@article{lee_lee_park_shin_frost_hsieh_shin_fischer_hsieh_choi_2023, title={Distinct regulatory pathways contribute to dynamic CHH methylation patterns in transposable elements throughout Arabidopsis embryogenesis}, volume={14}, ISSN={["1664-462X"]}, DOI={10.3389/fpls.2023.1204279}, abstractNote={CHH methylation (mCHH) increases gradually during embryogenesis across dicotyledonous plants, indicating conserved mechanisms of targeting and conferral. Although it is suggested that methylation increase during embryogenesis enhances transposable element silencing, the detailed epigenetic pathways underlying this process remain unclear. In Arabidopsis, mCHH is regulated by both small RNA-dependent DNA methylation (RdDM) and RNA-independent Chromomethylase 2 (CMT2) pathways. Here, we conducted DNA methylome profiling at five stages of Arabidopsis embryogenesis, and classified mCHH regions into groups based on their dependency on different methylation pathways. Our analysis revealed that the gradual increase in mCHH in embryos coincided with the expansion of small RNA expression and regional mCHH spreading to nearby sites at numerous loci. We identified distinct methylation dynamics in different groups of mCHH targets, which vary according to transposon length, location, and cytosine frequency. Finally, we highlight the characteristics of transposable element loci that are targeted by different mCHH machinery, showing that short, heterochromatic TEs with lower mCHG levels are enriched in loci that switch from CMT2 regulation in leaves, to RdDM regulation during embryogenesis. Our findings highlight the interplay between the length, location, and cytosine frequency of transposons and the mCHH machinery in modulating mCHH dynamics during embryogenesis.}, journal={FRONTIERS IN PLANT SCIENCE}, author={Lee, Jaehoon and Lee, Seunga and Park, Kyunghyuk and Shin, Sang-Yoon and Frost, Jennifer M. and Hsieh, Ping-Hung and Shin, Chanseok and Fischer, Robert L. and Hsieh, Tzung-Fu and Choi, Yeonhee}, year={2023}, month={Jun} }
 @article{hsieh_frost_choi_hsieh_zilberman_fischer_2023, title={Embryo-specific epigenetic mechanisms reconstitute the CHH methylation landscape during Arabidopsis embryogenesis}, url={https://doi.org/10.1101/2023.04.06.535361}, DOI={10.1101/2023.04.06.535361}, abstractNote={AbstractThe modification of flowering plant DNA by CHH methylation acts primarily to silence transposable elements, of which many active copies are present inArabidopsis thaliana. During embryogenesis, the CHH methylation landscape is dramatically reprogrammed, resulting in exceedingly high levels of this modification upon mature embryo formation. The mechanisms constituting the remodeling process, and its function in embryos, are unclear. Here, we isolate embryos from Arabidopsis plants harboring mutations for key components of the pathways that confer CHH methylation, namely RNA-directed DNA methylation (RdDM) and the Chromomethylase 2 (CMT2) pathways. We reveal that embryos are more methylated than leaves at shared CMT2 and RdDM targeting loci, accounting for most embryonic CHH hypermethylation. While the majority of embryo CHH methylated loci overlap with those in somatic tissues, a subset of conventional pericentric CMT2-methylated loci are instead targeted by RdDM in embryos. These loci, termed ‘embRdDM’ exhibit intermediate H3K9me2 levels, associated with increased chromatin accessibility. Strikingly, more than 50% of the embRdDM loci in pollen vegetative (nurse) cells andddm1mutant somatic tissues are also targeted by RdDM, and these tissues were also reported to exhibit increased chromatin accessibility in pericentric heterochromatin. Furthermore, the root columella stem cell niche also displays CHH hypermethylation and an enriched presence of small RNAs at embRdDM loci. Finally, we observe a significant overlap of CHH hypermethylated loci with endosperm DEMETER targeting sites, suggesting that non-cell autonomous communication within the seed may contribute to the epigenetic landscape of the embryo. However, similar overlap with vegetative cell DEMETER targets indicates that the chromatin landscape that allows DEMETER access is mirrored in developing embryos, permitting CHH methylation catalysis at the same loci. Our findings demonstrate that both conserved and embryo-specific epigenetic mechanisms reshape CHH methylation profiles in the dynamic chromatin environment of embryogenesis.}, author={Hsieh, Ping-Hung and Frost, Jennifer M. and Choi, Yeonhee and Hsieh, Tzung-Fu and Zilberman, Daniel and Fischer, Robert L}, year={2023}, month={Apr} }
 @article{frost_lee_hsieh_lin_min_bauer_runkel_cho_hsieh_fischer_et al._2023, title={H2A.X promotes endosperm-specific DNA methylation in <i>Arabidopsis thaliana</i>}, volume={23}, ISSN={["1471-2229"]}, url={http://dx.doi.org/10.1186/s12870-023-04596-y}, DOI={10.1186/s12870-023-04596-y}, abstractNote={Abstract
                Background
                H2A.X is an H2A variant histone in eukaryotes, unique for its ability to respond to DNA damage, initiating the DNA repair pathway. H2A.X replacement within the histone octamer is mediated by the FAcilitates Chromatin Transactions (FACT) complex, a key chromatin remodeler. FACT is required for DEMETER (DME)-mediated DNA demethylation at certain loci in Arabidopsis thaliana female gametophytes during reproduction. Here, we sought to investigate whether H2A.X is involved in DME- and FACT-mediated DNA demethylation during reproduction.
              
                Results
                H2A.X is encoded by two genes in Arabidopsis genome, HTA3 and HTA5. We generated h2a.x double mutants, which displayed a normal growth profile, whereby flowering time, seed development, and root tip organization, S-phase progression and proliferation were all normal. However, h2a.x mutants were more sensitive to genotoxic stress, consistent with previous reports. H2A.X fused to Green Fluorescent Protein (GFP) under the H2A.X promoter was highly expressed especially in newly developing Arabidopsis tissues, including in male and female gametophytes, where DME is also expressed. We examined DNA methylation in h2a.x developing seeds and seedlings using whole genome bisulfite sequencing, and found that CG DNA methylation is decreased genome-wide in h2a.x mutant endosperm. Hypomethylation was most striking in transposon bodies, and occurred on both parental alleles in the developing endosperm, but not the embryo or seedling. h2a.x-mediated hypomethylated sites overlapped DME targets, but also included other loci, predominately located in heterochromatic transposons and intergenic DNA.
              
                Conclusions
                Our genome-wide methylation analyses suggest that H2A.X could function in preventing access of the DME demethylase to non-canonical sites. Overall, our data suggest that H2A.X is required to maintain DNA methylation homeostasis in the unique chromatin environment of the Arabidopsis endosperm.
              }, number={1}, journal={BMC PLANT BIOLOGY}, author={Frost, Jennifer M. and Lee, Jaehoon and Hsieh, Ping-Hung and Lin, Samuel J. H. and Min, Yunsook and Bauer, Matthew and Runkel, Anne M. and Cho, Hyung-Taeg and Hsieh, Tzung-Fu and Fischer, Robert L. and et al.}, year={2023}, month={Nov} }
 @article{frost_lee_hsieh_lin_min_bauer_runkel_cho_hsieh_choi_et al._2023, title={H2A.Xmutants exhibit enhanced DNA demethylation inArabidopsis thaliana}, url={https://doi.org/10.1101/2023.01.08.523178}, DOI={10.1101/2023.01.08.523178}, abstractNote={AbstractH2A.Xis an H2A variant histone in eukaryotes, unique for its ability to respond to DNA damage, initiating the DNA repair pathway.H2A.Xreplacement within the histone octamer is mediated by the FAcilitates Chromatin Transactions (FACT) complex, a key chromatin remodeler. FACT is required for DEMETER (DME)-mediated DNA demethylation at certain loci inArabidopsis thalianafemale gametophytes during reproduction, though it is not known how FACT targets DME sites. Here, we investigated whether H2AX is involved in DME- and FACT-mediated DNA demethylation during Arabidopsis reproduction. We show thath2a.xmutants are more sensitive to genotoxic stress, consistent with previous reports.H2A.Xfused to theGreen Fluorescent Protein (GFP)gene under theH2A.Xpromoter was highly expressed in newly developingArabidopsistissues, including in male and female gametophytes. We examined DNA methylation inh2a.xdeveloping seeds using whole genome bisulfite sequencing, and found that CG DNA methylation in the developing endosperm, but not the embryo, is decreased genome-wide inh2a.xmutants, predominately in transposons and intergenic DNA. Hypomethylated sites overlapped 62 % with canonical DME loci. These data indicate that H2A.X is not required for DME function, but is important for DNA methylation homeostasis in the unique chromatin environment ofArabidopsisendosperm.}, author={Frost, Jennifer M. and Lee, Jaehoon and Hsieh, Ping-Hung and Lin, Samuel J. H. and Min, Yunsook and Bauer, Matthew and Runkel, Anne M. and Cho, Hyung-Taeg and Hsieh, Tzung-Fu and Choi, Yeonhee and et al.}, year={2023}, month={Jan} }
 @article{huang_wang_choong_huang_chen_hsieh_lin_2023, title={How ambient temperature affects the heading date of foxtail millet (Setaria italica)}, volume={14}, url={http://dx.doi.org/10.3389/fpls.2023.1147756}, DOI={10.3389/fpls.2023.1147756}, abstractNote={Foxtail millet (Setaria italica), a short-day plant, is one of the important crops for food security encountering climate change, particularly in regions where it is a staple food. Under the short-day condition in Taiwan, the heading dates (HDs) of foxtail millet accessions varied by genotypes and ambient temperature (AT). The allelic polymorphisms in flowering time (FT)–related genes were associated with HD variations. AT, in the range of 13°C–30°C that was based on field studies at three different latitudes in Taiwan and observations in the phytotron at four different AT regimes, was positively correlated with growth rate, and high AT promoted HD. To elucidate the molecular mechanism of foxtail millet HD, the expression of 14 key FT-related genes in four accessions at different ATs was assessed. We found that the expression levels of SiPRR95, SiPRR1, SiPRR59, SiGhd7-2, SiPHYB, and SiGhd7 were negatively correlated with AT, whereas the expression levels of SiEhd1, SiFT11, and SiCO4 were positively correlated with AT. Furthermore, the expression levels of SiGhd7-2, SiEhd1, SiFT, and SiFT11 were significantly associated with HD. A coexpression regulatory network was identified that shown genes involved in the circadian clock, light and temperature signaling, and regulation of flowering, but not those involved in photoperiod pathway, interacted and were influenced by AT. The results reveal how gene × temperature and gene × gene interactions affect the HD in foxtail millet and could serve as a foundation for breeding foxtail millet cultivars for shift production to increase yield in response to global warming.}, journal={Frontiers in Plant Science}, publisher={Frontiers Media SA}, author={Huang, Ya-Chen and Wang, Yu-tang and Choong, Yee-ching and Huang, Hsin-ya and Chen, Yu-ru and Hsieh, Tzung-Fu and Lin, Yann-rong}, year={2023}, month={Mar} }
 @article{shin_park_park_choi_yu_koh_kim_soh_waminal_belandres_et al._2022, title={Admixture of divergent genomes facilitates hybridization across species in the family Brassicaceae}, volume={4}, ISSN={["1469-8137"]}, DOI={10.1111/nph.18155}, abstractNote={Summary


Hybridization and polyploidization are pivotal to plant evolution. Genetic crosses between distantly related species are rare in nature due to reproductive barriers but how such hurdles can be overcome is largely unknown. Here we report the hybrid genome structure of xBrassicoraphanus, a synthetic allotetraploid of Brassica rapa and Raphanus sativus.

We performed cytogenetic analysis and de novo genome assembly to examine chromosome behaviors and genome integrity in the hybrid. Transcriptome analysis was conducted to investigate expression of duplicated genes in conjunction with epigenome analysis to address whether genome admixture entails epigenetic reconfiguration.

Allotetraploid xBrassicoraphanus retains both parental chromosomes without genome rearrangement. Meiotic synapsis formation and chromosome exchange are avoided between nonhomologous progenitor chromosomes. Reconfiguration of transcription network occurs, and less divergent cis‐elements of duplicated genes are associated with convergent expression. Genome‐wide DNA methylation asymmetry between progenitors is largely maintained but, notably, B. rapa‐originated transposable elements are transcriptionally silenced in xBrassicoraphanus through gain of DNA methylation.

Our results demonstrate that hybrid genome stabilization and transcription compatibility necessitate epigenome landscape adjustment and rewiring of cis–trans interactions. Overall, this study suggests that a certain extent of genome divergence facilitates hybridization across species, which may explain the great diversification and expansion of angiosperms during evolution.

}, journal={NEW PHYTOLOGIST}, author={Shin, Hosub and Park, Jeong Eun and Park, Hye Rang and Choi, Woo Lee and Yu, Seung Hwa and Koh, Wonjun and Kim, Seungill and Soh, Hye Yeon and Waminal, Nomar Espinosa and Belandres, Hadassah Roa and et al.}, year={2022}, month={Apr} }
 @article{hsieh_han_hung_zhang_bartels_rea_yang_park_zhang_fsicher_et al._2022, title={Loss of Linker Histone H1 in the Maternal Genome Influences DEMETER-Mediated Demethylation and Affects the Endosperm DNA Methylation Landscape}, url={https://doi.org/10.1101/2022.10.17.512625}, DOI={10.1101/2022.10.17.512625}, abstractNote={AbstractTheArabidopsisDEMETER (DME) DNA glycosylase demethylates the central cell genome prior to fertilization. This epigenetic reconfiguration of the female gamete companion cell establishes gene imprinting in the endosperm and is essential for seed viability. DME demethylates small and genic-flanking transposons as well as intergenic and heterochromatin sequences, but how DME is recruited to these target loci remains unknown. H1.2 was identified as a DME-interacting protein in a yeast two-hybrid screen, and maternal genome H1 loss affects DNA methylation and expression of selected imprinted genes in the endosperm. Yet, the extent to which how H1 influences DME demethylation and gene imprinting in theArabidopsisendosperm has not been investigated. Here, we showed that unlike in the vegetative cell, both canonical histone H1 variants are present in the central cell. Our endosperm methylome analysis revealed that without the maternal linker histones, DME-mediated demethylation is facilitated, particularly in the heterochromatin regions, indicating that H1-containing nucleosomes are barriers for DME demethylation. Loss of H1 in the maternal genome has a very limited effect on gene transcription or gene imprinting regulation in the endosperm; however, it variably influences euchromatin TE methylation and causes a slight hypermethylation and a reduced expression in selected imprinted genes. We conclude that loss of maternal H1 indirectly influences DME-mediated demethylation and endosperm DNA methylation landscape but does not appear to affect endosperm gene transcription and overall imprinting regulation.}, author={Hsieh, Tzung-Fu and Han, Qiang and Hung, Yu-Hung and Zhang, Changqing and Bartels, Arthur and Rea, Matthew and Yang, Hanwen and Park, Christine and Zhang, Xiang-Qian and Fsicher, Robert L. and et al.}, year={2022}, month={Oct} }
 @article{han_hung_zhang_bartels_rea_yang_park_zhang_fischer_xiao_et al._2022, title={Loss of linker histone H1 in the maternal genome influences DEMETER-mediated demethylation and affects the endosperm DNA methylation landscape}, volume={13}, ISSN={["1664-462X"]}, DOI={10.3389/fpls.2022.1070397}, abstractNote={The Arabidopsis DEMETER (DME) DNA glycosylase demethylates the central cell genome prior to fertilization. This epigenetic reconfiguration of the female gamete companion cell establishes gene imprinting in the endosperm and is essential for seed viability. DME demethylates small and genic-flanking transposons as well as intergenic and heterochromatin sequences, but how DME is recruited to these loci remains unknown. H1.2 was identified as a DME-interacting protein in a yeast two-hybrid screen, and maternal genome H1 loss affects DNA methylation and expression of selected imprinted genes in the endosperm. Yet, the extent to which H1 influences DME demethylation and gene imprinting in the Arabidopsis endosperm has not been investigated. Here, we showed that without the maternal linker histones, DME-mediated demethylation is facilitated, particularly in the heterochromatin regions, indicating that H1-bound heterochromatins are barriers for DME demethylation. Loss of H1 in the maternal genome has a very limited effect on gene transcription or gene imprinting regulation in the endosperm; however, it variably influences euchromatin TE methylation and causes a slight hypermethylation and a reduced expression in selected imprinted genes. We conclude that loss of maternal H1 indirectly influences DME-mediated demethylation and endosperm DNA methylation landscape but does not appear to affect endosperm gene transcription and overall imprinting regulation.}, journal={FRONTIERS IN PLANT SCIENCE}, author={Han, Qiang and Hung, Yu-Hung and Zhang, Changqing and Bartels, Arthur and Rea, Matthew and Yang, Hanwen and Park, Christine and Zhang, Xiang-Qian and Fischer, Robert L. L. and Xiao, Wenyan and et al.}, year={2022}, month={Dec} }
 @article{tan_wang_schneider_li_souza_tang_grimm_hsieh_wang_li_et al._2021, title={Comparative Phylogenomic Analysis Reveals Evolutionary Genomic Changes and Novel Toxin Families in Endophytic Liberibacter Pathogens}, volume={9}, ISSN={["2165-0497"]}, DOI={10.1128/Spectrum.00509-21}, abstractNote={
            Liberibacter
            pathogens are associated with several severe crop diseases, including citrus Huanglongbing, the most destructive disease to the citrus industry. Currently, no effective cure or treatments are available, and no resistant citrus variety has been found.
          }, number={2}, journal={MICROBIOLOGY SPECTRUM}, author={Tan, Yongjun and Wang, Cindy and Schneider, Theresa and Li, Huan and Souza, Robson Francisco and Tang, Xueming and Grimm, Kylie D. Swisher and Hsieh, Tzung-Fu and Wang, Xu and Li, Xu and et al.}, year={2021}, month={Oct} }
 @article{tan_wang_schneider_li_souza_tang_hsieh_wang_li_zhang_2021, title={Comparative phylogenomic analysis reveals evolutionary genomic changes and novel toxin families in endophytic Liberibacter pathogens}, volume={6}, url={https://doi.org/10.1101/2021.06.02.446850}, DOI={10.1101/2021.06.02.446850}, abstractNote={AbstractLiberibacterpathogens are the causative agents of several severe crop diseases worldwide, including citrus Huanglongbing and potato Zebra Chip. These bacteria are endophytic and non-culturable, which makes experimental approaches challenging and highlights the need for bioinformatic analysis in advancing our understanding aboutLiberibacterpathogenesis. Here, we performed an in-depth comparative phylogenomic analysis of theLiberibacterpathogens and their free-living, nonpathogenic, ancestral species, aiming to identify the major genomic changes and determinants associated with their evolutionary transitions in living habitats and pathogenicity. We found that prophage loci represent the most variable regions amongLiberibactergenomes. Using gene neighborhood analysis and phylogenetic classification, we systematically recovered, annotated, and classified all prophage loci into four types, including one previously unrecognized group. We showed that these prophages originated through independent gene transfers at different evolutionary stages ofLiberibacterand only the SC-type prophage was associated with the emergence of the pathogens. Using ortholog clustering, we vigorously identified two additional sets of genomic genes, which were either lost or gained in the ancestor of the pathogens. Consistent with the habitat change, the lost genes were enriched for biosynthesis of cellular building blocks. Importantly, among the gained genes, we uncovered several previously unrecognized toxins, including a novel class of polymorphic toxins, a YdjM phospholipase toxin, and a secreted EEP protein. Our results substantially extend the knowledge on the evolutionary events and potential determinants leading to the emergence of endophytic, pathogenicLiberibacterspecies and will facilitate the design of functional experiments and the development of new detection and blockage methods of these pathogens.ImportanceLiberibacterpathogens are associated with several severe crop diseases, including citrus Huanglongbing, the most destructive disease to the citrus industry. Currently, no effective cure or treatments are available, and no resistant citrus variety has been found. The fact that these obligate endophytic pathogens are not culturable has made it extremely challenging to experimentally uncover from the whole genome the genes/proteins important toLiberibacterpathogenesis. Further, earlier bioinformatics studies failed to identify the key genomic determinants, such as toxins and effector proteins, that underlie the pathogenicity of the bacteria. In this study, an in-depth comparative genomic analysis ofLiberibacterpathogens together with their ancestral non-pathogenic species identified the prophage loci and several novel toxins that are evolutionarily associated with the emergence of the pathogens. These results shed new lights on the disease mechanism ofLiberibacterpathogens and will facilitate the development of new detection and blockage methods targeting the toxins.}, publisher={Cold Spring Harbor Laboratory}, author={Tan, Yongjun and Wang, Cindy and Schneider, Theresa and Li, Huan and Souza, Robson Francisco and Tang, Xueming and Hsieh, Tzung-Fu and Wang, Xu and Li, Xu and Zhang, Dapeng}, year={2021}, month={Jun} }
 @article{li_cui_zhang_hsieh_2021, title={Epigenetic remodeling by DNA glycosylases during rice reproduction}, volume={14}, ISSN={["1752-9867"]}, url={https://doi.org/10.1016/j.molp.2021.07.009}, DOI={10.1016/j.molp.2021.07.009}, abstractNote={Cytosine methylation is a covalent modification of DNA that regulates important processes in eukaryotic genomes, including gene transcription, transposon silencing, and genomic imprinting (Law and Jacobsen, 2010Law J.A. Jacobsen S.E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals.Nat. Rev. Genet. 2010; 11: 204-220Crossref PubMed Scopus (2462) Google Scholar). DNA methylation patterns are faithfully duplicated upon cell division to ensure genome integrity and to maintain lineage-specific cell fate. However, DNA methylation also needs to be dynamically reprogramed or reconfigured during development to allow establishment of new cellular identity and transcriptional state, which plays a prominent role in animal development and reproduction, and is increasingly being appreciated for reproductive success in flowering plants (Walker et al., 2018Walker J. Gao H. Zhang J. Aldridge B. Vickers M. Higgins J.D. Feng X. Sexual-lineage-specific DNA methylation regulates meiosis in Arabidopsis.Nat. Genet. 2018; 50: 130-137https://doi.org/10.1038/s41588-017-0008-5Crossref PubMed Scopus (99) Google Scholar; Ono and Kinoshita, 2021Ono A. Kinoshita T. Epigenetics and plant reproduction: multiple steps for responsibly handling succession.Curr. Opin. Plant Biol. 2021; 61: 102032https://doi.org/10.1016/j.pbi.2021.102032Crossref PubMed Scopus (6) Google Scholar). In mammals, germline cells and zygotes undergo genome-wide methylation resets to obtain cellular pluripotency. In flowering plants, multiple waves of localized smaller-scale epigenetic dynamics and remodeling have also been documented during reproduction (Gehring, 2019Gehring M. Epigenetic dynamics during flowering plant reproduction: evidence for reprogramming?.New Phytol. 2019; 224: 91-96https://doi.org/10.1111/nph.15856Crossref PubMed Scopus (41) Google Scholar). For example, before fertilization localized demethylation in vegetative and central cells (VC and CC, companion cells of sperm and egg) was found to be essential for seed viability (Gehring, 2019Gehring M. Epigenetic dynamics during flowering plant reproduction: evidence for reprogramming?.New Phytol. 2019; 224: 91-96https://doi.org/10.1111/nph.15856Crossref PubMed Scopus (41) Google Scholar). Upon fertilization, the genomes of endosperm and embryo undergo methylation reconfiguration in Arabidopsis, soybean, and rice (Park et al., 2016Park K. Kim M.Y. Vickers M. Park J.S. Hyun Y. Okamoto T. Zilberman D. Fischer R.L. Feng X. Choi Y. et al.DNA demethylation is initiated in the central cells of Arabidopsis and rice.Proc. Natl. Acad. Sci. U S A. 2016; 113: 15138-15143https://doi.org/10.1073/pnas.1619047114Crossref PubMed Scopus (104) Google Scholar; Kim et al., 2019Kim M.Y. Ono A. Scholten S. Kinoshita T. Zilberman D. Okamoto T. Fischer R.L. DNA demethylation by ROS1a in rice vegetative cells promotes methylation in sperm.Proc. Natl. Acad. Sci. U S A. 2019; 116: 9652-9657https://doi.org/10.1073/pnas.1821435116Crossref PubMed Scopus (27) Google Scholar; Ono and Kinoshita, 2021Ono A. Kinoshita T. Epigenetics and plant reproduction: multiple steps for responsibly handling succession.Curr. Opin. Plant Biol. 2021; 61: 102032https://doi.org/10.1016/j.pbi.2021.102032Crossref PubMed Scopus (6) Google Scholar). Although DNA glycosylases and the de novo methylation pathways are implicated in some of these processes, many of their biological functions remain to be fully elucidated.Epigenome remodeling in gamete companion cells of Arabidopsis and riceIn Arabidopsis, the genome of the CC undergoes extensive demethylation at thousands of loci directed by the DEMETER (DME) glycosylase to establish parent-of-origin-specific expression of many imprinted genes in endosperm. DME also demethylates the genomes of VCs and the lack of DME activity in VCs resulted in CHH hypomethylation in corresponding loci in sperm (Ibarra et al., 2012Ibarra C.A. Feng X. Schoft V.K. Hsieh T.F. Uzawa R. Rodrigues J.A. Zemach A. Chumak N. Machlicova A. Nishimura T. et al.Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes.Science. 2012; 337: 1360-1364https://doi.org/10.1126/science.1224839Crossref PubMed Scopus (335) Google Scholar). Thus, the main functions of DME during Arabidopsis reproduction are to establish gene imprinting and to reinforce transgenerational TE silencing.Although no DME ortholog was detected in monocots, the rice ROS1a (DNG702) was shown to be the functional counterpart of DME in rice cultivar Nipponbare (Ono et al., 2012Ono A. Yamaguchi K. Fukada-Tanaka S. Terada R. Mitsui T. Iida S. A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny.Plant J. 2012; 71: 564-574https://doi.org/10.1111/j.1365-313X.2012.05009.xCrossref PubMed Scopus (74) Google Scholar). Similar to the VC of Arabidopsis, the rice VC genome is also extensively hypomethylated compared with the sperm genome in a ROS1a-dependent manner (Kim et al., 2019Kim M.Y. Ono A. Scholten S. Kinoshita T. Zilberman D. Okamoto T. Fischer R.L. DNA demethylation by ROS1a in rice vegetative cells promotes methylation in sperm.Proc. Natl. Acad. Sci. U S A. 2019; 116: 9652-9657https://doi.org/10.1073/pnas.1821435116Crossref PubMed Scopus (27) Google Scholar). In ROS1a/ros1a heterozygous plants, sperm CHH is hypomethylated at the loci where CG hypomethylation occurred in VCs, indicating that ROS1a activity in VCs is required for normal sperm CHH methylation. Furthermore, there is a large overlap between VC versus sperm and endosperm versus embryo hypomethylated DMRs, suggesting ROS1a also demethylates the rice CC genome (Kim et al., 2019Kim M.Y. Ono A. Scholten S. Kinoshita T. Zilberman D. Okamoto T. Fischer R.L. DNA demethylation by ROS1a in rice vegetative cells promotes methylation in sperm.Proc. Natl. Acad. Sci. U S A. 2019; 116: 9652-9657https://doi.org/10.1073/pnas.1821435116Crossref PubMed Scopus (27) Google Scholar). Thus, gamete companion cells epigenetic remodeling by DNA glycosylases is an evolutionarily conserved phenomenon in rice and Arabidopsis; species that diverged more than 150 million years ago.Despite this conservation, many distinct features exist between rice and Arabidopsis. For example, DME’s expression is primarily restricted to the gamete companion cells of Arabidopsis, whereas ROS1a is broadly expressed throughout rice development. This suggests that the gamete companion cell function of ROS1a was delegated to DME in Arabidopsis and ROS1a might also play a role in rice gamete formation and seed development.Epigenome remodeling of gametes and zygote in riceRice has a persistent endosperm that serves as a staple food for humans and its genome contains more TEs than Arabidopsis (International Rice Genome Sequencing, 2005International Rice Genome Sequencing, PThe map-based sequence of the rice genome.Nature. 2005; 436: 793-800https://doi.org/10.1038/nature03895Crossref PubMed Scopus (2937) Google Scholar). Understanding its reproductive epigenetic mechanisms has direct relevance to food production and can complement the knowledge gained from research in Arabidopsis and other plant models. The Nipponbare reference genome encodes at least four DNA demethylases, DNG701–DNG704. Except for DNG702/ROS1a’s roles in gamete companion cells, functions of these DNA glycosylases in germline cells and zygote were not known until now. A new report published in Molecular Plants attempted to address this question by conducting a comprehensive DNA methylome study in rice gamete, zygote, and developing embryos in wild-type and dng mutants (cultivar Dongjing) (Zhou et al., 2021Zhou S. Li X. Liu Q. Zhao Y. Jiang W. Wu A. Zhou D.X. DNA demethylases remodel DNA methylation in rice gametes and zygote and are required for reproduction.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.06.006Abstract Full Text Full Text PDF Scopus (11) Google Scholar). In addition, this new study also revealed methylation dynamics among these cell types/tissues. In wild-type plants, there is a moderate difference in bulk methylation level between egg and sperm, which is likely due to differential maintenance and do novo DNA methylase activities during male and female gametogenesis, a phenomenon consistent with what was observed during male sex lineage development in Arabidopsis (Walker et al., 2018Walker J. Gao H. Zhang J. Aldridge B. Vickers M. Higgins J.D. Feng X. Sexual-lineage-specific DNA methylation regulates meiosis in Arabidopsis.Nat. Genet. 2018; 50: 130-137https://doi.org/10.1038/s41588-017-0008-5Crossref PubMed Scopus (99) Google Scholar; Long et al., 2021Long J. Walker J. She W. Aldridge B. Gao H. Deans S. Vickers M. Feng X. Nurse cell-derived small RNAs define paternal epigenetic inheritance in Arabidopsis.Science. 2021; 373https://doi.org/10.1126/science.abh0556Crossref PubMed Scopus (27) Google Scholar). Inclusion of the unicellular zygote methylome (at 6.5 h after pollination) allowed for a direct interrogation into changes in parental genomes after they fused to form the zygote. Importantly, significant differential methylations were detected between zygote versus egg or sperm that were not a simple summation of the methylomes of the gametes, suggesting that the zygote epigenome is quickly reconfigured after fertilization. As the zygote develops, the globular embryo (GE) exhibited a slightly higher CG methylation but a lower CHH methylation level compared with the zygote. In the mature embryo, CHH methylation remains low as in GE, but CHG methylation showed a more significant decrease from GE. Taken together, these observations show that there is a substantial DNA methylation remodeling and reconfiguration during rice embryo development that is distinct from what was observed in Arabidopsis and soybean (Ono and Kinoshita, 2021Ono A. Kinoshita T. Epigenetics and plant reproduction: multiple steps for responsibly handling succession.Curr. Opin. Plant Biol. 2021; 61: 102032https://doi.org/10.1016/j.pbi.2021.102032Crossref PubMed Scopus (6) Google Scholar).To understand the contributions of gamete- and zygote-expressed glycosylases (DNG701, DNG702, and DNG704) to the rice epigenetic dynamics during reproduction, the authors generated loss-of-function mutations in these genes and profiled methylomes from mutant eggs, sperm, and zygotes. Self-pollination of homozygous dng702 plants can produce gametes but the zygotes failed to initiate embryogenesis. However, hybrid F1 seeds derived from reciprocal crosses between dng702 and wild-type plants produced viable embryos with defective endosperm. These observations showed that, for zygotes to initiate embryogenesis, at least one functional copy of DNG702 is needed; whereas for normal endosperm development, DNG702 activity is required in both the CC and sperm (Figure 1). This is intriguing as most known gametophytic-related endosperm failures are caused by CC defects (Huh et al., 2008Huh J.H. Bauer M.J. Hsieh T.-F. Fischer R.L. Cellular programming of plant gene imprinting.Cell. 2008; 132: 735-744Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). If validated, this would indicate DNG702 has a specific function in sperm that cannot be compensated by the wild-type CC genomes for endosperm development.For DNG701 and DNG704, reproductive defects were only observed in dng701/4 double mutants that produced 50% aborted, 35% normal, and 15% retarded seeds. This suggests that DNG701/4 also have a gamete formation function (see below) as well as a post-fertilization zygotic function that might be partially redundant with DNG702 (Figure 1). However, morphological phenotypes of F1 seeds derived from dng701/4 and wild-type reciprocal crosses were not presented, making it difficult to assess their functions accurately.Analysis of DMRs between mutant and wild-type gametes and zygotes enabled identification of loci targeted by each glycosylase. Interestingly, the DMRs of dng702 and dng701/704 versus wild-type egg and sperm are largely non-overlapping, indicating that DNG702 and DNG701/DNG704 have distinct targets in egg and sperm. Comparing targets of DNG701/4 in unicellular zygotes with those in eggs or sperm revealed that DNG701/4 have almost entirely new targets in zygotes, indicating that they have distinct functions before and after fertilization. Furthermore, loci demethylated by DNG702 in sperm or eggs are hypermethylated in wild-type zygotes, indicating that those gametic targets of DNG702 are quickly remethylated upon gamete fusion, presumably by the de novo methylation pathways. Likewise, DNG701/4 target sites in gametes were also remethylated in the unicellular zygotes.In conclusion, Zhou and colleagues provided a large amount of exciting new data that greatly enrich and extend our knowledge on the epigenetic dynamics during rice reproduction. Particularly, the generation of dng mutant plants and methylomes of gametes and zygotes provide the research community a rich epigenetic resource that will spark new discoveries and inspire motivations for deeper understanding of how these glycosylases function. This study strongly suggests that localized demethylation by these three DNG glycosylases in the sex cells likely serves to “prime” the gametes for successful fertilization and prepare the zygotes for rapid cellular division and differentiation during early embryogenesis. Once the gametes successfully fuse, parental imprints are quickly removed by de novo methylation and the glycosylases resume their zygotic functions by marking new target loci to ensure robust progression of embryogenesis. This notion is further supported by the transcriptomic analysis of zygote versus egg which revealed that the number of differentially expressed genes between zygote and egg is reduced by ∼50% in dng701/4, indicating that the zygotic function of DNG701/4 contributes substantially to the transcriptional state of the unicellular zygotes.FundingThis work is supported by the National Institute of Food and Agriculture ( Hatch-02413 ) and the National Science Foundation ( MCB-1715115 ). Cytosine methylation is a covalent modification of DNA that regulates important processes in eukaryotic genomes, including gene transcription, transposon silencing, and genomic imprinting (Law and Jacobsen, 2010Law J.A. Jacobsen S.E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals.Nat. Rev. Genet. 2010; 11: 204-220Crossref PubMed Scopus (2462) Google Scholar). DNA methylation patterns are faithfully duplicated upon cell division to ensure genome integrity and to maintain lineage-specific cell fate. However, DNA methylation also needs to be dynamically reprogramed or reconfigured during development to allow establishment of new cellular identity and transcriptional state, which plays a prominent role in animal development and reproduction, and is increasingly being appreciated for reproductive success in flowering plants (Walker et al., 2018Walker J. Gao H. Zhang J. Aldridge B. Vickers M. Higgins J.D. Feng X. Sexual-lineage-specific DNA methylation regulates meiosis in Arabidopsis.Nat. Genet. 2018; 50: 130-137https://doi.org/10.1038/s41588-017-0008-5Crossref PubMed Scopus (99) Google Scholar; Ono and Kinoshita, 2021Ono A. Kinoshita T. Epigenetics and plant reproduction: multiple steps for responsibly handling succession.Curr. Opin. Plant Biol. 2021; 61: 102032https://doi.org/10.1016/j.pbi.2021.102032Crossref PubMed Scopus (6) Google Scholar). In mammals, germline cells and zygotes undergo genome-wide methylation resets to obtain cellular pluripotency. In flowering plants, multiple waves of localized smaller-scale epigenetic dynamics and remodeling have also been documented during reproduction (Gehring, 2019Gehring M. Epigenetic dynamics during flowering plant reproduction: evidence for reprogramming?.New Phytol. 2019; 224: 91-96https://doi.org/10.1111/nph.15856Crossref PubMed Scopus (41) Google Scholar). For example, before fertilization localized demethylation in vegetative and central cells (VC and CC, companion cells of sperm and egg) was found to be essential for seed viability (Gehring, 2019Gehring M. Epigenetic dynamics during flowering plant reproduction: evidence for reprogramming?.New Phytol. 2019; 224: 91-96https://doi.org/10.1111/nph.15856Crossref PubMed Scopus (41) Google Scholar). Upon fertilization, the genomes of endosperm and embryo undergo methylation reconfiguration in Arabidopsis, soybean, and rice (Park et al., 2016Park K. Kim M.Y. Vickers M. Park J.S. Hyun Y. Okamoto T. Zilberman D. Fischer R.L. Feng X. Choi Y. et al.DNA demethylation is initiated in the central cells of Arabidopsis and rice.Proc. Natl. Acad. Sci. U S A. 2016; 113: 15138-15143https://doi.org/10.1073/pnas.1619047114Crossref PubMed Scopus (104) Google Scholar; Kim et al., 2019Kim M.Y. Ono A. Scholten S. Kinoshita T. Zilberman D. Okamoto T. Fischer R.L. DNA demethylation by ROS1a in rice vegetative cells promotes methylation in sperm.Proc. Natl. Acad. Sci. U S A. 2019; 116: 9652-9657https://doi.org/10.1073/pnas.1821435116Crossref PubMed Scopus (27) Google Scholar; Ono and Kinoshita, 2021Ono A. Kinoshita T. Epigenetics and plant reproduction: multiple steps for responsibly handling succession.Curr. Opin. Plant Biol. 2021; 61: 102032https://doi.org/10.1016/j.pbi.2021.102032Crossref PubMed Scopus (6) Google Scholar). Although DNA glycosylases and the de novo methylation pathways are implicated in some of these processes, many of their biological functions remain to be fully elucidated. Epigenome remodeling in gamete companion cells of Arabidopsis and riceIn Arabidopsis, the genome of the CC undergoes extensive demethylation at thousands of loci directed by the DEMETER (DME) glycosylase to establish parent-of-origin-specific expression of many imprinted genes in endosperm. DME also demethylates the genomes of VCs and the lack of DME activity in VCs resulted in CHH hypomethylation in corresponding loci in sperm (Ibarra et al., 2012Ibarra C.A. Feng X. Schoft V.K. Hsieh T.F. Uzawa R. Rodrigues J.A. Zemach A. Chumak N. Machlicova A. Nishimura T. et al.Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes.Science. 2012; 337: 1360-1364https://doi.org/10.1126/science.1224839Crossref PubMed Scopus (335) Google Scholar). Thus, the main functions of DME during Arabidopsis reproduction are to establish gene imprinting and to reinforce transgenerational TE silencing.Although no DME ortholog was detected in monocots, the rice ROS1a (DNG702) was shown to be the functional counterpart of DME in rice cultivar Nipponbare (Ono et al., 2012Ono A. Yamaguchi K. Fukada-Tanaka S. Terada R. Mitsui T. Iida S. A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny.Plant J. 2012; 71: 564-574https://doi.org/10.1111/j.1365-313X.2012.05009.xCrossref PubMed Scopus (74) Google Scholar). Similar to the VC of Arabidopsis, the rice VC genome is also extensively hypomethylated compared with the sperm genome in a ROS1a-dependent manner (Kim et al., 2019Kim M.Y. Ono A. Scholten S. Kinoshita T. Zilberman D. Okamoto T. Fischer R.L. DNA demethylation by ROS1a in rice vegetative cells promotes methylation in sperm.Proc. Natl. Acad. Sci. U S A. 2019; 116: 9652-9657https://doi.org/10.1073/pnas.1821435116Crossref PubMed Scopus (27) Google Scholar). In ROS1a/ros1a heterozygous plants, sperm CHH is hypomethylated at the loci where CG hypomethylation occurred in VCs, indicating that ROS1a activity in VCs is required for normal sperm CHH methylation. Furthermore, there is a large overlap between VC versus sperm and endosperm versus embryo hypomethylated DMRs, suggesting ROS1a also demethylates the rice CC genome (Kim et al., 2019Kim M.Y. Ono A. Scholten S. Kinoshita T. Zilberman D. Okamoto T. Fischer R.L. DNA demethylation by ROS1a in rice vegetative cells promotes methylation in sperm.Proc. Natl. Acad. Sci. U S A. 2019; 116: 9652-9657https://doi.org/10.1073/pnas.1821435116Crossref PubMed Scopus (27) Google Scholar). Thus, gamete companion cells epigenetic remodeling by DNA glycosylases is an evolutionarily conserved phenomenon in rice and Arabidopsis; species that diverged more than 150 million years ago.Despite this conservation, many distinct features exist between rice and Arabidopsis. For example, DME’s expression is primarily restricted to the gamete companion cells of Arabidopsis, whereas ROS1a is broadly expressed throughout rice development. This suggests that the gamete companion cell function of ROS1a was delegated to DME in Arabidopsis and ROS1a might also play a role in rice gamete formation and seed development. In Arabidopsis, the genome of the CC undergoes extensive demethylation at thousands of loci directed by the DEMETER (DME) glycosylase to establish parent-of-origin-specific expression of many imprinted genes in endosperm. DME also demethylates the genomes of VCs and the lack of DME activity in VCs resulted in CHH hypomethylation in corresponding loci in sperm (Ibarra et al., 2012Ibarra C.A. Feng X. Schoft V.K. Hsieh T.F. Uzawa R. Rodrigues J.A. Zemach A. Chumak N. Machlicova A. Nishimura T. et al.Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes.Science. 2012; 337: 1360-1364https://doi.org/10.1126/science.1224839Crossref PubMed Scopus (335) Google Scholar). Thus, the main functions of DME during Arabidopsis reproduction are to establish gene imprinting and to reinforce transgenerational TE silencing. Although no DME ortholog was detected in monocots, the rice ROS1a (DNG702) was shown to be the functional counterpart of DME in rice cultivar Nipponbare (Ono et al., 2012Ono A. Yamaguchi K. Fukada-Tanaka S. Terada R. Mitsui T. Iida S. A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny.Plant J. 2012; 71: 564-574https://doi.org/10.1111/j.1365-313X.2012.05009.xCrossref PubMed Scopus (74) Google Scholar). Similar to the VC of Arabidopsis, the rice VC genome is also extensively hypomethylated compared with the sperm genome in a ROS1a-dependent manner (Kim et al., 2019Kim M.Y. Ono A. Scholten S. Kinoshita T. Zilberman D. Okamoto T. Fischer R.L. DNA demethylation by ROS1a in rice vegetative cells promotes methylation in sperm.Proc. Natl. Acad. Sci. U S A. 2019; 116: 9652-9657https://doi.org/10.1073/pnas.1821435116Crossref PubMed Scopus (27) Google Scholar). In ROS1a/ros1a heterozygous plants, sperm CHH is hypomethylated at the loci where CG hypomethylation occurred in VCs, indicating that ROS1a activity in VCs is required for normal sperm CHH methylation. Furthermore, there is a large overlap between VC versus sperm and endosperm versus embryo hypomethylated DMRs, suggesting ROS1a also demethylates the rice CC genome (Kim et al., 2019Kim M.Y. Ono A. Scholten S. Kinoshita T. Zilberman D. Okamoto T. Fischer R.L. DNA demethylation by ROS1a in rice vegetative cells promotes methylation in sperm.Proc. Natl. Acad. Sci. U S A. 2019; 116: 9652-9657https://doi.org/10.1073/pnas.1821435116Crossref PubMed Scopus (27) Google Scholar). Thus, gamete companion cells epigenetic remodeling by DNA glycosylases is an evolutionarily conserved phenomenon in rice and Arabidopsis; species that diverged more than 150 million years ago. Despite this conservation, many distinct features exist between rice and Arabidopsis. For example, DME’s expression is primarily restricted to the gamete companion cells of Arabidopsis, whereas ROS1a is broadly expressed throughout rice development. This suggests that the gamete companion cell function of ROS1a was delegated to DME in Arabidopsis and ROS1a might also play a role in rice gamete formation and seed development. Epigenome remodeling of gametes and zygote in riceRice has a persistent endosperm that serves as a staple food for humans and its genome contains more TEs than Arabidopsis (International Rice Genome Sequencing, 2005International Rice Genome Sequencing, PThe map-based sequence of the rice genome.Nature. 2005; 436: 793-800https://doi.org/10.1038/nature03895Crossref PubMed Scopus (2937) Google Scholar). Understanding its reproductive epigenetic mechanisms has direct relevance to food production and can complement the knowledge gained from research in Arabidopsis and other plant models. The Nipponbare reference genome encodes at least four DNA demethylases, DNG701–DNG704. Except for DNG702/ROS1a’s roles in gamete companion cells, functions of these DNA glycosylases in germline cells and zygote were not known until now. A new report published in Molecular Plants attempted to address this question by conducting a comprehensive DNA methylome study in rice gamete, zygote, and developing embryos in wild-type and dng mutants (cultivar Dongjing) (Zhou et al., 2021Zhou S. Li X. Liu Q. Zhao Y. Jiang W. Wu A. Zhou D.X. DNA demethylases remodel DNA methylation in rice gametes and zygote and are required for reproduction.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.06.006Abstract Full Text Full Text PDF Scopus (11) Google Scholar). In addition, this new study also revealed methylation dynamics among these cell types/tissues. In wild-type plants, there is a moderate difference in bulk methylation level between egg and sperm, which is likely due to differential maintenance and do novo DNA methylase activities during male and female gametogenesis, a phenomenon consistent with what was observed during male sex lineage development in Arabidopsis (Walker et al., 2018Walker J. Gao H. Zhang J. Aldridge B. Vickers M. Higgins J.D. Feng X. Sexual-lineage-specific DNA methylation regulates meiosis in Arabidopsis.Nat. Genet. 2018; 50: 130-137https://doi.org/10.1038/s41588-017-0008-5Crossref PubMed Scopus (99) Google Scholar; Long et al., 2021Long J. Walker J. She W. Aldridge B. Gao H. Deans S. Vickers M. Feng X. Nurse cell-derived small RNAs define paternal epigenetic inheritance in Arabidopsis.Science. 2021; 373https://doi.org/10.1126/science.abh0556Crossref PubMed Scopus (27) Google Scholar). Inclusion of the unicellular zygote methylome (at 6.5 h after pollination) allowed for a direct interrogation into changes in parental genomes after they fused to form the zygote. Importantly, significant differential methylations were detected between zygote versus egg or sperm that were not a simple summation of the methylomes of the gametes, suggesting that the zygote epigenome is quickly reconfigured after fertilization. As the zygote develops, the globular embryo (GE) exhibited a slightly higher CG methylation but a lower CHH methylation level compared with the zygote. In the mature embryo, CHH methylation remains low as in GE, but CHG methylation showed a more significant decrease from GE. Taken together, these observations show that there is a substantial DNA methylation remodeling and reconfiguration during rice embryo development that is distinct from what was observed in Arabidopsis and soybean (Ono and Kinoshita, 2021Ono A. Kinoshita T. Epigenetics and plant reproduction: multiple steps for responsibly handling succession.Curr. Opin. Plant Biol. 2021; 61: 102032https://doi.org/10.1016/j.pbi.2021.102032Crossref PubMed Scopus (6) Google Scholar).To understand the contributions of gamete- and zygote-expressed glycosylases (DNG701, DNG702, and DNG704) to the rice epigenetic dynamics during reproduction, the authors generated loss-of-function mutations in these genes and profiled methylomes from mutant eggs, sperm, and zygotes. Self-pollination of homozygous dng702 plants can produce gametes but the zygotes failed to initiate embryogenesis. However, hybrid F1 seeds derived from reciprocal crosses between dng702 and wild-type plants produced viable embryos with defective endosperm. These observations showed that, for zygotes to initiate embryogenesis, at least one functional copy of DNG702 is needed; whereas for normal endosperm development, DNG702 activity is required in both the CC and sperm (Figure 1). This is intriguing as most known gametophytic-related endosperm failures are caused by CC defects (Huh et al., 2008Huh J.H. Bauer M.J. Hsieh T.-F. Fischer R.L. Cellular programming of plant gene imprinting.Cell. 2008; 132: 735-744Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). If validated, this would indicate DNG702 has a specific function in sperm that cannot be compensated by the wild-type CC genomes for endosperm development.For DNG701 and DNG704, reproductive defects were only observed in dng701/4 double mutants that produced 50% aborted, 35% normal, and 15% retarded seeds. This suggests that DNG701/4 also have a gamete formation function (see below) as well as a post-fertilization zygotic function that might be partially redundant with DNG702 (Figure 1). However, morphological phenotypes of F1 seeds derived from dng701/4 and wild-type reciprocal crosses were not presented, making it difficult to assess their functions accurately.Analysis of DMRs between mutant and wild-type gametes and zygotes enabled identification of loci targeted by each glycosylase. Interestingly, the DMRs of dng702 and dng701/704 versus wild-type egg and sperm are largely non-overlapping, indicating that DNG702 and DNG701/DNG704 have distinct targets in egg and sperm. Comparing targets of DNG701/4 in unicellular zygotes with those in eggs or sperm revealed that DNG701/4 have almost entirely new targets in zygotes, indicating that they have distinct functions before and after fertilization. Furthermore, loci demethylated by DNG702 in sperm or eggs are hypermethylated in wild-type zygotes, indicating that those gametic targets of DNG702 are quickly remethylated upon gamete fusion, presumably by the de novo methylation pathways. Likewise, DNG701/4 target sites in gametes were also remethylated in the unicellular zygotes.In conclusion, Zhou and colleagues provided a large amount of exciting new data that greatly enrich and extend our knowledge on the epigenetic dynamics during rice reproduction. Particularly, the generation of dng mutant plants and methylomes of gametes and zygotes provide the research community a rich epigenetic resource that will spark new discoveries and inspire motivations for deeper understanding of how these glycosylases function. This study strongly suggests that localized demethylation by these three DNG glycosylases in the sex cells likely serves to “prime” the gametes for successful fertilization and prepare the zygotes for rapid cellular division and differentiation during early embryogenesis. Once the gametes successfully fuse, parental imprints are quickly removed by de novo methylation and the glycosylases resume their zygotic functions by marking new target loci to ensure robust progression of embryogenesis. This notion is further supported by the transcriptomic analysis of zygote versus egg which revealed that the number of differentially expressed genes between zygote and egg is reduced by ∼50% in dng701/4, indicating that the zygotic function of DNG701/4 contributes substantially to the transcriptional state of the unicellular zygotes. Rice has a persistent endosperm that serves as a staple food for humans and its genome contains more TEs than Arabidopsis (International Rice Genome Sequencing, 2005International Rice Genome Sequencing, PThe map-based sequence of the rice genome.Nature. 2005; 436: 793-800https://doi.org/10.1038/nature03895Crossref PubMed Scopus (2937) Google Scholar). Understanding its reproductive epigenetic mechanisms has direct relevance to food production and can complement the knowledge gained from research in Arabidopsis and other plant models. The Nipponbare reference genome encodes at least four DNA demethylases, DNG701–DNG704. Except for DNG702/ROS1a’s roles in gamete companion cells, functions of these DNA glycosylases in germline cells and zygote were not known until now. A new report published in Molecular Plants attempted to address this question by conducting a comprehensive DNA methylome study in rice gamete, zygote, and developing embryos in wild-type and dng mutants (cultivar Dongjing) (Zhou et al., 2021Zhou S. Li X. Liu Q. Zhao Y. Jiang W. Wu A. Zhou D.X. DNA demethylases remodel DNA methylation in rice gametes and zygote and are required for reproduction.Mol. Plant. 2021; https://doi.org/10.1016/j.molp.2021.06.006Abstract Full Text Full Text PDF Scopus (11) Google Scholar). In addition, this new study also revealed methylation dynamics among these cell types/tissues. In wild-type plants, there is a moderate difference in bulk methylation level between egg and sperm, which is likely due to differential maintenance and do novo DNA methylase activities during male and female gametogenesis, a phenomenon consistent with what was observed during male sex lineage development in Arabidopsis (Walker et al., 2018Walker J. Gao H. Zhang J. Aldridge B. Vickers M. Higgins J.D. Feng X. Sexual-lineage-specific DNA methylation regulates meiosis in Arabidopsis.Nat. Genet. 2018; 50: 130-137https://doi.org/10.1038/s41588-017-0008-5Crossref PubMed Scopus (99) Google Scholar; Long et al., 2021Long J. Walker J. She W. Aldridge B. Gao H. Deans S. Vickers M. Feng X. Nurse cell-derived small RNAs define paternal epigenetic inheritance in Arabidopsis.Science. 2021; 373https://doi.org/10.1126/science.abh0556Crossref PubMed Scopus (27) Google Scholar). Inclusion of the unicellular zygote methylome (at 6.5 h after pollination) allowed for a direct interrogation into changes in parental genomes after they fused to form the zygote. Importantly, significant differential methylations were detected between zygote versus egg or sperm that were not a simple summation of the methylomes of the gametes, suggesting that the zygote epigenome is quickly reconfigured after fertilization. As the zygote develops, the globular embryo (GE) exhibited a slightly higher CG methylation but a lower CHH methylation level compared with the zygote. In the mature embryo, CHH methylation remains low as in GE, but CHG methylation showed a more significant decrease from GE. Taken together, these observations show that there is a substantial DNA methylation remodeling and reconfiguration during rice embryo development that is distinct from what was observed in Arabidopsis and soybean (Ono and Kinoshita, 2021Ono A. Kinoshita T. Epigenetics and plant reproduction: multiple steps for responsibly handling succession.Curr. Opin. Plant Biol. 2021; 61: 102032https://doi.org/10.1016/j.pbi.2021.102032Crossref PubMed Scopus (6) Google Scholar). To understand the contributions of gamete- and zygote-expressed glycosylases (DNG701, DNG702, and DNG704) to the rice epigenetic dynamics during reproduction, the authors generated loss-of-function mutations in these genes and profiled methylomes from mutant eggs, sperm, and zygotes. Self-pollination of homozygous dng702 plants can produce gametes but the zygotes failed to initiate embryogenesis. However, hybrid F1 seeds derived from reciprocal crosses between dng702 and wild-type plants produced viable embryos with defective endosperm. These observations showed that, for zygotes to initiate embryogenesis, at least one functional copy of DNG702 is needed; whereas for normal endosperm development, DNG702 activity is required in both the CC and sperm (Figure 1). This is intriguing as most known gametophytic-related endosperm failures are caused by CC defects (Huh et al., 2008Huh J.H. Bauer M.J. Hsieh T.-F. Fischer R.L. Cellular programming of plant gene imprinting.Cell. 2008; 132: 735-744Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). If validated, this would indicate DNG702 has a specific function in sperm that cannot be compensated by the wild-type CC genomes for endosperm development. For DNG701 and DNG704, reproductive defects were only observed in dng701/4 double mutants that produced 50% aborted, 35% normal, and 15% retarded seeds. This suggests that DNG701/4 also have a gamete formation function (see below) as well as a post-fertilization zygotic function that might be partially redundant with DNG702 (Figure 1). However, morphological phenotypes of F1 seeds derived from dng701/4 and wild-type reciprocal crosses were not presented, making it difficult to assess their functions accurately. Analysis of DMRs between mutant and wild-type gametes and zygotes enabled identification of loci targeted by each glycosylase. Interestingly, the DMRs of dng702 and dng701/704 versus wild-type egg and sperm are largely non-overlapping, indicating that DNG702 and DNG701/DNG704 have distinct targets in egg and sperm. Comparing targets of DNG701/4 in unicellular zygotes with those in eggs or sperm revealed that DNG701/4 have almost entirely new targets in zygotes, indicating that they have distinct functions before and after fertilization. Furthermore, loci demethylated by DNG702 in sperm or eggs are hypermethylated in wild-type zygotes, indicating that those gametic targets of DNG702 are quickly remethylated upon gamete fusion, presumably by the de novo methylation pathways. Likewise, DNG701/4 target sites in gametes were also remethylated in the unicellular zygotes. In conclusion, Zhou and colleagues provided a large amount of exciting new data that greatly enrich and extend our knowledge on the epigenetic dynamics during rice reproduction. Particularly, the generation of dng mutant plants and methylomes of gametes and zygotes provide the research community a rich epigenetic resource that will spark new discoveries and inspire motivations for deeper understanding of how these glycosylases function. This study strongly suggests that localized demethylation by these three DNG glycosylases in the sex cells likely serves to “prime” the gametes for successful fertilization and prepare the zygotes for rapid cellular division and differentiation during early embryogenesis. Once the gametes successfully fuse, parental imprints are quickly removed by de novo methylation and the glycosylases resume their zygotic functions by marking new target loci to ensure robust progression of embryogenesis. This notion is further supported by the transcriptomic analysis of zygote versus egg which revealed that the number of differentially expressed genes between zygote and egg is reduced by ∼50% in dng701/4, indicating that the zygotic function of DNG701/4 contributes substantially to the transcriptional state of the unicellular zygotes. FundingThis work is supported by the National Institute of Food and Agriculture ( Hatch-02413 ) and the National Science Foundation ( MCB-1715115 ). This work is supported by the National Institute of Food and Agriculture ( Hatch-02413 ) and the National Science Foundation ( MCB-1715115 ).}, number={9}, journal={MOLECULAR PLANT}, publisher={Elsevier BV}, author={Li, Mingzhuo and Cui, Qirui and Zhang, Xiang-Qian and Hsieh, Tzung-Fu}, year={2021}, month={Sep}, pages={1433–1435} }
 @article{fogarty_smith_sheridan_hu_islamovic_reid_jackson_maughan_ames_jellen_et al._2020, title={Identification of mixed linkage beta-glucan quantitative trait loci and evaluation of AsCslF6 homoeologs in hexaploid oat}, volume={60}, ISSN={["1435-0653"]}, DOI={10.1002/csc2.20015}, abstractNote={AbstractHexaploid oat (Avena sativa L.) is a commercially important cereal crop due in part to (1‐3,1‐4)‐β‐D‐glucan (β‐glucan), a hemicellulose important to human health. Cellulose synthase‐like (Csl) genes have been shown to contribute to β‐glucan production, with CslF6 likely being of major importance. Here, we report a genome‐wide association study (GWAS) to identify quantitative trait loci (QTLs) controlling β‐glucan production in oat. Three panels of elite accessions (Spring, Winter, and World Diversity) of oat were grown in multiple North American locations and genotyped using the Oat 6K Custom Infinium iSelect BeadChip. Independent GWAS were performed on each set of accessions and locations, with a meta‐analysis identifying 58 significantly associated markers. Synteny with the barley (Hordeum vulgare L.) genome identified four major regions of interest revealing the CslF and CslH gene families along with UGPase and AGPase as candidate genes. Subgenome‐specific expression of the A, C, and D AsCslF6 homoeologs revealed that AsCslF6_C is the least expressed in all tissue types and time points, with low‐β‐glucan varieties recording the highest proportion of AsCslF6_C expression. Linkage mapping of the homoeologs placed AsCslF6_D on consensus linkage group Mrg02 overlapping with QTL 2.2 and AsCslF6_A on Mrg12 flanked by markers associated with QTL 12.2. Many QTLs identified in this study were homoeologous, representing different gene copies duplicated in ancestral genomes, suggesting that multiple homoeologous copies of β‐glucan biosynthesis genes are contributing to the overall phenotype.}, number={2}, journal={CROP SCIENCE}, author={Fogarty, Melissa C. and Smith, Scott M. and Sheridan, Jaime L. and Hu, Gongshe and Islamovic, Emir and Reid, Rob and Jackson, Eric W. and Maughan, Peter J. and Ames, Nancy P. and Jellen, Eric N. and et al.}, year={2020}, pages={914–933} }
 @article{luan_liu_ke_dai_xie_hsieh_zhang_2019, title={Epigenetic modification of ESP, encoding a putative long noncoding RNA, affects panicle architecture in rice}, volume={12}, ISSN={1939-8425 1939-8433}, url={http://dx.doi.org/10.1186/s12284-019-0282-1}, DOI={10.1186/s12284-019-0282-1}, abstractNote={Epigenetic variants broaden phenotypic diversity in eukaryotes. Epialleles may also provide a new genetic source for crop breeding, but very few epialleles related to agricultural traits have been identified in rice. Here, we identified Epi-sp, a gain-of-function epiallele of the rice ESP (Epigenetic Short Panicle, Os01g0356951), which encodes a putative long noncoding RNA. The Epi-sp plants show a dense and short panicle phenotype, an agronomically important phenotypes that is inherited in a semidominant manner. We did not find any nucleotide sequence variation in ESP. Instead, we found hypomethylation in the transcriptional termination region (TTR) of ESP gene, which caused ectopic expression of ESP in Epi-sp plants. Bisulfite analysis revealed that the methylation status of 26 CGs and 13 CHGs within a continuous 313-bp region is essential for the regulation of ESP expression. Thus, our work identified a unique rice epiallele and demonstrated that epigenetic modification of ESP is associated with the regulation of panicle architecture in rice.}, number={1}, journal={Rice}, publisher={Springer Nature}, author={Luan, Xin and Liu, Shuchun and Ke, Shanwen and Dai, Hang and Xie, Xin-Ming and Hsieh, Tzung-Fu and Zhang, Xiang-Qian}, year={2019}, month={Apr} }
 @article{han_bartels_cheng_meyer_an_hsieh_xiao_2019, title={Epigenetics Regulates Reproductive Development in Plants}, volume={8}, ISSN={2223-7747}, url={http://dx.doi.org/10.3390/plants8120564}, DOI={10.3390/plants8120564}, abstractNote={Seed, resulting from reproductive development, is the main nutrient source for human beings, and reproduction has been intensively studied through genetic, molecular, and epigenetic approaches. However, how different epigenetic pathways crosstalk and integrate to regulate seed development remains unknown. Here, we review the recent progress of epigenetic changes that affect chromatin structure, such as DNA methylation, polycomb group proteins, histone modifications, and small RNA pathways in regulating plant reproduction. In gametogenesis of flowering plants, epigenetics is dynamic between the companion cell and gametes. Cytosine DNA methylation occurs in CG, CHG, CHH contexts (H = A, C, or T) of genes and transposable elements, and undergoes dynamic changes during reproduction. Cytosine methylation in the CHH context increases significantly during embryogenesis, reaches the highest levels in mature embryos, and decreases as the seed germinates. Polycomb group proteins are important transcriptional regulators during seed development. Histone modifications and small RNA pathways add another layer of complexity in regulating seed development. In summary, multiple epigenetic pathways are pivotal in regulating seed development. It remains to be elucidated how these epigenetic pathways interplay to affect dynamic chromatin structure and control reproduction.}, number={12}, journal={Plants}, publisher={MDPI AG}, author={Han, Qiang and Bartels, Arthur and Cheng, Xi and Meyer, Angela and An, Yong-Qiang Charles and Hsieh, Tzung-Fu and Xiao, Wenyan}, year={2019}, month={Dec}, pages={564} }
 @article{ke_liu_luan_xie_hsieh_zhang_2019, title={Mutation in a putative glycosyltransferase-like gene causes programmed cell death and early leaf senescence in rice}, volume={12}, ISSN={1939-8425 1939-8433}, url={http://dx.doi.org/10.1186/s12284-019-0266-1}, DOI={10.1186/s12284-019-0266-1}, abstractNote={Leaf senescence is a genetically regulated, highly complex and ordered process. Although it has been extensively studied, the mechanism of leaf senescence is not well understood. In this study, we isolated a rice mutant, designated as premature senescence leaf (psl), which exhibits early senescence and spontaneous lesion mimic phenotype after flowering. The psl mutant displays programmed cell death with elevated accumulation of reactive oxygen species (ROS). Molecular and genetic analyses revealed that the phenotypes were caused by a phenylalanine deletion in the OsPSL (LOC_Os12g42420) that encode a putative core 2/I branching beta-1,6-N-acetylglucosaminyl transferase predicted to be involved in protein glycosylation modification. OsPSL mRNA levels increased as senescence progressed, with maximum accumulation of transcripts at late senescence stages in WT plants. Moreover, remarkedly down-regulated transcriptional levels of O-linked N-acetylglucosamine (O-GlcNAc) transferases (OGTs) genes were observed in psl mutant, supporting the occurrence of impaired O-glycosylation modification. Proteomic analysis showed that ethylene-related metabolic enzymes including S-adenosyl methionine (SAM) synthetase (SAMS) were significantly upregulated in the psl mutant compared with WT. Consistent with the proteomic results, ethylene concentration is higher in psl mutant than in wild-type plants, and transcript levels of ethylene synthesis and signal transduction genes were induced in psl mutant. The early leaf senescence of psl can be partially rescued by ethylene biosynthesis inhibitor aminoethoxyvinylglycine treatment. These results highlight the importance of protein O-glycosylation in PCD and leaf senescence, and suggest a possible role of OsPSL in ethylene signaling.}, number={1}, journal={Rice}, publisher={Springer Science and Business Media LLC}, author={Ke, Shanwen and Liu, Shuchun and Luan, Xin and Xie, Xin-Ming and Hsieh, Tzung-Fu and Zhang, Xiang-Qian}, year={2019}, month={Feb} }
 @article{ke_luan_liang_hung_hsieh_zhang_2019, title={Rice OsPEX1, an extensin-like protein, affects lignin biosynthesis and plant growth}, volume={100}, ISSN={0167-4412 1573-5028}, url={http://dx.doi.org/10.1007/s11103-019-00849-3}, DOI={10.1007/s11103-019-00849-3}, abstractNote={Rice leucine-rich repeat extensin-like protein OsPEX1 mediates the intersection of lignin deposition and plant growth. Lignin, a major structural component of secondary cell wall, is essential for normal plant growth and development. However, the molecular and genetic regulation of lignin biosynthesis is not fully understood in rice. Here we report the identification and characterization of a rice semi-dominant dwarf mutant (pex1) with stiff culm. Molecular and genetic analyses revealed that the pex1 phenotype was caused by ectopic expression of a leucine-rich repeat extension-like gene, OsPEX1. Interestingly, the pex1 mutant showed significantly higher lignin content and increased expression levels of lignin-related genes compared with wild type plants. Conversely, OsPEX1-suppresssed transgenics displayed low lignin content and reduced transcriptional abundance of genes associated with lignin biosynthesis, indicating that the OsPEX1 mediates lignin biosynthesis and/or deposition in rice. When OsPEX1 was ectopically expressed in rice cultivars with tall stature that lacks the allele of semi-dwarf 1, well-known green revolution gene, the resulting transgenic plants displayed reduced height and enhanced lodging resistance. Our study uncovers a causative effect between the expression of OsPEX1 and lignin deposition. Lastly, we demonstrated that modulating OsPEX1 expression could provide a tool for improving rice lodging resistance.}, number={1-2}, journal={Plant Molecular Biology}, publisher={Springer Science and Business Media LLC}, author={Ke, Shanwen and Luan, Xin and Liang, Jiayan and Hung, Yu-Hung and Hsieh, Tzung-Fu and Zhang, Xiang-Qian}, year={2019}, month={Mar}, pages={151–161} }
 @article{zhang_hung_rim_zhang_frost_shin_jang_liu_xiao_iyer_et al._2019, title={The catalytic core of DEMETER guides active DNA demethylation in Arabidopsis}, volume={116}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.1907290116}, DOI={10.1073/pnas.1907290116}, abstractNote={
            The
            Arabidopsis
            DEMETER (DME) DNA glycosylase demethylates the maternal genome in the central cell prior to fertilization and is essential for seed viability. DME preferentially targets small transposons that flank coding genes, influencing their expression and initiating plant gene imprinting. DME also targets intergenic and heterochromatic regions, but how it is recruited to these differing chromatin landscapes is unknown. The C-terminal half of DME consists of 3 conserved regions required for catalysis in vitro. We show that this catalytic core guides active demethylation at endogenous targets, rescuing
            dme
            developmental and genomic hypermethylation phenotypes. However, without the N terminus, heterochromatin demethylation is significantly impeded, and abundant CG-methylated genic sequences are ectopically demethylated. Comparative analysis revealed that the conserved DME N-terminal domains are present only in flowering plants, whereas the domain architecture of DME-like proteins in nonvascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in plant lineages. We propose a bipartite model for DME protein action and suggest that the DME N terminus was acquired late during land plant evolution to improve specificity and facilitate demethylation at heterochromatin targets.
          }, number={35}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Zhang, Changqing and Hung, Yu-Hung and Rim, Hyun Jung and Zhang, Dapeng and Frost, Jennifer M. and Shin, Hosub and Jang, Hosung and Liu, Fang and Xiao, Wenyan and Iyer, Lakshminarayan M. and et al.}, year={2019}, month={Aug}, pages={17563–17571} }
 @article{bartels_han_nair_stacey_gaynier_mosley_huang_pearson_hsieh_an_et al._2018, title={Dynamic DNA Methylation in Plant Growth and Development}, volume={19}, ISSN={1422-0067}, url={http://dx.doi.org/10.3390/ijms19072144}, DOI={10.3390/ijms19072144}, abstractNote={DNA methylation is an epigenetic modification required for transposable element (TE) silencing, genome stability, and genomic imprinting. Although DNA methylation has been intensively studied, the dynamic nature of methylation among different species has just begun to be understood. Here we summarize the recent progress in research on the wide variation of DNA methylation in different plants, organs, tissues, and cells; dynamic changes of methylation are also reported during plant growth and development as well as changes in response to environmental stresses. Overall DNA methylation is quite diverse among species, and it occurs in CG, CHG, and CHH (H = A, C, or T) contexts of genes and TEs in angiosperms. Moderately expressed genes are most likely methylated in gene bodies. Methylation levels decrease significantly just upstream of the transcription start site and around transcription termination sites; its levels in the promoter are inversely correlated with the expression of some genes in plants. Methylation can be altered by different environmental stimuli such as pathogens and abiotic stresses. It is likely that methylation existed in the common eukaryotic ancestor before fungi, plants and animals diverged during evolution. In summary, DNA methylation patterns in angiosperms are complex, dynamic, and an integral part of genome diversity after millions of years of evolution.}, number={7}, journal={International Journal of Molecular Sciences}, publisher={MDPI AG}, author={Bartels, Arthur and Han, Qiang and Nair, Pooja and Stacey, Liam and Gaynier, Hannah and Mosley, Matthew and Huang, Qi and Pearson, Jacob and Hsieh, Tzung-Fu and An, Yong-Qiang and et al.}, year={2018}, month={Jul}, pages={2144} }
 @article{lowder_zhou_zhang_malzahn_zhong_hsieh_voytas_zhang_qi_2018, title={Robust Transcriptional Activation in Plants Using Multiplexed CRISPR-Act2.0 and mTALE-Act Systems}, volume={11}, ISSN={1674-2052}, url={http://dx.doi.org/10.1016/j.molp.2017.11.010}, DOI={10.1016/j.molp.2017.11.010}, abstractNote={User-friendly tools for robust transcriptional activation of endogenous genes are highly demanded in plants. We previously showed that a dCas9-VP64 system consisting of the deactivated CRISPR-associated protein 9 (dCas9) fused with four tandem repeats of the transcriptional activator VP16 (VP64) could be used for transcriptional activation of endogenous genes in plants. In this study, we developed a second generation of vector systems for enhanced transcriptional activation in plants. We tested multiple strategies for dCas9-based transcriptional activation, and found that simultaneous recruitment of VP64 by dCas9 and a modified guide RNA scaffold gRNA2.0 (designated CRISPR-Act2.0) yielded stronger transcriptional activation than the dCas9-VP64 system. Moreover, we developed a multiplex transcription activator-like effector activation (mTALE-Act) system for simultaneous activation of up to four genes in plants. Our results suggest that mTALE-Act is even more effective than CRISPR-Act2.0 in most cases tested. In addition, we explored tissue-specific gene activation using positive feedback loops. Interestingly, our study revealed that certain endogenous genes are more amenable than others to transcriptional activation, and tightly regulated genes may cause target gene silencing when perturbed by activation probes. Hence, these new tools could be used to investigate gene regulatory networks and their control mechanisms. Assembly of multiplex CRISPR-Act2.0 and mTALE-Act systems are both based on streamlined and PCR-independent Golden Gate and Gateway cloning strategies, which will facilitate transcriptional activation applications in both dicots and monocots.}, number={2}, journal={Molecular Plant}, publisher={Elsevier BV}, author={Lowder, Levi G. and Zhou, Jianping and Zhang, Yingxiao and Malzahn, Aimee and Zhong, Zhaohui and Hsieh, Tzung-Fu and Voytas, Daniel F. and Zhang, Yong and Qi, Yiping}, year={2018}, month={Feb}, pages={245–256} }
 @inbook{hung_liu_zhang_xiao_hsieh_2018, title={Sexual and Non-sexual Reproduction}, volume={88}, ISBN={9780128154038}, ISSN={0065-2296}, url={http://dx.doi.org/10.1016/bs.abr.2018.09.002}, DOI={10.1016/bs.abr.2018.09.002}, abstractNote={Although plant breeding has traditionally relied on exploiting genetic diversity, epigenetic variations are attracting new attentions in breeding design. Epigenetic modifications play a critical role in regulating gene expression during development and in response to environmental stimulation, and thereby contribute to phenotypic variation. When epigenetic modifications persist during reproduction and are stably transmitted to the next generation, transgenerational inheritance of epigenetic variations has a potential to produce heritable phenotypic diversity. In addition to naturally occurring epialleles, genetic variation due to organization of duplicated genes or transposon polymorphisms are a potential source for inducing epialleles. Furthermore, stress-induced activation and mobilization of transposable elements represent another promising avenue to create new genetic and epigenetic diversity that can be sexually or asexually propagated. Finally, methods for artificially creating epigenetic diversity in experimental models have been developed and applied to select crop species in some cases. With the advance in epigenome profiling techniques, dissection of epigenetic-based complex traits, and the development of molecular tools for locus-specific epigenome editing, the effects of epigenetics for underlying phenotypic traits can be unequivocally elucidated.}, booktitle={Advances in Botanical Research}, publisher={Elsevier}, author={Hung, Yu-Hung and Liu, Fang and Zhang, Xiang-Qian and Xiao, Wenyan and Hsieh, Tzung-Fu}, year={2018}, pages={117–163} }
 @article{zeng_tang_guo_ke_teng_hung_xu_xie_hsieh_zhang_et al._2017, title={A naturally occurring conditional albino mutant in rice caused by defects in the plastid-localized OsABCI8 transporter}, volume={94}, ISSN={0167-4412 1573-5028}, url={http://dx.doi.org/10.1007/s11103-017-0598-4}, DOI={10.1007/s11103-017-0598-4}, abstractNote={A wide range of molecules are transported across membranes by the ATP binding cassette (ABC) transporters. Plants possess a collection of ABC proteins bearing similarities to the components of prokaryotic multi subunit ABC transporters, designed as ABC group I. However the functions of most of them are not well understood. Here, we characterized a naturally occurring rice mutant that exhibited albino phenotype under continuous rainy days in the field, but gradually recovered to normal green after the rainy season. Molecular and genetic analyses revealed that the phenotypes were caused by a mutation in the OsABCI8 that encoded a member of the ABCI family. Subcellular localization demonstrated that OsABCI8 is a chloroplast ABC transporter. Expression of OsABCI8 is significantly enhanced in rainy days compared to sunny days. Besides defects in chloroplast development and chlorophyll biosynthesis, the mutant phenotype is accompanied by a higher accumulation of iron, suggesting that OsABCI8 is involved in iron transportation and/or homeostasis in rice. Our results demonstrate that OsABCI8 represents a conserved ABCI protein involved in transition metals transportation and/or homeostasis and suggest an important role of the plastid-localized OsABCI8 for chloroplast development.}, number={1-2}, journal={Plant Molecular Biology}, publisher={Springer Science and Business Media LLC}, author={Zeng, X. Y. and Tang, R. and Guo, H. R. and Ke, S. W. and Teng, B. and Hung, Y. H. and Xu, Z. J. and Xie, X. M. and Hsieh, Tzung-Fu and Zhang, X. Q. and et al.}, year={2017}, month={Mar}, pages={137–148} }
 @inbook{lin_hsieh_2017, title={Epigenetic Reprogramming During Plant Reproduction}, ISBN={9783319555195 9783319555201}, ISSN={2197-9731 2197-9758}, url={http://dx.doi.org/10.1007/978-3-319-55520-1_20}, DOI={10.1007/978-3-319-55520-1_20}, abstractNote={Epigenetics is the study of heritable change in gene expression state that is independent of DNA sequence variation. Such change can occur through DNA methylation or posttranscriptional modifications of histones. Epigenetic mechanisms play critical roles in regulating gene expression during development and in response to environmental stimulation. Such epigenetic information represents the transcriptional memory associated with cell fate decisions, developmental switches, or stress responses; memory that often needs to be erased and reset during reproduction. By contrast, transgenerational epigenetic information refers to more indelible marks that can be stably transmitted through meiosis and inherited in the subsequent generation. Epigenetic reprogramming, a global change in DNA and/or histone methylation, has been reported during reproduction in mammals and in flowering plants. Such reprogramming is thought to be essential for ensuring meiosis competence, establishing genomic imprinting, and silencing transposons. In Arabidopsis, gene imprinting is a consequence of a large-scale epigenetic reprogramming via DEMETER-mediated active DNA demethylation during gametogenesis. Such reprogramming is believed to be critical for the maintenance of trans-generational epigenome integrity.}, booktitle={Plant Epigenetics}, publisher={Springer International Publishing}, author={Lin, Jer-Young and Hsieh, Tzung-Fu}, year={2017}, pages={405–425} }
 @article{lin_le_chen_henry_hur_hsieh_chen_pelletier_pellegrini_fischer_et al._2017, title={Similarity between soybean and Arabidopsis seed methylomes and loss of non-CG methylation does not affect seed development}, volume={114}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.1716758114}, DOI={10.1073/pnas.1716758114}, abstractNote={Significance
          
            We describe the spatial and temporal profiles of soybean and
            Arabidopsis
            seed methylomes during development. CHH methylation increases globally from fertilization through dormancy in all seed parts, decreases following germination, and targets primarily transposons. By contrast, CG- and CHG-context methylation remains constant throughout seed development. Mutant seeds lacking non-CG methylation develop normally, but have a set of up-regulated transposon RNAs suggesting that the CHH methylation increase may be a failsafe mechanism to reinforce transposon silencing. Major classes of seed genes have similar methylation profiles, whether they are active or not. Our results suggest that soybean and
            Arabidopsis
            seed methylomes are similar, and that DNA methylation does not play a significant role in regulating many genes important for seed development.
          }, number={45}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Lin, Jer-Young and Le, Brandon H. and Chen, Min and Henry, Kelli F. and Hur, Jungim and Hsieh, Tzung-Fu and Chen, Pao-Yang and Pelletier, Julie M. and Pellegrini, Matteo and Fischer, Robert L. and et al.}, year={2017}, month={Oct}, pages={E9730–E9739} }
 @article{chaffin_huang_smith_bekele_babiker_gnanesh_foresman_blanchard_jay_reid_et al._2016, title={A Consensus Map in Cultivated Hexaploid Oat Reveals Conserved Grass Synteny with Substantial Subgenome Rearrangement}, volume={9}, ISSN={1940-3372}, url={http://dx.doi.org/10.3835/plantgenome2015.10.0102}, DOI={10.3835/plantgenome2015.10.0102}, abstractNote={Hexaploid oat (Avena sativa L., 2n = 6x = 42) is a member of the Poaceae family and has a large genome (∼12.5 Gb) containing 21 chromosome pairs from three ancestral genomes. Physical rearrangements among parental genomes have hindered the development of linkage maps in this species. The objective of this work was to develop a single high‐density consensus linkage map that is representative of the majority of commonly grown oat varieties. Data from a cDNA‐derived single‐nucleotide polymorphism (SNP) array and genotyping‐by‐sequencing (GBS) were collected from the progeny of 12 biparental recombinant inbred line populations derived from 19 parents representing oat germplasm cultivated primarily in North America. Linkage groups from all mapping populations were compared to identify 21 clusters of conserved collinearity. Linkage groups within each cluster were then merged into 21 consensus chromosomes, generating a framework consensus map of 7202 markers spanning 2843 cM. An additional 9678 markers were placed on this map with a lower degree of certainty. Assignment to physical chromosomes with high confidence was made for nine chromosomes. Comparison of homeologous regions among oat chromosomes and matches to orthologous regions of rice (Oryza sativa L.) reveal that the hexaploid oat genome has been highly rearranged relative to its ancestral diploid genomes as a result of frequent translocations among chromosomes. Heterogeneous chromosome rearrangements among populations were also evident, probably accounting for the failure of some linkage groups to match the consensus. This work contributes to a further understanding of the organization and evolution of hexaploid grass genomes.}, number={2}, journal={The Plant Genome}, publisher={Crop Science Society of America}, author={Chaffin, Ashley S. and Huang, Yung-Fen and Smith, Scott and Bekele, Wubishet A. and Babiker, Ebrahiem and Gnanesh, Belaghihalli N. and Foresman, Bradley J. and Blanchard, Steven G. and Jay, Jeremy J. and Reid, Robert W. and et al.}, year={2016}, pages={0} }
 @article{hsieh_2016, title={A Tug of war for DNA methylation}, volume={2}, number={11}, journal={Nature Plants}, author={Hsieh, T. F.}, year={2016} }
 @article{mendizabal_shi_keller_konopka_preuss_hsieh_hu_zhang_su_yi_et al._2016, title={Comparative Methylome Analyses Identify Epigenetic Regulatory Loci of Human Brain Evolution}, volume={33}, ISSN={0737-4038 1537-1719}, url={http://dx.doi.org/10.1093/molbev/msw176}, DOI={10.1093/molbev/msw176}, abstractNote={How do epigenetic modifications change across species and how do these modifications affect evolution? These are fundamental questions at the forefront of our evolutionary epigenomic understanding. Our previous work investigated human and chimpanzee brain methylomes, but it was limited by the lack of outgroup data which is critical for comparative (epi)genomic studies. Here, we compared whole genome DNA methylation maps from brains of humans, chimpanzees and also rhesus macaques (outgroup) to elucidate DNA methylation changes during human brain evolution. Moreover, we validated that our approach is highly robust by further examining 38 human-specific DMRs using targeted deep genomic and bisulfite sequencing in an independent panel of 37 individuals from five primate species. Our unbiased genome-scan identified human brain differentially methylated regions (DMRs), irrespective of their associations with annotated genes. Remarkably, over half of the newly identified DMRs locate in intergenic regions or gene bodies. Nevertheless, their regulatory potential is on par with those of promoter DMRs. An intriguing observation is that DMRs are enriched in active chromatin loops, suggesting human-specific evolutionary remodeling at a higher-order chromatin structure. These findings indicate that there is substantial reprogramming of epigenomic landscapes during human brain evolution involving noncoding regions.}, number={11}, journal={Molecular Biology and Evolution}, publisher={Oxford University Press (OUP)}, author={Mendizabal, I. and Shi, L. and Keller, T. E. and Konopka, G. and Preuss, T. M. and Hsieh, Tzung-Fu and Hu, E. Z. and Zhang, Z. and Su, B. and Yi, S. V. and et al.}, year={2016}, month={Aug}, pages={2947–2959} }
 @article{epigenetics: a tug of war for dna methylation._2016, url={https://doi.org/10.1038/nplants.2016.171}, DOI={10.1038/nplants.2016.171}, journal={Nature plants}, year={2016}, month={Nov} }
 @article{oliva_butenko_hsieh_hakim_katz_smorodinsky_michaeli_fischer_ohad_2016, title={FIE, a nuclear PRC2 protein, forms cytoplasmic complexes inArabidopsis thaliana}, volume={67}, ISSN={0022-0957 1460-2431}, url={http://dx.doi.org/10.1093/jxb/erw373}, DOI={10.1093/jxb/erw373}, abstractNote={Highlight FIE, a WD-40 subunit of the Arabidopsis PRC2 complexes known to take part in H3K27 methylation of nuclear chromatin, is also localized in cytoplasmic complexes.}, number={21}, journal={Journal of Experimental Botany}, publisher={Oxford University Press (OUP)}, author={Oliva, Moran and Butenko, Yana and Hsieh, Tzung-Fu and Hakim, Ofir and Katz, Aviva and Smorodinsky, Nechama I. and Michaeli, Daphna and Fischer, Robert L. and Ohad, Nir}, year={2016}, month={Oct}, pages={6111–6123} }
 @article{lowder_zhang_baltes_paul_tang_zheng_voytas_hsieh_zhang_qi_et al._2015, title={A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation}, volume={169}, ISSN={0032-0889 1532-2548}, url={http://dx.doi.org/10.1104/pp.15.00636}, DOI={10.1104/pp.15.00636}, abstractNote={A CRISPR/Cas9 toolbox enables multiplex genome editing and transcriptional regulation of genes in plants. The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied plant research.}, number={2}, journal={Plant Physiology}, publisher={American Society of Plant Biologists (ASPB)}, author={Lowder, L. G. and Zhang, D. W. and Baltes, N. J. and Paul, J. W. and Tang, X. and Zheng, X. L. and Voytas, D. F. and Hsieh, Tzung-Fu and Zhang, Y. and Qi, Y. P. and et al.}, year={2015}, month={Aug}, pages={971–985} }
 @article{jeong_park_yun_hsieh_choi_choi_lee_2015, title={Control of Paternally Expressed Imprinted UPWARD CURLY LEAF1, a Gene Encoding an F-Box Protein That Regulates CURLY LEAF Polycomb Protein, in the Arabidopsis Endosperm}, volume={10}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0117431}, DOI={10.1371/journal.pone.0117431}, abstractNote={Genomic imprinting, an epigenetic process in mammals and flowering plants, refers to the differential expression of alleles of the same genes in a parent-of-origin-specific manner. In Arabidopsis, imprinting occurs primarily in the endosperm, which nourishes the developing embryo. Recent high-throughput sequencing analyses revealed that more than 200 loci are imprinted in Arabidopsis; however, only a few of these imprinted genes and their imprinting mechanisms have been examined in detail. Whereas most imprinted loci characterized to date are maternally expressed imprinted genes (MEGs), PHERES1 (PHE1) and ADMETOS (ADM) are paternally expressed imprinted genes (PEGs). Here, we report that UPWARD CURLY LEAF1 (UCL1), a gene encoding an E3 ligase that degrades the CURLY LEAF (CLF) polycomb protein, is a PEG. After fertilization, paternally inherited UCL1 is expressed in the endosperm, but not in the embryo. The expression pattern of a β-glucuronidase (GUS) reporter gene driven by the UCL1 promoter suggests that the imprinting control region (ICR) of UCL1 is adjacent to a transposable element in the UCL1 5′-upstream region. Polycomb Repressive Complex 2 (PRC2) silences the maternal UCL1 allele in the central cell prior to fertilization and in the endosperm after fertilization. The UCL1 imprinting pattern was not affected in paternal PRC2 mutants. We found unexpectedly that the maternal UCL1 allele is reactivated in the endosperm of Arabidopsis lines with mutations in cytosine DNA METHYLTRANSFERASE 1 (MET1) or the DNA glycosylase DEMETER (DME), which antagonistically regulate CpG methylation of DNA. By contrast, maternal UCL1 silencing was not altered in mutants with defects in non-CpG methylation. Thus, silencing of the maternal UCL1 allele is regulated by both MET1 and DME as well as by PRC2, suggesting that divergent mechanisms for the regulation of PEGs evolved in Arabidopsis.}, number={2}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Jeong, Cheol Woong and Park, Guen Tae and Yun, Hyein and Hsieh, Tzung-Fu and Choi, Yang Do and Choi, Yeonhee and Lee, Jong Seob}, editor={Sun, Meng-xiangEditor}, year={2015}, month={Feb}, pages={e0117431} }
 @inbook{hsieh_2015, title={Whole-Genome DNA Methylation Profiling with Nucleotide Resolution}, volume={1284}, ISBN={9781493924431 9781493924448}, ISSN={1064-3745 1940-6029}, url={http://dx.doi.org/10.1007/978-1-4939-2444-8_2}, DOI={10.1007/978-1-4939-2444-8_2}, abstractNote={In many eukaryotic organisms, methylation at the fifth carbon of cytosine (5mC) is a stable epigenetic mark crucial for many biological processes, including cell differentiation, X-chromosome inactivation, transposon silencing, and genomic imprinting. DNA methylation can be stably inherited to the subsequent generation. It can also change dynamically in response to developmental cues or environmental stimuli, and is an important regulator for developmental switch and cell fate determination. Consequently, many human diseases are associated with aberrant DNA methylation. Gene-specific methylation analysis by sequencing of bisulfite-treated genomic DNA has been instrumental in understanding how DNA methylation affects gene transcription. In recent years, techniques have been developed for genome-wide 5mC detection, and complete methylome at single base resolution has been reported for several organisms, providing unprecedented details on the dynamic nature of DNA methylation during development. With the advance in high-throughput sequencing and the availability of genome sequences, mapping the methylome for species with complex genomes has become increasingly feasible.}, booktitle={Methods in Molecular Biology}, publisher={Springer New York}, author={Hsieh, Tzung-Fu}, year={2015}, pages={27–40} }
 @article{mérai_chumak_garcía-aguilar_hsieh_nishimura_schoft_bindics_ślusarz_arnoux_opravil_et al._2014, title={The AAA-ATPase molecular chaperone Cdc48/p97 disassembles sumoylated centromeres, decondenses heterochromatin, and activates ribosomal RNA genes}, volume={111}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.1418564111}, DOI={10.1073/pnas.1418564111}, abstractNote={Significance
          Centromeres are the fundamental unit required for segregation of chromosomes during mitosis and meiosis, and they are defined by the centromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A). In contrast to the relatively well-known process of de novo assembly of CenH3 at centromeres, little is known of how CenH3 is actively removed, leading to centromere disassembly, an essential biological process during the life of a cell. This study describes the process of centromere disassembly, demonstrating that it occurs via an active, proteolytic mechanism, which is also linked to major changes in chromosome dynamics: chromatin decondensation and bulk rRNA gene activation.}, number={45}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Mérai, Zsuzsanna and Chumak, Nina and García-Aguilar, Marcelina and Hsieh, Tzung-Fu and Nishimura, Toshiro and Schoft, Vera K. and Bindics, János and Ślusarz, Lucyna and Arnoux, Stéphanie and Opravil, Susanne and et al.}, year={2014}, month={Oct}, pages={16166–16171} }
 @inbook{ibarra_frost_shin_hsieh_fischer_2013, title={Epigenetic Control of Seed Gene Imprinting}, ISBN={9781118525524 9780470960158}, url={http://dx.doi.org/10.1002/9781118525524.ch4}, DOI={10.1002/9781118525524.ch4}, abstractNote={This chapter contains sections titled: Introduction Genomic Imprinting and Parental Conflict Theory Epigenetic Regulators of Arabidopsis Imprinting Mechanisms Establishing Arabidopsis Gene Imprinting Imprinting in the Embryo Imprinting in Monocots Evolution of Plant Imprinting Conclusion Acknowledgments References}, booktitle={Seed Genomics}, publisher={Wiley-Blackwell}, author={Ibarra, Christian A. and Frost, Jennifer M. and Shin, Juhyun and Hsieh, Tzung-Fu and Fischer, Robert L.}, year={2013}, month={Feb}, pages={63–82} }
 @article{zhang_hsieh_2013, title={Heritable Epigenetic Variation and its Potential Applications for Crop Improvement}, volume={1}, ISSN={2287-9366}, url={http://dx.doi.org/10.9787/PBB.2013.1.4.307}, DOI={10.9787/pbb.2013.1.4.307}, abstractNote={Phenotypic variation within organisms is driven primarily by genetic diversity. However, there is a growing appreciation that epigenetic variation, resulting from a multitude of diverse chemical modifications to the DNA and chromatin, can have profound effects on phenotype. Heritable epigenetic marks persist through meiosis and can be stably transmitted to the next generation, resulting in transgenerational epigenetic inheritance. Importantly, when epigenetic changes occur near coding genes, affecting their transcriptional state, heritable epigenetic variation can result in heritable phenotypic variation. Large-scale interrogation of epigenome inheritance in Arabidopsis has revealed that spontaneous variation in DNA methylation occurs at a rate that is orders of magnitude greater than genetic mutation, indicating the key importance of epigenetic variation during evolution. Thus, there is a potential for epigenetics to play a role in crop improvement, including regulation of transgene expression and creation of novel epialleles. Here, we review cases of naturally occurring and genetically induced epialleles, and discuss how the studies from two epigenetic populations are rapidly increasing our understanding of epigenetic diversity.}, number={4}, journal={Plant Breeding and Biotechnology}, publisher={Korean Society of Breeding Science}, author={Zhang, Changqing and Hsieh, Tzung-Fu}, year={2013}, month={Dec}, pages={307–319} }
 @article{ibarra_feng_schoft_hsieh_uzawa_rodrigues_zemach_chumak_machlicova_nishimura_et al._2012, title={Active DNA Demethylation in Plant Companion Cells Reinforces Transposon Methylation in Gametes}, volume={337}, ISSN={0036-8075 1095-9203}, url={http://dx.doi.org/10.1126/science.1224839}, DOI={10.1126/science.1224839}, abstractNote={Intergenerational Transposable Shutdown
          
            Transposable elements (TEs) are a potential threat, especially to the germline genome. In many eukaryotes, TEs are shut down by DNA methylation and/or small-RNA–mediated silencing. Therefore, it seems counterintuitive that results obtained by
            
              Ibarra
              et al.
            
            (p.
            1360
            ) on
            Arabidopsis
            showed that in the cells of this plant's sexual apparatus, many small TEs are demethylated by DEMETER (DME) DNA glycosylase and become activated. But it turns out that activation of the TEs triggers the formation of small-interfering RNAs, which in these experiments were seen to travel from the surrounding cells to the egg. Thus, activation of TEs in the companion cells “immunizes” the gametes via the interfering RNAs that shutdown the TEs in the gametes permanently.
          }, number={6100}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Ibarra, C. A. and Feng, X. and Schoft, V. K. and Hsieh, T.-F. and Uzawa, R. and Rodrigues, J. A. and Zemach, A. and Chumak, N. and Machlicova, A. and Nishimura, T. and et al.}, year={2012}, month={Sep}, pages={1360–1364} }
 @article{pedersen_hsieh_ibarra_fischer_2011, title={MethylCoder: software pipeline for bisulfite-treated sequences}, volume={27}, ISSN={1367-4803 1460-2059}, url={http://dx.doi.org/10.1093/bioinformatics/btr394}, DOI={10.1093/bioinformatics/btr394}, abstractNote={Abstract
               Motivation: MethylCoder is a software program that generates per-base methylation data given a set of bisulfite-treated reads. It provides the option to use either of two existing short-read aligners, each with different strengths. It accounts for soft-masked alignments and overlapping paired-end reads. MethylCoder outputs data in text and binary formats in addition to the final alignment in SAM format, so that common high-throughput sequencing tools can be used on the resulting output. It is more flexible than existing software and competitive in terms of speed and memory use.
               Availability: MethylCoder requires only a python interpreter and a C compiler to run. Extensive documentation and the full source code are available under the MIT license at: https://github.com/brentp/methylcode.
               Contact:  bpederse@gmail.com}, number={17}, journal={Bioinformatics}, publisher={Oxford University Press (OUP)}, author={Pedersen, B. and Hsieh, T.-F. and Ibarra, C. and Fischer, R. L.}, year={2011}, month={Jun}, pages={2435–2436} }
 @article{hsieh_shin_uzawa_silva_cohen_bauer_hashimoto_kirkbride_harada_zilberman_et al._2011, title={Regulation of imprinted gene expression in Arabidopsis endosperm}, volume={108}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.1019273108}, DOI={10.1073/pnas.1019273108}, abstractNote={
            Imprinted genes are expressed primarily or exclusively from either the maternal or paternal allele, a phenomenon that occurs in flowering plants and mammals. Flowering plant imprinted gene expression has been described primarily in endosperm, a terminal nutritive tissue consumed by the embryo during seed development or after germination. Imprinted expression in
            Arabidopsis thaliana
            endosperm is orchestrated by differences in cytosine DNA methylation between the paternal and maternal genomes as well as by Polycomb group proteins. Currently, only 11 imprinted
            A. thaliana
            genes are known. Here, we use extensive sequencing of cDNA libraries to identify 9 paternally expressed and 34 maternally expressed imprinted genes in
            A. thaliana
            endosperm that are regulated by the DNA-demethylating glycosylase DEMETER, the DNA methyltransferase MET1, and/or the core Polycomb group protein FIE. These genes encode transcription factors, proteins involved in hormone signaling, components of the ubiquitin protein degradation pathway, regulators of histone and DNA methylation, and small RNA pathway proteins. We also identify maternally expressed genes that may be regulated by unknown mechanisms or deposited from maternal tissues. We did not detect any imprinted genes in the embryo. Our results show that imprinted gene expression is an extensive mechanistically complex phenomenon that likely affects multiple aspects of seed development.
          }, number={5}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Hsieh, Tzung-Fu and Shin, Juhyun and Uzawa, Rie and Silva, Pedro and Cohen, Stephanie and Bauer, Matthew J. and Hashimoto, Meryl and Kirkbride, Ryan C. and Harada, John J. and Zilberman, Daniel and et al.}, year={2011}, month={Jan}, pages={1755–1762} }
 @article{hsieh_ibarra_silva_zemach_eshed-williams_fischer_zilberman_2009, title={Genome-Wide Demethylation of Arabidopsis Endosperm}, volume={324}, ISSN={0036-8075 1095-9203}, url={http://dx.doi.org/10.1126/science.1172417}, DOI={10.1126/science.1172417}, abstractNote={Dynamic Imprinting
          
            Gene imprinting—the silencing of either a maternally derived or paternally derived gene allele—is controlled in large part by DNA methylation. In plants, imprinting occurs in the endosperm, which nourishes the embryonic plant.
            
              Gehring
              et al.
            
            (p.
            1447
            ) and
            
              Hsieh
              et al.
            
            (p.
            1451
            ) analyzed the dynamics of DNA methylation in the endosperm and embryo of
            Arabidopsis
            and found extensive demethylation in the endosperm, suggesting that many imprinted genes are likely to exist. Gehring
            et al.
            characterized five imprinted genes in detail. Four of the 10 known imprinted genes are related homeodomain transcription factors. Furthermore, 5′ sequences demethylated in several of the genes were found to be derived from transposable elements, which supports the idea that imprinting arose as a by-product of silencing invading DNA.
          }, number={5933}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Hsieh, T.-F. and Ibarra, C. A. and Silva, P. and Zemach, A. and Eshed-Williams, L. and Fischer, R. L. and Zilberman, D.}, year={2009}, month={Jun}, pages={1451–1454} }
 @article{stone_braybrook_paula_kwong_meuser_pelletier_hsieh_fischer_goldberg_harada_2008, title={Arabidopsis LEAFY COTYLEDON2 induces maturation traits and auxin activity: Implications for somatic embryogenesis}, volume={105}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.0712364105}, DOI={10.1073/pnas.0712364105}, abstractNote={
            LEAFY COTYLEDON2 (LEC2) is a central regulator of embryogenesis sufficient to induce somatic cells to form embryos when expressed ectopically. Here, we analyze the cellular processes induced by LEC2, a B3 domain transcription factor, that may underlie its ability to promote somatic embryogenesis. We show auxin-responsive genes are induced after LEC2 activation in seedlings. Genes encoding enzymes involved in auxin biosynthesis,
            YUC2
            and
            YUC4
            , are activated within 1 h after induction of LEC2 activity, and
            YUC4
            appears to be a direct transcriptional target of LEC2. We also show ectopic
            LEC2
            expression induces accumulation of seed storage protein and oil bodies in vegetative and reproductive organs, events that normally occur during the maturation phase of embryogenesis. Furthermore, LEC2 activates seed protein genes before an increase in RNAs encoding LEC1 or FUS3 is observed. Thus, LEC2 causes rapid changes in auxin responses and induces cellular differentiation characteristic of the maturation phase. The relevance of these changes to the ability of LEC2 to promote somatic embryogenesis is discussed.
          }, number={8}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Stone, S. L. and Braybrook, S. A. and Paula, S. L. and Kwong, L. W. and Meuser, J. and Pelletier, J. and Hsieh, T.-F. and Fischer, R. L. and Goldberg, R. B. and Harada, J. J.}, year={2008}, month={Feb}, pages={3151–3156} }
 @article{huh_bauer_hsieh_fischer_2008, title={Cellular Programming of Plant Gene Imprinting}, volume={132}, ISSN={0092-8674}, url={http://dx.doi.org/10.1016/j.cell.2008.02.018}, DOI={10.1016/j.cell.2008.02.018}, abstractNote={Gene imprinting, the differential expression of maternal and paternal alleles, independently evolved in mammals and in flowering plants. A unique feature of flowering plants is a double-fertilization event in which the sperm fertilize not only the egg, which forms the embryo, but also the central cell, which develops into the endosperm (an embryo-supporting tissue). The distinctive mechanisms of gene imprinting in the endosperm, which involve DNA demethylation and histone methylation, begin in the central cell and sperm prior to fertilization. Flowering plants might have coevolved double fertilization and imprinting to prevent parthenogenetic development of the endosperm. Gene imprinting, the differential expression of maternal and paternal alleles, independently evolved in mammals and in flowering plants. A unique feature of flowering plants is a double-fertilization event in which the sperm fertilize not only the egg, which forms the embryo, but also the central cell, which develops into the endosperm (an embryo-supporting tissue). The distinctive mechanisms of gene imprinting in the endosperm, which involve DNA demethylation and histone methylation, begin in the central cell and sperm prior to fertilization. Flowering plants might have coevolved double fertilization and imprinting to prevent parthenogenetic development of the endosperm.}, number={5}, journal={Cell}, publisher={Elsevier BV}, author={Huh, Jin Hoe and Bauer, Matthew J. and Hsieh, Tzung-Fu and Fischer, Robert L.}, year={2008}, month={Mar}, pages={735–744} }
 @article{huh_bauer_hsieh_fischer_2007, title={Endosperm gene imprinting and seed development}, volume={17}, ISSN={0959-437X}, url={http://dx.doi.org/10.1016/j.gde.2007.08.011}, DOI={10.1016/j.gde.2007.08.011}, abstractNote={Imprinting occurs in the endosperm of flowering plants. Endosperm, produced by fertilization of the central cell in the female gametophyte, is essential for embryo and seed development. Several imprinted genes play an important role in endosperm development. The mechanism of gene imprinting involves DNA methylation and histone modification. DNA methylation is actively removed at the imprinted alleles to be activated. Histone methylation mediated by the Polycomb group complex provides another layer of epigenetic regulation at the silenced alleles. Endosperm gene imprinting can be uncoupled from seed development when fertilization of the central cell is prevented. Imprinting may be a mechanism to ensure fertilization of the central cell thereby preventing parthenogenic development of the endosperm.}, number={6}, journal={Current Opinion in Genetics & Development}, publisher={Elsevier BV}, author={Huh, Jin Hoe and Bauer, Matthew J and Hsieh, Tzung-Fu and Fischer, Robert}, year={2007}, month={Dec}, pages={480–485} }
 @inbook{penterman_huh_hsieh_fischer_2007, title={Genomic Imprinting in Arabidopsis thaliana and Zea mays}, ISBN={9783540712343}, url={http://dx.doi.org/10.1007/7089_2007_112}, DOI={10.1007/7089_2007_112}, booktitle={Plant Cell Monographs}, publisher={Springer Berlin Heidelberg}, author={Penterman, Jon and Huh, Jin Hoe and Hsieh, Tzung-Fu and Fischer, Robert L.}, year={2007}, month={May}, pages={219–239} }
 @article{gehring_huh_hsieh_penterman_choi_harada_goldberg_fischer_2006, title={DEMETER DNA Glycosylase Establishes MEDEA Polycomb Gene Self-Imprinting by Allele-Specific Demethylation}, volume={124}, ISSN={0092-8674}, url={http://dx.doi.org/10.1016/j.cell.2005.12.034}, DOI={10.1016/j.cell.2005.12.034}, abstractNote={MEDEA (MEA) is an Arabidopsis Polycomb group gene that is imprinted in the endosperm. The maternal allele is expressed and the paternal allele is silent. MEA is controlled by DEMETER (DME), a DNA glycosylase required to activate MEA expression, and METHYLTRANSFERASE I (MET1), which maintains CG methylation at the MEA locus. Here we show that DME is responsible for endosperm maternal-allele-specific hypomethylation at the MEA gene. DME can excise 5-methylcytosine in vitro and when expressed in E. coli. Abasic sites opposite 5-methylcytosine inhibit DME activity and might prevent DME from generating double-stranded DNA breaks. Unexpectedly, paternal-allele silencing is not controlled by DNA methylation. Rather, Polycomb group proteins that are expressed from the maternal genome, including MEA, control paternal MEA silencing. Thus, DME establishes MEA imprinting by removing 5-methylcytosine to activate the maternal allele. MEA imprinting is subsequently maintained in the endosperm by maternal MEA silencing the paternal allele.}, number={3}, journal={Cell}, publisher={Elsevier BV}, author={Gehring, Mary and Huh, Jin Hoe and Hsieh, Tzung-Fu and Penterman, Jon and Choi, Yeonhee and Harada, John J. and Goldberg, Robert B. and Fischer, Robert L.}, year={2006}, month={Feb}, pages={495–506} }
 @article{duval_hsieh_2006, title={Patenting Applied to Genetic Sequence Information}, volume={23}, ISSN={0264-8725 2046-5556}, url={http://dx.doi.org/10.1080/02648725.2006.10648091}, DOI={10.1080/02648725.2006.10648091}, number={1}, journal={Biotechnology and Genetic Engineering Reviews}, publisher={Informa UK Limited}, author={Duval, Manuel and Hsieh, Tzung-Fu}, year={2006}, month={Dec}, pages={317–330} }
 @article{hsieh_fischer_2005, title={BIOLOGY OF CHROMATIN DYNAMICS}, volume={56}, ISSN={1543-5008 1545-2123}, url={http://dx.doi.org/10.1146/annurev.arplant.56.032604.144118}, DOI={10.1146/annurev.arplant.56.032604.144118}, abstractNote={ During the development of a multicellular organism, cell differentiation involves activation and repression of transcription programs that must be stably maintained during subsequent cell divisions. Chromatin remodeling plays a crucial role in regulating chromatin states that conserve transcription programs and provide a mechanism for chromatin states to be maintained as cells proliferate, a process referred to as epigenetic inheritance inheritance of a gene activity state that is not specified by DNA sequence . A large number of factors and protein complexes are now known to be involved in regulating the dynamic states of chromatin structure. Their biological functions and molecular mechanisms are beginning to be revealed. }, number={1}, journal={Annual Review of Plant Biology}, publisher={Annual Reviews}, author={Hsieh, Tzung-Fu and Fischer, Robert L.}, year={2005}, month={Jun}, pages={327–351} }
 @article{hsieh_hakim_ohad_fischer_2003, title={From flour to flower: how Polycomb group proteins influence multiple aspects of plant development}, volume={8}, ISSN={1360-1385}, url={http://dx.doi.org/10.1016/S1360-1385(03)00189-4}, DOI={10.1016/s1360-1385(03)00189-4}, abstractNote={Cell identity and differentiation are determined by patterns of regulatory gene expression. Spatially and temporally regulated homeotic gene expression defines segment identities along the anterior-posterior axis of animal embryos. Polycomb group (PcG) proteins form a cellular memory system that maintains the repressed state of homeotic gene expression. Conserved PcG proteins control multiple aspects of Arabidopsis development and maintain homeotic gene repression. In animals, PcG proteins repress their target genes by modifying histone tails through deacetylation and methylation, generating a PcG-specific histone code that recruits other chromatin remodeling proteins to establish a stable, heritable mechanism of epigenetic expression control. Plant PcG proteins might function through a similar biochemical mechanism owing to their conserved structural and functional relationship to animal PcG proteins.}, number={9}, journal={Trends in Plant Science}, publisher={Elsevier BV}, author={Hsieh, T.-F. and Hakim, O. and Ohad, N. and Fischer, R.L.}, year={2003}, month={Sep}, pages={439–445} }
 @inbook{chuang_hsieh_duval_thomas_2003, title={Genomic analysis of Arabidopsis gene expression in response to a systemic fungicide}, booktitle={Genomics of Plants and Fungi}, publisher={CRC Press}, author={Chuang, Huey-wen and Hsieh, Tzung-Fu and Duval, Manuel and Thomas, Terry L}, year={2003}, pages={251–268} }
 @article{duval_hsieh_kim_thomas_2002, title={Molecular characterization of AtNAM: a member of the Arabidopsis NAC domain superfamily}, volume={50}, ISSN={0167-4412}, url={http://dx.doi.org/10.1023/A:1016028530943}, DOI={10.1023/a:1016028530943}, abstractNote={The petunia NAM and ArabidopsisATAF1 and CUC2 genes define the conserved NAC domain. In petunia, loss-of-function nam mutants result in embryos that fail to elaborate shoot apical meristems (SAM), and nam seedlings do not develop shoots and leaves. We have isolated a NAC domain gene, AtNAM, from an Arabidopsis developing seed cDNA library. Expression of AtNAM mRNA is restricted primarily to the region of the embryo including the SAM. The AtNAM gene contains three exons and is located on Chromosome 1. In vivo assays in yeast demonstrate that AtNAM encodes a transcription factor and that the NAC domain includes a specific DNA binding domain (DBD). The AtNAM DBD is contained within a 60 amino acid region which potentially folds into a helix-turn-helix motif that specifically binds to the CaMV 35S promoter. The putative transcriptional activation domain is located in the C-terminal region of the protein, a highly divergent region among NAC domain-containing genes. The Arabidopsis genome contains 90 predicted NAC domain genes; we refer to these collectively as the AtNAC superfamily. The first two exons of all members of this superfamily encode the NAC domain. Most AtNAC genes contain three exons with the last exon encoding an activation domain. A subfamily of AtNAC genes contains additional terminal exons coding for protein domains whose functions are unknown.}, number={2}, journal={Plant Molecular Biology}, publisher={Springer Nature}, author={Duval, Manuel and Hsieh, Tzung-Fu and Kim, Soo Young and Thomas, Terry L.}, year={2002}, pages={237–248} }
 @article{gene expression in the developing mouse retina by est sequencing and microarray analysis._2001, url={http://europepmc.org/articles/PMC97568}, DOI={10.1093/nar/29.24.4983}, abstractNote={Retinal development occurs in mice between embryonic day E11.5 and post-natal day P8 as uncommitted neuroblasts assume retinal cell fates. The genetic pathways regulating retinal development are being identified but little is understood about the global networks that link these pathways together or the complexity of the expressed gene set required to form the retina. At E14.5, the retina contains mostly uncommitted neuroblasts and newly differentiated neurons. Here we report a sequence analysis of an E14.5 retinal cDNA library. To date, we have archived 15 268 ESTs and have annotated 9035, which represent 5288 genes. The fraction of singly occurring ESTs as a function of total EST accrual suggests that the total number of expressed genes in the library could approach 27 000. The 9035 ESTs were categorized by their known or putative functions. Representation of the genes involved in eye development was significantly higher in the retinal clone set compared with the NIA mouse 15K cDNA clone set. Screening with a microarray containing 864 cDNA clones using wild-type and brn-3b (-/-) retinal cDNA probes revealed a potential regulatory linkage between the transcription factor Brn-3b and expression of GAP-43, a protein associated with axon growth. The retinal EST database will be a valuable platform for gene expression profiling and a new source for gene discovery.}, journal={Nucleic acids research}, year={2001}, month={Dec} }
 @article{chen_hsieh_thomas_safe_2001, title={Identification of estrogen-induced genes downregulated by AhR agonists in MCF-7 breast cancer cells using suppression subtractive hybridization}, volume={262}, ISSN={0378-1119}, url={http://dx.doi.org/10.1016/S0378-1119(00)00530-8}, DOI={10.1016/s0378-1119(00)00530-8}, abstractNote={Aryl hydrocarbon receptor (AhR) agonists inhibit 17β-estradiol (E2) induced growth of MCF-7 human breast cancer cells in vitro and rodent mammary tumor growth in vivo. Genes associated with inhibitory AhR-estrogen receptor (ER) crosstalk were investigated in MCF-7 human breast cancer cells using poly(A)+RNA from cells treated with either 1 nM E2 (target) or E2 plus 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (reference) or 25 μM diindolylmethane (DIM) as AhR agonists in MCF-7 cells. Suppression subtractive hybridization (SSH) was subsequently used to identify 33 genes with sequence homology to known human genes that are induced by E2 and inhibited by AhR agonists in MCF-7 cells; two unknown genes were also identified. Many of these genes are involved in cell proliferation and these include cell cycle regulators (cdc28/cdc2-associated protein), nucleotide synthases (thymidylate synthase), early intermediate genes (early growth response α, EGRα) and other proteins involved in signaling pathways (calmodulin, ATP synthase α subunit). Thus SSH has identified a diverse spectrum of new genes that are affected by inhibitory AhR-ER crosstalk and among this group are a subset of genes that may be critical for the in vivo antitumorigenic effects of AhR agonists.}, number={1-2}, journal={Gene}, publisher={Elsevier BV}, author={Chen, I and Hsieh, T and Thomas, T and Safe, S}, year={2001}, month={Jan}, pages={207–214} }
 @article{nuccio_hsieh_thomas_2000, title={The use of RT-PCR differential display in single-celled organisms and plant tissues}, number={224}, journal={Differential Display: A Practical Approach}, publisher={Courier Corporation}, author={NUCCIO, MICHAEL L and HSIEH, TZUNG-FU and THOMAS, TERRY L}, year={2000}, pages={83} }
 @article{characterization and subcellular compartmentation of recombinant 4-hydroxyphenylpyruvate dioxygenase from arabidopsis in transgenic tobacco._1999, url={https://doi.org/10.1104/pp.119.4.1507}, DOI={10.1104/pp.119.4.1507}, abstractNote={Abstract
               4-Hydroxyphenylpyruvate dioxygenase (4HPPD) catalyzes the formation of homogentisate (2,5-dihydroxyphenylacetate) fromp-hydroxyphenylpyruvate and molecular oxygen. In plants this enzyme activity is involved in two distinct metabolic processes, the biosynthesis of prenylquinones and the catabolism of tyrosine. We report here the molecular and biochemical characterization of an Arabidopsis 4HPPD and the compartmentation of the recombinant protein in chlorophyllous tissues. We isolated a 1508-bp cDNA with one large open reading frame of 1338 bp. Southern analysis strongly suggested that this Arabidopsis 4HPPD is encoded by a single-copy gene. We investigated the biochemical characteristics of this 4HPPD by overproducing the recombinant protein in Escherichia coli JM105. The subcellular localization of the recombinant 4HPPD in chlorophyllous tissues was examined by overexpressing its complete coding sequence in transgenic tobacco (Nicotiana tabacum), using Agrobacteriumtumefaciens transformation. We performed western analyses for the immunodetection of protein extracts from purified chloroplasts and total leaf extracts and for the immunocytochemistry on tissue sections. These analyses clearly revealed that 4HPPD was confined to the cytosol compartment, not targeted to the chloroplast. Western analyses confirmed the presence of a cytosolic form of 4HPPD in cultured green Arabidopsis cells.}, journal={Plant physiology}, year={1999}, month={Apr} }
 @inbook{garcia_rodgers_pépin_hsieh_matringe_1998, title={Plant P-Hydroxyphenylpyruvate Dioxygenase: A Target for New Bleaching Herbicides}, ISBN={9780792355472 9789401139533}, url={http://dx.doi.org/10.1007/978-94-011-3953-3_900}, DOI={10.1007/978-94-011-3953-3_900}, booktitle={Photosynthesis: Mechanisms and Effects}, publisher={Springer Netherlands}, author={Garcia, I. and Rodgers, M. and Pépin, R. and Hsieh, Tzung-Fu and Matringe, M.}, year={1998}, pages={3861–3864} }
 @article{molecular approaches to identify novel genes expressed in arabidopsis thaliana._1997, journal={SAAS bulletin, biochemistry and biotechnology}, year={1997}, month={Jan} }
 @inproceedings{hsieh_thomas_1996, title={Homeobox genes expressed in Arabidopsis embryo}, volume={111}, number={2}, booktitle={PLANT PHYSIOLOGY}, author={Hsieh, TF and Thomas, TL}, year={1996}, pages={733–733} }
 @article{identification of novel mrnas in immature seeds of arabidopsis thaliana._1996, journal={SAAS bulletin, biochemistry and biotechnology}, year={1996}, month={Jan} }
 @article{chung_hsieh_wang_1991, title={Identification of phospholipase activities in the exotoxin of Edwardsiella tarda}, volume={29}, journal={Reports On Fish Disease Research}, author={Chung, Y.W. and Hsieh, T.-F. and Wang, C.T.}, year={1991}, pages={39–46} }