 cultivar, 'Mikuriya-ao', due to the deletion of genomic DNA that results in the absence of a terminal Glc residue on the C-3 sugar chain. Sg-3 encodes a putative glycosyltransferase (UGT91H9), and its predicted protein sequence has a high homology with that of the product of GmSGT3 (Glyma.08G181000; UGT91H4), which conjugates rhamnose (Rha) to the third position of the C-3 sugar chain in vitro. A recombinant Glyma.10G104700 protein could utilize UDP-Glc as a substrate to conjugate the third Glc to the C-3 sugar chain, and introducing a functional Glyma.10G104700 transgene into the mutant complemented the sg-3 phenotype. Conversely, induction of a premature stop codon mutation in Glyma.10G104700 (W270*) resulted in the sg-3 phenotype, suggesting that Glyma.10G104700 was Sg-3. The gmsgt3 (R339H) mutant failed to accumulate soyasaponins with the third Rha at the C-3 sugar chain, and the third Glc and Rha conjugations were both disabled in the sg-3 gmsgt3 double mutant. These results demonstrated that Sg-3 and GmSGT3 are non-redundantly involved in conjugation of the third Glc and Rha at the C-3 sugar chain of soyasaponins, respectively.


Assuntos
Genes de Plantas , Variação Genética , Glycine max/genética , Proteínas de Plantas/genética , Saponinas/genética , Açúcares/metabolismo , Alelos , Sequência de Aminoácidos , Estudos de Associação Genética , Teste de Complementação Genética , Glicosiltransferases/metabolismo , Mutação/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/metabolismo , Saponinas/química , Saponinas/metabolismo , Transgenes
5.
PLoS Pathog ; 12(10): e1005921, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27711180

RESUMO

Magnaporthe oryzae, the fungus causing rice blast disease, should contend with host innate immunity to develop invasive hyphae (IH) within living host cells. However, molecular strategies to establish the biotrophic interactions are largely unknown. Here, we report the biological function of a M. oryzae-specific gene, Required-for-Focal-BIC-Formation 1 (RBF1). RBF1 expression was induced in appressoria and IH only when the fungus was inoculated to living plant tissues. Long-term successive imaging of live cell fluorescence revealed that the expression of RBF1 was upregulated each time the fungus crossed a host cell wall. Like other symplastic effector proteins of the rice blast fungus, Rbf1 accumulated in the biotrophic interfacial complex (BIC) and was translocated into the rice cytoplasm. RBF1-knockout mutants (Δrbf1) were severely deficient in their virulence to rice leaves, but were capable of proliferating in abscisic acid-treated or salicylic acid-deficient rice plants. In rice leaves, Δrbf1 inoculation caused necrosis and induced defense-related gene expression, which led to a higher level of diterpenoid phytoalexin accumulation than the wild-type fungus did. Δrbf1 showed unusual differentiation of IH and dispersal of the normally BIC-focused effectors around the short primary hypha and the first bulbous cell. In the Δrbf1-invaded cells, symplastic effectors were still translocated into rice cells but with a lower efficiency. These data indicate that RBF1 is a virulence gene essential for the focal BIC formation, which is critical for the rice blast fungus to suppress host immune responses.


Assuntos
Proteínas Fúngicas/metabolismo , Magnaporthe/patogenicidade , Micoses/microbiologia , Doenças das Plantas/microbiologia , Oryza , Reação em Cadeia da Polimerase , Virulência
6.
Biosci Biotechnol Biochem ; 80(1): 145-51, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26287768

RESUMO

We show that a rice GRAS family protein, CIGR2, is a bonafide transcriptional activator, and through this function, targets the B-type heat shock protein-encoding gene OsHsf23 (Os09g0456800). CIGR2 (Os07g0583600) is an N-acetylchitooligosaccharide elicitor-responsive gene whose activity, through the direct transcriptional control of OsHsf23, is required for mediating hypersensitive cell death activation during pathogen infection. RNAi lines of CIGR2 and OsHsf23 similarly exhibited the higher level of granulation in the epidermal cells of leaf sheath inoculated with an avirulent isolate of rice blast fungus. Interestingly, we did not observe altered levels of resistance, suggesting that CIGR2 suppresses excessive cell death in the incompatible interaction with blast fungus via activation of OsHsf23.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Magnaporthe/genética , Oryza/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Morte Celular/genética , Proteínas de Ligação a DNA/metabolismo , Resistência à Doença , Fatores de Transcrição de Choque Térmico , Interações Hospedeiro-Patógeno , Magnaporthe/metabolismo , Magnaporthe/patogenicidade , Oligossacarídeos/metabolismo , Oligossacarídeos/farmacologia , Oryza/metabolismo , Oryza/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/microbiologia , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica
7.
Plant J ; 67(5): 907-16, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21585570

RESUMO

In the present study, we investigated the function of the heterotrimeric G protein ß-subunit (Gß) gene (RGB1) in rice. RGB1 knock-down lines were generated in the wild type and d1-5, a mutant deficient for the heterotrimeric G protein α-subunit (Gα) gene (RGA1). Both transgenic lines showed browning of the lamina joint regions and nodes that could be attributed to a reduction of RGB1 function, as the abnormality was not observed in d1-5. The RGB1 knock-down lines generated in d1-5 were shorter, suggesting RGB1 to be a positive regulator of cellular proliferation, in addition to RGA1. The number of sterile seeds also increased in both RGB1 knock-down lines. These results suggest that Gßγ and Gα cooperatively function in cellular proliferation and seed fertility. We discuss the potential predominant role of RGB1 in G protein signaling in rice.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Oryza/fisiologia , Sementes/fisiologia , Proliferação de Células , DNA de Plantas/genética , Flores/anatomia & histologia , Flores/genética , Flores/fisiologia , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Oryza/anatomia & histologia , Oryza/genética , Fenótipo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Caules de Planta/anatomia & histologia , Caules de Planta/genética , Caules de Planta/fisiologia , Plantas Geneticamente Modificadas/anatomia & histologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Mensageiro/genética , RNA de Plantas/genética , Plântula/anatomia & histologia , Plântula/genética , Plântula/fisiologia , Sementes/anatomia & histologia , Sementes/genética , Deleção de Sequência , Transdução de Sinais/fisiologia
8.
Sci Rep ; 12(1): 14577, 2022 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-36028537

RESUMO

The planetary missions including the Venus Climate Orbiter 'Akatsuki' provide new information on various atmospheric phenomena. Nevertheless, it is difficult to elucidate their three-dimensional structures globally and continuously only from observations because satellite observations are considerably limited in time and space. We constructed the first 'objective analysis' of Venus' atmosphere by assimilating cloud-top horizontal winds on the dayside from the equator to mid-latitudes, which is frequently obtained from Akatsuki's Ultraviolet Imager (UVI). The three-dimensional structures of thermal tides, found recently to play a crucial role in maintaining the super rotation, are greatly improved by the data assimilation. This result is confirmed by comparison with Akatsuki's temperature observations. The momentum transport caused by the thermal tides and other disturbances are also modified by the wind assimilation and agrees well with those estimated from the UVI observations. The assimilated dataset is reliable and will be open to the public along with the Akatsuki observations for further investigation of Venus' atmospheric phenomena.

9.
Nat Commun ; 12(1): 3682, 2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140504

RESUMO

Gravity waves play essential roles in the terrestrial atmosphere because they propagate far from source regions and transport momentum and energy globally. Gravity waves are also observed in the Venus atmosphere, but their characteristics have been poorly understood. Here we demonstrate activities of small-scale gravity waves using a high-resolution Venus general circulation model with less than 20 and 0.25 km in the horizontal and vertical grid intervals, respectively. We find spontaneous gravity wave radiation from nearly balanced flows. In the upper cloud layer (~70 km), the thermal tides in the super-rotation are primary sources of small-scale gravity waves in the low-latitudes. Baroclinic/barotropic waves are also essential sources in the mid- and high-latitudes. The small-scale gravity waves affect the three-dimensional structure of the super-rotation and contribute to material mixing through their breaking processes. They propagate vertically and transport momentum globally, which decelerates the super-rotation in the upper cloud layer (~70 km) and accelerates it above ~80 km.

10.
Plant Cell Physiol ; 51(2): 271-81, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20040584

RESUMO

The d1 mutant, which is deficient for the heterotrimeric G-protein alpha subunit (Galpha) gene of rice, shows dwarfism and sets small round seeds. To determine whether dwarfism in d1 is due to a reduction in cell number or to shortened cell length, the cell number of the leaf sheath, the internode, the root and the lemma was compared between Nipponbare, a wild-type rice and d1-5, a d1 allele derived from Nipponbare. Our results indicate that the cell number was reduced in all organs analyzed in d1-5. In addition, cell enlargement was found in roots and lemma of d1-5, although the organ length in d1-5 was shorter than that of wild-type rice. These results suggest that rice Galpha participates in cell proliferation in rice. Western blot analyses using anti-Galpha antibody and RT-PCR analyses indicate that Galpha is mostly expressed in the developing organs. Galpha promoter activity studies using the GUS reporter gene confirmed that the expression of Galpha was highest in developing organs. We conclude that rice Galpha participates in the regulation of cell number in a developmental stage-dependent manner.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Oryza/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Contagem de Células , Proliferação de Células , Tamanho Celular , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , RNA de Plantas/genética
11.
Nat Commun ; 11(1): 5664, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199711

RESUMO

Triterpenoid saponins are specialised metabolites distributed widely in the plant kingdom that consist of one or more sugar moieties attached to triterpenoid aglycones. Despite the widely accepted view that glycosylation is catalysed by UDP-dependent glycosyltransferase (UGT), the UGT which catalyses the transfer of the conserved glucuronic acid moiety at the C-3 position of glycyrrhizin and various soyasaponins has not been determined. Here, we report that a cellulose synthase superfamily-derived glycosyltransferase (CSyGT) catalyses 3-O-glucuronosylation of triterpenoid aglycones. Gene co-expression analyses of three legume species (Glycyrrhiza uralensis, Glycine max, and Lotus japonicus) reveal the involvement of CSyGTs in saponin biosynthesis, and we characterise CSyGTs in vivo using Saccharomyces cerevisiae. CSyGT mutants of L. japonicus do not accumulate soyasaponin, but the ectopic expression of endoplasmic reticulum membrane-localised CSyGTs in a L. japonicus mutant background successfully complement soyasaponin biosynthesis. Finally, we produced glycyrrhizin de novo in yeast, paving the way for sustainable production of high-value saponins.


Assuntos
Biocatálise , Glucosiltransferases/metabolismo , Ácido Glucurônico/metabolismo , Saponinas/biossíntese , Vias Biossintéticas , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica de Plantas , Glicosilação , Glycyrrhiza uralensis/genética , Ácido Glicirrízico/metabolismo , Funções Verossimilhança , Lotus/genética , Filogenia , Saccharomyces cerevisiae/metabolismo , Saponinas/química , Glycine max/genética , Especificidade por Substrato , Triterpenos/metabolismo , Uridina Difosfato Ácido Glucurônico/metabolismo
12.
Plant Cell Physiol ; 50(1): 161-72, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19036785

RESUMO

The alpha subunit of plant heterotrimeric G proteins (Galpha) plays pivotal roles in multiple aspects of development and responses to plant hormones. Recently, several lines of evidence have shown that Galpha participates in brassinosteroid (BR) responses in Arabidopsis and rice plants. In this study, we conducted a comprehensive analysis of the roles of the rice Galpha in the responses to BR using a defective mutant of the Galpha gene, T65d1. Decreased sensitivity to 24-epi-brassinolide (24-epiBL) in the T65d1 mutant was observed in many processes examined, e.g. in the inhibition of root growth and the promotion of coleoptile elongation. The T65d1 mutant also showed similar phenotypes to those of BR-deficient mutants, such as the specifically shortened second internode and the constitutive photomorphogenic growth phenotype under dark conditions. However, a negative feedback effect by 24-epiBL on the expression of BR biosynthetic genes was observed in the T65d1 mutant, and the levels of BR intermediates did not fluctuate in this mutant. To determine the epistatic relationship between the T65d1 mutant and d61-7, a weak allele of a rice BR receptor mutant, the two mutants were crossed. The T65d1/d61-7 double mutant showed no epistasis in the elongation inhibition of the internodes, the internode elongation pattern, the leaf angle and the morphological abnormality of leaf, except for the vertical length of seed and the seed weight. Our results suggest that the rice Galpha affects the BR signaling cascade but the Galpha may not be a signaling molecule in BRI1-meditated perception/transduction.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Oryza/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Transdução de Sinais , Alelos , Brassinosteroides , Colestanóis/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Genes de Plantas , Mutagênese , Oryza/genética , Oryza/crescimento & desenvolvimento , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , RNA de Plantas/genética , Esteroides Heterocíclicos/metabolismo
13.
Genes Genet Syst ; 84(1): 35-42, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19420799

RESUMO

It has been shown that the disruption of the alpha-subunit gene of heterotorimeric G-proteins (Galpha) results in dwarf traits, the erection of leaves and the setting of small seeds in rice. These mutants are called d1. We have studied the expression profiles of the transcripts and translation products of rice Galpha in ten alleles of d1 including five additional alleles newly identified. By RT-PCR, the transcripts of the Galpha gene were detected in the all d1 alleles. By western blot, the Galpha proteins were not detected in the plasma membrane fractions of the d1 alleles with the exception of d1-4. In d1-4, one amino acid change in the GTP-binding box A of the Galpha protein was occurred and even in this case the Galpha protein was only just detectable in the plasma membrane fraction. Given that the Galpha protein did not accumulate in the plasma membrane fraction in d1-8 which has a deletion of just a single amino acid in the Galpha protein, it is likely that a proper conformation of the Galpha is necessary for accumulation of Galpha protein in the plasma membrane. Nine alleles of d1 showed a severer phenotype whilst d1-4 exhibited a mild phenotype with respect to seed size and elongation pattern of internodes. As brassinosteroid signaling was known to be partially impaired in d1s, the sensitivity to 24-epibrassinolide (24-epiBL) was compared among d1 alleles in a T65 genetic background. Only d1-4 showed responses similar to wild type rice. The results show that the d1-4 mutant is a mild allele in terms of the phenotype and mild hyposensitivity to the exogenously applied 24-epiBL.


Assuntos
Alelos , Membrana Celular/enzimologia , Subunidades alfa de Proteínas de Ligação ao GTP/biossíntese , Oryza/enzimologia , Proteínas de Plantas/biossíntese , Brassinosteroides , Membrana Celular/genética , Colestanóis/farmacologia , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Oryza/genética , Fenótipo , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Esteroides Heterocíclicos/farmacologia
14.
Phytochemistry ; 156: 96-105, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30261341

RESUMO

Triterpenoid saponins are specialized metabolites, which are abundant in soybean seeds. They have a wide variety of effects on human health and physiology. The composition of sugar chain attached to the aglycone moiety of saponins can be controlled by genetic loci, such as Sg-1, 3, and 4. Among these, the homozygous recessive sg-4 impairs the accumulation of saponins that have an arabinose moiety at the second position of the C-3 sugar chain (i.e., saponins Ad and ßa) in the hypocotyls. In this study, we found that sg-4 cultivars are disabled in Glyma.01G046300 expression in hypocotyls. This gene encodes a putative glycosyltransferase (UGT73P10) and is a homolog of GmSGT2 (UGT73P2) whose recombinant protein has been previously shown, in vitro, to conjugate the second galactose moiety at the C-3 position of soyasapogenol B monoglucuronide (SBMG). The sg-4 phenotype (absence of saponins Ad and ßa in hypocotyls) was restored by introducing the Glyma.01G046300 genomic DNA fragment that was obtained from the Sg-4 cultivar 'Ibarakimame 7'. Although Glyma.01G046300 is expressed in the cotyledons even in the sg-4 cultivars such as 'Enrei', the induced premature stop codon mutation (W244*) resulted in impaired accumulation of saponin ßa in this tissue also in the 'Enrei' genetic background. Furthermore, the recombinant Glyma.01G046300 protein was shown to conjugate the second Ara moiety at the C-3 position of SBMG using UDP-Ara as a sugar donor. These results demonstrate that Sg-4 is responsible for conjugation of the second Ara moiety at the C-3 position of soybean saponins.


Assuntos
Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Saponinas/biossíntese , Saponinas/química , Açúcares/metabolismo , Configuração de Carboidratos , Açúcares/química
15.
FEMS Microbiol Lett ; 255(1): 115-20, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16436070

RESUMO

We tested the possibility of nonconjugative lateral DNA transfer in a colony biofilm of mixed Escherichia coli strains. By simply coculturing a plasmid-free F(-) strain and another F(-) strain harboring a nonconjugative plasmid in a colony biofilm on antibiotic-free agar media, transformed cells were produced within 24-48 h at the frequency of 10(-10)-10(-9) per recipient cell. PCR analysis of the transformed cells demonstrated the occurrence of lateral plasmid transfer. These cells survived until at least day 7 under antibiotic-free conditions. Liquid cultures of the same strains in Luria-Bertani broth produced no or few transformants, suggesting the importance of colony-biofilm formation for plasmid transfer. This is a novel line of evidence indicating that nonconjugative, nonviral horizontal gene transfer can occur between E. coli cells.


Assuntos
Biofilmes/crescimento & desenvolvimento , Escherichia coli/genética , Transferência Genética Horizontal , Plasmídeos/genética , Transformação Bacteriana/genética , Meios de Cultura , Escherichia coli/crescimento & desenvolvimento
16.
Plant Physiol Biochem ; 42(6): 477-84, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15246060

RESUMO

Hormonal regulation of expression of alpha-amylase II-4 that lacks the gibberellin-response cis-element (GARE) in the promoter region of the gene was studied in germinating rice (Oryza sativa L.) seeds. Temporal and spatial expression of alpha-amylase II-4 in the aleurone layer were essentially identical to those of alpha-amylase I-1 whose gene contains GARE, although these were distinguishable in the embryo tissues at the early stage of germination. The gibberellin-responsible expression of alpha-amylase II-4 was also similar to that of alpha-amylase I-1. However, the level of alpha-amylase II-4 mRNA was not increased by gibberellin, indicating that the transcriptional enhancement of alpha-amylase II-4 expression did not occur in the aleurone. Gibberellin stimulated the accumulation of 45Ca2+ into the intracellular secretory membrane system. In addition, several inhibitors for Ca2+ signaling, such as EGTA, neomycin, ruthenium red (RuR), and W-7 prevented the gibberellin-induced expression of alpha-amylase II-4 effectively. While the gibberellin-induced expression of alpha-amylase II-4 occurred normally in the aleurone layer of a rice dwarf mutant d1 which is defective in the alpha subunit of the heterotrimeric G protein. Based on these results, it was concluded that the posttranscriptional regulation of alpha-amylase II-4 expression by gibberellin operates in the aleurone layer of germinating rice seed, which is mediated by Ca2+ but not the G protein.


Assuntos
Regulação da Expressão Gênica de Plantas/fisiologia , Giberelinas/metabolismo , Oryza/enzimologia , Processamento Pós-Transcricional do RNA/genética , alfa-Amilases/genética , Sequência de Bases , Northern Blotting , Primers do DNA , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Germinação , Mutação , Oryza/efeitos dos fármacos , Oryza/genética , Oryza/crescimento & desenvolvimento , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
Plant Signal Behav ; 7(4): 443-6, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22499179

RESUMO

The deficient mutant for the rice heterotrimeric G protein α subunit gene (RGA1), d1, showed dwarfism and set small seed due to a reduced cell number. Mutants for the rice heterotrimeric G protein ß subunit gene (RGB1) have not been isolated. To determine the functions of RGB1, transgenic rice plants with suppressed expression of RGB1 were studied using the RNAi method. RGB1 knock-down lines showed browning of the lamina joint regions and nodes and reduced fertility, but these abnormality were not observed in d1. Transgenic plants in which the G protein ß subunit was greatly decreased were not obtained, suggesting that the complete suppression of RGB1 mRNA may be lethal. In contrast, the d1 mutants, with complete loss of the G protein α subunit, were fertile and half the size of the WT. These studies suggest that RGB1 has different functions than RGA1.


Assuntos
Subunidades beta da Proteína de Ligação ao GTP/deficiência , Subunidades beta da Proteína de Ligação ao GTP/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Oryza/genética , Proteínas de Plantas/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Técnicas de Silenciamento de Genes , Mutação/genética , Oryza/anatomia & histologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
19.
Plant Signal Behav ; 4(2): 126-8, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19649188

RESUMO

The alpha subunit of heterotrimeric G-proteins (G alpha) is involved in a broad range of aspects of the brassinosteroid (BR) response, such as the enhancement of lamina bending. However, it has been suggested from epistatic analysis of d1 and d61, which are mutants deficient for G alpha and the BR receptor BRI1, that G alpha and BRI1 may function via distinct pathways in many cases. In this study, we investigated further the genetic interaction between G alpha and BRI1. We report the analysis of transformants of T65d1 and T65d1/d61-7 into which were introduced a constitutively active form of G alpha, Q223L. The application of 24-epi-brassinolide (24-epiBL) to T65d1 expressing Q223L still resulted in elongation of the coleoptile and, in fact, it was enhanced over the wild-type plant (WT) level in a concentration dependent manner. In T65d1/d61-7 expressing Q223L, the seed size was enlarged over that of d61-7 due to activation of G alpha. These results suggest that Q223L is able to augment the BR response in response to 24-epiBL and also that Q223L functions independently of BRI1 in the process of determining seed morphology, given that Q223L was functional in the BRI1-deficient mutant, d61-7.

20.
Plant Cell Physiol ; 46(2): 381-6, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15695461

RESUMO

We used site-directed mutagenesis to engineer two constitutively active forms of the alpha subunit of a rice heterotrimeric G protein. The recombinant proteins produced from these novel cDNAs had GTP-binding activity but no GTPase activity. A chimeric gene for a constitutively active form of the alpha subunit was introduced into the rice mutant d1, which is defective for the alpha-subunit gene. All the transformants essentially showed a wild-type phenotype compared with normal cultivars, although seed sizes were substantially increased and internode lengths also showed some increase.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Oryza/genética , Oryza/metabolismo , Sequência de Aminoácidos , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
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