RESUMO
Mitochondrial adrenodoxins (ADXs) are small iron-sulfur proteins with electron transfer properties. In animals, ADXs transfer electrons between an adrenodoxin reductase (ADXR) and mitochondrial P450s, which is crucial for steroidogenesis. Here we show that a plant mitochondrial steroidogenic pathway, dependent on an ADXR-ADX-P450 shuttle, is essential for female gametogenesis and early embryogenesis through a maternal effect. The steroid profile of maternal and gametophytic tissues of wild-type (WT) and adxr ovules revealed that homocastasterone is the main steroid present in WT gametophytes and that its levels are reduced in the mutant ovules. The application of exogenous homocastasterone partially rescued adxr and P450 mutant phenotypes, indicating that gametophytic homocastasterone biosynthesis is affected in the mutants and that a deficiency of this hormone causes the phenotypic alterations observed. These findings also suggest not only a remarkable similarity between steroid biosynthetic pathways in plants and animals but also a common function during sexual reproduction.
Assuntos
Adrenodoxina/metabolismo , Arabidopsis/embriologia , Ferredoxina-NADP Redutase/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/fisiologia , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/fisiologia , Desenvolvimento Embrionário/genética , Gametogênese/fisiologia , Células Germinativas Vegetais/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Fitosteróis/biossíntese , Ligação ProteicaRESUMO
The germination timing of seeds is of the utmost adaptive importance for plant populations. Light is one of the best characterized factors promoting seed germination in several species. The germination is also finely regulated by changes in hormones levels, mainly those of gibberellin (GA) and abscisic acid (ABA). Here, we performed physiological, pharmacological, and molecular analyses to uncover the role of ATHB2, an HD-ZIP II transcription factor, in germination of Arabidopsis seeds. Our study demonstrated that ATHB2 is a negative regulator and sustains the expression of transcription factors to block germination promoted by light. Besides, we found that ATHB2 increases ABA sensitivity. Moreover, ABA and auxin content in athb2-2 mutant is higher than wild-type in dry seeds, but the differences disappeared during the imbibition in darkness and the first hours of exposition to light, respectively. Some ABA and light transcription factors are up-regulated by ATHB2, such as ABI5, ABI3, XERICO, SOMNUS and PIL5/PIF1. In opposition, PIN7, an auxin transport, is down-regulated. The role of ATHB2 as a repressor of germination induced by light affecting the gemination timing, could have differential effects on the establishment of seedlings altering the competitiveness between crops and weeds in the field.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/embriologia , Germinação/fisiologia , Sementes/crescimento & desenvolvimento , Ácido Abscísico/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Genes de Plantas , Germinação/efeitos da radiação , Transdução de Sinais/fisiologia , Fatores de Transcrição/genéticaRESUMO
The major tissue types and stem-cell niches of plants are established during embryogenesis, and thus knowledge of embryo development is essential for a full understanding of plant development. Studies of seed development are also important for human health, because the nutrients stored in both the embryo and endosperm of plant seeds provide an essential part of our diet. Arabidopsis and maize have evolved different types of seeds, opening a range of experimental opportunities. Development of the Arabidopsis embryo follows an almost invariant pattern, while cell division patterns of maize embryos are variable. Embryo-endosperm interactions are also different between the two species: in Arabidopsis, the endosperm is consumed during seed development, while mature maize seeds contain an enormous endosperm. Genetic screens have provided important insights into seed development in both species. In the genomic era, genetic analysis will continue to provide important tools for understanding embryo and endosperm biology in plants, because single gene functional studies can now be integrated with genome-wide information. Here, we lay out important factors to consider when designing genetic screens to identify new genes or to probe known pathways in seed development. We then highlight the technical details of two previous genetic screens that may serve as useful examples for future experiments.
Assuntos
Arabidopsis/embriologia , Endosperma/embriologia , Zea mays/embriologia , Arabidopsis/genética , Endosperma/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Mutagênese , Sementes/embriologia , Sementes/genética , Zea mays/genéticaRESUMO
Venation patterning is a taxonomic attribute for classification of plants and it also plays a role in the interaction of plants with the environment. Despite its importance, the molecular physiology controlling this aspect of plant development is still poorly understood. Auxin plays a central role modulating the final vein network and patterning. This addendum discusses recent findings on the role of homeodomain-leucine zipper (HD-Zip) transcription factors on the regulation of leaf venation patterning. Moreno-Piovano et al. reported that ectopic expression of a sunflower HD-Zip I gene, HaHB4, increased the asymmetry of leaf venation. Even more, this work showed that auxin transport in the leaf through LAX carriers controls venation patterning. Here, we provide evidence indicating that some Arabidopsis thaliana HD-Zip I genes play a role in the determination of the final leaf venation patterning. We propose that these genes contribute to regulate vein patterning, likely controlling auxin homeostasis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Padronização Corporal , Proteínas de Homeodomínio/metabolismo , Zíper de Leucina , Folhas de Planta/embriologia , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Padronização Corporal/genética , Genes de Plantas , Mutação/genética , Folhas de Planta/genéticaRESUMO
The endosperm is a transitory structure involved in proper embryo elongation. The cell walls of mature seed endosperm are generally composed of a uniform distribution of cellulose, unesterified homogalacturonans, and arabinans. Recent studies suggest that changes in cell wall properties during endosperm development could be related to embryo growth. The degree of methyl esterification of homogalacturonans may be involved in this endosperm tissue remodelling. The relevance of the degree of homogalacturonan methyl esterification during seed development was determined by immunohistochemical analyses using a panel of probes with specificity for homogalaturonans with different degrees of methyl esterification. Low-esterified and un-esterified homogalacturonans were abundant in endosperm cells during embryo bending and were also detected in mature embryos. BIDXII (BDX) could be involved in seed development, because bdx-1 mutants had misshapen embryos. The methyl esterification pattern described for WT seeds was different during bdx-1 seed development; un-esterified homogalacturonans were scarcely present in the cell walls of endosperm in bending embryos and mature seeds. Our results suggested that the degree of methyl esterification of homogalacturonans in the endosperm cell wall may be involved in proper embryo development.
Assuntos
Arabidopsis/embriologia , Arabidopsis/fisiologia , Endosperma/embriologia , Endosperma/metabolismo , Pectinas/metabolismo , Sementes/fisiologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Desenvolvimento Embrionário/fisiologia , EsterificaçãoRESUMO
During early embryo development, profound changes in chromatin structure and regulation take place. It is difficult to study these changes in plant embryos however, largely because of their relative inaccessibility, which impedes the application of current epigenomic and biochemistry protocols. To circumvent this issue and to analyze the epigenetic status of the embryo at both the cellular and subcellular level, we describe here a simple method to immunolocalize chromatin marks in whole mount early Arabidopsis embryos, either within maternal tissues or isolated from seeds. We show that this protocol can be combined with fluorescent protein markers, allowing for the simultaneous detection of several chromatin components and/or cell fate markers. This new protocol will facilitate deciphering the epigenetic circuits controlling early embryogenesis in plants.
Assuntos
Arabidopsis/embriologia , Cromatina/metabolismo , Epigenômica/métodos , Sementes/metabolismo , Arabidopsis/química , Cromatina/química , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Código das Histonas , Histonas/metabolismo , Conformação Molecular , Sementes/químicaRESUMO
miRNAs are essential regulators of cell identity, yet their role in early embryo development in plants remains largely unexplored. To determine the earliest stage at which miRNAs act to promote pattern formation in embryogenesis, we examined a series of mutant alleles in the Arabidopsis thaliana miRNA biogenesis enzymes DICER-LIKE 1 (DCL1), SERRATE (SE), and HYPONASTIC LEAVES 1 (HYL1). Cellular and patterning defects were observed in dcl1, se and hyl1 embryos from the zygote through the globular stage of embryogenesis. To identify miRNAs that are expressed in early embryogenesis, we sequenced mRNAs from globular stage Columbia wild type (wt) and se-1 embryos, and identified transcripts potentially corresponding to 100 miRNA precursors. Considering genome location and transcript increase between wt and se-1, 39 of these MIRNAs are predicted to be bona fide early embryo miRNAs. Among these are conserved miRNAs such as miR156, miR159, miR160, miR161, miR164, miR165, miR166, miR167, miR168, miR171, miR319, miR390 and miR394, as well as miRNAs whose function has never been characterized. Our analysis demonstrates that miRNAs promote pattern formation beginning in the zygote, and provides a comprehensive dataset for functional studies of individual miRNAs in Arabidopsis embryogenesis.
Assuntos
Arabidopsis/embriologia , Arabidopsis/genética , Padronização Corporal/genética , MicroRNAs/metabolismo , Sementes/embriologia , Sementes/genética , Zigoto/metabolismo , Arabidopsis/citologia , Divisão Celular , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Morfogênese/genética , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para Cima/genéticaRESUMO
Cytochrome c oxidase (CcO) biogenesis requires several accessory proteins implicated, among other processes, in copper and haem a insertion. In yeast, the farnesyltransferase Cox10p that catalyses the conversion of haem b to haem o is the limiting factor in haem a biosynthesis and is essential for haem a insertion in CcO. In this work, we characterized AtCOX10, a putative Cox10p homologue from Arabidopsis thaliana. AtCOX10 was localized in mitochondria and was able to restore growth of a yeast Δcox10 null mutant on non-fermentable carbon sources, suggesting that it also participates in haem o synthesis. Plants with T-DNA insertions in the coding region of both copies of AtCOX10 could not be recovered, and heterozygous mutant plants showed seeds with embryos arrested at early developmental stages that lacked CcO activity. Heterozygous mutant plants exhibited lower levels of CcO activity and cyanide-sensitive respiration but normal levels of total respiration at the expense of an increase in alternative respiration. AtCOX10 seems to be implicated in the onset and progression of senescence, since heterozygous mutant plants showed a faster decrease in chlorophyll content and photosynthetic performance than wild-type plants after natural and dark-induced senescence. Furthermore, complementation of mutants by expressing AtCOX10 under its own promoter allowed us to obtain plants with T-DNA insertions in both AtCOX10 copies, which showed phenotypic characteristics comparable to those of wild type. Our results highlight the relevance of haem o synthesis in plants and suggest that this process is a limiting factor that influences CcO activity levels, mitochondrial respiration, and plant senescence.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Farnesiltranstransferase/genética , Heme/metabolismo , Proteínas Mitocondriais/genética , Arabidopsis/embriologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Respiração Celular , Farnesiltranstransferase/metabolismo , Proteínas Mitocondriais/metabolismo , Organismos Geneticamente Modificados/genética , Saccharomyces cerevisiae/genéticaRESUMO
Pollen tubes elongate by tip growth toward the ovule to deliver the sperm cells during fertilization. Since pollen tubes from several species can be grown in vitro maintaining their polarity, pollen tube growth is a suitable model system to study cell polarity and tip growth. A. thaliana pollen tubes germinated in vitro for 6 h can reach up to 800 µm. By studying the phenotype of mutants of T-DNA insertion lines, genes involved in pollen tube growth can be identified. Moreover, components involved in the regulation of pollen tube growth such as calcium ions and reactive oxygen species (ROS) can be analyzed.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/embriologia , Botânica/métodos , Tubo Polínico/crescimento & desenvolvimento , Arabidopsis/fisiologia , Polaridade Celular , DNA Bacteriano/análise , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Germinação , MutaçãoRESUMO
Cytoplasmic calcium [(Ca(2+))cyt] is a central component of cellular signal transduction pathways. In plants, many external and internal stimuli transiently elevate (Ca(2+))cyt, initiating downstream responses that control different features of plant development. In pollen tubes the establishment of an oscillatory gradient of calcium at the tip is essential for polarized growth. Disruption of the cytosolic Ca(2+) gradient by chelators or channel blockers inhibits pollen tube growth. To quantify the physiological role of (Ca(2+))cyt in cellular systems, genetically encoded Ca(2+) indicators such as Yellow Cameleons (YCs) have been developed. The Cameleons are based on a fluorescence resonance energy transfer (FRET) process. Here, we describe a method for imaging cytoplasmic Ca(2+) dynamics in growing pollen tubes that express the fluorescent calcium indicator Yellow Cameleon 3.6 (YC 3.6), using laser-scanning confocal microscopy.