RESUMEN
Given the prevalence of the malignant weed Chinese Sprangletop (Leptochloa chinensis (L.) Nees) in rice fields, the development of novel herbicides against this weed has aroused wide interest. Here, we report a novel diphenyl ether-pyrimidine hybrid, DEP-5, serving as a systematic pre/postemergence herbicide candidate for broad-spectrum weed control in rice fields, specifically for L. chinensis. Notably, DEP-5 exhibits over 80% herbicidal activity against the resistant biotypes even at 37.5 g a.i./ha under greenhouse conditions and has complete control of L. chinensis at 150 g a.i./ha in the rice fields. We uncover that DEP-5 acts as a noncompetitive inhibitor of acetohydroxyacid synthase (AHAS) with an inhibition constant (Ki) of 39.4 µM. We propose that DEP-5 binds to AHAS in two hydrophobic-driven binding modes that differ from commercial AHAS inhibitors. Overall, these findings demonstrate that DEP-5 has great potential to be developed into a herbicide for L. chinensis control and inspire fresh concepts for novel AHAS-inhibiting herbicide design.
Asunto(s)
Acetolactato Sintasa , Herbicidas , Oryza , Proteínas de Plantas , Malezas , Poaceae , Control de Malezas , Herbicidas/farmacología , Herbicidas/química , Oryza/química , Malezas/efectos de los fármacos , Malezas/enzimología , Acetolactato Sintasa/metabolismo , Acetolactato Sintasa/antagonistas & inhibidores , Acetolactato Sintasa/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/antagonistas & inhibidores , Poaceae/química , Poaceae/enzimología , Poaceae/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Pirimidinas/farmacología , Cinética , Éteres Fenílicos/farmacología , Éteres Fenílicos/químicaRESUMEN
This study confirmed a field population of American sloughgrass (Beckmannia syzigachne (Steud.) Fernald) that developed simultaneously high levels of resistance (resistance index >10) to three divergent modes of action herbicides: fenoxaprop-P-ethyl, mesosulfuron-methyl, and isoproturon. The resistance phenotype observed in this population was not attributed to target-site alterations; rather, the resistant plants exhibited a significant increase in the activity of cytochrome P450s (P450s) and enhanced metabolism rates for all three herbicides. RNA sequencing revealed significant upregulation of two P450s, CYP709B1 and CYP704C1, in the resistant plants both before and after herbicide treatments. Molecular docking predicted that the homology models of these P450s should exhibit a binding affinity for a range of herbicides. The heterologous expression of the identified P450s in yeast cells indicated improved growth in the presence of all three of the aforementioned herbicides. Collectively, the increased expression of CYP709B1 and CYP704C1 likely contributed to the P450s-mediated enhanced metabolism, thereby conferring multiple herbicide resistance in B. syzigachne.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Resistencia a los Herbicidas , Herbicidas , Proteínas de Plantas , Resistencia a los Herbicidas/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/química , Herbicidas/farmacología , Herbicidas/metabolismo , Herbicidas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Simulación del Acoplamiento Molecular , Poaceae/genética , Poaceae/metabolismo , Poaceae/enzimología , Poaceae/efectos de los fármacos , Poaceae/químicaRESUMEN
Photoreactivation enzyme that repairs cyclobutane pyrimidine dimer (CPD) induced by ultraviolet-B radiation, commonly called CPD photolyase (PHR) is essential for plants living under sunlight. Rice (Oryza sativa) PHR (OsPHR) is a unique triple-targeting protein. The signal sequences required for its translocation to the nucleus or mitochondria are located in the C-terminal region but have yet to be identified for chloroplasts. Here, we identified sequences located in the N-terminal region, including the serine-phosphorylation site at position 7 of OsPHR, and found that OsPHR is transported/localized to chloroplasts via a vesicle transport system under the control of serine-phosphorylation. However, the sequence identified in this study is only conserved in some Poaceae species, and in many other plants, PHR is not localized to the chloroplasts. Therefore, we reasoned that Poaceae species need the ability to repair CPD in the chloroplast genome to survive under sunlight and have uniquely acquired this mechanism for PHR chloroplast translocation.
Asunto(s)
Cloroplastos , Desoxirribodipirimidina Fotoliasa , Oryza , Rayos Ultravioleta , Cloroplastos/metabolismo , Desoxirribodipirimidina Fotoliasa/metabolismo , Desoxirribodipirimidina Fotoliasa/genética , Oryza/genética , Oryza/enzimología , Oryza/efectos de la radiación , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Dímeros de Pirimidina/metabolismo , Poaceae/genética , Poaceae/enzimología , Poaceae/efectos de la radiación , Poaceae/metabolismo , Secuencia de Aminoácidos , Transporte de ProteínasRESUMEN
BACKGROUND: Glutathione transferases (GSTs) are enzymes with a wide range of functions, including herbicide detoxification. Up-regulation of GSTs and their detoxification activity enables the grass weed black-grass (Alopecurus myosuroides Huds.) to metabolize the very-long-chain fatty acid synthesis inhibitor flufenacet and other herbicides leading to multiple herbicide resistance. However, the genomic organization and regulation of GSTs genes is still poorly understood. RESULTS: In this genome-wide study the location and expression of 115 GSTs were investigated using a recently published black-grass genome. Particularly, the most abundant GSTs of class tau and phi were typically clustered and often followed similar expression patterns but possessed divergent upstream regulatory regions. Similarities were found in the promoters of the most up-regulated GSTs, which are located next to each other in a cluster. The binding motif of the E2F/DP transcription factor complex in the promoter of an up-regulated GST was identical in susceptible and resistant plants, however, adjacent sequences differed. This led to a stronger binding of proteins to the motif of the susceptible plant, indicating repressor activity. CONCLUSIONS: This study constitutes the first analysis dealing with the genomic investigation of GST genes found in black-grass and their transcriptional regulation. It highlights the complexity of the evolution of GSTs in black-grass, their duplication and divergence over time. The large number of GSTs allows weeds to detoxify a broad spectrum of herbicides. Ultimately, more research is needed to fully elucidate the regulatory mechanisms of GST expression. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Asunto(s)
Acetamidas , Regulación de la Expresión Génica de las Plantas , Glutatión Transferasa , Resistencia a los Herbicidas , Herbicidas , Poaceae , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Resistencia a los Herbicidas/genética , Poaceae/genética , Poaceae/enzimología , Herbicidas/farmacología , Acetamidas/farmacología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Genoma de Planta , Estudio de Asociación del Genoma Completo , TiadiazolesRESUMEN
Lignin biosynthesis enzymes form complexes for metabolic channelling during lignification and these enzymes also play an essential role in biotic and abiotic stress response. Cinnamyl alcohol dehydrogenase (CAD) is a vital enzyme that catalyses the reduction of aldehydes to alcohols, which is the final step in the lignin biosynthesis pathway. In the present study, we identified 49 CAD enzymes in five Bambusoideae species and analysed their phylogenetic relationships and conserved domains. Expression analysis of Moso bamboo PheCAD genes in several developmental tissues and stages revealed that among the PheCAD genes, PheCAD2 has the highest expression level and is expressed in many tissues and PheCAD1, PheCAD6, PheCAD8 and PheCAD12 were also expressed in most of the tissues studied. Co-expression analysis identified that the PheCAD2 positively correlates with most lignin biosynthesis enzymes, indicating that PheCAD2 might be the key enzyme involved in lignin biosynthesis. Further, more than 35% of the co-expressed genes with PheCADs were involved in biotic or abiotic stress responses. Abiotic stress transcriptomic data (SA, ABA, drought, and salt) analysis identified that PheCAD2, PheCAD3 and PheCAD5 genes were highly upregulated, confirming their involvement in abiotic stress response. Through yeast two-hybrid analysis, we found that PheCAD1, PheCAD2 and PheCAD8 form homo-dimers. Interestingly, BiFC and pull-down experiments identified that these enzymes form both homo- and hetero- dimers. These data suggest that PheCAD genes are involved in abiotic stress response and PheCAD2 might be a key lignin biosynthesis pathway enzyme. Moreover, this is the first report to show that three PheCAD enzymes form complexes and that the formation of PheCAD homo- and hetero- dimers might be tissue specific.
Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Regulación de la Expresión Génica de las Plantas , Lignina/biosíntesis , Poaceae/enzimología , Estrés Fisiológico , Oxidorreductasas de Alcohol/genética , Dimerización , Poaceae/genética , Multimerización de ProteínaRESUMEN
Miscanthus, a member of the Saccharinae subtribe that includes sorghum and sugarcane, has been widely studied as a feedstock for cellulosic biofuel production. Here, we report the sequencing and assembly of the Miscanthus floridulus genome by the integration of PacBio sequencing and Hi-C mapping, resulting in a chromosome-scale, high-quality reference genome of the genus Miscanthus. Comparisons among Saccharinae genomes suggest that Sorghum split first from the common ancestor of Saccharum and Miscanthus, which subsequently diverged from each other, with two successive whole-genome duplication events occurring independently in the Saccharum genus and one whole-genome duplication occurring in the Miscanthus genus. Fusion of two chromosomes occurred during rediploidization in M. floridulus and no significant subgenome dominance was observed. A survey of cellulose synthases (CesA) in M. floridulus revealed quite high expression of most CesA genes in growing stems, which is in agreement with the high cellulose content of this species. Resequencing and comparisons of 75 Miscanthus accessions suggest that M. lutarioriparius is genetically close to M. sacchariflorus and that M. floridulus is more distantly related to other species and is more genetically diverse. This study provides a valuable genomic resource for molecular breeding and improvement of Miscanthus and Saccharinae crops.
Asunto(s)
Genoma de Planta/genética , Poaceae/genética , Saccharum/genética , Cromosomas de las Plantas/genética , Evolución Molecular , Duplicación de Gen/genética , Genética de Población , Glucosiltransferasas/genética , Filogenia , Poaceae/enzimología , Alineación de Secuencia , Análisis de Secuencia de ADN , Sorghum/genética , Sintenía/genéticaRESUMEN
Imperata cylindrica is known to produce a pair of triterpenes, isoarborinol and fernenol, that exhibit identical planar structures but possess opposite stereochemistry at six of the nine chiral centers. These differences arise from a boat or a chair cyclization of the B-ring of the substrate. Herein, we report the characterization of three OSC genes from I. cylindrica. IcOSC1 and IcOSC5 were identified as isoarborinol and fernenol synthases, respectively, while IcOSC3 was characterized as a multifunctional enzyme that produces glutinol and friedelin as its major products. Mutational studies of isoarborinol and fernenol synthases revealed that the residues surrounding the DCTAE motif partially affected the conformation of the B-ring during cyclization. Additionally, the IcOSC1-W255H mutant produced the rare triterpene boehmerol. The introduced histidine residue presumably abstracted a proton from the intermediary carbocation at C18 during the 1,2-rearrangement. Expression analysis indicated that all OSC genes were highly expressed in stems.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Poaceae/enzimología , Triterpenos/metabolismo , Biocatálisis , Ciclización , Estructura Molecular , Estereoisomerismo , Triterpenos/químicaRESUMEN
The cryoprotection of cell activity is a key determinant in frozen-dough technology. Although several factors that contribute to freezing tolerance have been reported, the mechanism underlying the manner in which yeast cells respond to freezing and thawing (FT) stress is not well established. Therefore, the present study demonstrated the relationship between DaMDHAR encoding monodehydroascorbate reductase from Antarctic hairgrass Deschampsia antarctica and stress tolerance to repeated FT cycles (FT2) in transgenic yeast Saccharomyces cerevisiae. DaMDHAR-expressing yeast (DM) cells identified by immunoblotting analysis showed high tolerance to FT stress conditions, thereby causing lower damage for yeast cells than wild-type (WT) cells with empty vector alone. To detect FT2 tolerance-associated genes, 3'-quant RNA sequencing was employed using mRNA isolated from DM and WT cells exposed to FT (FT2) conditions. Approximately 332 genes showed ≥2-fold changes in DM cells and were classified into various groups according to their gene expression. The expressions of the changed genes were further confirmed using western blot analysis and biochemical assay. The upregulated expression of 197 genes was associated with pentose phosphate pathway, NADP metabolic process, metal ion homeostasis, sulfate assimilation, ß-alanine metabolism, glycerol synthesis, and integral component of mitochondrial and plasma membrane (PM) in DM cells under FT2 stress, whereas the expression of the remaining 135 genes was partially related to protein processing, selenocompound metabolism, cell cycle arrest, oxidative phosphorylation, and α-glucoside transport under the same condition. With regard to transcription factors in DM cells, MSN4 and CIN5 were activated, but MSN2 and MGA1 were not. Regarding antioxidant systems and protein kinases in DM cells under FT stress, CTT1, GTO, GEX1, and YOL024W were upregulated, whereas AIF1, COX2, and TRX3 were not. Gene activation represented by transcription factors and enzymatic antioxidants appears to be associated with FT2-stress tolerance in transgenic yeast cells. RCK1, MET14, and SIP18, but not YPK2, have been known to be involved in the protein kinase-mediated signalling pathway and glycogen synthesis. Moreover, SPI18 and HSP12 encoding hydrophilin in the PM were detected. Therefore, it was concluded that the genetic network via the change of gene expression levels of multiple genes contributing to the stabilization and functionality of the mitochondria and PM, not of a single gene, might be the crucial determinant for FT tolerance in DaMDAHR-expressing transgenic yeast. These findings provide a foundation for elucidating the DaMDHAR-dependent molecular mechanism of the complex functional resistance in the cellular response to FT stress.
Asunto(s)
Congelación/efectos adversos , NADH NADPH Oxidorreductasas/genética , Saccharomyces cerevisiae/genética , Regulación Fúngica de la Expresión Génica/genética , Redes Reguladoras de Genes/genética , Poaceae/enzimología , Estrés Fisiológico/genética , Factores de Transcripción/genéticaRESUMEN
C4-like plants represent the penultimate stage of evolution from C3 to C4 plants. Although Coleataenia prionitis (formerly Panicum prionitis) has been described as a C4 plant, its leaf anatomy and gas exchange traits suggest that it may be a C4-like plant. Here, we reexamined the leaf structure and biochemical and physiological traits of photosynthesis in this grass. The large vascular bundles were surrounded by two layers of bundle sheath (BS): a colorless outer BS and a chloroplast-rich inner BS. Small vascular bundles, which generally had a single BS layer with various vascular structures, also occurred throughout the mesophyll together with BS cells not associated with vascular tissue. The mesophyll cells did not show a radial arrangement typical of Kranz anatomy. These features suggest that the leaf anatomy of C. prionitis is on the evolutionary pathway to a complete C4 Kranz type. Phosphoenolpyruvate carboxylase (PEPC) and pyruvate, Pi dikinase occurred in the mesophyll and outer BS. Glycine decarboxylase was confined to the inner BS. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) accumulated in the mesophyll and both BSs. C. prionitis had biochemical traits of NADP-malic enzyme type, whereas its gas exchange traits were close to those of C4-like intermediate plants rather than C4 plants. A gas exchange study with a PEPC inhibitor suggested that Rubisco in the mesophyll could fix atmospheric CO2. These data demonstrate that C. prionitis is not a true C4 plant but should be considered as a C4-like plant.
Asunto(s)
Dióxido de Carbono/metabolismo , Fotosíntesis , Poaceae/fisiología , Cloroplastos/enzimología , Cloroplastos/fisiología , Cloroplastos/ultraestructura , Glicina-Deshidrogenasa (Descarboxilante)/metabolismo , Malato Deshidrogenasa/metabolismo , Células del Mesófilo/enzimología , Células del Mesófilo/fisiología , Células del Mesófilo/ultraestructura , Fenotipo , Fosfoenolpiruvato Carboxilasa/antagonistas & inhibidores , Fosfoenolpiruvato Carboxilasa/metabolismo , Hojas de la Planta/enzimología , Hojas de la Planta/fisiología , Hojas de la Planta/ultraestructura , Proteínas de Plantas/metabolismo , Poaceae/enzimología , Poaceae/ultraestructura , Ribulosa-Bifosfato Carboxilasa/metabolismoRESUMEN
Multiple-herbicide resistance (MHR) is a global threat to weed control in cereal crops. MHR weeds express a specific phi class glutathione transferase (MHR-GSTF) that confers resistance against multiple herbicides and therefore represents a promising target against MHR weeds. Kinetics inhibition analysis of MHR-GSTFs from grass weeds Lolium rigidum (LrGSTF) Alopecurus myosuroides (AmGSTF) and crops Hordeum vulgare (HvGSTF) and Triticum aestivum (TaGSTF) allowed the identification of the acetanilide herbicide butachlor as a potent and selective inhibitor towards MHR-GSTFs. Also, butachlor is a stronger inhibitor for LrGSTF and AmGSTF compared to HvGSTF and TaGSTF from crops. The crystal structure of LrGSTF was determined at 1.90 Å resolution in complex with the inhibitor S-(4-nitrobenzyl)glutathione. A specific 3D pharmacophore targeting the MHR-GSTFs was designed and used to identify structural elements important for potent and selective inhibition. Structural analysis of GSTFs revealed a decisive role of conserved Tyr118 in ligand binding and pharmacophore design. Its positioning is dependent on an outer patch of adjacent residues that span from position 132 to 134 which are similar for both LrGSTF and AmGSTF but different in HvGSTF and TaGSTF. The results presented here provide new knowledge that may be adopted to cope with MHR weeds.
Asunto(s)
Glutatión Transferasa/genética , Resistencia a los Herbicidas , Herbicidas , Malezas/enzimología , Poaceae/enzimología , Malezas/genética , Poaceae/genéticaRESUMEN
Pogonatherum crinitum is a promising lead (Pb) hyperaccumulator; however, the effects of Pb contamination on P. crinitum rhizosphere soil enzymatic activities and microbial composition remain largely unexplored. Thus, an indoor experiment was conducted by cultivating P. crinitum seedlings and exposing them to four Pb concentrations (0, 1,000, 2000 and 3000 mg/kg Pb). Protease, urease, acid phosphatase and invertase activities were determined using standard methods while soil bacterial composition was determined by 16 S rDNA sequencing. The results showed that rhizosphere soil acid phosphatase activity significantly increased with increasing Pb concentration, while urease activity was significantly greater in rhizosphere soil contaminated with 1000 and 2000 mg/kg than in the control. There was a clear shift in bacterial composition during phytoremediation by P. crinitum. Compared to the control, Bacteroidetes was more abundant in all Pb-contaminated soils, Actinobacteria was more abundant in 1000 mg/kg Pb-treated soil, and Firmicutes was more abundant in 3000 mg/kg Pb-treated soil. Positive correlations were observed between dominant bacterial phyla and soil enzyme activities. Metabolic pathways, such as ABC transporter, quinine reductase, and ATP-binding protein were significantly increased in rhizosphere soil bacteria with Pb contamination. In conclusion, Pb contamination differentially influenced the activities of rhizosphere soil enzymes, specifically increasing acid phosphatase and urease activities, and alters the dominance of soil bacteria through up-regulation of genes related to some metabolic pathways. The strong correlations between dominant bacterial phyla and enzymatic activities suggest synergetic effects on the growth of P. crinitum during Pb contamination.
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Bioacumulación , Plomo/toxicidad , Poaceae/efectos de los fármacos , Poaceae/enzimología , Rizosfera , Microbiología del Suelo , Contaminantes del Suelo/toxicidad , Fosfatasa Ácida/metabolismo , Actinobacteria/efectos de los fármacos , Actinobacteria/enzimología , Biodegradación Ambiental , Plomo/metabolismo , Péptido Hidrolasas/metabolismo , Poaceae/crecimiento & desarrollo , Plantones/efectos de los fármacos , Plantones/metabolismo , Suelo/química , Contaminantes del Suelo/metabolismo , Ureasa/metabolismoRESUMEN
Salt stress negatively affects plant growth, and the fungal endophyte Epichloëgansuensis increases the tolerance of its host grass species, Achnatherum inebrians, to abiotic stresses. In this work, we first evaluated the effects of E. gansuensis on glucose-6-phosphate dehydrogenase (G6PDH) and plasma membrane (PM) H+-ATPase activity of Achnatherum inebrians plants under varying NaCl concentrations. Our results showed that the presence of E. gansuensis increased G6PDH, PM H+-ATPase, superoxide dismutase and catalase activity to decrease O2â¢-, H2O2 and Na+ contents in A. inebrians under NaCl stress, resulting in enhanced salt tolerance. In addition, the PM NADPH oxidase activity and NADPH/NADP+ ratios were all lower in A. inebrians with E. ganusensis plants than A. inebrians plants without this endophyte under NaCl stress. In conclusion, E. gansuensis has a positive role in improving host grass yield under NaCl stress by enhancing the activity of G6PDH and PM H+-ATPase to decrease ROS content. This provides a new way for the selection of stress-resistant and high-quality forage varieties by the use of systemic fungal endophytes.
Asunto(s)
Endófitos/enzimología , Epichloe/enzimología , Glucosafosfato Deshidrogenasa/metabolismo , Poaceae/enzimología , ATPasas de Translocación de Protón/metabolismo , Cloruro de Sodio/metabolismo , Membrana CelularRESUMEN
We characterized an Na+ transporter SvHKT1;1 from a halophytic turf grass, Sporobolus virginicus. SvHKT1;1 mediated inward and outward Na+ transport in Xenopus laevis oocytes and did not complement K+ transporter-defective mutant yeast. SvHKT1;1 did not complement athkt1;1 mutant Arabidopsis, suggesting its distinguishable function from other typical HKT1 transporters. The transcript was abundant in the shoots compared with the roots in S. virginicus and was upregulated by severe salt stress (500 mM NaCl), but not by lower stress. SvHKT1;1-expressing Arabidopsis lines showed higher shoot Na+ concentrations and lower salt tolerance than wild type (WT) plants under nonstress and salt stress conditions and showed higher Na+ uptake rate in roots at the early stage of salt treatment. These results suggested that constitutive expression of SvHKT1;1 enhanced Na+ uptake in root epidermal cells, followed by increased Na+ transport to shoots, which led to reduced salt tolerance. However, Na+ concentrations in phloem sap of the SvHKT1;1 lines were higher than those in WT plants under salt stress. Based on this result, together with the induction of the SvHKT1;1 transcription under high salinity stress, it was suggested that SvHKT1;1 plays a role in preventing excess shoot Na+ accumulation in S. virginicus.
Asunto(s)
Magnoliopsida , Brotes de la Planta/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Sodio/metabolismo , Sodio/farmacología , Arabidopsis/genética , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Magnoliopsida/enzimología , Magnoliopsida/genética , Magnoliopsida/metabolismo , Brotes de la Planta/genética , Plantas Modificadas Genéticamente , Poaceae/enzimología , Poaceae/genética , Poaceae/metabolismo , Estrés Salino/genética , Tolerancia a la Sal , Plantas Tolerantes a la Sal/genética , Plantas Tolerantes a la Sal/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
MAIN CONCLUSION: The changes in the expression of key sugar metabolism enzymes (SPS and SUS), sucrose content and arrangement of chloroplast starch may play a significant role in the cold response in M. giganteus and maize plants. To understand the mechanism of the chilling-response of two closely-related C4 plants, we investigated the changes in the expression of sucrose phosphate synthase (SPS) and sucrose synthase (SUS) as well as changes in their potential products: sucrose, cellulose and starch in the leaves of Miscanthus × giganteus and Zea mays. Low temperature (12-14 °C) increased SPS content in Miscanthus (MG) and chilling-sensitive maize line (Zm-S), but not in chilling-tolerant one (Zm-T). In Zm-S line, chilling also caused the higher intensity of labelling of SPS in the cytoplasm of mesophyll cells, as demonstrated by electron microscopy. SUS labelling was also increased by cold stress only in MG plants what was observed in the secondary wall between mesophyll and bundle sheath cells, as well as in the vacuoles of companion cells. Cold led to a marked increase in total starch grain area in the chloroplasts of Zm-S line. In turn, Fourier transform infrared spectroscopy (FTIR) showed a slight shift in the cellulose band position, which may indicate the formation of more compact cellulose arrangement in Zm-T maize line. In conclusion, this work presents new findings supporting diversified cold-response, not only between two C4 plant species but also within one species of maize.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Glucosiltransferasas/metabolismo , Poaceae/enzimología , Zea mays/enzimología , Celulosa/metabolismo , Cloroplastos/metabolismo , Frío , Inmunohistoquímica , Hojas de la Planta/enzimología , Hojas de la Planta/fisiología , Hojas de la Planta/ultraestructura , Proteínas de Plantas/metabolismo , Poaceae/fisiología , Poaceae/ultraestructura , Almidón/metabolismo , Estrés Fisiológico , Sacarosa/metabolismo , Zea mays/fisiología , Zea mays/ultraestructuraRESUMEN
KEY MESSAGE: Distinct catalytic features of the Poaceae TPS-a subfamily arose early in grass evolution and the reactions catalyzed have become more complex with time. The structural diversity of terpenes found in nature is mainly determined by terpene synthases (TPS). TPS enzymes accept ubiquitous prenyl diphosphates as substrates and convert them into the various terpene skeletons by catalyzing a carbocation-driven reaction. Based on their sequence similarity, terpene synthases from land plants can be divided into different subfamilies, TPS-a to TPS-h. In this study, we aimed to understand the evolution and functional diversification of the TPS-a subfamily in the Poaceae (the grass family), a plant family that contains important crops such as maize, wheat, rice, and sorghum. Sequence comparisons showed that aside from one clade shared with other monocot plants, the Poaceae TPS-a subfamily consists of five well-defined clades I-V, the common ancestor of which probably originated very early in the evolution of the grasses. A survey of the TPS literature and the characterization of representative TPS enzymes from clades I-III revealed clade-specific substrate and product specificities. The enzymes in both clade I and II function as sesquiterpene synthases with clade I enzymes catalyzing initial C10-C1 or C11-C1 ring closures and clade II enzymes catalyzing C6-C1 closures. The enzymes of clade III mainly act as monoterpene synthases, forming cyclic and acyclic monoterpenes. The reconstruction and characterization of clade ancestors demonstrated that the differences among clades I-III were already present in their ancestors. However, the ancestors generally catalyzed simpler reactions with less double-bond isomerization and fewer cyclization steps. Overall, our data indicate an early origin of key enzymatic features of TPS-a enzymes in the Poaceae, and the development of more complex reactions over the course of evolution.
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Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Poaceae/enzimología , Poaceae/genética , Transferasas Alquil y Aril/clasificación , Clonación Molecular , Escherichia coli/genética , Evolución Molecular , Genes de Plantas/genética , Liasas Intramoleculares/metabolismo , Proteínas de Plantas/genética , Análisis de Secuencia , Terpenos/metabolismoRESUMEN
The adaptation of proteins for novel functions often requires changes in their kinetics via amino acid replacement. This process can require multiple mutations, and therefore extended periods of selection. The transfer of genes among distinct species might speed up the process, by providing proteins already adapted for the novel function. However, this hypothesis remains untested in multicellular eukaryotes. The grass Alloteropsis is an ideal system to test this hypothesis due to its diversity of genes encoding phosphoenolpyruvate carboxylase, an enzyme that catalyzes one of the key reactions in the C4 pathway. Different accessions of Alloteropsis either use native isoforms relatively recently co-opted from other functions or isoforms that were laterally acquired from distantly related species that evolved the C4 trait much earlier. By comparing the enzyme kinetics, we show that native isoforms with few amino acid replacements have substrate KM values similar to the non-C4 ancestral form, but exhibit marked increases in catalytic efficiency. The co-option of native isoforms was therefore followed by rapid catalytic improvements, which appear to rely on standing genetic variation observed within one species. Native C4 isoforms with more amino acid replacements exhibit additional changes in affinities, suggesting that the initial catalytic improvements are followed by gradual modifications. Finally, laterally acquired genes show both strong increases in catalytic efficiency and important changes in substrate handling. We conclude that the transfer of genes among distant species sharing the same physiological novelty creates an evolutionary shortcut toward more efficient enzymes, effectively accelerating evolution.
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Evolución Biológica , Transferencia de Gen Horizontal , Fosfoenolpiruvato Carboxilasa/genética , Fotosíntesis/genética , Poaceae/genética , Sustitución de Aminoácidos , Poaceae/enzimologíaRESUMEN
Changing climatic scenarios affect plant growth and consequences are more malicious in drought conditions. This study was performed for better understanding of tolerance mechanisms under prevailing drought stress and succeeding recovery in Axonopus compressus by exogenously applied abscisic acid (ABA) and glycine betaine (GB). Three A. compressus accessions (A-38, A-58 and A-59) were subjected to well-watered (100% field capacity) and drought (40% field capacity) conditions. Two weeks later, plants were recovered from drought by re-watering. Water (control), GB, ABA and their combination were foliar applied on plants under drought twice a week until recovery. Drought stress decreased photosynthetic pigments and increased reactive oxygen species, lipid peroxidation, osmolytes and antioxidants in all accessions of A. compressus. Nonetheless, exogenous ABA and GB alone or in combination improved drought tolerance in all accessions which was maintained even after recovery. Maximum decrease in hydrogen peroxide and malondialdehyde, and increase in soluble sugars, proteins, proline, phenolics and chlorophyll contents, and superoxide dismutase, catalase, peroxidase and ascorbate peroxidase activity was recorded when GB was applied alone under drought. Order of improvement in drought tolerance among accessions was A-58 > A-59 > A-38. In conclusion, improved drought tolerance mechanisms by ABA and GB in A. compressus were retained even after recovery.
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Ácido Abscísico/metabolismo , Adaptación Fisiológica , Betaína/metabolismo , Sequías , Poaceae/fisiología , Estrés Fisiológico , Antioxidantes/metabolismo , Catalasa/metabolismo , Membrana Celular/metabolismo , Peroxidación de Lípido , Peroxidasa/metabolismo , Fotosíntesis , Pigmentos Biológicos/metabolismo , Poaceae/enzimología , Prolina/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Multiple-herbicide resistant (MHR) weeds are a global problem and a looming threat to weed control in crops. MHR weeds express a specific phi class glutathione transferase (MHR-GSTF) which seems to contribute to herbicide resistance. The present work aims to investigate the structure and catalytic properties of the MHR-GSTFs from different grass weeds and crops (Alopecurus myosuroides, Lolium rigidum, Hordeum vulgare, Triticum aestivum). Recombinant MHR-GSTFs were expressed in E. coli and purified by affinity chromatography. Kinetic analysis of substrate specificity using a range of thiol substrates and xenobiotic compounds suggested that all enzymes display a broad range of specificity and are capable of detoxifying major stress-induced toxic products. Notably, all tested enzymes exhibited high activity towards organic hydroperoxides. The crystal structure of MHR-GSTF from Alopecurus myosuroides (AmGSTF) was determined by molecular replacement at 1.33 Å resolution. The enzyme was resolved with bound glutathione sulfenic acid (GSOH) at the G-site and succinic acid at the H-site. The enzyme shows conserved structural features compared to other Phi class GSTs. However, some differences were observed at the C-terminal helix H9 that may affect substrate specificity. The structural and functional features of AmGSTF were compared with those of the homologue crop enzymes (HvGSTF and TaGSTF) and discussed in light of their contribution to the MHR mechanism.
Asunto(s)
Resistencia a Medicamentos , Glutatión Transferasa , Resistencia a los Herbicidas , Poaceae , Resistencia a Medicamentos/genética , Escherichia coli , Glutatión Transferasa/química , Glutatión Transferasa/metabolismo , Resistencia a los Herbicidas/genética , Cinética , Poaceae/enzimología , Poaceae/genéticaRESUMEN
Three subtypes of C4 photosynthesis exist (NADP-ME, NAD-ME and PEPCK), each known to be beneficial under specific environmental conditions. However, the influence of photosynthetic subtype on transcriptomic plasticity, as well as the genes underpinning this variability, remain largely unknown. Here, we comprehensively investigate the responses of six C4 grass species, spanning all three C4 subtypes, to two controlled environmental stresses: low light (200 µmol m-2 sec-1 ) and glacial CO2 (subambient; 180 ppm). We identify a susceptibility within NADP-ME species to glacial CO2 . Notably, although glacial CO2 phenotypes could be tied to C4 subtype, biochemical and transcriptomic responses to glacial CO2 were largely species specific. Nevertheless, we were able to identify subtype specific subsets of significantly differentially expressed transcripts which link resource acquisition and allocation to NADP-ME species susceptibility to glacial CO2 . Here, low light phenotypes were comparable across species with no clear subtype response, while again, transcriptomic responses to low light were largely species specific. However, numerous functional similarities were noted within the transcriptomic responses to low light, suggesting these responses are functionally relatively conserved. Additionally, PEPCK species exhibited heightened regulation of transcripts related to metabolism in response to both stresses, likely tied to their C4 metabolic pathway. These results highlight the influence that both species and subtype can have on plant responses to abiotic stress, building on our mechanistic understanding of acclimation within C4 grasses and highlighting avenues for future crop improvements.
Asunto(s)
Dióxido de Carbono/metabolismo , Fosfoenolpiruvato Carboxilasa/metabolismo , Poaceae/genética , Transcriptoma , Aclimatación , Perfilación de la Expresión Génica , Luz , Redes y Vías Metabólicas , Fenotipo , Fosfoenolpiruvato Carboxilasa/genética , Fotosíntesis , Poaceae/enzimología , Poaceae/fisiología , Poaceae/efectos de la radiación , Especificidad de la EspecieRESUMEN
Deschampsiaantarctica inhabits the maritime territory of Antarctica and South Patagonia. It grows under very harsh environmental conditions. The survival of this species in low freezing temperatures and under high levels of UV-B radiation may constitute some of the most remarkable adaptive plant responses and suggests that this plant possesses genes associated with cold and UV tolerance. Frequently, increased levels of flavonoids have been linked to highly UV-B irradiated plants. Studies examining the biosynthesis of flavonoids in D. antarctica may provide clues to its success in this extreme environment. In this study, we characterized the family of genes encoding chalcone synthase, a key enzyme of the flavonoid biosynthetic pathway. DaCHS was cloned, sequenced and characterized by using software tools. CHS contains two domains, the N-terminal domain ranges from amino acid 8 to 231 and the C-terminal domain ranges from amino acid 241 to 391. Sequence analysis of the three family members revealed a high degree of identity after comparison with other monocotyledons such as Oryza sativa L., Zea mays L. and Hordeum vulgare L. According to these results, DaCHS can be grouped together with H. vulgare CHS1 in the same branch. The phylogenetic tree was built using MEGA software and the neighbour join method with 1000 bootstrap replicates. A model of DaCHS was constructed by way of structural tools and key amino acid residues were identified at the active motif site.