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1.
Methods Mol Biol ; 2122: 141-150, 2020.
Article in English | MEDLINE | ID: mdl-31975301

ABSTRACT

Cells differentiate from undifferentiated precursors in order to establish the tissues of vascular plants. The different cell types and stem cells are first specified in the early embryo. How cell type specification is instructed by transcriptional control on a genome-wide level is poorly understood. A major hurdle has been the technical challenge associated with obtaining cellular transcriptomes in this inaccessible tissue. Recently, we adapted a two-component genetic labeling system called INTACT to isolate nuclei and generate a microarray-based expression atlas of the cell types in the early Arabidopsis thaliana embryo. Here we present a step-by-step description of the adapted INTACT protocol, as well as the approach to generate transcriptomic profiles. This protocol has been adapted to account for using seeds with embryos of various developmental stages as a starting material, and the relatively few cell type-specific nuclei that can be isolated from embryos.


Subject(s)
Arabidopsis/genetics , Gene Expression Profiling/methods , Seeds/genetics , Transcriptome , Arabidopsis/embryology , Gene Expression Regulation, Plant , Seeds/embryology
2.
Curr Top Dev Biol ; 131: 401-434, 2019.
Article in English | MEDLINE | ID: mdl-30612625

ABSTRACT

Flowering plants constitute an indispensable basis for the existence of most organisms, including humans. In a world characterized by rapid population growth and climate changes, understanding plant reproduction becomes increasingly important in order to respond to the resource shortage associated with this development. New technologies enabling powerful forward genetic approaches, comprehensive genome and transcriptome analyses, and sophisticated cell isolation and imaging have advanced our understanding of the molecular mechanisms underlying gamete formation and fertilization. In addition, these techniques have allowed us to explore the fascinating cellular crosstalk, which coordinates the intra- and interorganismic interactions that secure reproductive success. Here we review the basic principles underlying development of the germ cell-harboring female gametophyte in flowering plants. We start with the selection of the founder cells and end with the formation of a few-celled, highly specialized structure that operates on the basis of division of labor in order to generate the next generation.


Subject(s)
Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Magnoliopsida/growth & development , Ovule/growth & development , Plant Proteins/genetics , Magnoliopsida/genetics , Ovule/genetics
3.
Plant Reprod ; 32(1): 113-121, 2019 03.
Article in English | MEDLINE | ID: mdl-30430248

ABSTRACT

In the early embryo of vascular plants, the different cell types and stem cells of the seedling are specified as the embryo develops from a zygote towards maturity. How the key steps in cell and tissue specification are instructed by genome-wide transcriptional activity is poorly understood. Progress in defining transcriptional regulation at the genome-wide level in plant embryos has been hampered by difficulties associated with capturing cell-type-specific transcriptomes in this small and inaccessible structure. We recently adapted a two-component genetic nucleus labelling system called INTACT to isolate nuclei from distinct cell types at different stages of Arabidopsis thaliana embryogenesis. We have used these to generate a transcriptomic atlas of embryo development following microarray-based expression profiling. Here, we present a general description of the adapted INTACT procedure, including the two-component labelling system, seed isolation, nuclei preparation and purification, as well as transcriptomic profiling. We also compare nuclear and cellular transcriptomes from the early Arabidopsis embryo to assess nucleocytoplasmic differences and discuss how these differences can be used to infer regulation of gene activity.


Subject(s)
Arabidopsis/genetics , Gene Expression Profiling/methods , RNA, Messenger/metabolism , RNA, Plant/metabolism , Seeds/genetics , Staining and Labeling/methods , Arabidopsis/cytology , Cell Nucleus/metabolism , Cytoplasm/metabolism , Plant Cells , Seeds/metabolism
4.
Nat Plants ; 4(2): 128, 2018 02.
Article in English | MEDLINE | ID: mdl-29326478

ABSTRACT

In the version of this Resource originally published, the author information was incorrect. Jos R. Wendrich should have had a present address: Department of Plant Biotechnology and Bioinformatics and VIB Center for Plant Systems Biology, Ghent University, Technologiepark 927, 9052 Ghent, Belgium. Mark Boekschoten and Guido J. Hooiveld should have been affiliated to the Nutrition, Metabolism and Genomics Group, Division of Human Nutrition, Wageningen University, 6708 WE Wageningen, The Netherlands. In addition, the version of Supplementary Table 5 originally published with this Resource was not the intended final version and included inaccurate citations to the display items of the Resource, and the file format and extension did not match. These errors have now been corrected in all versions of the Resource.

5.
Nat Plants ; 3(11): 894-904, 2017 Nov.
Article in English | MEDLINE | ID: mdl-29116234

ABSTRACT

During early plant embryogenesis, precursors for all major tissues and stem cells are formed. While several components of the regulatory framework are known, how cell fates are instructed by genome-wide transcriptional activity remains unanswered-in part because of difficulties in capturing transcriptome changes at cellular resolution. Here, we have adapted a two-component transgenic labelling system to purify cell-type-specific nuclear RNA and generate a transcriptome atlas of early Arabidopsis embryo development, with a focus on root stem cell niche formation. We validated the dataset through gene expression analysis, and show that gene activity shifts in a spatio-temporal manner, probably signifying transcriptional reprogramming, to induce developmental processes reflecting cell states and state transitions. This atlas provides the most comprehensive tissue- and cell-specific description of genome-wide gene activity in the early plant embryo, and serves as a valuable resource for understanding the genetic control of early plant development.


Subject(s)
Arabidopsis/genetics , Seeds/genetics , Transcriptome , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Genetic Techniques , Plant Cells , Plant Roots/cytology , Plant Roots/genetics , Plants, Genetically Modified , Staining and Labeling/methods
6.
Front Microbiol ; 7: 1670, 2016.
Article in English | MEDLINE | ID: mdl-27818655

ABSTRACT

To decipher the response of mesopelagic prokaryotic communities to input of nutrients, we tracked changes in prokaryotic abundance, extracellular enzymatic activities, heterotrophic production, dark dissolved inorganic carbon (DIC) fixation, community composition (16S rRNA sequencing) and community gene expression (metatranscriptomics) in 3 microcosm experiments with water from the mesopelagic North Atlantic. Responses in 3 different treatments amended with thiosulfate, ammonium or organic matter (i.e., pyruvate plus acetate) were compared to unamended controls. The strongest stimulation was found in the organic matter enrichments, where all measured rates increased >10-fold. Strikingly, in the organic matter treatment, the dark DIC fixation rates-assumed to be related to autotrophic metabolisms-were equally stimulated as all the other heterotrophic-related parameters. This increase in DIC fixation rates was paralleled by an up-regulation of genes involved in DIC assimilation via anaplerotic pathways. Alkaline phosphatase was the metabolic rate most strongly stimulated and its activity seemed to be related to cross-activation by nonpartner histidine kinases, and/or the activation of genes involved in the regulation of elemental balance during catabolic processes. These findings suggest that episodic events such as strong sedimentation of organic matter into the mesopelagic might trigger rapid increases of originally rare members of the prokaryotic community, enhancing heterotrophic and autotrophic carbon uptake rates, ultimately affecting carbon cycling. Our experiments highlight a number of fairly unstudied microbial processes of potential importance in mesopelagic waters that require future attention.

7.
Annu Rev Cell Dev Biol ; 32: 47-75, 2016 10 06.
Article in English | MEDLINE | ID: mdl-27576120

ABSTRACT

Land plants can grow to tremendous body sizes, yet even the most complex architectures are the result of iterations of the same developmental processes: organ initiation, growth, and pattern formation. A central question in plant biology is how these processes are regulated and coordinated to allow for the formation of ordered, 3D structures. All these elementary processes first occur in early embryogenesis, during which, from a fertilized egg cell, precursors for all major tissues and stem cells are initiated, followed by tissue growth and patterning. Here we discuss recent progress in our understanding of this phase of plant life. We consider the cellular basis for multicellular development in 3D and focus on the genetic regulatory mechanisms that direct specific steps during early embryogenesis.


Subject(s)
Morphogenesis , Seeds/embryology , Body Patterning , Stem Cell Niche
8.
ISME J ; 10(3): 568-81, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26262814

ABSTRACT

To test whether protist grazing selectively affects the composition of aquatic bacterial communities, we combined high-throughput sequencing to determine bacterial community composition with analyses of grazing rates, protist and bacterial abundances and bacterial cell sizes and physiological states in a mesocosm experiment in which nutrients were added to stimulate a phytoplankton bloom. A large variability was observed in the abundances of bacteria (from 0.7 to 2.4 × 10(6) cells per ml), heterotrophic nanoflagellates (from 0.063 to 2.7 × 10(4) cells per ml) and ciliates (from 100 to 3000 cells per l) during the experiment (∼3-, 45- and 30-fold, respectively), as well as in bulk grazing rates (from 1 to 13 × 10(6) bacteria per ml per day) and bacterial production (from 3 to 379 µg per C l per day) (1 and 2 orders of magnitude, respectively). However, these strong changes in predation pressure did not induce comparable responses in bacterial community composition, indicating that bacterial community structure was resilient to changes in protist predation pressure. Overall, our results indicate that peaks in protist predation (at least those associated with phytoplankton blooms) do not necessarily trigger substantial changes in the composition of coastal marine bacterioplankton communities.


Subject(s)
Bacteria/isolation & purification , Eukaryota/physiology , Phytoplankton/growth & development , Bacteria/classification , Bacteria/genetics , Bacteria/growth & development , Biodiversity , Heterotrophic Processes
9.
FEMS Microbiol Ecol ; 91(6)2015 Jun.
Article in English | MEDLINE | ID: mdl-26032602

ABSTRACT

Bacterioplankton communities are made up of a small set of abundant taxa and a large number of low-abundant organisms (i.e. 'rare biosphere'). Despite the critical role played by bacteria in marine ecosystems, it remains unknown how this large diversity of organisms are affected by human-induced perturbations, or what controls the responsiveness of rare compared to abundant bacteria. We studied the response of a Mediterranean bacterioplankton community to two anthropogenic perturbations (i.e. nutrient enrichment and/or acidification) in two mesocosm experiments (in winter and summer). Nutrient enrichment increased the relative abundance of some operational taxonomic units (OTUs), e.g. Polaribacter, Tenacibaculum, Rhodobacteraceae and caused a relative decrease in others (e.g. Croceibacter). Interestingly, a synergistic effect of acidification and nutrient enrichment was observed on specific OTUs (e.g. SAR86). We analyzed the OTUs that became abundant at the end of the experiments and whether they belonged to the rare (<0.1% of relative abundance), the common (0.1-1.0% of relative abundance) or the abundant (>1% relative abundance) fractions. Most of the abundant OTUs at the end of the experiments were abundant, or at least common, in the original community of both experiments, suggesting that ecosystem alterations do not necessarily call for rare members to grow.


Subject(s)
Bacteria/classification , Bacteria/growth & development , Eutrophication , Plankton/growth & development , Aquatic Organisms/microbiology , Bacteria/genetics , Base Sequence , Biodiversity , DNA, Bacterial/genetics , Ecosystem , Humans , Hydrogen-Ion Concentration , Mediterranean Region , Microbiota/genetics , RNA, Ribosomal, 16S/genetics , Seasons , Sequence Analysis, DNA
10.
Science ; 345(6197): 1255215, 2014 Aug 08.
Article in English | MEDLINE | ID: mdl-25104393

ABSTRACT

Coordination of cell division and pattern formation is central to tissue and organ development, particularly in plants where walls prevent cell migration. Auxin and cytokinin are both critical for division and patterning, but it is unknown how these hormones converge upon tissue development. We identify a genetic network that reinforces an early embryonic bias in auxin distribution to create a local, nonresponding cytokinin source within the root vascular tissue. Experimental and theoretical evidence shows that these cells act as a tissue organizer by positioning the domain of oriented cell divisions. We further demonstrate that the auxin-cytokinin interaction acts as a spatial incoherent feed-forward loop, which is essential to generate distinct hormonal response zones, thus establishing a stable pattern within a growing vascular tissue.


Subject(s)
Arabidopsis/growth & development , Body Patterning/physiology , Indoleacetic Acids/metabolism , Plant Vascular Bundle/growth & development , Aminohydrolases , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Body Patterning/drug effects , Body Patterning/genetics , Cell Division/genetics , Cell Division/physiology , Cytokines/biosynthesis , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Gene Regulatory Networks , Indoleacetic Acids/pharmacology , Nuclear Proteins/genetics , Plant Vascular Bundle/drug effects , Trans-Activators/metabolism
11.
Proc Natl Acad Sci U S A ; 111(35): E3650-8, 2014 Sep 02.
Article in English | MEDLINE | ID: mdl-25136122

ABSTRACT

Proteorhodopsin (PR) is present in half of surface ocean bacterioplankton, where its light-driven proton pumping provides energy to cells. Indeed, PR promotes growth or survival in different bacteria. However, the metabolic pathways mediating the light responses remain unknown. We analyzed growth of the PR-containing Dokdonia sp. MED134 (where light-stimulated growth had been found) in seawater with low concentrations of mixed [yeast extract and peptone (YEP)] or single (alanine, Ala) carbon compounds as models for rich and poor environments. We discovered changes in gene expression revealing a tightly regulated shift in central metabolic pathways between light and dark conditions. Bacteria showed relatively stronger light responses in Ala compared with YEP. Notably, carbon acquisition pathways shifted toward anaplerotic CO2 fixation in the light, contributing 31 ± 8% and 24 ± 6% of the carbon incorporated into biomass in Ala and YEP, respectively. Thus, MED134 was a facultative double mixotroph, i.e., photo- and chemotrophic for its energy source and using both bicarbonate and organic matter as carbon sources. Unexpectedly, relative expression of the glyoxylate shunt genes (isocitrate lyase and malate synthase) was >300-fold higher in the light--but only in Ala--contributing a more efficient use of carbon from organic compounds. We explored these findings in metagenomes and metatranscriptomes and observed similar prevalence of the glyoxylate shunt compared with PR genes and highest expression of the isocitrate lyase gene coinciding with highest solar irradiance. Thus, regulatory interactions between dissolved organic carbon quality and central metabolic pathways critically determine the fitness of surface ocean bacteria engaging in PR phototrophy.


Subject(s)
Flavobacteriaceae/metabolism , Phototrophic Processes/physiology , Plankton/metabolism , Rhodopsin/metabolism , Adaptation, Physiological/physiology , Bicarbonates/metabolism , Biomass , Carbon/metabolism , Carbon Dioxide/metabolism , Flavobacteriaceae/genetics , Gene Expression Regulation, Bacterial , Light , Marine Biology , Metabolic Networks and Pathways/genetics , Plankton/genetics , Rhodopsin/genetics , Rhodopsins, Microbial , Seawater/microbiology
12.
Trends Plant Sci ; 18(9): 514-21, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23726727

ABSTRACT

Early plant embryogenesis condenses the fundamental processes underlying plant development into a short sequence of predictable steps. The main tissues, as well as stem cells for their post-embryonic maintenance, are specified through genetic control networks. A key question is how cell fates are instructed by unique cellular transcriptomes, and important insights have recently been gained through cell type-specific transcriptomics during post-embryonic development. However, the poor accessibility and small size of Arabidopsis (Arabidopsis thaliana) embryos have obstructed similar progress during embryogenesis. Here, we review the current situation in plant embryo transcriptomics, and discuss how the recent development of novel cell-specific analysis technologies will enable the identification of cellular transcriptomes in the early Arabidopsis embryo.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/embryology , Arabidopsis/genetics , Gene Expression Profiling/methods , Gene Expression Regulation, Plant , Cell Differentiation , Organ Specificity , Seeds/embryology , Seeds/genetics
13.
Environ Microbiol ; 15(5): 1400-15, 2013 May.
Article in English | MEDLINE | ID: mdl-23379752

ABSTRACT

Proteorhodopsin (PR), a ubiquitous membrane photoprotein in marine environments, acts as a light-driven proton pump and can provide energy for bacterial cellular metabolism. However, knowledge of factors that regulate PR gene expression in different bacteria remains strongly limited. Here, experiments with Vibrio sp. AND4 showed that PR phototrophy promoted survival only in cells from stationary phase and not in actively growing cells. PR gene expression was tightly regulated, with very low values in exponential phase, a pronounced peak at the exponential/stationary phase intersection, and a marked decline in stationary phase. Thus, PR gene expression at the entry into stationary phase preceded, and could therefore largely explain, the stationary phase light-induced survival response in AND4. Further experiments revealed nutrient limitation, not light exposure, regulated this differential PR expression. Screening of available marine vibrios showed that the PR gene, and thus the potential for PR phototrophy, is found in at least three different clusters in the genus Vibrio. In an ecological context, our findings suggest that some PR-containing bacteria adapted to the exploitation of nutrient-rich micro-environments rely on a phase of relatively slowly declining resources to mount a cellular response preparing them for adverse conditions dispersed in the water column.


Subject(s)
Gene Expression Regulation, Bacterial , Rhodopsin/genetics , Rhodopsin/metabolism , Vibrio/genetics , Vibrio/metabolism , Culture Media/chemistry , Gene Order , Light , Mutation , Phylogeny , Rhodopsins, Microbial , Vibrio/classification , Vibrio/growth & development
14.
New Phytol ; 188(1): 122-35, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20561212

ABSTRACT

• In seed plants, current knowledge concerning embryonic pattern formation by polar auxin transport (PAT) and WUSCHEL-related homeobox (WOX) gene activity is primarily derived from studies on angiosperms, while less is known about these processes in gymnosperms. In view of the differences in their embryogeny, and the fact that somatic embryogenesis is used for mass propagation of conifers, a better understanding of embryo development is vital. • The expression patterns of PaWOX2 and PaWOX8/9 were followed with quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and in situ hybridization (ISH) during seed and somatic embryo development in Norway spruce (Picea abies), and in somatic embryos treated with the PAT inhibitor N-1-naphthylphthalamic acid (NPA). • Both PaWOX2 and PaWOX8/9 were highly expressed at the early growth stages of zygotic and somatic embryos, and shared a similar expression pattern over the entire embryo. At later embryo stages, high expression of PaWOX8/9 became restricted to cotyledon primordia, epidermis, procambium and root apical meristem (RAM), which became most evident in NPA-treated somatic embryos, while expression of PaWOX2 was much lower. • Our results suggest an ancestral role of WOX in seed plant embryo development, and strengthen the proposed connection between PAT, PIN-FORMED (PIN) and WOX in the regulation of embryo patterning in seed plants.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Plant , Homeodomain Proteins/genetics , Picea/embryology , Picea/genetics , Plant Proteins/genetics , Seeds/genetics , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Plant/drug effects , Genes, Plant/genetics , Homeodomain Proteins/metabolism , In Situ Hybridization , Phthalimides/pharmacology , Phylogeny , Picea/drug effects , Plant Proteins/metabolism , Plant Shoots/drug effects , Plant Shoots/genetics , Reverse Transcriptase Polymerase Chain Reaction , Seedlings/drug effects , Seedlings/genetics , Seeds/drug effects , Seeds/embryology
15.
Tree Physiol ; 30(4): 479-89, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20129931

ABSTRACT

In seed plants, the body organization is established during embryogenesis and is uniform across gymnosperms and angiosperms, despite differences during early embryogeny. Evidence from angiosperms implicates the plant hormone auxin and its polar transport, mainly established by the PIN family of auxin efflux transporters, in the patterning of embryos. Here, PaPIN1 from Norway spruce (Picea abies [L.] Karst.), a gene widely expressed in conifer tissues and organs, was characterized and its expression and localization patterns were determined with reverse transcription polymerase chain reaction and in situ hybridization during somatic embryo development and in seedlings. PaPIN1 shares the predicted structure of other PIN proteins, but its central hydrophilic loop is longer than most PINs. In phylogenetic analyses, PaPIN1 clusters with Arabidopsis thaliana (L.) Heynh. PIN3, PIN4 and PIN7, but its expression pattern also suggests similarity to PIN1. The PaPIN1 expression signal was high in the protoderm of pre-cotyledonary embryos, but not if embryos were pre-treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). This, together with a high auxin immunolocalization signal in this cell layer, suggests a role of PaPIN1 during cotyledon formation. At later stages, high PaPIN1 expression was observed in differentiating procambium, running from the tip of incipient cotyledons down through the embryo axis and to the root apical meristem (RAM), although the mode of RAM specification in conifer embryos differs from that of most angiosperms. Also, the PaPIN1 in situ signal was high in seedling root tips including root cap columella cells. The results thus suggest that PaPIN1 provides an ancient function associated with auxin transport and embryo pattern formation prior to the separation of angiosperms and gymnosperms, in spite of some morphological differences.


Subject(s)
Indoleacetic Acids/metabolism , Picea/genetics , Plant Proteins/genetics , Seedlings/metabolism , Seeds/metabolism , Arabidopsis Proteins/genetics , Cotyledon/growth & development , Embryonic Development , Gene Expression , In Situ Hybridization , Membrane Transport Proteins/genetics , Multigene Family , Phylogeny , Picea/embryology , Picea/metabolism , Plant Proteins/metabolism , Plant Roots/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Seeds/embryology
16.
Plant Signal Behav ; 4(2): 153-5, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19649198

ABSTRACT

The WOX family of transcription factors and polar auxin transport (PAT) are both essential for embryonic patterning and thus normal embryo development in angiosperms. Recent analysis by us of WOX-related genes in Picea and Pinus suggests that they play fundamental roles during embryo development also in conifers. It has been proposed that there is a connection between the spatial separation of WOX2 and WOX8, and PAT in the formation of the apical-basal axis in Arabidopsis embryos and that both are involved in the regulation of the auxin efflux carrier PIN1. Auxin also seems to play a crucial role in apical-basal axis formation in conifer embryos based on studies using the polar auxin inhibitor NPA. We recently analyzed the expression of a PIN1-like gene in NPA-treated and untreated precotyledonary somatic spruce embryos and could see a significant upregulation of the PIN1-like gene in the NPA-treated embryos.2 Here we show that PaWOX2 is also significantly upregulated in the same embryos. Taken together, this suggests that PAT is involved in regulating both PIN1 and WOX2 expression in conifers and strengthens the evidence for the proposed connection between WOX and PIN genes in seed plants.

17.
Tree Physiol ; 29(4): 483-96, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19203973

ABSTRACT

Auxin and polar auxin transport have been implicated in controlling embryo patterning and development in angiosperms but less is known from the gymnosperms. The aims of this study were to determine at what stages of conifer embryo development auxin and polar auxin transport are the most important for normal development and to analyze the changes in embryos after treatment with the polar auxin inhibitor N-1-naphthylphthalamic acid (NPA). For these studies, somatic embryos of Norway spruce (Picea abies L. Karst) were used. Growth on medium containing NPA leads to the formation of embryos with poor shoot apical meristem (SAM) and fused cotyledons, and to a pin-formed phenotype of the regenerated plantlets. The effect of NPA on embryo morphology was most severe if embryos were transferred to NPA-containing medium immediately before cotyledon initiation and SAM specification. Indole-3-acetic acid (IAA) was identified by immunolocalization in developing embryos. The highest staining intensity was seen in early staged embryos and then decreased as the embryos matured. No clear IAA-maxima was seen, although the apical parts of embryos, particularly the protoderm, and the suspensor cells appear to accumulate more IAA, as reflected by the staining pattern. The NPA treatment also caused expanded procambium and a broader root apical meristem in embryos, and a significant increase in the expression of a PIN1-like gene. Taken together, our results show that, for proper cotyledon initiation, correct auxin transport is needed only during a short period at the transition stage of embryo development, probably involving PIN efflux proteins and that a common mechanism is behind proper cotyledon formation within the species of angiosperms and conifers, despite their cotyledon number which normally differs.


Subject(s)
Indoleacetic Acids/metabolism , Membrane Transport Proteins/metabolism , Phthalimides/pharmacology , Picea/metabolism , Plant Growth Regulators/pharmacology , Plant Proteins/metabolism , Seeds/metabolism , Cell Line , Embryonic Development/drug effects , Gene Expression Regulation, Plant , Indoleacetic Acids/analysis , Indoleacetic Acids/antagonists & inhibitors , Membrane Transport Proteins/genetics , Picea/anatomy & histology , Picea/drug effects , Picea/embryology , Plant Proteins/genetics , Seeds/anatomy & histology , Seeds/drug effects , Seeds/growth & development
18.
Plant Mol Biol ; 66(5): 533-49, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18209956

ABSTRACT

In angiosperms, the WOX family of transcription factors has important functions in meristem regulation and in control of the partitioning of developing embryos into functional domains. In this study, a putative WOX2 homologous gene was isolated from Picea abies, and its expression pattern during somatic embryo development was followed using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). We used strategies of both absolute and relative quantification of gene expression, and benefits and disadvantages of the two methods are presented and discussed. During embryogenesis, PaWOX2 expression was highest at the earliest stages of development, but low levels were also detected in seedling tissues. No PaWOX2 expression was detected in a non-embryogenic cell culture, indicating that PaWOX2 plays a fundamental role during early somatic embryo development, and can be used as a possible marker for embryogenic potential. Additional results show that conifers, like angiosperms, contain a large number of WOX-related genes, many of them expressed during embryo development. In phylogenetic analysis based on the deduced homeodomain of retrieved pine and spruce EST sequences, no conifer WUS homolog was found. Neither did we find any homeodomain to cluster with WOX5. Interestingly, a clade including only conifer sequences derived from various tissues was resolved as sister to a Physcomitrella WOX-like gene, suggestive of the early origin of this gene family. Our results thus provide basic information for further studies of the evolution of this gene family and of their function in relation to meristem dynamics and specification of stem cells in gymnosperms.


Subject(s)
Gene Expression Regulation, Plant , Homeodomain Proteins/metabolism , Picea/growth & development , Picea/metabolism , Transcription Factors/metabolism , Cell Line , Cotyledon/genetics , Cotyledon/growth & development , Cotyledon/metabolism , Databases, Nucleic Acid , Homeodomain Proteins/classification , Homeodomain Proteins/genetics , Homeodomain Proteins/isolation & purification , Phylogeny , Picea/genetics , RNA, Messenger/genetics , Seedlings/genetics , Seedlings/growth & development , Seedlings/metabolism , Transcription Factors/classification , Transcription Factors/genetics , Transcription Factors/isolation & purification
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