Sabine Zachgo

Ergothioneine (EGT) is a sulphur‐containing histidine derivative and a potent antioxidant that exhibits beneficial effects on human health. Thus far, only fungi and certain bacteria have been reported to produce EGT, whereas plants are assumed to rely on an uptake of EGT. Here, the presence of EGT biosynthetic genes and their functionality were investigated in Viridiplantae. The biosynthetic genes EGT1 and EGT2 from yeast were used for transcriptome and genome analyses in evolutionarily informative species across Viridiplantae. Targeted metabolomics (HPLC‐MRM/MS) was used to quantify EGT in selected algae and land plants grown under control conditions and exposed to abiotic stress. EGT1 and EGT2 genes were identified in streptophyte algae, bryophytes, lycophytes, monilophytes, and gymnosperms. Targeted metabolomic profiling demonstrated endogenous EGT production in diverse algae and land plants, refuting the long‐standing view that plants cannot synthesize this antioxidant. Notably, EGT1 genes do not exist in angiosperms, which likely lost this gene and the capability to synthesize EGT. After high light and heat stress exposure, EGT synthesis increases significantly in the streptophyte algae Klebsormidium nitens and the moss Physcomitrium patens , suggesting that EGT also exerts an antioxidant function in plants. Contrary to previous assumptions, various plants possess EGT genes and are capable of synthesizing EGT. Abiotic stress experiments reveal a link between EGT and the plant stress response, opening new avenues for research in stress signalling and adaptation – areas that are also relevant for enhancing crop resilience and nutritional quality.

Societal Impact Statement: Ambitious targets have been set to backup seeds of threatened plants by the global strategy for plant conservation (GSPC), but it is unclear in how far these targets have been met and how seed collection should be organized to meet future challenges. Here, we provide an overview of the status of 44 countries in achieving seed conservation targets. We show that progress varies strongly across countries, but in general, targets of the 2011–2020 GSCP have not been reached. By a regional example, we illustrate how seed collection could be organized to safeguard our threatened flora. Summary: Seed banking of wild plants is a central pillar of conservation strategies for threatened plants. Target 8 of the Global Strategy for Plant Conservation (GSPC) called for at least 75% of threatened plant species to be conserved ex situ by 2020, but to what extent countries have achieved this target is unclear. Consequently, it is unknown how seed banking should be organized in future to safeguard plant diversity and support the increasing demand for seeds for habitat restoration. We conducted a comprehensive survey across 44 countries in Europe and western Asia and collected information on seed bank stocks of native taxa. We harmonized the taxonomic names across the collections and matched them against a database of national plant Red Lists to assign each taxon its IUCN (International Union for Conservation of Nature) threat status in its country of origin. We analysed each country's achievement in terms of the conservation of its threatened flora and evaluated the quality of the collections in terms of genetic representativeness and seed quantity. We found that none of the studied countries had reached Target 8 of the GSPC by 2020. While a few countries had come close to it, on average only 21% of threatened taxa were conserved across countries. Our results demonstrate that the current seed conservation strategy has not met conservation demands. Using an example from Belgium, where considerable progress has been achieved by investing in a network of volunteer seed collectors, we show how future seed collection could be organized in collaborative community‐based approaches.

Plant root development is tightly regulated and depends on the coordination among different cell types located in the root stem cell niche (SCN). A complex network of regulators includes the gasotransmitter nitric oxide (NO) known to inhibit primary root growth, modify root architecture and alter meristem organisation. However, little is known about the molecular targets of this signalling molecule during root SCN homeostasis. Here, genetic, molecular and physiological evidence support that TGA transcription factors (TFs) of the bZIP family, and specifically PERIANTHIA (PAN), contribute to the regulation of the root SCN through NO sensing. This gasotransmitter induces reversible S-nitrosylation of PAN, thereby inhibiting its binding to DNA and associated transcriptional activation; whilst the protein-SNO reductase activity of TRXh5 can denitrosylate PAN. This switch on and off mechanism for PAN activity modulates gene expression in the SCN and the functioning of the quiescent centre, thereby altering columella stem cell maintenance through a NO-dependent mechanism. Furthermore, PAN interacts selectively with several TGA members which are susceptible to differential S-nitrosylation. Given the functional redundancy within the TGA family, analysis of quadruple and pentuple mutants revealed close coordination between PAN and other members of this family of TFs, which contributes to the NO sensing during the development of the root apical meristem. Collectively, these findings reveal a molecular framework in which the TGA family, primarily coordinated by PAN activity, acts as a master NO sensor in the root SCN.

SUMMARY: Redox processes and reactive oxygen species (ROS) signaling play not only a crucial role in stress responses but also in angiosperm development. However, the specific mechanisms by which redox homeostasis regulates meristems and growth in non‐vascular plants remain poorly understood. Here, we demonstrate the applicability of the roGFP2‐hGrx1 and HyPer7 redox‐biosensors for imaging dynamic glutathione (GSH) and H₂O₂ redox states in the liverwort Marchantia polymorpha. RoGFP2‐hGrx1 microscopy, together with analysis of knockdown plants of the GAMMA GLUTAMYLCYSTEINE SYNTHETASE gene MpGSH1, unveiled a more reduced GSH redox potential (E_GSH) in the meristematic region and a more oxidized state in differentiated thallus tissues. Rather than absolute E_GSH values, maintenance of a GSH redox gradient is crucial for proper vegetative development. High‐resolution HyPer7 analysis detected a heterogenous H₂O₂ accumulation. Overall, the meristematic region exhibits lower H₂O₂ levels. Notably, a small zone with higher sensor oxidation is localized in the center of the meristem, likely comprising stem cells and proliferating derivatives. In differentiated thallus tissue, higher levels of H₂O₂ were detected. External H₂O₂ application revealed dose‐dependent effects that promote or arrest growth. Overproliferation in the meristematic region, driven by treatment with the CLAVATA3/EMBRYO SURROUNDING REGION‐related peptide (MpCLE2p) increased H₂O₂ levels in expanded meristems, supporting the importance of H₂O₂ signaling in balancing cell proliferation and differentiation in M. polymorpha. Further comparative high‐resolution redox sensor studies in bryophytes and vascular plants can shed light on the contribution of redox processes to the regulation of developmental processes and the formation of increasingly complex land plants.

Key message: We link key aspects of land plant reproductive evolution and detail how successive molecular changes leading to novel tissues and organs require co-evolution of communication systems between tissues. The transition of water-dependent reproduction of algae to mechanisms with very limited water dependence in many land plant lineages allowed plants to colonize diverse terrestrial environments, leading to the vast variety of extant plant species. The emergence of modified cell types, novel tissues, and organs enabled this transition; their origin is associated with the co-evolution of novel or adapted molecular communication systems and gene regulatory networks. In the light of an increasing number of genome sequences in combination with the establishment of novel genetic model organisms from diverse green plant lineages, our knowledge and understanding about the origin and evolution of individual traits that arose in a concerted way increases steadily. For example, novel members of gene families in signaling pathways emerged for communication between gametes and gametophytes with additional tissues surrounding the gametes. Here, we provide a comprehensive overview on the origin and evolution of reproductive novelties such as pollen grains, immobile sperms, ovules and seeds, carpels, gamete/gametophytic communication systems, double fertilization, and the molecular mechanisms that have arisen anew or have been co-opted during evolution, including but not limited to the incorporation of phytohormones, reactive oxygen species and redox signaling as well as small RNAs in regulatory modules that contributed to the evolution of land plant sexual reproduction.

Plant adaptation to terrestrial life started 450 million years ago and has played a major role in the evolution of life on Earth. The genetic mechanisms allowing this adaptation to a diversity of terrestrial constraints have been mostly studied by focusing on flowering plants. Here, we gathered a collection of 133 accessions of the model bryophyte Marchantia polymorpha and studied its intraspecific diversity using selection signature analyses, a genome–environment association study and a pangenome. We identified adaptive features, such as peroxidases or nucleotide-binding and leucine-rich repeats (NLRs), also observed in flowering plants, likely inherited from the first land plants. The M. polymorpha pangenome also harbors lineage-specific accessory genes absent from seed plants. We conclude that different land plant lineages still share many elements from the genetic toolkit evolved by their most recent common ancestor to adapt to the terrestrial habitat, refined by lineage-specific polymorphisms and gene family evolution.

SUMMARY: DICER‐LIKE (DCL) proteins play a central role in plant small RNA (sRNA) biogenesis. The genome of the early land plant Marchantia polymorpha encodes four DCL proteins: MpDCL1a, MpDCL1b, MpDCL3, and MpDCL4. While MpDCL1a, MpDCL3 and MpDCL4 show high similarities to their orthologs in Physcomitrium patens and Arabidopsis thaliana, MpDCL1b shares only a limited homology with PpDCL1b, but it is very similar, in terms of functional domains, to orthologs in other moss and fern species. We generated Mpdcl^ge mutant lines for all MpDCL genes with the CRISPR/Cas9 system and conducted phenotypic analyses under control, salt stress, and phytohormone treatments to uncover specific MpDCL functions. The mutants displayed severe developmental aberrations, altered responses to salt and phytohormones, and disturbed sexual organ development. By combining mRNA and sRNA analyses, we demonstrate that MpDCLs and their associated sRNAs play pivotal roles in regulating development, abiotic stress tolerance and phytohormone response in M. polymorpha. We identified MpDCL1a in microRNA biogenesis, MpDCL4 in trans‐acting small interfering RNA generation, and MpDCL3 in the regulation of pathogen‐related genes. Notably, salt sensitivity in M. polymorpha is dependent on MpDCL1b and Mpdcl1b^ge mutants display enhanced tolerance and reduced miRNA expression in response to salt stress. We propose that M. polymorpha employs specific mechanisms for regulating MpDCL1b associated miRNAs under high salinity conditions, potentially shared with other species harboring MpDCL1b homologs.

Summary: 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.

Summary: 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 ( At CER4) and bifunctional wax ester synthase/acyl‐CoA:diacylglycerol acyltransferase 1 ( At WSD1) 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 Mp FAR2 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 Pp WSD1 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.

Summary: 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.