RESUMEN

With the emergence of multidrug-resistant microorganisms, microbial agents have become a serious global threat, affecting human health and various plants. Therefore, new therapeutic alternatives, such as chitin-binding proteins, are necessary. Chitin is an essential component of the fungal cell wall, and chitin-binding proteins exhibit antifungal activity. In the present study, chitin-binding peptides isolated from Capsicum chinense seeds were characterized and evaluated for their in vitro antimicrobial effect against the growth of Candida and Fusarium fungi. Proteins were extracted from the seeds and subsequently the chitin-binding proteins were separated by chitin affinity chromatography. After chromatography, two fractions, Cc-F1 (not retained on the column) and Cc-F2 (retained on the column), were obtained. Electrophoresis revealed major protein bands between 6.5 and 26.6 kDa for Cc-F1 and only a ~ 6.5 kDa protein band for Cc-F2, which was subsequently subjected to mass spectrometry. The protein showed similarity with hevein-like and endochitinase and was then named Cc-Hev. Data are available via ProteomeXchange with identifier PXD054607. Next, we predicted the three-dimensional structure of the peptides and performed a peptide docking with (NAG)3. Subsequently, growth inhibition assays were performed to evaluate the ability of the peptides to inhibit microorganism growth. Cc-Hev inhibited the growth of C. albicans (up to 75% inhibition) and C. tropicalis (100% inhibition) and induced a 65% decrease in cell viability for C. albicans and 100% for C. tropicalis. Based on these results, new techniques to combat fungal diseases could be developed through biotechnological applications; therefore, further studies are needed.

Asunto(s)

Antifúngicos , Candida , Capsicum , Quitina , Quitinasas , Fusarium , Semillas , Semillas/química , Antifúngicos/farmacología , Antifúngicos/aislamiento & purificación , Antifúngicos/química , Antifúngicos/metabolismo , Quitina/metabolismo , Quitina/farmacología , Fusarium/efectos de los fármacos , Quitinasas/farmacología , Quitinasas/metabolismo , Quitinasas/química , Quitinasas/aislamiento & purificación , Candida/efectos de los fármacos , Candida/enzimología , Lectinas de Plantas/farmacología , Lectinas de Plantas/química , Lectinas de Plantas/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Péptidos/farmacología , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Simulación del Acoplamiento Molecular , Proteínas de Plantas/farmacología , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Péptidos Catiónicos Antimicrobianos

RESUMEN

Cowpea weevil, Callosobruchus maculatus, is the primary pest of stored cowpea seeds. The management of this infestation currently relies on insecticides, resulting in environmental pollution and selection of insecticide-resistant pests. Consequently, research efforts are being devoted to identify natural insecticides as sustainable and environment friendly alternatives for the control of C. maculatus. In this study, we explore the toxic effects of the nonhost seeds Parkia multijuga, Copaifera langsdorffii, Ormosia arborea, Amburana cearensis, Lonchocarpus guilleminianus, Sapindus saponaria, and Myroxylon peruiferum, on the cowpea weevil C. maculatus. Notably, all nonhost seeds led to reductions between 60 and 100% in oviposition by C. maculatus females. Additionally, the larvae were unable to penetrate the nonhost seeds. Artificial seeds containing 0.05% to 10% of cotyledon flour were toxic to C. maculatus larvae. Approximately 40% of larvae that consumed seeds containing 0.05% of O. arborea failed to develop, in contrast to control larvae. Proteomic analysis of A. cearensis and O. arborea seeds identify revealed a total of 371 proteins. From those, 237 are present in both seeds, 91 were exclusive to O. arborea seeds, and 43 were specific to A. cearensis seeds. Some of these proteins are related to defense, such as proteins containing the cupin domain and 11S seed storage protein. The in silico docking of cupin domain-containing proteins and 11S storage protein with N-acetylglucosamine (NAG)4 showed negative values of affinity energy, indicating spontaneous binding. These results showed that nonhost seeds have natural insecticide compounds with potential to control C. maculatus infestation.

Asunto(s)

Escarabajos , Insecticidas , Vigna , Gorgojos , Animales , Femenino , Insecticidas/toxicidad , Proteómica , Larva , Semillas/química

RESUMEN

The guava tree (Psidium guajava) is a tropical species native to South America and is recognized as the 11th most economically important fruit tree in Brazil. However, the presence of the nematode Meloidogyne enterolobii and the fungus Fusarium solani in the roots of guava plants leads to the development of root galls, causing significant damage. In contrast, the species P. guineense and P. cattleianum have been identified as resistant and immune to the nematode, respectively. In this study, the researchers aimed to compare the metabolomic profiles of infected and uninfected roots of P. guajava, P. cattleianum, and P. guineense using mass spectrometry coupled with liquid chromatography (LC-MS). The goal was to identify secondary metabolites that could potentially be utilized as biochemical resources for nematode control. The findings of the study demonstrated that the plant metabolism of all three species undergoes alterations in response to the phytopathogen inoculation. By employing molecular networks, the researchers identified that the secondary metabolites affected by the infection, whether produced or suppressed, are primarily of a polar chemical nature. Further analysis of the database confirmed the polar nature of the regulated substances after infection, specifically hydrolysable tannins and lignans in P. guineense and P. cattleianum. Interestingly, a group of non-polar substances belonging to the terpene class was also identified in the resistant and immune species. This suggests that these terpenes may act as inhibitors of M. enterolobii, working as repellents or as molecules that can reduce oxidative stress during the infection process, thus enhancing the guava resistance to the nematode. Overall, this study provides valuable insights into the metabolic alterations occurring in different Psidium spp. in response to M. enterolobii infection. The identification of specific secondary metabolites, particularly terpenes, opens up new possibilities for developing effective strategies to control the nematode and enhance guava resistance.

RESUMEN

Non-domesticated species may represent a treasure chest of defensive molecules which must be investigated and rescued. Clitoria fairchildiana R. Howard is a non-domesticated Fabacea, native from the Amazonian Forest whose seeds are exquisitely refractory to insect predation. Secondary metabolites from these seeds were fractionated by different organic solvents and the CH2Cl2 fraction (CFD - Clitoria fairchildiana dichloromethane fraction), as the most toxic to 3rd instar Aedes aegypti larvae (LC50 180 PPM), was subjected to silica gel chromatography, eluted with a gradient of CH2Cl2: MeOH and sub fractioned in nine fractions (CFD1 - CFD9). All obtained fractions were tested in their toxicity to the insect larvae. Two rotenoids, a 11α-O-ß-D-glucopyranosylrotenoid and a 6-deoxyclitoriacetal 11-O-n-glucopyranoside, were identified in the mixture of CFD 7.4 and CFD 7.5, and they were toxic (LC50 120 PPM) to 3rd instar Ae. aegypti larvae, leading to exoskeleton changes, cuticular detachment and perforations in larval thorax and abdomen. These C. fairchildiana rotenoids interfered with the acidification process of cell vesicles in larvae midgut and caused inhibition of 55% of V-ATPases activity of larvae treated with 80 PPM of the compounds, when compared to control larvae. The rotenoids also led to a significant increase in the production of reactive oxygen species (ROS) in treated larvae, especially in the hindgut region of larvae intestines, indicating a triggering of an oxidative stress process to these insects.

Asunto(s)

Aedes , Clitoria , Fabaceae , Insecticidas , Animales , Clitoria/química , Insecticidas/química , Larva , Extractos Vegetales/química , Extractos Vegetales/toxicidad , Semillas/química

RESUMEN

Proteins extracted from Capsicum annuum L. fruits were initially subjected to reversed-phase chromatography on HPLC, resulting in eight peptide-rich fractions. All the fractions obtained were tested for their ability to inhibit porcine trypsin and amylase from both human saliva and from larval insect in vitro. All fractions were also tested for their ability to inhibit growth of the phytopathogenic fungi. Several fractions inhibited the activity of human salivary amylase and larval insect amylase, especially fraction Fa5. No fraction tested was found to inhibit trypsin activity, being Fa2 fraction an exception. Interestingly fraction Fa5 also displayed high antimicrobial activity against the species of the Fusarium genus. Fraction Fa5 was found to have two major protein bands of 17 and 6.5 kDa, and these were sequenced by mass spectrometry. Two peptides were obtained from the 6.5-kDa band, which showed similarity to antimicrobial peptides. Fraction Fa5 was also tested for its ability to permeabilize membranes and induce ROS. Fraction Fa5 was able to permeabilize the membranes of all the fungi tested. Fungi belonging to the genus Fusarium also showed an increase in the endogenous production of ROS when treated with this fraction. Antimicrobial peptides were also identified in the fruits from other Capsicum species.

Asunto(s)

Antiinfecciosos , Capsicum/química , Inhibidores Enzimáticos , Frutas/química , Fusarium/crecimiento & desarrollo , Péptidos , Proteínas de Plantas , alfa-Amilasas/antagonistas & inhibidores , Animales , Antiinfecciosos/química , Antiinfecciosos/aislamiento & purificación , Antiinfecciosos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Humanos , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/farmacología , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/farmacología , Porcinos

RESUMEN

Soybean (Glycine max) is a major legume crop worldwide, providing a critical source of protein and oil. The release of the soybean genome fuelled several transcriptome projects comprising multiple developmental stages and environmental conditions. Nevertheless, the global transcriptional patterns of embryonic axes during germination remain unknown. Here we report the analysis of ~1.58 billion RNA-Seq reads from soybean embryonic axes at five germination stages. Our results support the early activation of processes that are critical for germination, such as glycolysis, Krebs cycle and cell wall remodelling. Strikingly, only 3 hours after imbibition there is a preferential up-regulation of protein kinases and transcription factors, particularly from the LOB domain family, implying that transcriptional and post-transcriptional regulation play major roles early after imbibition. Lipid mobilization and glyoxylate pathways are also transcriptionally active in the embryonic axes, indicating that the local catabolism of oil reserves in the embryonic axes contributes to energy production during germination. We also present evidence supporting abscisic acid inactivation and the up-regulation of gibberellin, ethylene and brassinosteroid pathways. Further, there is a remarkable differential activation of paralogous genes in these hormone signalling pathways. Taken together, our results provide insights on the regulation and biochemistry of soybean germination.

Asunto(s)

Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Germinación , Glycine max/crecimiento & desarrollo , Redes y Vías Metabólicas/genética , Análisis de Secuencia de ARN

RESUMEN

The seed coat develops primarily from maternal tissues and comprises multiple cell layers at maturity, providing a metabolically dynamic interface between the developing embryo and the environment during embryogenesis, dormancy and germination of seeds. Seed coat development involves dramatic cellular changes, and the aim of this research was to investigate the role of programmed cell death (PCD) events during the development of seed coats of cowpea [Vigna unguiculata (L.) Walp.]. We demonstrate that cells of the developing cowpea seed coats undergo a programme of autolytic cell death, detected as cellular morphological changes in nuclei, mitochondria, chloroplasts and vacuoles, DNA fragmentation and oligonucleosome accumulation in the cytoplasm, and loss of membrane viability. We show for the first time that classes 6 and 8 caspase-like enzymes are active during seed coat development, and that these activities may be compartmentalized by translocation between vacuoles and cytoplasm during PCD events.

Asunto(s)

Apoptosis , Fabaceae/fisiología , Semillas/fisiología , Caspasa 6/genética , Caspasa 6/metabolismo , Caspasa 8/genética , Caspasa 8/metabolismo , Fabaceae/enzimología , Fabaceae/crecimiento & desarrollo , Germinación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Semillas/crecimiento & desarrollo

RESUMEN

Bruchid beetles infest various seeds. The seed coat is the first protective barrier against bruchid infestation. Although non-host seed coats often impair the oviposition, eclosion and survival of the bruchid Callosobruchus maculatus larvae, morphological and biochemical aspects of this phenomenon remain unclear. Here we show that Phaseolus vulgaris (non-host) seed coat reduced C. maculatus female oviposition about 48%, increased 83% the seed penetration time, reduced larval mass and survival about 62 % and 40 % respectively. Interestingly, we found no visible effect on the major events of insect embryogenesis, namely the formation of the cellular blastoderm, germ band extension/retraction, embryo segmentation, appendage formation and dorsal closure. Larvae fed on P. vulgaris seed coat have greater FITC fluorescence signal in the midgut than in the feces, as opposed to what is observed in control larvae fed on Vigna unguiculata. Cysteine protease, α-amylase and α-glucosidase activities were reduced in larvae fed on P. vulgaris natural seed coat. Taken together, our results suggest that although P. vulgaris seed coat does not interfere with C. maculatus embryonic development, food digestion was clearly compromised, impacting larval fitness (e.g. body mass and survivability).

Asunto(s)

Phaseolus/fisiología , Semillas/fisiología , Gorgojos/crecimiento & desarrollo , Animales , Digestión , Desarrollo Embrionario , Femenino , Larva/crecimiento & desarrollo , Oviposición , Gorgojos/embriología

RESUMEN

F-box proteins constitute a large gene family that regulates processes from hormone signaling to stress response. F-box proteins are the substrate recognition modules of SCF E3 ubiquitin ligases. Here we report very distinct trends in family size, duplication, synteny and transcription of F-box genes in two nitrogen-fixing legumes, Glycine max (soybean) and Medicago truncatula (alfafa). While the soybean FBX genes emerged mainly through segmental duplications (including whole-genome duplications), M. truncatula genome is dominated by locally-duplicated (tandem) F-box genes. Many of these young FBX genes evolved complex transcriptional patterns, including preferential transcription in different tissues, suggesting that they have probably been recruited to important biochemical pathways (e.g. nodulation and seed development).

Asunto(s)

Proteínas F-Box/genética , Duplicación de Gen , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Glycine max/genética , Medicago truncatula/genética , Proteínas de Plantas/genética , Evolución Molecular , Proteínas F-Box/metabolismo , Perfilación de la Expresión Génica , Medicago truncatula/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Glycine max/metabolismo , Sintenía , Secuencias Repetidas en Tándem , Transcripción Genética

RESUMEN

In eukaryotes, protein kinases catalyze the transfer of a gamma-phosphate from ATP (or GTP) to specific amino acids in protein targets. In plants, protein kinases have been shown to participate in signaling cascades driving responses to environmental stimuli and developmental processes. Plant meristems are undifferentiated tissues that provide the major source of cells that will form organs throughout development. However, non-dividing specialized cells can also dedifferentiate and re-initiate cell division if exposed to appropriate conditions. Mps1 (Monopolar spindle) is a dual-specificity protein kinase that plays a critical role in monitoring the accuracy of chromosome segregation in the mitotic checkpoint mechanism. Although Mps1 functions have been clearly demonstrated in animals and fungi, its role in plants is so far unclear. Here, using structural and biochemical analyses here we show that Mps1 has highly similar homologs in many plant genomes across distinct lineages (e.g. AtMps1 in Arabidopsis thaliana). Several structural features (i.e. catalytic site, DFG motif and threonine triad) are clearly conserved in plant Mps1 kinases. Structural and sequence analysis also suggest that AtMps1 interact with other cell cycle proteins, such as Mad2 and MAPK1. By using a very specific Mps1 inhibitor (SP600125) we show that compromised AtMps1 activity hampers the development of A. thaliana seedlings in a dose-dependent manner, especially in secondary roots. Moreover, concomitant administration of the auxin IAA neutralizes the AtMps1 inhibition phenotype, allowing secondary root development. These observations let us to hypothesize that AtMps1 might be a downstream regulator of IAA signaling in the formation of secondary roots. Our results indicate that Mps1 might be a universal component of the Spindle Assembly Checkpoint machinery across very distant lineages of eukaryotes.

Asunto(s)

Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/química , Regulación de la Expresión Génica de las Plantas , Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Proteínas Tirosina Quinasas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Catálisis , Inhibidores Enzimáticos/farmacología , Evolución Molecular , Genoma de Planta , Conformación Molecular , Datos de Secuencia Molecular , Fosforilación , Filogenia , Raíces de Plantas/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal

RESUMEN

Cysteine peptidases are the best characterized peptidases among those involved with storage protein mobilization during seed germination. In the present work we show two major groups of cysteine peptidase activities, one of higher (55 to 97 kDa) and other with lower (15 to 20 kDa) molecular masses which are temporally activated after 24 and 48 HAI, respectively, in germinating cowpeas. The former group is found both in protein bodies and in cytoplasmic fraction, while the latter is mostly present outside protein bodies. A third cysteine peptidase activity of ~37 kDa was specifically active at quiescent cotyledons and at 12 and 60 hours after imbibition (HAI). Main peptidase activities of albumin fractions were synchronizedly detected with radicle emergence at 36 HAI. Major vicilin mobilization was more pronounced from 60 HAI onwards and steadily increased until 144 HAI, when low levels of the smallest vicilin subunit were present. Cysteine peptidases were susceptible to iodoacetamide, E-64, iodoacetic acid, pCMB and ß-mercaptoethanol, except for the ~37 kDa peptidase, which was not affected by any of the inhibitors. By a two-dimensional native/SDS-PAGE combination it was observed an apparent linear arrangement of protein breakdown products as well as of peptidase activity spots. The finding may indicate a complex set of sequential proteolytic events where peptidases induce or activate new peptidases, which may act upon different aggregates or zymogens, and these hydrolysis products appear in a line of constant decreasing Rf x Mr ratio.

Asunto(s)

Cotiledón/química , Cisteína Endopeptidasas/metabolismo , Fabaceae/química , Proteínas de Plantas/metabolismo , Albúminas/química , Albúminas/metabolismo , Cotiledón/enzimología , Cisteína Endopeptidasas/química , Citoplasma/química , Citoplasma/metabolismo , Electroforesis en Gel de Poliacrilamida , Fabaceae/enzimología , Germinación , Globulinas/química , Globulinas/metabolismo , Proteínas de Plantas/análisis , Proteínas de Plantas/química

RESUMEN

Seeds sprouts have been used as a good source of basic nutrients and nutraceutical compounds. The high nutritional value of seeds derives from the deposition of compounds during development. However some of these molecules are used in metabolic processes like germination, which leads to a considerable variation in their concentrations once these events are completed. In this work, we investigate the levels of inositols (myo-inositol, D-pinitol and ononitol), soluble carbohydrates and proteins in cotyledons of Phaseolus vulgaris and Vigna unguiculata sprouts. Sprouting increased myo-inositol and glucose content and reduction of raffinose and ononitol was observed. The protein levels increased in P. vulgaris and decreased in V. unguiculata sprouting. The level of sucrose was maintained in both sprouts. D-Pinitol was detected only in quiescent seeds. Our results suggested that bean sprout is an important source of proteins, sucrose, glucose and myo-inositol. Additionally, bean sprouts have low levels of raffinose, an antinutritional compound.

Asunto(s)

Carbohidratos/análisis , Ciclitoles/análisis , Fabaceae/química , Phaseolus/química , Proteínas de Plantas/análisis , Cromatografía de Gases y Espectrometría de Masas

RESUMEN

The major Ricinus communis allergens are the 2S albumins, Ric c 1 and Ric c 3. These proteins contain a trypsin/α-amylase inhibitor family domain, suggesting that they have a role in insect resistance. In this study, we verified that Ric c 1 and Ric c 3 inhibited the α-amylase activity of Callosobruchus maculatus, Zabrotes subfasciatus, and Tenebrio molitor (TMA) larvae as well as mammalian α-amylase. The toxicity of 2S albumin was determined through its incorporation in C. maculatus larvae as part of an artificial diet. Bioassays revealed that 2S albumin reduced larval growth by 20%. We also analyzed the tridimensional structures of Ric c 1 and Ric c 3 by (a) constructing a comparative model of Ric c 1 based on Ric c 3 NMR structure and (b) constructing the theoretical structure of the Ric c 1-TMA and Ric c 3-TMA complexes. Our biological and theoretical results revealed that Ric c 1 and Ric c 3 are a new class of α-amylase inhibitors. They could potentially be used to help design inhibitors that would be useful in diverse fields, ranging from diabetes treatment to crop protection.

Asunto(s)

Albuminas 2S de Plantas/química , Antígenos de Plantas/química , Escarabajos/enzimología , Inhibidores Enzimáticos/química , Proteínas de Insectos/antagonistas & inhibidores , Ricinus communis/química , alfa-Amilasas/antagonistas & inhibidores , Albuminas 2S de Plantas/metabolismo , Albuminas 2S de Plantas/farmacología , Secuencia de Aminoácidos , Animales , Antígenos de Plantas/metabolismo , Antígenos de Plantas/farmacología , Ricinus communis/metabolismo , Escarabajos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Cinética , Datos de Secuencia Molecular , Semillas/química , Semillas/metabolismo , Alineación de Secuencia , alfa-Amilasas/química , alfa-Amilasas/metabolismo

RESUMEN

Studies have shown that vicilins (7S storage proteins) from seeds were able to bind to the surface of the Callosobruchus maculatus larval midgut and to the peritrophic matrices of the midguts of Diatraea saccharalis and Tenebrio molitor , inhibiting larval development. Vicilins were also shown to inhibit yeast growth and bind to yeast cells through the association with chitin-containing structures. The present work studies the association of peptides from vicilins of genotypes of Vigna unguiculata (susceptible and resistant to bruchid) with acetylated chitin and the toxicity of vicilin fragments and chitin-binding vicilin fragments to C. maculatus and phytopathogenic fungi. Hydrolysis of vicilins with alpha-chymotrypsin results in a complex mixture of fragments that were separated by chitin-affinity chromatography. Chitin-binding peptides from both genotypes were toxic to C. maculatus larvae, and alpha-chymotrypsin-hydrolyzed vicilins were deleterious to the above insect and to Fusarium oxysporum , Colletotrichum musae , and Saccharomyces cerevisiae fungi.

Asunto(s)

Escarabajos , Fungicidas Industriales/farmacología , Insecticidas , Proteínas de Almacenamiento de Semillas/farmacología , Semillas/química , Animales , Quitina/metabolismo , Quimotripsina/metabolismo , Colletotrichum/efectos de los fármacos , Fabaceae/química , Fusarium/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Almacenamiento de Semillas/metabolismo

RESUMEN

An insulin-binding protein was isolated from Canavalia ensiformis seed coat, by using an insulin-Sepharose 4B affinity chromatography, and the protein was identified as canavalin (Canavalia 7S globulin) by mass spectrometry analysis. The major novelty of these data is the acidic nature of this globulin insulin-binding, in contrast to the basic Bg-like insulin-binding proteins so far reported in plants.

Asunto(s)

Canavalia/química , Insulina/química , Proteínas de Plantas/química , Proteínas Portadoras , Cromatografía de Afinidad , Globulinas/química , Semillas/química