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1.
J Biol Chem ; 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32414842

RESUMO

Cyclotides are plant-derived peptides characterized by a ~30-amino-acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and one known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, M, and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide-membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity.

2.
ACS Chem Biol ; 15(4): 962-969, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32203656

RESUMO

Cyclotides are a class of cyclic disulfide-rich peptides found in plants that have been adopted as a molecular scaffold for pharmaceutical applications due to their inherent stability and ability to penetrate cell membranes. For research purposes, they are usually produced and cyclized synthetically, but there are concerns around the cost and environmental impact of large-scale chemical synthesis. One strategy to improve this is to combine a recombinant production system with native enzyme-mediated cyclization. Asparaginyl endopeptidases (AEPs) are enzymes that can act as peptide ligases in certain plants to facilitate cyclotide maturation. One of these ligases, OaAEP1b, originates from the cyclotide-producing plant, Oldenlandia affinis, and can be produced recombinantly for use in vitro as an alternative to chemical cyclization of recombinant substrates. However, not all engineered cyclotides are compatible with AEP-mediated cyclization because new pharmaceutical epitopes often replace the most flexible region of the peptide, where the native cyclization site is located. Here we redesign a popular cyclotide grafting scaffold, MCoTI-II, to incorporate an AEP cyclization site located away from the usual grafting region. We demonstrate the incorporation of a bioactive peptide sequence in the most flexible region of MCoTI-II while maintaining AEP compatibility, where the two were previously mutually exclusive. We anticipate that our AEP-compatible scaffold, based on the most popular cyclotide for pharmaceutical applications, will be useful in designing bioactive cyclotides that are compatible with AEP-mediated cyclization and will therefore open up the possibility of larger scale enzyme-mediated production of recombinant or synthetic cyclotides alike.

3.
Nat Commun ; 11(1): 1575, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221295

RESUMO

Asparaginyl endopeptidases (AEPs) catalyze the key backbone cyclization step during the biosynthesis of plant-derived cyclic peptides. Here, we report the identification of two AEPs from Momordica cochinchinensis and biochemically characterize MCoAEP2 that catalyzes the maturation of trypsin inhibitor cyclotides. Recombinantly produced MCoAEP2 catalyzes the backbone cyclization of a linear cyclotide precursor (MCoTI-II-NAL) with a kcat/Km of 620 mM-1 s-1, making it one of the fastest cyclases reported to date. We show that MCoAEP2 can mediate both the N-terminal excision and C-terminal cyclization of cyclotide precursors in vitro. The rate of cyclization/hydrolysis is primarily influenced by varying pH, which could potentially control the succession of AEP-mediated processing events in vivo. Furthermore, MCoAEP2 efficiently catalyzes the backbone cyclization of an engineered MCoTI-II analog with anti-angiogenic activity. MCoAEP2 provides enhanced synthetic access to structures previously inaccessible by direct chemistry approaches and enables the wider application of trypsin inhibitor cyclotides in biotechnology applications.

4.
Mar Biotechnol (NY) ; 22(2): 285-307, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32016679

RESUMO

Regeneration of a limb or tissue can be achieved through multiple different pathways and mechanisms. The sea anemone Exaiptasia pallida has been observed to have excellent regenerative proficiency, but this has not yet been described transcriptionally. In this study, we examined the genetic expression changes during a regenerative timecourse and reported key genes involved in regeneration and wound healing. We found that the major response was an early (within the first 8 h) upregulation of genes involved in cellular movement and cell communication, which likely contribute to a high level of tissue plasticity resulting in the rapid regeneration response observed in this species. We find the immune system was only transcriptionally active in the first 8 h post-amputation and conclude, in accordance with previous literature, that the immune system and regeneration have an inverse relationship. Fifty-nine genes (3.8% of total) differentially expressed during regeneration were identified as having no orthologues in other species, indicating that regeneration in E. pallida may rely on the activation of species-specific novel genes. Additionally, taxonomically restricted novel genes, including species-specific novels, and highly conserved genes were identified throughout the regenerative timecourse, showing that both may work in concert to achieve complete regeneration.

5.
Sci Rep ; 9(1): 10820, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31346249

RESUMO

Asparaginyl endopeptidases (AEPs) are a class of enzymes commonly associated with proteolysis in the maturation of seed storage proteins. However, a subset of AEPs work preferentially as peptide ligases, coupling release of a leaving group to formation of a new peptide bond. These "ligase-type" AEPs require only short recognition motifs to ligate a range of targets, making them useful tools in peptide and protein engineering for cyclisation of peptides or ligation of separate peptides into larger products. Here we report the recombinant expression, ligase activity and cyclisation kinetics of three new AEPs from the cyclotide producing plant Oldenlandia affinis with superior kinetics to the prototypical recombinant AEP ligase OaAEP1b. These AEPs work preferentially as ligases at both acidic and neutral pH and we term them "canonical AEP ligases" to distinguish them from other AEPs where activity preferences shift according to pH. We show that these ligases intrinsically favour ligation over hydrolysis, are highly efficient at cyclising two unrelated peptides and are compatible with organic co-solvents. Finally, we demonstrate the broad scope of recombinant AEPs in biotechnology by the backbone cyclisation of an intrinsically disordered protein, the 25 kDa malarial vaccine candidate Plasmodium falciparum merozoite surface protein 2 (MSP2).

6.
Front Plant Sci ; 10: 602, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31156672

RESUMO

The backbone cyclic and disulfide bridged sunflower trypsin inhibitor-1 (SFTI-1) peptide is a proven effective scaffold for a range of peptide therapeutics. For production at laboratory scale, solid phase peptide synthesis techniques are widely used, but these synthetic approaches are costly and environmentally taxing at large scale. Here, we developed a plant-based approach for the recombinant production of SFTI-1-based peptide drugs. We show that transient expression in Nicotiana benthamiana allows for rapid peptide production, provided that asparaginyl endopeptidase enzymes with peptide-ligase functionality are co-expressed with the substrate peptide gene. Without co-expression, no target cyclic peptides are detected, reflecting rapid in planta degradation of non-cyclized substrate. We test this recombinant production system by expressing a SFTI-1-based therapeutic candidate that displays potent and selective inhibition of human plasmin. By using an innovative multi-unit peptide expression cassette, we show that in planta yields reach ~60 µg/g dry weight at 6 days post leaf infiltration. Using nuclear magnetic resonance structural analysis and functional in vitro assays, we demonstrate the equivalence of plant and synthetically derived plasmin inhibitor peptide. The methods and insights gained in this study provide opportunities for the large scale, cost effective production of SFTI-1-based therapeutics.

7.
Front Plant Sci ; 10: 645, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31191573

RESUMO

The perennial leguminous herb Clitoria ternatea (butterfly pea) has attracted significant interest based on its agricultural and medical applications, which range from use as a fodder and nitrogen fixing crop, to applications in food coloring and cosmetics, traditional medicine and as a source of an eco-friendly insecticide. In this article we provide a broad multidisciplinary review that includes descriptions of the physical appearance, distribution, taxonomy, habitat, growth and propagation, phytochemical composition and applications of this plant. Notable amongst its repertoire of chemical components are anthocyanins which give C. ternatea flowers their characteristic blue color, and cyclotides, ultra-stable macrocyclic peptides that are present in all tissues of this plant. The latter are potent insecticidal molecules and are implicated as the bioactive agents in a plant extract used commercially as an insecticide. We include a description of the genetic origin of these peptides, which interestingly involve the co-option of an ancestral albumin gene to produce the cyclotide precursor protein. The biosynthesis step in which the cyclic peptide backbone is formed involves an asparaginyl endopeptidase, of which in C. ternatea is known as butelase-1. This enzyme is highly efficient in peptide ligation and has been the focus of many recent studies on peptide ligation and cyclization for biotechnological applications. The article concludes with some suggestions for future studies on this plant, including the need to explore possible synergies between the various peptidic and non-peptidic phytochemicals.

8.
Proc Natl Acad Sci U S A ; 116(16): 7831-7836, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30944220

RESUMO

Cyclotides are plant defense peptides that have been extensively investigated for pharmaceutical and agricultural applications, but key details of their posttranslational biosynthesis have remained elusive. Asparaginyl endopeptidases are crucial in the final stage of the head-to-tail cyclization reaction, but the enzyme(s) involved in the prerequisite steps of N-terminal proteolytic release were unknown until now. Here we use activity-guided fractionation to identify specific members of papain-like cysteine proteases involved in the N-terminal cleavage of cyclotide precursors. Through both characterization of recombinantly produced enzymes and in planta peptide cyclization assays, we define the molecular basis of the substrate requirements of these enzymes, including the prototypic member, here termed kalatase A. The findings reported here will pave the way for improving the efficiency of plant biofactory approaches for heterologous production of cyclotide analogs of therapeutic or agricultural value.


Assuntos
Ciclotídeos , Cisteína Proteases , Papaína , Proteínas de Plantas , Ciclotídeos/química , Ciclotídeos/metabolismo , Cisteína Proteases/química , Cisteína Proteases/metabolismo , Defensinas/química , Defensinas/metabolismo , Modelos Moleculares , Papaína/química , Papaína/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo
9.
J Exp Bot ; 69(3): 633-641, 2018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29309615

RESUMO

Cyclotides are ultra-stable, backbone-cyclized plant defence peptides that have attracted considerable interest in the pharmaceutical industry. This is due to their range of native bioactivities as well as their ability to stabilize other bioactive peptides within their framework. However, a hindrance to their widespread application is the lack of scalable, cost-effective production strategies. Plant-based production is an attractive, benign option since all biosynthetic steps are performed in planta. Nonetheless, cyclization in non-cyclotide-producing plants is poor. Here, we show that cyclic peptides can be produced efficiently in Nicotiana benthamiana, one of the leading plant-based protein production platforms, by co-expressing cyclotide precursors with asparaginyl endopeptidases that catalyse peptide backbone cyclization. This approach was successful in a range of other plants (tobacco, bush bean, lettuce, and canola), either transiently or stably expressed, and was applicable to both native and engineered cyclic peptides. We also describe the use of the transgenic system to rapidly identify new asparaginyl endopeptidase cyclases and interrogate their substrate sequence requirements. Our results pave the way for exploiting cyclotides for pest protection in transgenic crops as well as large-scale production of cyclic peptide pharmaceuticals in plants.


Assuntos
Cisteína Endopeptidases/metabolismo , Peptídeos Cíclicos/metabolismo , Proteínas de Plantas/metabolismo , Tabaco/metabolismo , Cisteína Endopeptidases/genética , Perfilação da Expressão Gênica , Peptídeos Cíclicos/genética , Proteínas de Plantas/genética , Tabaco/genética
10.
Plant Biotechnol J ; 14(12): 2240-2253, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27155090

RESUMO

Next-generation sequencing of complete genomes has given researchers unprecedented levels of information to study the multifaceted evolutionary changes that have shaped elite plant germplasm. In conjunction with population genetic analytical techniques and detailed online databases, we can more accurately capture the effects of domestication on entire biological pathways of agronomic importance. In this study, we explore the genetic diversity and signatures of selection in all predicted gene models of the storage starch synthesis pathway of Sorghum bicolor, utilizing a diversity panel containing lines categorized as either 'Landraces' or 'Wild and Weedy' genotypes. Amongst a total of 114 genes involved in starch synthesis, 71 had at least a single signal of purifying selection and 62 a signal of balancing selection and others a mix of both. This included key genes such as STARCH PHOSPHORYLASE 2 (SbPHO2, under balancing selection), PULLULANASE (SbPUL, under balancing selection) and ADP-glucose pyrophosphorylases (SHRUNKEN2, SbSH2 under purifying selection). Effectively, many genes within the primary starch synthesis pathway had a clear reduction in nucleotide diversity between the Landraces and wild and weedy lines indicating that the ancestral effects of domestication are still clearly identifiable. There was evidence of the positional rate variation within the well-characterized primary starch synthesis pathway of sorghum, particularly in the Landraces, whereby low evolutionary rates upstream and high rates downstream in the metabolic pathway were expected. This observation did not extend to the wild and weedy lines or the minor starch synthesis pathways.


Assuntos
Genoma de Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sorghum/genética , Sorghum/metabolismo , Amido/metabolismo
11.
New Phytol ; 210(2): 717-30, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26668107

RESUMO

Plants have evolved many strategies to protect themselves from attack, including peptide toxins that are ribosomally synthesized and thus adaptable directly by genetic polymorphisms. Certain toxins in Clitoria ternatea (butterfly pea) are cyclic cystine-knot peptides of c. 30 residues, called cyclotides, which have co-opted the plant's albumin-1 gene family for their production. How butterfly pea albumin-1 genes were commandeered and how these cyclotides are utilized in defence remain unclear. The role of cyclotides in host plant ecology and biotechnological applications requires exploration. We characterized the sequence diversity and expression dynamics of precursor and processing proteins implicated in butterfly pea cyclotide biosynthesis by expression profiling through RNA-sequencing (RNA-seq). Peptide-enriched extracts from various organs were tested for activity against insect-like membranes and the model nematode Caenorhabditis elegans. We found that the evolution and deployment of cyclotides involved their diversification to exhibit different chemical properties and expression between organs facing different defensive challenges. Cyclotide-enriched fractions from soil-contacting organs were effective at killing nematodes, whereas similar enriched fractions from aerial organs contained cyclotides that exhibited stronger interactions with insect-like membrane lipids. Cyclotides are employed as versatile and combinatorial mediators of defence in C. ternatea and have specialized to affect different classes of attacking organisms.


Assuntos
Evolução Molecular , Genes de Plantas , Peptídeos Cíclicos/metabolismo , Plantas/genética , Plantas/imunologia , Sequência de Aminoácidos , Análise por Conglomerados , Sequência Conservada , Regulação da Expressão Gênica de Plantas , Especificidade de Órgãos/genética , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/química , Proteínas de Plantas/metabolismo , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Solo/química , Especificidade da Espécie , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Água
12.
Nat Commun ; 6: 10199, 2015 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-26680698

RESUMO

Cyclotides are diverse plant backbone cyclized peptides that have attracted interest as pharmaceutical scaffolds, but fundamentals of their biosynthetic origin remain elusive. Backbone cyclization is a key enzyme-mediated step of cyclotide biosynthesis and confers a measure of stability on the resultant cyclotide. Furthermore, cyclization would be desirable for engineered peptides. Here we report the identification of four asparaginyl endopeptidases (AEPs), proteases implicated in cyclization, from the cyclotide-producing plant Oldenlandia affinis. We recombinantly express OaAEP1b and find it functions preferably as a cyclase by coupling C-terminal cleavage of propeptide substrates with backbone cyclization. Interestingly, OaAEP1b cannot cleave at the N-terminal site of O. affinis cyclotide precursors, implicating additional proteases in cyclotide biosynthesis. Finally, we demonstrate the broad utility of this enzyme by cyclization of peptides unrelated to cyclotides. We propose that recombinant OaAEP1b is a powerful tool for use in peptide engineering applications where increased stability of peptide products is desired.


Assuntos
Cisteína Endopeptidases/metabolismo , Proteínas de Plantas/metabolismo , RNA Mensageiro/metabolismo , Ciclização , Cisteína Endopeptidases/genética , Perfilação da Expressão Gênica , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Oldenlandia , Peptídeos/metabolismo , Proteínas Recombinantes
13.
J Allergy Clin Immunol ; 135(1): 133-42, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25129679

RESUMO

BACKGROUND: Genomic data are lacking for many allergen sources. To circumvent this limitation, we implemented a strategy to reveal the repertoire of pollen allergens of a grass with clinical importance in subtropical regions, where an increasing proportion of the world's population resides. OBJECTIVE: We sought to identify and immunologically characterize the allergenic components of the Panicoideae Johnson grass pollen (JGP; Sorghum halepense). METHODS: The total pollen transcriptome, proteome, and allergome of JGP were documented. Serum IgE reactivities with pollen and purified allergens were assessed in 64 patients with grass pollen allergy from a subtropical region. RESULTS: Purified Sor h 1 and Sor h 13 were identified as clinically important allergen components of JGP with serum IgE reactivity in 49 (76%) and 28 (43.8%), respectively, of patients with grass pollen allergy. Within whole JGP, multiple cDNA transcripts and peptide spectra belonging to grass pollen allergen families 1, 2, 4, 7, 11, 12, 13, and 25 were identified. Pollen allergens restricted to subtropical grasses (groups 22-24) were also present within the JGP transcriptome and proteome. Mass spectrometry confirmed the IgE-reactive components of JGP included isoforms of Sor h 1, Sor h 2, Sor h 13, and Sor h 23. CONCLUSION: Our integrated molecular approach revealed qualitative differences between the allergenic components of JGP and temperate grass pollens. Knowledge of these newly identified allergens has the potential to improve specific diagnosis and allergen immunotherapy treatment for patients with grass pollen allergy in subtropical regions and reduce the burden of allergic respiratory disease globally.


Assuntos
Alérgenos/imunologia , Pólen/imunologia , Rinite Alérgica/imunologia , Sorghum/imunologia , Adulto , Antígenos de Plantas/imunologia , Feminino , Humanos , Imunoglobulina E/sangue , Masculino , Pessoa de Meia-Idade , Extratos Vegetais/farmacologia , Proteínas de Plantas/imunologia , Proteoma , Rinite Alérgica/sangue , Testes Cutâneos , Transcriptoma , Clima Tropical
14.
J Agric Food Chem ; 62(40): 9819-31, 2014 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-25177767

RESUMO

Grain protein composition determines quality traits, such as value for food, feedstock, and biomaterials uses. The major storage proteins in sorghum are the prolamins, known as kafirins. Located primarily on the periphery of the protein bodies surrounding starch, cysteine-rich ß- and γ-kafirins may limit enzymatic access to internally positioned α-kafirins and starch. An integrated approach was used to characterize sorghum with allelic variation at the kafirin loci to determine the effects of this genetic diversity on protein expression. Reversed-phase high performance liquid chromatography and lab-on-a-chip analysis showed reductions in alcohol-soluble protein in ß-kafirin null lines. Gel-based separation and liquid chromatography-tandem mass spectrometry identified a range of redox active proteins affecting storage protein biochemistry. Thioredoxin, involved in the processing of proteins at germination, has reported impacts on grain digestibility and was differentially expressed across genotypes. Thus, redox states of endosperm proteins, of which kafirins are a subset, could affect quality traits in addition to the expression of proteins.


Assuntos
Endosperma/química , Proteínas de Plantas/análise , Sorghum/química , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese em Gel de Poliacrilamida , Genótipo , Glutarredoxinas/metabolismo , Dispositivos Lab-On-A-Chip , Mutação , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prolaminas/metabolismo , Proteômica , Proteínas de Armazenamento de Sementes/metabolismo , Sorghum/genética , Espectrometria de Massas em Tandem , Tiorredoxinas/metabolismo
15.
BMC Res Notes ; 7: 488, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-25084827

RESUMO

BACKGROUND: The sequencing, de novo assembly and annotation of transcriptome datasets generated with next generation sequencing (NGS) has enabled biologists to answer genomic questions in non-model species with unprecedented ease. Reliable and accurate de novo assembly and annotation of transcriptomes, however, is a critically important step for transcriptome assemblies generated from short read sequences. Typical benchmarks for assembly and annotation reliability have been performed with model species. To address the reliability and accuracy of de novo transcriptome assembly in non-model species, we generated an RNAseq dataset for an intertidal gastropod mollusc species, Nerita melanotragus, and compared the assembly produced by four different de novo transcriptome assemblers; Velvet, Oases, Geneious and Trinity, for a number of quality metrics and redundancy. RESULTS: Transcriptome sequencing on the Ion Torrent PGM™ produced 1,883,624 raw reads with a mean length of 133 base pairs (bp). Both the Trinity and Oases de novo assemblers produced the best assemblies based on all quality metrics including fewer contigs, increased N50 and average contig length and contigs of greater length. Overall the BLAST and annotation success of our assemblies was not high with only 15-19% of contigs assigned a putative function. CONCLUSIONS: We believe that any improvement in annotation success of gastropod species will require more gastropod genome sequences, but in particular an increase in mollusc protein sequences in public databases. Overall, this paper demonstrates that reliable and accurate de novo transcriptome assemblies can be generated from short read sequencers with the right assembly algorithms.


Assuntos
Gastrópodes/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Anotação de Sequência Molecular , Transcriptoma/genética , Animais , Mapeamento de Sequências Contíguas , Ontologia Genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Especificidade da Espécie
16.
Nat Commun ; 4: 2320, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23982223

RESUMO

Sorghum is a food and feed cereal crop adapted to heat and drought and a staple for 500 million of the world's poorest people. Its small diploid genome and phenotypic diversity make it an ideal C4 grass model as a complement to C3 rice. Here we present high coverage (16-45 × ) resequenced genomes of 44 sorghum lines representing the primary gene pool and spanning dimensions of geographic origin, end-use and taxonomic group. We also report the first resequenced genome of S. propinquum, identifying 8 M high-quality SNPs, 1.9 M indels and specific gene loss and gain events in S. bicolor. We observe strong racial structure and a complex domestication history involving at least two distinct domestication events. These assembled genomes enable the leveraging of existing cereal functional genomics data against the novel diversity available in sorghum, providing an unmatched resource for the genetic improvement of sorghum and other grass species.


Assuntos
Produtos Agrícolas/genética , Grão Comestível/genética , Genoma de Planta/genética , Análise de Sequência de DNA , Sorghum/genética , África , Genótipo , Desequilíbrio de Ligação/genética , Mutação/genética , Filogenia , Polimorfismo de Nucleotídeo Único/genética , Seleção Genética
17.
Nat Commun ; 4: 1483, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23403584

RESUMO

The production of adequate agricultural outputs to support the growing human population places great demands on agriculture, especially in light of ever-greater restrictions on input resources. Sorghum is a drought-adapted cereal capable of reliable production where other cereals fail, and thus represents a good candidate to address food security as agricultural inputs of water and arable land grow scarce. A long-standing issue with sorghum grain is that it has an inherently lower digestibility. Here we show that a low-frequency allele type in the starch metabolic gene, pullulanase, is associated with increased digestibility, regardless of genotypic background. We also provide evidence that the beneficial allele type is not associated with deleterious pleiotropic effects in the modern field environment. We argue that increasing the digestibility of an adapted crop is a viable way forward towards addressing food security while maximizing water and land-use efficiency.


Assuntos
Adaptação Fisiológica/genética , Alelos , Secas , Abastecimento de Alimentos , Genes de Plantas/genética , Variação Genética , Sorghum/genética , Sequência de Aminoácidos , Grão Comestível/genética , Grão Comestível/fisiologia , Frequência do Gene/genética , Genótipo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Humanos , Endogamia , Dados de Sequência Molecular , Mutação/genética , Peptídeos/química , Fenótipo , Estrutura Terciária de Proteína , Sorghum/fisiologia
18.
PLoS One ; 6(8): e23041, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21853065

RESUMO

Detecting artificial selection in the genome of domesticated species can not only shed light on human history but can also be beneficial to future breeding strategies. Evidence for selection has been documented in domesticated species including maize and rice, but few studies have to date detected signals of artificial selection in the Sorghum bicolor genome. Based on evidence that domesticated S. bicolor and its wild relatives show significant differences in endosperm structure and quality, we sequenced three candidate seed storage protein (kafirin) loci and three candidate starch biosynthesis loci to test whether these genes show non-neutral evolution resulting from the domestication process. We found strong evidence of non-neutral selection at the starch synthase IIa gene, while both starch branching enzyme I and the beta kafirin gene showed weaker evidence of non-neutral selection. We argue that the power to detect consistent signals of non-neutral selection in our dataset is confounded by the absence of low frequency variants at four of the six candidate genes. A future challenge in the detection of positive selection associated with domestication in sorghum is to develop models that can accommodate for skewed frequency spectrums.


Assuntos
Produtos Agrícolas/genética , Evolução Molecular , Deriva Genética , Mutação/genética , Sorghum/genética , Sequência de Bases , Loci Gênicos/genética , Haplótipos/genética , Humanos
19.
Plant J ; 64(2): 304-17, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21070410

RESUMO

Transcriptome analysis using the Affymetrix ATH1 platform has been completed on purified trichomes from the gl3-sst mutant. These trichomes display immature features, such as glassy cell walls and blunted branches. The gl3-sst trichome transcriptome was greatly enriched for genes involved in lipid biosynthesis, including those mediating the synthesis of fatty acids and wax. In addition, gl3-sst trichomes displayed reduced expression of the R3 MYBs TRY and CPC, which normally function to limit trichome development. The expression of the MIXTA-like MYB gene NOK was elevated. Members of the MIXTA-like family promote conical cell outgrowth, and in some cases, trichome initiation in diverse plant species. In contrast, NOK limits trichome outgrowth in wild-type Arabidopsis plants. Similar to other MIXTA-like genes, NOK was required for the expansion of gl3-sst trichomes, as the gl3-sst nok double mutant trichomes were greatly reduced in size. Expression of NOK in nok mutants reduced branch formation, whereas in gl3-sst nok, NOK expression promoted trichome cell outgrowth, illustrating duel roles for NOK in both promoting and limiting trichome development. MIXTA-like genes from phylogenetically diverse plant species could substitute for NOK in both nok and gl3-sst nok backgrounds. These findings suggest that certain aspects of NOK and MIXTA-like gene function have been conserved.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Morfogênese , Folhas de Planta/metabolismo , Fatores de Transcrição/metabolismo , Antirrhinum/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Dendrobium/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Medicago/genética , Mutação , Fenótipo , Folhas de Planta/crescimento & desenvolvimento , Fatores de Transcrição/genética , Regulação para Cima
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