RESUMEN
Abscisic acid (ABA)-based chemically induced proximity (CIP) is primarily mediated by the interaction of the ABA receptor pyrabactin resistance 1-like 1 (PYL1) and the 2C-type protein phosphatase ABI1, which confers ABA-induced proximity to their fusion proteins, and offers precise temporal control of a wide array of biological processes. However, broad application of ABA-based CIP has been limited by ABA response intensity. In this study, we demonstrated that ABA-induced interaction between another ABA receptor pyrabactin resistance 1 (PYR1) and ABI1 exhibited higher ABA response intensity than that between PYL1 and ABI1 in HEK293T cells. We engineered PYR1-ABI1 and PYL1-ABI1 into ABA-induced transcriptional activation tools in mammalian cells by integration with CRISPR/dCas9 and found that the tool based on PYR1-ABI1 demonstrated better ABA response intensity than that based on PYL1-ABI1 for both exogenous and endogenous genes in mammalian cells. We further achieved ABA-induced RNA m6A modification installation and erasure by combining ABA-induced PYR1-ABI1 interaction with CRISPR/dCas13, successfully inhibiting tumor cell proliferation. We subsequently improved the interaction of PYR1-ABI1 through phage-assisted continuous evolution (PACE), successfully generating a PYR1 mutant (PYR1m) whose interaction with ABI1 exhibited a higher ABA response intensity than that of the wild-type. In addition, we tested the transcriptional activation tool based on PYRm-ABI1 and found that it also showed a higher ABA response intensity than that of the wild type. These results demonstrate that we have developed a novel ABA-based CIP and further improved upon it using PACE, providing a new approach for the modification of other CIP systems.
RESUMEN
Apoptosis, a programmed cell death mechanism, is a regulatory process controlling cell proliferation as cells undergo demise. Caspase-8 serves as a pivotal apoptosis-inducing factor that initiates the death receptor-mediated apoptosis pathway. In this investigation, we have devised an optogenetic method to swiftly modulate caspase-8 activation in response to blue light. The cornerstone of our optogenetic tool relies on the PHR domain of Arabidopsis thaliana cryptochrome 2, which self-oligomerizes upon exposure to blue light. In this study, we have developed two optogenetic approaches for rapidly controlling caspase-8 activation in response to blue light in cellular systems. The first strategy, denoted as Opto-Casp8-V1, entails the fusion expression of the Arabidopsis blue light receptor CRY2 N-terminal PHR domain with caspase-8. The second strategy, referred to as Opto-Casp8-V2, involves the independent fusion expression of caspase-8 with the PHR domain and the CRY2 blue light-interacting protein CIB1 N-terminal CIB1N. Upon induction with blue light, PHR undergoes aggregation, leading to caspase-8 aggregation. Additionally, the blue light-dependent interaction between PHR and CIB1N also results in caspase-8 aggregation. We have validated these strategies in both HEK293T and HeLa cells. The findings reveal that both strategies are capable of inducing apoptosis, with Opto-Casp8-V2 demonstrating significantly superior efficiency compared to Opto-Casp8-V1.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Humanos , Arabidopsis/genética , Arabidopsis/metabolismo , Criptocromos/genética , Criptocromos/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Optogenética/métodos , Caspasa 8/genética , Caspasa 8/metabolismo , Células HeLa , Células HEK293 , Luz , Factores de Transcripción/metabolismo , ApoptosisRESUMEN
Catalase (CAT) is often phosphorylated and activated by protein kinases to maintain hydrogen peroxide (H2O2) homeostasis and protect cells against stresses, but whether and how CAT is switched off by protein phosphatases remains inconclusive. Here, we identified a manganese (Mn2+)-dependent protein phosphatase, which we named PHOSPHATASE OF CATALASE 1 (PC1), from rice (Oryza sativa L.) that negatively regulates salt and oxidative stress tolerance. PC1 specifically dephosphorylates CatC at Ser-9 to inhibit its tetramerization and thus activity in the peroxisome. PC1 overexpressing lines exhibited hypersensitivity to salt and oxidative stresses with a lower phospho-serine level of CATs. Phosphatase activity and seminal root growth assays indicated that PC1 promotes growth and plays a vital role during the transition from salt stress to normal growth conditions. Our findings demonstrate that PC1 acts as a molecular switch to dephosphorylate and deactivate CatC and negatively regulate H2O2 homeostasis and salt tolerance in rice. Moreover, knockout of PC1 not only improved H2O2-scavenging capacity and salt tolerance but also limited rice grain yield loss under salt stress conditions. Together, these results shed light on the mechanisms that switch off CAT and provide a strategy for breeding highly salt-tolerant rice.
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Oryza , Catalasa/genética , Catalasa/metabolismo , Oryza/metabolismo , Peróxido de Hidrógeno/metabolismo , Proteína Fosfatasa 1/metabolismo , Tolerancia a la Sal/genética , Homeostasis , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Moso bamboo is a rapidly growing species with significant economic, social, and cultural value. Transplanting moso bamboo container seedlings for afforestation has become a cost-effective method. The growth and development of the seedlings is greatly affected by the quality of light, including light morphogenesis, photosynthesis, and secondary metabolite production. Therefore, studies on the effects of specific light wavelengths on the physiology and proteome of moso bamboo seedlings are crucial. In this study, moso bamboo seedlings were germinated in darkness and then exposed to blue and red light conditions for 14 days. The effects of these light treatments on seedling growth and development were observed and compared through proteomics analysis. Results showed that moso bamboo has higher chlorophyll content and photosynthetic efficiency under blue light, while it displays longer internode and root length, more dry weight, and higher cellulose content under red light. Proteomics analysis reveals that these changes under red light are likely caused by the increased content of cellulase CSEA, specifically expressed cell wall synthetic proteins, and up-regulated auxin transporter ABCB19 in red light. Additionally, blue light is found to promote the expression of proteins constituting photosystem II, such as PsbP and PsbQ, more than red light. These findings provide new insights into the growth and development of moso bamboo seedlings regulated by different light qualities.
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Proteómica , Plantones , Poaceae/metabolismo , Ácidos Indolacéticos/metabolismo , Crecimiento y Desarrollo , Regulación de la Expresión Génica de las PlantasRESUMEN
Cryptochromes are blue light receptors that regulate plant growth and development. They also act as the core components of the central clock oscillator in animals. Although plant cryptochromes have been reported to regulate the circadian clock in blue light, how they do so is unclear. Here we show that Arabidopsis cryptochrome 2 (CRY2) forms photobodies with the TCP22 transcription factor in response to blue light in plant cells. We provide evidence that PPK kinases influence the characteristics of these photobodies and that together these components, along with LWD transcriptional regulators, can positively regulate the expression of CCA1 encoding a central component of the circadian oscillator.
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Proteínas de Arabidopsis , Arabidopsis , Relojes Circadianos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ritmo Circadiano , Criptocromos/genética , Criptocromos/metabolismo , Regulación de la Expresión Génica de las Plantas , Luz , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Maize yield is significantly influenced by low temperature, particularly chilling stress at the maize seedling stage. Various physiological approaches have been established to resist chilling stress; however, the detailed proteins change patterns underlying the maize chilling stress response at the seedling stage remain unknown, preventing the development of breeding-based methods to resist chilling stress in maize. Thus, we performed comprehensive physiological, comparative proteomics and specific phytohormone abscisic acid (ABA) assay on different maize inbred lines (tolerant-line KR701 and sensitive-line hei8834) at different seedling stages (the first leaf stage and third leaf stage) under chilling stress. The results revealed several signalling proteins and pathways in response to chilling stress at the maize seedling stage. Meanwhile, we found ABA pathway was important for chilling resistance of tolerant-line KR701 at the first leaf stage. Related chilling-responsive proteins were further catalogued and analysed, providing a resource for further investigation and maize breeding.
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Proteómica , Zea mays , Ácido Abscísico/metabolismo , Genotipo , Proteómica/métodos , Plantones/genética , Zea mays/genéticaRESUMEN
In vascular plants, cryptochromes acting as blue-light photoreceptors have various functions to adapt plants to the fluctuating light conditions on land, while the roles of cryptochromes in bryophytes have been rarely reported. In this study, we investigated functions of a single-copy ortholog of cryptochrome (MpCRY) in the liverwort Marchantia polymorpha. Knock-out of MpCRY showed that a large number of the mutant plants exhibited asymmetric growth of thalli under blue light. Transcriptome analyses indicated that MpCRY is mainly involved in photosynthesis and sugar metabolism. Further physiological analysis showed that Mpcry mutant exhibited a reduction in CO2 uptake and sucrose metabolism. In addition, exogenous application of sucrose or glucose partially restored the symmetrical growth of the Mpcry mutant thalli. Together, these results suggest that MpCRY is involved in the symmetrical growth of thallus and the regulation of carbon fixation and sucrose metabolism in M. polymorpha.
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Ciclo del Carbono , Criptocromos/metabolismo , Marchantia/metabolismo , Sacarosa/metabolismo , Secuencia de Aminoácidos , Ciclo del Carbono/efectos de la radiación , Criptocromos/química , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Glucosa/metabolismo , Luz , Marchantia/efectos de la radiación , Mutación/genética , Fotosíntesis/genética , Fotosíntesis/efectos de la radiación , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Transcripción Genética/efectos de la radiaciónRESUMEN
Phytohormone ABA regulates the expression of numerous genes to significantly affect seed dormancy, seed germination and early seedling responses to biotic and abiotic stresses. However, the function of many ABA-responsive genes remains largely unknown. In order to improve the ABA-related signaling network, we conducted a large-scale ABA phenotype screening. LSH, an important transcription factor family, extensively participates in seedling development and floral organogenesis in plants, but whether its family genes are involved in the ABA signaling pathway has not been reported. Here we describe a new function of the transcription factor LSH8 in an ABA signaling pathway. In this study, we found that LSH8 was localized in the nucleus, and the expression level of LSH8 was significantly induced by exogenous ABA at the transcription level and protein level. Meanwhile, seed germination and root length measurements revealed that lsh8 mutant lines were ABA insensitive, whereas LSH8 overexpression lines showed an ABA-hypersensitive phenotype. With further TMT labeling quantitative proteomic analysis, we found that under ABA treatment, ABA-responsive proteins (ARPs) in the lsh8 mutant presented different changing patterns with those in wild-type Col4. Additionally, the number of ARPs contained in the lsh8 mutant was 397, six times the number in wild-type Col4. In addition, qPCR analysis found that under ABA treatment, LSH8 positively mediated the expression of downstream ABA-related genes of ABI3, ABI5, RD29B and RAB18. These results indicate that in Arabidopsis, LSH8 is a novel ABA regulator that could specifically change the expression pattern of APRs to positively mediate ABA responses.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Germinación/fisiología , Fenotipo , Proteómica/métodos , Semillas/metabolismoRESUMEN
Phytochromobilin (PΦB) participates in the regulation of plant growth and development as an important synthetase of photoreceptor phytochromes (phy). In addition, Arabidopsis long hypocotyl 2 (HY2) appropriately works as a key PΦB synthetase. However, whether HY2 takes part in the plant stress response signal network remains unknown. Here, we described the function of HY2 in NaCl signaling. The hy2 mutant was NaCl-insensitive, whereas HY2-overexpressing lines showed NaCl-hypersensitive phenotypes during seed germination. The exogenous NaCl induced the transcription and the protein level of HY2, which positively mediated the expression of downstream stress-related genes of RD29A, RD29B, and DREB2A. Further quantitative proteomics showed the patterns of 7391 proteins under salt stress. HY2 was then found to specifically mediate 215 differentially regulated proteins (DRPs), which, according to GO enrichment analysis, were mainly involved in ion homeostasis, flavonoid biosynthetic and metabolic pathways, hormone response (SA, JA, ABA, ethylene), the reactive oxygen species (ROS) metabolic pathway, photosynthesis, and detoxification pathways to respond to salt stress. More importantly, ANNAT1-ANNAT2-ANNAT3-ANNAT4 and GSTU19-GSTF10-RPL5A-RPL5B-AT2G32060, two protein interaction networks specifically regulated by HY2, jointly participated in the salt stress response. These results direct the pathway of HY2 participating in salt stress, and provide new insights for the plant to resist salt stress.
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Arabidopsis/genética , Arabidopsis/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Sequías , Germinación/fisiología , Oxidorreductasas/fisiología , Fitocromo/metabolismo , Desarrollo de la Planta/efectos de los fármacos , Plantas Modificadas Genéticamente , Estrés Salino/efectos de los fármacos , Estrés Salino/genética , Estrés Salino/fisiología , Semillas/metabolismo , Transducción de Señal/fisiología , Cloruro de Sodio/metabolismo , Estrés Fisiológico/genéticaRESUMEN
De novo shoot organogenesis is an important biotechnological tool for fundamental studies in plant. However, it is difficult in most bamboo species, and the genetic control of this highly dynamic and complicated regeneration process remains unclear. In this study, based on an in-depth analysis at the cellular level, the shoot organogenesis from calli of Ma bamboo (Dendrocalamus latiflorus Munro) was divided into five stages. Subsequently, single-molecule long-read isoform sequencing of tissue samples pooled from all five stages was performed to generate a full-length transcript landscape. A total of 83 971 transcripts, including 73 209 high-quality full-length transcripts, were captured, which served as an annotation reference for the subsequent RNA sequencing analysis. Time-course transcriptome analysis of samples at the abovementioned five stages was conducted to investigate the global gene expression atlas showing genome-wide expression of transcripts during the course of bamboo shoot organogenesis. K-means clustering analysis and stage-specific transcript identification revealed important dynamically expressed transcription regulators that function in bamboo shoot organogenesis. The majority of abiotic stress-responsive genes altered their expression levels during this process, and further experiments demonstrated that exogenous application of moderate but not severe abiotic stress increased the shoot regeneration efficiency. In summary, our study provides an overview of the genetic flow dynamics during bamboo shoot organogenesis. Full-length cDNA sequences generated in this study can serve as a valuable resource for fundamental and applied research in bamboo in the future.
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Bambusa/genética , Organogénesis de las Plantas/genética , Estrés Fisiológico , Transcriptoma , Bambusa/crecimiento & desarrollo , Bambusa/fisiología , ADN Complementario/genética , Perfilación de la Expresión Génica , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Brotes de la Planta/genética , Brotes de la Planta/crecimiento & desarrollo , Brotes de la Planta/fisiología , ARN de Planta/genética , Análisis de Secuencia de ARN , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
For rapid growth, moso bamboo (Phyllostachys edulis) requires large amounts of nutrients. Nitrate is an indispensable molecular signal to regulate nitrogen absorption and assimilation, which are regulated by group III NIN-LIKE PROTEINs (NLPs). However, no Phyllostachys edulis NLP (PeNLP) has been characterized. Here, eight PeNLPs were identified, which showed dynamic expression patterns in bamboo tissues. Nitrate did not affect PeNLP mRNA levels, and PeNLP1, -2, -5, -6, -7, and -8 successfully restored nitrate signaling in Arabidopsis atnlp7-1 protoplasts through recovering AtNiR and AtNRT2.1 expression. Four group I and II PeNLPs (PeNLP1, -2, -5, and -8) interacted with the nitrate-responsive cis-element of PeNiR. Moreover, nitrate triggered the nuclear retention of PeNLP8. PeNLP8 overexpression in Arabidopsis significantly increased the primary root length, lateral root number, leaf area, and dry and wet weight of the transgenic plants, and PeNLP8 expression rescued the root architectural defect phenotype of atnlp7-1 mutants. Interestingly, PeNLP8 overexpression dramatically reduced nitrate content but elevated total amino acid content in Arabidopsis. Overall, the present study unveiled the potential involvement of group I and II NLPs in nitrate signaling regulation and provided genetic resources for engineering plants with high nitrogen use efficiency.
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Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Nitratos/metabolismo , Proteínas de Plantas/metabolismo , Poaceae/genética , Poaceae/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Genes de Plantas , Genoma de Planta , Mutación , Filogenia , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Tallos de la Planta/genética , Tallos de la Planta/metabolismo , Plantas Modificadas Genéticamente/genéticaRESUMEN
The evolutionarily conserved target of rapamycin (TOR) kinase acts as a master regulator that coordinates cell proliferation and growth by integrating nutrient, energy, hormone and stress signals in all eukaryotes1,2. Research has focused mainly on TOR-regulated translation, but how TOR orchestrates the global transcriptional network remains unclear. Here we identify ethylene-insensitive protein 2 (EIN2), a central integrator3-5 that shuttles between the cytoplasm and the nucleus, as a direct substrate of TOR in Arabidopsis thaliana. Glucose-activated TOR kinase directly phosphorylates EIN2 to prevent its nuclear localization. Notably, the rapid global transcriptional reprogramming that is directed by glucose-TOR signalling is largely compromised in the ein2-5 mutant, and EIN2 negatively regulates the expression of a wide range of target genes of glucose-activated TOR that are involved in DNA replication, cell wall and lipid synthesis and various secondary metabolic pathways. Chemical, cellular and genetic analyses reveal that cell elongation and proliferation processes that are controlled by the glucose-TOR-EIN2 axis are decoupled from canonical ethylene-CTR1-EIN2 signalling, and mediated by different phosphorylation sites. Our findings reveal a molecular mechanism by which a central signalling hub is shared but differentially modulated by diverse signalling pathways using distinct phosphorylation codes that can be specified by upstream protein kinases.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Desarrollo de la Planta , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Arabidopsis/citología , Arabidopsis/genética , Dominio Catalítico , Proteínas de Unión al ADN/metabolismo , Etilenos/metabolismo , Glucosa/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Meristema/metabolismo , Fosforilación , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas Quinasas/metabolismo , Especificidad por Sustrato , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
COP1/SPA1 complex in Arabidopsis inhibits photomorphogenesis through the ubiquitination of multiple photo-responsive transcription factors in darkness, but such inhibiting function of COP1/SPA1 complex would be suppressed by cryptochromes in blue light. Extensive studies have been conducted on these mechanisms in Arabidopsis whereas little attention has been focused on whether another branch of land plants bryophyte utilizes this blue-light regulatory pathway. To study this problem, we conducted a study in the liverwort Marchantia polymorpha and obtained a MpSPA knock-out mutant, in which Mpspa exhibits the phenotype of an increased percentage of individuals with asymmetrical thallus growth, similar to MpCRY knock-out mutant. We also verified interactions of MpSPA with MpCRY (in a blue light-independent way) and with MpCOP1. Concomitantly, both MpSPA and MpCOP1 could interact with MpHY5, and MpSPA can promote MpCOP1 to ubiquitinate MpHY5 but MpCRY does not regulate the ubiquitination of MpHY5 by MpCOP1/MpSPA complex. These data suggest that COP1/SPA ubiquitinating HY5 is conserved in Marchantia polymorpha, but dissimilar to CRY in Arabidopsis, MpCRY is not an inhibitor of this process under blue light.
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Proteínas de Arabidopsis/metabolismo , Marchantia/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Criptocromos/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , LuzRESUMEN
PD1/PDL1 pathway plays a critical role in cancer immune responses. The immune checkpoint inhibitors of PD1/PDL1 have been well explored and developed for immunotherapies of solid tumors. Recently, various monoclonal antibodies targeting the PD1/PDL1 pathway have emerged and achieved remarkable success in clinical trials. However, challenges with these monoclonal antibodies have appeared during cancer therapies, including predictors of response, patient selection, and innate resistance. Thus, a competitive antagonist of native PD1/PDL1, with smaller size and lower side-effect, is required for future cancer therapies. In this study, we utilized a protein evolution system of phage-assisted continuous evolution (PACE) to evolve PD1 continuously. Our results indicated that the newly evolved PD1 bound to PDL1 with higher affinity. The interactome analysis further suggested that these evolved PD1s exhibited higher specificity with PDL1. Therefore, these evolved PD1s may be applied as a new tool for tumor immunotherapy.
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Antígeno B7-H1/metabolismo , Evolución Molecular Dirigida/métodos , Receptor de Muerte Celular Programada 1/metabolismo , Antígeno B7-H1/química , Antígeno B7-H1/genética , Sitios de Unión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica/efectos de los fármacos , Genes Reporteros , Células HEK293 , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida/métodos , Mutágenos/farmacología , Biblioteca de Péptidos , Plásmidos/química , Plásmidos/metabolismo , Receptor de Muerte Celular Programada 1/química , Receptor de Muerte Celular Programada 1/genética , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The conversion of light energy into chemical energy in leaves is very important for plant growth and development. During this process, chlorophylls and their derivatives are indispensable as their fundamental role in the energy absorption and transduction activities. Chlorophyll variation mutants are important materials for studying chlorophyll metabolism, chloroplast biogenesis, photosynthesis and related physiological processes. Here, a chlorophyll-reduced mutant (crm1) was isolated from ethyl methanesulfonate (EMS) mutagenized Brassica napus. Compared to wild type, crm1 showed yellow leaves, reduced chlorophyll content, fewer thylakoid stacks and retarded growth. Quantitative mass spectrometry analysis with Tandem Mass Tag (TMT) isobaric labeling showed that totally 4575 proteins were identified from the chloroplast of Brassica napus leaves, and 466 of which displayed differential accumulations between wild type and crm1. The differential abundance proteins were found to be involved in chlorophyll metabolism, photosynthesis, phagosome and proteasome. Our results suggest that the decreased abundance of chlorophyll biosynthetic enzymes, proteins involved in photosynthesis might account for the reduced chlorophyll content, impaired thylakoid structure, and reduction of plant productivity. The increased abundance of proteins involved in phagosome and proteasome pathways might allow plants to adapt the proteome to environmental conditions to ensure growth and survival due to chlorophyll reduction. BIOLOGICAL SIGNIFICANCE: Photosynthesis, which consists of light and dark reactions, is fundamental to biomass production. Chloroplast is regarded as the main site for photosynthesis. During photosynthesis, the pigment chlorophyll is essential for light harvesting and energy transfer. This work provides new insights into protein expression patterns, and enables the identification of many attractive candidates for investigation of chlorophyll biosynthesis, chloroplast structure and photosynthesis in Brassica napus. These findings may be applied to improve the photosynthetic efficiency by genetic engineering in crops.
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Brassica napus , Brassica napus/genética , Brassica napus/metabolismo , Clorofila , Cloroplastos/metabolismo , Fenotipo , Fotosíntesis , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ProteómicaRESUMEN
Moso bamboo is an important forest species with a variety of ecological, economic, and cultural values. However, the gene annotation information of moso bamboo is only based on the transcriptome sequencing, lacking the evidence of proteome. The lignification and fiber in moso bamboo leads to a difficulty in the extraction of protein using conventional methods, which seriously hinders research on the proteomics of moso bamboo. The purpose of this study is to establish efficient methods for extracting the total proteins from moso bamboo for following mass spectrometry-based quantitative proteome identification. Here, we have successfully established a set of efficient methods for extracting total proteins of moso bamboo followed by mass spectrometry-based label-free quantitative proteome identification, which further improved the protein annotation of moso bamboo genes. In this study, 10,376 predicted coding genes were confirmed by quantitative proteomics, accounting for 35.8% of all annotated protein-coding genes. Proteome analysis also revealed the protein-coding potential of 1015 predicted long noncoding RNA (lncRNA), accounting for 51.03% of annotated lncRNAs. Thus, mass spectrometry-based proteomics provides a reliable method for gene annotation. Especially, quantitative proteomics revealed the translation patterns of proteins in moso bamboo. In addition, the 3284 transcript isoforms from 2663 genes identified by Pacific BioSciences (PacBio) single-molecule real-time long-read isoform sequencing (Iso-Seq) was confirmed on the protein level by mass spectrometry. Furthermore, domain analysis of mass spectrometry-identified proteins encoded in the same genomic locus revealed variations in domain composition pointing towards a functional diversification of protein isoform. Finally, we found that part transcripts targeted by nonsense-mediated mRNA decay (NMD) could also be translated into proteins. In summary, proteomic analysis in this study improves the proteomics-assisted genome annotation of moso bamboo and is valuable to the large-scale research of functional genomics in moso bamboo. In summary, this study provided a theoretical basis and technical support for directional gene function analysis at the proteomics level in moso bamboo.
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Genoma de Planta , Proteínas de Plantas , Poaceae/genética , Estabilidad del ARN , Empalme Alternativo , Anotación de Secuencia Molecular , Degradación de ARNm Mediada por Codón sin Sentido , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Isoformas de Proteínas/genética , Proteogenómica , ARN Largo no CodificanteRESUMEN
Peptides could have specific tastes or bioactivities depending on the length and sequence of amino acids. Till date it remains unknown what peptides are formed during the white tea manufacturing process and whether they contribute to the flavor or bio-activities of white tea. As a first step to address these questions, we applied ultra-high pressure liquid chromatography coupled with quadrupole-orbitrap ultra-high resolution mass spectrometry (UPLC-Quadrupole-Orbitrap-UHRMS) to monitor peptides dynamic changes during the withering process. A total of 196 abundant peptides were identified. Most of them were oligopeptides within a molecular weight of 1000â¯Da. Four of them were randomly selected, synthesized peptides were applied for further confirmation and quantification. Sequence analysis suggested that some of them were potential taste contributors. Proteinase cleave site analysis identified two separate periods of active proteins degradation at 0-12â¯h and 30-42â¯h of the withering processes. Further analysis of cleavage sites also suggested that protein degradation during withering steps were random rather than a stepwise reaction.
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Cromatografía Líquida de Alta Presión , Espectrometría de Masas , Oligopéptidos/análisis , Té/química , Análisis de los Alimentos , Manipulación de Alimentos , Calidad de los Alimentos , Límite de DetecciónRESUMEN
Soybean (Glycine max (L.) Merr.), grown for its plant oils and proteins, is one of the most important crops throughout the world. Generating stable and heritable transgenic soybeans is relatively inefficient; therefore, there is an urgent need for a simple and high-efficient transient transformation method by which to enable the investigation of gene functions in soybeans, which will facilitate the elucidation and improvement of the molecular mechanisms regulating the associated agronomic traits. We established a system of transient expression in soybean mesophyll protoplasts and obtained a high level of protoplast transfection efficiency (up to 83.5%). The subcellular activity of the protoplasts was well preserved, as demonstrated by the dynamic formation of GmCRY nucleus photobodies (NPs) and/or cytoplasmic photobody-like structures (CPs) in response to blue light. In addition, we showed that GmCRY1b CPs colocalized with GmCOP1b, a co-ortholog of Arabidopsis thaliana CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), which provided new insight into the potential roles of GmCRY1s in the cytoplasm.
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Glycine max/genética , Glycine max/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Protoplastos/metabolismo , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Citoplasma/metabolismo , Regulación de la Expresión Génica de las Plantas , TransfecciónRESUMEN
In response to blue light, cryptochromes photoexcite and interact with signal partners to transduce signal almost synchronously in plants. The detailed mechanism of CRY-mediated light signaling remains unclear: the photobiochemical reactions of cryptochrome are transient and synchronous, thus making the monitoring and analysis of each step difficult in plant cells. In this study, we reconstituted the Arabidopsis CRY2 signaling pathway in mammalian cells and investigated the biological role of Arabidopsis CRY2 in this heterologous system, eliminating the interferences of other plant proteins. Our results demonstrated that, besides being the light receptor, Arabidopsis CRY2 binds to DNA directly and acts as a transcriptional activator in a blue-light-enhanced manner. Similar to classic transcription factors, we found that the transcriptional activity of CRY2 is regulated by its dimerization and phosphorylation. In addition, CRY2 cooperates with CIB1 to regulate transcription by enhancing the DNA affinity and transcriptional activity of CIB1 under blue light.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Criptocromos/metabolismo , ADN/metabolismo , Transducción de Señal , Transcripción Genética , Arabidopsis/efectos de la radiación , Células HEK293 , Humanos , Luz , Fototransducción/efectos de la radiación , Fosforilación/efectos de la radiación , Unión Proteica/efectos de la radiación , Multimerización de Proteína/efectos de la radiación , Transcripción Genética/efectos de la radiación , Activación Transcripcional/genéticaRESUMEN
Cryptochromes (CRYs) are blue light receptors that mediate light regulation of plant growth and development. Land plants possess various numbers of cryptochromes, CRY1 and CRY2, which serve overlapping and partially redundant functions in different plant species. Cryptochromes exist as physiologically inactive monomers in darkness; photoexcited cryptochromes undergo homodimerization to increase their affinity to the CRY-signaling proteins, such as CIBs (CRY2-interacting bHLH), PIFs (Phytochrome-Interacting Factors), AUX/IAA (Auxin/INDOLE-3-ACETIC ACID), and the COP1-SPAs (Constitutive Photomorphogenesis 1-Suppressors of Phytochrome A) complexes. These light-dependent protein-protein interactions alter the activity of the CRY-signaling proteins to change gene expression and developmental programs in response to light. In the meantime, photoexcitation also changes the affinity of cryptochromes to the CRY-regulatory proteins, such as BICs (Blue-light Inhibitors of CRYs) and PPKs (Photoregulatory Protein Kinases), to modulate the activity, modification, or abundance of cryptochromes and photosensitivity of plants in response to the changing light environment.