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.

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.

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.

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.

Light Response Curves in Land Plants.

Light is the driving force for photosynthesis. Two techniques are commonly employed to help characterize the relationship between the light environment and photosynthesis in plants.Chlorophyll a fluorescence analysis is used to examine both the capacity for and the efficiency of the conversion of absorbed light into energy for photosynthesis. Additionally, gas exchange analysis is used to assess the utilization of that energy for carbon fixation. These techniques are used either in isolation or in combination to acquire light response curves that measure the response of the plant to sequential changes in irradiance. Light response curves can help users understand photosynthetic mechanisms, evaluate how plants respond to light conditions, or assess the extent of physiological plasticity within plants. In this chapter, we provide a generalized method for acquiring light response curves suitable for both chlorophyll a fluorescence and gas exchange techniques using commercially available apparatus. Depending on the equipment available, these methods can be applied individually or combined to acquire data simultaneously. The methods are broadly applicable to most land plants but are ideally suited to help those that are unfamiliar with these techniques.

Photosynthetic Gas Exchange in Land Plants at the Leaf Level.

Leaf-level gas exchange enables insights into the physiology and in vivo biochemical processes of plants. Advances in infrared gas analysis have resulted in user-friendly off-the-shelf gas exchange systems that allow researchers to collect physiological measurements with the push of a few buttons. Here, I describe how to set up the gas exchange equipment, what to pay attention to while making measurements, and provide some guidelines on how to analyze and interpret the data obtained.

Evolution of endosymbiosis-mediated nuclear calcium signaling in land plants.

The ability of fungi to establish mycorrhizal associations with plants and enhance the acquisition of mineral nutrients stands out as a key feature of terrestrial life. Evidence indicates that arbuscular mycorrhizal (AM) association is a trait present in the common ancestor of land plants,1,2,3,4 suggesting that AM symbiosis was an important adaptation for plants in terrestrial environments.5 The activation of nuclear calcium signaling in roots is essential for AM within flowering plants.6 Given that the earliest land plants lacked roots, whether nuclear calcium signals are required for AM in non-flowering plants is unknown. To address this question, we explored the functional conservation of symbiont-induced nuclear calcium signals between the liverwort Marchantia paleacea and the legume Medicago truncatula. In M. paleacea, AM fungi penetrate the rhizoids and form arbuscules in the thalli.7 Here, we demonstrate that AM germinating spore exudate (GSE) activates nuclear calcium signals in the rhizoids of M. paleacea and that this activation is dependent on the nuclear-localized ion channel DOES NOT MAKE INFECTIONS 1 (MpaDMI1). However, unlike flowering plants, MpaDMI1-mediated calcium signaling is only required for the thalli colonization but not for the AM penetration within rhizoids. We further demonstrate that the mechanism of regulation of DMI1 has diverged between M. paleacea and M. truncatula, including a key amino acid residue essential to sustain DMI1 in an inactive state. Our study reveals functional evolution of nuclear calcium signaling between liverworts and flowering plants and opens new avenues of research into the mechanism of endosymbiosis signaling.

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.

Cryogenian Origins of Multicellularity in Archaeplastida.

Earth was impacted by global glaciations during the Cryogenian (720 to 635 million years ago; Ma), events invoked to explain both the origins of multicellularity in Archaeplastida and radiation of the first land plants. However, the temporal relationship between these environmental and biological events is poorly established, due to a paucity of molecular and fossil data, precluding resolution of the phylogeny and timescale of archaeplastid evolution. We infer a time-calibrated phylogeny of early archaeplastid evolution based on a revised molecular dataset and reappraisal of the fossil record. Phylogenetic topology testing resolves deep archaeplastid relationships, identifying two clades of Viridiplantae and placing Bryopsidales as sister to the Chlorophyceae. Our molecular clock analysis infers an origin of Archaeplastida in the late-Paleoproterozoic to early-Mesoproterozoic (1712 to 1387 Ma). Ancestral state reconstruction of cytomorphological traits on this time-calibrated tree reveals many of the independent origins of multicellularity span the Cryogenian, consistent with the Cryogenian multicellularity hypothesis. Multicellular rhodophytes emerged 902 to 655 Ma while crown-Anydrophyta (Zygnematophyceae and Embryophyta) originated 796 to 671 Ma, broadly compatible with the Cryogenian plant terrestrialization hypothesis. Our analyses resolve the timetree of Archaeplastida with age estimates for ancestral multicellular archaeplastids coinciding with the Cryogenian, compatible with hypotheses that propose a role of Snowball Earth in plant evolution.

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.

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.

Plant evolution: Streptophyte multicellularity, ecology, and the acclimatisation of plants to life on land.

Land plants are celebrated as one of the three great instances of complex multicellularity, but new phylogenomic and phenotypic analyses are revealing deep evolutionary roots of multicellularity among algal relatives, prompting questions about the causal basis of this major evolutionary transition.

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.

Comparative analysis of SPL transcription factors from streptophyte algae and embryophytes reveals evolutionary trajectories of SPL family in streptophytes.

SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes encode plant-specific transcription factors which are important regulators of diverse plant developmental processes. We took advantage of available genome sequences of streptophyte algae representatives to investigate the relationships of SPL genes between freshwater green algae and land plants. Our analysis showed that streptophyte algae, hornwort and liverwort genomes encode from one to four SPL genes which is the smallest set, in comparison to other land plants studied to date. Based on the phylogenetic analysis, four major SPL phylogenetic groups were distinguished with Group 3 and 4 being sister to Group 1 and 2. Comparative motif analysis revealed conserved protein motifs within each phylogenetic group and unique bryophyte-specific motifs within Group 1 which suggests lineage-specific protein speciation processes. Moreover, the gene structure analysis also indicated the specificity of each by identifying differences in exon-intron structures between the phylogenetic groups, suggesting their evolutionary divergence. Since current understanding of SPL genes mostly arises from seed plants, the presented comparative and phylogenetic analyzes from freshwater green algae and land plants provide new insights on the evolutionary trajectories of the SPL gene family in different classes of streptophytes.

d-amino acids metabolism reflects the evolutionary origin of higher plants and their adaptation to the environment.

d-amino acids are the d stereoisomers of the common l-amino acids found in proteins. Over the past two decades, the occurrence of d-amino acids in plants has been reported and circumstantial evidence for a role in various processes, including interaction with soil microorganisms or interference with cellular signalling, has been provided. However, examples are not numerous and d-amino acids can also be detrimental, some of them inhibiting growth and development. Thus, the persistence of d-amino acid metabolism in plants is rather surprising, and the evolutionary origins of d-amino acid metabolism are currently unclear. Systemic analysis of sequences associated with d-amino acid metabolism enzymes shows that they are not simply inherited from cyanobacterial metabolism. In fact, the history of plant d-amino acid metabolism enzymes likely involves multiple steps, cellular compartments, gene transfers and losses. Regardless of evolutionary steps, enzymes of d-amino acid metabolism, such as d-amino acid transferases or racemases, have been retained by higher plants and have not simply been eliminated, so it is likely that they fulfil important metabolic roles such as serine, folate or plastid peptidoglycan metabolism. We suggest that d-amino acid metabolism may have been critical to support metabolic functions required during the evolution of land plants.