Our genetic and molecular studies have indicated that FIE a WD-repeat Polycomb group (PcG) protei... more Our genetic and molecular studies have indicated that FIE a WD-repeat Polycomb group (PcG) protein takes part in multi-component protein complexes. We have shown that FIE PcG protein represses inappropriate programs of development during the reproductive and vegetative phases of the Arabidopsis life cycle. Moreover, we have shown that FIE represses the expression of key regulatory genes that promote flowering (AG and LFY), embryogenesis (LEC1), and shoot formation (KNAT1). These results suggest that the FIE PcG protein participates in the formation of distinct PcG complexes that repress inappropriate gene expression at different stages of plant development. PcG complexes modulate chromatin compactness by modifying histones and thereby regulate gene expression and imprinting. The main goals of our original project were to elucidate the biological functions of PcG proteins, and to understand the molecular mechanisms used by FIE PcG complexes to repress the expression of its gene targe...
The Snf2 chromatin remodeler, DECREASE IN DNA METHYLATION 1 (DDM1) facilitates DNA methylation. I... more The Snf2 chromatin remodeler, DECREASE IN DNA METHYLATION 1 (DDM1) facilitates DNA methylation. In flowering plants, DDM1 mediates methylation in heterochromatin, which is targeted primarily by MET1 and CMT methylases and is necessary for silencing transposons and for proper development. DNA methylation mechanisms evolved throughout plant evolution, whereas the role of DDM1 in early terrestrial plants remains elusive. Here, we studied the function of DDM1 in the moss, Physcomitrium (Physcomitrella) patens, which has robust DNA methylation that suppresses transposons and is mediated by a MET1, a CMT, and a DNMT3 methylases. To elucidate the role of DDM1 in P. patens, we have generated a knockout mutant and found DNA methylation to be strongly disrupted at any of its sequence contexts. Symmetric CG and CHG sequences were affected stronger than asymmetric CHH sites. Furthermore, despite their separate targeting mechanisms, CG (MET) and CHG (CMT) methylation were similarly depleted by about 75%. CHH (DNMT3) methylation was overall reduced by about 25%, with an evident hyper-methylation activity within lowly-methylated euchromatic transposon sequences. Despite the strong hypomethylation effect, only a minute number of transposons were transcriptionally activated in Ppddm1. Finally, Ppddm1 was found to develop normally throughout the plant life cycle. These results demonstrate that DNA methylation is strongly dependent on DDM1 in a nonflowering plant; that DDM1 is required for plant-DNMT3 (CHH) methylases, though to a lower extent than for MET1 and CMT enzymes; and that distinct and separate methylation pathways (e.g. MET1-CG and CMT-CHG), can be equally regulated by the chromatin and that DDM1 plays a role in it. Finally, our data suggest that the biological significance of DDM1 in terms of transposon regulation and plant development, is species dependent.
Arabidopsis mutants that we have isolated, encode for fertilization-independent endosperm (fie), ... more Arabidopsis mutants that we have isolated, encode for fertilization-independent endosperm (fie), fertilization-independent seed2 (fis2) and medea (mea) genes, act in the female gametophyte and allow endosperm to develop without fertilization when mutated. We cloned the FIE and MEA genes and showed that they encode WD and SET domain polycomb (Pc G) proteins, respectively. Homologous proteins of FIE and MEA in other organisms are known to regulate gene transcription by modulating chromatin structure. Based on our results, we proposed a model whereby both FIE and MEA interact to suppress transcription of regulatory genes. These genes are transcribed only at proper developmental stages, as in the central cell of the female gametophyte after fertilization, thus activating endosperm development. To test our model, the following questions were addressed: What is the Composition and Function of the Polycomb Complex? Molecular, biochemical, genetic and genomic approaches were offered to iden...
Endosperm is a key nutritive tissue that supports the developing embryo or seedling, and serves a... more Endosperm is a key nutritive tissue that supports the developing embryo or seedling, and serves as a major nutritional source for human and livestock feed. In sexually-reproducing flowering plants, it generally develops after fertilization. However, autonomous endosperm (AE) formation (i.e. independent of fertilization) is also possible. Recent findings of AE loci/ genes and aberrant imprinting in native apomicts, together with a successful initiation of parthenogenesis in rice and lettuce, have enhanced our understanding of the mechanisms bridging sexual and apomictic seed formation. However, the mechanisms driving AE development are not well understood. This review presents novel aspects related to AE development in sexual and asexual plants underlying stress conditions as the primary trigger for AE. Both application of hormones to unfertilized ovules and mutations that impair epigenetic regulation lead to AE development in sexual Arabidopsis thaliana, which may point to a common ...
Background Circular RNA (circRNA) is an emerging class of RNA molecules attracting researchers du... more Background Circular RNA (circRNA) is an emerging class of RNA molecules attracting researchers due to its potential for serving as markers for diagnosis, prognosis, or therapeutic targets of cancer, cardiovascular, and autoimmune diseases. Current methods for detection of circRNA from RNA sequencing (RNA-seq) focus mostly on improving mapping quality of reads supporting the back-splicing junction (BSJ) of a circRNA to eliminate false positives (FPs). We show that mapping information alone often cannot predict if a BSJ-supporting read is derived from a true circRNA or not, thus increasing the rate of FP circRNAs. Results We have developed Circall, a novel circRNA detection method from RNA-seq. Circall controls the FPs using a robust multidimensional local false discovery rate method based on the length and expression of circRNAs. It is computationally highly efficient by using a quasi-mapping algorithm for fast and accurate RNA read alignments. We applied Circall on two simulated dat...
A fundamental problem in biology is to understand how fertilization initiates reproductive develo... more A fundamental problem in biology is to understand how fertilization initiates reproductive development. During plant reproduction, one sperm cell fuses with the egg to form an embryo, whereas a second sperm cell fuses with the adjacent central cell nucleus to form the endosperm tissue that supports embryo and/or seedling development. To understand the mechanisms that initiate reproduction, we have isolated mutants of Arabidopsis that allow for replication of the central cell and subsequent endosperm development without fertilization. In this project we have cloned the MEA gene and showed that it encode a SET- domain polycomb protein. Such proteins are known to form chromatin-protein complexes that repress homeotic gene transcription and influence cell proliferation from Drosophylla to mammals. We propose a model whereby MEA and an additional polycomb protein we have cloned, FIE , function to suppress a critical aspect of early plant reproduction and endosperm development, until fert...
Circall is a novel method for fast and accurate discovery of circular RNAs from paired-end RNA-se... more Circall is a novel method for fast and accurate discovery of circular RNAs from paired-end RNA-sequencing data. The method controls false positives by two-dimensional local false discovery method and employs quasi-mapping for fast and accurate alignments.
Proceedings of the National Academy of Sciences, 2020
Significance While DNA methylation in plants occurs at CG, CHG, and CHH sequence contexts, their ... more Significance While DNA methylation in plants occurs at CG, CHG, and CHH sequence contexts, their comparative roles in genome regulation remain elusive. Here, we utilized Physcomitrella patens as a plant model suitable to study this question. By profiling transcriptomes in a series of context-specific methylation mutants, we found redundancy between asymmetric (CHH) methylation and symmetric methylation in silencing transposons, and that CHH methylation regulates the expression of CG/CHG-depleted transposons. Specific elimination of CG methylation had a significantly smaller effect on transcription than elimination of non-CG methylation. Additionally, we found CHG methylation as a stronger silencer than CG methylation. These results disentangle the transcriptional roles of the individual methylation contexts and elucidate the crucial role of non-CG methylation in genome regulation.
DNA methylation in plants occurs in CG, CHG, and CHH sites. While depletion of CG methylation in ... more DNA methylation in plants occurs in CG, CHG, and CHH sites. While depletion of CG methylation in transposons is associated with ample transcriptional activation, it was mainly studied in species with limited non-CG methylation that is linked to CG methylation. Here we profiled transcription in the moss plant, Physcomitrella patens, that has robust non-CG methylation with similar symmetrical CG and CHG methylation levels. Separated contextual methylation mechanisms in Physcomitrella patens enabled generation of numerous context-specific hypomethylated mutants. Our transcriptome data show that specific elimination of CG methylation is fully complemented by non-CG methylation. Conversely, exclusive removal of non-CG methylation massively dysregulated genes and transposons. Moreover, CHG methylation silenced transposons stronger than CG methylation. Lastly, we found non-CG methylation as crucial for silencing CG-depleted transposons. These results demonstrate the potency of non-CG methy...
The Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) polycomb group (PcG) protein, a WD40 ho... more The Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) polycomb group (PcG) protein, a WD40 homologue of Drosophila extra sex comb (ESC), regulates endosperm and embryo development and represses flowering during embryo and seedling development. As fie alleles are not transmitted maternally, homozygous mutant plants cannot be obtained. To study FIE function during the entire plant life cycle, we used Arabidopsis FIE co-suppressed plants. Low FIE level in these plants produced dramatic morphological aberrations, including loss of apical dominance, curled leaves, early flowering and homeotic conversion of leaves, flower organs and ovules into carpel-like structures. These morphological aberrations are similar to those exhibited by plants overexpressing AGAMOUS (AG) or CURLY LEAF (clf) mutants. Furthermore, the aberrant leaf morphology of FIE-silenced and clf plants correlates with de-repression of the class I KNOTTED-like homeobox (KNOX) genes including KNOTTED-like from Arabidopsis thaliana 2 (KNAT2) and SHOOTMERISTEMLESS (STM), whereas BREVIPEDICELLUS (BP) was upregulated in FIE-silenced plants, but not in the clf mutant. Thus, FIE is essential for the control of shoot and leaf development. Yeast two-hybrid and pull-down assays demonstrate that FIE interacts with CLF. Collectively, the morphological characteristics, together with the molecular and biochemical data presented in this work, strongly suggest that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Our findings demonstrate that the versatility of the plant FIE function, which is derived from association with different SET (SU (VAR)3-9, E (Z), Trithorax) domain PcG proteins, results in differential regulation of gene expression throughout the plant life cycle.
Plants possess a single gene for the structurally related HETEROCHROMATIN PROTEIN1 (HP1), termed ... more Plants possess a single gene for the structurally related HETEROCHROMATIN PROTEIN1 (HP1), termed LIKE-HP1 (LHP1). We investigated the subnuclear localization, binding properties, and dynamics of LHP1 proteins in Arabidopsis thaliana cells. Transient expression assays showed that tomato (Solanum lycopersicum) LHP1 fused to green fluorescent protein (GFP; Sl LHP1-GFP) and Arabidopsis LHP1 (At LHP1-GFP) localized to heterochromatic chromocenters and showed punctuated distribution within the nucleus; tomato but not Arabidopsis LHP1 was also localized within the nucleolus. Mutations of aromatic cage residues that recognize methyl K9 of histone H3 abolished their punctuated distribution and localization to chromocenters. Sl LHP1-GFP plants displayed cell type–dependent subnuclear localization. The diverse localization pattern of tomato LHP1 did not require the chromo shadow domain (CSD), whereas the chromodomain alone was insufficient for localization to chromocenters; a nucleolar localiz...
The high-concentration C0,-requiring mutant N5 of Synechococcus sp. PCC 7942 was obtained by the ... more The high-concentration C0,-requiring mutant N5 of Synechococcus sp. PCC 7942 was obtained by the insertion of a kanamycinresistant gene at the fcoRl site, 12.4 kb upstream of rbc. l h e mutant is unable to accumulate inorganic carbon internally and exhibits very low apparent photosynthetic affinity for inorganic carbon but a photosynthetic VmaX similar to that of the wild type. Sequence and northern analyses showed that the insertion inactivated a gene highly homologous to ndhl, encoding subunit II of NADH dehydrogenase in Synechocystis sp. PCC 6803 (T. Ogawa [1991] Proc Natl Acad Sci USA 88: 4275-4279). When the mutant and the wild-type cells were exposed to 5% COz in air, their photosynthetic electron transfer capabilities, as revealed by fluorescence and thermoluminescence measurements, were similar. On the other hand, a significant decrease in variable fluorescence was observed when the mutant (but not the wild-type) cells were exposed tolow COz under continuous light. l h e same treatment also resulted in a shift (from 38-27'C) in the temperature at which the maximal thermoluminescence emission signal was obtained in the mutant but not in the wild type. These results may indicate that subunit II of NADH dehydrogenase is essential for the fundional operation of the photosynthetic electron transport in Synechococcus under low but not high levels of COz. We suggest that the inability to accumulate inorganic carbon under air conditions stems from disrupture of electron transport in this mutant.
Protein function is often mediated via formation of stable or transient complexes. Here we report... more Protein function is often mediated via formation of stable or transient complexes. Here we report the determination of protein-protein interactions in plants using bimolecular fluorescence complementation (BiFC). The yellow fluorescent protein (YFP) was split into two non-overlapping N-terminal (YN) and C-terminal (YC) fragments. Each fragment was cloned in-frame to a gene of interest, enabling expression of fusion proteins. To demonstrate the feasibility of BiFC in plants, two pairs of interacting proteins were utilized: (i) the alpha and beta subunits of the Arabidopsis protein farnesyltransferase (PFT), and (ii) the polycomb proteins, FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA). Members of each protein pair were transiently co-expressed in leaf epidermal cells of Nicotiana benthamiana or Arabidopsis. Reconstitution of a fluorescing YFP chromophore occurred only when the inquest proteins interacted. No fluorescence was detected following co-expression of free non-fused YN and YC or non-interacting protein pairs. Yellow fluorescence was detected in the cytoplasm of cells that expressed PFT alpha and beta subunits, or in nuclei and cytoplasm of cells that expressed FIE and MEA. In vivo measurements of fluorescence spectra emitted from reconstituted YFPs were identical to that of a non-split YFP, confirming reconstitution of the chromophore. Expression of the inquest proteins was verified by immunoblot analysis using monoclonal antibodies directed against tags within the hybrid proteins. In addition, protein interactions were confirmed by immunoprecipitations. These results demonstrate that plant BiFC is a simple, reliable and relatively fast method for determining protein-protein interactions in plants.
Following the complete genome sequencing of different plant species such as Arabidopsis (Arabidop... more Following the complete genome sequencing of different plant species such as Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Physcomitrella (Physcomitrella patens), as well as advances toward deciphering entire proteomes, the need for a reliable way to identify protein-protein interactions is becoming a major task for the future. Bimolecular fluorescent complementation (BiFC) is a noninvasive fluorescentbased technique that allows detection of protein-protein interactions in living cells, and furthermore can be used to determine subcellular localization of the interacting proteins, and if it changes over time, without requiring addition of external agents. BiFC is based upon reconstitution of split nonfluorescent GFP variants, primarily yellow fluorescent protein (YFP), to form a fluorescent fluorophore (Ghosh et al., 2000; Hu et al., 2002). The technique has become increasingly popular due to its simplicity, ease of use, and the capability to carry out experiments with ...
The original version of this Article contained an error in Fig. 5, in which the evolutionary orig... more The original version of this Article contained an error in Fig. 5, in which the evolutionary origin of DRM2 was incorrectly placed prior to the divergence between gymnosperms and angiosperms. The correct evolutionary origin of DRM2 should be in angiosperms. The correct version of Fig. 5 is shown here as Fig. 1, which replaces the incorrect version which is shown here as Fig. 2. In addition, in the "Percent methylation change" section of the Methods, Equation 1 was incorrectly given as: WT mCHH À cmt mCHHWT mCHH 100 if WT mCHH > cmt mCHH The correct form of Equation 1 is as follows: WT mCHH À cmt mCHH WT mCHH 100 if WT mCHH > cmt mCHH ð1Þ This has been corrected in both the PDF and HTML versions of the Article.
Sustainable diets: linking nutrition and food systems, 2019
The evolutionary history of the concepts of food security (FS) and sustainability have run in par... more The evolutionary history of the concepts of food security (FS) and sustainability have run in parallel for many years. After the food crisis of 2008, stability was added to definition of FS as a short-term time dimension to express the ability to withstand shocks to the food system caused by natural or man-made disasters. We have proposed that sustainability be added as a fifth long-term time dimension, thus bringing together FS and sustainability. In 2015, the United Nations described the seventeen sustainable development goals. We believe that FS involves all the goals to a greater or lesser extent. The challenge ahead is to build and integrate FS on the sustainability agenda and vice versa. The final common pathway for all these efforts is for countries to develop their most appropriate sustainable food systems. As a practical exercise towards this aim, we have reviewed what eight different countries (United States, Brazil, France, Greece, Spain, the Netherlands, United Kingdom, ...
To properly regulate the genome, cytosine methylation is established by animal DNA methyltransfer... more To properly regulate the genome, cytosine methylation is established by animal DNA methyltransferase 3 s (DNMT3s). While altered DNMT3 homologs, Domains rearranged methyltransferases (DRMs), have been shown to establish methylation via the RNA directed DNA methylation (RdDM) pathway, the role of true-plant DNMT3 orthologs remains elusive. Here, we profile de novo (RPS transgene) and genomic methylation in the basal plant, Physcomitrella patens, mutated in each of its PpDNMTs. We show that PpDNMT3b mediates CG and CHH de novo methylation, independently of PpDRMs. Complementary de novo CHG methylation is specifically mediated by the CHROMOMETHYLASE, PpCMT. Intragenomically, PpDNMT3b functions preferentially within heterochromatin and is affected by PpCMT. In comparison, PpDRMs target active-euchromatic transposons. Overall, our data resolve how DNA methylation in plants can be established in heterochromatin independently of RdDM; suggest that DRMs have emerged to target euchromatin; and link DNMT3 loss in angiosperms to the initiation of heterochromatic CHH methylation by CMT2.
Posttranslational modification (PTM) of histone tails plays a critical role in the dynamic of chr... more Posttranslational modification (PTM) of histone tails plays a critical role in the dynamic of chromatin and chromosomes. However, emerging evidence suggests that individual histone modifications do not reliably predict a single functional output [Sims and Reinberg (Nat Rev Mol Cell Biol 9:815–820, 2008)]. In plants, the cell cycle-dependent phosphorylation of histone H3 has been described best; it is hyperphosphorylated at serines 10/28 and at threonines 3/11/32 during both mitosis and meiosis in patterns that are specifically coordinated in both space and time. Although this posttranslational modification is highly conserved, data show that the chromosomal distribution of individual modifications can differ between groups of eukaryotes. We describe the function of plant Aurora and Haspin kinases which have the capacity to phosphorylate H3 and discuss the cross talk between phosphorylation and other PTMs.
Our genetic and molecular studies have indicated that FIE a WD-repeat Polycomb group (PcG) protei... more Our genetic and molecular studies have indicated that FIE a WD-repeat Polycomb group (PcG) protein takes part in multi-component protein complexes. We have shown that FIE PcG protein represses inappropriate programs of development during the reproductive and vegetative phases of the Arabidopsis life cycle. Moreover, we have shown that FIE represses the expression of key regulatory genes that promote flowering (AG and LFY), embryogenesis (LEC1), and shoot formation (KNAT1). These results suggest that the FIE PcG protein participates in the formation of distinct PcG complexes that repress inappropriate gene expression at different stages of plant development. PcG complexes modulate chromatin compactness by modifying histones and thereby regulate gene expression and imprinting. The main goals of our original project were to elucidate the biological functions of PcG proteins, and to understand the molecular mechanisms used by FIE PcG complexes to repress the expression of its gene targe...
The Snf2 chromatin remodeler, DECREASE IN DNA METHYLATION 1 (DDM1) facilitates DNA methylation. I... more The Snf2 chromatin remodeler, DECREASE IN DNA METHYLATION 1 (DDM1) facilitates DNA methylation. In flowering plants, DDM1 mediates methylation in heterochromatin, which is targeted primarily by MET1 and CMT methylases and is necessary for silencing transposons and for proper development. DNA methylation mechanisms evolved throughout plant evolution, whereas the role of DDM1 in early terrestrial plants remains elusive. Here, we studied the function of DDM1 in the moss, Physcomitrium (Physcomitrella) patens, which has robust DNA methylation that suppresses transposons and is mediated by a MET1, a CMT, and a DNMT3 methylases. To elucidate the role of DDM1 in P. patens, we have generated a knockout mutant and found DNA methylation to be strongly disrupted at any of its sequence contexts. Symmetric CG and CHG sequences were affected stronger than asymmetric CHH sites. Furthermore, despite their separate targeting mechanisms, CG (MET) and CHG (CMT) methylation were similarly depleted by about 75%. CHH (DNMT3) methylation was overall reduced by about 25%, with an evident hyper-methylation activity within lowly-methylated euchromatic transposon sequences. Despite the strong hypomethylation effect, only a minute number of transposons were transcriptionally activated in Ppddm1. Finally, Ppddm1 was found to develop normally throughout the plant life cycle. These results demonstrate that DNA methylation is strongly dependent on DDM1 in a nonflowering plant; that DDM1 is required for plant-DNMT3 (CHH) methylases, though to a lower extent than for MET1 and CMT enzymes; and that distinct and separate methylation pathways (e.g. MET1-CG and CMT-CHG), can be equally regulated by the chromatin and that DDM1 plays a role in it. Finally, our data suggest that the biological significance of DDM1 in terms of transposon regulation and plant development, is species dependent.
Arabidopsis mutants that we have isolated, encode for fertilization-independent endosperm (fie), ... more Arabidopsis mutants that we have isolated, encode for fertilization-independent endosperm (fie), fertilization-independent seed2 (fis2) and medea (mea) genes, act in the female gametophyte and allow endosperm to develop without fertilization when mutated. We cloned the FIE and MEA genes and showed that they encode WD and SET domain polycomb (Pc G) proteins, respectively. Homologous proteins of FIE and MEA in other organisms are known to regulate gene transcription by modulating chromatin structure. Based on our results, we proposed a model whereby both FIE and MEA interact to suppress transcription of regulatory genes. These genes are transcribed only at proper developmental stages, as in the central cell of the female gametophyte after fertilization, thus activating endosperm development. To test our model, the following questions were addressed: What is the Composition and Function of the Polycomb Complex? Molecular, biochemical, genetic and genomic approaches were offered to iden...
Endosperm is a key nutritive tissue that supports the developing embryo or seedling, and serves a... more Endosperm is a key nutritive tissue that supports the developing embryo or seedling, and serves as a major nutritional source for human and livestock feed. In sexually-reproducing flowering plants, it generally develops after fertilization. However, autonomous endosperm (AE) formation (i.e. independent of fertilization) is also possible. Recent findings of AE loci/ genes and aberrant imprinting in native apomicts, together with a successful initiation of parthenogenesis in rice and lettuce, have enhanced our understanding of the mechanisms bridging sexual and apomictic seed formation. However, the mechanisms driving AE development are not well understood. This review presents novel aspects related to AE development in sexual and asexual plants underlying stress conditions as the primary trigger for AE. Both application of hormones to unfertilized ovules and mutations that impair epigenetic regulation lead to AE development in sexual Arabidopsis thaliana, which may point to a common ...
Background Circular RNA (circRNA) is an emerging class of RNA molecules attracting researchers du... more Background Circular RNA (circRNA) is an emerging class of RNA molecules attracting researchers due to its potential for serving as markers for diagnosis, prognosis, or therapeutic targets of cancer, cardiovascular, and autoimmune diseases. Current methods for detection of circRNA from RNA sequencing (RNA-seq) focus mostly on improving mapping quality of reads supporting the back-splicing junction (BSJ) of a circRNA to eliminate false positives (FPs). We show that mapping information alone often cannot predict if a BSJ-supporting read is derived from a true circRNA or not, thus increasing the rate of FP circRNAs. Results We have developed Circall, a novel circRNA detection method from RNA-seq. Circall controls the FPs using a robust multidimensional local false discovery rate method based on the length and expression of circRNAs. It is computationally highly efficient by using a quasi-mapping algorithm for fast and accurate RNA read alignments. We applied Circall on two simulated dat...
A fundamental problem in biology is to understand how fertilization initiates reproductive develo... more A fundamental problem in biology is to understand how fertilization initiates reproductive development. During plant reproduction, one sperm cell fuses with the egg to form an embryo, whereas a second sperm cell fuses with the adjacent central cell nucleus to form the endosperm tissue that supports embryo and/or seedling development. To understand the mechanisms that initiate reproduction, we have isolated mutants of Arabidopsis that allow for replication of the central cell and subsequent endosperm development without fertilization. In this project we have cloned the MEA gene and showed that it encode a SET- domain polycomb protein. Such proteins are known to form chromatin-protein complexes that repress homeotic gene transcription and influence cell proliferation from Drosophylla to mammals. We propose a model whereby MEA and an additional polycomb protein we have cloned, FIE , function to suppress a critical aspect of early plant reproduction and endosperm development, until fert...
Circall is a novel method for fast and accurate discovery of circular RNAs from paired-end RNA-se... more Circall is a novel method for fast and accurate discovery of circular RNAs from paired-end RNA-sequencing data. The method controls false positives by two-dimensional local false discovery method and employs quasi-mapping for fast and accurate alignments.
Proceedings of the National Academy of Sciences, 2020
Significance While DNA methylation in plants occurs at CG, CHG, and CHH sequence contexts, their ... more Significance While DNA methylation in plants occurs at CG, CHG, and CHH sequence contexts, their comparative roles in genome regulation remain elusive. Here, we utilized Physcomitrella patens as a plant model suitable to study this question. By profiling transcriptomes in a series of context-specific methylation mutants, we found redundancy between asymmetric (CHH) methylation and symmetric methylation in silencing transposons, and that CHH methylation regulates the expression of CG/CHG-depleted transposons. Specific elimination of CG methylation had a significantly smaller effect on transcription than elimination of non-CG methylation. Additionally, we found CHG methylation as a stronger silencer than CG methylation. These results disentangle the transcriptional roles of the individual methylation contexts and elucidate the crucial role of non-CG methylation in genome regulation.
DNA methylation in plants occurs in CG, CHG, and CHH sites. While depletion of CG methylation in ... more DNA methylation in plants occurs in CG, CHG, and CHH sites. While depletion of CG methylation in transposons is associated with ample transcriptional activation, it was mainly studied in species with limited non-CG methylation that is linked to CG methylation. Here we profiled transcription in the moss plant, Physcomitrella patens, that has robust non-CG methylation with similar symmetrical CG and CHG methylation levels. Separated contextual methylation mechanisms in Physcomitrella patens enabled generation of numerous context-specific hypomethylated mutants. Our transcriptome data show that specific elimination of CG methylation is fully complemented by non-CG methylation. Conversely, exclusive removal of non-CG methylation massively dysregulated genes and transposons. Moreover, CHG methylation silenced transposons stronger than CG methylation. Lastly, we found non-CG methylation as crucial for silencing CG-depleted transposons. These results demonstrate the potency of non-CG methy...
The Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) polycomb group (PcG) protein, a WD40 ho... more The Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) polycomb group (PcG) protein, a WD40 homologue of Drosophila extra sex comb (ESC), regulates endosperm and embryo development and represses flowering during embryo and seedling development. As fie alleles are not transmitted maternally, homozygous mutant plants cannot be obtained. To study FIE function during the entire plant life cycle, we used Arabidopsis FIE co-suppressed plants. Low FIE level in these plants produced dramatic morphological aberrations, including loss of apical dominance, curled leaves, early flowering and homeotic conversion of leaves, flower organs and ovules into carpel-like structures. These morphological aberrations are similar to those exhibited by plants overexpressing AGAMOUS (AG) or CURLY LEAF (clf) mutants. Furthermore, the aberrant leaf morphology of FIE-silenced and clf plants correlates with de-repression of the class I KNOTTED-like homeobox (KNOX) genes including KNOTTED-like from Arabidopsis thaliana 2 (KNAT2) and SHOOTMERISTEMLESS (STM), whereas BREVIPEDICELLUS (BP) was upregulated in FIE-silenced plants, but not in the clf mutant. Thus, FIE is essential for the control of shoot and leaf development. Yeast two-hybrid and pull-down assays demonstrate that FIE interacts with CLF. Collectively, the morphological characteristics, together with the molecular and biochemical data presented in this work, strongly suggest that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Our findings demonstrate that the versatility of the plant FIE function, which is derived from association with different SET (SU (VAR)3-9, E (Z), Trithorax) domain PcG proteins, results in differential regulation of gene expression throughout the plant life cycle.
Plants possess a single gene for the structurally related HETEROCHROMATIN PROTEIN1 (HP1), termed ... more Plants possess a single gene for the structurally related HETEROCHROMATIN PROTEIN1 (HP1), termed LIKE-HP1 (LHP1). We investigated the subnuclear localization, binding properties, and dynamics of LHP1 proteins in Arabidopsis thaliana cells. Transient expression assays showed that tomato (Solanum lycopersicum) LHP1 fused to green fluorescent protein (GFP; Sl LHP1-GFP) and Arabidopsis LHP1 (At LHP1-GFP) localized to heterochromatic chromocenters and showed punctuated distribution within the nucleus; tomato but not Arabidopsis LHP1 was also localized within the nucleolus. Mutations of aromatic cage residues that recognize methyl K9 of histone H3 abolished their punctuated distribution and localization to chromocenters. Sl LHP1-GFP plants displayed cell type–dependent subnuclear localization. The diverse localization pattern of tomato LHP1 did not require the chromo shadow domain (CSD), whereas the chromodomain alone was insufficient for localization to chromocenters; a nucleolar localiz...
The high-concentration C0,-requiring mutant N5 of Synechococcus sp. PCC 7942 was obtained by the ... more The high-concentration C0,-requiring mutant N5 of Synechococcus sp. PCC 7942 was obtained by the insertion of a kanamycinresistant gene at the fcoRl site, 12.4 kb upstream of rbc. l h e mutant is unable to accumulate inorganic carbon internally and exhibits very low apparent photosynthetic affinity for inorganic carbon but a photosynthetic VmaX similar to that of the wild type. Sequence and northern analyses showed that the insertion inactivated a gene highly homologous to ndhl, encoding subunit II of NADH dehydrogenase in Synechocystis sp. PCC 6803 (T. Ogawa [1991] Proc Natl Acad Sci USA 88: 4275-4279). When the mutant and the wild-type cells were exposed to 5% COz in air, their photosynthetic electron transfer capabilities, as revealed by fluorescence and thermoluminescence measurements, were similar. On the other hand, a significant decrease in variable fluorescence was observed when the mutant (but not the wild-type) cells were exposed tolow COz under continuous light. l h e same treatment also resulted in a shift (from 38-27'C) in the temperature at which the maximal thermoluminescence emission signal was obtained in the mutant but not in the wild type. These results may indicate that subunit II of NADH dehydrogenase is essential for the fundional operation of the photosynthetic electron transport in Synechococcus under low but not high levels of COz. We suggest that the inability to accumulate inorganic carbon under air conditions stems from disrupture of electron transport in this mutant.
Protein function is often mediated via formation of stable or transient complexes. Here we report... more Protein function is often mediated via formation of stable or transient complexes. Here we report the determination of protein-protein interactions in plants using bimolecular fluorescence complementation (BiFC). The yellow fluorescent protein (YFP) was split into two non-overlapping N-terminal (YN) and C-terminal (YC) fragments. Each fragment was cloned in-frame to a gene of interest, enabling expression of fusion proteins. To demonstrate the feasibility of BiFC in plants, two pairs of interacting proteins were utilized: (i) the alpha and beta subunits of the Arabidopsis protein farnesyltransferase (PFT), and (ii) the polycomb proteins, FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) and MEDEA (MEA). Members of each protein pair were transiently co-expressed in leaf epidermal cells of Nicotiana benthamiana or Arabidopsis. Reconstitution of a fluorescing YFP chromophore occurred only when the inquest proteins interacted. No fluorescence was detected following co-expression of free non-fused YN and YC or non-interacting protein pairs. Yellow fluorescence was detected in the cytoplasm of cells that expressed PFT alpha and beta subunits, or in nuclei and cytoplasm of cells that expressed FIE and MEA. In vivo measurements of fluorescence spectra emitted from reconstituted YFPs were identical to that of a non-split YFP, confirming reconstitution of the chromophore. Expression of the inquest proteins was verified by immunoblot analysis using monoclonal antibodies directed against tags within the hybrid proteins. In addition, protein interactions were confirmed by immunoprecipitations. These results demonstrate that plant BiFC is a simple, reliable and relatively fast method for determining protein-protein interactions in plants.
Following the complete genome sequencing of different plant species such as Arabidopsis (Arabidop... more Following the complete genome sequencing of different plant species such as Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and Physcomitrella (Physcomitrella patens), as well as advances toward deciphering entire proteomes, the need for a reliable way to identify protein-protein interactions is becoming a major task for the future. Bimolecular fluorescent complementation (BiFC) is a noninvasive fluorescentbased technique that allows detection of protein-protein interactions in living cells, and furthermore can be used to determine subcellular localization of the interacting proteins, and if it changes over time, without requiring addition of external agents. BiFC is based upon reconstitution of split nonfluorescent GFP variants, primarily yellow fluorescent protein (YFP), to form a fluorescent fluorophore (Ghosh et al., 2000; Hu et al., 2002). The technique has become increasingly popular due to its simplicity, ease of use, and the capability to carry out experiments with ...
The original version of this Article contained an error in Fig. 5, in which the evolutionary orig... more The original version of this Article contained an error in Fig. 5, in which the evolutionary origin of DRM2 was incorrectly placed prior to the divergence between gymnosperms and angiosperms. The correct evolutionary origin of DRM2 should be in angiosperms. The correct version of Fig. 5 is shown here as Fig. 1, which replaces the incorrect version which is shown here as Fig. 2. In addition, in the "Percent methylation change" section of the Methods, Equation 1 was incorrectly given as: WT mCHH À cmt mCHHWT mCHH 100 if WT mCHH > cmt mCHH The correct form of Equation 1 is as follows: WT mCHH À cmt mCHH WT mCHH 100 if WT mCHH > cmt mCHH ð1Þ This has been corrected in both the PDF and HTML versions of the Article.
Sustainable diets: linking nutrition and food systems, 2019
The evolutionary history of the concepts of food security (FS) and sustainability have run in par... more The evolutionary history of the concepts of food security (FS) and sustainability have run in parallel for many years. After the food crisis of 2008, stability was added to definition of FS as a short-term time dimension to express the ability to withstand shocks to the food system caused by natural or man-made disasters. We have proposed that sustainability be added as a fifth long-term time dimension, thus bringing together FS and sustainability. In 2015, the United Nations described the seventeen sustainable development goals. We believe that FS involves all the goals to a greater or lesser extent. The challenge ahead is to build and integrate FS on the sustainability agenda and vice versa. The final common pathway for all these efforts is for countries to develop their most appropriate sustainable food systems. As a practical exercise towards this aim, we have reviewed what eight different countries (United States, Brazil, France, Greece, Spain, the Netherlands, United Kingdom, ...
To properly regulate the genome, cytosine methylation is established by animal DNA methyltransfer... more To properly regulate the genome, cytosine methylation is established by animal DNA methyltransferase 3 s (DNMT3s). While altered DNMT3 homologs, Domains rearranged methyltransferases (DRMs), have been shown to establish methylation via the RNA directed DNA methylation (RdDM) pathway, the role of true-plant DNMT3 orthologs remains elusive. Here, we profile de novo (RPS transgene) and genomic methylation in the basal plant, Physcomitrella patens, mutated in each of its PpDNMTs. We show that PpDNMT3b mediates CG and CHH de novo methylation, independently of PpDRMs. Complementary de novo CHG methylation is specifically mediated by the CHROMOMETHYLASE, PpCMT. Intragenomically, PpDNMT3b functions preferentially within heterochromatin and is affected by PpCMT. In comparison, PpDRMs target active-euchromatic transposons. Overall, our data resolve how DNA methylation in plants can be established in heterochromatin independently of RdDM; suggest that DRMs have emerged to target euchromatin; and link DNMT3 loss in angiosperms to the initiation of heterochromatic CHH methylation by CMT2.
Posttranslational modification (PTM) of histone tails plays a critical role in the dynamic of chr... more Posttranslational modification (PTM) of histone tails plays a critical role in the dynamic of chromatin and chromosomes. However, emerging evidence suggests that individual histone modifications do not reliably predict a single functional output [Sims and Reinberg (Nat Rev Mol Cell Biol 9:815–820, 2008)]. In plants, the cell cycle-dependent phosphorylation of histone H3 has been described best; it is hyperphosphorylated at serines 10/28 and at threonines 3/11/32 during both mitosis and meiosis in patterns that are specifically coordinated in both space and time. Although this posttranslational modification is highly conserved, data show that the chromosomal distribution of individual modifications can differ between groups of eukaryotes. We describe the function of plant Aurora and Haspin kinases which have the capacity to phosphorylate H3 and discuss the cross talk between phosphorylation and other PTMs.
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Papers by Nir Ohad