@article{ma_li_wu_jiang_chen_xing_zhao_liu_jiang_xia_et al._2024, title={<i>Camellia</i><i> sinensis</i> CsMYB4a participates in regulation of stamen growth by interaction with auxin signaling transduction repressor CsAUX/IAA4}, volume={12}, ISSN={["2214-5141"]}, DOI={10.1016/j.cj.2023.11.006}, abstractNote={Subgroup 4 (Sg4) members of the R2R3-MYB are generally known as negative regulators of the phenylpropanoid pathway in plants. Our previous research showed that a R2R3-MYB Sg4 member from Camellia sinensis (CsMYB4a) inhibits expression of some genes in the phenylpropanoid pathway, but its physiological function in the tea plant remained unknown. Here, CsMYB4a was found to be highly expressed in anther and filaments, and participated in regulating filament growth. Transcriptome analysis and exogenous auxin treatment showed that the target of CsMYB4a might be the auxin signal pathway. Auxin/indole-3-acetic acid 4 (AUX/IAA4), a repressor in auxin signal transduction, was detected from a yeast two-hybrid screen using CsMYB4a as bait. Gene silencing assays showed that both CsIAA4 and CsMYB4a regulate filament growth. Tobacco plants overexpressing CsIAA4 were insensitive to exogenous α-NAA, consistent with overexpression of CsMYB4a. Protein-protein interaction experiments revealed that CsMYB4a interacts with N-terminal of CsIAA4 to prevent CsIAA4 degradation. Knock out of the endogenous NtIAA4 gene, a CsIAA4 homolog, in tobacco alleviated filament growth inhibition and α-NAA insensitivity in plants overexpressing CsMYB4a. All results strongly suggest that CsMYB4a works synergistically with CsIAA4 and participates in regulation of the auxin pathway in stamen.}, number={1}, journal={CROP JOURNAL}, author={Ma, Guoliang and Li, Mingzhuo and Wu, Yingling and Jiang, Changjuan and Chen, Yifan and Xing, Dawei and Zhao, Yue and Liu, Yajun and Jiang, Xiaolan and Xia, Tao and et al.}, year={2024}, month={Feb}, pages={188–201} }
 @article{li_wang_wang_guo_liu_jiang_gao_xia_2024, title={Duplicated chalcone synthase (CHS) genes modulate flavonoid production in tea plants in response to light stress}, volume={23}, ISSN={["2352-3425"]}, DOI={10.1016/j.jia.2024.03.060}, abstractNote={In tea plants, the abundant flavonoid compounds are responsible for the health benefits for the human body and define the astringent flavor profile. While the downstream mechanisms of flavonoid biosynthesis have been extensively studied, the role of chalcone synthase (CHS) in this secondary metabolic process within tea plants remains less clear. In our current study, we compared the evolutionary profile of the flavonoid metabolism pathway and discovered that gene duplication of CHS occurred in tea plants. We identified three CsCHS genes, along with a CsCHS-like gene, as potential candidates for further functional investigation. Unlike the CsCHS-like gene, the CsCHS genes effectively restored flavonoid production in Arabidopsis chs-mutants. Additionally, CsCHS transgenic tobacco plants exhibited higher flavonoid compound accumulation compared to their wild-type counterparts. Most notably, our examination of promoter and gene expression levels for the selected CHS genes revealed distinct responses to UV-B stress in tea plants. Our findings suggest that environmental factors such as UV-B exposure could be key drivers behind the gene duplication events in CHS.}, number={6}, journal={JOURNAL OF INTEGRATIVE AGRICULTURE}, author={Li, Mingzhuo and Wang, Wenzhao and Wang, Yeru and Guo, Lili and Liu, Yajun and Jiang, Xiaolan and Gao, Liping and Xia, Tao}, year={2024}, month={Jun}, pages={1940–1955} }
 @article{judd_dong_sun_zhu_li_xie_2023, title={Metabolic engineering of the anthocyanin biosynthetic pathway in Artemisia annua and relation to the expression of the artemisinin biosynthetic pathway}, volume={257}, ISSN={["1432-2048"]}, url={https://doi.org/10.1007/s00425-023-04091-6}, DOI={10.1007/s00425-023-04091-6}, abstractNote={Four types of cells were engineered from Artemisia annua to produce approximately 17 anthocyanins, four of which were elucidated structurally. All of them expressed the artemisinin pathway. Artemisia annua is the only medicinal crop to produce artemisinin for the treatment of malignant malaria. Unfortunately, hundreds of thousands of people still lose their life every year due to the lack of sufficient artemisinin. Artemisinin is considered to result from the spontaneous autoxidation of dihydroartemisinic acid in the presence of reactive oxygen species (ROS) in an oxidative condition of glandular trichomes (GTs); however, whether increasing antioxidative compounds can inhibit artemisinin biosynthesis in plant cells is unknown. Anthocyanins are potent antioxidants that can remove ROS in plant cells. To date, no anthocyanins have been structurally elucidated from A. annua. In this study, we had two goals: (1) to engineer anthocyanins in A. annua cells and (2) to understand the artemisinin biosynthesis in anthocyanin-producing cells. Arabidopsis Production of Anthocyanin Pigment 1 was used to engineer four types of transgenic anthocyanin-producing A. annua (TAPA1-4) cells. Three wild-type cell types were developed as controls. TAPA1 cells produced the highest contents of total anthocyanins. LC-MS analysis detected 17 anthocyanin or anthocyanidin compounds. Crystallization, LC/MS/MS, and NMR analyses identified cyanidin, pelargonidin, one cyanin, and one pelargonin. An integrative analysis characterized that four types of TAPA cells expressed the artemisinin pathway and TAPA1 cells produced the highest artemisinin and artemisinic acid. The contents of arteannuin B were similar in seven cell types. These data showed that the engineering of anthocyanins does not eliminate the biosynthesis of artemisinin in cells. These data allow us to propose a new hypothesis that enzymes catalyze the formation of artemisinin from dihydroartemisinic acid in non-GT cells. These findings show a new platform to increase artemisinin production via non-GT cells of A. annua.}, number={3}, journal={PLANTA}, author={Judd, Rika and Dong, Yilun and Sun, Xiaoyan and Zhu, Yue and Li, Mingzhuo and Xie, De-Yu}, year={2023}, month={Mar} }
 @article{li_he_la hovary_zhu_dong_liu_xing_liu_jie_ma_et al._2022, title={A de novo regulation design shows an effectiveness in altering plant secondary metabolism}, volume={37}, ISSN={["2090-1224"]}, url={http://dx.doi.org/10.1016/j.jare.2021.06.017}, DOI={10.1016/j.jare.2021.06.017}, abstractNote={Transcription factors (TFs) and cis-regulatory elements (CREs) control gene transcripts involved in various biological processes. We hypothesize that TFs and CREs can be effective molecular tools for De Novo regulation designs to engineer plants. We selected two Arabidopsis TF types and two tobacco CRE types to design a De Novo regulation and evaluated its effectiveness in plant engineering. G-box and MYB recognition elements (MREs) were identified in four Nicotiana tabacum JAZs (NtJAZs) promoters. MRE-like and G-box like elements were identified in one nicotine pathway gene promoter. TF screening led to select Arabidopsis Production of Anthocyanin Pigment 1 (PAP1/MYB) and Transparent Testa 8 (TT8/bHLH). Two NtJAZ and two nicotine pathway gene promoters were cloned from commercial Narrow Leaf Madole (NL) and KY171 (KY) tobacco cultivars. Electrophoretic mobility shift assay (EMSA), cross-linked chromatin immunoprecipitation (ChIP), and dual-luciferase assays were performed to test the promoter binding and activation by PAP1 (P), TT8 (T), PAP1/TT8 together, and the PAP1/TT8/Transparent Testa Glabra 1 (TTG1) complex. A DNA cassette was designed and then synthesized for stacking and expressing PAP1 and TT8 together. Three years of field trials were performed by following industrial and GMO protocols. Gene expression and metabolic profiling were completed to characterize plant secondary metabolism. PAP1, TT8, PAP1/TT8, and the PAP1/TT8/TTG1 complex bound to and activated NtJAZ promoters but did not bind to nicotine pathway gene promoters. The engineered red P + T plants significantly upregulated four NtJAZs but downregulated the tobacco alkaloid biosynthesis. Field trials showed significant reduction of five tobacco alkaloids and four carcinogenic tobacco specific nitrosamines in most or all cured leaves of engineered P + T and PAP1 genotypes. G-boxes, MREs, and two TF types are appropriate molecular tools for a De Novo regulation design to create a novel distant-pathway cross regulation for altering plant secondary metabolism.}, journal={JOURNAL OF ADVANCED RESEARCH}, publisher={Elsevier BV}, author={Li, Mingzhuo and He, Xianzhi and La Hovary, Christophe and Zhu, Yue and Dong, Yilun and Liu, Shibiao and Xing, Hucheng and Liu, Yajun and Jie, Yucheng and Ma, Dongming and et al.}, year={2022}, month={Mar}, pages={43–60} }
 @article{li_guo_wang_li_jiang_liu_xie_gao_xia_2022, title={Molecular and biochemical characterization of two 4-coumarate: CoA ligase genes in tea plant (Camellia sinensis)}, volume={109}, ISSN={["1573-5028"]}, url={https://doi.org/10.1007/s11103-022-01269-6}, DOI={10.1007/s11103-022-01269-6}, abstractNote={Two 4-coumarate: CoA ligase genes in tea plant involved in phenylpropanoids biosynthesis and response to environmental stresses. Tea plant is rich in flavonoids benefiting human health. Lignin is essential for tea plant growth. Both flavonoids and lignin defend plants from stresses. The biosynthesis of lignin and flavonoids shares a key intermediate, 4-coumaroyl-CoA, which is formed from 4-coumaric acid catalyzed by 4-coumaric acid: CoA ligase (4CL). Herein, we report two 4CL paralogs from tea plant, Cs4CL1 and Cs4CL2, which are a member of class I and II of this gene family, respectively. Cs4CL1 was mainly expressed in roots and stems, while Cs4CL2 was mainly expressed in leaves. The promoter of Cs4CL1 had AC, nine types of light sensitive (LSE), four types of stress-inducible (SIE), and two types of meristem-specific elements (MSE). The promoter of Cs4CL2 also had AC and nine types of LSEs, but only had two types of SIEs and did not have MSEs. In addition, the LSEs varied in the two promoters. Based on the different features of regulatory elements, three stress treatments were tested to understand their expression responses to different conditions. The resulting data indicated that the expression of Cs4CL1 was sensitive to mechanical wounding, while the expression of Cs4CL2 was UV-B-inducible. Enzymatic assays showed that both recombinant Cs4CL1 and Cs4CL2 transformed 4-coumaric acid (CM), ferulic acid (FR), and caffeic acid (CF) to their corresponding CoA ethers. Kinetic analysis indicated that the recombinant Cs4CL1 preferred to catalyze CF, while the recombinant Cs4CL2 favored to catalyze CM. The overexpression of both Cs4CL1 and Cs4CL2 increased the levels of chlorogenic acid and total lignin in transgenic tobacco seedlings. In addition, the overexpression of Cs4CL2 consistently increased the levels of three flavonoid compounds. These findings indicate the differences of Cs4CL1 and Cs4CL2 in the phenylpropanoid metabolism.}, number={4-5}, journal={PLANT MOLECULAR BIOLOGY}, publisher={Springer Science and Business Media LLC}, author={Li, Mingzhuo and Guo, Lili and Wang, Yeru and Li, Yanzhi and Jiang, Xiaolan and Liu, Yajun and Xie, De-Yu and Gao, Liping and Xia, Tao}, year={2022}, month={May} }
 @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{wang_zhou_wu_dai_liu_qian_li_jiang_wang_gao_et al._2018, title={Insight into Catechins Metabolic Pathways of Camellia sinensis Based on Genome and Transcriptome Analysis}, volume={66}, ISSN={["1520-5118"]}, DOI={10.1021/acs.jafc.8b00946}, abstractNote={Tea is an important economic crop with a 3.02 Gb genome. It accumulates various bioactive compounds, especially catechins, which are closely associated with tea flavor and quality. Catechins are biosynthesized through the phenylpropanoid and flavonoid pathways, with 12 structural genes being involved in their synthesis. However, we found that in Camellia sinensis the understanding of the basic profile of catechins biosynthesis is still unclear. The gene structure, locus, transcript number, transcriptional variation, and function of multigene families have not yet been clarified. Our previous studies demonstrated that the accumulation of flavonoids in tea is species, tissue, and induction specific, which indicates that gene coexpression patterns may be involved in tea catechins and flavonoids biosynthesis. In this paper, we screened candidate genes of multigene families involved in the phenylpropanoid and flavonoid pathways based on an analysis of genome and transcriptome sequence data. The authenticity of candidate genes was verified by PCR cloning, and their function was validated by reverse genetic methods. In the present study, 36 genes from 12 gene families were identified and were accessed in the NCBI database. During this process, some intron retention events of the CsCHI and CsDFR genes were found. Furthermore, the transcriptome sequencing of various tea tissues and subcellular location assays revealed coexpression and colocalization patterns. The correlation analysis showed that CsCHIc, CsF3'H, and CsANRb expression levels are associated significantly with the concentration of soluble PA as well as the expression levels of CsPALc and CsPALf with the concentration of insoluble PA. This work provides insights into catechins metabolism in tea and provides a foundation for future studies.}, number={16}, journal={JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY}, author={Wang, Wenzhao and Zhou, Yihui and Wu, Yingling and Dai, Xinlong and Liu, Yajun and Qian, Yumei and Li, Mingzhuo and Jiang, Xiaolan and Wang, Yunsheng and Gao, Liping and et al.}, year={2018}, month={Apr}, pages={4281–4293} }
 @article{li_xi_ji_li_xie_2018, title={Non-plastidial expression of a synthetic insect geranyl pyrophosphate synthase effectively increases tobacco plant biomass}, volume={221}, ISSN={["1618-1328"]}, DOI={10.1016/j.jplph.2017.12.014}, abstractNote={Designing effective synthetic genes of interest is a fundamental step in plant synthetic biology for biomass. Geranyl pyrophosphate (diphosphate) synthase (GPPS) catalyzes a bottleneck step toward terpenoid metabolism. We previously designed and synthesized a plant (Arabidopsis thaliana)-insect (Myzus persicae, Mp) GPPS- human influenza hemagglutinin (HA) cDNA, namely PTP-MpGPPS-HA (or PTP-sMpGPPS-HA, s: synthetic), to localize the protein in plastids and improve plant biomass. To better understand the effects of different subcellular localizations on plant performance, herein we report PTP-sMpGPPS-HA re-design to synthesize a new MpGPPS-HA cDNA, namely sMpGPPS-HA, to express a non-plastidial sMpGPPS-HA protein. The sMpGPPS-HA cDNA driven by a 2 × S 35S promoter was introduced into Nicotiana tabacum Xanthi. PTP-MpGPPS-HA and PMDC84 vector transgenic plants were also generated as positive and negative controls, respectively. Eighteen to twenty transgenic T0 lines were generated for each sMpGPPS-HA, PTP-sMpGPPS-HA, and PMDC84. Transcriptional genotyping analysis demonstrated the expression of sMpGPPS-HA in transgenic plants. Confocal microscopy analysis of transgenic progeny demonstrated the non-plastidial localization of sMpGPPS-HA. Growth of T1 transgenic and wild-type control plants showed that the expression of sMpGPPS-HA effectively increased plant height by 50-80%, leaf numbers and sizes, and dry biomass by 60-80%. Calculation of the vegetative growth rates showed that the expression of sMpGPPS-HA increased plant height each week. Moreover, sMpGPPS-HA expression promoted early flowering and reduced leaf carotenoid levels. In conclusion, non-plastidial expression of the novel sMpGPPS-HA was effective for improving tobacco growth and biomass. Our data indicate that research examining different subcellular localizations facilitates a better understanding of in planta functions of proteins encoded by synthetic cDNAs.}, journal={JOURNAL OF PLANT PHYSIOLOGY}, author={Li, Gui and Xi, Jing and Ji, Xiaoming and Li, Ming-Zhuo and Xie, De-Yu}, year={2018}, month={Feb}, pages={144–155} }