RESUMO
The fusion of male and female gametes is a fundamental process in the perpetuation and diversification of species. During the last 50 years, significant efforts have been made to isolate and characterize sperm cells from flowering plants, and to identify how these cells interact with female gametes to achieve double fertilization. The first techniques and analytical approaches not only provided structural and biochemical characterizations of plant sperm cells but also paved the way for in vitro fertilization studies. Further technological advances then led to unique insights into sperm biology at the transcriptomic, proteomic, and epigenetic level. Starting with a historical overview of sperm cell isolation techniques, we provide examples of how these contributed to create our current knowledge of sperm cell biology, and point out remaining challenges.
Assuntos
Proteômica , Sementes , Animais , Espermatozoides , Fertilização , Separação CelularRESUMO
Because it is the precursor for various essential cellular components, the amino acid serine is indispensable for every living organism. In plants, serine is synthesized by two major pathways: photorespiration and the phosphorylated pathway of serine biosynthesis (PPSB). However, the importance of these pathways in providing serine for plant development is not fully understood. In this study, we examine the relative contributions of photorespiration and PPSB to providing serine for growth and metabolism in the C3 model plant Arabidopsis thaliana. Our analyses of cell proliferation and elongation reveal that PPSB-derived serine is indispensable for plant growth and its loss cannot be compensated by photorespiratory serine biosynthesis. Using isotope labeling, we show that PPSB-deficiency impairs the synthesis of proteins and purine nucleotides in plants. Furthermore, deficiency in PPSB-mediated serine biosynthesis leads to a strong accumulation of metabolites related to nitrogen metabolism. This result corroborates 15N-isotope labeling in which we observed an increased enrichment in labeled amino acids in PPSB-deficient plants. Expression studies indicate that elevated ammonium uptake and higher glutamine synthetase/glutamine oxoglutarate aminotransferase (GS/GOGAT) activity causes this phenotype. Metabolic analyses further show that elevated nitrogen assimilation and reduced amino acid turnover into proteins and nucleotides are the most likely driving forces for changes in respiratory metabolism and amino acid catabolism in PPSB-deficient plants. Accordingly, we conclude that even though photorespiration generates high amounts of serine in plants, PPSB-derived serine is more important for plant growth and its deficiency triggers the induction of nitrogen assimilation, most likely as an amino acid starvation response.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Proliferação de Células/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Nitrogênio/metabolismo , Desenvolvimento Vegetal/fisiologia , Reguladores de Crescimento de Plantas/metabolismo , Serina/biossíntese , Vias Biossintéticas , FosforilaçãoRESUMO
Flowering plants (angiosperms) are characterized by pollen tubes (PTs; male gametophytes) carrying two immobile sperm cells that grow over long distances through the carpel toward the ovules, where double fertilization is executed. It is not understood how these reproductive structures evolved, which genes occur de novo in male gametophytes of angiosperms, and to which extent PT functions are conserved among angiosperms. To contribute to a deeper understanding of the evolution of gametophyte functions, we generated RNA sequencing data from seven reproductive and two vegetative control tissues of the basal angiosperm Amborella trichopoda and complemented these with proteomic data of pollen grains (PGs) and PTs. The eudicot model plant Arabidopsis (Arabidopsis thaliana) served as a reference organism for data analysis, as more than 200 genes have been associated with male gametophyte functions in this species. We describe methods to collect bicellular A. trichopoda PGs, to induce their germination in vitro, and to monitor PT growth and germ cell division. Transcriptomic and proteomic analyses indicate that A. trichopoda PGs are prepared for germination requiring lipids, energy, but likely also reactive oxygen species, while PTs are especially characterized by catabolic/biosynthetic and transport processes including cell wall biosynthesis and gene regulation. Notably, a number of pollen-specific genes were lacking in Arabidopsis, and the number of genes involved in pollen signaling is significantly reduced in A. trichopoda In conclusion, we provide insight into male gametophyte functions of the most basal angiosperm and establish a valuable resource for future studies on the evolution of flowering plants.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Germinação/genética , Magnoliopsida/crescimento & desenvolvimento , Magnoliopsida/genética , Pólen/crescimento & desenvolvimento , Pólen/genética , Evolução Biológica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Germinação/fisiologia , Tubo Polínico/genética , Tubo Polínico/crescimento & desenvolvimento , Proteômica , TranscriptomaRESUMO
In eukaryotes, the regulated transport of mRNAs from the nucleus to the cytosol through nuclear pore complexes represents an important step in the expression of protein-coding genes. In plants, the mechanism of nucleocytosolic mRNA transport and the factors involved are poorly understood. The Arabidopsis (Arabidopsis thaliana) genome encodes two likely orthologs of UAP56-interacting factor, which acts as mRNA export factor in mammalian cells. In yeast and plant cells, both proteins interact directly with the mRNA export-related RNA helicase UAP56 and the interaction was mediated by an N-terminal UAP56-binding motif. Accordingly, the two proteins were termed UAP56-INTERACTING EXPORT FACTOR1 and 2 (UIEF1/2). Despite lacking a known RNA-binding motif, recombinant UIEF1 interacted with RNA, and the C-terminal part of UIEF1 mainly contributed to the RNA interaction. Mutation of UIEF1, UIEF2, or both in the double-mutant 2xuief caused modest growth defects. A cross between the 2xuief and 4xaly (defective in the four ALY1-4 mRNA export factors) mutants produced the sextuple mutant 4xaly 2xuief, which displayed more severe growth impairment than the 4xaly plants. Developmental defects including delayed bolting and reduced seed set were observed in the 4xaly but not the 2xuief plants. Analysis of the cellular distribution of polyadenylated mRNAs revealed more pronounced nuclear mRNA accumulation in 4xaly 2xuief than in 2xuief and 4xaly cells. In conclusion, the results indicate that UIEF1 and UIEF2 act as mRNA export factors in plants and that they cooperate with ALY1-ALY4 to mediate efficient nucleocytosolic mRNA transport.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Genoma de Planta , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Although the plant Phosphorylated Pathway of l-Ser Biosynthesis (PPSB) is essential for embryo and pollen development, and for root growth, its metabolic implications have not been fully investigated. A transcriptomics analysis of Arabidopsis (Arabidopsis thaliana) PPSB-deficient mutants at night, when PPSB activity is thought to be more important, suggested interaction with the sulfate assimilation process. Because sulfate assimilation occurs mainly in the light, we also investigated it in PPSB-deficient lines in the day. Key genes in the sulfate starvation response, such as the adenosine 5'phosphosulfate reductase genes, along with sulfate transporters, especially those involved in sulfate translocation in the plant, were induced in the PPSB-deficient lines. However, sulfate content was not reduced in these lines as compared with wild-type plants; besides the glutathione (GSH) steady-state levels in roots of PPSB-deficient lines were even higher than in wild type. This suggested that PPSB deficiency perturbs the sulfate assimilation process between tissues/organs. Alteration of thiol distribution in leaves from different developmental stages, and between aerial parts and roots in plants with reduced PPSB activity, provided evidence supporting this idea. Diminished PPSB activity caused an enhanced flux of 35S into thiol biosynthesis, especially in roots. GSH turnover also accelerated in the PPSB-deficient lines, supporting the notion that not only biosynthesis, but also transport and allocation, of thiols were perturbed in the PPSB mutants. Our results suggest that PPSB is required for sulfide assimilation in specific heterotrophic tissues and that a lack of PPSB activity perturbs sulfur homeostasis between photosynthetic and nonphotosynthetic tissues.
Assuntos
Arabidopsis/metabolismo , Serina/biossíntese , Transdução de Sinais/genética , Enxofre/metabolismo , Arabidopsis/genética , Oxirredução , Fosforilação , TranscriptomaRESUMO
In plants, phosphoglycerate kinase (PGK) converts 1,3-bisphosphoglycerate into 3-phosphoglycerate in glycolysis but also participates in the reverse reaction in gluconeogenesis and the Calvin-Benson cycle. In the databases, we found three genes that encode putative PGKs. Arabidopsis (Arabidopsis thaliana) PGK1 was localized exclusively in the chloroplasts of photosynthetic tissues, while PGK2 was expressed in the chloroplast/plastid of photosynthetic and nonphotosynthetic cells. PGK3 was expressed ubiquitously in the cytosol of all studied cell types. Measurements of carbohydrate content and photosynthetic activities in PGK mutants and silenced lines corroborated that PGK1 was the photosynthetic isoform, while PGK2 and PGK3 were the plastidial and cytosolic glycolytic isoforms, respectively. The pgk1.1 knockdown mutant displayed reduced growth, lower photosynthetic capacity, and starch content. The pgk3.2 knockout mutant was characterized by reduced growth but higher starch levels than the wild type. The pgk1.1 pgk3.2 double mutant was bigger than pgk3.2 and displayed an intermediate phenotype between the two single mutants in all measured biochemical and physiological parameters. Expression studies in PGK mutants showed that PGK1 and PGK3 were down-regulated in pgk3.2 and pgk1.1, respectively. These results indicate that the down-regulation of photosynthetic activity could be a plant strategy when glycolysis is impaired to achieve metabolic adjustment and optimize growth. The double mutants of PGK3 and the triose-phosphate transporter (pgk3.2 tpt3) displayed a drastic growth phenotype, but they were viable. This implies that other enzymes or nonspecific chloroplast transporters could provide 3-phosphoglycerate to the cytosol. Our results highlight both the complexity and the plasticity of the plant primary metabolic network.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Fosfoglicerato Quinase/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Citosol/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Glicéricos/metabolismo , Metabolômica/métodos , Família Multigênica , Mutação , Fosfoglicerato Quinase/genética , Componentes Aéreos da Planta/genética , Componentes Aéreos da Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Plásticos/metabolismoRESUMO
The regulated transport of mRNAs from the cell nucleus to the cytosol is a critical step linking transcript synthesis and processing with translation. However, in plants, only a few of the factors that act in the mRNA export pathway have been functionally characterized. Flowering plant genomes encode several members of the ALY protein family, which function as mRNA export factors in other organisms. Arabidopsis (Arabidopsis thaliana) ALY1 to ALY4 are commonly detected in root and leaf cells, but they are differentially expressed in reproductive tissue. Moreover, the subnuclear distribution of ALY1/2 differs from that of ALY3/4. ALY1 binds with higher affinity to single-stranded RNA than double-stranded RNA and single-stranded DNA and interacts preferentially with 5-methylcytosine-modified single-stranded RNA. Compared with the full-length protein, the individual RNA recognition motif of ALY1 binds RNA only weakly. ALY proteins interact with the RNA helicase UAP56, indicating a link to the mRNA export machinery. Consistently, ALY1 complements the lethal phenotype of yeast cells lacking the ALY1 ortholog Yra1. Whereas individual aly mutants have a wild-type appearance, disruption of ALY1 to ALY4 in 4xaly plants causes vegetative and reproductive defects, including strongly reduced growth, altered flower morphology, as well as abnormal ovules and female gametophytes, causing reduced seed production. Moreover, polyadenylated mRNAs accumulate in the nuclei of 4xaly cells. Our results highlight the requirement of efficient mRNA nucleocytosolic transport for proper plant growth and development and indicate that ALY1 to ALY4 act partly redundantly in this process; however, differences in expression and subnuclear localization suggest distinct functions.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Transporte de RNA , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The presence of two glycolytic pathways working in parallel in plastids and cytosol has complicated the understanding of this essential process in plant cells, especially the integration of the plastidial pathway into the metabolism of heterotrophic and autotrophic organs. It is assumed that this integration is achieved by transport systems, which exchange glycolytic intermediates across plastidial membranes. However, it is unknown whether plastidial and cytosolic pools of 3-phosphoglycerate (3-PGA) can equilibrate in non-photosynthetic tissues. To resolve this question, we employed Arabidopsis mutants of the plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) that express the triose phosphate translocator (TPT) under the control of the 35S (35S:TPT) or the native GAPCp1 (GAPCp1:TPT) promoters. TPT expression under the control of both promoters complemented the vegetative developmental defects and metabolic disorders of the GAPCp double mutants (gapcp1gapcp2). However, as the 35S is poorly expressed in the tapetum, full vegetative and reproductive complementation of gapcp1gapcp2 was achieved only by transforming this mutant with the GAPCp1:TPT construct. Our results indicate that the main function of GAPCp is to supply 3-PGA for anabolic pathways in plastids of heterotrophic cells and suggest that the plastidial glycolysis may contribute to fatty acid biosynthesis in seeds. They also suggest a 3-PGA deficiency in the plastids of gapcp1gapcp2, and that 3-PGA pools between cytosol and plastid do not equilibrate in heterotrophic cells.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Plastídeos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Ácidos Glicéricos/metabolismo , Glicólise/genética , Glicólise/fisiologia , Plastídeos/genéticaRESUMO
This study functionally characterizes the Arabidopsis (Arabidopsis thaliana) plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in photosynthetic and heterotrophic cells. We expressed the enzyme in gapcp double mutants (gapcp1gapcp2) under the control of photosynthetic (Rubisco small subunit RBCS2B [RBCS]) or heterotrophic (phosphate transporter PHT1.2 [PHT]) cell-specific promoters. Expression of GAPCp1 under the control of RBCS in gapcp1gapcp2 had no significant effect on the metabolite profile or growth in the aerial part (AP). GAPCp1 expression under the control of the PHT promoter clearly affected Arabidopsis development by increasing the number of lateral roots and having a major effect on AP growth and metabolite profile. Our results indicate that GAPCp1 is not functionally important in photosynthetic cells but plays a fundamental role in roots and in heterotrophic cells of the AP. Specifically, GAPCp activity may be required in root meristems and the root cap for normal primary root growth. Transcriptomic and metabolomic analyses indicate that the lack of GAPCp activity affects nitrogen and carbon metabolism as well as mineral nutrition and that glycerate and glutamine are the main metabolites responding to GAPCp activity. Thus, GAPCp could be an important metabolic connector of glycolysis with other pathways, such as the phosphorylated pathway of serine biosynthesis, the ammonium assimilation pathway, or the metabolism of γ-aminobutyrate, which in turn affect plant development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Carbono/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Nitrogênio/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica/fisiologia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Isoenzimas , Regiões Promotoras GenéticasRESUMO
This study characterizes the phosphorylated pathway of Ser biosynthesis (PPSB) in Arabidopsis thaliana by targeting phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Lack of PSP1 activity delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of psp1 mutants could be complemented with PSP1 cDNA under the control of Pro35S (Pro35S:PSP1). However, this construct, which was poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in psp1.1/psp1.1 Pro35S:PSP1 arrested at the polarized stage. The tapetum from these lines displayed delayed and irregular development. The expression of PSP1 in the tapetum at critical stages of microspore development suggests that PSP1 activity in this cell layer is essential in pollen development. In addition to embryo death and male sterility, conditional psp1 mutants displayed a short-root phenotype, which was reverted in the presence of Ser. A metabolomic study demonstrated that the PPSB plays a crucial role in plant metabolism by affecting glycolysis, the tricarboxylic acid cycle, and the biosynthesis of amino acids. We provide evidence of the crucial role of the PPSB in embryo, pollen, and root development and suggest that this pathway is an important link connecting primary metabolism with development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Raízes de Plantas/metabolismo , Pólen/metabolismo , Sementes/metabolismo , Serina/biossíntese , Aminoácidos/biossíntese , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Vias Biossintéticas/genética , Ciclo do Ácido Cítrico/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Glicólise/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Mutação , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/crescimento & desenvolvimento , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sementes/genética , Sementes/crescimento & desenvolvimentoRESUMO
This work contributes to unraveling the role of the phosphorylated pathway of serine (Ser) biosynthesis in Arabidopsis (Arabidopsis thaliana) by functionally characterizing genes coding for the first enzyme of this pathway, 3-phosphoglycerate dehydrogenase (PGDH). We identified two Arabidopsis plastid-localized PGDH genes (3-PGDH and EMBRYO SAC DEVELOPMENT ARREST9 [EDA9]) with a high percentage of amino acid identity with a previously identified PGDH. All three genes displayed a different expression pattern indicating that they are not functionally redundant. pgdh and 3-pgdh mutants presented no drastic visual phenotypes, but eda9 displayed delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of eda9 was complemented with an EDA9 complementary DNA under the control of a 35S promoter (Pro-35S:EDA9). However, this construct, which is poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in eda9.1eda9.1 Pro-35S:EDA9 was arrested at the polarized stage. Pollen from these lines lacked tryphine in the interstices of the exine layer, displayed shrunken and collapsed forms, and were unable to germinate when cultured in vitro. A metabolomic analysis of PGDH mutant and overexpressing plants revealed that all three PGDH family genes can regulate Ser homeostasis, with PGDH being quantitatively the most important in the process of Ser biosynthesis at the whole-plant level. By contrast, the essential role of EDA9 could be related to its expression in very specific cell types. We demonstrate the crucial role of EDA9 in embryo and pollen development, suggesting that the phosphorylated pathway of Ser biosynthesis is an important link connecting primary metabolism with development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Família Multigênica , Fosfoglicerato Desidrogenase/metabolismo , Plastídeos/enzimologia , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Isoenzimas/classificação , Isoenzimas/genética , Isoenzimas/metabolismo , Metabolômica/métodos , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Fosfoglicerato Desidrogenase/classificação , Fosfoglicerato Desidrogenase/genética , Fosforilação , Filogenia , Componentes Aéreos da Planta/enzimologia , Componentes Aéreos da Planta/genética , Componentes Aéreos da Planta/metabolismo , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Pólen/enzimologia , Pólen/genética , Pólen/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sementes/enzimologia , Sementes/genética , Sementes/metabolismo , Homologia de Sequência de Aminoácidos , Serina/genética , Serina/metabolismoRESUMO
Mutations in numerous genes affect reproduction in Arabidopsis leading to sterility and abortion of seed development, respectively. These include mutations in regulators of reproductive development and fertilization, but also in house-keeping genes lacking mutant phenotypes during vegetative development. However, during the haploid phase of germline development or during seed development, lethality or failures become visible when gene activity is needed. Plant reproduction is complex and includes many processes from flowering and flower organ development toward the formation of seeds after a double fertilization process. For those who are less familiar with the various reproductive processes in Arabidopsis and who aim to study the cause of reproductive defects during germline development and function, fertilization, or embryogenesis in a given mutant, we provide here a step-by-step guideline and basic protocols to elucidate the reproductive process affected.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Mutação/genética , Sementes/genéticaRESUMO
KEY MESSAGE: Analyses of secretomes of in vitro grown pollen tubes from Amborella, maize and tobacco identified many components of processes associated with the cell wall, signaling and metabolism as well as novel small secreted peptides. Flowering plants (angiosperms) generate pollen grains that germinate on the stigma and produce tubes to transport their sperm cells cargo deep into the maternal reproductive tissues toward the ovules for a double fertilization process. During their journey, pollen tubes secrete many proteins (secreted proteome or secretome) required, for example, for communication with the maternal reproductive tissues, to build a solid own cell wall that withstands their high turgor pressure while softening simultaneously maternal cell wall tissue. The composition and species specificity or family specificity of the pollen tube secretome is poorly understood. Here, we provide a suitable method to obtain the pollen tube secretome from in vitro grown pollen tubes of the basal angiosperm Amborella trichopoda (Amborella) and the Poaceae model maize. The previously published secretome of tobacco pollen tubes was used as an example of eudicotyledonous plants in this comparative study. The secretome of the three species is each strongly different compared to the respective protein composition of pollen grains and tubes. In Amborella and maize, about 40% proteins are secreted by the conventional "classic" pathway and 30% by unconventional pathways. The latter pathway is expanded in tobacco. Proteins enriched in the secretome are especially involved in functions associated with the cell wall, cell surface, energy and lipid metabolism, proteolysis and redox processes. Expansins, pectin methylesterase inhibitors and RALFs are enriched in maize, while tobacco secretes many proteins involved, for example, in proteolysis and signaling. While the majority of proteins detected in the secretome occur also in pollen grains and pollen tubes, and correlate in the number of mapped peptides with relative gene expression levels, some novel secreted small proteins were identified. Moreover, the identification of secreted proteins containing pro-peptides indicates that these are processed in the apoplast. In conclusion, we provide a proteome resource from three distinct angiosperm clades that can be utilized among others to study the localization, abundance and processing of known secreted proteins and help to identify novel pollen tube secreted proteins for functional studies.
Assuntos
Magnoliopsida , Tubo Polínico , Óvulo Vegetal , Peptídeos , Nicotiana , Zea maysRESUMO
The appearance of plant organs mediated the explosive radiation of land plants, which shaped the biosphere and allowed the establishment of terrestrial animal life. The evolution of organs and immobile gametes required the coordinated acquisition of novel gene functions, the co-option of existing genes and the development of novel regulatory programmes. However, no large-scale analyses of genomic and transcriptomic data have been performed for land plants. To remedy this, we generated gene expression atlases for various organs and gametes of ten plant species comprising bryophytes, vascular plants, gymnosperms and flowering plants. A comparative analysis of the atlases identified hundreds of organ- and gamete-specific orthogroups and revealed that most of the specific transcriptomes are significantly conserved. Interestingly, our results suggest that co-option of existing genes is the main mechanism for evolving new organs. In contrast to female gametes, male gametes showed a high number and conservation of specific genes, which indicates that male reproduction is highly specialized. The expression atlas capturing pollen development revealed numerous transcription factors and kinases essential for pollen biogenesis and function.
Assuntos
Embriófitas/crescimento & desenvolvimento , Embriófitas/genética , Perfilação da Expressão Gênica , Magnoliopsida/crescimento & desenvolvimento , Magnoliopsida/genética , Organogênese Vegetal/genética , Reprodução/genética , Regulação da Expressão Gênica de Plantas , Variação Genética , Genótipo , Organogênese Vegetal/fisiologia , Fenótipo , Proteínas de Plantas/metabolismo , Reprodução/fisiologia , Análise de Sequência de RNA , Fatores de Transcrição/metabolismoRESUMO
The article Transcriptomics of manually isolated Amborella trichopoda egg apparatus cells, written by María Flores-Tornero, Sebastian Proost, Marek Mutwil, Charles P. Scutt, Thomas Dresselhaus, Stefanie Sprunck, was originally published electronically on the publisher's internet portal (currently SpringerLink) on 1 February 2019 without open access.
RESUMO
KEY MESSAGE: A protocol for the isolation of egg apparatus cells from the basal angiosperm Amborella trichopoda to generate RNA-seq data for evolutionary studies of fertilization-associated genes. Sexual reproduction is particularly complex in flowering plants (angiosperms). Studies in eudicot and monocot model species have significantly contributed to our knowledge on cell fate specification of gametophytic cells and on the numerous cellular communication events necessary to deliver the two sperm cells into the embryo sac and to accomplish double fertilization. However, for a deeper understanding of the evolution of these processes, morphological, genomic and gene expression studies in extant basal angiosperms are inevitable. The basal angiosperm Amborella trichopoda is of special importance for evolutionary studies, as it is likely sister to all other living angiosperms. Here, we report about a method to isolate Amborella egg apparatus cells and on genome-wide gene expression profiles in these cells. Our transcriptomics data revealed Amborella-specific genes and genes conserved in eudicots and monocots. Gene products include secreted proteins, such as small cysteine-rich proteins previously reported to act as extracellular signaling molecules with important roles during double fertilization. The detection of transcripts encoding EGG CELL 1 (EC1) and related prolamin-like family proteins in Amborella egg cells demonstrates the potential of the generated data set to study conserved molecular mechanisms and the evolution of fertilization-related genes and their encoded proteins.
Assuntos
Separação Celular/métodos , Genoma de Planta , Magnoliopsida/citologia , Magnoliopsida/genética , Óvulo Vegetal/genética , Óvulo Vegetal/citologia , RNA de Plantas , TranscriptomaRESUMO
Photorespiration is an essential pathway in photosynthetic organisms and is particularly important to detoxify and recycle 2-phosphoglycolate (2-PG), a by-product of oxygenic photosynthesis. The enzymes that catalyze the reactions in the photorespiratory core cycle and closely associated pathways have been identified; however, open questions remain concerning the metabolic network in which photorespiration is embedded. The amino acid serine represents one of the major intermediates in the photorespiratory pathway and photorespiration is thought to be the major source of serine in plants. The restriction of photorespiration to autotrophic cells raises questions concerning the source of serine in heterotrophic tissues. Recently, the phosphorylated pathway of serine biosynthesis has been found to be extremely important for plant development and metabolism. In this protocol, we describe a detailed methodological workflow to analyze the generative and vegetative phenotypes of plants deficient in the phosphorylated pathway of serine biosynthesis, which together allow a better understanding of its function in plants.
Assuntos
Arabidopsis/metabolismo , Dióxido de Carbono/metabolismo , Consumo de Oxigênio/fisiologia , Fotossíntese/fisiologia , Folhas de Planta/metabolismo , Serina/biossíntese , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Bases de Dados Genéticas , Expressão Gênica , Glicolatos/metabolismo , Redes e Vias Metabólicas , Mutação , Oxigênio/metabolismo , Fenótipo , Fosfoglicerato Desidrogenase/deficiência , Fosfoglicerato Desidrogenase/genética , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/metabolismo , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismoRESUMO
The cellular compartmentalization of metabolic processes is an important feature in plants where the same pathways could be simultaneously active in different compartments. Plant glycolysis occurs in the cytosol and plastids of green and non-green cells in which the requirements of energy and precursors may be completely different. Because of this, the relevance of plastidial glycolysis could be very different depending on the cell type. In the associated study, we investigated the function of plastidial glycolysis in photosynthetic and heterotrophic cells by specifically driving the expression of plastidial glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in a glyceraldehyde-3-phosphate dehydrogenase double mutant background (gapcp1gapcp2). We showed that GAPCp is not functionally significant in photosynthetic cells, while it plays a crucial function in heterotrophic cells. We also showed that (i) GAPCp activity expression in root tips is necessary for primary root growth, (ii) its expression in heterotrophic cells of aerial parts and roots is necessary for plant growth and development, and (iii) GAPCp is an important metabolic connector of carbon and nitrogen metabolism through the phosphorylated pathway of serine biosynthesis (PPSB). We discuss here the role that this pathway could play in the control of plant growth and development.
Assuntos
Arabidopsis/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/fisiologia , Glicólise , Plastídeos/metabolismo , Arabidopsis/citologia , Arabidopsis/fisiologia , Carbono/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Mutação , Nitrogênio/metabolismo , Fosforilação , Fotossíntese , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Raízes de Plantas/fisiologia , Serina/biossínteseRESUMO
Three different pathways of serine (Ser) biosynthesis have been described in plants: the Glycolate pathway, which is part of the Photorespiratory pathway, and 2 non-Photorespiratory pathways, the Glycerate and the Phosphorylated pathways. The Phosphorylated Pathway of Ser Biosynthesis (PPSB) has been known to exist since the 1950s, but its biological relevance was not revealed until quite recently when the last enzyme of the pathway, the Phosphoserine Phosphatase, was functionally characterized. In the associated study (1), we characterized a family of genes coding for putatite phosphoglycerate dehydrogenases (PGDH, 3-PGDH, and EDA9), the first enzyme of the PPSB. A metabolomics study using overexpressing plants indicated that all PGDH family genes were able to regulate Ser homeostasis but only lacking of EDA9 expression caused drastic developmental defects. We provided genetic and molecular evidence for the essential role of EDA9 for embryo and pollen development. Here, some new insights into the physiological/molecular function of PPSB and Ser are presented and discussed.