The sexual lability hypothesis for the origin of the land plant generation cycle.

The evolution of the land plant alternation of generations has been an open question for the past 150 years. Two hypotheses have dominated the discussion: the antithetic hypothesis, which posits that the diploid sporophyte generation arose de novo and gradually increased in complexity, and the homologous hypothesis, which holds that land plant ancestors had independently living sporophytes and haploid gametophytes of similar complexity. Changes in ploidy levels were unknown to early researchers. The antithetic hypothesis is contradicted by generation cycles in Lower Devonian Rhynie chert plants, whose sporophytes and gametophytes have similar morphologies and by some Silurian sporophytes whose complexity exceeds that of Rhynie chert sporophytes. The oldest unambiguous bryophyte gametophytes (thalli) are from the upper Middle Devonian, with an unconnected sporophyte nearby. Based on the 2024 discovery that conjugate algae are paraphyletic to land plants, we present a new hypothesis for the evolution of the land plant generation cycle, focusing on labile ploidy levels and types of reproduction found in conjugate algae. Our 'sexual lability' hypothesis assumes a period of unstable generation cycles (as regards ploidy), likely with predominant clonal growth, as is common in conjugate algae, resulting in sporophytes and gametophytes of similar morphology. When sexual reproduction became stabilized, the timing of gamete fusion, meiosis, and resistant wall formation, which are heterochronic in some conjugate algae, became standardized, with wall formation permanently delayed. In our scenario, independently living adult sporophytes are the land plant ancestral condition, and life-long sporophyte retention on the gametophyte is a bryophyte apomorphy.

The receptor-like kinase ARK controls symbiotic balance across land plants.

The mutualistic arbuscular mycorrhizal (AM) symbiosis arose in land plants more than 450 million years ago and is still widely found in all major land plant lineages. Despite its broad taxonomic distribution, little is known about the molecular components underpinning symbiosis outside of flowering plants. The ARBUSCULAR RECEPTOR-LIKE KINASE (ARK) is required for sustaining AM symbiosis in distantly related angiosperms. Here, we demonstrate that ARK has an equivalent role in symbiosis maintenance in the bryophyte Marchantia paleacea and is part of a broad AM genetic program conserved among land plants. In addition, our comparative transcriptome analysis identified evolutionarily conserved expression patterns for several genes in the core symbiotic program required for presymbiotic signaling, intracellular colonization, and nutrient exchange. This study provides insights into the molecular pathways that consistently associate with AM symbiosis across land plants and identifies an ancestral role for ARK in governing symbiotic balance.

Evolutionary assembly of the plant terrestrialization toolkit from protein domains.

Land plants (embryophytes) came about in a momentous evolutionary singularity: plant terrestrialization. This event marks not only the conquest of land by plants but also the massive radiation of embryophytes into a diverse array of novel forms and functions. The unique suite of traits present in the earliest land plants is thought to have been ushered in by a burst in genomic novelty. Here, we asked the question of how these bursts were possible. For this, we explored: (i) the initial emergence and (ii) the reshuffling of domains to give rise to hallmark environmental response genes of land plants. We pinpoint that a quarter of the embryophytic genes for stress physiology are specific to the lineage, yet a significant portion of this novelty arises not de novo but from reshuffling and recombining of pre-existing domains. Our data suggest that novel combinations of old genomic substrate shaped the plant terrestrialization toolkit, including hallmark processes in signalling, biotic interactions and specialized metabolism.

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.

Genomes of multicellular algal sisters to land plants illuminate signaling network evolution.

Zygnematophyceae are the algal sisters of land plants. Here we sequenced four genomes of filamentous Zygnematophyceae, including chromosome-scale assemblies for three strains of Zygnema circumcarinatum. We inferred traits in the ancestor of Zygnematophyceae and land plants that might have ushered in the conquest of land by plants: expanded genes for signaling cascades, environmental response, and multicellular growth. Zygnematophyceae and land plants share all the major enzymes for cell wall synthesis and remodifications, and gene gains shaped this toolkit. Co-expression network analyses uncover gene cohorts that unite environmental signaling with multicellular developmental programs. Our data shed light on a molecular chassis that balances environmental response and growth modulation across more than 600 million years of streptophyte evolution.

Molecular Evolution of RAMOSA1 (RA1) in Land Plants.

RAMOSA1 (RA1) is a Cys2-His2-type (C2H2) zinc finger transcription factor that controls plant meristem fate and identity and has played an important role in maize domestication. Despite its importance, the origin of RA1 is unknown, and the evolution in plants is only partially understood. In this paper, we present a well-resolved phylogeny based on 73 amino acid sequences from 48 embryophyte species. The recovered tree topology indicates that, during grass evolution, RA1 arose from two consecutive SUPERMAN duplications, resulting in three distinct grass sequence lineages: RA1-like A, RA1-like B, and RA1; however, most of these copies have unknown functions. Our findings indicate that RA1 and RA1-like play roles in the nucleus despite lacking a traditional nuclear localization signal. Here, we report that copies diversified their coding region and, with it, their protein structure, suggesting different patterns of DNA binding and protein-protein interaction. In addition, each of the retained copies diversified regulatory elements along their promoter regions, indicating differences in their upstream regulation. Taken together, the evidence indicates that the RA1 and RA1-like gene families in grasses underwent subfunctionalization and neofunctionalization enabled by gene duplication.

Cytochrome b5 diversity in green lineages preceded the evolution of syringyl lignin biosynthesis.

Lignin production marked a milestone in vascular plant evolution, and the emergence of syringyl (S) lignin is lineage specific. S-lignin biosynthesis in angiosperms, mediated by ferulate 5-hydroxylase (F5H, CYP84A1), has been considered a recent evolutionary event. F5H uniquely requires the cytochrome b5 protein CB5D as an obligatory redox partner for catalysis. However, it remains unclear how CB5D functionality originated and whether it coevolved with F5H. We reveal here the ancient evolution of CB5D-type function supporting F5H-catalyzed S-lignin biosynthesis. CB5D emerged in charophyte algae, the closest relatives of land plants, and is conserved and proliferated in embryophytes, especially in angiosperms, suggesting functional diversification of the CB5 family before terrestrialization. A sequence motif containing acidic amino residues in Helix 5 of the CB5 heme-binding domain contributes to the retention of CB5D function in land plants but not in algae. Notably, CB5s in the S-lignin-producing lycophyte Selaginella lack these residues, resulting in no CB5D-type function. An independently evolved S-lignin biosynthetic F5H (CYP788A1) in Selaginella relies on NADPH-dependent cytochrome P450 reductase as sole redox partner, distinct from angiosperms. These results suggest that angiosperm F5Hs coopted the ancient CB5D, forming a modern cytochrome P450 monooxygenase system for aromatic ring meta-hydroxylation, enabling the reemergence of S-lignin biosynthesis in angiosperms.

Diversification of Plastid Structure and Function in Land Plants.

Plastids represent a largely diverse group of organelles in plant and algal cells that have several common features but also a broad spectrum of morphological, ultrastructural, biochemical, and physiological differences. Plastids and their structural and metabolic diversity significantly contribute to the functionality and developmental flexibility of the plant body throughout its lifetime. In addition to the multiple roles of given plastid types, this diversity is accomplished in some cases by interconversions between different plastids as a consequence of developmental and environmental signals that regulate plastid differentiation and specialization. In addition to basic plastid structural features, the most important plastid types, the newly characterized peculiar plastids, and future perspectives in plastid biology are also provided in this chapter.

Sexual reproduction: Is the genetic pathway for female germ cell specification conserved in land plants?

Land plants share several core factors responsible for female gametophyte development, despite their differing structures and developmental programs. New work providing molecular dissection of reproductive phases in non-angiosperm plants is a powerful tool for elucidating the underlying genetic network.

The ALOG domain defines a family of plant-specific transcription factors acting during Arabidopsis flower development.

The ALOG (Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 (LSH1) and Oryza G1) proteins are conserved plant-specific Transcription Factors (TFs). They play critical roles in the development of various plant organs (meristems, inflorescences, floral organs, and nodules) from bryophytes to higher flowering plants. Despite the fact that the first members of this family were originally discovered in Arabidopsis, their role in this model plant has remained poorly characterized. Moreover, how these transcriptional regulators work at the molecular level is unknown. Here, we study the redundant function of the ALOG proteins LSH1,3,4 from Arabidopsis. We uncover their role in the repression of bract development and position them within a gene regulatory network controlling this process and involving the floral regulators LEAFY, BLADE-ON-PETIOLE, and PUCHI. Next, using in vitro genome-wide studies, we identified the conserved DNA motif bound by ALOG proteins from evolutionarily distant species (the liverwort Marchantia polymorpha and the flowering plants Arabidopsis, tomato, and rice). Resolution of the crystallographic structure of the ALOG DNA-binding domain in complex with DNA revealed the domain is a four-helix bundle with a disordered NLS and a zinc ribbon insertion between helices 2 and 3. The majority of DNA interactions are mediated by specific contacts made by the third alpha helix and the NLS. Taken together, this work provides the biochemical and structural basis for DNA-binding specificity of an evolutionarily conserved TF family and reveals its role as a key player in Arabidopsis flower development.

Research Progress of Group II Intron Splicing Factors in Land Plant Mitochondria.

Mitochondria are important organelles that provide energy for the life of cells. Group II introns are usually found in the mitochondrial genes of land plants. Correct splicing of group II introns is critical to mitochondrial gene expression, mitochondrial biological function, and plant growth and development. Ancestral group II introns are self-splicing ribozymes that can catalyze their own removal from pre-RNAs, while group II introns in land plant mitochondria went through degenerations in RNA structures, and thus they lost the ability to self-splice. Instead, splicing of these introns in the mitochondria of land plants is promoted by nuclear- and mitochondrial-encoded proteins. Many proteins involved in mitochondrial group II intron splicing have been characterized in land plants to date. Here, we present a summary of research progress on mitochondrial group II intron splicing in land plants, with a major focus on protein splicing factors and their probable functions on the splicing of mitochondrial group II introns.

Plant evolution: A tapetum is now effectively present in all land plant lineages.

The tapetum, a tissue that elsewhere ensures correct spore development, is missing in some bryophytes. A new study shows that, in the liverwort, Marchantia polymorpha, a gene controlling spore wall deposition is expressed in the capsule lining, so these cells essentially function as a tapetum.

Phylogenomic insights into the first multicellular streptophyte.

Streptophytes are best known as the clade containing the teeming diversity of embryophytes (land plants).1,2,3,4 Next to embryophytes are however a range of freshwater and terrestrial algae that bear important information on the emergence of key traits of land plants. Among these, the Klebsormidiophyceae stand out. Thriving in diverse environments-from mundane (ubiquitous occurrence on tree barks and rocks) to extreme (from the Atacama Desert to the Antarctic)-Klebsormidiophyceae can exhibit filamentous body plans and display remarkable resilience as colonizers of terrestrial habitats.5,6 Currently, the lack of a robust phylogenetic framework for the Klebsormidiophyceae hampers our understanding of the evolutionary history of these key traits. Here, we conducted a phylogenomic analysis utilizing advanced models that can counteract systematic biases. We sequenced 24 new transcriptomes of Klebsormidiophyceae and combined them with 14 previously published genomic and transcriptomic datasets. Using an analysis built on 845 loci and sophisticated mixture models, we establish a phylogenomic framework, dividing the six distinct genera of Klebsormidiophyceae in a novel three-order system, with a deep divergence more than 830 million years ago. Our reconstructions of ancestral states suggest (1) an evolutionary history of multiple transitions between terrestrial-aquatic habitats, with stem Klebsormidiales having conquered land earlier than embryophytes, and (2) that the body plan of the last common ancestor of Klebsormidiophyceae was multicellular, with a high probability that it was filamentous whereas the sarcinoids and unicells in Klebsormidiophyceae are likely derived states. We provide evidence that the first multicellular streptophytes likely lived about a billion years ago.

Immunobiodiversity: Conserved and specific immunity across land plants and beyond.

Angiosperms represent most plants that humans cultivate, grow, and eat. However, angiosperms are only one of five major land plant lineages. As a whole lineage, plants also include algal groups. All these clades represent a tremendous genetic diversity that can be investigated to reveal the evolutionary history of any given mechanism. In this review, we describe the current model of the plant immune system, discuss its evolution based on the recent literature, and propose future directions for the field. In angiosperms, plant-microbe interactions have been intensively studied, revealing essential cell surface and intracellular immune receptors, as well as metabolic and hormonal defense pathways. Exploring diversity at the genomic and functional levels demonstrates the conservation of these pathways across land plants, some of which are beyond plants. On basis of the conserved mechanisms, lineage-specific variations have occurred, leading to diversified reservoirs of immune mechanisms. In rare cases, this diversity has been harnessed and successfully transferred to other species by integration of wild immune receptors or engineering of novel forms of receptors for improved resistance to pathogens. We propose that exploring further the diversity of immune mechanisms in the whole plant lineage will reveal completely novel sources of resistance to be deployed in crops.

RHO of plant signaling was established early in streptophyte evolution.

The algal ancestors of land plants underwent a transition from a unicellular to a multicellular body plan.1 This transition likely took place early in streptophyte evolution, sometime after the divergence of the Chlorokybophyceae/Mesostigmatophyceae lineage, but before the divergence of the Klebsormidiophyceae lineage.2 How this transition was brought about is unknown; however, it was likely facilitated by the evolution of novel mechanisms to spatially regulate morphogenesis. In land plants, RHO of plant (ROP) signaling plays a conserved role in regulating polarized cell growth and cell division orientation to orchestrate morphogenesis.3,4,5,6,7,8 ROP constitutes a plant-specific subfamily of the RHO GTPases, which are more widely conserved throughout eukaryotes.9,10 Although the RHO family originated in early eukaryotes,11,12 how and when the ROP subfamily originated had remained elusive. Here, we demonstrate that ROP signaling was established early in the streptophyte lineage, sometime after the divergence of the Chlorokybophyceae/Mesostigmatophyceae lineage, but before the divergence of the Klebsormidiophyceae lineage. This period corresponds to when the unicellular-to-multicellular transition likely took place in the streptophytes. In addition to being critical for the complex morphogenesis of extant land plants, we speculate that ROP signaling contributed to morphological evolution in early streptophytes.

Conserved and non-conserved RNA-target modules in plants: lessons for a better understanding of Marchantia development.

A wide variety of functional regulatory non-coding RNAs (ncRNAs) have been identified as essential regulators of plant growth and development. Depending on their category, ncRNAs are not only involved in modulating target gene expression at the transcriptional and post-transcriptional levels but also are involved in processes like RNA splicing and RNA-directed DNA methylation. To fulfill their molecular roles properly, ncRNAs must be precisely processed by multiprotein complexes. In the case of small RNAs, DICER-LIKE (DCL) proteins play critical roles in the production of mature molecules. Land plant genomes contain at least four distinct classes of DCL family proteins (DCL1-DCL4), of which DCL1, DCL3 and DCL4 are also present in the genomes of bryophytes, indicating the early divergence of these genes. The liverwort Marchantia polymorpha has become an attractive model species for investigating the evolutionary history of regulatory ncRNAs and proteins that are responsible for ncRNA biogenesis. Recent studies on Marchantia have started to uncover the similarities and differences in ncRNA production and function between the basal lineage of bryophytes and other land plants. In this review, we summarize findings on the essential role of regulatory ncRNAs in Marchantia development. We provide a comprehensive overview of conserved ncRNA-target modules among M. polymorpha, the moss Physcomitrium patens and the dicot Arabidopsis thaliana, as well as Marchantia-specific modules. Based on functional studies and data from the literature, we propose new connections between regulatory pathways involved in Marchantia's vegetative and reproductive development and emphasize the need for further functional studies to understand the molecular mechanisms that control ncRNA-directed developmental processes.

A bHLH heterodimer regulates germ cell differentiation in land plant gametophytes.

Land plants are a monophyletic group of photosynthetic eukaryotes that diverged from streptophyte algae about 470 million years ago. During both the alternating haploid and diploid stages of the life cycle, land plants form multicellular bodies.1,2,3,4 The haploid multicellular body (gametophyte) produces progenitor cells that give rise to gametes and the reproductive organs.5,6,7,8 In the liverwort Marchantia polymorpha, differentiation of the initial cells of gamete-producing organs (gametangia) from the gametophyte is regulated by MpBONOBO (MpBNB), a member of the basic helix-loop-helix (bHLH) transcription factor subfamily VIIIa. In Arabidopsis thaliana, specification of generative cells in developing male gametophytes (pollen) requires redundant action of BNB1 and BNB2.9 Subfamily XI bHLHs, such as LOTUS JAPONICUS ROOTHAIRLESS LIKE1 (LRL1)/DEFECTIVE REGION OF POLLEN1 (DROP1) and LRL2/DROP2 in A. thaliana and the single LRL/DROP protein MpLRL in M. polymorpha, are the evolutionarily conserved regulators of rooting system development.10 Although the role of LRL1/DROP1 and LRL2/DROP2 in gametogenesis remains unclear, their loss leads to the formation of abnormal pollen devoid of sperm cells.11 Here, we show that BNBs and LRL/DROPs co-localize to gametophytic cell nuclei and form heterodimers. LRL1/DROP1 and LRL2/DROP2 act redundantly to regulate BNB expression for generative cell specification in A. thaliana after asymmetric division of the haploid microspore. MpLRL is required for differentiation of MpBNB-expressing gametangium initial cells in M. polymorpha gametophytes. Our findings suggest that broadly expressed LRL/DROP stabilizes BNB expression, leading to the formation of an evolutionarily conserved bHLH heterodimer, which regulates germ cell differentiation in the haploid gametophyte of land plants.