The evolution of plant responses underlying specialized metabolism in host-pathogen interactions.

In the course of plant evolution from aquatic to terrestrial environments, land plants (embryophytes) acquired a diverse array of specialized metabolites, including phenylpropanoids, flavonoids and cuticle components, enabling adaptation to various environmental stresses. While embryophytes and their closest algal relatives share candidate enzymes responsible for producing some of these compounds, the complete genetic network for their biosynthesis emerged in embryophytes. In this review, we analysed genomic data from chlorophytes, charophytes and embryophytes to identify genes related to phenylpropanoid, flavonoid and cuticle biosynthesis. By integrating published research, transcriptomic data and metabolite studies, we provide a comprehensive overview on how these specialized metabolic pathways have contributed to plant defence responses to pathogens in non-vascular bryophytes and vascular plants throughout evolution. The evidence suggests that these biosynthetic pathways have provided land plants with a repertoire of conserved and lineage-specific compounds, which have shaped immunity against invading pathogens. The discovery of additional enzymes and metabolites involved in bryophyte responses to pathogen infection will provide evolutionary insights into these versatile pathways and their impact on environmental terrestrial challenges.This article is part of the theme issue 'The evolution of plant metabolism'.

Zygospore formation in Zygnematophyceae predates several land plant traits.

Recent research on a special type of sexual reproduction and zygospore formation in Zygnematophyceae, the sister group of land plants, is summarized. Within this group, gamete fusion occurs by conjugation. Zygospore development in Mougeotia, Spirogyra and Zygnema is highlighted, which has recently been studied using Raman spectroscopy, allowing chemical imaging and detection of changes in starch and lipid accumulation. Three-dimensional reconstructions after serial block-face scanning electron microscopy (SBF-SEM) or focused ion beam SEM (FIB-SEM) made it possible to visualize and quantify cell wall and organelle changes during zygospore development. The zygospore walls undergo strong modifications starting from uniform thin cell walls to a multilayered structure. The mature cell wall is composed of a cellulosic endospore and exospore and a central mesospore built up by aromatic compounds. In Spirogyra, the exospore and endospore consist of thick layers of helicoidally arranged cellulose fibrils, which are otherwise only known from stone cells of land plants. While starch is degraded during maturation, providing building blocks for cell wall formation, lipid droplets accumulate and fill large parts of the ripe zygospores, similar to spores and seeds of land plants. Overall, data show similarities between streptophyte algae and embryophytes, suggesting that the genetic toolkit for many land plant traits already existed in their shared algal ancestor. This article is part of the theme issue 'The evolution of plant metabolism'.

Biotic interactions, evolutionary forces and the pan-plant specialized metabolism.

Plant specialized metabolism has a complex evolutionary history. Some aspects are conserved across the green lineage, but many metabolites are unique to certain lineages. The network of specialized metabolism continuously diversified, simplified or reshaped during the evolution of streptophytes. Many routes of pan-plant specialized metabolism are involved in plant defence. Biotic interactions are recalled as major drivers of lineage-specific metabolomic diversification. However, the consequences of this diversity of specialized metabolism in the context of plant terrestrialization and land plant diversification into the major lineages of bryophytes, lycophytes, ferns, gymnosperms and angiosperms remain only little explored. Overall, this hampers conclusions on the evolutionary scenarios that shaped specialized metabolism. Recent efforts have brought forth new streptophyte model systems, an increase in genetically accessible species from distinct major plant lineages, and new functional data from a diversity of land plants on specialized metabolic pathways. In this review, we will integrate the recent data on the evolution of the plant immune system with the molecular data of specialized metabolism and its recognition. Based on this we will provide a contextual framework of the pan-plant specialized metabolism, the evolutionary aspects that shape it and the impact on adaptation to the terrestrial environment.This article is part of the theme issue 'The evolution of plant metabolism'.

The southwesternmost record of late Silurian (Pridolian) early land plants of Gondwana.

The Silurian-Devonian transition played a crucial role in the development of early terrestrial ecosystems due to the rapid diversification of early vascular plants. However, records of Pridolian plants in western Gondwana are scarce, limited to outcrops located in southern Bolivia. In this contribution, an association of fossil plants housed in the Rinconada Formation is presented. This association corresponds to primitive fossil flora with reproductive structures and sterile axes linked to basal tracheophytes. The fossil assemblage is composed of Aberlemnia caledonica, Caia langii Cooksonia cf. cambrensis, C. paranensis, C. cf. pertoni, Hostinella sp, Cf. Isidrophyton sp, Salopella marcensis, Steganoteca striata, two morphotypes of doubtful taxonomy, and graptolites colonies. The association between flora remains and graptolites, represents a parautochthonous assemblage in an inner marine platform, dominated by gravity flows. This record has paleophytogeographic importance indicating the extension of the northwest Gondwana-southern Laurusia unit to more southern areas of Gondwana. This expansion would have been favored by the post-glacial climatic improvement of the Late Silurian, together with a great radiation capacity and environmental flexibility of the flora. Furthermore, the biochron is extended of three taxa (A. caledonica, C. paranensis and Cf. Isidrophyton sp) first known from the Lochkovian, to the Pridoli.

Evolution of small molecule-mediated regulation of arbuscular mycorrhiza symbiosis.

The arbuscular mycorrhizal (AM) symbiosis formed by most extant land plants with symbiotic fungi evolved 450 Ma. AM promotes plant growth by improving mineral nutrient and water uptake, while the symbiotic fungi obtain carbon in return. A number of plant genes regulating the steps leading to an efficient symbiosis have been identified; however, our understanding of the metabolic processes involved in the symbiosis and how they were wired to symbiosis regulation during plant evolution remains limited. Among them, the exchange of chemical signals, the activation of dedicated biosynthesis pathways and the production of secondary metabolites regulating late stages of the AM symbiosis begin to be well described across several land plant clades. Here, we review our current understanding of these processes and propose future directions to fully grasp the phylogenetic distribution and role played by small molecules during this ancient plant symbiosis. This article is part of the theme issue 'The evolution of plant metabolism'.

Presence of vitamin B12 metabolism in the last common ancestor of land plants.

Vitamin B12, also known as cobalamin, is an essential organic cofactor for methionine synthase (METH), and is only synthesized by a subset of bacteria. Plants and fungi have an alternative methionine synthase (METE) that does not need B12 and are typically considered not to utilize it. Some algae facultatively utilize B12 because they encode both METE and METH, while other algae are dependent on B12 as they encode METH only. We performed phylogenomic analyses of METE, METH and 11 further proteins involved in B12 metabolism across more than 1600 plant and algal genomes and transcriptomes (e.g. from OneKp), demonstrating the presence of B12-associated metabolism deep into the streptophytes. METH and five further accessory proteins (MTRR, CblB, CblC, CblD and CblJ) were detected in the hornworts (Anthocerotophyta), and two (CblB and CblJ) were identified in liverworts (Marchantiophyta) in the bryophytes, suggesting a retention of B12-metabolism in the last common land plant ancestor. Our data further show more limited distributions for other B12-related proteins (MCM and RNR-II) and B12 dependency in several algal orders. Finally, considering the collection sites of algae that have lost B12 metabolism, we propose freshwater-to-land transitions and symbiotic associations to have been constraining factors for B12 availability in early plant evolution. This article is part of the theme issue 'The evolution of plant metabolism'.

The evolution of flavonoid biosynthesis.

The flavonoid pathway is characteristic of land plants and a central biosynthetic component enabling life in a terrestrial environment. Flavonoids provide tolerance to both abiotic and biotic stresses and facilitate beneficial relationships, such as signalling to symbiont microorganisms, or attracting pollinators and seed dispersal agents. The biosynthetic pathway shows great diversity across species, resulting principally from repeated biosynthetic gene duplication and neofunctionalization events during evolution. Such events may reflect a selection for new flavonoid structures with novel functions that enable occupancy of varied ecological niches. However, the biochemical and genetic diversity of the pathway also likely resulted from evolution along parallel trends across land plant lineages, producing variant compounds with similar biological functions. Analyses of the wide range of whole-plant genome sequences now available, particularly for archegoniate plants, have enabled proposals on which genes were ancestral to land plants and which arose within the land plant lineages. In this review, we discuss the emerging proposals for how the flavonoid pathway may have evolved and diversified. This article is part of the theme issue 'The evolution of plant metabolism'.

Plant terrestrialization: an environmental pull on the evolution of multi-sourced streptophyte phenolics.

Phenolic compounds of land plants are varied: they are chemodiverse, are sourced from different biosynthetic routes and fulfil a broad spectrum of functions that range from signalling phytohormones, to protective shields against stressors, to structural compounds. Their action defines the biology of land plants as we know it. Often, their roles are tied to environmental responses that, however, impacted already the algal progenitors of land plants, streptophyte algae. Indeed, many streptophyte algae successfully dwell in terrestrial habitats and have homologues for enzymatic routes for the production of important phenolic compounds, such as the phenylpropanoid pathway. Here, we synthesize what is known about the production of specialized phenolic compounds across hundreds of millions of years of streptophyte evolution. We propose an evolutionary scenario in which selective pressures borne out of environmental cues shaped the chemodiversity of phenolics in streptophytes. This article is part of the theme issue 'The evolution of plant metabolism'.

An ancient role for CYP73 monooxygenases in phenylpropanoid biosynthesis and embryophyte development.

The phenylpropanoid pathway is one of the plant metabolic pathways most prominently linked to the transition to terrestrial life, but its evolution and early functions remain elusive. Here, we show that activity of the t-cinnamic acid 4-hydroxylase (C4H), the first plant-specific step in the pathway, emerged concomitantly with the CYP73 gene family in a common ancestor of embryophytes. Through structural studies, we identify conserved CYP73 residues, including a crucial arginine, that have supported C4H activity since the early stages of its evolution. We further demonstrate that impairing C4H function via CYP73 gene inactivation or inhibitor treatment in three bryophyte species-the moss Physcomitrium patens, the liverwort Marchantia polymorpha and the hornwort Anthoceros agrestis-consistently resulted in a shortage of phenylpropanoids and abnormal plant development. The latter could be rescued in the moss by exogenous supply of p-coumaric acid, the product of C4H. Our findings establish the emergence of the CYP73 gene family as a foundational event in the development of the plant phenylpropanoid pathway, and underscore the deep-rooted function of the C4H enzyme in embryophyte biology.

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.

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.

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.

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 evolution and development of sporangia-The fundamental reproductive organ of land plant sporophytes.

A key innovation of land plants is the origin and evolution of the sporangium, the fundamental reproductive structure of the diploid sporophyte. In vascular plants, whether the structure is a cone, fertile leaf, or flower-all are clusters of sporangia. The evolution of morphologically distinct sporangia (heterospory) and retention of the gametophyte evolved three times independently as a prerequisite for the evolution of seeds. This review summarizes the development of vascular plant sporangia, molecular genetics of angiosperm sporangia, and provides a framework to investigate evolution and development in vascular plant sporangia.

Reaction-diffusion modeling provides insights into biophysical carbon-concentrating mechanisms in land plants.

Carbon-concentrating mechanisms (CCMs) have evolved numerous times in photosynthetic organisms. They elevate the concentration of CO2 around the carbon-fixing enzyme rubisco, thereby increasing CO2 assimilatory flux and reducing photorespiration. Biophysical CCMs, like the pyrenoid-based CCM (PCCM) of Chlamydomonas reinhardtii or carboxysome systems of cyanobacteria, are common in aquatic photosynthetic microbes, but in land plants appear only among the hornworts. To predict the likely efficiency of biophysical CCMs in C3 plants, we used spatially resolved reaction-diffusion models to predict rubisco saturation and light use efficiency. We found that the energy efficiency of adding individual CCM components to a C3 land plant is highly dependent on the permeability of lipid membranes to CO2, with values in the range reported in the literature that are higher than those used in previous modeling studies resulting in low light use efficiency. Adding a complete PCCM into the leaf cells of a C3 land plant was predicted to boost net CO2 fixation, but at higher energetic costs than those incurred by photorespiratory losses without a CCM. Two notable exceptions were when substomatal CO2 levels are as low as those found in land plants that already use biochemical CCMs and when gas exchange is limited, such as with hornworts, making the use of a biophysical CCM necessary to achieve net positive CO2 fixation under atmospheric CO2 levels. This provides an explanation for the uniqueness of hornworts' CCM among land plants and the evolution of pyrenoids multiple times.

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.

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.

Unearthing the soil-borne microbiome of land plants.

Plant-soil biodiversity interactions are fundamental for the functioning of terrestrial ecosystems. Yet, the existence of a set of globally distributed topsoil microbial and small invertebrate organisms consistently associated with land plants (i.e., their consistent soil-borne microbiome), together with the environmental preferences and functional capabilities of these organisms, remains unknown. We conducted a standardized field survey under 150 species of land plants, including 58 species of bryophytes and 92 of vascular plants, across 124 locations from all continents. We found that, despite the immense biodiversity of soil organisms, the land plants evaluated only shared a small fraction (less than 1%) of all microbial and invertebrate taxa that were present across contrasting climatic and soil conditions and vegetation types. These consistent taxa were dominated by generalist decomposers and phagotrophs and their presence was positively correlated with the abundance of functional genes linked to mineralization. Finally, we showed that crossing environmental thresholds in aridity (aridity index of 0.65, i.e., the transition from mesic to dry ecosystems), soil pH (5.5; i.e., the transition from acidic to strongly acidic soils), and carbon (less than 2%, the lower limit of fertile soils) can result in drastic disruptions in the associations between land plants and soil organisms, with potential implications for the delivery of soil ecosystem processes under ongoing global environmental change.

Malondialdehyde Assays in Higher Plants.

Malondialdehyde is a three-carbon dialdehyde produced as a byproduct of polyunsaturated fatty acid peroxidation widely used as a marker of the extent of lipid peroxidation in plants. There are several methodological approaches to quantify malondialdehyde contents in higher plants, ranging from the simplest, cheapest, and quickest spectrophotometric approaches to the more complex ones using tandem mass spectrometry. This chapter summarizes the advantages and limitations of approaches followed and provides brief protocols with some tips to facilitate the selection of the best method for each experimental condition and application.