|Friday, July 09|
Clade III TGACG-motif binding basic leucine zipper transcription factors mediate BLADE-ON-PETIOLE dependent regulation of plant development
* Sibei Li, Carleton University, Canada
Shelley Hepworth, Carleton University
Plant growth and development rely on meristems. Lateral organs formed by the shoot apical meristem (SAM) are separated from surrounding stem cells by regions of low growth called boundaries. Impairment of boundaries can lead to SAM termination and/or structural defects at the base of organs. Specific aspects of plant architecture controlled by boundaries include leaf shape, branching complexity, and abscission. In the model plant species Arabidopsis thaliana, BLADE-ON-PETIOLE 1 and 2 (BOP1/2) represent a class of genes important for boundary patterning in land plants. Members of this family lack a DNA binding domain and interact with TGACG-motif binding (TGA) basic leucine zipper (bZIP) transcription factors for recruitment to DNA. Here, we show that clade III TGA transcription factors, TGA3 and TGA7, previously associated with plant defense, are important partners of BOP1/2 in regulation of plant development. TGA3 and TGA7 are expressed at organ boundaries and function in the same genetic pathway as BOP1 and BOP2 required for SAM maintenance, flowering, and inflorescence architecture. Further, BOP1 and BOP2 require TGA3 and TGA7 for the deposition of lignin in vascular tissues of the stem. Yeast two-hybrid assays show that TGA3 and TGA7 proteins form a complex with BOP1 and BOP2, possibly involved in the co-activation of genes required for boundary establishment or lignin biosynthesis. These studies identity a dual role for clade III TGA factors in development and defense.
Discerning Biological Function of the Cytosine Methyltransferase DNMT2 in the Moss Physcomitrium patens
Meenu Kapoor, Guru Gobind Singh Indraprastha University, India
* Darshika Singh, Guru Gobind Singh Indraprastha University
Radha Yadav, Guru Gobind Singh Indraprastha University
Nikita Wadhwa, Guru Gobind Singh Indraprastha University
DNA Methyltransferase2/tRNA Methyltransferase1 (DNMT2/TRDMT1) is a m5C RNA methyltransferase that methylates cytosine-38 in the anticodon stem loop of Aspartic acid, Valine and Glycine tRNAs. This modification is crucial for stability of tRNAs and for translation of poly Asp containing proteins under stress. DNMT2 is highly conserved in eukaryotes. Unlike animal systems where its role in cellular response to heat, oxidative and arsenite stress is well established, its biological function in land plants has remained enigmatic. In this study, we describe the role of DNMT2 in the moss Physcomitrium patens. On the basis of reverse genetics, biochemical, genome-wide transcriptomic and quantitative proteomic studies we show that PpDNMT2 plays a crucial role in oxidative stress management, ion homeostasis, chromatin and epigenetic gene regulation in moss protonemata. PpDNMT2-deficient plants are indistinguishable from wild type plants under standard laboratory conditions. However, under salt and osmotic stress the mutants are unable to maintain stable tRNAAsp, transcript levels and biochemical activities of antioxidant enzymes, levels of apoptosis associated protein encoding genes, levels of genes encoding Ubiquitin-26S proteasome components and genome stability. Our work also reveals that PpDNMT2 exists in complex with CuZn-SOD in vivo and in yeast nuclei. On the basis of biochemical, quantitative gene expression and protein deletion studies we further show that PpDNMT2 function possibly affects activity of other tRNA modifying enzymes as levels of genes encoding tRNA-Guanine-N-7-methyltransferases (Trm8) that catalyze formation of m7G46 in many tRNAs are differentially expressed in ppdnmt2. Further, PpDNMT2 also physically interacts with Trm8, Trm8L1 and Trm8L2 in vivo.
Extensive N4 Cytosine Methylation is Essential for Marchantia Sperm Function
* James Walker, John Innes Centre, United Kingdom
Jingyi Zhang, John Innes Centre
Yalin Liu, John Innes Centre
Martin Vickers, John Innes Centre
Liam Dolan, Gregor Mendel Institute
Keiji Nakajima, Nara Institute of Science and Technology
Xiaoqi Feng, John Innes Centre
4-methylcytosine (4mC) is an important DNA modification in prokaryotes, but its relevance, and even presence in eukaryotes have been mysterious. Here we show that spermatogenesis in the liverwort Marchantia polymorpha involves two waves of extensive DNA methylation reprogramming. First, 5-methylcytosine (5mC), a well-known eukaryotic DNA modification, expands from transposons to the entire genome. Notably, the second wave installs 4mC throughout genic regions, covering over 50% of CG sites in sperm. 4mC is catalyzed by two novel methyltransferases (MpDN4MT1a and MpDN4MT1b) specifically expressed during late spermiogenesis. Deletion of MpDN4MT1s eliminates 4mC, alters the sperm transcriptome, and produces sperm with swimming defects. Our results reveal extensive 4mC in a eukaryote and define a new family of eukaryotic methyltransferases, thereby expanding the repertoire of functional eukaryotic DNA modifications.
Novel de novo DNA methylation by CMT and DNMT3 orthologs in Physcomitrella patens and their role in genome regulation
* Nir Ohad, Tel Aviv University, Israel
Rafael Yaari , Tel Aviv University
Katherine Domb , Tel Aviv University
Aviva Katz , Tel Aviv University
Keith Harris , Tel Aviv University
Assaf Zemach , Tel Aviv University
Cytosine methylation, a key process in regulating genome function, is established and maintained by DNA methyltransferases (DNMTs). DNMT3 known to establish methylation in mammals. In plants, altered DNMT3 homologs, DOMAINS REARRANGED METHYLTRANSFERASEs (DRMs), was shown to establish methylation via the RNA directed DNA methylation (RdDM) pathway, yet the role of true plant DNMT3 orthologs remained elusive. To elucidate the role of plant DNMTs, we profiled both genomic and de novo methylation in the basal moss plant, Physcomitrella patens, mutated in each of its PpDNMTs. To evaluate P. patens DNMTs role in de novo methylation, we introduced the repetitive DNA sequence (RPS) from Petunia hybrida, uncommon to P. patens. Methylation analysis of RPS at the first transgenic generation (T1), using bisulfite sequencing, reveal that RPS is methylated in WT cells in all three methylation contexts, CG, CHG, and CHH, implying on its ability to be de novo methylated in P. patens. Methylation status of Ppdnmt mutants reveals, that PpDNMT3b mediates CG and CHH de novo methylation, independently of PpDRMs. Complementary de novo CHG methylation is specifically mediated by the CHROMOMETHYLASE, PpCMT. Intragenomically, PpDNMT3b functions preferentially within heterochromatin and is affected by PpCMT. In comparison, PpDRMs target active-euchromatic transposons. Our data resolve how DNA methylation in plants can be established in heterochromatin independently of RdDM pathway; suggesting that DRMs have emerged to target euchromatin; and link DNMT3 loss in angiosperms to the initiation of heterochromatic CHH methylation by CMT2. The role of CG and non-CG methylation in regulating transcription will be discussed.
Riccia fluitans, an informative amphibious liverwort to study plant terrestrialization
* Felix Althoff, Botany Department, School of Biology and Chemistry, Osnabrück University, Germany
Sabine Zachgo, Botany Department, School of Biology and Chemistry, Osnabrück University
The colonization of land by streptophyte algae, ancestors of embryophyte plants, was a fundamental event in the history of life on earth. Bryophytes are evolutionary informative as they enable to investigate adaptation mechanisms from freshwater to terrestrial ecosystems. The amphibious liverwort Riccia fluitans, a member of the most species-rich genus in complex liverworts, can thrive in aquatic and terrestrial habitats by undergoing morphological transitions resulting in distinctive water and land form features. The R. fluitans water form develops slender, elongated thalli that branch occasionally and lack air pores and rhizoids. Thallus of the R. fluitans land form is wider, frequently branched and forms air pores and rhizoids. This morphological plasticity realized by one genotype makes R. fluitans ideal to study the adaptive molecular mechanisms enabling the colonialization of land by aquatic plants. To make R. fluitans amenable for genetic analyses, we established a transformation protocol using R. fluitans callus tissue and generated the first transgenic R. fluitans lines. Furthermore, for comprehensive studies spanning all R. fluitans life stages, the switch from vegetative to reproductive development can be induced by flooding or starvation of land form thalli. These new transformation and sexual induction protocols will make R. fluitans accessible in the future for investigating adaptations to a terrestrial plant life style. Althoff, F., Zachgo, S. (2020). Transformation of Riccia fluitans, an amphibious liverwort dynamically responding to environmental changes. International Journal of Molecular Sciences 21 (15): 5410
Role of Phytochelatin Synthase in metal detoxification in Marchantia polymorpha
Mingai Li, Fondazione Edmund Mach
Martina Leso, Fondazione Edmund Mach
Erika Bellini, Università di Pisa
Matteo Buti, Università di Firenze
Alesandro Saba, Università di Pisa
Luigi Sanità di Toppi, Università di Pisa
* Claudio Varotto, Fondazione Edmund Mach, Italy
Marchantia polymorpha has an active Phytochelatin synthase (PCS), but its actual contribution to heavy metal tolerance in vivo remains an open question. The aim of the present work was to elucidate functionally the role of MpPCS in the detoxification of different heavy metals and to characterize the effects that its knockout has on the plant transcriptome. We obtained by CRISPR/Cas9 genome editing two independent mutant alleles of PCS, Mppcsge_1 and Mppcsge_2 and tested them for susceptibility to different metal(loid)s. We further compared the WT and Mppcsge_1 transcriptomes by Illumina RNA-Seq analysis of differentially expressed genes followed by pathway analysis. Thiol-peptide quantification demonstrated the complete lack of phytochelatins in the mutants, which showed no visible phenotype in normal growth conditions. However, they resulted extremely sensitive to treatment with cadmium, but tolerant to zinc. Noteworthy, mutant plants were highly tolerant to the metalloid arsenic. Whole transcriptome analysis of WT and one of the mutant lines identified a range of differentially expressed genes in different metabolic pathways that clearly distinguish the mutant even in the absence of heavy metal stress and of a visible phenotype. These results demonstrate that the phyochelatin-mediated detoxification of heavy metals is an ancestral trait in land plants. Despite PCS plays a major role in the detoxification of divalent, non-essential heavy metals, the results further suggest that phytochelatin synthase may participate to metal homeostasis in normal conditions. The mutant genotypes obtained further represent highly sensitive bioindicators of Cd contamination, and as such they could find application in biomonitoring.
Finetuning the in vitro growth of the model hornwort Anthoceros agrestis
* Andika Gunadi, Boyce Thompson Institute, United States
Joyce Van Eck, Boyce Thompson Institute
Fay-Wei Li, Boyce Thompson Institute
Hornworts are an understudied lineage of bryophytes with unique sets of evolutionarily and agriculturally relevant genes. As part of a concerted effort to develop effective functional genetic tools in hornworts, we evaluated the contribution of several culture medium addenda on the gametophytic growth of the model hornwort Anthoceros agrestis (Oxford strain). Tissue development was rapidly quantified using time-lapse imaging spanning four weeks of culture of homogenized tissue grown on modifications of Hatcher’s solidified medium. Equal amounts of starting tissue material were added to each medium at four petri dishes (replicates). Aside from microscopic observations, whole petri dish images were captured and analyzed through trainable image segmentation to obtain two-dimensional area of total green tissue (thalli) per dish. Incremental addition of exogenous activated charcoal, ammonium nitrate, sucrose, MES buffering, Gamborg vitamins and growth regulators (6-benzylaminopurine, 2,4-dichlorophenoxyacetic acid and thidiazuron) were evaluated, resulting in a gradient of phenotypic effects that include changes in gametophyte tissue survival, tissue color, patterns and speed of thalli growth, as well as frequency of rhizoid formation. Based on these findings, an improved medium composition and growth regimen for A. agrestis gametophyte tissue was formulated, which maintained tissue vitality while increasing the average area of thalli by more than five times following three weeks of culture. Importantly, this amount of thalli growth was more than double the amount after four weeks of culture on various baseline hornwort solidified media (Hatcher’s, BCD and KNOP). These results provide critical groundwork towards establishing effective tools for studying gene function in hornworts.