RESUMEN
Centromeres are the chromosomal domains, where the kinetochore protein complex is formed, mediating proper segregation of chromosomes during cell division. Although the function of centromeres has remained conserved during evolution, centromeric DNA is highly variable, even in closely related species. In addition, the composition of the kinetochore complexes varies among organisms. Therefore, it is assumed that the centromeric position is determined epigenetically, and the centromeric histone H3 (CENH3) serves as an epigenetic marker. The loading of CENH3 onto centromeres depends on centromere-licensing factors, chaperones, and transcription of centromeric repeats. Several proteins that regulate CENH3 loading and kinetochore assembly interact with the centromeric transcripts and DNA in a sequence-independent manner. However, the functional aspects of these interactions are not fully understood. This review discusses the variability of centromeric sequences in different organisms and the regulation of their transcription through the RNA Pol II and RNAi machinery. The data suggest that the interaction of proteins involved in CENH3 loading and kinetochore assembly with centromeric DNA and transcripts plays a role in centromere, and possibly neocentromere, formation in a sequence-independent manner.
RESUMEN
The centromere is the chromosome region where microtubules attach during cell division. In contrast to monocentric chromosomes with one centromere, holocentric species usually distribute hundreds of centromere units along the entire chromatid. We assembled the chromosome-scale reference genome and analyzed the holocentromere and (epi)genome organization of the lilioid Chionographis japonica. Remarkably, each of its holocentric chromatids consists of only 7 to 11 evenly spaced megabase-sized centromere-specific histone H3-positive units. These units contain satellite arrays of 23 and 28 bp-long monomers capable of forming palindromic structures. Like monocentric species, C. japonica forms clustered centromeres in chromocenters at interphase. In addition, the large-scale eu- and heterochromatin arrangement differs between C. japonica and other known holocentric species. Finally, using polymer simulations, we model the formation of prometaphase line-like holocentromeres from interphase centromere clusters. Our findings broaden the knowledge about centromere diversity, showing that holocentricity is not restricted to species with numerous and small centromere units.
Asunto(s)
Proteínas de Ciclo Celular , Centrómero , Centrómero/genética , División Celular , Cromátides , Heterocromatina/genéticaRESUMEN
Double haploid production is the most effective way to create true-breeding lines in a single generation. In Arabidopsis, haploid induction via mutation of the centromere-specific histone H3 (cenH3) has been shown when the mutant is outcrossed to the wild-type, and the wild-type genome remains in the haploid progeny. However, factors that affect haploid induction are still poorly understood. Here, we report that a mutant of the cenH3 assembly factor Kinetochore Null2 (KNL2) can be used as a haploid inducer when pollinated by the wild-type. We discovered that short-term temperature stress of the knl2 mutant increased the efficiency of haploid induction 10-fold. We also demonstrated that a point mutation in the CENPC-k motif of KNL2 is sufficient to generate haploid-inducing lines, suggesting that haploid-inducing lines in crops can be identified in a naturally occurring or chemically induced mutant population, avoiding the generic modification (GM) approach at any stage. Furthermore, a cenh3-4 mutant functioned as a haploid inducer in response to short-term heat stress, even though it did not induce haploids under standard conditions. Thus, we identified KNL2 as a new target gene for the generation of haploid-inducer lines and showed that exposure of centromeric protein mutants to high temperature strongly increases their haploid induction efficiency.
Asunto(s)
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Haploidia , Temperatura , Centrómero/genética , CinetocorosRESUMEN
The generation of haploid plants accelerates the crop breeding process. One of the haploidization strategies is based on the genetic manipulation of endogenous centromere-specific histone 3 (CENH3). To extend the haploidization toolbox, we tested whether targeted in vivo degradation of CENH3 protein can be harnessed to generate haploids in Arabidopsis thaliana. We show that a recombinant anti-GFP nanobody fused to either heterologous F-box (NSlmb) or SPOP/BTB ligase proteins can recognize maternally derived enhanced yellow fluorescent protein (EYFP)-tagged CENH3 in planta and make it accessible for the ubiquitin-proteasome pathway. Outcrossing of the genomic CENH3-EYFP-complemented cenh3.1 mother with plants expressing the GFP-nanobody-targeted E3 ubiquitin ligase resulted in a haploid frequency of up to 7.6% in pooled F1 seeds. EYFP-CENH3 degradation occurred independently in embryo and endosperm cells. In reciprocal crosses, no haploid induction occurred. We propose that the uniparental degradation of EYFP-fused genomic CENH3 during early embryogenesis leads to a decrease in its level at centromeres and subsequently weakens the centromeres. The male-derived wild type CENH3 containing centromere outcompetes the CENH3-EYFP depleted centromere. Consequently, maternal chromosomes undergo elimination, resulting in haploids.
Asunto(s)
Arabidopsis , Ubiquitina , Arabidopsis/genética , Complejo de la Endopetidasa Proteasomal , GenómicaRESUMEN
Rapeseed (Brassica napus) is an allopolyploid hybrid (AACC genome) of turnip rape (B. rapa, genome: AA) and vegetable cabbage (B. oleraceae, genome: CC). Rapeseed oil is one of the main vegetable oils used worldwide for food and other technical purposes. Therefore, breeding companies worldwide are interested in developing rapeseed varieties with high yields and increased adaptation to harsh climatic conditions such as heat and prolonged drought. One approach to studying the mechanism of the epigenetically regulated stress response is to analyze the transcriptional changes it causes. In addition, comparing the expression of certain genes between stress- and non-stress-tolerant varieties will help guide breeding in the desired direction. Quantitative reverse transcription PCR (RT-qPCR) has been intensively used for gene expression analysis for several decades. However, the transfer of this method from model plants to crop species has several limitations due to the high accumulation of secondary metabolites, the higher water content in some tissues and therefore problems with their grinding and other factors. For allopolyploid rapeseed, the presence of two genomes, often with different levels of expression of homeologous genes, must also be considered. In this study, we describe the optimization of transcriptional RT-qPCR analysis of low-expression epigenetic genes in rapeseed, using Kinetochore Null2 (KNL2), a regulator of kinetochore complex assembly, as an example. We demonstrated that a combination of various factors, such as tissue homogenization and RNA extraction with TRIzol, synthesis of cDNA with gene-specific primers, and RT-qPCR in white plates, significantly increased the sensitivity of RT-qPCR for the detection of BnKNL2A and BnKNL2C gene expression.
RESUMEN
Determining the function of proteins remains a key task of modern biology. Classical genetic approaches to knocking out protein function in plants still face limitations, such as the time-consuming nature of generating homozygous transgenic lines or the risk of non-viable loss-of-function phenotypes. We aimed to overcome these limitations by acting downstream of the protein level. Chimeric E3 ligases degrade proteins of interest in mammalian cell lines, Drosophila melanogaster embryos, and transgenic tobacco. We successfully recruited the 26S proteasome pathway to directly degrade a protein of interest located in plant nuclei. This success was achieved via replacement of the interaction domain of the E3 ligase adaptor protein SPOP (Speckle-type POZ adapter protein) with a specific anti-GFP nanobody (VHHGFP4). For proof of concept, the target protein CENH3 of A. thaliana fused to EYFP was subjected to nanobody-guided proteasomal degradation in planta. Our results show the potential of the modified E3-ligase adapter protein VHHGFP4-SPOP in this respect. We were able to point out its capability for nucleus-specific protein degradation in plants.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Histonas/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Ingeniería de Proteínas , Proteolisis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Línea Celular , Histonas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
In most diploids the centromere-specific histone H3 (CENH3), the assembly site of active centromeres, is encoded by a single copy gene. Persistance of two CENH3 paralogs in diploids species raises the possibility of subfunctionalization. Here we analysed both CENH3 genes of the diploid dryland crop cowpea. Phylogenetic analysis suggests that gene duplication of CENH3 occurred independently during the speciation of Vigna unguiculata. Both functional CENH3 variants are transcribed, and the corresponding proteins are intermingled in subdomains of different types of centromere sequences in a tissue-specific manner together with the kinetochore protein CENPC. CENH3.2 is removed from the generative cell of mature pollen, while CENH3.1 persists. CRISPR/Cas9-based inactivation of CENH3.1 resulted in delayed vegetative growth and sterility, indicating that this variant is needed for plant development and reproduction. By contrast, CENH3.2 knockout individuals did not show obvious defects during vegetative and reproductive development. Hence, CENH3.2 of cowpea is likely at an early stage of pseudogenization and less likely undergoing subfunctionalization.
Asunto(s)
Proteína A Centromérica/genética , Centrómero/genética , Variación Genética , Vigna/genética , Centrómero/metabolismo , Proteína A Centromérica/metabolismo , Evolución Molecular , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica de las Plantas , Hibridación Fluorescente in Situ , Especificidad de Órganos , Fenotipo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Vigna/clasificaciónRESUMEN
TPX2 (Targeting Protein for Xklp2) is an evolutionary conserved microtubule-associated protein important for microtubule nucleation and mitotic spindle assembly. The protein was described as an activator of the mitotic kinase Aurora A in humans and the Arabidopsis AURORA1 (AUR1) kinase. In contrast to animal genomes that encode only one TPX2 gene, higher plant genomes encode a family with several TPX2-LIKE gene members (TPXL). TPXL genes of Arabidopsis can be divided into two groups. Group A proteins (TPXL2, 3, 4, and 8) contain Aurora binding and TPX2_importin domains, while group B proteins (TPXL1, 5, 6, and 7) harbor an Xklp2 domain. Canonical TPX2 contains all the above-mentioned domains. We confirmed using in vitro kinase assays that the group A proteins contain a functional Aurora kinase binding domain. Transient expression of Arabidopsis TPX2-like proteins in Nicotiana benthamiana revealed preferential localization to microtubules and nuclei. Co-expression of AUR1 together with TPX2-like proteins changed the localization of AUR1, indicating that these proteins serve as targeting factors for Aurora kinases. Taken together, we visualize the various localizations of the TPX2-LIKE family in Arabidopsis as a proxy to their functional divergence and provide evidence of their role in the targeted regulation of AUR1 kinase activity.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Secuencia de Aminoácidos , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/análisis , Proteínas de Arabidopsis/genética , Aurora Quinasas/metabolismo , Genes de Plantas , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Asociadas a Microtúbulos/genética , Unión Proteica , Dominios ProteicosRESUMEN
Several histone variants are posttranslationally phosphorylated. Little is known about phosphorylation of the centromere-specific histone 3 (CENH3) variant in plants. We show that CENH3 of Arabidopsis thaliana is phosphorylated in vitro by Aurora3, predominantly at serine 65. Interaction of Aurora3 and CENH3 was found by immunoprecipitation (IP) in A. thaliana and by bimolecular fluorescence complementation. Western blotting with an anti-CENH3 pS65 antibody showed that CENH3 pS65 is more abundant in flower buds than elsewhere in the plant. Substitution of serine 65 by either alanine or aspartic acid resulted in a range of phenotypic abnormalities, especially in reproductive tissues. We conclude that Aurora3 phosphorylates CENH3 at S65 and that this post-translational modification is required for the proper development of the floral meristem.
RESUMEN
Aurora kinases are key regulators of mitosis. Multicellular eukaryotes generally possess two functionally diverged types of Aurora kinases. In plants, including Arabidopsis (Arabidopsis thaliana), these are termed α- and ß-Auroras. As the functional specification of Aurora kinases is determined by their specific interaction partners, we initiated interactomics analyses using both Arabidopsis α-Aurora kinases (AUR1 and AUR2). Proteomics results revealed that TPX2-LIKE PROTEINS2 and 3 (TPXL2/3) prominently associated with α-Auroras, as did the conserved TPX2 to a lower degree. Like TPX2, TPXL2 and TPXL3 strongly activated the AUR1 kinase but exhibited cell-cycle-dependent localization differences on microtubule arrays. The separate functions of TPX2 and TPXL2/3 were also suggested by their different influences on AUR1 localization upon ectopic expressions. Furthermore, genetic analyses showed that TPXL3, but not TPX2 and TPXL2, acts nonredundantly to enable proper embryo development. In contrast to vertebrates, plants have an expanded TPX2 family and these family members have both redundant and unique functions. Moreover, as neither TPXL2 nor TPXL3 contains the C-terminal Kinesin-5 binding domain present in the canonical TPX2, the targeting and activity of this kinesin must be organized differently in plants.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Proteínas Serina-Treonina Quinasas/genética , Semillas/genética , Secuencia de Aminoácidos , Arabidopsis/embriología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Activación Enzimática/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Microscopía Confocal , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Plantas Modificadas Genéticamente , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteómica/métodos , Semillas/embriología , Semillas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The ability to generate (doubled) haploid plants significantly accelerates the crop breeding process. Haploids have been induced mainly through the generation of plants from cultivated gametophic (haploid) cells and tissues, i.e., in vitro haploid technologies, or through the selective loss of a parental chromosome set upon inter- or intraspecific hybridization. Here, we focus our review on the mechanisms responsible for the in vivo formation of haploids in the context of inter- and intraspecific hybridization. The application of a modified CENH3 for uniparental genome elimination, the IG1 system used for paternal as well as the BBM-like and the patatin-like phospholipase essential for maternal haploidy induction are discussed in detail.
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Haploidia , Fitomejoramiento/métodos , Centrómero/metabolismo , Productos Agrícolas/genética , Hibridación Genética , PartenogénesisRESUMEN
KINETOCHORE NULL2 (KNL2) is involved in recognition of centromeres and in centromeric localization of the centromere-specific histone cenH3. Our study revealed a cenH3 nucleosome binding CENPC-k motif at the C terminus of Arabidopsis thaliana KNL2, which is conserved among a wide spectrum of eukaryotes. Centromeric localization of KNL2 is abolished by deletion of the CENPC-k motif and by mutating single conserved amino acids, but can be restored by insertion of the corresponding motif of Arabidopsis CENP-C. We showed by electrophoretic mobility shift assay that the C terminus of KNL2 binds DNA sequence-independently and interacts with the centromeric transcripts in vitro. Chromatin immunoprecipitation with anti-KNL2 antibodies indicated that in vivo KNL2 is preferentially associated with the centromeric repeat pAL1 Complete deletion of the CENPC-k motif did not influence its ability to interact with DNA in vitro. Therefore, we suggest that KNL2 recognizes centromeric nucleosomes, similar to CENP-C, via the CENPC-k motif and binds adjoining DNA.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Centrómero/genética , Proteínas Cromosómicas no Histona/genética , ADN de Plantas/genética , ADN de Plantas/metabolismo , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica de las Plantas , Cinetocoros/metabolismo , Modelos Genéticos , Mutación , Nucleosomas/genética , Nucleosomas/metabolismo , Plantas Modificadas Genéticamente , Unión ProteicaRESUMEN
The effect of plant ploidy level on the rate of cytomixis in microsporogenesis has been analyzed with the help of a unique model, the collection of tobacco plants of different ploidies (2n = 2x = 24, 4x = 48, 6x = 72, and 8x = 96). As has been shown, the rate of cytomixis proportionally increases in 6x and 8x cytotypes, being rather similar in 2x and 4x plants. The rate of cytomixis is highly variable, differing even in the genetically identical plants grown under the same conditions. The cytological pattern of cytomixis in the microsporogenesis of control 4x plants has been compared with the corresponding patterns of 2x, 6x, and 8x plants. Involvement of cytomixis in production of unreduced gametes and stabilization of the newly formed hybrid and polyploidy genomes is discussed.
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Gametogénesis en la Planta , Meiosis , Nicotiana/citología , Ploidias , Flores/anatomía & histología , CariotipificaciónRESUMEN
Aurora kinases are evolutionarily conserved key mitotic determinants in all eukaryotes. Yeasts contain a single Aurora kinase, whereas multicellular eukaryotes have at least two functionally diverged members. The involvement of Aurora kinases in human cancers has provided an in-depth mechanistic understanding of their roles throughout cell division in animal and yeast models. By contrast, understanding Aurora kinase function in plants is only starting to emerge. Nevertheless, genetic, cell biological, and biochemical approaches have revealed functional diversification between the plant Aurora kinases and suggest a role in formative (asymmetric) divisions, chromatin modification, and genome stability. This review provides an overview of the accumulated knowledge on the function of plant Aurora kinases as well as some major challenges for the future.
Asunto(s)
Aurora Quinasas/metabolismo , Desarrollo de la Planta , Plantas/enzimología , Unión Proteica , Transporte de Proteínas , Especificidad por SustratoRESUMEN
Cytomixis is a poorly studied process of nuclear migration between plant cells. It is so far unknown what drives cytomixis and what is the functional state of the chromatin migrating between cells. Using immunostaining, we have analyzed the distribution of posttranslational histone modifications (methylation, acetylation, and phosphorylation) that reflect the functional state of chromatin in the tobacco microsporocytes involved in cytomixis. We demonstrate that the chromatin in the cytomictic cells does not differ from the chromatin in intact microsporocytes according to all 14 analyzed histone modification types. We have also for the first time demonstrated that the migrating chromatin contains normal structures of the synaptonemal complex (SC) and lacks any signs of apoptosis. As has been shown, the chromatin migrating between cells in cytomixis is neither selectively heterochromatized nor degraded both before its migration to another cell and after it enters a recipient cell as micronuclei. We also showed that cytomictic chromatin contains marks typical for transcriptionally active chromatin as well as heterochromatin. Moreover, marks typical for chromosome condensation, SC formation and key proteins required for the formation of bivalents were also detected at migrated chromatin.
RESUMEN
The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called "CENP-A") is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923-937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest.
Asunto(s)
Autoantígenos/genética , Centrómero/genética , Proteínas Cromosómicas no Histona/genética , Haploidia , Plantas/genética , Mutación Puntual , Arabidopsis/genética , Arabidopsis/metabolismo , Autoantígenos/metabolismo , Western Blotting , Centrómero/metabolismo , Proteína A Centromérica , Proteínas Cromosómicas no Histona/metabolismo , Genotipo , Histonas/genética , Hordeum/genética , Hordeum/metabolismo , Cinetocoros/metabolismo , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Modelos Genéticos , Fenotipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semillas/genética , Semillas/metabolismo , Semillas/ultraestructuraRESUMEN
Aurora is an evolutionary conserved protein kinase family involved in monitoring of chromosome segregation via phosphorylation of different substrates. In plants, however, the involvement of Aurora proteins in meiosis and in sensing microtubule attachment remains to be proven, although the downstream components leading to the targeting of spindle assembly checkpoint signals to anaphase-promoting complex have been described. To analyze the three members of Aurora family (AtAurora1, -2, and -3) of Arabidopsis we employed different combinations of T-DNA insertion mutants and/or RNAi transformants. Meiotic defects and the formation of unreduced pollen were revealed including plants with an increased ploidy level. The effect of reduced expression of Aurora was mimicked by application of the ATP-competitive Aurora inhibitor II. In addition, strong overexpression of any member of the AtAurora family is not possible. Only tagged or truncated forms of Aurora kinases can be overexpressed. Expression of truncated AtAurora1 resulted in a high number of aneuploids in Arabidopsis, while expression of AtAurora1-TAPi construct in tobacco resulted in 4C (possible tetraploid) progeny. In conclusion, our data demonstrate an essential role of Aurora kinases in the monitoring of meiosis in plants.
Asunto(s)
Arabidopsis/enzimología , Aurora Quinasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Arabidopsis/citología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Aurora Quinasas/genética , Segregación Cromosómica , Flores/citología , Flores/enzimología , Flores/genética , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Meiosis , Microtúbulos/metabolismo , Mitosis , Familia de Multigenes , Mutagénesis Insercional , Fenotipo , Fosforilación , Plantas Modificadas Genéticamente , Poliploidía , Plantones/citología , Plantones/enzimología , Plantones/genética , Semillas/citología , Semillas/enzimología , Semillas/genéticaRESUMEN
The centromere-the primary constriction of monocentric chromosomes-is essential for correct segregation of chromosomes during mitosis and meiosis. Centromeric DNA varies between different organisms in sequence composition and extension. The main components of centromeric and pericentromeric DNA of Brassicaceae species are centromeric satellite repeats. Centromeric DNA initiates assembly of the kinetochore, the large protein complex where the spindle fibers attach during nuclear division to pull sister chromatids apart. Kinetochore assembly is initiated by incorporation of the centromeric histone H3 cenH3 into centromeric nucleosomes. The spindle assembly checkpoint acts during mitosis and meiosis at centromeres and maintains genome stability by preventing chromosome segregation before all kinetochores are correctly attached to microtubules. The function of the spindle assembly checkpoint in plants is still poorly understood. Here, we review recent advances of studies on structure and functional importance of centromeric DNA of Brassicaceae, assembly and function of cenH3 in Arabidopsis thaliana and characterization of core SAC proteins of A. thaliana in comparison with non-plant homologues.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Centrómero/metabolismo , Genes de Plantas , Cinetocoros/metabolismo , Arabidopsis/clasificación , Proteínas de Arabidopsis/metabolismo , Segregación Cromosómica/genética , Cromosomas de las Plantas/genética , Cromosomas de las Plantas/metabolismo , ADN de Plantas/genética , Ingeniería Genética , Inestabilidad Genómica , Histonas/genética , Histonas/metabolismo , Meiosis , Microtúbulos/metabolismo , Mitosis , Análisis de Secuencia de ADN , Activación TranscripcionalRESUMEN
The centromeric histone H3 variant cenH3 is an essential centromeric protein required for assembly, maintenance, and proper function of kinetochores during mitosis and meiosis. We identified a kinetochore null2 (KNL2) homolog in Arabidopsis thaliana and uncovered features of its role in cenH3 loading at centromeres. We show that Arabidopsis KNL2 colocalizes with cenH3 and is associated with centromeres during all stages of the mitotic cell cycle, except from metaphase to mid-anaphase. KNL2 is regulated by the proteasome degradation pathway. The KNL2 promoter is mainly active in meristematic tissues, similar to the cenH3 promoter. A knockout mutant for KNL2 shows a reduced level of cenH3 expression and reduced amount of cenH3 protein at chromocenters of meristematic nuclei, anaphase bridges during mitosis, micronuclei in pollen tetrads, and 30% seed abortion. Moreover, knl2 mutant plants display reduced expression of suppressor of variegation 3-9 homologs2, 4, and 9 and reduced DNA methylation, suggesting an impact of KNL2 on the epigenetic environment for centromere maintenance.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Centrómero/metabolismo , Regulación de la Expresión Génica de las Plantas , Histonas/metabolismo , Cinetocoros/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Metilación de ADN , Epigénesis Genética , Flores/citología , Flores/genética , Flores/metabolismo , Redes Reguladoras de Genes , Genes Reporteros , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Meiosis , Mitosis , Mutagénesis Insercional , Fenotipo , Regiones Promotoras Genéticas , Complejo de la Endopetidasa Proteasomal , Plantones/citología , Plantones/genética , Plantones/metabolismoRESUMEN
To establish three-dimensional structures/organs, plant cells continuously have to adapt the orientation of their division plane in a highly regulated manner. However, mechanisms underlying switches in division plane orientation remain elusive. Here, we characterize a viable double knockdown mutant in Arabidopsis thaliana group α Aurora (AUR) kinases, AUR1 and AUR2, (aur1-2 aur2-2), with a primary defect in lateral root formation and outgrowth. Mutant analysis revealed that aur1-2 aur2-2 lateral root primordia are built from randomly oriented cell divisions instead of distinct cell layers. This phenotype could be traced back to cytokinesis defects and misoriented cell plates during the initial anticlinal pericycle cell divisions that give rise to lateral root primordia. Complementation assays showed that the Arabidopsis α group Aurora kinases are functionally divergent from the single ß group member AUR3 and that AUR1 functions in division plane orientation prior to cytokinesis. In addition to defective lateral root patterning, aur1-2 aur2-2 plants also show defects in orienting formative divisions during embryogenesis, divisions surrounding the main root stem cell niche, and divisions surrounding stomata formation. Taken together, our results put forward a central role for α Aurora kinases in regulating formative division plane orientation throughout development.