RESUMO
In plants, salicylic acid (SA) hydroxylation regulates SA homoeostasis, playing an essential role during plant development and response to pathogens. This reaction is catalysed by SA hydroxylase enzymes, which hydroxylate SA producing 2,3-dihydroxybenzoic acid (2,3-DHBA) and/or 2,5-dihydroxybenzoic acid (2,5-DHBA). Several SA hydroxylases have recently been identified and characterised from different plant species, but no such activity has yet been reported in maize. In this work, we describe the identification and characterisation of a new SA hydroxylase in maize plants. This enzyme, with high sequence similarity to previously described SA hydroxylases from Arabidopsis and rice, converts SA into 2,5-DHBA; however, it has different kinetic properties to those of previously characterised enzymes, and it also catalysers the conversion of the flavonoid dihydroquercetin into quercetin in in vitro activity assays, suggesting that the maize enzyme may have different roles in vivo to those previously reported from other species. Despite this, ZmS5H can complement the pathogen resistance and the early senescence phenotypes of Arabidopsis s3h mutant plants. Finally, we characterised a maize mutant in the S5H gene (s5hMu) that has altered growth, senescence and increased resistance against Colletotrichum graminicola infection, showing not only alterations in SA and 2,5-DHBA but also in flavonol levels. Together, the results presented here provide evidence that SA hydroxylases in different plant species have evolved to show differences in catalytic properties that may be important to fine tune SA levels and other phenolic compounds such as flavonols, to regulate different aspects of plant development and pathogen defence.
Assuntos
Colletotrichum , Resistência à Doença , Doenças das Plantas , Proteínas de Plantas , Ácido Salicílico , Zea mays , Zea mays/genética , Zea mays/enzimologia , Zea mays/microbiologia , Ácido Salicílico/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Resistência à Doença/genética , Doenças das Plantas/microbiologia , Doenças das Plantas/imunologia , Colletotrichum/fisiologia , Cinética , Ácidos Cetoglutáricos/metabolismo , Oxigenases de Função Mista/metabolismo , Oxigenases de Função Mista/genética , Regulação da Expressão Gênica de Plantas , Arabidopsis/genética , Arabidopsis/enzimologia , Arabidopsis/microbiologia , Gentisatos/metabolismo , Filogenia , Quercetina/metabolismo , HidroxibenzoatosRESUMO
This experiment evaluated the interactive effects among xylanase (XL; 0, 8,000, 16,000, and 32,000 BXU/kg), amino acid density (AA; high and low 10% difference), and additional fat (AF; 0 or +1.17%) applied postpellet in corn-soybean meal diets with dried distillers grains with solubles on performance, energy utilization, digestibility, and carcass traits in Ross 708 male broilers. A completely randomized block (pen location) design with 16 treatments arranged factorially (4 XL levels, 2 AA, and 2 AF) was analyzed using mixed models. No significant interactions or main effects were observed for feed intake at 49 d (P > 0.05) but chicks were heavier when consuming diets containing 0 or 8,000 BXU/kg (P = 0.015), high AA (P < 0.001), and 1.17% AF (P < 0.001). Feed efficiency did not vary with XL supplementation (P > 0.05) but was improved in broilers fed the higher AA and AF diet (P = 0.015 for AA × AF). AME, GE, and CP digestibility were assessed at days 17 and 42. There were multiple interactions observed at day 17 with a significant three-way showing that AME and CP digestibility improved when increasing the XL and AF levels in the high AA fed birds compared with the low-density diets. At day 42, XL and AF significantly affected AMEn, GE, or CP digestibility; however, there was a significant interaction between XL and AF. Diets supplemented with 1.17% AF improved AMEn significantly in broilers fed the highest XL level. Breast yield was not affected by treatments, but wing yield decreased with high AA density when diets contained 16,000 BXU/kg without differences for the other diets (P = 0.04 for XL × AA). Effects of XL, AA, and AF interactions on performance and cut-up-part yields have to be considered until day 42 for most of the variables studied. However, at 49 d of age, the dietary AA density and AF did not markedly influence the response to XL in maize-based diets.
Assuntos
Aminoácidos/administração & dosagem , Galinhas , Gorduras na Dieta/administração & dosagem , Endo-1,4-beta-Xilanases/administração & dosagem , Glycine max/enzimologia , Zea mays/enzimologia , Ração Animal/análise , Fenômenos Fisiológicos da Nutrição Animal , Animais , Galinhas/crescimento & desenvolvimento , Galinhas/metabolismo , Dieta/veterinária , Suplementos Nutricionais , Digestão , MasculinoRESUMO
The use of plant growth-promoting bacteria as agricultural inoculants of plants should be encouraged because of their prominent role in biological nitrogen fixation, the increase of nutrient uptake by roots, abiotic stress mitigation, and disease control. The complex mechanisms underlying the association between plant and beneficial bacteria have been increasingly studied, and proteomic tools can expand our perception regarding the fundamental molecular processes modulated by the interaction. In this study, we investigated the changes in protein expression in maize roots in response to treatment with the endophytic diazotrophic Herbaspirillum seropedicae and the activities of enzymes related to nitrogen metabolism. To identify maize proteins whose expression levels were altered in the presence of bacteria, a label-free quantitative proteomic approach was employed. Using this approach, we identified 123 differentially expressed proteins, of which 34 were upregulated enzymes, in maize roots cultivated with H. seropedicae. The maize root colonization of H. seropedicae modulated the differential expression of enzymes involved in the stress response, such as peroxidases, phenylalanine ammonia-lyase, and glutathione transferase. The differential protein profile obtained in the inoculated roots reflects the effect of colonization on plant growth and development compared with control plants.
Assuntos
Herbaspirillum/fisiologia , Proteínas de Plantas/metabolismo , Zea mays/enzimologia , Zea mays/microbiologia , Raízes de Plantas/enzimologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Proteômica , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismoRESUMO
BACKGROUND: The study of cold tolerance in maize seeds and seedlings through physiological quality assessments, as well as the genetic control associated with this trait, allows an early characterization of genotypes. Here we studied the genetic control for cold tolerance during the germination process in maize seeds and genes influenced by this stress. RESULTS: Six maize lines were used, three classified as tolerant and three as susceptible to low germination temperature. A field was developed to produce the hybrid seeds, in a partial diallel scheme including the reciprocal crosses. For the expression analysis, seeds from two contrasting lines were used, as well as their hybrid combination and their reciprocal crosses, on dried and moistened seeds at 10 °C for 4 and 7 days. It was evaluated the catalase (CAT) and esterase (EST) enzymes, heat-resistant proteins and the genes Putative stearoyl-ACP desaturase (SAD), Ascorbate Peroxidase (APX), Superoxide Dismutase (SOD) and Mitogen Activated Protein Kinase (ZmMPK5). The estimated values ââfor heterosis, general and specific combining abilities and reciprocal maternal and non-maternal effects were carried out and it showed that there is heterosis for germination at low temperatures, also the non-additive genes were more important and there was a reciprocal effect. CONCLUSIONS: There is a greater expression of the CAT and EST enzymes in moistened seeds at seven days and there is less expression of heat-resistant proteins and the SAD gene at seven days of moistening. Also, there are variations in the expression of the APX, SOD and ZmMPK5 genes in dried and moistened seeds, as well as among the genotypes studied.
Assuntos
Regulação da Expressão Gênica de Plantas , Germinação/genética , Termotolerância/genética , Zea mays/genética , Água , Zea mays/embriologia , Zea mays/enzimologiaRESUMO
Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic, site-directed mutagenesis and crystallographic results are consistent with allosteric activation, but the existence of a G6P-allosteric site was questioned and competitive activation-in which G6P would bind to the active site eliciting the same positive homotropic effect as the substrate phosphoenolpyruvate (PEP)-was proposed. Here, we report the crystal structure of the PEPC-C4 isozyme from Zea mays with G6P well bound into the previously proposed allosteric site, unambiguously confirming its existence. To test its functionality, Asp239-which participates in a web of interactions of the protein with G6P-was changed to alanine. The D239A variant was not activated by G6P but, on the contrary, inhibited. Inhibition was also observed in the wild-type enzyme at concentrations of G6P higher than those producing activation, and probably arises from G6P binding to the active site in competition with PEP. The lower activity and cooperativity for the substrate PEP, lower activation by glycine and diminished response to malate of the D239A variant suggest that the heterotropic allosteric activation effects of free-PEP are also abolished in this variant. Together, our findings are consistent with both the existence of the G6P-allosteric site and its essentiality for the activation of PEPC enzymes by phosphorylated compounds. Furthermore, our findings suggest a central role of the G6P-allosteric site in the overall kinetics of these enzymes even in the absence of G6P or other phospho-sugars, because of its involvement in activation by free-PEP.
Assuntos
Glucose-6-Fosfato/química , Fosfoenolpiruvato Carboxilase/química , Fosfoenolpiruvato/química , Proteínas de Plantas/química , Zea mays/enzimologia , Regulação Alostérica , Domínio Catalítico , Glucose-6-Fosfato/metabolismo , Cinética , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Zea mays/genéticaRESUMO
BACKGROUND: Plants reprogram metabolism and development to rapidly adapt to biotic and abiotic stress. Protein kinases play a significant role in this process by phosphorylating protein substrates that activate or inactivate signaling cascades that regulate cellular and metabolic adaptations. Despite their importance in plant biology, a notably small fraction of the plant kinomes has been studied to date. RESULTS: In this report, we describe ZmDRIK1, a stress-responsive receptor-like pseudokinase whose expression is downregulated under water restriction. We show the structural features and molecular basis of the absence of ATP binding exhibited by ZmDRIK1. The ZmDRIK1 kinase domain lacks conserved amino acids that are essential for phosphorylation activity. The crystal structure of the ZmDRIK1 kinase domain revealed the presence of a spine formed by the side chain of the triad Leu240, Tyr363, and Leu375 that occludes the ATP binding pocket. Although ZmDRIK1 is unable to bind nucleotides, it does bind the small molecule ENMD-2076 which, in a cocrystal structure, revealed the potential to serve as a ZmDRIK1 inhibitor. CONCLUSION: ZmDRIK1 is a novel receptor-like pseudokinase responsive to biotic and abiotic stress. The absence of ATP binding and consequently, the absence of phosphorylation activity, was proven by the crystal structure of the apo form of the protein kinase domain. The expression profiling of the gene encoding ZmDRIK1 suggests this kinase may play a role in downregulating the expression of stress responsive genes that are not necessary under normal conditions. Under biotic and abiotic stress, ZmDRIK1 is down-regulated to release the expression of these stress-responsive genes.
Assuntos
Trifosfato de Adenosina/química , Proteínas de Plantas/química , Receptores Proteína Tirosina Quinases/química , Zea mays/enzimologia , Cristalografia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Inibidores de Proteínas Quinases/química , Pirazóis/química , Pirimidinas/química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Estresse Fisiológico/genética , Zea mays/genéticaRESUMO
Cyclin dependent kinase A; 1 (CDKA; 1) is essential in G1/S transition of cell cycle and its oxidation has been implicated in cell cycle arrest during plant abiotic stress. In the present study, an evaluation at the molecular level was performed to find possible sites of protein oxidative modifications. In vivo studies demonstrated that carbonylation of maize CDKA,1 is associated with a decrease in complex formation with maize cyclin D (CycD). Control and in vitro oxidized recombinant CDKA; 1 were sequenced by mass spectrometry. Proline at the PSTAIRE cyclin-binding motif was identified as the most susceptible oxidation site by comparative analysis of the resulted peptides. The specific interaction between CDKA; 1 and CycD6; 1, measured by surface plasmon resonance (SPR), demonstrated that the affinity and the kinetic of the interaction depended on the reduced-oxidized state of the CDKA; 1. CDKA; 1 protein oxidative modification would be in part responsible for affecting cell cycle progression, and thus producing plant growth inhibition under oxidative stress.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Prolina/metabolismo , Zea mays/enzimologia , Sequência de Aminoácidos , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Ciclinas/química , Modelos Moleculares , Oxirredução , Prolina/química , Alinhamento de SequênciaRESUMO
In C4 grasses of agronomical interest, malate shuttled into the bundle sheath cells is decarboxylated mainly by nicotinamide adenine dinucleotide phosphate (NADP)-malic enzyme (C4-NADP-ME). The activity of C4-NADP-ME was optimized by natural selection to efficiently deliver CO2 to Rubisco. During its evolution from a plastidic non-photosynthetic NADP-ME, C4-NADP-ME acquired increased catalytic efficiency, tetrameric structure and pH-dependent inhibition by its substrate malate. Here, we identified specific amino acids important for these C4 adaptions based on strict differential conservation of amino acids, combined with solving the crystal structures of maize and sorghum C4-NADP-ME. Site-directed mutagenesis and structural analyses show that Q503, L544 and E339 are involved in catalytic efficiency; E339 confers pH-dependent regulation by malate, F140 is critical for the stabilization of the oligomeric structure and the N-terminal region is involved in tetramerization. Together, the identified molecular adaptations form the basis for the efficient catalysis and regulation of one of the central biochemical steps in C4 metabolism.
Assuntos
Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Sorghum/enzimologia , Zea mays/enzimologia , Motivos de Aminoácidos , Biocatálise , Domínio Catalítico , Concentração de Íons de Hidrogênio , Malato Desidrogenase/genética , Malatos/metabolismo , Fotossíntese , Proteínas de Plantas/genética , Sorghum/química , Sorghum/genética , Zea mays/química , Zea mays/genéticaRESUMO
Detoxification of methylglyoxal, a toxic by-product of central sugar metabolism, is a major issue for all forms of life. The glyoxalase pathway evolved to effectively convert methylglyoxal into d-lactate via a glutathione hemithioacetal intermediate. Recently, we have shown that the monomeric glyoxalase I from maize exhibits a symmetric fold with two cavities, potentially harboring two active sites, in analogy with homodimeric enzyme surrogates. Here we confirm that only one of the two cavities exhibits glyoxalase I activity and show that it adopts a tunnel-shaped structure upon substrate binding. Such conformational change gives rise to independent binding sites for glutathione and methylglyoxal in the same active site, with important implications for the molecular reaction mechanism, which has been a matter of debate for several decades. DATABASE: Structural data are available in The Protein Data Bank database under the accession numbers 6BNN, 6BNX, and 6BNZ.
Assuntos
Lactoilglutationa Liase/química , Substâncias Macromoleculares/química , Conformação Proteica , Zea mays/enzimologia , Sequência de Aminoácidos/genética , Domínio Catalítico/genética , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/ultraestrutura , Substâncias Macromoleculares/ultraestrutura , Dobramento de Proteína , Aldeído Pirúvico/química , Especificidade por Substrato , Açúcares/metabolismoRESUMO
The Proliferating Cell Nuclear Antigen, PCNA, has roles in both G1 and S phases of the cell cycle. Here we show that maize PCNA can be found in cells in structures of a trimer or a dimer of trimer, in complexes of high molecular mass that change in size as germination proceeds, co-eluting with cell cycle proteins as CycD3;1 and CDKs (A/B1;1). Using different methodological strategies, we show that PCNA actually interacts with CycD3;1, CDKA, CDKB1;1, KRP1;1 and KRP4;1, all of which contain PIP or PIP-like motifs. Anti-PCNA immunoprecipitates show kinase activity that is inhibited by KRP1;1 and KRP4;2, indicating the formation of quaternary complexes PCNA-CycD/CDKs-KRPs in which PCNA would act as a platform. This inhibitory effect seems to be differential during the germination process, more pronounced as germination advances, suggesting a complex regulatory mechanism in which PCNA could bind different sets of cyclins/CDKs, some more susceptible to inhibition by KRPs than others.