RESUMEN
Cytokinins are important for in vitro shoot regeneration in plants. Cytokinin N-glucosides are produced via an irreversible glycosylation pathway, which regulates the endogenous cytokinin content. Although cytokinin N-glucoside pathways have been uncovered in higher plants, no regulator has been identified to date. We performed a metabolome genome-wide association study (mGWAS), weighted gene co-expression network analysis (WGCNA), and expression quantitative trait nucleotide (eQTN) mappings to build a core triple genetic network (mGWAS-gene expression-phenotype) for the trans-zeatin N-glucoside (ZNG) metabolite using data from 435 unrelated Populus tomentosa individuals. Variation of the ZNG level in poplar is attributed to the differential transcription of PtoWRKY42, a member of WRKY multigene family group IIb. Functional analysis revealed that PtoWRKY42 negatively regulated ZNG accumulation by binding directly to the W-box of the UDP-glycosyltransferase 76C 1-1 (PtoUGT761-1) promoter. Also, PtoWRKY42 was strongly induced by leaf senescence, 6-BA, wounding, and salt stress, resulting in a reduced ZNG level. We identified PtoWRKY42, a negative regulator of cytokinin N-glucosides, which contributes to the natural variation in ZNG level and mediates ZNG accumulation by directly modulating the key glycosyltransferase gene PtoUGT76C1-1.
Asunto(s)
Citocininas , Populus , Redes Reguladoras de Genes , Estudio de Asociación del Genoma Completo , Populus/genética , ZeatinaRESUMEN
Photosynthesis and wood formation underlie the ability of trees to provide renewable resources and perform ecological functions; however, the genetic basis and regulatory pathways coordinating these two linked processes remain unclear. Here, we used a systems genetics strategy, integrating genome-wide association studies, transcriptomic analyses, and transgenic experiments, to investigate the genetic architecture of photosynthesis and wood properties among 435 unrelated individuals of Populus tomentosa, and unravel the coordinated regulatory networks resulting in two trait categories. We detected 222 significant single-nucleotide polymorphisms, annotated to 177 candidate genes, for 10 traits of photosynthesis and wood properties. Epistasis uncovered 74 epistatic interactions for phenotypes. Strikingly, we deciphered the coordinated regulation patterns of pleiotropic genes underlying phenotypic variations for two trait categories. Furthermore, expression quantitative trait nucleotide mapping and coexpression analysis were integrated to unravel the potential transcriptional regulatory networks of candidate genes coordinating photosynthesis and wood properties. Finally, heterologous expression of two pleiotropic genes, PtoMYB62 and PtoMYB80, in Arabidopsis thaliana demonstrated that they control regulatory networks balancing photosynthesis and stem secondary cell wall components, respectively. Our study provides insights into the regulatory mechanisms coordinating photosynthesis and wood formation in poplar, and should facilitate genetic breeding in trees via molecular design.
Asunto(s)
Populus , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes , Estudio de Asociación del Genoma Completo , Fotosíntesis/genética , Fitomejoramiento , Polimorfismo de Nucleótido Simple , Populus/genética , Madera/genéticaRESUMEN
Chlorogenic acid (CGA) plays a crucial role in defense response, immune regulation, and the response to abiotic stress in plants. However, the genetic regulatory network of CGA biosynthesis pathways in perennial plants remains unclear. Here, we investigated the genetic architecture for CGA biosynthesis using a metabolite-based genome-wide association study (mGWAS) and expression quantitative trait nucleotide (eQTN) mapping in a population of 300 accessions of Populus tomentosa. In total, we investigated 204 SNPs which were significantly associated with 11 metabolic traits, corresponding to 206 genes, and were mainly involved in metabolism and cell growth processes of P. tomentosa. We identified 874 eQTNs representing 1066 genes, in which the expression and interaction of causal genes affected phenotypic variation. Of these, 102 genes showed significant signatures of selection in three geographical populations, which provided insights into the adaptation of CGA biosynthesis to the local environment. Finally, we constructed a genetic network of six causal genes that coordinately regulate CGA biosynthesis, revealing the multiple regulatory patterns affecting CGA accumulation in P. tomentosa. Our study provides a multiomics strategy for understanding the genetic basis underlying the natural variation in the CGA biosynthetic metabolites of Populus, which will enhance the genetic development of abiotic-resistance varieties in forest trees.
Asunto(s)
Ácido Clorogénico/metabolismo , Redes Reguladoras de Genes , Metaboloma , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleótido Simple , Populus/metabolismo , Sitios de Carácter Cuantitativo , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Estudio de Asociación del Genoma Completo , Fenotipo , Proteínas de Plantas/genética , Populus/genética , Populus/crecimiento & desarrolloRESUMEN
Photosynthesis is a key reaction that ultimately generates the carbohydrates needed to form woody tissues in trees. However, the genetic regulatory network of protein-encoding genes (PEGs) and regulatory noncoding RNAs (ncRNAs), including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), underlying the photosynthetic pathway is unknown. Here, we integrated data from coexpression analysis, association studies (additive, dominance and epistasis), and expression quantitative trait nucleotide (eQTN) mapping to dissect the causal variants and genetic interaction network underlying photosynthesis in Populus. We initially used 30 PEGs, 6 miRNAs and 12 lncRNAs to construct a coexpression network based on the tissue-specific gene expression profiles of 15 Populus samples. Then, we performed association studies using a natural population of 435 unrelated Populus tomentosa individuals, and identified 72 significant associations (P ≤ 0.001, q ≤ 0.05) with diverse additive and dominance patterns underlying photosynthesis-related traits. Analysis of epistasis and eQTNs revealed that the complex genetic interactions in the coexpression network contribute to phenotypes at various levels. Finally, we demonstrated that heterologously expressing the most highly linked gene (PtoPsbX1) in this network significantly improved photosynthesis in Arabidopsis thaliana, pointing to the functional role of PtoPsbX1 in the photosynthetic pathway. This study provides an integrated strategy for dissecting a complex genetic interaction network, which should accelerate marker-assisted breeding efforts to genetically improve woody plants.
Asunto(s)
Redes Reguladoras de Genes , Fotosíntesis/genética , Populus/genética , Populus/fisiología , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Polimorfismo de Nucleótido SimpleRESUMEN
Increasing evidence indicates that DNA methylation is heritable and serves as an essential marker contributing to phenotypic variation. Linkage-linkage disequilibrium mapping was used to decipher the epigenetic architecture underlying nine growth and wood property traits in a linkage population (550 F1 progeny) and a natural population (435 unrelated individuals) of Populus using methylation-sensitive amplification polymorphism (MSAP)-based analysis. The interactions between genetic and epigenetic variants in the causative genes was further unveiled using expression quantitative trait methylation (eQTM) and nucleotide (eQTN) mapping strategies. A total of 163 epigenetic quantitative trait loci (epiQTLs; LOD ≥ 3.0), explaining 1.7-44.5% of phenotypic variations, were mapped to a high-resolution epigenetic map with 19 linkage groups, which was supported by the significant MSAP associations (P < 0.001) in the two populations. There were 23 causal genes involved in growth regulation and wood formation, whose markers were located in epiQTLs and associated with the same traits in both populations. Further eQTN and eQTM mapping showed that causal genetic and epigenetic variants within the 23 candidate genes may interact more in trans in gene expression and phenotype. The present study provides strategies for investigating epigenetic architecture and the interaction between genetic and epigenetic variants modulating complex traits in forest trees.
Asunto(s)
Epigénesis Genética , Desequilibrio de Ligamiento/genética , Populus/crecimiento & desarrollo , Populus/genética , Sitios de Carácter Cuantitativo/genética , Madera/crecimiento & desarrollo , Madera/genética , Mapeo Cromosómico , Metilación de ADN/genética , Regulación de la Expresión Génica de las Plantas , Estudios de Asociación Genética , Marcadores Genéticos , Genoma de Planta , Polimorfismo GenéticoRESUMEN
Lignin provides structural support in perennial woody plants and is a complex phenolic polymer derived from phenylpropanoid pathway. Lignin biosynthesis is regulated by coordinated networks involving transcription factors (TFs), microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). However, the genetic networks underlying the lignin biosynthesis pathway for tree growth and wood properties remain unknown. Here, we used association genetics (additive, dominant and epistasis) and expression quantitative trait nucleotide (eQTN) mapping to decipher the genetic networks for tree growth and wood properties in 435 unrelated individuals of Populus tomentosa. We detected 124 significant associations (P ≤ 6.89E-05) for 10 growth and wood property traits using 30 265 single nucleotide polymorphisms from 203 lignin biosynthetic genes, 81 TF genes, 36 miRNA genes and 71 lncRNA loci, implying their common roles in wood formation. Epistasis analysis uncovered 745 significant pairwise interactions, which helped to construct proposed genetic networks of lignin biosynthesis pathway and found that these regulators might affect phenotypes by linking two lignin biosynthetic genes. eQTNs were used to interpret how causal genes contributed to phenotypes. Lastly, we investigated the possible functions of the genes encoding 4-coumarate: CoA ligase and cinnamate-4-hydroxylase in wood traits using epistasis, eQTN mapping and enzymatic activity assays. Our study provides new insights into the lignin biosynthesis pathway in poplar and enables the novel genetic factors as biomarkers for facilitating genetic improvement of trees.
Asunto(s)
Genes de Plantas/genética , Lignina/biosíntesis , Populus/genética , ARN no Traducido/genética , Factores de Transcripción/genética , Madera/crecimiento & desarrollo , Genes de Plantas/fisiología , Desequilibrio de Ligamiento/genética , Redes y Vías Metabólicas/genética , MicroARNs/genética , MicroARNs/fisiología , Polimorfismo de Nucleótido Simple/genética , Populus/crecimiento & desarrollo , Populus/metabolismo , Sitios de Carácter Cuantitativo/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/fisiología , ARN no Traducido/fisiología , Factores de Transcripción/fisiología , Transcriptoma , Madera/metabolismoRESUMEN
The acrosome is a specialized organelle that covers the anterior part of the sperm nucleus and plays an essential role in mammalian fertilization. However, the regulatory mechanisms controlling acrosome biogenesis and acrosome exocytosis during fertilization are largely unknown. Equatorin (Eqtn) is a membrane protein that is specifically localized to the acrosomal membrane. In the present study, the physiological functions of Eqtn were investigated using a gene knockout mouse model. We found that Eqtn(-/-) males were subfertile. Only approximately 50% of plugged females were pregnant after mating with Eqtn(-/-) males, whereas more than 90% of plugged females were pregnant after mating with control males. Sperm and acrosomes from Eqtn(-/-) mice presented normal motility and morphology. However, the fertilization and induced acrosome exocytosis rates of Eqtn-deficient sperm were dramatically reduced. Further studies revealed that the Eqtn protein might interact with Syntaxin1a and SNAP25, but loss of Eqtn did not affect the protein levels of these genes. Therefore, our study demonstrates that Eqtn is not essential for acrosome biogenesis but is required for the acrosome reaction. Eqtn is involved in the fusion of the outer acrosomal membrane and the sperm plasma membrane during the acrosome reaction, most likely via an interaction with the SNARE complex.
Asunto(s)
Reacción Acrosómica , Acrosoma/fisiología , Proteínas de la Membrana/metabolismo , Acrosoma/ultraestructura , Animales , Femenino , Fertilización , Técnicas de Inactivación de Genes , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sintaxina 1/metabolismoRESUMEN
Drought frequency and severity are exacerbated by global climate change, which could compromise forest ecosystems. However, there have been minimal efforts to systematically investigate the genetic basis of the response to drought stress in perennial trees. Here, we implemented a systems genetics approach that combines co-expression analysis, association genetics, and expression quantitative trait nucleotide (eQTN) mapping to construct an allelic genetic regulatory network comprising four key regulators (PtoeIF-2B, PtoABF3, PtoPSB33, and PtoLHCA4) under drought stress conditions. Furthermore, Hap_01PtoeIF-2B, a superior haplotype associated with the net photosynthesis, was revealed through allelic frequency and haplotype analysis. In total, 75 candidate genes related to drought stress were identified through transcriptome analyses of five Populus cultivars (P. tremula × P. alba, P. nigra, P. simonii, P. trichocarpa, and P. tomentosa). Through association mapping, we detected 92 unique SNPs from 38 genes and 104 epistatic gene pairs that were associated with six drought-related traits by association mapping. eQTN mapping unravels drought stress-related gene loci that were significantly associated with the expression levels of candidate genes for drought stress. In summary, we have developed an integrated strategy for dissecting a complex genetic network, which facilitates an integrated population genomics approach that can assess the effects of environmental threats.
RESUMEN
Transcription factors (TFs) play crucial roles in the regulation of photosynthesis; elucidating these roles will facilitate our understanding of photosynthesis and thus accelerate its improvement for enhancing crop yield. Promoter analysis of 52 nuclear-encoded Populus tomentosa Carr. genes involved in the Calvin-Benson-Bassham (CBB) cycle revealed 706 motifs and 326 potentially interacting TFs. A backward elimination random forest (BWERF) algorithm reduced the number of TFs to 40, involved in a three-layer gene regulatory network (GRN) including 46 photosynthesis genes (bottom layer), 25 TFs (second layer) and 15 TFs (top layer). Phenotype-genotype association identified 248 single-nucleotide polymorphisms (SNPs) within 72 genes associated with 11 photosynthesis traits. Of the regulatory pairs identified by the BWERF (202 pairs), 77 TF-target combinations harbored SNPs associated with the same trait, supporting similar mechanisms of phenotype modulation. We used expression quantitative trait nucleotide (eQTN) analysis to identify causal SNPs affecting gene expression, identifying 1851 eQTN signals for 50 eGenes (genes whose expressions are regulated by eQTNs). Distribution patterns identified 14 eQTNs from seven TFs associated with eight expression levels of their downstream targets (defined in the GRN), whereas seven TF-target pairs were also identified by phenotype-genotype associations. To further validate the roles of TFs at the metabolic level, we selected 6764 SNPs from 55 genes (identified by GRN-association or GRN-eQTN pairs or both) for metabolic association, identifying variants within 10 TFs affecting metabolic processes underlying the CBB cycle. Our study provides new insights into the photosynthesis pathway in poplar and may facilitate understanding of processes underlying photosynthesis improvement.
Asunto(s)
Fotosíntesis , Populus , Redes Reguladoras de Genes , Regiones Promotoras Genéticas , Factores de TranscripciónRESUMEN
The acrosome is a Golgi-derived sperm cell organelle enclosed by a continuous acrosomal membrane. The acrosomal membrane complexes with surrounding matrices containing molecules necessary for fertilization; however, the complex of acrosomal membrane and associating matrices (CAMAM) has not been visualized in detail under living conditions. Here, we analyzed the CAMAM at the nanometer level using super-resolution stimulated emission depletion (STED) fluorescence microscopy and equatorin-enhanced green fluorescent protein transgenic mice. The STED images were compared with the corresponding images taken by immunoelectron microscopy. Consequently, the substructure of CAMAM could be differentiated at nanometer-scale resolution by STED microscopy without the need for sectioning. The information obtained in this study will be beneficial not only for understanding the molecular mechanism of fertilization but also for cell imaging under living conditions.