RESUMO
Pre-harvest sprouting of cereals greatly reduces yield and quality of the grains. Abscisic acid (ABA) is an essential phytohormone for the induction and maintenance of seed dormancy. In this study, the ABA responsive promoter-driven ABA biosynthesis gene system was introduced to common wheat (Triticum aestivum L.) to enhance ABA production in the embryos and pre-harvest sprouting tolerance of the grains. This system consists of a wheat ABA responsive element containing Early-Methionine-labelled (EM) promoter and a sorghum 9-cis-epoxycarotenoid dioxygenase (SbNCED) gene which encodes an ABA biosynthesis rate-limiting enzyme. Twenty-three independent single-insertion lines were obtained, from which five homozygous lines showing various SbNCED expression levels were selected. Correlations were observed between SbNCED expression, ABA accumulation in the embryos and enhanced dormancy levels of the grains. The engineered wheat grains exhibited a few day-delay in germination, which should be effective in reducing pre-harvest sprouting damage. However, the increase in ABA levels in the recombinant grains was moderate, which explains why germination was not completely suppressed. Further analysis indicated a concomitant increase in the expression of the ABA catabolic enzyme gene TaABA8'OH1 and in the levels of isoleucine-conjugated jasmonic acid, implying the presence of possible negative feedback regulation in the innate system, which should be overcome for future technology development. These findings advance an understanding of the regulatory mechanisms of hormone metabolism in seeds and facilitate the development of pre-harvest sprouting tolerance in cereal grains.
RESUMO
The DELAY OF GERMINATION1 (DOG1) family genes (DFGs) in Arabidopsis thaliana are involved in seed dormancy, reserve accumulation, and desiccation tolerance. Decoding the molecular evolution of DFGs is key to understanding how these seed programs evolved. This article demonstrates that DFGs have diverged in the four lineages DOG1, DOG1-LIKE4 (DOGL4), DOGL5 and DOGL6, whereas DOGL1, DOGL2 and DOGL3 arose separately within the DOG1 lineage. The systematic DFG nomenclature proposed in this article addresses the current issues of inconsistent DFG annotation and highlights DFG genomic synteny in angiosperms. DFG pseudogenes, or collapsed coding sequences, hidden in the genomes of early-diverging angiosperms are documented here. They suggest ancient birth and loss of DFGs over the course of angiosperm evolution. The proposed models suggest that the origin of DFG diversification dates back to the most recent common ancestor of living angiosperms. The presence of a single form of DFG in nonflowering plants is discussed. Phylogenetic analysis of gymnosperm, lycophyte, and liverwort DFGs and similar genes found in mosses and algae suggests that DFGs diverged from the TGACG motif-binding transcription factor genes before the divergence of the bryophyte lineage.
Assuntos
Proteínas de Arabidopsis , Proteínas de Arabidopsis/metabolismo , Evolução Molecular , Duplicação Gênica , Regulação da Expressão Gênica de Plantas , Germinação , Filogenia , Sementes/metabolismoRESUMO
Genetic studies of seed maturation regulators, combining transcriptomics and network analysis, suggest the significance of genetic diversification for maturation programs, particularly in seed plants. By contrast, analogs of the maturation programs, such as dormancy and desiccation tolerance, are also found in non-seed plants. It is thus conceivable that seeds recalled or renovated ancient programs of stress-induced growth arrest that were already present in ferns, bryophytes, and possibly in algae. This opinion article discusses the origins and genetic diversification of the abscisic acid (ABA)-dependent seed maturation programs by addressing questions provoked by recent findings about the DELAY OF GERMINATION1 (DOG1) family proteins, which regulate reserve accumulation, dormancy, and/or desiccation tolerance in seeds in a gene-specific or redundant manner.
Assuntos
Ácido Abscísico/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Germinação , Dormência de Plantas , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Sementes/crescimento & desenvolvimento , Prova Pericial , Sementes/efeitos dos fármacos , Sementes/genéticaRESUMO
More than 70% of global food supply depends on seeds. The major seed reserves, such as proteins, lipids, and polysaccharides, are produced during seed maturation. Here, we report that DELAY OF GERMINATION 1-LIKE 4 (DOGL4) is a major inducer of reserve accumulation during seed maturation. The DOGL family proteins are plant-specific proteins of largely unknown biochemical function. DOGL4 shares only limited homology in amino acid sequence with DOG1, a major regulator of seed dormancy. DOGL4 was identified as one of the outstanding abscisic acid (ABA)-induced genes in our RNA sequencing analysis, whereas DOG1 was not induced by ABA. Induction of DOGL4 caused the expression of 70 seed maturation-specific genes, even in germinating seeds, including the major seed reserves ALBUMIN, CRUCIFERIN and OLEOSIN. Although DOG1 affects the expression of many seed maturation genes, the major seed reserve genes induced by DOGL4 are not altered by the dog1 mutation. Furthermore, the reduced dormancy and longevity phenotypes observed in the dog1 seeds were not observed in the dogl4 mutants, suggesting that these two genes have limited functional overlap. Taken together, these results suggest that DOGL4 is a central factor mediating reserve accumulation in seeds, and that the two DOG1 family proteins have diverged over the course of evolution into independent regulators of seed maturation, but retain some overlapping function.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Germinação/genética , Sementes/genética , Fatores de Transcrição/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/classificação , Proteínas de Ligação a DNA/classificação , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Germinação/efeitos dos fármacos , Fenótipo , Filogenia , Reguladores de Crescimento de Plantas/farmacologia , Plantas Geneticamente Modificadas , Sementes/crescimento & desenvolvimento , Fatores de Transcrição/classificaçãoRESUMO
Vivipary, germination of seeds on the maternal plant, is observed in nature and provides ecological advantages in certain wild species, such as mangroves. However, precocious seed germination in agricultural species, such as preharvest sprouting (PHS) in cereals, is a serious issue for food security. PHS reduces grain quality and causes economical losses to farmers. PHS can be prevented by translating the basic knowledge of hormone biology in seeds into technologies. Biosynthesis of abscisic acid (ABA), which is an essential hormone for seed dormancy, can be engineered to enhance dormancy and prevent PHS. Enhancing nine-cis-epoxycarotenoid dioxygenase (NCED), a rate-limiting enzyme of ABA biosynthesis, through a chemically induced gene expression system, has successfully been used to suppress germination of Arabidopsis seeds. The more advanced system NCED positive-feedback system, which amplifies ABA biosynthesis in a seed-specific manner without chemical induction, has also been developed. The proofs of concept established in the model species are now ready to be applied to crops. A potential problem is recovery of germination from hyperdormant crop grains. Hyperdormancy induced by the NCED systems can be reversed by inducing counteracting genes, such as NCED RNA interference or gibberellin (GA) biosynthesis genes. Alternatively, seed sensitivity to ABA can be modified to rescue germination using the knowledge of chemical biology. ABA antagonists, which were developed recently, have great potential to recover germination from the hyperdormant seeds. Combination of the dormancy-imposing and -releasing approaches will establish a comprehensive technology for PHS prevention and germination recovery.
RESUMO
Vibrio cholerae, an etiological agent of cholera, circulates between aquatic reservoirs and the human gastrointestinal tract. The type II secretion (T2S) system plays a pivotal role in both stages of the lifestyle by exporting multiple proteins, including cholera toxin. Here, we studied the kinetics of expression of genes encoding the T2S system and its cargo proteins. We have found that under laboratory growth conditions, the T2S complex was continuously expressed throughout V. cholerae growth, whereas there was growth phase-dependent transcriptional activity of genes encoding different cargo proteins. Moreover, exposure of V. cholerae to different environmental cues encountered by the bacterium in its life cycle induced transcriptional expression of T2S. Subsequent screening of a V. cholerae genomic library suggested that σ(E) stress response, phosphate metabolism, and the second messenger 3',5'-cyclic diguanylic acid (c-di-GMP) are involved in regulating transcriptional expression of T2S. Focusing on σ(E), we discovered that the upstream region of the T2S operon possesses both the consensus σ(E) and σ(70) signatures, and deletion of the σ(E) binding sequence prevented transcriptional activation of T2S by RpoE. Ectopic overexpression of σ(E) stimulated transcription of T2S in wild-type and isogenic ΔrpoE strains of V. cholerae, providing additional support for the idea that the T2S complex belongs to the σ(E) regulon. Together, our results suggest that the T2S pathway is characterized by the growth phase-dependent expression of genes encoding cargo proteins and requires a multifactorial regulatory network to ensure appropriate kinetics of the secretory traffic and the fitness of V. cholerae in different ecological niches.
Assuntos
Proteínas de Bactérias/metabolismo , Fator sigma/metabolismo , Vibrio cholerae/metabolismo , Proteínas de Bactérias/genética , Clonagem Molecular , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/fisiologia , Fator sigma/genética , Fatores de Tempo , Transcrição GênicaRESUMO
Abscisic acid is an essential hormone for seed dormancy. Our previous study using the plant gene switch system, a chemically induced gene expression system, demonstrated that induction of 9-cis-epoxycarotenoid dioxygenase (NCED), a rate-limiting ABA biosynthesis gene, was sufficient to suppress germination in imbibed Arabidopsis seeds. Here, we report development of an efficient experimental system that causes amplification of NCED expression during seed maturation. The system was created with a Triticum aestivum promoter containing ABA responsive elements (ABREs) and a Sorghum bicolor NCED to cause ABA-stimulated ABA biosynthesis and signaling, through a positive feedback mechanism. The chimeric gene pABRE:NCED enhanced NCED and ABF (ABRE-binding factor) expression in Arabidopsis Columbia-0 seeds, which caused 9- to 73-fold increases in ABA levels. The pABRE:NCED seeds exhibited unusually deep dormancy which lasted for more than 3 months. Interestingly, the amplified ABA pathways also caused enhanced expression of Arabidopsis NCED5, revealing the presence of positive feedback in the native system. These results demonstrated the robustness of positive feedback mechanisms and the significance of NCED expression, or single metabolic change, during seed maturation. The pABRE:NCED system provides an excellent experimental system producing dormant and non-dormant seeds of the same maternal origin, which differ only in zygotic ABA. The pABRE:NCED seeds contain a GFP marker which enables seed sorting between transgenic and null segregants and are ideal for comparative analysis. In addition to its utility in basic research, the system can also be applied to prevention of pre-harvest sprouting during crop production, and therefore contributes to translational biology.
Assuntos
Ácido Abscísico/metabolismo , Dioxigenases/genética , Retroalimentação Fisiológica/fisiologia , Proteínas de Plantas/genética , Sementes/metabolismo , Sorghum/genética , Triticum/genética , Ácido Abscísico/biossíntese , Arabidopsis/genética , Arabidopsis/metabolismo , Dioxigenases/metabolismo , Germinação/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Dormência de Plantas , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Sementes/genética , Sementes/crescimento & desenvolvimentoRESUMO
The ascomycete fungus Tolypocladium inflatum, a pathogen of beetle larvae, is best known as the producer of the immunosuppressant drug cyclosporin. The draft genome of T. inflatum strain NRRL 8044 (ATCC 34921), the isolate from which cyclosporin was first isolated, is presented along with comparative analyses of the biosynthesis of cyclosporin and other secondary metabolites in T. inflatum and related taxa. Phylogenomic analyses reveal previously undetected and complex patterns of homology between the nonribosomal peptide synthetase (NRPS) that encodes for cyclosporin synthetase (simA) and those of other secondary metabolites with activities against insects (e.g., beauvericin, destruxins, etc.), and demonstrate the roles of module duplication and gene fusion in diversification of NRPSs. The secondary metabolite gene cluster responsible for cyclosporin biosynthesis is described. In addition to genes necessary for cyclosporin biosynthesis, it harbors a gene for a cyclophilin, which is a member of a family of immunophilins known to bind cyclosporin. Comparative analyses support a lineage specific origin of the cyclosporin gene cluster rather than horizontal gene transfer from bacteria or other fungi. RNA-Seq transcriptome analyses in a cyclosporin-inducing medium delineate the boundaries of the cyclosporin cluster and reveal high levels of expression of the gene cluster cyclophilin. In medium containing insect hemolymph, weaker but significant upregulation of several genes within the cyclosporin cluster, including the highly expressed cyclophilin gene, was observed. T. inflatum also represents the first reference draft genome of Ophiocordycipitaceae, a third family of insect pathogenic fungi within the fungal order Hypocreales, and supports parallel and qualitatively distinct radiations of insect pathogens. The T. inflatum genome provides additional insight into the evolution and biosynthesis of cyclosporin and lays a foundation for further investigations of the role of secondary metabolite gene clusters and their metabolites in fungal biology.
Assuntos
Besouros/microbiologia , Ciclosporina/metabolismo , Hypocreales/genética , Complexos Multienzimáticos/genética , Peptídeo Sintases/genética , Animais , Evolução Molecular , Transferência Genética Horizontal , Genoma , Hypocreales/enzimologia , Complexos Multienzimáticos/metabolismo , Família Multigênica , Peptídeo Sintases/metabolismo , Filogenia , Análise de Sequência de RNARESUMO
Chmp2b is closely related to Vps2, a key component of the yeast protein complex that creates the intralumenal vesicles of multivesicular bodies. Dominant negative mutations in Chmp2b cause autophagosome accumulation and neurodegenerative disease. Loss of Chmp2b causes failure of dendritic spine maturation in cultured neurons. The homeobox gene Lbx1 plays an essential role in specifying postmitotic dorsal interneuron populations during late pattern formation in the neural tube. We have discovered that Chmp2b is one of the most highly regulated cell-autonomous targets of Lbx1 in the embryonic mouse neural tube. Chmp2b was expressed and depended on Lbx1 in only two of the five nascent, Lbx1-expressing, postmitotic, dorsal interneuron populations. It was also expressed in neural tube cell populations that lacked Lbx1 protein. The observed population-specific expression of Chmp2b indicated that only certain population-specific combinations of sequence specific transcription factors allow Chmp2b expression. The cell populations that expressed Chmp2b corresponded, in time and location, to neurons that make the first synapses of the spinal cord. Chmp2b protein was transported into neurites within the motor- and association-neuropils, where the first synapses are known to form between E11.5 and E12.5 in mouse neural tubes. Selective, developmentally-specified gene expression of Chmp2b may therefore be used to endow particular neuronal populations with the ability to mature dendritic spines. Such a mechanism could explain how mammalian embryos reproducibly establish the disynaptic cutaneous reflex only between particular cell populations.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas Musculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Animais , Dendritos/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos ICR , Neurônios Motores/metabolismo , Fibras Nervosas/metabolismo , Proteínas do Tecido Nervoso/genética , Tubo Neural/citologia , Tubo Neural/embriologia , Tubo Neural/metabolismoRESUMO
Full understanding of mechanisms that control seed dormancy and germination remains elusive. Whereas it has been proposed that translational control plays a predominant role in germination, other studies suggest the importance of specific gene expression patterns in imbibed seeds. Transgenic plants were developed to permit conditional expression of a gene encoding 9-cis-epoxycarotenoid dioxygenase 6 (NCED6), a rate-limiting enzyme in abscisic acid (ABA) biosynthesis, using the ecdysone receptor-based plant gene switch system and the ligand methoxyfenozide. Induction of NCED6 during imbibition increased ABA levels more than 20-fold and was sufficient to prevent seed germination. Germination suppression was prevented by fluridone, an inhibitor of ABA biosynthesis. In another study, induction of the NCED6 gene in transgenic seeds of nondormant mutants tt3 and tt4 reestablished seed dormancy. Furthermore, inducing expression of NCED6 during seed development suppressed vivipary, precocious germination of developing seeds. These results indicate that expression of a hormone metabolism gene in seeds can be a sole determinant of dormancy. This study opens the possibility of developing a robust technology to suppress or promote seed germination through engineering pathways of hormone metabolism.