Rubisco is evolving for improved catalytic efficiency and CO2 assimilation in plants.

Rubisco is the primary entry point for carbon into the biosphere. However, rubisco is widely regarded as inefficient leading many to question whether the enzyme can adapt to become a better catalyst. Through a phylogenetic investigation of the molecular and kinetic evolution of Form I rubisco we uncover the evolutionary trajectory of rubisco kinetic evolution in angiosperms. We show that rbcL is among the 1% of slowest-evolving genes and enzymes on Earth, accumulating one nucleotide substitution every 0.9 My and one amino acid mutation every 7.2 My. Despite this, rubisco catalysis has been continually evolving toward improved CO2/O2 specificity, carboxylase turnover, and carboxylation efficiency. Consistent with this kinetic adaptation, increased rubisco evolution has led to a concomitant improvement in leaf-level CO2 assimilation. Thus, rubisco has been slowly but continually evolving toward improved catalytic efficiency and CO2 assimilation in plants.

The Quest for Orthologs orthology benchmark service in 2022.

The Orthology Benchmark Service (https://orthology.benchmarkservice.org) is the gold standard for orthology inference evaluation, supported and maintained by the Quest for Orthologs consortium. It is an essential resource to compare existing and new methods of orthology inference (the bedrock for many comparative genomics and phylogenetic analysis) over a standard dataset and through common procedures. The Quest for Orthologs Consortium is dedicated to maintaining the resource up to date, through regular updates of the Reference Proteomes and increasingly accessible data through the OpenEBench platform. For this update, we have added a new benchmark based on curated orthology assertion from the Vertebrate Gene Nomenclature Committee, and provided an example meta-analysis of the public predictions present on the platform.

Conditional stomatal closure in a fern shares molecular features with flowering plant active stomatal responses.

Stomata evolved as plants transitioned from water to land, enabling carbon dioxide uptake and water loss to be controlled. In flowering plants, the most recently divergent land plant lineage, stomatal pores actively close in response to drought. In this response, the phytohormone abscisic acid (ABA) triggers signaling cascades that lead to ion and water loss in the guard cells of the stomatal complex, causing a reduction in turgor and pore closure. Whether this stimulus-response coupling pathway acts in other major land plant lineages is unclear, with some investigations reporting that stomatal closure involves ABA but others concluding that closure is passive. Here, we show that in the model fern Ceratopteris richardii active stomatal closure is conditional on sensitization by pre-exposure to either low humidity or exogenous ABA and is promoted by ABA. RNA-seq analysis and de novo transcriptome assembly reconstructed the protein-coding complement of the C. richardii genome, with coverage comparable to other plant models, enabling transcriptional signatures of stomatal sensitization and closure to be inferred. In both cases, changes in abundance of homologs of ABA, Ca2+, and ROS-related signaling components were observed, suggesting that the closure-response pathway is conserved in ferns and flowering plants. These signatures further suggested that sensitization is achieved by lowering the threshold required for a subsequent closure-inducing signal to trigger a response. We conclude that the canonical signaling network for active stomatal closure functioned in at least a rudimentary form in the stomata of the last common ancestor of ferns and flowering plants.

Rubisco Adaptation Is More Limited by Phylogenetic Constraint Than by Catalytic Trade-off.

Rubisco assimilates CO2 to form the sugars that fuel life on earth. Correlations between rubisco kinetic traits across species have led to the proposition that rubisco adaptation is highly constrained by catalytic trade-offs. However, these analyses did not consider the phylogenetic context of the enzymes that were analyzed. Thus, it is possible that the correlations observed were an artefact of the presence of phylogenetic signal in rubisco kinetics and the phylogenetic relationship between the species that were sampled. Here, we conducted a phylogenetically resolved analysis of rubisco kinetics and show that there is a significant phylogenetic signal in rubisco kinetic traits. We re-evaluated the extent of catalytic trade-offs accounting for this phylogenetic signal and found that all were attenuated. Following phylogenetic correction, the largest catalytic trade-offs were observed between the Michaelis constant for CO2 and carboxylase turnover (∼21-37%), and between the Michaelis constants for CO2 and O2 (∼9-19%), respectively. All other catalytic trade-offs were substantially attenuated such that they were marginal (<9%) or non-significant. This phylogenetically resolved analysis of rubisco kinetic evolution also identified kinetic changes that occur concomitant with the evolution of C4 photosynthesis. Finally, we show that phylogenetic constraints have played a larger role than catalytic trade-offs in limiting the evolution of rubisco kinetics. Thus, although there is strong evidence for some catalytic trade-offs, rubisco adaptation has been more limited by phylogenetic constraint than by the combined action of all catalytic trade-offs.

NO GAMETOPHORES 2 Is a Novel Regulator of the 2D to 3D Growth Transition in the Moss Physcomitrella patens.

The colonization of land by plants was one of the most transformative events in the history of life on Earth. The transition from water, which coincided with and was likely facilitated by the evolution of three-dimensional (3D) growth, enabled the generation of morphological diversity on land. In many plants, the transition from two-dimensional (2D) to 3D growth occurs during embryo development. However, in the early divergent moss Physcomitrella patens, 3D growth is preceded by an extended filamentous phase that can be maintained indefinitely. Here, we describe the identification of the cytokinin-responsive NO GAMETOPHORES 2 (PpNOG2) gene, which encodes a shikimate o-hydroxycinnamoyltransferase. In mutants lacking PpNOG2 function, transcript levels of CLAVATA and SCARECROW genes are significantly reduced, excessive gametophore initial cells are produced, and buds undergo premature developmental arrest. Mutants also exhibit misregulation of auxin-responsive genes. Our results suggest that PpNOG2 functions in the ascorbic acid pathway leading to cuticle formation and that NOG2-related genes were co-opted into the lignin biosynthesis pathway after the divergence of bryophytes and vascular plants. We present a revised model of 3D growth in which PpNOG2 comprises part of a feedback mechanism that is required for the modulation of gametophore initial cell frequency. We also propose that the 2D to 3D growth transition in P. patens is underpinned by complex auxin-cytokinin crosstalk that is regulated, at least in part, by changes in flavonoid metabolism.

Gene expression data support the hypothesis that Isoetes rootlets are true roots and not modified leaves.

Rhizomorphic lycopsids are the land plant group that includes the first giant trees to grow on Earth and extant species in the genus Isoetes. Two mutually exclusive hypotheses account for the evolution of terminal rooting axes called rootlets among the rhizomorphic lycopsids. One hypothesis states that rootlets are true roots, like roots in other lycopsids. The other states that rootlets are modified leaves. Here we test predictions of each hypothesis by investigating gene expression in the leaves and rootlets of Isoetes echinospora. We assembled the de novo transcriptome of axenically cultured I. echinospora. Gene expression signatures of I. echinospora rootlets and leaves were different. Furthermore, gene expression signatures of I. echinospora rootlets were similar to gene expression signatures of true roots of Selaginella moellendorffii and Arabidopsis thaliana. RSL genes which positively regulate cell differentiation in roots were either exclusively or preferentially expressed in the I. echinospora rootlets, S. moellendorffii roots and A. thaliana roots compared to the leaves of each respective species. Taken together, gene expression data from the de-novo transcriptome of I. echinospora are consistent with the hypothesis that Isoetes rootlets are true roots and not modified leaves.

Benchmarking Orthogroup Inference Accuracy: Revisiting Orthobench.

Orthobench is the standard benchmark to assess the accuracy of orthogroup inference methods. It contains 70 expert-curated reference orthogroups (RefOGs) that span the Bilateria and cover a range of different challenges for orthogroup inference. Here, we leveraged improvements in tree inference algorithms and computational resources to reinterrogate these RefOGs and carry out an extensive phylogenetic delineation of their composition. This phylogenetic revision altered the membership of 31 of the 70 RefOGs, with 24 subject to extensive revision and 7 that required minor changes. We further used these revised and updated RefOGs to provide an assessment of the orthogroup inference accuracy of widely used orthogroup inference methods. Finally, we provide an open-source benchmarking suite to support the future development and use of the Orthobench benchmark.

Agricultural Selection of Wheat Has Been Shaped by Plant-Microbe Interactions.

The influence of wheat (modern wheat, both bread and pasta, their wild ancestors and synthetic hybrids) on the microbiota of their roots and surrounding soil is characterized. We isolated lines of bread wheat by hybridizing diploid (Aegilops tauschii) with tetraploid Triticum durum and crossed it with a modern cultivar of Triticum aestivum. The newly created, synthetic hybrid wheat, which recapitulate the breeding history of wheat through artificial selection, is found to support a microbiome enriched in beneficial Glomeromycetes fungi, but also in, potentially detrimental, Nematoda. We hypothesize that during wheat domestication this plant-microbe interaction diminished, suggesting an evolutionary tradeoff; sacrificing advantageous nutrient acquisition through fungal interactions to minimize interaction with pathogenic fungi. Increased plant selection for Glomeromycetes and Nematoda is correlated with the D genome derived from A. tauschii. Despite differences in their soil microbiota communities, overall wheat plants consistently show a low ratio of eukaryotes to prokaryotes. We propose that this is a mechanism for protection against soil-borne fungal disease and appears to be deeply rooted in the wheat genome. We suggest that the influence of plants on the composition of their associated microbiota is an integral factor, hitherto overlooked, but intrinsic to selection during wheat domestication.

Multiple Metabolic Innovations and Losses Are Associated with Major Transitions in Land Plant Evolution.

Investigating the evolution of plant biochemistry is challenging because few metabolites are preserved in fossils and because metabolic networks are difficult to experimentally characterize in diverse extant organisms. We report a comparative computational approach based on whole-genome metabolic pathway databases of eight species representative of major plant lineages, combined with homologous relationships among genes of 72 species from streptophyte algae to angiosperms. We use this genomic approach to identify metabolic gains and losses during land plant evolution. We extended our findings with additional analysis of 305 non-angiosperm plant transcriptomes. Our results revealed that genes encoding the complete biosynthetic pathway for brassinosteroid phytohormones and enzymes for brassinosteroid inactivation are present only in spermatophytes. Genes encoding only part of the biosynthesis pathway are present in ferns and lycophytes, indicating a stepwise evolutionary acquisition of this pathway. Nevertheless, brassinosteroids are ubiquitous in land plants, suggesting that brassinosteroid biosynthetic pathways differ between earlier- and later-diverging lineages. Conversely, genes for gibberellin biosynthesis and inactivation using methyltransferases are found in all land plant lineages. This suggests that bioactive gibberellins might be present in bryophytes, although they have yet to be detected experimentally. We also found that cytochrome P450 oxidases involved in cutin and suberin production are absent in genomes of non-angiosperm plants that nevertheless do contain these biopolymers. Overall, we identified significant differences in crucial metabolic processes between angiosperms and earlier-diverging land plants and resolve details of the evolutionary history of several phytohormone and structural polymer biosynthetic pathways in land plants.

Gene Duplication Accelerates the Pace of Protein Gain and Loss from Plant Organelles.

Organelle biogenesis and function is dependent on the concerted action of both organellar-encoded (if present) and nuclear-encoded proteins. Differences between homologous organelles across the Plant Kingdom arise, in part, as a result of differences in the cohort of nuclear-encoded proteins that are targeted to them. However, neither the rate at which differences in protein targeting accumulate nor the evolutionary consequences of these changes are known. Using phylogenomic approaches coupled to ancestral state estimation, we show that the plant organellar proteome has diversified in proportion with molecular sequence evolution such that the proteomes of plant chloroplasts and mitochondria lose or gain on average 3.6 proteins per million years. We further demonstrate that changes in organellar protein targeting are associated with an increase in the rate of molecular sequence evolution and that such changes predominantly occur in genes with regulatory rather than metabolic functions. Finally, we show that gain and loss of protein target signals occurs at a higher rate following gene duplication, revealing that gene and genome duplication are a key facilitator of plant organelle evolution.

OrthoFinder: phylogenetic orthology inference for comparative genomics.

Here, we present a major advance of the OrthoFinder method. This extends OrthoFinder's high accuracy orthogroup inference to provide phylogenetic inference of orthologs, rooted gene trees, gene duplication events, the rooted species tree, and comparative genomics statistics. Each output is benchmarked on appropriate real or simulated datasets, and where comparable methods exist, OrthoFinder is equivalent to or outperforms these methods. Furthermore, OrthoFinder is the most accurate ortholog inference method on the Quest for Orthologs benchmark test. Finally, OrthoFinder's comprehensive phylogenetic analysis is achieved with equivalent speed and scalability to the fastest, score-based heuristic methods. OrthoFinder is available at https://github.com/davidemms/OrthoFinder.

STRIDE: Species Tree Root Inference from Gene Duplication Events.

The correct interpretation of any phylogenetic tree is dependent on that tree being correctly rooted. We present STRIDE, a fast, effective, and outgroup-free method for identification of gene duplication events and species tree root inference in large-scale molecular phylogenetic analyses. STRIDE identifies sets of well-supported in-group gene duplication events from a set of unrooted gene trees, and analyses these events to infer a probability distribution over an unrooted species tree for the location of its root. We show that STRIDE correctly identifies the root of the species tree in multiple large-scale molecular phylogenetic data sets spanning a wide range of timescales and taxonomic groups. We demonstrate that the novel probability model implemented in STRIDE can accurately represent the ambiguity in species tree root assignment for data sets where information is limited. Furthermore, application of STRIDE to outgroup-free inference of the origin of the eukaryotic tree resulted in a root probability distribution that provides additional support for leading hypotheses for the origin of the eukaryotes.

The Stepwise Increase in the Number of Transcription Factor Families in the Precambrian Predated the Diversification of Plants On Land.

The colonization of the land by streptophytes and their subsequent radiation is a major event in Earth history. We report a stepwise increase in the number of transcription factor (TF) families and subfamilies in Archaeplastida before the colonization of the land. The subsequent increase in TF number on land was through duplication within existing TF families and subfamilies. Almost all subfamilies of the Homeodomain (HD) and basic Helix-Loop-Helix (bHLH) had evolved before the radiation of extant land plant lineages from a common ancestor. We demonstrate that the evolution of these TF families independently followed similar trends in both plants and metazoans; almost all extant HD and bHLH subfamilies were present in the first land plants and in the last common ancestor of bilaterians. These findings reveal that the majority of innovation in plant and metazoan TF families occurred in the Precambrian before the Phanerozoic radiation of land plants and metazoans.

Independent and Parallel Evolution of New Genes by Gene Duplication in Two Origins of C4 Photosynthesis Provides New Insight into the Mechanism of Phloem Loading in C4 Species.

UNLABELLED: C4 photosynthesis is considered one of the most remarkable examples of evolutionary convergence in eukaryotes. However, it is unknown whether the evolution of C4 photosynthesis required the evolution of new genes. Genome-wide gene-tree species-tree reconciliation of seven monocot species that span two origins of C4 photosynthesis revealed that there was significant parallelism in the duplication and retention of genes coincident with the evolution of C4 photosynthesis in these lineages. Specifically, 21 orthologous genes were duplicated and retained independently in parallel at both C4 origins. Analysis of this gene cohort revealed that the set of parallel duplicated and retained genes is enriched for genes that are preferentially expressed in bundle sheath cells, the cell type in which photosynthesis was activated during C4 evolution. Furthermore, functional analysis of the cohort of parallel duplicated genes identified SWEET-13 as a potential key transporter in the evolution of C4 photosynthesis in grasses, and provides new insight into the mechanism of phloem loading in these C4 species. KEY WORDS: C4 photosynthesis, gene duplication, gene families, parallel evolution.

OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy.

Identifying homology relationships between sequences is fundamental to biological research. Here we provide a novel orthogroup inference algorithm called OrthoFinder that solves a previously undetected gene length bias in orthogroup inference, resulting in significant improvements in accuracy. Using real benchmark datasets we demonstrate that OrthoFinder is more accurate than other orthogroup inference methods by between 8 % and 33 %. Furthermore, we demonstrate the utility of OrthoFinder by providing a complete classification of transcription factor gene families in plants revealing 6.9 million previously unobserved relationships.