Control of sporophyte secondary cell wall development in Marchantia by a Class II KNOX gene.

Land plants evolved from an ancestral alga around 470 mya, evolving complex multicellularity in both haploid gametophyte and diploid sporophyte generations. The evolution of water-conducting tissues in the sporophyte generation was crucial for the success of land plants, paving the way for the colonization of a variety of terrestrial habitats. Class II KNOX (KNOX2) genes are major regulators of secondary cell wall formation and seed mucilage (pectin) deposition in flowering plants. Here, we show that, in the liverwort Marchantia polymorpha, loss-of-function alleles of the KNOX2 ortholog, MpKNOX2, or its dimerization partner, MpBELL1, have defects in capsule wall secondary cell wall and spore pectin biosynthesis. Both genes are expressed in the gametophytic calyptra surrounding the sporophyte and exert maternal effects, suggesting intergenerational regulation from the maternal gametophyte to the sporophytic embryo. These findings also suggest the presence of a secondary wall genetic program in the non-vascular liverwort capsule wall, with attributes of secondary walls in vascular tissues.

Contrasting and conserved roles of NPR pathways in diverged land plant lineages.

The NPR proteins function as salicylic acid (SA) receptors in Arabidopsis thaliana. AtNPR1 plays a central role in SA-induced transcriptional reprogramming whereby positively regulates SA-mediated defense. NPRs are found in the genomes of nearly all land plants. However, we know little about the molecular functions and physiological roles of NPRs in most plant species. We conducted phylogenetic and alignment analyses of NPRs from 68 species covering the significant lineages of land plants. To investigate NPR functions in bryophyte lineages, we generated and characterized NPR loss-of-function mutants in the liverwort Marchantia polymorpha. Brassicaceae NPR1-like proteins have characteristically gained or lost functional residues identified in AtNPRs, pointing to the possibility of a unique evolutionary trajectory for the Brassicaceae NPR1-like proteins. We find that the only NPR in M. polymorpha, MpNPR, is not the master regulator of SA-induced transcriptional reprogramming and negatively regulates bacterial resistance in this species. The Mpnpr transcriptome suggested roles of MpNPR in heat and far-red light responses. We identify both Mpnpr and Atnpr1-1 display enhanced thermomorphogenesis. Interspecies complementation analysis indicated that the molecular properties of AtNPR1 and MpNPR are partially conserved. We further show that MpNPR has SA-binding activity. NPRs and NPR-associated pathways have evolved distinctively in diverged land plant lineages to cope with different terrestrial environments.

Divergent evolution of the alcohol-forming pathway of wax biosynthesis among bryophytes.

The plant cuticle is a hydrophobic barrier, which seals the epidermal surface of most aboveground organs. While the cuticle biosynthesis of angiosperms has been intensively studied, knowledge about its existence and composition in nonvascular plants is scarce. Here, we identified and characterized homologs of Arabidopsis thaliana fatty acyl-CoA reductase (FAR) ECERIFERUM 4 (AtCER4) and bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase 1 (AtWSD1) in the liverwort Marchantia polymorpha (MpFAR2 and MpWSD1) and the moss Physcomitrium patens (PpFAR2A, PpFAR2B, and PpWSD1). Although bryophyte harbor similar compound classes as described for angiosperm cuticles, their biosynthesis may not be fully conserved between the bryophytes M. polymorpha and P. patens or between these bryophytes and angiosperms. While PpFAR2A and PpFAR2B contribute to the production of primary alcohols in P. patens, loss of MpFAR2 function does not affect the wax profile of M. polymorpha. By contrast, MpWSD1 acts as the major wax ester-producing enzyme in M. polymorpha, whereas mutations of PpWSD1 do not affect the wax ester levels of P. patens. Our results suggest that the biosynthetic enzymes involved in primary alcohol and wax ester formation in land plants have either evolved multiple times independently or undergone pronounced radiation followed by the formation of lineage-specific toolkits.

MpTGA, together with MpNPR, regulates sexual reproduction and independently affects oil body formation in Marchantia polymorpha.

In angiosperms, basic leucine-zipper (bZIP) TGACG-motif-binding (TGA) transcription factors (TFs) regulate developmental and stress-related processes, the latter often involving NON EXPRESSOR OF PATHOGENESIS-RELATED GENES (NPR) coregulator interactions. To gain insight into their functions in an early diverging land-plant lineage, the single MpTGA and sole MpNPR genes were investigated in the liverwort Marchantia polymorpha. We generated Marchantia MpTGA and MpNPR knockout and overexpression mutants and conducted morphological, transcriptomic and expression studies. Furthermore, we investigated MpTGA interactions with wild-type and mutagenized MpNPR and expanded our analyses including TGA TFs from two streptophyte algae. Mptga mutants fail to induce the switch from vegetative to reproductive development and lack gametangiophore formation. MpTGA and MpNPR proteins interact and Mpnpr mutant analysis reveals a novel coregulatory NPR role in sexual reproduction. Additionally, MpTGA acts independently of MpNPR as a repressor of oil body (OB) formation and can thereby affect herbivory. The single MpTGA TF exerts a dual role in sexual reproduction and OB formation in Marchantia. Common activities of MpTGA/MpNPR in sexual development suggest that coregulatory interactions were established after emergence of land-plant-specific NPR genes and contributed to the diversification of TGA TF functions during land-plant evolution.

Whole-Genome Sequence of Paenibacillus marchantiae Isolated from the Liverwort Marchantia polymorpha subsp. ruderalis Ecotype BoGa.

The bacterium Paenibacillus marchantiae was isolated from male plants of the liverwort Marchantia polymorpha subsp. ruderalis ecotype BoGa. Here, we report on the complete genome sequence generated from long Nanopore reads. The genome sequence comprises 6,983,959 bp with a GC content of 46.02% and 6,195 predicted protein-coding genes.

Whole-Genome Sequence of Aneurinibacillus sp. Ricciae_BoGa-3, Isolated from Riccia fluitans.

Here, we present the Nanopore-only genome sequence of Aneurinibacillus sp. Ricciae_BoGa-3. It was isolated from Riccia fluitans ecotype BoGa-3 and its source was Botanical Garden Osnabrück (Germany). The complete circular genome is 4,981,254 bp with a GC content of 44.8%.

Nuclear redox processes in land plant development and stress adaptation.

Recent findings expanded our knowledge about plant redox regulation in stress responses by demonstrating that redox processes exert crucial nuclear regulatory functions in meristems and other developmental processes. Analyses of redox-modulated transcription factor functions and coregulatory ROXYs, CC-type land-plant specific glutaredoxins, reveal new insights into the redox control of plant transcription factors and participation of ROXYs in plant development. The role for ROS and redox signaling in response to low-oxygen conditions further strengthens the importance of redox processes in meristems and tissue differentiation as well as for adaptation to changing environments effecting food crop productivity.

The renaissance and enlightenment of Marchantia as a model system.

The liverwort Marchantia polymorpha has been utilized as a model for biological studies since the 18th century. In the past few decades, there has been a Renaissance in its utilization in genomic and genetic approaches to investigating physiological, developmental, and evolutionary aspects of land plant biology. The reasons for its adoption are similar to those of other genetic models, e.g. simple cultivation, ready access via its worldwide distribution, ease of crossing, facile genetics, and more recently, efficient transformation, genome editing, and genomic resources. The haploid gametophyte dominant life cycle of M. polymorpha is conducive to forward genetic approaches. The lack of ancient whole-genome duplications within liverworts facilitates reverse genetic approaches, and possibly related to this genomic stability, liverworts possess sex chromosomes that evolved in the ancestral liverwort. As a representative of one of the three bryophyte lineages, its phylogenetic position allows comparative approaches to provide insights into ancestral land plants. Given the karyotype and genome stability within liverworts, the resources developed for M. polymorpha have facilitated the development of related species as models for biological processes lacking in M. polymorpha.

Developmental Plasticity of the Amphibious Liverwort Riccia fluitans.

The colonization of land by ancestors of embryophyte plants was one of the most significant evolutionary events in the history of life on earth. The lack of a buffering aquatic environment necessitated adaptations for coping with novel abiotic challenges, particularly high light intensities and desiccation as well as the formation of novel anchoring structures. Bryophytes mark the transition from freshwater to terrestrial habitats and form adaptive features such as rhizoids for soil contact and water uptake, devices for gas exchange along with protective and repellent surface layers. The amphibious liverwort Riccia fluitans can grow as a land form (LF) or water form (WF) and was employed to analyze these critical traits in two different habitats. A combination of light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) studies was conducted to characterize and compare WF and LF morphologies. A complete phenotypic adaptation of a WF plant to a terrestrial habitat is accomplished within 15 days after the transition. Stable transgenic R. fluitans lines expressing GFP-TUBULIN and mCherry proteins were generated to study cell division and differentiation processes and revealed a higher cell division activity in enlarged meristematic regions at LF apical notches. Morphological studies demonstrated that the R. fluitans WF initiates air pore formation. However, these pores are arrested at an early four cell stage and do not develop further into open pores that could mediate gas exchange. Similarly, also arrested rhizoid initial cells are formed in the WF, which exhibit a distinctive morphology compared to other ventral epidermal cells. Furthermore, we detected that the LF thallus has a reduced surface permeability compared to the WF, likely mediated by formation of thicker LF cell walls and a distinct cuticle compared to the WF. Our R. fluitans developmental plasticity studies can serve as a basis to further investigate in a single genotype the molecular mechanisms of adaptations essential for plants during the conquest of land.

Gamete expression of TALE class HD genes activates the diploid sporophyte program in Marchantia polymorpha.

Eukaryotic life cycles alternate between haploid and diploid phases and in phylogenetically diverse unicellular eukaryotes, expression of paralogous homeodomain genes in gametes primes the haploid-to-diploid transition. In the unicellular chlorophyte alga Chlamydomonas, KNOX and BELL TALE-homeodomain genes mediate this transition. We demonstrate that in the liverwort Marchantia polymorpha, paternal (sperm) expression of three of five phylogenetically diverse BELL genes, MpBELL234, and maternal (egg) expression of both MpKNOX1 and MpBELL34 mediate the haploid-to-diploid transition. Loss-of-function alleles of MpKNOX1 result in zygotic arrest, whereas a loss of either maternal or paternal MpBELL234 results in variable zygotic and early embryonic arrest. Expression of MpKNOX1 and MpBELL34 during diploid sporophyte development is consistent with a later role for these genes in patterning the sporophyte. These results indicate that the ancestral mechanism to activate diploid gene expression was retained in early diverging land plants and subsequently co-opted during evolution of the diploid sporophyte body.

Dual-color 3D-dSTORM colocalization and quantification of ROXY1 and RNAPII variants throughout the transcription cycle in root meristem nuclei.

To unravel the function of a protein of interest, it is crucial to asses to what extent it associates via direct interactions or by overlapping expression with other proteins. ROXY1, a land plant-specific glutaredoxin, exerts a function in Arabidopsis flower development and interacts with TGA transcription factors in the nucleus. We detected a novel ROXY1 function in the root meristem. Root cells that lack chlorophyll reducing plant-specific background problems that can hamper colocalization 3D microscopy. Thus far, a super-resolution three-dimensional stochastic optical reconstruction microscopy (3D-dSTORM) approach has mainly been applied in animal studies. We established 3D-dSTORM using the roxy1 mutant complemented with green fluorescence protein-ROXY1 and investigated its colocalization with three distinct RNAPII isoforms. To quantify the colocalization results, 3D-dSTORM was coupled with the coordinate-based colocalization method. Interestingly, ROXY1 proteins colocalize with different RNA polymerase II (RNAPII) isoforms that are active at distinct transcription cycle steps. Our colocalization data provide new insights on nuclear glutaredoxin activities suggesting that ROXY1 is not only required in early transcription initiation events via interaction with transcription factors but likely also participates throughout further transcription processes until late termination steps. Furthermore, we showed the applicability of the combined approaches to detect and quantify responses to altered growth conditions, exemplified by analysis of H2 O2 treatment, causing a dissociation of ROXY1 and RNAPII isoforms. We envisage that the powerful dual-color 3D-dSTORM/coordinate-based colocalization combination offers plant cell biologists the opportunity to colocalize and quantify root meristem proteins at an increased, unprecedented resolution level <50 nm, which will enable the detection of novel subcellular protein associations and functions.

Transformation of Riccia fluitans, an Amphibious Liverwort Dynamically Responding to Environmental Changes.

The colonization of land by streptophyte algae, ancestors of embryophyte plants, was a fundamental event in the history of life on earth. Bryophytes are early diversifying land plants that mark the transition from freshwater to terrestrial ecosystems. The amphibious liverwort Riccia fluitans can thrive in aquatic and terrestrial environments and thus represents an ideal organism to investigate this major transition. Therefore, we aimed to establish a transformation protocol for R. fluitans to make it amenable for genetic analyses. An Agrobacterium transformation procedure using R. fluitans callus tissue allows to generate stably transformed plants within 10 weeks. Furthermore, for comprehensive studies spanning all life stages, we demonstrate that the switch from vegetative to reproductive development can be induced by both flooding and poor nutrient availability. Interestingly, a single R. fluitans plant can consecutively adapt to different growth environments and forms distinctive and reversible features of the thallus, photosynthetically active tissue that is thus functionally similar to leaves of vascular plants. The morphological plasticity affecting vegetative growth, air pore formation, and rhizoid development realized by one genotype in response to two different environments makes R. fluitans ideal to study the adaptive molecular mechanisms enabling the colonialization of land by aquatic plants.

Oil Body Formation in Marchantia polymorpha Is Controlled by MpC1HDZ and Serves as a Defense against Arthropod Herbivores.

The origin of a terrestrial flora in the Ordovician required adaptation to novel biotic and abiotic stressors. Oil bodies, a synapomorphy of liverworts, accumulate secondary metabolites, but their function and development are poorly understood. Oil bodies of Marchantia polymorpha develop within specialized cells as one single large organelle. Here, we show that a class I homeodomain leucine-zipper (C1HDZ) transcription factor controls the differentiation of oil body cells in two different ecotypes of the liverwort M. polymorpha, a model genetic system for early divergent land plants. In flowering plants, these transcription factors primarily modulate responses to abiotic stress, including drought. However, loss-of-function alleles of the single ortholog gene, MpC1HDZ, in M. polymorpha did not exhibit phenotypes associated with abiotic stress. Rather, Mpc1hdz mutant plants were more susceptible to herbivory, and total plant extracts of the mutant exhibited reduced antibacterial activity. Transcriptomic analysis of the mutant revealed a reduction in expression of genes related to secondary metabolism that was accompanied by a specific depletion of oil body terpenoid compounds. Through time-lapse imaging, we observed that MpC1HDZ expression maxima precede oil body formation, indicating that MpC1HDZ mediates differentiation of oil body cells. Our results indicate that M. polymorpha oil bodies, and MpC1HDZ, are critical for defense against herbivory, but not for abiotic stress tolerance. Thus, C1HDZ genes were co-opted to regulate separate responses to biotic and abiotic stressors in two distinct land plant lineages.

MpTCP1 controls cell proliferation and redox processes in Marchantia polymorpha.

TCP transcription factors are key regulators of angiosperm cell proliferation processes. It is unknown whether their regulatory growth capacities are conserved across land plants, which we examined in liverworts, one of the earliest diverging land plant lineages. We generated knockout mutants for MpTCP1, the single TCP-P clade gene in Marchantia polymorpha, and characterized its function by conducting cell proliferation and morphological analyses as well as messenger RNA expression, transcriptome, chemical, and DNA binding studies. Mptcp1ge lines show a reduced vegetative thallus growth and extra tissue formation in female reproductive structures. Additionally, mutant plants reveal increased hydrogen peroxide (H2 O2 ) levels and an enhanced pigmentation in the thallus caused by formation of secondary metabolites, such as aminochromes. MpTCP1 proteins interact redox dependently with DNA and regulate the expression of a comprehensive redox network, comprising enzymes involved in H2 O2 metabolism. MpTCP1 regulates Marchantia growth in a context-dependent manner. Redox sensitivity of the DNA binding capacity of MpTCP1 proteins provides a mechanism to respond to altered redox conditions. Our data suggest that MpTCP1 activity could thereby have contributed to diversification of land plant morphologies and to adaptations to abiotic and biotic challenges, as experienced by liverworts during early land plant colonization.

Transient genetic transformation of Mougeotia scalaris (Zygnematophyceae) mediated by the endogenous α-tubulin1 promoter.

Mougeotia scalaris is a filamentous streptophyte alga renowned for light-inducible plastid rotation and microtubule-dependent polarity establishment. As a first step toward transgenic approaches we determined the 5,825 base pair genomic sequence encoding the α-tubulin1 gene (MsTUA1) of M. scalaris (strain SAG 164.80). The subcloned MsTUA1 promoter facilitated strong transgene expression in M. scalaris and tobacco leaf cells, as shown by particle bombardment and the subsequent visualization of expressed fluorescent protein markers. Our results provide a route for the genetic transformation of the filamentous streptophyte alga M. scalaris based on the endogenous TUA1 promoter.

TALEN-mediated genome-editing approaches in the liverwort Marchantia polymorpha yield high efficiencies for targeted mutagenesis.

BACKGROUND: The liverwort Marchantia polymorpha occupies a crucial position in land plant evolution and provides the opportunity to investigate adaptations to a terrestrial plant life style. Marchantia reverse genetic analyses have thus far been conducted by employing a homologous recombination approach, which yields an efficiency of around 3%. Availability of the characterized and suitable endogenous MpEF1α promoter prompted us to establish the TALEN gene targeting technique for Marchantia. RESULTS: Here, two different TALEN techniques, using custom and self-assembled TALEN constructs, were applied and compared. The MpNOP1 gene was selected as a candidate gene, as the respective knockout mutant has been shown to lack air chamber formation, representing an easily traceable phenotype. We demonstrate that both TALEN approaches are successful in Marchantia yielding high gene targeting efficiencies of over 20%. Investigation of selected G1 up to G4 generations proved the stability of the knockout mutants. In 392 analyzed T1 plants, no additional phenotypes were observed and only one chimeric knockout plant was detected after an extended cultivation period. Interestingly, two out of the 24 sequenced mutants harbored indels causing in-frame mutations and revealed novel Mpnop1-related phenotypes. This demonstrates the potential to detect crucial amino acids and motives of targeted proteins, which is of special interest for essential genes where full knockouts are lethal. The FastTALE™ TALEN assembly kit enables the rapid assembly and ligation of the TALEN arms within half a day. For transformations, custom and assembled constructs were subcloned into Marchantia binary vectors possessing the MpEF1α promoter. CONCLUSION: Considering time, costs and practicability, the assembly TALEN approach represents a rapid and highly efficient gene targeting system to generate Marchantia knockout mutants, which can be further adapted for future advanced genome-editing applications.

Conserved redox-dependent DNA binding of ROXY glutaredoxins with TGA transcription factors.

The Arabidopsis thaliana CC-type glutaredoxin (GRX) ROXY1 and the bZIP TGA transcription factor (TF) PERIANTHIA (PAN) interact in the nucleus and together regulate petal development. The CC-type GRXs exist exclusively in land plants, and in contrast to the ubiquitously occurring CPYC and CGFS GRX classes, only the CC-type GRXs expanded strongly during land plant evolution. Phylogenetic analyses show that TGA TFs evolved before the CC-type GRXs in charophycean algae. MpROXY1/2 and MpTGA were isolated from the liverwort Marchantia polymorpha to analyze regulatory ROXY/TGA interactions in a basal land plant. Homologous and heterologous protein interaction studies demonstrate that nuclear ROXY/TGA interactions are conserved since the occurrence of CC-type GRXs in bryophytes and mediated by a conserved ROXY C-terminus. Redox EMSA analyses show a redox-sensitive binding of MpTGA to the cis-regulatory as-1-like element. Furthermore, we demonstrate that MpTGA binds together with MpROXY1/2 to this motif under reducing conditions, whereas this interaction is not observed under oxidizing conditions. Remarkably, heterologous complementation studies reveal a strongly conserved land plant ROXY activity, suggesting an ancestral role for CC-type GRXs in modulating the activities of TGA TFs. Super-resolution microscopy experiments detected a strong colocalization of ROXY1 with the active form of the RNA polymerase II in the nucleus. Together, these data shed new light on the function of ROXYs and TGA TFs and the evolution of redox-sensitive transcription regulation processes, which likely contributed to adapt land plants to novel terrestrial habitats.

The Evolution of Cell Division: From Streptophyte Algae to Land Plants.

The mechanism of cell division has undergone significant alterations during the evolution from aquatic streptophyte algae to land plants. Two new structures evolved, the cytokinetic phragmoplast and the preprophase band (PPB) of microtubules, whereas the ancestral mechanism of cleavage and the centrosomes disappeared. We map cell biological data onto the recently emerged phylogenetic tree of streptophytes. The tree suggests that, after the establishment of the phragmoplast mechanism, several groups independently lost their centrosomes. Surprisingly, the phragmoplast shows reductions in the Zygnematophyceae (the sister to land plants), many of which returned to cleavage. The PPB by contrast evolved stepwise and, most likely, originated in the algae. The phragmoplast/PPB mechanism established in this way served as a basis for the 3D development of land plants.

The N-Terminus of the Floral Arabidopsis TGA Transcription Factor PERIANTHIA Mediates Redox-Sensitive DNA-Binding.

The Arabidopsis TGA transcription factor (TF) PERIANTHIA (PAN) regulates the formation of the floral organ primordia as revealed by the pan mutant forming an abnormal pentamerous arrangement of the outer three floral whorls. The Arabidopsis TGA bZIP TF family comprises 10 members, of which PAN and TGA9/10 control flower developmental processes and TGA1/2/5/6 participate in stress-responses. For the TGA1 protein it was shown that several cysteines can be redox-dependently modified. TGA proteins interact in the nucleus with land plant-specific glutaredoxins, which may alter their activities posttranslationally. Here, we investigated the DNA-binding of PAN to the AAGAAT motif under different redox-conditions. The AAGAAT motif is localized in the second intron of the floral homeotic regulator AGAMOUS (AG), which controls stamen and carpel development as well as floral determinacy. Whereas PAN protein binds to this regulatory cis-element under reducing conditions, the interaction is strongly reduced under oxidizing conditions in EMSA studies. The redox-sensitive DNA-binding is mediated via a special PAN N-terminus, which is not present in other Arabidopsis TGA TFs and comprises five cysteines. Two N-terminal PAN cysteines, Cys68 and Cys87, were shown to form a disulfide bridge and Cys340, localized in a C-terminal putative transactivation domain, can be S-glutathionylated. Comparative land plant analyses revealed that the AAGAAT motif exists in asterid and rosid plant species. TGA TFs with N-terminal extensions of variable length were identified in all analyzed seed plants. However, a PAN-like N-terminus exists only in the rosids and exclusively Brassicaceae homologs comprise four to five of the PAN N-terminal cysteines. Redox-dependent modifications of TGA cysteines are known to regulate the activity of stress-related TGA TFs. Here, we show that the N-terminal PAN cysteines participate in a redox-dependent control of the PAN interaction with a highly conserved regulatory AG cis-element, emphasizing the importance of redox-modifications in the regulation of flower developmental processes.

Identification of miRNAs and Their Targets in the Liverwort Marchantia polymorpha by Integrating RNA-Seq and Degradome Analyses.

Bryophytes (liverworts, hornworts and mosses) comprise the three earliest diverging lineages of land plants (embryophytes). Marchantia polymorpha, a complex thalloid Marchantiopsida liverwort that has been developed into a model genetic system, occupies a key phylogenetic position. Therefore, M. polymorpha is useful in studies aiming to elucidate the evolution of gene regulation mechanisms in plants. In this study, we used computational, transcriptomic, small RNA and degradome analyses to characterize microRNA (miRNA)-mediated pathways of gene regulation in M. polymorpha. The data have been integrated into the open access ContigViews-miRNA platform for further reference. In addition to core components of the miRNA pathway, 129 unique miRNA sequences, 11 of which could be classified into seven miRNA families that are conserved in embryophytes (miR166a, miR390, miR529c, miR171-3p, miR408a, miR160 and miR319a), were identified. A combination of computational and degradome analyses allowed us to identify and experimentally validate 249 targets. In some cases, the target genes are orthologous to those of other embryophytes, but in other cases, the conserved miRNAs target either paralogs or members of different gene families. In addition, the newly discovered Mpo-miR11707.1 and Mpo-miR11707.2 are generated from a common precursor and target MpARGONAUTE1 (LW1759). Two other newly discovered miRNAs, Mpo-miR11687.1 and Mpo-miR11681.1, target the MADS-box transcription factors MpMADS1 and MpMADS2, respectively. Interestingly, one of the pentatricopeptide repeat (PPR) gene family members, MpPPR_66 (LW9825), the protein products of which are generally involved in various steps of RNA metabolism, has a long stem-loop transcript that can generate Mpo-miR11692.1 to autoregulate MpPPR_66 (LW9825) mRNA. This study provides a foundation for further investigations of the RNA-mediated silencing mechanism in M. polymorpha as well as of the evolution of this gene silencing pathway in embryophytes.